Bio

Bio


Thomas Christian Südhof was born in Göttingen, Germany, on Dec. 22 in 1955, obtained his M.D. and doctoral degrees from the University of Göttingen in 1982. He performed his doctoral thesis work at the Max-Planck-Institut für biophysikalische Chemie in Göttingen with Prof. Victor P. Whittaker on the biophysical structure of secretory granules. From 1983-1986, Südhof trained as a postdoctoral fellow with Drs. Mike Brown and Joe Goldstein at UT Southwestern in Dallas, TX, and elucidated the structure, expression and cholesterol-dependent regulation of the LDL receptor gene. Südhof began his independent career as an assistant professor at UT Southwestern in 1986. When Südhof started his laboratory, he decided to switch from cholesterol metabolism to neuroscience, and to pursue a molecular characterization of synaptic transmission. His work initially focused on the mechanism of neurotransmitter release which is the first step in synaptic transmission, and whose molecular basis was completely unknown in 1986. Later on, Südhof's work increasingly turned to the analysis of synapse formation and specification, processes that mediate the initial assembly of synapses, regulate their maintenance and elimination, and determine their properties. Südhof served on the faculty of UT Southwestern in Dallas until 2008, and among others was the founding chair of the Department of Neuroscience at that institution. In 2008, Südhof moved to Stanford, and became the Avram Goldstein Professor in the School of Medicine at Stanford University. In addition, Südhof has been an Investigator of the Howard Hughes Medical Institute since 1986.

Academic Appointments


Honors & Awards


  • Nobel Prize in Physiology or Medicine, Nobel Foundation (2013)
  • Lasker~DeBakey Basic Medical Research Award, Albert and Mary Lasker Foundation (2013)
  • Elected member, American Academy of Arts and Sciences (2010)
  • Kavli Prize in Neuroscience, Kavli Foundation (2010)
  • Elected member, Institute of Medicine (2008)
  • Elected member, National Academy of Sciences (2002)

Research & Scholarship

Current Research and Scholarly Interests


Human thought and perception, emotions and actions universally depend on signaling between neurons in the brain. This signalling largely happens at synapses, specialized intercellular junctions formed by pre- and postsynaptic neurons. When stimulated, a presynaptic neuron releases chemical messages—called neurotransmitters— that is recognized by a postsynaptic neuron.

For decades, the majority of neuroscientists focused their research on the postsynaptic neuron and its role in learning and memory. But throughout his career, Thomas Südhof has studied the presynaptic neuron. His collective findings have provided much of our current scientific understanding of presynaptic neuron behavior in neurotransmission and synapse formation. His work also has revealed the role of presynaptic neurons in neuropsychiatric illnesses, such as autism or neurodegenerative disorders.

Born in Germany, Südhof obtained a medical degree from the University of Gottingen in 1982. He became familiar with neuroscience when he performed research for his doctoral degree at the Max Planck Institute for Biophysical Chemistry. His thesis dealt with the release of hormones from adrenal cells, a model of neurotransmitter release.

To expand his knowledge of biochemistry and molecular biology, Südhof started to work in 1983 as a postdoctoral fellow at the laboratories of Michael Brown and Joseph Goldstein at the University of Texas Southwestern Medical Center at Dallas. He cloned the gene for the receptor of LDL (the low-density lipoprotein), a particle in the blood that transports cholesterol. Moreover, his work identified the sequences that mediate the regulation of the LDL receptor gene expression by cholesterol.

In 1986, Südhof started his own laboratory at UT Southwestern. He began his inquiry into the presynaptic neuron. At the time, what scientists mainly knew about the presynaptic neuron was that calcium ions stimulate the release of neurotransmitters from membrane-bound sacs called vesicles into the synapse, in a process that takes less than a millisecond.

But much was unknown: What allowed rapid neurotransmitter release? How did release occur at the specific region of the neuron—the synapse? How did repeated activity change the presynaptic neuron? How did the pre- and postsynaptic neurons come together at the synapse?

Südhof decided to try to answer these questions. Among the discoveries in his 20 years of research, Südhof revealed how synaptotagmin proteins sense calcium and mediate neurotransmitter release from presynaptic neurons. He also defined the molecules that organize release in space and time at a synapse, such as RIMs and Munc13's, and identified central components of the presynaptic machinery that mediate the fusion of synaptic vesicles containing neurotransmitters with the presynaptic plasma membrane, the process that ultimately causes neurotransmitter release, and that is controlled by synaptotagmins.

Südhof's work also revealed how pre- and postsynaptic proteins form physical connections, permitting neurotransmission. Specifically, he identified proteins on presynaptic neurons, called neurexins, and proteins on the postsynaptic neuron, called neuroligins, that bind to each other at the synapse. There are many types of neurexins and neuroligins. Their variable pairing shapes the wide variability in the types of synapses in the brain. Mutations in these proteins severely impair synapse function in mice, and contribute to the pathogenesis of disease such as autism and schizophrenia in humans.

At present, Südhof's lab attempts to build on these findings in defining the relationship between specific synaptic proteins and information processing in the brain, with its concordant manifestations in behavior. This large-scale project attempts to provide insight both into the mechanisms undelying synaptic communication, and the processes causing human disease.

Publications

Journal Articles


  • Ultrahigh-resolution imaging reveals formation of neuronal SNARE/Munc18 complexes in situ PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Pertsinidis, A., Mukherjee, K., Sharma, M., Pang, Z. P., Park, S. R., Zhang, Y., Brunger, A. T., Suedhof, T. C., Chu, S. 2013; 110 (30): E2812-E2820

    Abstract

    Membrane fusion is mediated by complexes formed by SNAP-receptor (SNARE) and Secretory 1 (Sec1)/mammalian uncoordinated-18 (Munc18)-like (SM) proteins, but it is unclear when and how these complexes assemble. Here we describe an improved two-color fluorescence nanoscopy technique that can achieve effective resolutions of up to 7.5-nm full width at half maximum (3.2-nm localization precision), limited only by stochastic photon emission from single molecules. We use this technique to dissect the spatial relationships between the neuronal SM protein Munc18-1 and SNARE proteins syntaxin-1 and SNAP-25 (25 kDa synaptosome-associated protein). Strikingly, we observed nanoscale clusters consisting of syntaxin-1 and SNAP-25 that contained associated Munc18-1. Rescue experiments with syntaxin-1 mutants revealed that Munc18-1 recruitment to the plasma membrane depends on the Munc18-1 binding to the N-terminal peptide of syntaxin-1. Our results suggest that in a primary neuron, SNARE/SM protein complexes containing syntaxin-1, SNAP-25, and Munc18-1 are preassembled in microdomains on the presynaptic plasma membrane. Our superresolution imaging method provides a framework for investigating interactions between the synaptic vesicle fusion machinery and other subcellular systems in situ.

    View details for DOI 10.1073/pnas.1310654110

    View details for Web of Science ID 000322112300011

    View details for PubMedID 23821748

  • Presynaptic Neurexin-3 Alternative Splicing trans-Synaptically Controls Postsynaptic AMPA Receptor Trafficking CELL Aoto, J., Martinelli, D. C., Malenka, R. C., Tabuchi, K., Suedhof, T. C. 2013; 154 (1): 75-88

    Abstract

    Neurexins are essential presynaptic cell adhesion molecules that are linked to schizophrenia and autism and are subject to extensive alternative splicing. Here, we used a genetic approach to test the physiological significance of neurexin alternative splicing. We generated knockin mice in which alternatively spliced sequence #4 (SS4) of neuexin-3 is constitutively included but can be selectively excised by cre-recombination. SS4 of neurexin-3 was chosen because it is highly regulated and controls neurexin binding to neuroligins, LRRTMs, and other ligands. Unexpectedly, constitutive inclusion of SS4 in presynaptic neurexin-3 decreased postsynaptic AMPA, but not NMDA receptor levels, and enhanced postsynaptic AMPA receptor endocytosis. Moreover, constitutive inclusion of SS4 in presynaptic neurexin-3 abrogated postsynaptic AMPA receptor recruitment during NMDA receptor-dependent LTP. These phenotypes were fully rescued by constitutive excision of SS4 in neurexin-3. Thus, alternative splicing of presynaptic neurexin-3 controls postsynaptic AMPA receptor trafficking, revealing an unanticipated alternative splicing mechanism for trans-synaptic regulation of synaptic strength and long-term plasticity.

    View details for DOI 10.1016/j.cell.2013.05.060

    View details for Web of Science ID 000321327900011

    View details for PubMedID 23827676

  • Synaptotagmin-12 Phosphorylation by cAMP-Dependent Protein Kinase Is Essential for Hippocampal Mossy Fiber LTP JOURNAL OF NEUROSCIENCE Kaeser-Woo, Y. J., Younts, T. J., Yang, X., Zhou, P., Wu, D., Castillo, P. E., Suedhof, T. C. 2013; 33 (23): 9769-9780

    Abstract

    Synaptotagmin-12 (Syt12) is an abundant synaptic vesicle protein that-different from other synaptic vesicle-associated synaptotagmins-does not bind Ca(2+). Syt12 is phosphorylated by cAMP-dependent protein kinase-A at serine-97 in an activity-dependent manner, suggesting a function for Syt12 in cAMP-dependent synaptic plasticity. To test this hypothesis, we here generated (1) Syt12 knock-out mice and (2) Syt12 knockin mice carrying a single amino-acid substitution [the serine-97-to-alanine- (S97A)-substitution]. Both Syt12 knock-out mice and Syt12 S97A-knockin mice were viable and fertile, and exhibited no measurable change in basal synaptic strength or short-term plasticity as analyzed in cultured cortical neurons or in acute hippocampal slices. However, both Syt12 knock-out and Syt12 S97A-knockin mice displayed a major impairment in cAMP-dependent mossy-fiber long-term potentiation (LTP) in the CA3 region of the hippocampus. This impairment was observed using different experimental configurations for inducing and monitoring mossy-fiber LTP. Moreover, although the Syt12 knock-out had no effect on the short-term potentiation of synaptic transmission induced by the adenylate-cyclase activator forskolin in cultured cortical neurons and in the CA1 region of the hippocampus, both the Syt12 knock-out and the Syt12 S97A-knockin impaired the long-term increase in mossy-fiber synaptic transmission induced by forskolin. Thus, Syt12 is essential for cAMP-dependent presynaptic LTP at mossy-fiber synapses, and a single amino-acid substitution that blocks the cAMP-dependent phosphorylation of Syt12 is sufficient to impair the function of Syt12 in mossy-fiber LTP, suggesting that cAMP-dependent phosphorylation of Syt12 on serine-97 contributes to the induction of mossy-fiber LTP.

    View details for DOI 10.1523/JNEUROSCI.5814-12.2013

    View details for Web of Science ID 000319963300020

    View details for PubMedID 23739973

  • Rapid single-step induction of functional neurons from human pluripotent stem cells. Neuron Zhang, Y., Pak, C., Han, Y., Ahlenius, H., Zhang, Z., Chanda, S., Marro, S., Patzke, C., Acuna, C., Covy, J., Xu, W., Yang, N., Danko, T., Chen, L., Wernig, M., Südhof, T. C. 2013; 78 (5): 785-798

    Abstract

    Available methods for differentiating human embryonic stem cells (ESCs) and induced pluripotent cells (iPSCs) into neurons are often cumbersome, slow, and variable. Alternatively, human fibroblasts can be directly converted into induced neuronal (iN) cells. However, with present techniques conversion is inefficient, synapse formation is limited, and only small amounts of neurons can be generated. Here, we show that human ESCs and iPSCs can be converted into functional iN cells with nearly 100% yield and purity in less than 2 weeks by forced expression of a single transcription factor. The resulting ES-iN or iPS-iN cells exhibit quantitatively reproducible properties independent of the cell line of origin, form mature pre- and postsynaptic specializations, and integrate into existing synaptic networks when transplanted into mouse brain. As illustrated by selected examples, our approach enables large-scale studies of human neurons for questions such as analyses of human diseases, examination of human-specific genes, and drug screening.

    View details for DOI 10.1016/j.neuron.2013.05.029

    View details for PubMedID 23764284

  • Autism-associated neuroligin-3 mutations commonly disrupt tonic endocannabinoid signaling. Neuron Földy, C., Malenka, R. C., Südhof, T. C. 2013; 78 (3): 498-509

    Abstract

    Neuroligins are postsynaptic cell-adhesion molecules that interact with presynaptic neurexins. Rare mutations in neuroligins and neurexins predispose to autism, including a neuroligin-3 amino acid substitution (R451C) and a neuroligin-3 deletion. Previous analyses showed that neuroligin-3 R451C-knockin mice exhibit robust synaptic phenotypes but failed to uncover major changes in neuroligin-3 knockout mice, questioning the notion that a common synaptic mechanism mediates autism pathogenesis in patients with these mutations. Here, we used paired recordings in mice carrying these mutations to measure synaptic transmission at GABAergic synapses formed by hippocampal parvalbumin- and cholecystokinin-expressing basket cells onto pyramidal neurons. We demonstrate that in addition to unique gain-of-function effects produced by the neuroligin-3 R451C-knockin but not the neuroligin-3 knockout mutation, both mutations dramatically impaired tonic but not phasic endocannabinoid signaling. Our data thus suggest that neuroligin-3 is specifically required for tonic endocannabinoid signaling, raising the possibility that alterations in endocannabinoid signaling may contribute to autism pathophysiology.

    View details for DOI 10.1016/j.neuron.2013.02.036

    View details for PubMedID 23583622

  • A neural circuit for memory specificity and generalization. Science Xu, W., Südhof, T. C. 2013; 339 (6125): 1290-1295

    Abstract

    Increased fear memory generalization is associated with posttraumatic stress disorder, but the circuit mechanisms that regulate memory specificity remain unclear. Here, we define a neural circuit-composed of the medial prefrontal cortex, the nucleus reuniens (NR), and the hippocampus-that controls fear memory generalization. Inactivation of prefrontal inputs into the NR or direct silencing of NR projections enhanced fear memory generalization, whereas constitutive activation of NR neurons decreased memory generalization. Direct optogenetic activation of phasic and tonic action-potential firing of NR neurons during memory acquisition enhanced or reduced memory generalization, respectively. We propose that the NR determines the specificity and generalization of memory attributes for a particular context by processing information from the medial prefrontal cortex en route to the hippocampus.

    View details for DOI 10.1126/science.1229534

    View details for PubMedID 23493706

  • LTP Requires a Unique Postsynaptic SNARE Fusion Machinery NEURON Jurado, S., Goswami, D., Zhang, Y., Minano Molina, A. J., Suedhof, T. C., Malenka, R. C. 2013; 77 (3): 542-558

    Abstract

    Membrane fusion during exocytosis is mediated by assemblies of SNARE (soluble NSF-attachment protein receptor) and SM (Sec1/Munc18-like) proteins. The SNARE/SM proteins involved in vesicle fusion during neurotransmitter release are well understood, whereas little is known about the protein machinery that mediates activity-dependent AMPA receptor (AMPAR) exocytosis during long-term potentiation (LTP). Using direct measurements of LTP in acute hippocampal slices and an in vitro LTP model of stimulated AMPAR exocytosis, we demonstrate that the Q-SNARE proteins syntaxin-3 and SNAP-47 are required for regulated AMPAR exocytosis during LTP but not for constitutive basal AMPAR exocytosis. In contrast, the R-SNARE protein synaptobrevin-2/VAMP2 contributes to both regulated and constitutive AMPAR exocytosis. Both the central complexin-binding and the N-terminal Munc18-binding sites of syntaxin-3 are essential for its postsynaptic role in LTP. Thus, postsynaptic exocytosis of AMPARs during LTP is mediated by a unique fusion machinery that is distinct from that used during presynaptic neurotransmitter release.

    View details for DOI 10.1016/j.neuron.2012.11.029

    View details for Web of Science ID 000317030800015

    View details for PubMedID 23395379

  • Complexin Activates Exocytosis of Distinct Secretory Vesicles Controlled by Different Synaptotagmins JOURNAL OF NEUROSCIENCE Cao, P., Yang, X., Suedhof, T. C. 2013; 33 (4): 1714-?

    Abstract

    Complexins are SNARE-complex binding proteins essential for the Ca(2+)-triggered exocytosis mediated by synaptotagmin-1, -2, -7, or -9, but the possible role of complexins in other types of exocytosis controlled by other synaptotagmin isoforms remains unclear. Here we show that, in mouse olfactory bulb neurons, synaptotagmin-1 localizes to synaptic vesicles and to large dense-core secretory vesicles as reported previously, whereas synaptotagmin-10 localizes to a distinct class of peptidergic secretory vesicles containing IGF-1. Both synaptotagmin-1-dependent synaptic vesicle exocytosis and synaptotagmin-10-dependent IGF-1 exocytosis were severely impaired by knockdown of complexins, demonstrating that complexin acts as a cofactor for both synaptotagmin-1 and synaptotagmin-10 despite the functional differences between these synaptotagmins. Rescue experiments revealed that only the activating but not the clamping function of complexins was required for IGF-1 exocytosis controlled by synaptotagmin-10. Thus, our data indicate that complexins are essential for activation of multiple types of Ca(2+)-induced exocytosis that are regulated by different synaptotagmin isoforms. These results suggest that different types of regulated exocytosis are mediated by similar synaptotagmin-dependent fusion mechanisms, that particular synaptotagmin isoforms confer specificity onto different types of regulated exocytosis, and that complexins serve as universal synaptotagmin adaptors for all of these types of exocytosis independent of which synaptotagmin isoform is involved.

    View details for DOI 10.1523/JNEUROSCI.4087-12.2013

    View details for Web of Science ID 000313956900041

    View details for PubMedID 23345244

  • Syntaxin-1 N-peptide and H-abc-domain perform distinct essential functions in synaptic vesicle fusion EMBO JOURNAL Zhou, P., Pang, Z. P., Yang, X., Zhang, Y., Rosenmund, C., Bacaj, T., Suedhof, T. C. 2013; 32 (1): 159-171

    Abstract

    Among SNARE proteins mediating synaptic vesicle fusion, syntaxin-1 uniquely includes an N-terminal peptide ('N-peptide') that binds to Munc18-1, and a large, conserved H(abc)-domain that also binds to Munc18-1. Previous in vitro studies suggested that the syntaxin-1 N-peptide is functionally important, whereas the syntaxin-1 H(abc)-domain is not, but limited information is available about the in vivo functions of these syntaxin-1 domains. Using rescue experiments in cultured syntaxin-deficient neurons, we now show that the N-peptide and the H(abc)-domain of syntaxin-1 perform distinct and independent roles in synaptic vesicle fusion. Specifically, we found that the N-peptide is essential for vesicle fusion as such, whereas the H(abc)-domain regulates this fusion, in part by forming the closed syntaxin-1 conformation. Moreover, we observed that deletion of the H(abc)-domain but not deletion of the N-peptide caused a loss of Munc18-1 which results in a decrease in the readily releasable pool of vesicles at a synapse, suggesting that Munc18 binding to the H(abc)-domain stabilizes Munc18-1. Thus, the N-terminal syntaxin-1 domains mediate different functions in synaptic vesicle fusion, probably via formation of distinct Munc18/SNARE-protein complexes.

    View details for DOI 10.1038/emboj.2012.307

    View details for Web of Science ID 000314141900015

    View details for PubMedID 23188083

  • MDGAs interact selectively with neuroligin-2 but not other neuroligins to regulate inhibitory synapse development PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Lee, K., Kim, Y., Lee, S., Qiang, Y., Lee, D., Lee, H. W., Kim, H., Je, H. S., Suedhof, T. C., Ko, J. 2013; 110 (1): 336-341

    Abstract

    The MAM domain-containing GPI anchor proteins MDGA1 and MDGA2 are Ig superfamily adhesion molecules composed of six IG domains, a fibronectin III domain, a MAM domain, and a GPI anchor. MDGAs contribute to the radial migration and positioning of a subset of cortical neurons during early neural development. However, MDGAs continue to be expressed in postnatal brain, and their functions during postnatal neural development remain unknown. Here, we demonstrate that MDGAs specifically and with a nanomolar affinity bind to neuroligin-2, a cell-adhesion molecule of inhibitory synapses, but do not bind detectably to neuroligin-1 or neuroligin-3. We observed no cell adhesion between cells expressing neuroligin-2 and MDGA1, suggesting a cis interaction. Importantly, RNAi-mediated knockdown of MDGAs increased the abundance of inhibitory but not excitatory synapses in a neuroligin-2-dependent manner. Conversely, overexpression of MDGA1 decreased the numbers of functional inhibitory synapses. Likewise, coexpression of both MDGA1 and neuroligin-2 reduced the synaptogenic capacity of neuroligin-2 in an artificial synapse-formation assay by abolishing the ability of neuroligin-2 to form an adhesion complex with neurexins. Taken together, our data suggest that MDGAs inhibit the activity of neuroligin-2 in controlling the function of inhibitory synapses and that MDGAs do so by binding to neuroligin-2.

    View details for DOI 10.1073/pnas.1219987110

    View details for Web of Science ID 000313630300073

    View details for PubMedID 23248271

  • Native a-synuclein induces clustering of synaptic-vesicle mimics via binding to phospholipids and synaptobrevin-2/VAMP2. eLife Diao, J., Burré, J., Vivona, S., Cipriano, D. J., Sharma, M., Kyoung, M., Südhof, T. C., Brunger, A. T. 2013; 2

    Abstract

    ?-Synuclein is a presynaptic protein that is implicated in Parkinson's and other neurodegenerative diseases. Physiologically, native ?-synuclein promotes presynaptic SNARE-complex assembly, but its molecular mechanism of action remains unknown. Here, we found that native ?-synuclein promotes clustering of synaptic-vesicle mimics, using a single-vesicle optical microscopy system. This vesicle-clustering activity was observed for both recombinant and native ?-synuclein purified from mouse brain. Clustering was dependent on specific interactions of native ?-synuclein with both synaptobrevin-2/VAMP2 and anionic lipids. Out of the three familial Parkinson's disease-related point mutants of ?-synuclein, only the lipid-binding deficient mutation A30P disrupted clustering, hinting at a possible loss of function phenotype for this mutant. ?-Synuclein had little effect on Ca(2+)-triggered fusion in our reconstituted single-vesicle system, consistent with in vivo data. ?-Synuclein may therefore lead to accumulation of synaptic vesicles at the active zone, providing a 'buffer' of synaptic vesicles, without affecting neurotransmitter release itself. DOI:http://dx.doi.org/10.7554/eLife.00592.001.

    View details for DOI 10.7554/eLife.00592

    View details for PubMedID 23638301

  • Local use of geographic information systems to improve data utilisation and health services: mapping caesarean section coverage in rural Rwanda TROPICAL MEDICINE & INTERNATIONAL HEALTH Sudhof, L., Amoroso, C., Barebwanuwe, P., Munyaneza, F., Karamaga, A., Zambotti, G., Drobac, P., Hirschhorn, L. R. 2013; 18 (1): 18-26

    Abstract

    To show the utility of combining routinely collected data with geographic location using a Geographic Information System (GIS) in order to facilitate a data-driven approach to identifying potential gaps in access to emergency obstetric care within a rural Rwandan health district.Total expected births in 2009 at sub-district levels were estimated using community health worker collected population data. Clinical data were extracted from birth registries at eight health centres (HCs) and the district hospital (DH). C-section rates as a proportion of total expected births were mapped by cell. Peri-partum foetal mortality rates per facility-based births, as well as the rate of uterine rupture as an indication for C-section, were compared between areas of low and high C-section rates.The lowest C-section rates were found in the more remote part of the hospital catchment area. The sector with significantly lower C-section rates had significantly higher facility-based peri-partum foetal mortality and incidence of uterine rupture than the sector with the highest C-section rates (P < 0.034).This simple approach for geographic monitoring and evaluation leveraging existing health service and GIS data facilitated evidence-based decision making and represents a feasible approach to further strengthen local data-driven decisions for resource allocation and quality improvement.

    View details for DOI 10.1111/tmi.12016

    View details for Web of Science ID 000312739700004

    View details for PubMedID 23279379

  • Candidate autism gene screen identifies critical role for cell-adhesion molecule CASPR2 in dendritic arborization and spine development PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Anderson, G. R., Galfin, T., Xu, W., Aoto, J., Malenka, R. C., Suedhof, T. C. 2012; 109 (44): 18120-18125

    Abstract

    Mutations in the contactin-associated protein 2 (CNTNAP2) gene encoding CASPR2, a neurexin-related cell-adhesion molecule, predispose to autism, but the function of CASPR2 in neural circuit assembly remains largely unknown. In a knockdown survey of autism candidate genes, we found that CASPR2 is required for normal development of neural networks. RNAi-mediated knockdown of CASPR2 produced a cell-autonomous decrease in dendritic arborization and spine development in pyramidal neurons, leading to a global decline in excitatory and inhibitory synapse numbers and a decrease in synaptic transmission without a detectable change in the properties of these synapses. Our data suggest that in addition to the previously described role of CASPR2 in mature neurons, where CASPR2 organizes nodal microdomains of myelinated axons, CASPR2 performs an earlier organizational function in developing neurons that is essential for neural circuit assembly and operates coincident with the time of autism spectrum disorder (ASD) pathogenesis.

    View details for DOI 10.1073/pnas.1216398109

    View details for Web of Science ID 000311149900087

    View details for PubMedID 23074245

  • Systematic mutagenesis of a-synuclein reveals distinct sequence requirements for physiological and pathological activities. journal of neuroscience Burré, J., Sharma, M., Südhof, T. C. 2012; 32 (43): 15227-15242

    Abstract

    -Synuclein is an abundant presynaptic protein that binds to phospholipids and synaptic vesicles. Physiologically, ?-synuclein functions as a SNARE-protein chaperone that promotes SNARE-complex assembly for neurotransmitter release. Pathologically, ?-synuclein mutations and ?-synuclein overexpression cause Parkinson's disease, and aggregates of ?-synuclein are found as Lewy bodies in multiple neurodegenerative disorders ("synucleinopathies"). The relation of the physiological functions to the pathological effects of ?-synuclein remains unclear. As an initial avenue of addressing this question, we here systematically examined the effect of ?-synuclein mutations on its physiological and pathological activities. We generated 26 ?-synuclein mutants spanning the entire molecule, and analyzed them compared with wild-type ?-synuclein in seven assays that range from biochemical studies with purified ?-synuclein, to analyses of ?-synuclein expression in cultured neurons, to examinations of the effects of virally expressed ?-synuclein introduced into the mouse substantia nigra by stereotactic injections. We found that both the N-terminal and C-terminal sequences of ?-synuclein were required for its physiological function as SNARE-complex chaperone, but that these sequences were not essential for its neuropathological effects. In contrast, point mutations in the central region of ?-synuclein, referred to as nonamyloid ? component (residues 61-95), as well as point mutations linked to Parkinson's disease (A30P, E46K, and A53T) increased the neurotoxicity of ?-synuclein but did not affect its physiological function in SNARE-complex assembly. Thus, our data show that the physiological function of ?-synuclein, although protective of neurodegeneration in some contexts, is fundamentally distinct from its neuropathological effects, thereby dissociating the two activities of ?-synuclein.

    View details for DOI 10.1523/JNEUROSCI.3545-12.2012

    View details for PubMedID 23100443

  • Proteasome Inhibition Alleviates SNARE-Dependent Neurodegeneration SCIENCE TRANSLATIONAL MEDICINE Sharma, M., Burre, J., Suedhof, T. C. 2012; 4 (147)

    Abstract

    Activation of the proteasomal degradation of misfolded proteins has been proposed as a therapeutic strategy for treating neurodegenerative diseases, but it is unclear whether proteasome dysfunction contributes to neurodegeneration. We tested the role of proteasome activity in neurodegeneration developed by mice lacking cysteine string protein-? (CSP?). Unexpectedly, we found that proteasome inhibitors alleviated neurodegeneration in CSP?-deficient mice, reversing impairment of SNARE (soluble N-ethylmaleimide-sensitive factor attachment protein receptor)-complex assembly and extending life span. We tested whether dysfunctional SNARE-complex assembly could contribute to neurodegeneration in Alzheimer's and Parkinson's disease by analyzing postmortem brain tissue from these patients; we found reduced SNARE-complex assembly in the brain tissue samples. Our results suggest that proteasomal activation may not always be beneficial for alleviating neurodegeneration and that blocking the proteasome may represent a potential therapeutic avenue for treating some forms of neurodegenerative disease.

    View details for DOI 10.1126/scitranslmed.3004028

    View details for Web of Science ID 000307958800008

    View details for PubMedID 22896677

  • Inositol hexakisphosphate suppresses excitatory neurotransmission via synaptotagmin-1 C2B domain in the hippocampal neuron PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Yang, S., Shi, Y., Yang, G., Li, Y., Yu, L., Shin, O., Bacaj, T., Suedhof, T. C., Yu, J., Berggren, P. 2012; 109 (30): 12183-12188

    Abstract

    Inositol hexakisphosphate (InsP(6)) levels rise and fall with neuronal excitation and silence, respectively, in the hippocampus, suggesting potential signaling functions of this inositol polyphosphate in hippocampal neurons. We now demonstrate that intracellular application of InsP(6) caused a concentration-dependent inhibition of autaptic excitatory postsynaptic currents (EPSCs) in cultured hippocampal neurons. The treatment did not alter the size and replenishment rate of the readily releasable pool in autaptic neurons. Intracellular exposure to InsP(6) did not affect spontaneous EPSCs or excitatory amino acid-activated currents in neurons lacking autapses. The InsP(6)-induced inhibition of autaptic EPSCs was effectively abolished by coapplication of an antibody to synaptotagmin-1 C2B domain. Importantly, preabsorption of the antibody with a GST-WT synaptotagmin-1 C2B domain fragment but not with a GST-mutant synaptotagmin-1 C2B domain fragment that poorly reacted with the antibody impaired the activity of the antibody on the InsP(6)-induced inhibition of autaptic EPSCs. Furthermore, K(+) depolarization significantly elevated endogenous levels of InsP(6) and occluded the inhibition of autaptic EPSCs by exogenous InsP(6). These data reveal that InsP(6) suppresses excitatory neurotransmission via inhibition of the presynaptic synaptotagmin-1 C2B domain-mediated fusion via an interaction with the synaptotagmin Ca(2+)-binding sites rather than via interference with presynaptic Ca(2+) levels, synaptic vesicle trafficking, or inactivation of postsynaptic ionotropic glutamate receptors. Therefore, elevated InsP(6) in activated neurons serves as a unique negative feedback signal to control hippocampal excitatory neurotransmission.

    View details for DOI 10.1073/pnas.1115070109

    View details for Web of Science ID 000306992700063

    View details for PubMedID 22778403

  • RIM genes differentially contribute to organizing presynaptic release sites PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Kaeser, P. S., Deng, L., Fan, M., Suedhof, T. C. 2012; 109 (29): 11830-11835

    Abstract

    Tight coupling of Ca(2+) channels to the presynaptic active zone is critical for fast synchronous neurotransmitter release. RIMs are multidomain proteins that tether Ca(2+) channels to active zones, dock and prime synaptic vesicles for release, and mediate presynaptic plasticity. Here, we use conditional knockout mice targeting all RIM isoforms expressed by the Rims1 and Rims2 genes to examine the contributions and mechanism of action of different RIMs in neurotransmitter release. We show that acute single deletions of each Rims gene decreased release and impaired vesicle priming but did not alter the extracellular Ca(2+)-responsiveness of release (which for Rims gene mutants is a measure of presynaptic Ca(2+) influx). Moreover, single deletions did not affect the synchronization of release (which depends on the close proximity of Ca(2+) channels to release sites). In contrast, deletion of both Rims genes severely impaired the Ca(2+) responsiveness and synchronization of release. RIM proteins may act on Ca(2+) channels in two modes: They tether Ca(2+) channels to active zones, and they directly modulate Ca(2+)-channel inactivation. The first mechanism is essential for localizing presynaptic Ca(2+) influx to nerve terminals, but the role of the second mechanism remains unknown. Strikingly, we find that although the RIM2 C(2)B domain by itself significantly decreased Ca(2+)-channel inactivation in transfected HEK293 cells, it did not rescue any aspect of the RIM knockout phenotype in cultured neurons. Thus, RIMs primarily act in release as physical Ca(2+)-channel tethers and not as Ca(2+)-channel modulators. Different RIM proteins compensate for each other in recruiting Ca(2+) channels to active zones, but contribute independently and incrementally to vesicle priming.

    View details for DOI 10.1073/pnas.1209318109

    View details for Web of Science ID 000306837100069

    View details for PubMedID 22753485

  • The Presynaptic Active Zone NEURON Suedhof, T. C. 2012; 75 (1): 11-25

    Abstract

    Neurotransmitters are released by synaptic vesicle exocytosis at the active zone of a presynaptic nerve terminal. In this review, I discuss the molecular composition and function of the active zone. Active zones are composed of an evolutionarily conserved protein complex containing as core constituents RIM, Munc13, RIM-BP, ?-liprin, and ELKS proteins. This complex docks and primes synaptic vesicles for exocytosis, recruits Ca(2+) channels to the site of exocytosis, and positions the active zone exactly opposite to postsynaptic specializations via transsynaptic cell-adhesion molecules. Moreover, this complex mediates short- and long-term plasticity in response to bursts of action potentials, thus critically contributing to the computational power of a synapse.

    View details for DOI 10.1016/j.neuron.2012.06.012

    View details for Web of Science ID 000306539600005

    View details for PubMedID 22794257

  • Neurotransmitter Release at the Thalamocortical Synapse Instructs Barrel Formation But Not Axon Patterning in the Somatosensory Cortex JOURNAL OF NEUROSCIENCE Narboux-Neme, N., Evrard, A., Ferezou, I., Erzurumlu, R. S., Kaeser, P. S., Laine, J., Rossier, J., Ropert, N., Suedhof, T. C., Gaspar, P. 2012; 32 (18): 6183-6196

    Abstract

    To assess the impact of synaptic neurotransmitter release on neural circuit development, we analyzed barrel cortex formation after thalamic or cortical ablation of RIM1 and RIM2 proteins, which control synaptic vesicle fusion. Thalamus-specific deletion of RIMs reduced neurotransmission efficacy by 67%. A barrelless phenotype was found with a dissociation of effects on the presynaptic and postsynaptic cellular elements of the barrel. Presynaptically, thalamocortical axons formed a normal whisker map, whereas postsynaptically the cytoarchitecture of layer IV neurons was altered as spiny stellate neurons were evenly distributed and their dendritic trees were symmetric. Strikingly, cortex-specific deletion of the RIM genes did not modify barrel development. Adult mice with thalamic-specific RIM deletion showed a lack of activity-triggered immediate early gene expression and altered sensory-related behaviors. Thus, efficient synaptic release is required at thalamocortical but not at corticocortical synapses for building the whisker to barrel map and for efficient sensory function.

    View details for DOI 10.1523/JNEUROSCI.0343-12.2012

    View details for Web of Science ID 000303598900014

    View details for PubMedID 22553025

  • Synapses and Alzheimer's disease. Cold Spring Harbor perspectives in biology Sheng, M., Sabatini, B. L., Südhof, T. C. 2012; 4 (5)

    Abstract

    Alzheimer's disease (AD) is a major cause of dementia in the elderly. Pathologically, AD is characterized by the accumulation of insoluble aggregates of A?-peptides that are proteolytic cleavage products of the amyloid-? precursor protein ("plaques") and by insoluble filaments composed of hyperphosphorylated tau protein ("tangles"). Familial forms of AD often display increased production of A? peptides and/or altered activity of presenilins, the catalytic subunits of ?-secretase that produce A? peptides. Although the pathogenesis of AD remains unclear, recent studies have highlighted two major themes that are likely important. First, oligomeric A? species have strong detrimental effects on synapse function and structure, particularly on the postsynaptic side. Second, decreased presenilin function impairs synaptic transmission and promotes neurodegeneration. The mechanisms underlying these processes are beginning to be elucidated, and, although their relevance to AD remains debated, understanding these processes will likely allow new therapeutic avenues to AD.

    View details for DOI 10.1101/cshperspect.a005777

    View details for PubMedID 22491782

  • Synaptic cell adhesion. Cold Spring Harbor perspectives in biology Missler, M., Südhof, T. C., Biederer, T. 2012; 4 (4)

    Abstract

    Chemical synapses are asymmetric intercellular junctions that mediate synaptic transmission. Synaptic junctions are organized by trans-synaptic cell adhesion molecules bridging the synaptic cleft. Synaptic cell adhesion molecules not only connect pre- and postsynaptic compartments, but also mediate trans-synaptic recognition and signaling processes that are essential for the establishment, specification, and plasticity of synapses. A growing number of synaptic cell adhesion molecules that include neurexins and neuroligins, Ig-domain proteins such as SynCAMs, receptor phosphotyrosine kinases and phosphatases, and several leucine-rich repeat proteins have been identified. These synaptic cell adhesion molecules use characteristic extracellular domains to perform complementary roles in organizing synaptic junctions that are only now being revealed. The importance of synaptic cell adhesion molecules for brain function is highlighted by recent findings implicating several such molecules, notably neurexins and neuroligins, in schizophrenia and autism.

    View details for DOI 10.1101/cshperspect.a005694

    View details for PubMedID 22278667

  • A novel evolutionarily conserved domain of cell-adhesion GPCRs mediates autoproteolysis EMBO JOURNAL Arac, D., Boucard, A. A., Bolliger, M. F., Nguyen, J., Soltis, S. M., Suedhof, T. C., Brunger, A. T. 2012; 31 (6): 1364-1378

    Abstract

    The G protein-coupled receptor (GPCR) Proteolysis Site (GPS) of cell-adhesion GPCRs and polycystic kidney disease (PKD) proteins constitutes a highly conserved autoproteolysis sequence, but its catalytic mechanism remains unknown. Here, we show that unexpectedly the ?40-residue GPS motif represents an integral part of a much larger ?320-residue domain that we termed GPCR-Autoproteolysis INducing (GAIN) domain. Crystal structures of GAIN domains from two distantly related cell-adhesion GPCRs revealed a conserved novel fold in which the GPS motif forms five ?-strands that are tightly integrated into the overall GAIN domain. The GAIN domain is evolutionarily conserved from tetrahymena to mammals, is the only extracellular domain shared by all human cell-adhesion GPCRs and PKD proteins, and is the locus of multiple human disease mutations. Functionally, the GAIN domain is both necessary and sufficient for autoproteolysis, suggesting an autoproteolytic mechanism whereby the overall GAIN domain fine-tunes the chemical environment in the GPS to catalyse peptide bond hydrolysis. Thus, the GAIN domain embodies a unique, evolutionarily ancient and widespread autoproteolytic fold whose function is likely relevant for GPCR signalling and for multiple human diseases.

    View details for DOI 10.1038/emboj.2012.26

    View details for Web of Science ID 000302131600005

    View details for PubMedID 22333914

  • High Affinity Neurexin Binding to Cell Adhesion G-protein-coupled Receptor CIRL1/Latrophilin-1 Produces an Intercellular Adhesion Complex JOURNAL OF BIOLOGICAL CHEMISTRY Boucard, A. A., Ko, J., Suedhof, T. C. 2012; 287 (12): 9399-9413

    Abstract

    The G-protein-coupled receptor CIRL1/latrophilin-1 (CL1) and the type-1 membrane proteins neurexins represent distinct neuronal cell adhesion molecules that exhibit no similarities except for one common function: both proteins are receptors for ?-latrotoxin, a component of black widow spider venom that induces massive neurotransmitter release at synapses. Unexpectedly, we have now identified a direct binding interaction between the extracellular domains of CL1 and neurexins that is regulated by alternative splicing of neurexins at splice site 4 (SS4). Using saturation binding assays, we showed that neurexins lacking an insert at SS4 bind to CL1 with nanomolar affinity, whereas neurexins containing an insert at SS4 are unable to bind. CL1 competed for neurexin binding with neuroligin-1, a well characterized neurexin ligand. The extracellular sequences of CL1 contain five domains (lectin, olfactomedin-like, serine/threonine-rich, hormone-binding, and G-protein-coupled receptor autoproteolysis-inducing (GAIN) domains). Of these domains, the olfactomedin-like domain mediates neurexin binding as shown by deletion mapping. Cell adhesion assays using cells expressing neurexins and CL1 revealed that their interaction produces a stable intercellular adhesion complex, indicating that their interaction can be trans-cellular. Thus, our data suggest that CL1 constitutes a novel ligand for neurexins that may be localized postsynaptically based on its well characterized interaction with intracellular SH3 and multiple ankyrin repeats adaptor proteins (SHANK) and could form a trans-synaptic complex with presynaptic neurexins.

    View details for DOI 10.1074/jbc.M111.318659

    View details for Web of Science ID 000301797800070

    View details for PubMedID 22262843

  • Distinct Neuronal Coding Schemes in Memory Revealed by Selective Erasure of Fast Synchronous Synaptic Transmission NEURON Xu, W., Morishita, W., Buckmaster, P. S., Pang, Z. P., Malenka, R. C., Suedhof, T. C. 2012; 73 (5): 990-1001

    Abstract

    Neurons encode information by firing spikes in isolation or bursts and propagate information by spike-triggered neurotransmitter release that initiates synaptic transmission. Isolated spikes trigger neurotransmitter release unreliably but with high temporal precision. In contrast, bursts of spikes trigger neurotransmission reliably (i.e., boost transmission fidelity), but the resulting synaptic responses are temporally imprecise. However, the relative physiological importance of different spike-firing modes remains unclear. Here, we show that knockdown of synaptotagmin-1, the major Ca(2+) sensor for neurotransmitter release, abrogated neurotransmission evoked by isolated spikes but only delayed, without abolishing, neurotransmission evoked by bursts of spikes. Nevertheless, knockdown of synaptotagmin-1 in the hippocampal CA1 region did not impede acquisition of recent contextual fear memories, although it did impair the precision of such memories. In contrast, knockdown of synaptotagmin-1 in the prefrontal cortex impaired all remote fear memories. These results indicate that different brain circuits and types of memory employ distinct spike-coding schemes to encode and transmit information.

    View details for DOI 10.1016/j.neuron.2011.12.036

    View details for Web of Science ID 000301558600013

    View details for PubMedID 22405208

  • Region-specific deletions of RIM1 reproduce a subset of global RIM1a-/- phenotypes GENES BRAIN AND BEHAVIOR Haws, M. E., Kaeser, P. S., Jarvis, D. L., SUEDHOF, T. C., Powell, C. M. 2012; 11 (2): 201-213

    Abstract

    The presynaptic protein RIM1? mediates multiple forms of presynaptic plasticity at both excitatory and inhibitory synapses. Previous studies of mice lacking RIM1? (RIM1?(-/-) throughout the brain showed that deletion of RIM1? results in multiple behavioral abnormalities. In an effort to begin to delineate the brain regions in which RIM1 deletion mediates these abnormal behaviors, we used conditional (floxed) RIM1 knockout mice (fRIM1). By crossing these fRIM1 mice to previously characterized transgenic cre lines, we aimed to delete RIM1 selectively in the dentate gyrus (DG), using a specific preproopiomelanocortin promoter driving cre recombinase (POMC-cre) line , and in pyramidal neurons of the CA3 region of hippocampus, using the kainate receptor subunit 1 promoter driving cre recombinase (KA-cre). Neither of these cre driver lines was uniquely selective to the targeted regions. In spite of this, we were able to reproduce a subset of the global RIM1?(-/-) behavioral abnormalities, thereby narrowing the brain regions in which loss of RIM1 is sufficient to produce these behavioral differences. Most interestingly, hypersensitivity to the pyschotomimetic MK-801 was shown in mice lacking RIM1 selectively in the DG, arcuate nucleus of the hypothalamus and select cerebellar neurons, implicating novel brain regions and neuronal subtypes in this behavior.

    View details for DOI 10.1111/j.1601-183X.2011.00755.x

    View details for Web of Science ID 000299634400009

    View details for PubMedID 22103334

  • C-Terminal Complexin Sequence Is Selectively Required for Clamping and Priming But Not for Ca2+ Triggering of Synaptic Exocytosis JOURNAL OF NEUROSCIENCE Kaeser-Woo, Y. J., Yang, X., Suedhof, T. C. 2012; 32 (8): 2877-2885

    Abstract

    Complexins are small soluble proteins that bind to assembling SNARE complexes during synaptic vesicle exocytosis, which in turn mediates neurotransmitter release. Complexins are required for clamping of spontaneous "mini " release and for the priming and synaptotagmin-dependent Ca(2+) triggering of evoked release. Mammalian genomes encode four complexins that are composed of an N-terminal unstructured sequence that activates synaptic exocytosis, an accessory ?-helix that clamps exocytosis, an essential central ?-helix that binds to assembling SNARE complexes and is required for all of its functions, and a long, apparently unstructured C-terminal sequence whose function remains unclear. Here, we used cultured mouse neurons to show that the C-terminal sequence of complexin-1 is not required for its synaptotagmin-activating function but is essential for its priming and clamping functions. Wild-type complexin-3 did not clamp exocytosis but nevertheless fully primed and activated exocytosis. Strikingly, exchanging the complexin-1 C terminus for the complexin-3 C terminus abrogated clamping, whereas exchanging the complexin-3 C terminus for the complexin-1 C terminus enabled clamping. Analysis of point mutations in the complexin-1 C terminus identified two single amino-acid substitutions that impaired clamping without altering the activation function of complexin-1. Examination of release induced by stimulus trains revealed that clamping-deficient C-terminal complexin mutants produced a modest relative increase in delayed release. Overall, our results show that the relatively large C-terminal complexin-1 sequence acts in priming and clamping synaptic exocytosis and demonstrate that the clamping function is not conserved in complexin-3, presumably because of its distinct C-terminal sequences.

    View details for DOI 10.1523/JNEUROSCI.3360-11.2012

    View details for Web of Science ID 000300716600030

    View details for PubMedID 22357870

  • CSPa knockout causes neurodegeneration by impairing SNAP-25 function. EMBO journal Sharma, M., Burré, J., Bronk, P., Zhang, Y., Xu, W., Südhof, T. C. 2012; 31 (4): 829-841

    Abstract

    At a synapse, the synaptic vesicle protein cysteine-string protein-? (CSP?) functions as a co-chaperone for the SNARE protein SNAP-25. Knockout (KO) of CSP? causes fulminant neurodegeneration that is rescued by ?-synuclein overexpression. The CSP? KO decreases SNAP-25 levels and impairs SNARE-complex assembly; only the latter but not the former is reversed by ?-synuclein. Thus, the question arises whether the CSP? KO phenotype is due to decreased SNAP-25 function that then causes neurodegeneration, or due to the dysfunction of multiple as-yet uncharacterized CSP? targets. Here, we demonstrate that decreasing SNAP-25 levels in CSP? KO mice by either KO or knockdown of SNAP-25 aggravated their phenotype. Conversely, increasing SNAP-25 levels by overexpression rescued their phenotype. Inactive SNAP-25 mutants were unable to rescue, showing that the rescue was specific. Under all conditions, the neurodegenerative phenotype precisely correlated with SNARE-complex assembly, indicating that impaired SNARE-complex assembly due to decreased SNAP-25 levels is the ultimate correlate of neurodegeneration. Our findings suggest that the neurodegeneration in CSP? KO mice is primarily produced by defective SNAP-25 function, which causes neurodegeneration by impairing SNARE-complex assembly.

    View details for DOI 10.1038/emboj.2011.467

    View details for PubMedID 22187053

  • Direct conversion of mouse fibroblasts to self-renewing, tripotent neural precursor cells PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Lujan, E., Chanda, S., Ahlenius, H., Suedhof, T. C., Wernig, M. 2012; 109 (7): 2527-2532

    Abstract

    We recently showed that defined sets of transcription factors are sufficient to convert mouse and human fibroblasts directly into cells resembling functional neurons, referred to as "induced neuronal" (iN) cells. For some applications however, it would be desirable to convert fibroblasts into proliferative neural precursor cells (NPCs) instead of neurons. We hypothesized that NPC-like cells may be induced using the same principal approach used for generating iN cells. Toward this goal, we infected mouse embryonic fibroblasts derived from Sox2-EGFP mice with a set of 11 transcription factors highly expressed in NPCs. Twenty-four days after transgene induction, Sox2-EGFP(+) colonies emerged that expressed NPC-specific genes and differentiated into neuronal and astrocytic cells. Using stepwise elimination, we found that Sox2 and FoxG1 are capable of generating clonal self-renewing, bipotent induced NPCs that gave rise to astrocytes and functional neurons. When we added the Pou and Homeobox domain-containing transcription factor Brn2 to Sox2 and FoxG1, we were able to induce tripotent NPCs that could be differentiated not only into neurons and astrocytes but also into oligodendrocytes. The transcription factors FoxG1 and Brn2 alone also were capable of inducing NPC-like cells; however, these cells generated less mature neurons, although they did produce astrocytes and even oligodendrocytes capable of integration into dysmyelinated Shiverer brain. Our data demonstrate that direct lineage reprogramming using target cell-type-specific transcription factors can be used to induce NPC-like cells that potentially could be used for autologous cell transplantation-based therapies in the brain or spinal cord.

    View details for DOI 10.1073/pnas.1121003109

    View details for Web of Science ID 000300489200073

    View details for PubMedID 22308465

  • Postsynaptic Complexin Controls AMPA Receptor Exocytosis during LTP NEURON Ahmad, M., Polepalli, J. S., Goswami, D., Yang, X., Kaeser-Woo, Y. J., Suedhof, T. C., Malenka, R. C. 2012; 73 (2): 260-267

    Abstract

    Long-term potentiation (LTP) is a compelling synaptic correlate of learning and memory. LTP induction requires NMDA receptor (NMDAR) activation, which triggers SNARE-dependent exocytosis of AMPA receptors (AMPARs). However, the molecular mechanisms mediating AMPAR exocytosis induced by NMDAR activation remain largely unknown. Here, we show that complexin, a protein that regulates neurotransmitter release via binding to SNARE complexes, is essential for AMPAR exocytosis during LTP but not for the constitutive AMPAR exocytosis that maintains basal synaptic strength. The regulated postsynaptic AMPAR exocytosis during LTP requires binding of complexin to SNARE complexes. In hippocampal neurons, presynaptic complexin acts together with synaptotagmin-1 to mediate neurotransmitter release. However, postsynaptic synaptotagmin-1 is not required for complexin-dependent AMPAR exocytosis during LTP. These results suggest a complexin-dependent molecular mechanism for regulating AMPAR delivery to synapses, a mechanism that is surprisingly similar to presynaptic exocytosis but controlled by regulators other than synaptotagmin-1.

    View details for DOI 10.1016/j.neuron.2011.11.020

    View details for Web of Science ID 000299761600008

    View details for PubMedID 22284181

  • The membrane fusion enigma: SNAREs, Sec1/Munc18 proteins, and their accomplices--guilty as charged? Annual review of cell and developmental biology Rizo, J., Südhof, T. C. 2012; 28: 279-308

    Abstract

    Neurotransmitter release is governed by proteins that have homo-logs in most types of intracellular membrane fusion, including the Sec1/Munc18 protein Munc18-1 and the SNARE proteins syntaxin-1, synaptobrevin/VAMP, and SNAP-25. The SNAREs initiate fusion by forming tight SNARE complexes that bring the vesicle and plasma membranes together. SNARE maintenance in a functional state depends on two chaperone systems (Hsc70/?CSP/SGT and synuclein); defects in these systems lead to neurodegeneration. Munc18-1 binds to an autoinhibitory closed conformation of syntaxin-1, gating formation of SNARE complexes, and also binds to SNARE complexes, which likely underlies the crucial function of Munc18-1 in membrane fusion by an as-yet unclear mechanism. Syntaxin-1 opening is mediated by Munc13s through their MUN domain, which is homologous to diverse tethering factors and may also have a general role in fusion. MUN domain activity is likely modulated in diverse presynaptic plasticity processes that depend on Ca(2+) and RIM proteins, among others.

    View details for DOI 10.1146/annurev-cellbio-101011-155818

    View details for PubMedID 23057743

  • Calcium control of neurotransmitter release. Cold Spring Harbor perspectives in biology Südhof, T. C. 2012; 4 (1)

    Abstract

    Upon entering a presynaptic terminal, an action potential opens Ca(2+) channels, and transiently increases the local Ca(2+) concentration at the presynaptic active zone. Ca(2+) then triggers neurotransmitter release within a few hundred microseconds by activating synaptotagmins Ca(2+). Synaptotagmins bind Ca(2+) via two C2-domains, and transduce the Ca(2+) signal into a nanomechanical activation of the membrane fusion machinery; this activation is mediated by the Ca(2+)-dependent interaction of the synaptotagmin C2-domains with phospholipids and SNARE proteins. In triggering exocytosis, synaptotagmins do not act alone, but require an obligatory cofactor called complexin, a small protein that binds to SNARE complexes and simultaneously activates and clamps the SNARE complexes, thereby positioning the SNARE complexes for subsequent synaptotagmin action. The conserved function of synaptotagmins and complexins operates generally in most, if not all, Ca(2+)-regulated forms of exocytosis throughout the body in addition to synaptic vesicle exocytosis, including in the degranulation of mast cells, acrosome exocytosis in sperm cells, hormone secretion from endocrine cells, and neuropeptide release.

    View details for DOI 10.1101/cshperspect.a011353

    View details for PubMedID 22068972

  • Comprehensive qPCR profiling of gene expression in single neuronal cells NATURE PROTOCOLS Citri, A., Pang, Z. P., Suedhof, T. C., Wernig, M., Malenka, R. C. 2012; 7 (1): 118-127

    Abstract

    A major challenge in neuronal stem cell biology lies in characterization of lineage-specific reprogrammed human neuronal cells, a process that necessitates the use of an assay sensitive to the single-cell level. Single-cell gene profiling can provide definitive evidence regarding the conversion of one cell type into another at a high level of resolution. The protocol we describe uses Fluidigm Biomark dynamic arrays for high-throughput expression profiling from single neuronal cells, assaying up to 96 independent samples with up to 96 quantitative PCR (qPCR) probes (equivalent to 9,216 reactions) in a single experiment, which can be completed within 2-3 d. The protocol enables simple and cost-effective profiling of several hundred transcripts from a single cell, and it could have numerous utilities.

    View details for DOI 10.1038/nprot.2011.430

    View details for Web of Science ID 000299108900011

    View details for PubMedID 22193304

  • Induced Neuronal Cells: How to Make and Define a Neuron CELL STEM CELL Yang, N., Ng, Y. H., Pang, Z. P., Suedhof, T. C., Wernig, M. 2011; 9 (6): 517-525

    Abstract

    Cellular plasticity is a major focus of investigation in developmental biology. The recent discovery that induced neuronal (iN) cells can be generated from mouse and human fibroblasts by expression of defined transcription factors suggested that cell fate plasticity is much wider than previously anticipated. In this review, we summarize the most recent developments in this nascent field and suggest criteria to help define and categorize iN cells that take into account the complexity of neuronal identity.

    View details for DOI 10.1016/j.stem.2011.11.015

    View details for Web of Science ID 000297895000012

    View details for PubMedID 22136927

  • Synaptic Vesicle Exocytosis COLD SPRING HARBOR PERSPECTIVES IN BIOLOGY Suedhof, T. C., Rizo, J. 2011; 3 (12)

    Abstract

    Presynaptic nerve terminals release neurotransmitters by synaptic vesicle exocytosis. Membrane fusion mediating synaptic exocytosis and other intracellular membrane traffic is affected by a universal machinery that includes SNARE (for "soluble NSF-attachment protein receptor") and SM (for "Sec1/Munc18-like") proteins. During fusion, vesicular and target SNARE proteins assemble into an ?-helical trans-SNARE complex that forces the two membranes tightly together, and SM proteins likely wrap around assembling trans-SNARE complexes to catalyze membrane fusion. After fusion, SNARE complexes are dissociated by the ATPase NSF (for "N-ethylmaleimide sensitive factor"). Fusion-competent conformations of SNARE proteins are maintained by chaperone complexes composed of CSP?, Hsc70, and SGT, and by nonenzymatically acting synuclein chaperones; dysfunction of these chaperones results in neurodegeneration. The synaptic membrane-fusion machinery is controlled by synaptotagmin, and additionally regulated by a presynaptic protein matrix (the "active zone") that includes Munc13 and RIM proteins as central components.

    View details for DOI 10.1101/cshperspect.a005637

    View details for Web of Science ID 000298135700008

    View details for PubMedID 22026965

  • Direct Lineage Conversion of Terminally Differentiated Hepatocytes to Functional Neurons CELL STEM CELL Marro, S., Pang, Z. P., Yang, N., Tsai, M., Qu, K., Chang, H. Y., Suedhof, T. C., Wernig, M. 2011; 9 (4): 374-382

    Abstract

    Several recent studies have showed that mouse and human fibroblasts can be directly reprogrammed into induced neuronal (iN) cells, bypassing a pluripotent intermediate state. However, fibroblasts represent heterogeneous mesenchymal progenitor cells that potentially contain neural crest lineages, and the cell of origin remained undefined. This raises the fundamental question of whether lineage reprogramming is possible between cell types derived from different germ layers. Here, we demonstrate that terminally differentiated hepatocytes can be directly converted into functional iN cells. Importantly, single-cell and genome-wide expression analyses showed that fibroblast- and hepatocyte-derived iN cells not only induced a neuronal transcriptional program, but also silenced their donor transcriptome. The remaining donor signature decreased over time and could not support functional hepatocyte properties. Thus, the reprogramming factors lead to a binary lineage switch decision rather than an induction of hybrid phenotypes, but iN cells retain a small but detectable epigenetic memory of their donor cells.

    View details for DOI 10.1016/j.stem.2011.09.002

    View details for Web of Science ID 000296041200015

    View details for PubMedID 21962918

  • The neurexin ligands, neuroligins and leucine-rich repeat transmembrane proteins, perform convergent and divergent synaptic functions in vivo PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Soler-Llavina, G. J., Fuccillo, M. V., Ko, J., Suedhof, T. C., Malenka, R. C. 2011; 108 (40): 16502-16509

    Abstract

    Synaptic cell adhesion molecules, including the neurexin ligands, neuroligins (NLs) and leucine-rich repeat transmembrane proteins (LRRTMs), are thought to organize synapse assembly and specify synapse function. To test the synaptic role of these molecules in vivo, we performed lentivirally mediated knockdown of NL3, LRRTM1, and LRRTM2 in CA1 pyramidal cells of WT and NL1 KO mice at postnatal day (P)0 (when synapses are forming) and P21 (when synapses are largely mature). P0 knockdown of NL3 in WT or NL1 KO neurons did not affect excitatory synaptic transmission, whereas P0 knockdown of LRRTM1 and LRRTM2 selectively reduced AMPA receptor-mediated synaptic currents. P0 triple knockdown of NL3 and both LRRTMs in NL1 KO mice yielded greater reductions in AMPA and NMDA receptor-mediated currents, suggesting functional redundancy between NLs and LRRTMs during early synapse development. In contrast, P21 knockdown of LRRTMs did not alter excitatory transmission, whereas NL manipulations supported a role for NL1 in maintaining NMDA receptor-mediated transmission. These results show that neurexin ligands in vivo form a dynamic synaptic cell adhesion network, with compensation between NLs and LRRTMs during early synapse development and functional divergence upon synapse maturation.

    View details for DOI 10.1073/pnas.1114028108

    View details for Web of Science ID 000295536000012

    View details for PubMedID 21953696

  • Rab3B protein is required for long-term depression of hippocampal inhibitory synapses and for normal reversal learning PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Tsetsenis, T., Younts, T. J., Chiu, C. Q., Kaeser, P. S., Castillo, P. E., Suedhof, T. C. 2011; 108 (34): 14300-14305

    Abstract

    Rab3B, similar to other Rab3 isoforms, is a synaptic vesicle protein that interacts with the Rab3-interacting molecule (RIM) isoforms RIM1? and RIM2? as effector proteins in a GTP-dependent manner. Previous studies showed that at excitatory synapses, Rab3A and RIM1? are essential for presynaptically expressed long-term potentiation (LTP), whereas at inhibitory synapses RIM1? is required for endocannabinoid-dependent long-term depression (referred to as "i-LTD"). However, it remained unknown whether i-LTD also involves a Rab3 isoform and whether i-LTD, similar to other forms of long-term plasticity, is important for learning and memory. Here we show that Rab3B is highly enriched in inhibitory synapses in the CA1 region of the hippocampus. Using electrophysiological recordings in acute slices, we demonstrate that knockout (KO) of Rab3B does not alter the strength or short-term plasticity of excitatory or inhibitory synapses but does impair i-LTD significantly without changing classical NMDA receptor-dependent LTP. Behaviorally, we found that Rab3B KO mice exhibit no detectable changes in all basic parameters tested, including the initial phase of learning and memory. However, Rab3B KO mice did display a selective enhancement in reversal learning, as measured using Morris water-maze and fear-conditioning assays. Our data support the notion that presynaptic forms of long-term plasticity at excitatory and inhibitory synapses generally are mediated by a common Rab3/RIM-dependent pathway, with various types of synapses using distinct Rab3 isoforms. Moreover, our results suggest that i-LTD contributes to learning and memory, presumably by stabilizing circuits established in previous learning processes.

    View details for DOI 10.1073/pnas.1112237108

    View details for Web of Science ID 000294163500084

    View details for PubMedID 21844341

  • Autism-linked neuroligin-3 R451C mutation differentially alters hippocampal and cortical synaptic function PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Etherton, M., Foeldy, C., Sharma, M., Tabuchi, K., Liu, X., Shamloo, M., Malenka, R. C., Suedhof, T. C. 2011; 108 (33): 13764-13769

    Abstract

    Multiple independent mutations in neuroligin genes were identified in patients with familial autism, including the R451C substitution in neuroligin-3 (NL3). Previous studies showed that NL3(R451C) knock-in mice exhibited modestly impaired social behaviors, enhanced water maze learning abilities, and increased synaptic inhibition in the somatosensory cortex, and they suggested that the behavioral changes in these mice may be caused by a general shift of synaptic transmission to inhibition. Here, we confirm that NL3(R451C) mutant mice behaviorally exhibit social interaction deficits and electrophysiologically display increased synaptic inhibition in the somatosensory cortex. Unexpectedly, however, we find that the NL3(R451C) mutation produced a strikingly different phenotype in the hippocampus. Specifically, in the hippocampal CA1 region, the NL3(R451C) mutation caused an ?1.5-fold increase in AMPA receptor-mediated excitatory synaptic transmission, dramatically altered the kinetics of NMDA receptor-mediated synaptic responses, induced an approximately twofold up-regulation of NMDA receptors containing NR2B subunits, and enhanced long-term potentiation almost twofold. NL3 KO mice did not exhibit any of these changes. Quantitative light microscopy and EM revealed that the NL3(R451C) mutation increased dendritic branching and altered the structure of synapses in the stratum radiatum of the hippocampus. Thus, in NL3(R451C) mutant mice, a single point mutation in a synaptic cell adhesion molecule causes context-dependent changes in synaptic transmission; these changes are consistent with the broad impact of this mutation on murine and human behaviors, suggesting that NL3 controls excitatory and inhibitory synapse properties in a region- and circuit-specific manner.

    View details for DOI 10.1073/pnas.1111093108

    View details for Web of Science ID 000293895100079

    View details for PubMedID 21808020

  • Induction of human neuronal cells by defined transcription factors NATURE Pang, Z. P., Yang, N., Vierbuchen, T., Ostermeier, A., Fuentes, D. R., Yang, T. Q., Citri, A., Sebastiano, V., Marro, S., Suedhof, T. C., Wernig, M. 2011; 476 (7359): 220-U122

    Abstract

    Somatic cell nuclear transfer, cell fusion, or expression of lineage-specific factors have been shown to induce cell-fate changes in diverse somatic cell types. We recently observed that forced expression of a combination of three transcription factors, Brn2 (also known as Pou3f2), Ascl1 and Myt1l, can efficiently convert mouse fibroblasts into functional induced neuronal (iN) cells. Here we show that the same three factors can generate functional neurons from human pluripotent stem cells as early as 6?days after transgene activation. When combined with the basic helix-loop-helix transcription factor NeuroD1, these factors could also convert fetal and postnatal human fibroblasts into iN cells showing typical neuronal morphologies and expressing multiple neuronal markers, even after downregulation of the exogenous transcription factors. Importantly, the vast majority of human iN cells were able to generate action potentials and many matured to receive synaptic contacts when co-cultured with primary mouse cortical neurons. Our data demonstrate that non-neural human somatic cells, as well as pluripotent stem cells, can be converted directly into neurons by lineage-determining transcription factors. These methods may facilitate robust generation of patient-specific human neurons for in vitro disease modelling or future applications in regenerative medicine.

    View details for DOI 10.1038/nature10202

    View details for Web of Science ID 000293731900039

    View details for PubMedID 21617644

  • Neuroligins/LRRTMs prevent activity- and Ca2+/calmodulin-dependent synapse elimination in cultured neurons JOURNAL OF CELL BIOLOGY Ko, J., Soler-Llavina, G. J., Fuccillo, M. V., Malenka, R. C., Suedhof, T. C. 2011; 194 (2): 323-334

    Abstract

    Neuroligins (NLs) and leucine-rich repeat transmembrane proteins (LRRTMs) are postsynaptic cell adhesion molecules that bind to presynaptic neurexins. In this paper, we show that short hairpin ribonucleic acid-mediated knockdowns (KDs) of LRRTM1, LRRTM2, and/or NL-3, alone or together as double or triple KDs (TKDs) in cultured hippocampal neurons, did not decrease synapse numbers. In neurons cultured from NL-1 knockout mice, however, TKD of LRRTMs and NL-3 induced an ?40% loss of excitatory but not inhibitory synapses. Strikingly, synapse loss triggered by the LRRTM/NL deficiency was abrogated by chronic blockade of synaptic activity as well as by chronic inhibition of Ca(2+) influx or Ca(2+)/calmodulin (CaM) kinases. Furthermore, postsynaptic KD of CaM prevented synapse loss in a cell-autonomous manner, an effect that was reversed by CaM rescue. Our results suggest that two neurexin ligands, LRRTMs and NLs, act redundantly to maintain excitatory synapses and that synapse elimination caused by the absence of NLs and LRRTMs is promoted by synaptic activity and mediated by a postsynaptic Ca(2+)/CaM-dependent signaling pathway.

    View details for DOI 10.1083/jcb.201101072

    View details for Web of Science ID 000293121700014

    View details for PubMedID 21788371

  • An autism-associated point mutation in the neuroligin cytoplasmic tail selectively impairs AMPA receptor-mediated synaptic transmission in hippocampus EMBO JOURNAL Etherton, M. R., Tabuchi, K., Sharma, M., Ko, J., Suedhof, T. C. 2011; 30 (14): 2908-2919

    Abstract

    Neuroligins are evolutionarily conserved postsynaptic cell-adhesion molecules that function, at least in part, by forming trans-synaptic complexes with presynaptic neurexins. Different neuroligin isoforms perform diverse functions and exhibit distinct intracellular localizations, but contain similar cytoplasmic sequences whose role remains largely unknown. Here, we analysed the effect of a single amino-acid substitution (R704C) that targets a conserved arginine residue in the cytoplasmic sequence of all neuroligins, and that was associated with autism in neuroligin-4. We introduced the R704C mutation into mouse neuroligin-3 by homologous recombination, and examined its effect on synapses in vitro and in vivo. Electrophysiological and morphological studies revealed that the neuroligin-3 R704C mutation did not significantly alter synapse formation, but dramatically impaired synapse function. Specifically, the R704C mutation caused a major and selective decrease in AMPA receptor-mediated synaptic transmission in pyramidal neurons of the hippocampus, without similarly changing NMDA or GABA receptor-mediated synaptic transmission, and without detectably altering presynaptic neurotransmitter release. Our results suggest that the cytoplasmic tail of neuroligin-3 has a central role in synaptic transmission by modulating the recruitment of AMPA receptors to postsynaptic sites at excitatory synapses.

    View details for DOI 10.1038/emboj.2011.182

    View details for Web of Science ID 000293970100016

    View details for PubMedID 21642956

  • Doc2 Supports Spontaneous Synaptic Transmission by a Ca2+-Independent Mechanism NEURON Pang, Z. P., Bacaj, T., Yang, X., Zhou, P., Xu, W., Suedhof, T. C. 2011; 70 (2): 244-251

    Abstract

    Two families of Ca(2+)-binding proteins have been proposed as Ca(2+) sensors for spontaneous release: synaptotagmins and Doc2s, with the intriguing possibility that Doc2s may represent high-affinity Ca(2+) sensors that are activated by deletion of synaptotagmins, thereby accounting for the increased spontaneous release in synaptotagmin-deficient synapses. Here, we use an shRNA-dependent quadruple knockdown of all four Ca(2+)-binding proteins of the Doc2 family to confirm that Doc2-deficient synapses exhibit a marked decrease in the frequency of spontaneous release events. Knockdown of Doc2s in synaptotagmin-1-deficient synapses, however, failed to reduce either the increased spontaneous release or the decreased evoked release of these synapses, suggesting that Doc2s do not constitute Ca(2+) sensors for asynchronous release. Moreover, rescue experiments revealed that the decrease in spontaneous release induced by the Doc2 knockdown in wild-type synapses is fully reversed by mutant Doc2B lacking Ca(2+)-binding sites. Thus, our data suggest that Doc2s are modulators of spontaneous synaptic transmission that act by a Ca(2+)-independent mechanism.

    View details for DOI 10.1016/j.neuron.2011.03.011

    View details for Web of Science ID 000291073700007

    View details for PubMedID 21521611

  • Activity-Dependent IGF-1 Exocytosis Is Controlled by the Ca2+-Sensor Synaptotagmin-10 CELL Cao, P., Maximov, A., Suedhof, T. C. 2011; 145 (2): 300-311

    Abstract

    Synaptotagmins Syt1, Syt2, Syt7, and Syt9 act as Ca(2+)-sensors for synaptic and neuroendocrine exocytosis, but the function of other synaptotagmins remains unknown. Here, we show that olfactory bulb neurons secrete IGF-1 by an activity-dependent pathway of exocytosis, and that Syt10 functions as the Ca(2+)-sensor that triggers IGF-1 exocytosis in these neurons. Deletion of Syt10 impaired activity-dependent IGF-1 secretion in olfactory bulb neurons, resulting in smaller neurons and an overall decrease in synapse numbers. Exogenous IGF-1 completely reversed the Syt10 knockout phenotype. Syt10 colocalized with IGF-1 in somatodendritic vesicles of olfactory bulb neurons, and Ca(2+)-binding to Syt10 caused these vesicles to undergo exocytosis, thereby secreting IGF-1. Thus, Syt10 controls a previously unrecognized pathway of Ca(2+)-dependent exocytosis that is spatially and temporally distinct from Ca(2+)-dependent synaptic vesicle exocytosis controlled by Syt1. Our findings thereby reveal that two different synaptotagmins can regulate functionally distinct Ca(2+)-dependent membrane fusion reactions in the same neuron.

    View details for DOI 10.1016/j.cell.2011.03.034

    View details for Web of Science ID 000289549900017

    View details for PubMedID 21496647

  • Cerebellins meet neurexins (Commentary on Matsuda & Yuzaki) EUROPEAN JOURNAL OF NEUROSCIENCE Martinelli, D. C., Suedhof, T. C. 2011; 33 (8): 1445-1446
  • The cell-adhesion G protein-coupled receptor BAI3 is a high-affinity receptor for C1q-like proteins PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Bolliger, M. F., Martinelli, D. C., Suedhof, T. C. 2011; 108 (6): 2534-2539

    Abstract

    C1q-like genes (C1ql1-C1ql4) encode small, secreted proteins that are expressed in differential patterns in the brain but whose receptors and functions remain unknown. BAI3 protein, in contrast, is a member of the cell-adhesion class of G protein-coupled receptors that are expressed at high levels in the brain but whose ligands have thus far escaped identification. Using a biochemical approach, we show that all four C1ql proteins bind to the extracellular thrombospondin-repeat domain of BAI3 with high affinity, and that this binding is mediated by the globular C1q domains of the C1ql proteins. Moreover, we demonstrate that addition of submicromolar concentrations of C1ql proteins to cultured neurons causes a significant decrease in synapse density, and that this decrease was prevented by simultaneous addition of the thrombospondin-repeat fragment of BAI3, which binds to C1ql proteins. Our data suggest that C1ql proteins are secreted signaling molecules that bind to BAI3 and act, at least in part, to regulate synapse formation and/or maintenance.

    View details for DOI 10.1073/pnas.1019577108

    View details for Web of Science ID 000287084500066

    View details for PubMedID 21262840

  • Delayed onset of hyperglycaemia in a mouse model with impaired glucagon secretion demonstrates that dysregulated glucagon secretion promotes hyperglycaemia and type 2 diabetes DIABETOLOGIA Gustavsson, N., SEAH, T., Lao, Y., Radda, G. K., SUEDHOF, T. C., Han, W. 2011; 54 (2): 415-422

    Abstract

    Type 2 diabetes is caused by relative deficiency of insulin secretion and is associated with dysregulation of glucagon secretion during the late stage of diabetes development. Like insulin secretion from beta cells, glucagon secretion is dependent on calcium signals and a calcium sensing protein, synaptotagmin-7. In this study, we tested the relative contribution of dysregulated glucagon secretion and reduced insulin release in the development of hyperglycaemia and type 2 diabetes by using synaptotagmin-7 knockout (KO) mice, which exhibit glucose intolerance, reduced insulin secretion and nearly abolished Ca(2+)-stimulated glucagon secretion.We fed the synaptotagmin-7 KO and control mice with a high-fat diet (HFD) for 14 weeks, and compared their body weight, glucose levels, glucose and insulin tolerance, and insulin and glucagon secretion.On the HFD, synaptotagmin-7 KO mice showed progressive impairment of glucose tolerance and insulin secretion, along with continued maintenance of a low glucagon level. The control mice were less affected in terms of glucose intolerance, and showed enhanced insulin secretion with a concurrent increase in glucagon levels. Unexpectedly, after 14 weeks of HFD feeding, only the control mice displayed resting hyperglycaemia, whereas in synaptotagmin-7 KO mice defective insulin secretion and reduced insulin sensitivity were not sufficient to cause hyperglycaemia in the absence of enhanced glucagon secretion.Our data uncover a previously overlooked role of dysregulated glucagon secretion in promoting hyperglycaemia and the ensuing diabetes, and strongly suggest maintenance of adequate regulation of glucagon secretion as an important therapeutic target in addition to the preservation of beta cell function and mass in the prevention and treatment of diabetes.

    View details for DOI 10.1007/s00125-010-1950-2

    View details for Web of Science ID 000286001800027

    View details for PubMedID 20978738

  • RIM Proteins Activate Vesicle Priming by Reversing Autoinhibitory Homodimerization of Munc13 NEURON Deng, L., Kaeser, P. S., Xu, W., Suedhof, T. C. 2011; 69 (2): 317-331

    Abstract

    At a synapse, the presynaptic active zone mediates synaptic vesicle exocytosis. RIM proteins are active zone scaffolding molecules that--among others--mediate vesicle priming and directly or indirectly interact with most other essential presynaptic proteins. In particular, the Zn²+ finger domain of RIMs binds to the C?A domain of the priming factor Munc13, which forms a homodimer in the absence of RIM but a heterodimer with it. Here, we show that RIMs mediate vesicle priming not by coupling Munc13 to other active zone proteins as thought but by directly activating Munc13. Specifically, we found that the isolated Zn²+ finger domain of RIMs autonomously promoted vesicle priming by binding to Munc13, thereby relieving Munc13 homodimerization. Strikingly, constitutively monomeric mutants of Munc13 rescued priming in RIM-deficient synapses, whereas wild-type Munc13 did not. Both mutant and wild-type Munc13, however, rescued priming in Munc13-deficient synapses. Thus, homodimerization of Munc13 inhibits its priming function, and RIMs activate priming by disrupting Munc13 homodimerization.

    View details for DOI 10.1016/j.neuron.2011.01.005

    View details for Web of Science ID 000286792900014

    View details for PubMedID 21262469

  • RIM Determines Ca2+ Channel Density and Vesicle Docking at the Presynaptic Active Zone NEURON Han, Y., Kaeser, P. S., Suedhof, T. C., Schneggenburger, R. 2011; 69 (2): 304-316

    Abstract

    At presynaptic active zones, neurotransmitter release is initiated by the opening of voltage-gated Ca²+ channels close to docked vesicles. The mechanisms that enrich Ca²+ channels at active zones are, however, largely unknown, possibly because of the limited presynaptic accessibility of most synapses. Here, we have established a Cre-lox based conditional knockout approach at a presynaptically accessible central nervous system synapse, the calyx of Held, to directly study the functions of RIM proteins. Removal of all RIM1/2 isoforms strongly reduced the presynaptic Ca²+ channel density, revealing a role of RIM proteins in Ca²+ channel targeting. Removal of RIMs also reduced the readily releasable pool, paralleled by a similar reduction of the number of docked vesicles, and the Ca²+ channel-vesicle coupling was decreased. Thus, RIM proteins co-ordinately regulate key functions for fast transmitter release, enabling a high presynaptic Ca²+ channel density and vesicle docking at the active zone.

    View details for DOI 10.1016/j.neuron.2010.12.014

    View details for Web of Science ID 000286792900013

    View details for PubMedID 21262468

  • RIM Proteins Tether Ca2+ Channels to Presynaptic Active Zones via a Direct PDZ-Domain Interaction CELL Kaeser, P. S., Deng, L., Wang, Y., Dulubova, I., Liu, X., Rizo, J., Suedhof, T. C. 2011; 144 (2): 282-295

    Abstract

    At a synapse, fast synchronous neurotransmitter release requires localization of Ca(2+) channels to presynaptic active zones. How Ca(2+) channels are recruited to active zones, however, remains unknown. Using unbiased yeast two-hybrid screens, we here identify a direct interaction of the central PDZ domain of the active-zone protein RIM with the C termini of presynaptic N- and P/Q-type Ca(2+) channels but not L-type Ca(2+) channels. To test the physiological significance of this interaction, we generated conditional knockout mice lacking all multidomain RIM isoforms. Deletion of RIM proteins ablated most neurotransmitter release by simultaneously impairing the priming of synaptic vesicles and by decreasing the presynaptic localization of Ca(2+) channels. Strikingly, rescue of the decreased Ca(2+)-channel localization required the RIM PDZ domain, whereas rescue of vesicle priming required the RIM N terminus. We propose that RIMs tether N- and P/Q-type Ca(2+) channels to presynaptic active zones via a direct PDZ-domain-mediated interaction, thereby enabling fast, synchronous triggering of neurotransmitter release at a synapse.

    View details for DOI 10.1016/j.cell.2010.12.029

    View details for Web of Science ID 000286459900012

    View details for PubMedID 21241895

  • CSP alpha promotes SNARE-complex assembly by chaperoning SNAP-25 during synaptic activity NATURE CELL BIOLOGY Sharma, M., Burre, J., Suedhof, T. C. 2011; 13 (1): 30-U74

    Abstract

    A neuron forms thousands of presynaptic nerve terminals on its axons, far removed from the cell body. The protein CSP? resides in presynaptic terminals, where it forms a chaperone complex with Hsc70 and SGT. Deletion of CSP? results in massive neurodegeneration that impairs survival in mice and flies. In CSP?-knockout mice, levels of presynaptic SNARE complexes and the SNARE protein SNAP-25 are reduced, suggesting that CSP? may chaperone SNARE proteins, which catalyse synaptic vesicle fusion. Here, we show that the CSP?-Hsc70-SGT complex binds directly to monomeric SNAP-25 to prevent its aggregation, enabling SNARE-complex formation. Deletion of CSP? produces an abnormal SNAP-25 conformer that inhibits SNARE-complex formation, and is subject to ubiquitylation and proteasomal degradation. Even in wild-type mouse terminals, SNAP-25 degradation is regulated by synaptic activity; this degradation is decreased by CSP? overexpression, and enhanced by CSP? deletion. Thus, SNAP-25 function is maintained during rapid SNARE cycles by equilibrium between CSP?-dependent chaperoning and ubiquitin-dependent degradation, revealing unique protein quality-control machinery within the presynaptic compartment.

    View details for DOI 10.1038/ncb2131

    View details for Web of Science ID 000285502200008

    View details for PubMedID 21151134

  • Testing the SNARE/SM protein model of membrane fusion PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Bacaj, T., Pang, Z. P., Suedhof, T. C. 2010; 107 (52): 22365-22366

    View details for DOI 10.1073/pnas.1017268108

    View details for Web of Science ID 000285684200006

    View details for PubMedID 21169506

  • SynCAM 1 Adhesion Dynamically Regulates Synapse Number and Impacts Plasticity and Learning NEURON Robbins, E. M., Krupp, A. J., de Arce, K. P., Ghosh, A. K., Fogel, A. I., Boucard, A., Suedhof, T. C., Stein, V., Biederer, T. 2010; 68 (5): 894-906

    Abstract

    Synaptogenesis is required for wiring neuronal circuits in the developing brain and continues to remodel adult networks. However, the molecules organizing synapse development and maintenance in vivo remain incompletely understood. We now demonstrate that the immunoglobulin adhesion molecule SynCAM 1 dynamically alters synapse number and plasticity. Overexpression of SynCAM 1 in transgenic mice promotes excitatory synapse number, while loss of SynCAM 1 results in fewer excitatory synapses. By turning off SynCAM 1 overexpression in transgenic brains, we show that it maintains the newly induced synapses. SynCAM 1 also functions at mature synapses to alter their plasticity by regulating long-term depression. Consistent with these effects on neuronal connectivity, SynCAM 1 expression affects spatial learning, with knock-out mice learning better. The reciprocal effects of increased SynCAM 1 expression and loss reveal that this adhesion molecule contributes to the regulation of synapse number and plasticity, and impacts how neuronal networks undergo activity-dependent changes.

    View details for DOI 10.1016/j.neuron.2010.11.003

    View details for Web of Science ID 000285664900011

    View details for PubMedID 21145003

  • Complexin Clamps Asynchronous Release by Blocking a Secondary Ca2+ Sensor via Its Accessory alpha Helix NEURON Yang, X., Kaeser-Woo, Y. J., Pang, Z. P., Xu, W., Suedhof, T. C. 2010; 68 (5): 907-920

    Abstract

    Complexin activates and clamps neurotransmitter release; impairing complexin function decreases synchronous, but increases spontaneous and asynchronous synaptic vesicle exocytosis. Here, we show that complexin-different from the Ca(2+) sensor synaptotagmin-1-activates synchronous exocytosis by promoting synaptic vesicle priming, but clamps spontaneous and asynchronous exocytosis-similar to synaptotagmin-1-by blocking a secondary Ca(2+) sensor. Activation and clamping functions of complexin depend on distinct, autonomously acting sequences, namely its N-terminal region and accessory ? helix, respectively. Mutations designed to test whether the accessory ? helix of complexin clamps exocytosis by inserting into SNARE-complexes support this hypothesis, suggesting that the accessory ? helix blocks completion of trans-SNARE-complex assembly until Ca(2+) binding to synaptotagmin relieves this block. Moreover, a juxtamembranous mutation in the SNARE-protein synaptobrevin-2, which presumably impairs force transfer from nascent trans-SNARE complexes onto fusing membranes, also unclamps spontaneous fusion by disinhibiting a secondary Ca(2+) sensor. Thus, complexin performs mechanistically distinct activation and clamping functions that operate in conjunction with synaptotagmin-1 by controlling trans-SNARE-complex assembly.

    View details for DOI 10.1016/j.neuron.2010.11.001

    View details for Web of Science ID 000285664900012

    View details for PubMedID 21145004

  • Inactivation of presenilins causes pre-synaptic impairment prior to post-synaptic dysfunction JOURNAL OF NEUROCHEMISTRY Zhang, D., Zhang, C., Ho, A., Kirkwood, A., Suedhof, T. C., Shen, J. 2010; 115 (5): 1215-1221

    Abstract

    J. Neurochem. (2010) 115, 1215-1221. ABSTRACT: Synaptic dysfunction is widely thought to be a pathogenic precursor to neurodegeneration in Alzheimer's disease (AD), and the extent of synaptic loss provides the best correlate for the severity of dementia in AD patients. Presenilins 1 and 2 are the major causative genes of early-onset familial AD. Conditional inactivation of presenilins in the adult cerebral cortex results in synaptic dysfunction and memory impairment, followed by age-dependent neurodegeneration. To characterize further the consequence of presenilin inactivation in the synapse, we evaluated the temporal development of pre-synaptic and post-synaptic deficits in the Schaeffer-collateral pathway of presenilin conditional double knockout (PS cDKO) mice prior to onset of neurodegeneration. Following presenilin inactivation at 4?weeks, synaptic facilitation and probability of neurotransmitter release are impaired in PS cDKO mice at 5?weeks of age, whereas post-synaptic NMDA receptor (NMDAR)-mediated responses are normal at 5?weeks but impaired at 6?weeks of age. Long-term potentiation induced by theta burst stimulation is also reduced in PS cDKO mice at 6?weeks of age. These results show that loss of presenilins results in pre-synaptic deficits in short-term plasticity and probability of neurotransmitter release prior to post-synaptic NMDAR dysfunction, raising the possibility that presenilins may regulate post-synaptic NMDAR function in part via a trans-synaptic mechanism.

    View details for DOI 10.1111/j.1471-4159.2010.07011.x

    View details for Web of Science ID 000283951300013

    View details for PubMedID 20854432

  • Neuronal Calcium Sensor Synaptotagmin-9 Is Not Involved in the Regulation of Glucose Homeostasis or Insulin Secretion PLOS ONE Gustavsson, N., Wang, X., Wang, Y., Seah, T., Xu, J., Radda, G. K., Suedhof, T. C., Han, W. 2010; 5 (11)

    Abstract

    Insulin secretion is a complex and highly regulated process. It is well established that cytoplasmic calcium is a key regulator of insulin secretion, but how elevated intracellular calcium triggers insulin granule exocytosis remains unclear, and we have only begun to define the identities of proteins that are responsible for sensing calcium changes and for transmitting the calcium signal to release machineries. Synaptotagmins are primarily expressed in brain and endocrine cells and exhibit diverse calcium binding properties. Synaptotagmin-1, -2 and -9 are calcium sensors for fast neurotransmitter release in respective brain regions, while synaptotagmin-7 is a positive regulator of calcium-dependent insulin release. Unlike the three neuronal calcium sensors, whose deletion abolished fast neurotransmitter release, synaptotagmin-7 deletion resulted in only partial loss of calcium-dependent insulin secretion, thus suggesting that other calcium-sensors must participate in the regulation of insulin secretion. Of the other synaptotagmin isoforms that are present in pancreatic islets, the neuronal calcium sensor synaptotagmin-9 is expressed at the highest level after synaptotagmin-7.In this study we tested whether synaptotagmin-9 participates in the regulation of glucose-stimulated insulin release by using pancreas-specific synaptotagmin-9 knockout (p-S9X) mice. Deletion of synaptotagmin-9 in the pancreas resulted in no changes in glucose homeostasis or body weight. Glucose tolerance, and insulin secretion in vivo and from isolated islets were not affected in the p-S9X mice. Single-cell capacitance measurements showed no difference in insulin granule exocytosis between p-S9X and control mice.Thus, synaptotagmin-9, although a major calcium sensor in the brain, is not involved in the regulation of glucose-stimulated insulin release from pancreatic ?-cells.

    View details for DOI 10.1371/journal.pone.0015414

    View details for Web of Science ID 000284035900029

    View details for PubMedID 21085706

  • Push-and-pull regulation of the fusion pore by synaptotagmin-7 PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Segovia, M., Ales, E., Angeles Montes, M., Bonifas, I., Jemal, I., Lindau, M., Maximov, A., Suedhof, T. C., de Toledo, G. A. 2010; 107 (44): 19032-19037

    Abstract

    In chromaffin cells, Ca(2+) binding to synaptotagmin-1 and -7 triggers exocytosis by promoting fusion pore opening and fusion pore expansion. Synaptotagmins contain two C2 domains that both bind Ca(2+) and contribute to exocytosis; however, it remains unknown whether the C2 domains act similarly or differentially to promote opening and expansion of fusion pores. Here, we use patch amperometry measurements in WT and synaptotagmin-7-mutant chromaffin cells to analyze the role of Ca(2+) binding to the two synaptotagmin-7 C2 domains in exocytosis. We show that, surprisingly, Ca(2+) binding to the C2A domain suffices to trigger fusion pore opening but that the resulting fusion pores are unstable and collapse, causing a dramatic increase in kiss-and-run fusion events. Thus, synaptotagmin-7 controls fusion pore dynamics during exocytosis via a push-and-pull mechanism in which Ca(2+) binding to both C2 domains promotes fusion pore opening, but the C2B domain is selectively essential for continuous expansion of an otherwise unstable fusion pore.

    View details for DOI 10.1073/pnas.1014070107

    View details for Web of Science ID 000283749000054

    View details for PubMedID 20956309

  • Rab3 Proteins Involved in Vesicle Biogenesis and Priming in Embryonic Mouse Chromaffin Cells TRAFFIC Schonn, J., van Weering, J. R., Mohrmann, R., Schlueter, O. M., Suedhof, T. C., de Wit, H., Verhage, M., Sorensen, J. B. 2010; 11 (11): 1415-1428

    Abstract

    The four Rab3 paralogs A-D are involved in exocytosis, but their mechanisms of action are hard to study due to functional redundancy. Here, we used a quadruple Rab3 knockout (KO) (rab3a, rab3b, rab3c, rab3d null, here denoted as ABCD(-/-) ) mouse line to investigate Rab3 function in embryonic mouse adrenal chromaffin cells by electron microscopy and electrophysiological measurements. We show that in cells from ABCD(-/-) animals large dense-core vesicles (LDCVs) are less abundant, while the number of morphologically docked granules is normal. By capacitance measurements, we show that deletion of Rab3s reduces the size of the releasable vesicle pools but does not alter their fusion kinetics, consistent with an altered function in vesicle priming. The sustained release component has a sigmoid shape in ABCD(-/-) cells when normalized to the releasable pool size, indicating that vesicle priming follows at a higher rate after an initial delay. Rescue experiments showed that short-term (4-6 h) overexpression of Rab3A or Rab3C suffices to rescue vesicle priming and secretion, but it does not restore the number of secretory vesicles. We conclude that Rab3 proteins play two distinct stimulating roles for LDCV fusion in embryonic chromaffin cells, by facilitating vesicle biogenesis and stabilizing the primed vesicle state.

    View details for DOI 10.1111/j.1600-0854.2010.01107.x

    View details for Web of Science ID 000282571500004

    View details for PubMedID 20716109

  • Calmodulin Suppresses Synaptotagmin-2 Transcription in Cortical Neurons JOURNAL OF BIOLOGICAL CHEMISTRY Pang, Z. P., Xu, W., Cao, P., Suedhof, T. C. 2010; 285 (44): 33930-33939

    Abstract

    Calmodulin (CaM) is a ubiquitous Ca(2+) sensor protein that plays a pivotal role in regulating innumerable neuronal functions, including synaptic transmission. In cortical neurons, most neurotransmitter release is triggered by Ca(2+) binding to synaptotagmin-1; however, a second delayed phase of release, referred to as asynchronous release, is triggered by Ca(2+) binding to an unidentified secondary Ca(2+) sensor. To test whether CaM could be the enigmatic Ca(2+) sensor for asynchronous release, we now use in cultured neurons short hairpin RNAs that suppress expression of ?70% of all neuronal CaM isoforms. Surprisingly, we found that in synaptotagmin-1 knock-out neurons, the CaM knockdown caused a paradoxical rescue of synchronous release, instead of a block of asynchronous release. Gene and protein expression studies revealed that both in wild-type and in synaptotagmin-1 knock-out neurons, the CaM knockdown altered expression of >200 genes, including that encoding synaptotagmin-2. Synaptotagmin-2 expression was increased several-fold by the CaM knockdown, which accounted for the paradoxical rescue of synchronous release in synaptotagmin-1 knock-out neurons by the CaM knockdown. Interestingly, the CaM knockdown primarily activated genes that are preferentially expressed in caudal brain regions, whereas it repressed genes in rostral brain regions. Consistent with this correlation, quantifications of protein levels in adult mice uncovered an inverse relationship of CaM and synaptotagmin-2 levels in mouse forebrain, brain stem, and spinal cord. Finally, we employed molecular replacement experiments using a knockdown rescue approach to show that Ca(2+) binding to the C-lobe but not the N-lobe of CaM is required for suppression of synaptotagmin-2 expression in cortical neurons. Our data describe a previously unknown, Ca(2+)/CaM-dependent regulatory pathway that controls the expression of synaptic proteins in the rostral-caudal neuraxis.

    View details for DOI 10.1074/jbc.M110.150151

    View details for Web of Science ID 000283354000040

    View details for PubMedID 20729199

  • QnAs with Thomas C. Südhof. Interview by Prashant Nair. Proceedings of the National Academy of Sciences of the United States of America Südhof, T. C. 2010; 107 (42): 17863-?

    View details for DOI 10.1073/pnas.1012628107

    View details for PubMedID 20855597

  • Soluble amyloid precursor protein (APP) regulates transthyretin and Klotho gene expression without rescuing the essential function of APP PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Li, H., Wang, B., Wang, Z., Guo, Q., Tabuchi, K., Hammer, R. E., Suedhof, T. C., Zheng, H. 2010; 107 (40): 17362-17367

    Abstract

    Amyloidogenic processing of the amyloid precursor protein (APP) generates a large secreted ectodomain fragment (APPs?), ?-amyloid (A?) peptides, and an APP intracellular domain (AICD). Whereas A? is viewed as critical for Alzheimer's disease pathogenesis, the role of other APP processing products remains enigmatic. Of interest, the AICD has been implicated in transcriptional regulation, and N-terminal cleavage of APPs? has been suggested to produce an active fragment that may mediate axonal pruning and neuronal cell death. We previously reported that mice deficient in APP and APP-like protein 2 (APLP2) exhibit early postnatal lethality and neuromuscular synapse defects, whereas mice with neuronal conditional deletion of APP and APLP2 are viable. Using transcriptional profiling, we now identify transthyretin (TTR) and Klotho as APP/APLP2-dependent genes whose expression is decreased in loss-of-function states but increased in gain-of-function states. Significantly, by creating an APP knockin allele that expresses only APPs? protein, we demonstrate that APPs? is not normally cleaved in vivo and is fully capable of mediating the APP-dependent regulation of TTR and Klotho gene expression. Despite being an active regulator of gene expression, APPs? did not rescue the lethality and neuromuscular synapse defects of APP and APLP2 double-KO animals. Our studies identify TTR and Klotho as physiological targets of APP that are regulated by soluble APPs? independent of developmental APP functions. This unexpected APP-mediated signaling pathway may play an important role in maintaining TTR and Klotho levels and their respective functions in A? sequestration and aging.

    View details for DOI 10.1073/pnas.1012568107

    View details for Web of Science ID 000282512000057

    View details for PubMedID 20855613

  • Genetic Dissection of the Amyloid Precursor Protein in Developmental Function and Amyloid Pathogenesis JOURNAL OF BIOLOGICAL CHEMISTRY Li, H., Wang, Z., Wang, B., Guo, Q., Dolios, G., Tabuchi, K., Hammer, R. E., Suedhof, T. C., Wang, R., Zheng, H. 2010; 285 (40): 30598-30605

    Abstract

    Proteolytic processing of the amyloid precursor protein (APP) generates large soluble APP derivatives, ?-amyloid (A?) peptides, and APP intracellular domain. Expression of the extracellular sequences of APP or its Caenorhabditis elegans counterpart has been shown to be sufficient in partially rescuing the CNS phenotypes of the APP-deficient mice and the lethality of the apl-1 null C. elegans, respectively, leaving open the question as what is the role of the highly conserved APP intracellular domain? To address this question, we created an APP knock-in allele in which the mouse A? sequence was replaced by the human A?. A frameshift mutation was introduced that replaced the last 39 residues of the APP sequence. We demonstrate that the C-terminal mutation does not overtly affect APP processing and amyloid pathology. In contrast, crossing the mutant allele with APP-like protein 2 (APLP2)-null mice results in similar neuromuscular synapse defects and early postnatal lethality as compared with mice doubly deficient in APP and APLP2, demonstrating an indispensable role of the APP C-terminal domain in these development activities. Our results establish an essential function of the conserved APP intracellular domain in developmental regulation, and this activity can be genetically uncoupled from APP processing and A? pathogenesis.

    View details for DOI 10.1074/jbc.P110.137729

    View details for Web of Science ID 000282135500028

    View details for PubMedID 20693289

  • An interview with the Kavli prize winners. Interview by Claudia Wiedemann. Nature reviews. Neuroscience Rothman, J., Scheller, R., Südhof, T. 2010; 11 (10): 669-673

    View details for PubMedID 21080540

  • alpha-Synuclein Promotes SNARE-Complex Assembly in Vivo and in Vitro SCIENCE Burre, J., Sharma, M., Tsetsenis, T., Buchman, V., Etherton, M. R., Suedhof, T. C. 2010; 329 (5999): 1663-1667

    Abstract

    Presynaptic nerve terminals release neurotransmitters repeatedly, often at high frequency, and in relative isolation from neuronal cell bodies. Repeated release requires cycles of soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE)-complex assembly and disassembly, with continuous generation of reactive SNARE-protein intermediates. Although many forms of neurodegeneration initiate presynaptically, only few pathogenic mechanisms are known, and the functions of presynaptic proteins linked to neurodegeneration, such as ?-synuclein, remain unclear. Here, we show that maintenance of continuous presynaptic SNARE-complex assembly required a nonclassical chaperone activity mediated by synucleins. Specifically, ?-synuclein directly bound to the SNARE-protein synaptobrevin-2/vesicle-associated membrane protein 2 (VAMP2) and promoted SNARE-complex assembly. Moreover, triple-knockout mice lacking synucleins developed age-dependent neurological impairments, exhibited decreased SNARE-complex assembly, and died prematurely. Thus, synucleins may function to sustain normal SNARE-complex assembly in a presynaptic terminal during aging.

    View details for DOI 10.1126/science.1195227

    View details for Web of Science ID 000282098100047

    View details for PubMedID 20798282

  • Structural and Mutational Analysis of Functional Differentiation between Synaptotagmins-1 and-7 PLOS ONE Xue, M., Craig, T. K., Shin, O., Li, L., Brautigam, C. A., Tomchick, D. R., Suedhof, T. C., Rosenmund, C., Rizo, J. 2010; 5 (9)

    Abstract

    Synaptotagmins are known to mediate diverse forms of Ca2+-triggered exocytosis through their C2 domains, but the principles underlying functional differentiation among them are unclear. Synaptotagmin-1 functions as a Ca2+ sensor in neurotransmitter release at central nervous system synapses, but synaptotagmin-7 does not, and yet both isoforms act as Ca2+ sensors in chromaffin cells. To shed light into this apparent paradox, we have performed rescue experiments in neurons from synaptotagmin-1 knockout mice using a chimera that contains the synaptotagmin-1 sequence with its C2B domain replaced by the synaptotagmin-7 C2B domain (Syt1/7). Rescue was not achieved either with the WT Syt1/7 chimera or with nine mutants where residues that are distinct in synaptotagmin-7 were restored to those present in synaptotagmin-1. To investigate whether these results arise because of unique conformational features of the synaptotagmin-7 C2B domain, we determined its crystal structure at 1.44 A resolution. The synaptotagmin-7 C2B domain structure is very similar to that of the synaptotagmin-1 C2B domain and contains three Ca2+-binding sites. Two of the Ca2+-binding sites of the synaptotagmin-7 C2B domain are also present in the synaptotagmin-1 C2B domain and have analogous ligands to those determined for the latter by NMR spectroscopy, suggesting that a discrepancy observed in a crystal structure of the synaptotagmin-1 C2B domain arose from crystal contacts. Overall, our results suggest that functional differentiation in synaptotagmins arises in part from subtle sequence changes that yield dramatic functional differences.

    View details for DOI 10.1371/journal.pone.0012544

    View details for Web of Science ID 000281480900025

    View details for PubMedID 20824061

  • Cell biology of Ca2+-triggered exocytosis CURRENT OPINION IN CELL BIOLOGY Pang, Z. P., Suedhof, T. C. 2010; 22 (4): 496-505

    Abstract

    Ca(2+) triggers many forms of exocytosis in different types of eukaryotic cells, for example synaptic vesicle exocytosis in neurons, granule exocytosis in mast cells, and hormone exocytosis in endocrine cells. Work over the past two decades has shown that synaptotagmins function as the primary Ca(2+)-sensors for most of these forms of exocytosis, and that synaptotagmins act via Ca(2+)-dependent interactions with both the fusing phospholipid membranes and the membrane fusion machinery. However, some forms of Ca(2+)-induced exocytosis may utilize other, as yet unidentified Ca(2+)-sensors, for example, slow synaptic exocytosis mediating asynchronous neurotransmitter release. In the following overview, we will discuss the synaptotagmin-based mechanism of Ca(2+)-triggered exocytosis in neurons and neuroendocrine cells, and its potential extension to other types of Ca(2+)-stimulated exocytosis for which no synaptotagmin Ca(2+)-sensor has been identified.

    View details for DOI 10.1016/j.ceb.2010.05.001

    View details for Web of Science ID 000280945500011

    View details for PubMedID 20561775

  • Neurexins Physically and Functionally Interact with GABA(A) Receptors NEURON Zhang, C., Atasoy, D., Arac, D., Yang, X., Fucillo, M. V., Robison, A. J., Ko, J., Brunger, A. T., Sudhof, T. C. 2010; 66 (3): 403-416

    Abstract

    Neurexins are presynaptic cell-adhesion molecules that form trans-synaptic complexes with postsynaptic neuroligins. When overexpressed in nonneuronal cells, neurexins induce formation of postsynaptic specializations in cocultured neurons, suggesting that neurexins are synaptogenic. However, we find that when overexpressed in neurons, neurexins do not increase synapse density, but instead selectively suppressed GABAergic synaptic transmission without decreasing GABAergic synapse numbers. This suppression was mediated by all subtypes of neurexins tested, in a cell-autonomous and neuroligin-independent manner. Strikingly, addition of recombinant neurexin to cultured neurons at submicromolar concentrations induced the same suppression of GABAergic synaptic transmission as neurexin overexpression. Moreover, experiments with native brain proteins and purified recombinant proteins revealed that neurexins directly and stoichiometrically bind to GABA(A) receptors, suggesting that they decrease GABAergic synaptic responses by interacting with GABA(A) receptors. Our findings suggest that besides their other well-documented interactions, presynaptic neurexins directly act on postsynaptic GABA(A) receptors, which may contribute to regulate the excitatory/inhibitory balance in brain.

    View details for DOI 10.1016/j.neuron.2010.04.008

    View details for Web of Science ID 000277825200009

    View details for PubMedID 20471353

  • Climate Change and the Integrity of Science SCIENCE Gleick, P. H., ADAMS, R. M., Amasino, R. M., ANDERS, E., ANDERSON, D. J., Anderson, W. W., Anselin, L. E., Arroyo, M. K., Asfaw, B., Ayala, F. J., Bax, A., Bebbington, A. J., Bell, G., Bennett, M. V., Bennetzen, J. L., Berenbaum, M. R., Berlin, O. B., BJORKMAN, P. J., Blackburn, E., Blamont, J. E., Botchan, M. R., Boyer, J. S., Boyle, E. A., Branton, D., Briggs, S. P., Briggs, W. R., BRILL, W. J., Britten, R. J., Broecker, W. S., Brown, J. H., Brown, P. O., Brunger, A. T., Cairns, J., Canfield, D. E., Carpenter, S. R., Carrington, J. C., Cashmore, A. R., CASTILLA, J. C., Cazenave, A., Chapin, F. S., CIECHANOVER, A. J., Clapham, D. E., CLARK, W. C., Clayton, R. N., Coe, M. D., Conwell, E. M., Cowling, E. B., Cowling, R. M., Cox, C. S., Croteau, R. B., Crothers, D. M., Crutzen, P. J., Daily, G. C., Dalrymple, G. B., Dangl, J. L., Darst, S. A., Davies, D. R., Davis, M. B., De Camilli, P. V., Dean, C., DeFries, R. S., Deisenhofer, J., Delmer, D. P., DeLong, E. F., DeRosier, D. J., Diener, T. O., Dirzo, R., Dixon, J. E., Donoghue, M. J., Doolittle, R. F., Dunne, T., Ehrlich, P. R., Eisenstadt, S. N., Eisner, T., Emanuel, K. A., Englander, S. W., Ernst, W. G., Falkowski, P. G., Feher, G., FEREJOHN, J. A., Fersht, A., Fischer, E. H., Fischer, R., Flannery, K. V., Frank, J., Frey, P. A., Fridovich, I., FRIEDEN, C., Futuyma, D. J., Gardner, W. R., GARRETT, C. J., Gilbert, W., Goldberg, R. B., GOODENOUGH, W. H., Goodman, C. S., Goodman, M., Greengard, P., Hake, S., HAMMEL, G., Hanson, S., HARRISON, S. C., Hart, S. R., Hartl, D. L., Haselkorn, R., Hawkes, K., Hayes, J. M., Hille, B., Hoekfelt, T., House, J. S., HOUT, M., Hunten, D. M., Izquierdo, I. A., Jagendorf, A. T., Janzen, D. H., Jeanloz, R., Jencks, C. S., Jury, W. A., Kaback, H. R., KAILATH, T., Kay, P., Kay, S. A., Kennedy, D., Kerr, A., Kessler, R. C., Khush, G. S., Kieffer, S. W., Kirch, P. V., Kirk, K., Kivelson, M. G., Klinman, J. P., Klug, A., Knopoff, L., Kornberg, H., Kutzbach, J. E., Lagarias, J. C., Lambeck, K., Landy, A., Langmuir, C. H., Larkins, B. A., Le Pichon, X. T., Lenski, R. E., Leopold, E. B., Levin, S. A., Levitt, M., Likens, G. E., Lippincott-Schwartz, J., Lorand, L., Lovejoy, C. O., Lynch, M., Mabogunje, A. L., Malone, T. F., Manabe, S., Marcus, J., Massey, D. S., McWilliams, J. C., Medina, E., Melosh, H. J., Meltzer, D. J., Michener, C. D., Miles, E. L., Mooney, H. A., Moore, P. B., Morel, F. M., Mosley-Thompson, E. S., Moss, B., Munk, W. H., Myers, N., Nair, G. B., Nathans, J., Nester, E. W., Nicoll, R. A., Novick, R. P., O'Connell, J. F., Olsen, P. E., Opdyke, N. D., Oster, G. F., Ostrom, E., Pace, N. R., Paine, R. T., Palmiter, R. D., Pedlosky, J., Petsko, G. A., Pettengill, G. H., Philander, S. G., Piperno, D. R., Pollard, T. D., Price, P. B., REICHARD, P. A., Reskin, B. F., Ricklefs, R. E., Rivest, R. L., Roberts, J. D., Romney, A. K., Rossmann, M. G., Russell, D. W., Rutter, W. J., Sabloff, J. A., SAGDEEV, R. Z., Sahlins, M. D., Salmond, A., Sanes, J. R., Schekman, R., Schellnhuber, J., Schindler, D. W., Schmitt, J., Schneider, S. H., Schramm, V. L., SEDEROFF, R. R., Shatz, C. J., Sherman, F., Sidman, R. L., Sieh, K., Simons, E. L., Singer, B. H., Singer, M. F., Skyrms, B., Sleep, N. H., Smith, B. D., Snyder, S. H., Sokal, R. R., Spencer, C. S., Steitz, T. A., Strier, K. B., SUEDHOF, T. C., Taylor, S. S., Terborgh, J., Thomas, D. H., Thompson, L. G., Jian, R. T., Turner, M. G., Uyeda, S., Valentine, J. W., Valentine, J. S., Van Etten, J. L., van Holde, K. E., Vaughan, M., Verba, S., von Hippel, P. H., Wake, D. B., Walker, A., Walker, J. E., Watson, E. B., Watson, P. J., Weigel, D., Wessler, S. R., West-Eberhard, M. J., White, T. D., Wilson, W. J., Wolfenden, R. V., Wood, J. A., Woodwell, G. M., Wright, H. E., Wu, C., Wunsch, C., Zoback, M. L. 2010; 328 (5979): 689-690

    View details for Web of Science ID 000277357100011

    View details for PubMedID 20448167

  • Evolution of CASK into a Mg2+-Sensitive Kinase SCIENCE SIGNALING Mukherjee, K., Sharma, M., Jahn, R., Wahl, M. C., Suedhof, T. C. 2010; 3 (119)

    Abstract

    All known protein kinases, except CASK [calcium/calmodulin (CaM)-activated serine-threonine kinase], require magnesium ions (Mg(2+)) to stimulate the transfer of a phosphate from adenosine 5'-triphosphate (ATP) to a protein substrate. The CaMK (calcium/calmodulin-dependent kinase) domain of CASK shows activity in the absence of Mg(2+); indeed, it is inhibited by divalent ions including Mg(2+). Here, we converted the Mg(2+)-inhibited wild-type CASK kinase (CASK(WT)) into a Mg(2+)-stimulated kinase (CASK(4M)) by substituting four residues within the ATP-binding pocket. Crystal structures of CASK(4M) with and without bound nucleotide and Mn(2+), together with kinetic analyses, demonstrated that Mg(2+) accelerates catalysis of CASK(4M) by stabilizing the transition state, enhancing the leaving group properties of adenosine 5'-diphosphate, and indirectly shifting the position of the gamma-phosphate of ATP. Phylogenetic analysis revealed that the four residues conferring Mg(2+)-mediated stimulation were substituted from CASK during early animal evolution, converting a primordial, Mg(2+)-coordinating form of CASK into a Mg(2+)-inhibited kinase. This emergence of Mg(2+) sensitivity (inhibition by Mg(2+)) conferred regulation of CASK activity by divalent cations, in parallel with the evolution of the animal nervous systems.

    View details for DOI 10.1126/scisignal.2000800

    View details for Web of Science ID 000277111000006

    View details for PubMedID 20424264

  • RIM1 alpha and Interacting Proteins Involved in Presynaptic Plasticity Mediate Prepulse Inhibition and Additional Behaviors Linked to Schizophrenia JOURNAL OF NEUROSCIENCE Blundell, J., Kaeser, P. S., Sudhof, T. C., Powell, C. M. 2010; 30 (15): 5326-5333

    Abstract

    Several presynaptic proteins involved in neurotransmitter release in the CNS have been implicated in schizophrenia in human clinical genetic studies, in postmortem studies, and in studies of putative animal models of schizophrenia. The presynaptic protein RIM1alpha mediates presynaptic plasticity and cognitive function. We now demonstrate that mice deficient in RIM1alpha exhibit abnormalities in multiple schizophrenia-relevant behavioral tasks including prepulse inhibition, response to psychotomimetic drugs, and social interaction. These schizophrenia-relevant behavioral findings are relatively selective to RIM1alpha-deficient mice, as mice bearing mutations in the RIM1alpha binding partners Rab3A or synaptotagmin 1 only show decreased prepulse inhibition. In addition to RIM1alpha's involvement in multiple behavioral abnormalities, these data suggest that alterations in presynaptic forms of short-term plasticity are linked to alterations in prepulse inhibition, a measure of sensorimotor gating.

    View details for DOI 10.1523/JNEUROSCI.0328-10.2010

    View details for Web of Science ID 000276685100020

    View details for PubMedID 20392954

  • Piccolo and bassoon maintain synaptic vesicle clustering without directly participating in vesicle exocytosis PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Mukherjee, K., Yang, X., Gerber, S. H., Kwon, H., Ho, A., Castillo, P. E., Liu, X., Suedhof, T. C. 2010; 107 (14): 6504-6509

    Abstract

    Piccolo and bassoon are highly homologous multidomain proteins of the presynaptic cytomatrix whose function is unclear. Here, we generated piccolo knockin/knockout mice that either contain wild-type levels of mutant piccolo unable to bind Ca(2+) (knockin), approximately 60% decreased levels of piccolo that is C-terminally truncated (partial knockout), or <5% levels of piccolo (knockout). All piccolo mutant mice were viable and fertile, but piccolo knockout mice exhibited increased postnatal mortality. Unexpectedly, electrophysiology and electron microscopy of piccolo-deficient synapses failed to uncover a major phenotype either in acute hippocampal slices or in cultured cortical neurons. To unmask potentially redundant functions of piccolo and bassoon, we thus acutely knocked down expression of bassoon in wild-type and piccolo knockout neurons. Despite a nearly complete loss of piccolo and bassoon, however, we still did not detect an electrophysiological phenotype in cultured piccolo- and bassoon-deficient neurons in either GABAergic or glutamatergic synaptic transmission. In contrast, electron microscopy revealed a significant reduction in synaptic vesicle clustering in double bassoon/piccolo-deficient synapses. Thus, we propose that piccolo and bassoon play a redundant role in synaptic vesicle clustering in nerve terminals without directly participating in neurotransmitter release.

    View details for DOI 10.1073/pnas.1002307107

    View details for Web of Science ID 000276374400068

    View details for PubMedID 20332206

  • Calmodulin Controls Synaptic Strength via Presynaptic Activation of Calmodulin Kinase II JOURNAL OF NEUROSCIENCE Pang, Z. P., Cao, P., Xu, W., Suedhof, T. C. 2010; 30 (11): 4132-4142

    Abstract

    Calmodulin regulates multifarious cellular processes via a panoply of target interactions. However, the central role, multiple isoforms, and complex target interactions of calmodulin make it difficult to examine its precise functions. Here, we analyzed calmodulin function in neurons using lentivirally delivered short-hairpin RNAs that suppressed expression of all calmodulin isoforms by approximately 70%. Calmodulin knockdown did not significantly alter neuronal survival or synapse formation but depressed spontaneous neuronal network activity. Strikingly, calmodulin knockdown decreased the presynaptic release probability almost twofold, without altering the presynaptic readily-releasable vesicle pool or postsynaptic neurotransmitter reception. In calmodulin knockdown neurons, presynaptic release was restored to wild-type levels by expression of constitutively active calmodulin-dependent kinase-IIalpha (CaMKIIalpha); in contrast, in control neurons, expression of constitutively active CaMKIIalpha had no effect on presynaptic release. Viewed together, these data suggest that calmodulin performs a major function in boosting synaptic strength via direct activation of presynaptic calmodulin-dependent kinase II.

    View details for DOI 10.1523/JNEUROSCI.3129-09.2010

    View details for Web of Science ID 000275724800029

    View details for PubMedID 20237283

  • Munc13 C2B domain is an activity-dependent Ca2+ regulator of synaptic exocytosis NATURE STRUCTURAL & MOLECULAR BIOLOGY Shin, O., Lu, J., Rhee, J., Tomchick, D. R., Pang, Z. P., Wojcik, S. M., Camacho-Perez, M., Brose, N., Machius, M., Rizo, J., Rosenmund, C., Suedhof, T. C. 2010; 17 (3): 280-U42

    Abstract

    Munc13 is a multidomain protein present in presynaptic active zones that mediates the priming and plasticity of synaptic vesicle exocytosis, but the mechanisms involved remain unclear. Here we use biophysical, biochemical and electrophysiological approaches to show that the central C(2)B domain of Munc13 functions as a Ca(2+) regulator of short-term synaptic plasticity. The crystal structure of the C(2)B domain revealed an unusual Ca(2+)-binding site with an amphipathic alpha-helix. This configuration confers onto the C(2)B domain unique Ca(2+)-dependent phospholipid-binding properties that favor phosphatidylinositolphosphates. A mutation that inactivated Ca(2+)-dependent phospholipid binding to the C(2)B domain did not alter neurotransmitter release evoked by isolated action potentials, but it did depress release evoked by action-potential trains. In contrast, a mutation that increased Ca(2+)-dependent phosphatidylinositolbisphosphate binding to the C(2)B domain enhanced release evoked by isolated action potentials and by action-potential trains. Our data suggest that, during repeated action potentials, Ca(2+) and phosphatidylinositolphosphate binding to the Munc13 C(2)B domain potentiate synaptic vesicle exocytosis, thereby offsetting synaptic depression induced by vesicle depletion.

    View details for DOI 10.1038/nsmb.1758

    View details for Web of Science ID 000275182700006

    View details for PubMedID 20154707

  • Direct conversion of fibroblasts to functional neurons by defined factors NATURE Vierbuchen, T., Ostermeier, A., Pang, Z. P., Kokubu, Y., Suedhof, T. C., Wernig, M. 2010; 463 (7284): 1035-U50

    Abstract

    Cellular differentiation and lineage commitment are considered to be robust and irreversible processes during development. Recent work has shown that mouse and human fibroblasts can be reprogrammed to a pluripotent state with a combination of four transcription factors. This raised the question of whether transcription factors could directly induce other defined somatic cell fates, and not only an undifferentiated state. We hypothesized that combinatorial expression of neural-lineage-specific transcription factors could directly convert fibroblasts into neurons. Starting from a pool of nineteen candidate genes, we identified a combination of only three factors, Ascl1, Brn2 (also called Pou3f2) and Myt1l, that suffice to rapidly and efficiently convert mouse embryonic and postnatal fibroblasts into functional neurons in vitro. These induced neuronal (iN) cells express multiple neuron-specific proteins, generate action potentials and form functional synapses. Generation of iN cells from non-neural lineages could have important implications for studies of neural development, neurological disease modelling and regenerative medicine.

    View details for DOI 10.1038/nature08797

    View details for Web of Science ID 000275108400027

    View details for PubMedID 20107439

  • Neuroligin-1 Deletion Results in Impaired Spatial Memory and Increased Repetitive Behavior JOURNAL OF NEUROSCIENCE Blundell, J., Blaiss, C. A., Etherton, M. R., Espinosa, F., Tabuchi, K., Walz, C., Bolliger, M. F., Suedhof, T. C., Powell, C. M. 2010; 30 (6): 2115-2129

    Abstract

    Neuroligins (NLs) are a family of neural cell-adhesion molecules that are involved in excitatory/inhibitory synapse specification. Multiple members of the NL family (including NL1) and their binding partners have been linked to cases of human autism and mental retardation. We have now characterized NL1-deficient mice in autism- and mental retardation-relevant behavioral tasks. NL1 knock-out (KO) mice display deficits in spatial learning and memory that correlate with impaired hippocampal long-term potentiation. In addition, NL1 KO mice exhibit a dramatic increase in repetitive, stereotyped grooming behavior, a potential autism-relevant abnormality. This repetitive grooming abnormality in NL1 KO mice is associated with a reduced NMDA/AMPA ratio at corticostriatal synapses. Interestingly, we further demonstrate that the increased repetitive grooming phenotype can be rescued in adult mice by administration of the NMDA receptor partial coagonist d-cycloserine. Broadly, these data are consistent with a role of synaptic cell-adhesion molecules in general, and NL1 in particular, in autism and implicate reduced excitatory synaptic transmission as a potential mechanism and treatment target for repetitive behavioral abnormalities.

    View details for DOI 10.1523/JNEUROSCI.4517-09.2010

    View details for Web of Science ID 000274398200013

    View details for PubMedID 20147539

  • beta-Neurexin Is a Ligand for the Staphylococcus aureus MSCRAMM SdrC PLOS PATHOGENS Barbu, E. M., Ganesh, V. K., Gurusiddappa, S., Mackenzie, R. C., Foster, T. J., Sudhof, T. C., Hoeoek, M. 2010; 6 (1)

    Abstract

    Gram-positive bacteria contain a family of surface proteins that are covalently anchored to the cell wall of the organism. These cell-wall anchored (CWA) proteins appear to play key roles in the interactions between pathogenic organisms and the host. A subfamily of the CWA has a common structural organization with multiple domains adopting characteristic IgG-like folds. The identified microbial surface components recognizing adhesive matrix molecules (MSCRAMMs) belong to this subfamily, as does SdrC from S. aureus. However, an interactive host ligand for the putative MSCRAMM SdrC was not previously identified. We have screened a phage display peptide library and identified a peptide sequence found in beta-neurexin that binds SdrC. A synthetic peptide corresponding to the identified sequence as well as a recombinant form of the beta-neurexin 1 exodomain binds SdrC with high affinity and specificity. Furthermore, expression of SdrC on bacteria greatly enhances microbial adherence to cultured mammalian cells expressing beta-neurexin on their surface. Taken together, our experimental results demonstrate that beta-neurexin is a ligand for SdrC. This interaction involves a specific sequence located in the N-terminal region of the mammalian protein and the N(2)N(3) domain of the MSCRAMM. The fact that these two proteins interact when expressed on the appropriate cells demonstrates the functionality of the interaction. Possible implications of this interaction are discussed.

    View details for DOI 10.1371/journal.ppat.1000726

    View details for Web of Science ID 000274227100017

    View details for PubMedID 20090838

  • LRRTM2 Functions as a Neurexin Ligand in Promoting Excitatory Synapse Formation NEURON Ko, J., Fuccillo, M. V., Malenka, R. C., Suedhof, T. C. 2009; 64 (6): 791-798

    Abstract

    Recently, leucine-rich repeat transmembrane proteins (LRRTMs) were found to be synaptic cell-adhesion molecules that, when expressed in nonneuronal cells, induce presynaptic differentiation in contacting axons. We now demonstrate that LRRTM2 induces only excitatory synapses, and that it also acts to induce synapses in transfected neurons similarly to neuroligin-1. Using affinity chromatography, we identified alpha- and beta-neurexins as LRRTM2 ligands, again rendering LRRTM2 similar to neuroligin-1. However, whereas neuroligins bind neurexins containing or lacking an insert in splice site #4, LRRTM2 only binds neurexins lacking an insert in splice site #4. Binding of neurexins to LRRTM2 can produce cell-adhesion junctions, consistent with a trans-interaction regulated by neurexin alternative splicing, and recombinant neurexin-1beta blocks LRRTM2's ability to promote presynaptic differentiation. Thus, our data suggest that two unrelated postsynaptic cell-adhesion molecules, LRRTMs and neuroligins, unexpectedly bind to neurexins as the same presynaptic receptor, but that their binding is subject to distinct regulatory mechanisms.

    View details for DOI 10.1016/j.neuron.2009.12.012

    View details for Web of Science ID 000273425800007

    View details for PubMedID 20064387

  • Neuroligin-2 Deletion Selectively Decreases Inhibitory Synaptic Transmission Originating from Fast-Spiking but Not from Somatostatin-Positive Interneurons JOURNAL OF NEUROSCIENCE Gibson, J. R., Huber, K. M., Suedhof, T. C. 2009; 29 (44): 13883-13897

    Abstract

    Neuroligins are cell adhesion molecules involved in synapse formation and/or function. Neurons express four neuroligins (NL1-NL4), of which NL1 is specific to excitatory and NL2 to inhibitory synapses. Excitatory and inhibitory synapses include numerous subtypes. However, it is unknown whether NL1 performs similar functions in all excitatory and NL2 in all inhibitory synapses, or whether they regulate the formation and/or function of specific subsets of synapses. To address this central question, we performed paired recordings in primary somatosensory cortex of mice lacking NL1 or NL2. Using this system, we examined neocortical microcircuits formed by reciprocal synapses between excitatory neurons and two subtypes of inhibitory interneurons, namely, fast-spiking and somatostatin-positive interneurons. We find that the NL1 deletion had little effect on inhibitory synapses, whereas the NL2 deletion decreased (40-50%) the unitary (cell-to-cell) IPSC amplitude evoked from single fast-spiking interneurons. Strikingly, the NL2 deletion had no effect on IPSC amplitude evoked from single somatostatin-positive inhibitory interneurons. Moreover, the frequency of unitary synaptic connections between individual fast-spiking and somatostatin-positive interneurons and excitatory neurons was unchanged. The decrease in unitary IPSC amplitude originating from fast-spiking interneurons in NL2-deficient mice was due to a multiplicative and uniform downscaling of the amplitude distribution, which in turn was mediated by a decrease in both synaptic quantal amplitude and quantal content, the latter inferred from an increase in the coefficient of variation. Thus, NL2 is not necessary for establishing unitary inhibitory synaptic connections but is selectively required for "scaling up" unitary connections originating from a subset of interneurons.

    View details for DOI 10.1523/JNEUROSCI.2457-09.2009

    View details for Web of Science ID 000271471400015

    View details for PubMedID 19889999

  • ELKS2 alpha/CAST Deletion Selectively Increases Neurotransmitter Release at Inhibitory Synapses NEURON Kaeser, P. S., Deng, L., Chavez, A. E., Liu, X., Castillo, P. E., Suedhof, T. C. 2009; 64 (2): 227-239

    Abstract

    The presynaptic active zone is composed of a protein network that contains ELKS2alpha (a.k.a. CAST) as a central component. Here we demonstrate that in mice, deletion of ELKS2alpha caused a large increase in inhibitory, but not excitatory, neurotransmitter release, and potentiated the size, but not the properties, of the readily-releasable pool of vesicles at inhibitory synapses. Quantitative electron microscopy revealed that the ELKS2alpha deletion did not change the number of docked vesicles or other ultrastructural parameters of synapses, except for a small decrease in synaptic vesicle numbers. The ELKS2alpha deletion did, however, alter the excitatory/inhibitory balance and exploratory behaviors, possibly as a result of the increased synaptic inhibition. Thus, as opposed to previous studies indicating that ELKS2alpha is essential for mediating neurotransmitter release, our results suggest that ELKS2alpha normally restricts release and limits the size of the readily-releasable pool of synaptic vesicles at the active zone of inhibitory synapses.

    View details for DOI 10.1016/j.neuron.2009.09.019

    View details for Web of Science ID 000271889900012

    View details for PubMedID 19874790

  • Neuroligin-1 performs neurexin-dependent and neurexin-independent functions in synapse validation EMBO JOURNAL Ko, J., Zhang, C., Arac, D., Boucard, A. A., Brunger, A. T., Suedhof, T. C. 2009; 28 (20): 3244-3255

    Abstract

    Postsynaptic neuroligins are thought to perform essential functions in synapse validation and synaptic transmission by binding to, and dimerizing, presynaptic alpha- and beta-neurexins. To test this hypothesis, we examined the functional effects of neuroligin-1 mutations that impair only alpha-neurexin binding, block both alpha- and beta-neurexin binding, or abolish neuroligin-1 dimerization. Abolishing alpha-neurexin binding abrogated neuroligin-induced generation of neuronal synapses onto transfected non-neuronal cells in the so-called artificial synapse-formation assay, even though beta-neurexin binding was retained. Thus, in this assay, neuroligin-1 induces apparent synapse formation by binding to presynaptic alpha-neurexins. In transfected neurons, however, neither alpha- nor beta-neurexin binding was essential for the ability of postsynaptic neuroligin-1 to dramatically increase synapse density, suggesting a neurexin-independent mechanism of synapse formation. Moreover, neuroligin-1 dimerization was not required for either the non-neuronal or the neuronal synapse-formation assay. Nevertheless, both alpha-neurexin binding and neuroligin-1 dimerization were essential for the increase in apparent synapse size that is induced by neuroligin-1 in transfected neurons. Thus, neuroligin-1 performs diverse synaptic functions by mechanisms that include as essential components of alpha-neurexin binding and neuroligin dimerization, but extend beyond these activities.

    View details for DOI 10.1038/emboj.2009.249

    View details for Web of Science ID 000271008200016

    View details for PubMedID 19730411

  • Mouse neurexin-1 alpha deletion causes correlated electrophysiological and behavioral changes consistent with cognitive impairments PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Etherton, M. R., Blaiss, C. A., Powell, C. M., Suedhof, T. C. 2009; 106 (42): 17998-18003

    Abstract

    Deletions in the neurexin-1alpha gene were identified in large-scale unbiased screens for copy-number variations in patients with autism or schizophrenia. To explore the underlying biology, we studied the electrophysiological and behavioral phenotype of mice lacking neurexin-1alpha. Hippocampal slice physiology uncovered a defect in excitatory synaptic strength in neurexin-1alpha deficient mice, as revealed by a decrease in miniature excitatory postsynaptic current (EPSC) frequency and in the input-output relation of evoked postsynaptic potentials. This defect was specific for excitatory synaptic transmission, because no change in inhibitory synaptic transmission was observed in the hippocampus. Behavioral studies revealed that, compared with littermate control mice, neurexin-1alpha deficient mice displayed a decrease in prepulse inhibition, an increase in grooming behaviors, an impairment in nest-building activity, and an improvement in motor learning. However, neurexin-1alpha deficient mice did not exhibit any obvious changes in social behaviors or in spatial learning. Together, these data indicate that the neurexin-1alpha deficiency induces a discrete neural phenotype whose extent correlates, at least in part, with impairments observed in human patients.

    View details for DOI 10.1073/pnas.0910297106

    View details for Web of Science ID 000270963100071

    View details for PubMedID 19822762

  • Differential but convergent functions of Ca2+ binding to synaptotagmin-1 C-2 domains mediate neurotransmitter release PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Shin, O., Xu, J., Rizo, J., Suedhof, T. C. 2009; 106 (38): 16469-16474

    Abstract

    Neurotransmitter release is triggered by cooperative Ca2+-binding to the Ca2+-sensor protein synaptotagmin-1. Synaptotagmin-1 contains two C2 domains, referred to as the C2A and C2B domains, that bind Ca2+ with similar properties and affinities. However, Ca2+ binding to the C2A domain is not required for release, whereas Ca2+ binding to the C2B domain is essential for release. We now demonstrate that despite its expendability, Ca2+-binding to the C2A domain significantly contributes to the overall triggering of neurotransmitter release, and determines its Ca2+ cooperativity. Biochemically, Ca2+ induces more tight binding of the isolated C2A domain than of the isolated C2B domain to standard liposomes composed of phosphatidylcholine and phosphatidylserine. However, here we show that surprisingly, the opposite holds true when the double C2A/B-domain fragment of synaptotagmin-1 is used instead of isolated C2 domains, and when liposomes containing a physiological lipid composition are used. Under these conditions, Ca2+ binding to the C2B domain but not the C2A domain becomes the primary determinant of phospholipid binding. Thus, the unique requirement for Ca2+ binding to the C2B domain for synaptotagmin-1 in Ca2+-triggered neurotransmitter release may be accounted for, at least in part, by the unusual phospholipid-binding properties of its double C2A/B-domain fragment.

    View details for DOI 10.1073/pnas.0908798106

    View details for Web of Science ID 000270071600084

    View details for PubMedID 19805322

  • A Neuroligin-4 Missense Mutation Associated with Autism Impairs Neuroligin-4 Folding and Endoplasmic Reticulum Export JOURNAL OF NEUROSCIENCE Zhang, C., Milunsky, J. M., Newton, S., Ko, J., Zhao, G., Maher, T. A., Tager-Flusberg, H., Bolliger, M. F., Carter, A. S., Boucard, A. A., Powell, C. M., Suedhof, T. C. 2009; 29 (35): 10843-10854

    Abstract

    Neuroligins (NLs) are postsynaptic cell-adhesion molecules essential for normal synapse function. Mutations in neuroligin-4 (NL4) (gene symbol: NLGN4) have been reported in some patients with autism spectrum disorder (ASD) and other neurodevelopmental impairments. However, the low frequency of NL4 mutations and the limited information about the affected patients and the functional consequences of their mutations cast doubt on the causal role of NL4 mutations in these disorders. Here, we describe two brothers with classical ASD who carry a single amino-acid substitution in NL4 (R87W). This substitution was absent from the brothers' asymptomatic parents, suggesting that it arose in the maternal germ line. R87 is conserved in all NL isoforms, and the R87W substitution is not observed in control individuals. At the protein level, the R87W substitution impaired glycosylation processing of NL4 expressed in HEK293 and COS cells, destabilized NL4, caused NL4 retention in the endoplasmic reticulum in non-neuronal cells and neurons, and blocked NL4 transport to the cell surface. As a result, the R87W substitution inactivated the synapse-formation activity of NL4 and abolished the functional effect of NL4 on synapse strength. Viewed together, these observations suggest that a point mutation in NL4 can cause ASD by a loss-of-function mechanism.

    View details for DOI 10.1523/JNEUROSCI.1248-09.2009

    View details for Web of Science ID 000269518500008

    View details for PubMedID 19726642

  • Presenilins are essential for regulating neurotransmitter release NATURE Zhang, C., Wu, B., Beglopoulos, V., Wines-Samuelson, M., Zhang, D., Dragatsis, I., Suedhof, T. C., Shen, J. 2009; 460 (7255): 632-U100

    Abstract

    Mutations in the presenilin genes are the main cause of familial Alzheimer's disease. Loss of presenilin activity and/or accumulation of amyloid-beta peptides have been proposed to mediate the pathogenesis of Alzheimer's disease by impairing synaptic function. However, the precise site and nature of the synaptic dysfunction remain unknown. Here we use a genetic approach to inactivate presenilins conditionally in either presynaptic (CA3) or postsynaptic (CA1) neurons of the hippocampal Schaeffer-collateral pathway. We show that long-term potentiation induced by theta-burst stimulation is decreased after presynaptic but not postsynaptic deletion of presenilins. Moreover, we found that presynaptic but not postsynaptic inactivation of presenilins alters short-term plasticity and synaptic facilitation. The probability of evoked glutamate release, measured with the open-channel NMDA (N-methyl-D-aspartate) receptor antagonist MK-801, is reduced by presynaptic inactivation of presenilins. Notably, depletion of endoplasmic reticulum Ca(2+) stores by thapsigargin, or blockade of Ca(2+) release from these stores by ryanodine receptor inhibitors, mimics and occludes the effects of presynaptic presenilin inactivation. Collectively, these results indicate a selective role for presenilins in the activity-dependent regulation of neurotransmitter release and long-term potentiation induction by modulation of intracellular Ca(2+) release in presynaptic terminals, and further suggest that presynaptic dysfunction might be an early pathogenic event leading to dementia and neurodegeneration in Alzheimer's disease.

    View details for DOI 10.1038/nature08177

    View details for Web of Science ID 000268454300051

    View details for PubMedID 19641596

  • alpha-Latrotoxin Stimulates a Novel Pathway of Ca2+-Dependent Synaptic Exocytosis Independent of the Classical Synaptic Fusion Machinery JOURNAL OF NEUROSCIENCE Deak, F., Liu, X., Khvotchev, M., Li, G., Kavalali, E. T., Sugita, S., Sudhof, T. C. 2009; 29 (27): 8639-8648

    Abstract

    Alpha-latrotoxin induces neurotransmitter release by stimulating synaptic vesicle exocytosis via two mechanisms: (1) A Ca(2+)-dependent mechanism with neurexins as receptors, in which alpha-latrotoxin acts like a Ca(2+) ionophore, and (2) a Ca(2+)-independent mechanism with CIRL/latrophilins as receptors, in which alpha-latrotoxin directly stimulates the transmitter release machinery. Here, we show that the Ca(2+)-independent release mechanism by alpha-latrotoxin requires the synaptic SNARE-proteins synaptobrevin/VAMP and SNAP-25, and, at least partly, the synaptic active-zone protein Munc13-1. In contrast, the Ca(2+)-dependent release mechanism induced by alpha-latrotoxin does not require any of these components of the classical synaptic release machinery. Nevertheless, this type of exocytotic neurotransmitter release appears to fully operate at synapses, and to stimulate exocytosis of the same synaptic vesicles that participate in physiological action potential-triggered release. Thus, synapses contain two parallel and independent pathways of Ca(2+)-triggered exocytosis, a classical, physiological pathway that operates at the active zone, and a novel reserve pathway that is recruited only when Ca(2+) floods the synaptic terminal.

    View details for DOI 10.1523/JNEUROSCI.0898-09.2009

    View details for Web of Science ID 000267818400004

    View details for PubMedID 19587270

  • Conditional Forebrain Inactivation of Nicastrin Causes Progressive Memory Impairment and Age-Related Neurodegeneration JOURNAL OF NEUROSCIENCE Tabuchi, K., Chen, G., Suedhof, T. C., Shen, J. 2009; 29 (22): 7290-7301

    Abstract

    Loss of presenilin function in adult mouse brains causes memory loss and age-related neurodegeneration. Since presenilin possesses gamma-secretase-dependent and -independent activities, it remains unknown which activity is required for presenilin-dependent memory formation and neuronal survival. To address this question, we generated postnatal forebrain-specific nicastrin conditional knock-out (cKO) mice, in which nicastrin, a subunit of gamma-secretase, is inactivated selectively in mature excitatory neurons of the cerebral cortex. nicastrin cKO mice display progressive impairment in learning and memory and exhibit age-dependent cortical neuronal loss, accompanied by astrocytosis, microgliosis, and hyperphosphorylation of the microtubule-associated protein Tau. The neurodegeneration observed in nicastrin cKO mice likely occurs via apoptosis, as evidenced by increased numbers of apoptotic neurons. These findings demonstrate an essential role of nicastrin in the execution of learning and memory and the maintenance of neuronal survival in the brain and suggest that presenilin functions in memory and neuronal survival via its role as a gamma-secretase subunit.

    View details for DOI 10.1523/JNEUROSCI.1320-09.2009

    View details for Web of Science ID 000266632400020

    View details for PubMedID 19494151

  • Common circuit defect of excitatory-inhibitory balance in mouse models of autism JOURNAL OF NEURODEVELOPMENTAL DISORDERS Gogolla, N., LeBlanc, J. J., Quast, K. B., Sudhof, T. C., Fagiolini, M., Hensch, T. K. 2009; 1 (2): 172-181

    Abstract

    One unifying explanation for the complexity of Autism Spectrum Disorders (ASD) may lie in the disruption of excitatory/inhibitory (E/I) circuit balance during critical periods of development. We examined whether Parvalbumin (PV)-positive inhibitory neurons, which normally drive experience-dependent circuit refinement (Hensch Nat Rev Neurosci 6:877-888, 1), are disrupted across heterogeneous ASD mouse models. We performed a meta-analysis of PV expression in previously published ASD mouse models and analyzed two additional models, reflecting an embryonic chemical insult (prenatal valproate, VPA) or single-gene mutation identified in human patients (Neuroligin-3, NL-3 R451C). PV-cells were reduced in the neocortex across multiple ASD mouse models. In striking contrast to controls, both VPA and NL-3 mouse models exhibited an asymmetric PV-cell reduction across hemispheres in parietal and occipital cortices (but not the underlying area CA1). ASD mouse models may share a PV-circuit disruption, providing new insight into circuit development and potential prevention by treatment of autism.The online version of this article (doi:10.1007/s11689-009-9023-x) contains supplementary material, which is available to authorized users.

    View details for DOI 10.1007/s11689-009-9023-x

    View details for Web of Science ID 000274848700007

    View details for PubMedID 20664807

  • Synaptotagmin-1 functions as a Ca2+ sensor for spontaneous release NATURE NEUROSCIENCE Xu, J., Pang, Z. P., Shin, O., Suedhof, T. C. 2009; 12 (6): 759-U111

    Abstract

    Spontaneous 'mini' release occurs at all synapses, but its nature remains enigmatic. We found that >95% of spontaneous release in murine cortical neurons was induced by Ca2+-binding to synaptotagmin-1 (Syt1), the Ca2+ sensor for fast synchronous neurotransmitter release. Thus, spontaneous and evoked release used the same Ca2+-dependent release mechanism. As a consequence, Syt1 mutations that altered its Ca2+ affinity altered spontaneous and evoked release correspondingly. Paradoxically, Syt1 deletions (as opposed to point mutations) massively increased spontaneous release. This increased spontaneous release remained Ca2+ dependent but was activated at lower Ca2+ concentrations and with a lower Ca2+ cooperativity than synaptotagmin-driven spontaneous release. Thus, in addition to serving as a Ca2+ sensor for spontaneous and evoked release, Syt1 clamped a second, more sensitive Ca2+ sensor for spontaneous release that resembles the Ca2+ sensor for evoked asynchronous release. These data suggest that Syt1 controls both evoked and spontaneous release at a synapse as a simultaneous Ca2+-dependent activator and clamp of exocytosis.

    View details for DOI 10.1038/nn.2320

    View details for Web of Science ID 000266380900020

    View details for PubMedID 19412166

  • Synaptotagmin-7 is a principal Ca2+ sensor for Ca2+-induced glucagon exocytosis in pancreas JOURNAL OF PHYSIOLOGY-LONDON Gustavsson, N., Wei, S., Hoang, D. N., Lao, Y., Zhang, Q., Radda, G. K., Rorsman, P., Sudhof, T. C., Han, W. 2009; 587 (6): 1169-1178

    Abstract

    Hormones such as glucagon are secreted by Ca(2+)-induced exocytosis of large dense-core vesicles, but the mechanisms involved have only been partially elucidated. Studies of pancreatic beta-cells secreting insulin revealed that synaptotagmin-7 alone is not sufficient to mediate Ca(2+)-dependent insulin granule exocytosis, and studies of chromaffin cells secreting neuropeptides and catecholamines showed that synaptotagmin-1 and -7 collaborate as Ca(2+) sensors for exocytosis, and that both are equally involved. As no other peptide secretion was analysed, it remains unclear whether synaptotagmins generally act as Ca(2+) sensors in large dense-core vesicle exocytosis in endocrine cells, and if so, whether synaptotagmin-7 always functions with a partner in that role. In particular, far less is known about the mechanisms underlying Ca(2+)-triggered glucagon release from alpha-cells than insulin secretion from beta-cells, even though insulin and glucagon together regulate blood glucose levels. To address these issues, we analysed the role of synaptotagmins in Ca(2+)-triggered glucagon exocytosis. Surprisingly, we find that deletion of a single synaptotagmin isoform, synaptotagmin-7, nearly abolished Ca(2+)-triggered glucagon secretion. Moreover, single-cell capacitance measurements confirmed that pancreatic alpha-cells lacking synaptotagmin-7 exhibited little Ca(2+)-induced exocytosis, whereas all other physiological and morphological parameters of the alpha-cells were normal. Our data thus identify synaptotagmin-7 as a principal Ca(2+) sensor for glucagon secretion, and support the notion that synaptotagmins perform a universal but selective function as individually acting Ca(2+) sensors in neurotransmitter, neuropeptide, and hormone secretion.

    View details for DOI 10.1113/jphysiol.2008.168005

    View details for Web of Science ID 000264188400005

    View details for PubMedID 19171650

  • Munc18-1 binding to the neuronal SNARE complex controls synaptic vesicle priming JOURNAL OF CELL BIOLOGY Deak, F., Xu, Y., Chang, W., Dulubova, I., Khvotchev, M., Liu, X., Suedhof, T. C., Rizo, J. 2009; 184 (5): 751-764

    Abstract

    Munc18-1 and soluble NSF attachment protein receptors (SNAREs) are critical for synaptic vesicle fusion. Munc18-1 binds to the SNARE syntaxin-1 folded into a closed conformation and to SNARE complexes containing open syntaxin-1. Understanding which steps in fusion depend on the latter interaction and whether Munc18-1 competes with other factors such as complexins for SNARE complex binding is critical to elucidate the mechanisms involved. In this study, we show that lentiviral expression of Munc18-1 rescues abrogation of release in Munc18-1 knockout mice. We describe point mutations in Munc18-1 that preserve tight binding to closed syntaxin-1 but markedly disrupt Munc18-1 binding to SNARE complexes containing open syntaxin-1. Lentiviral rescue experiments reveal that such disruption selectively impairs synaptic vesicle priming but not Ca(2+)-triggered fusion of primed vesicles. We also find that Munc18-1 and complexin-1 bind simultaneously to SNARE complexes. These results suggest that Munc18-1 binding to SNARE complexes mediates synaptic vesicle priming and that the resulting primed state involves a Munc18-1-SNARE-complexin macromolecular assembly that is poised for Ca(2+) triggering of fusion.

    View details for DOI 10.1083/jcb.200812026

    View details for Web of Science ID 000264016700012

    View details for PubMedID 19255244

  • Increased anxiety-like behavior in mice lacking the inhibitory synapse cell adhesion molecule neuroligin 2 GENES BRAIN AND BEHAVIOR Blundell, J., Tabuchi, K., Bolliger, M. F., Blaiss, C. A., Brose, N., Liu, X., Sudhof, T. C., Powell, C. M. 2009; 8 (1): 114-126

    Abstract

    Neuroligins (NL) are postsynaptic cell adhesion molecules that are thought to specify synapse properties. Previous studies showed that mutant mice carrying an autism-associated point mutation in NL3 exhibit social interaction deficits, enhanced inhibitory synaptic function and increased staining of inhibitory synaptic puncta without changes in overall inhibitory synapse numbers. In contrast, mutant mice lacking NL2 displayed decreased inhibitory synaptic function. These studies raised two relevant questions. First, does NL2 deletion impair inhibitory synaptic function by altering the number of inhibitory synapses, or by changing their efficacy? Second, does this effect of NL2 deletion on inhibition produce behavioral changes? We now show that although NL2-deficient mice exhibit an apparent decrease in number of inhibitory synaptic puncta, the number of symmetric synapses as determined by electron microscopy is unaltered, suggesting that NL2 deletion impairs the function of inhibitory synapses without decreasing their numbers. This decrease in inhibitory synaptic function in NL2-deficient mice correlates with a discrete behavioral phenotype that includes a marked increase in anxiety-like behavior, a decrease in pain sensitivity and a slight decrease in motor co-ordination. This work confirms that NL2 modulates inhibitory synaptic function and is the first demonstration that global deletion of NL2 can lead to a selective behavioral phenotype.

    View details for DOI 10.1111/j.1601-183X.2008.00455.x

    View details for Web of Science ID 000262888000015

    View details for PubMedID 19016888

  • SV2 Renders Primed Synaptic Vesicles Competent for Ca2+-Induced Exocytosis JOURNAL OF NEUROSCIENCE Chang, W., Suedhof, T. C. 2009; 29 (4): 883-897

    Abstract

    Synaptic vesicle protein 2 (SV2), one of the first synaptic vesicle proteins identified, is characterized by multiple transmembrane regions that exhibit homology to sugar transporters, and by a highly glycosylated intravesicular sequence. Deletion of SV2 causes postnatal lethality in mice, primarily because of fulminant epilepsy. At the cellular level, deletion of SV2 impairs neurotransmitter release, but its function is unknown, and even the exact point at which release is affected in SV2-deleted synapses remains unclear. Using electrophysiological approaches, we now examine at what step in exocytosis the deletion of SV2 impairs release. Our data demonstrate that deletion of SV2 produces a decrease in evoked synaptic responses without causing changes in mini frequency, mini amplitude, the readily releasable pool of vesicles, or the apparent Ca(2+) sensitivity of vesicle fusion. These findings indicate that a previously unidentified step may couple priming of synaptic vesicles to Ca(2+) triggering of fusion, and that SV2 acts in this step to render primed synaptic vesicles fully Ca(2+) responsive. To investigate the structural requirements for this function of SV2, we used rescue experiments. We demonstrate that conserved charged residues within the transmembrane regions and the intravesicular glycosylation of SV2 are required for its normal folding and trafficking. In contrast, the conserved putative synaptotagmin-binding sequence of SV2 is fully dispensable. Viewed together, these observations suggest that SV2 functions in a maturation step of primed vesicles that converts the vesicles into a Ca(2+)- and synaptotagmin-responsive state.

    View details for DOI 10.1523/JNEUROSCI.4521-08.2009

    View details for Web of Science ID 000262859000001

    View details for PubMedID 19176798

  • Membrane Fusion: Grappling with SNARE and SM Proteins SCIENCE Sudhof, T. C., Rothman, J. E. 2009; 323 (5913): 474-477

    Abstract

    The two universally required components of the intracellular membrane fusion machinery, SNARE and SM (Sec1/Munc18-like) proteins, play complementary roles in fusion. Vesicular and target membrane-localized SNARE proteins zipper up into an alpha-helical bundle that pulls the two membranes tightly together to exert the force required for fusion. SM proteins, shaped like clasps, bind to trans-SNARE complexes to direct their fusogenic action. Individual fusion reactions are executed by distinct combinations of SNARE and SM proteins to ensure specificity, and are controlled by regulators that embed the SM-SNARE fusion machinery into a physiological context. This regulation is spectacularly apparent in the exquisite speed and precision of synaptic exocytosis, where synaptotagmin (the calcium-ion sensor for fusion) cooperates with complexin (the clamp activator) to control the precisely timed release of neurotransmitters that initiates synaptic transmission and underlies brain function.

    View details for DOI 10.1126/science.1161748

    View details for Web of Science ID 000262587900033

    View details for PubMedID 19164740

  • Complexin Controls the Force Transfer from SNARE Complexes to Membranes in Fusion SCIENCE Maximov, A., Tang, J., Yang, X., Pang, Z. P., Sudhof, T. C. 2009; 323 (5913): 516-521

    Abstract

    Trans-SNAP receptor (SNARE, where SNAP is defined as soluble NSF attachment protein, and NSF is defined as N-ethylmaleimide-sensitive factor) complexes catalyze synaptic vesicle fusion and bind complexin, but the function of complexin binding to SNARE complexes remains unclear. Here we show that in neuronal synapses, complexin simultaneously suppressed spontaneous fusion and activated fast calcium ion-evoked fusion. The dual function of complexin required SNARE binding and also involved distinct amino-terminal sequences of complexin that localize to the point where trans-SNARE complexes insert into the fusing membranes, suggesting that complexin controls the force that trans-SNARE complexes apply onto the fusing membranes. Consistent with this hypothesis, a mutation in the membrane insertion sequence of the v-SNARE synaptobrevin/vesicle-associated membrane protein (VAMP) phenocopied the complexin loss-of-function state without impairing complexin binding to SNARE complexes. Thus, complexin probably activates and clamps the force transfer from assembled trans-SNARE complexes onto fusing membranes.

    View details for DOI 10.1126/science.1166505

    View details for Web of Science ID 000262587900046

    View details for PubMedID 19164751

  • Deletion of Mint Proteins Decreases Amyloid Production in Transgenic Mouse Models of Alzheimer's Disease JOURNAL OF NEUROSCIENCE Ho, A., Liu, X., Sudhof, T. C. 2008; 28 (53): 14392-14400

    Abstract

    Mints/X11s are neuronal adaptor proteins that bind to amyloid-beta precursor protein (APP). Previous studies suggested that Mint/X11 proteins influence APP cleavage and affect production of pathogenic amyloid-beta (Abeta) peptides in Alzheimer's disease; however, the biological significance of Mint/X11 binding to APP and their possible role in Abeta production remain unclear. Here, we crossed conditional and constitutive Mint1, Mint2, and Mint3 knock-out mice with transgenic mouse models of Alzheimer's disease overproducing human Abeta peptides. We show that deletion of all three individual Mint proteins delays the age-dependent production of amyloid plaque numbers and Abeta40 and Abeta42 levels with loss of Mint2 having the largest effect. Acute conditional deletion of all three Mints in cultured neurons suppresses the accumulation of APP C-terminal fragments and the secretion of ectodomain APP by decreasing beta-cleavage but does not impair subsequent gamma-cleavage. These results suggest that the three Mint/X11 proteins regulate Abeta production by a novel mechanism that may have implications for therapeutic approaches to altering APP cleavage in Alzheimer's disease.

    View details for DOI 10.1523/JNEUROSCI.2481-08.2008

    View details for Web of Science ID 000262137800008

    View details for PubMedID 19118172

  • RIM1 alpha and RIM1 beta Are Synthesized from Distinct Promoters of the RIM1 Gene to Mediate Differential But Overlapping Synaptic Functions JOURNAL OF NEUROSCIENCE Kaeser, P. S., Kwon, H., Chiu, C. Q., Deng, L., Castillo, P. E., Suedhof, T. C. 2008; 28 (50): 13435-13447

    Abstract

    At a synapse, presynaptic terminals form a specialized area of the plasma membrane called the active zone that mediates neurotransmitter release. RIM1alpha is a multidomain protein that constitutes a central component of the active zone by binding to other active zone proteins such as Munc13 s, alpha-liprins, and ELKS, and to synaptic vesicle proteins such as Rab3 and synaptotagmin-1. In mice, knockout of RIM1alpha significantly impairs synaptic vesicle priming and presynaptic long-term plasticity, but is not lethal. We now find that the RIM1 gene encodes a second, previously unknown RIM1 isoform called RIM1beta that is upregulated in RIM1alpha knock-out mice. RIM1beta is identical to RIM1alpha except for the N terminus where RIM1beta lacks the N-terminal Rab3-binding sequence of RIM1alpha. Using newly generated knock-out mice lacking both RIM1alpha and RIM1beta, we demonstrate that different from the deletion of only RIM1alpha, deletion of both RIM1alpha and RIM1beta severely impairs mouse survival. Electrophysiological analyses show that the RIM1alphabeta deletion abolishes long-term presynaptic plasticity, as does RIM1alpha deletion alone. In contrast, the impairment in synaptic strength and short-term synaptic plasticity that is caused by the RIM1alpha deletion is aggravated by the deletion of both RIM1alpha and RIM1beta. Thus, our data indicate that the RIM1 gene encodes two different isoforms that perform overlapping but distinct functions in neurotransmitter release.

    View details for DOI 10.1523/JNEUROSCI.3235-08.2008

    View details for Web of Science ID 000261601800009

    View details for PubMedID 19074017

  • Understanding Synapses: Past, Present, and Future NEURON Suedhof, T. C., Malenka, R. C. 2008; 60 (3): 469-476

    Abstract

    Classical physiological work by Katz, Eccles, and others revealed the central importance of synapses in brain function, and characterized the mechanisms involved in synaptic transmission. Building on this work, major advances in the past two decades have elucidated how synapses work molecularly. In the present perspective, we provide a short description of our personal view of these advances, suggest a series of important future questions about synapses, and discuss ideas about how best to achieve further progress in the field.

    View details for DOI 10.1016/j.neuron.2008.10.011

    View details for Web of Science ID 000260983800018

    View details for PubMedID 18995821

  • Neuroligins and neurexins link synaptic function to cognitive disease NATURE Sudhof, T. C. 2008; 455 (7215): 903-911

    Abstract

    The brain processes information by transmitting signals at synapses, which connect neurons into vast networks of communicating cells. In these networks, synapses not only transmit signals but also transform and refine them. Neurexins and neuroligins are synaptic cell-adhesion molecules that connect presynaptic and postsynaptic neurons at synapses, mediate signalling across the synapse, and shape the properties of neural networks by specifying synaptic functions. In humans, alterations in genes encoding neurexins or neuroligins have recently been implicated in autism and other cognitive diseases, linking synaptic cell adhesion to cognition and its disorders.

    View details for DOI 10.1038/nature07456

    View details for Web of Science ID 000260038300038

    View details for PubMedID 18923512

  • RiM1 alpha phosphorylation at serine-413 by protein kinase A is not required for presynaptic long-term plasticity or learning PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Kaeser, P. S., Kwon, H., Blundell, J., Chevaleyre, V., Morishita, W., Malenka, R. C., Powell, C. M., Castillo, P. E., Sudhof, T. C. 2008; 105 (38): 14680-14685

    Abstract

    Activation of presynaptic cAMP-dependent protein kinase A (PKA) triggers presynaptic long-term plasticity in synapses such as cerebellar parallel fiber and hippocampal mossy fiber synapses. RIM1alpha, a large multidomain protein that forms a scaffold at the presynaptic active zone, is essential for presynaptic long-term plasticity in these synapses and is phosphorylated by PKA at serine-413. Previous studies suggested that phosphorylation of RIM1alpha at serine-413 is required for presynaptic long-term potentiation in parallel fiber synapses formed in vitro by cultured cerebellar neurons and that this type of presynaptic long-term potentiation is mediated by binding of 14-3-3 proteins to phosphorylated serine-413. To test the role of serine-413 phosphorylation in vivo, we have now produced knockin mice in which serine-413 is mutated to alanine. Surprisingly, we find that in these mutant mice, three different forms of presynaptic PKA-dependent long-term plasticity are normal. Furthermore, we observed that in contrast to RIM1alpha KO mice, RIM1 knockin mice containing the serine-413 substitution exhibit normal learning capabilities. The lack of an effect of the serine-413 mutation of RIM1alpha is not due to compensation by RIM2alpha because mice carrying both the serine-413 substitution and a RIM2alpha deletion still exhibited normal long-term presynaptic plasticity. Thus, phosphorylation of serine-413 of RIM1alpha is not essential for PKA-dependent long-term presynaptic plasticity in vivo, suggesting that PKA operates by a different mechanism despite the dependence of long-term presynaptic plasticity on RIM1alpha.

    View details for DOI 10.1073/pnas.0806679105

    View details for Web of Science ID 000259592400081

    View details for PubMedID 18799741

  • Conformational switch of syntaxin-1 controls synaptic vesicle fusion SCIENCE Gerber, S. H., Rah, J., Min, S., Liu, X., de Wit, H., Dulubova, I., Meyer, A. C., Rizo, J., Arancillo, M., Hammer, R. E., Verhage, M., Rosenmund, C., Sudhof, T. C. 2008; 321 (5895): 1507-1510

    Abstract

    During synaptic vesicle fusion, the soluble N-ethylmaleimide-sensitive factor-attachment protein receptor (SNARE) protein syntaxin-1 exhibits two conformations that both bind to Munc18-1: a "closed" conformation outside the SNARE complex and an "open" conformation in the SNARE complex. Although SNARE complexes containing open syntaxin-1 and Munc18-1 are essential for exocytosis, the function of closed syntaxin-1 is unknown. We generated knockin/knockout mice that expressed only open syntaxin-1B. Syntaxin-1B(Open) mice were viable but succumbed to generalized seizures at 2 to 3 months of age. Binding of Munc18-1 to syntaxin-1 was impaired in syntaxin-1B(Open) synapses, and the size of the readily releasable vesicle pool was decreased; however, the rate of synaptic vesicle fusion was dramatically enhanced. Thus, the closed conformation of syntaxin-1 gates the initiation of the synaptic vesicle fusion reaction, which is then mediated by SNARE-complex/Munc18-1 assemblies.

    View details for DOI 10.1126/science.1163174

    View details for Web of Science ID 000259121800046

    View details for PubMedID 18703708

  • Cysteine string protein-alpha is essential for the high calcium sensitivity of exocytosis in a vertebrate synapse EUROPEAN JOURNAL OF NEUROSCIENCE Ruiz, R., Casanas, J. J., Sudhof, T. C., Tabares, L. 2008; 27 (12): 3118-3131

    Abstract

    Cysteine string protein (CSPalpha) is a synaptic vesicle protein present in most central and peripheral nervous system synapses. Previous studies demonstrated that the deletion of CSPalpha results in postnatal sensorial and motor impairment and premature lethality. To understand the participation of CSPalpha in neural function in vertebrates, we have studied the properties of synaptic transmission of motor terminals in wild-type and CSPalpha knockout mice. Our results demonstrate that, in the absence of CSPalpha, fast Ca2+-triggered release was not affected at postnatal day (P)14 but was dramatically reduced at P18 and P30 without a change in release kinetics. Although mutant terminals also exhibited a reduction in functional vesicle pool size by P30, further analysis showed that neurotransmission could be 'rescued' by high extracellular [Ca2+] or by the presence of a phorbol ester, suggesting that an impairment in the fusion machinery, or in vesicle recycling, was not the primary cause of the dysfunction of this synapse. The specific shift to the right of the Ca2+ dependence of synchronous release, and the lineal dependence of secretion on extracellular [Ca2+] in mutant terminals after P18, suggests that CSPalpha is indispensable for a normal Ca2+ sensitivity of exocytosis in vertebrate mature synapses.

    View details for DOI 10.1111/j.1460-9568.2008.06301.x

    View details for Web of Science ID 000256853800004

    View details for PubMedID 18598257

  • Unusually rapid evolution of Neuroligin-4 in mice PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Bolliger, M. F., Pei, J., Maxeiner, S., Boucard, A. A., Grishin, N. V., Sudhof, T. C. 2008; 105 (17): 6421-6426

    Abstract

    Neuroligins (NLs) are postsynaptic cell-adhesion molecules that are implicated in humans in autism spectrum disorders because the genes encoding NL3 and NL4 are mutated in rare cases of familial autism. NLs are highly conserved evolutionarily, except that no NL4 was detected in the currently available mouse genome sequence assemblies. We now demonstrate that mice express a distant NL4 variant that rapidly evolved from other mammalian NL4 genes and that exhibits sequence variations even between different mouse strains. Despite its divergence, mouse NL4 binds neurexins and is transported into dendritic spines, suggesting that the core properties of NLs are retained in this divergent NL isoform. The selectively rapid evolution of NL4 in mice suggests that its function in the brain is under less stringent control than that of other NLs, shedding light on why its mutation in autism spectrum disorder patients is not lethal, but instead leads to a discrete developmental brain disorder.

    View details for DOI 10.1073/pnas.0801383105

    View details for Web of Science ID 000255534100038

    View details for PubMedID 18434543

  • CASK functions as a Mg2+-independent neurexin kinase CELL Mukherjee, K., Sharma, M., Urlaub, H., Bourenkov, G. P., Jahn, R., Suedhof, T. C., Wahl, M. C. 2008; 133 (2): 328-339

    Abstract

    CASK is a unique MAGUK protein that contains an N-terminal CaM-kinase domain besides the typical MAGUK domains. The CASK CaM-kinase domain is presumed to be a catalytically inactive pseudokinase because it lacks the canonical DFG motif required for Mg2+ binding that is thought to be indispensable for kinase activity. Here we show, however, that CASK functions as an active protein kinase even without Mg2+ binding. High-resolution crystal structures reveal that the CASK CaM-kinase domain adopts a constitutively active conformation that binds ATP and catalyzes phosphotransfer without Mg2+. The CASK CaM-kinase domain phosphorylates itself and at least one physiological interactor, the synaptic protein neurexin-1, to which CASK is recruited via its PDZ domain. Thus, our data indicate that CASK combines the scaffolding activity of MAGUKs with an unusual kinase activity that phosphorylates substrates recuited by the scaffolding activity. Moreover, our study suggests that other pseudokinases (10% of the kinome) could also be catalytically active.

    View details for DOI 10.1016/j.cell.2008.02.036

    View details for Web of Science ID 000255052000021

    View details for PubMedID 18423203

  • Synaptotagmin-1 and-7 are functionally overlapping Ca2+ sensors for exocytosis in adrenal chromaffin cells PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Schonn, J., Maximov, A., Lao, Y., Suedhof, T. C., Sorensen, J. B. 2008; 105 (10): 3998-4003

    Abstract

    Synaptotagmin-1, the canonical isoform of the synaptotagmin family, is a Ca(2+) sensor for fast synchronous neurotransmitter release in forebrain neurons and chromaffin cells. Even though deletion of synaptotagmin-1 abolishes fast exocytosis in chromaffin cells, it reduces overall secretion by only 20% because of the persistence of slow exocytosis. Therefore, another Ca(2+) sensor dominates release in these cells. Synaptotagmin-7 has a higher Ca(2+) affinity and slower binding kinetics than synaptotagmin-1, matching the proposed properties for the second, slower Ca(2+) sensor. Here, we examined Ca(2+)-triggered exocytosis in chromaffin cells from KO mice lacking synaptotagmin-7, and from knockin mice containing normal levels of a mutant synaptotagmin-7 whose C(2)B domain does not bind Ca(2+). In both types of mutant chromaffin cells, Ca(2+)-triggered exocytosis was decreased dramatically. Moreover, in chromaffin cells lacking both synaptotagmin-1 and -7, only a very slow release component, accounting for approximately 30% of WT exocytosis, persisted. These data establish synaptotagmin-7 as a major Ca(2+) sensor for exocytosis in chromaffin cells, which, together with synaptotagmin-1, mediates almost all of the Ca(2+) triggering of exocytosis in these cells, a surprising result, considering the lack of a role of synaptotagmin-7 in synaptic vesicle exocytosis.

    View details for DOI 10.1073/pnas.0712373105

    View details for Web of Science ID 000253930600061

    View details for PubMedID 18308932

  • Genetic analysis of synaptotagmin-7 function in synaptic vesicle exocytosis PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Maximov, A., Lao, Y., Li, H., Chen, X., Rizo, J., Sorensen, J. B., Suedhof, T. C. 2008; 105 (10): 3986-3991

    Abstract

    Synaptotagmin-7 is a candidate Ca(2+) sensor for exocytosis that is at least partly localized to synapses. Similar to synaptotagmin-1, which functions as a Ca(2+) sensor for fast synaptic vesicle (SV) exocytosis, synaptotagmin-7 contains C(2)A and C(2)B domains that exhibit Ca(2+)-dependent phospholipid binding. However, synaptotagmin-7 cannot replace synaptotagmin-1 as a Ca(2+) sensor for fast SV exocytosis, raising questions about the physiological significance of its Ca(2+)-binding properties. Here, we examine how synaptotagmin-7 binds Ca(2+) and test whether this Ca(2+) binding regulates Ca(2+)-triggered SV exocytosis. We show that the synaptotagmin-7 C(2)A domain exhibits a Ca(2+)-binding mode similar to that of the synaptotagmin-1 C(2)A domain, suggesting that the synaptotagmin-1 and -7 C(2) domains generally employ comparable Ca(2+)-binding mechanisms. We then generated mutant mice that lack synaptotagmin-7 or contain point mutations inactivating Ca(2+) binding either to both C(2) domains of synaptotagmin-7 or only to its C(2)B domain. Synaptotagmin-7-mutant mice were viable and fertile. Inactivation of Ca(2+) binding to both C(2) domains caused an approximately 70% reduction in synaptotagmin-7 levels, whereas inactivation of Ca(2+) binding to only the C(2)B domain did not alter synaptotagmin-7 levels. The synaptotagmin-7 deletion did not change fast synchronous release, slow asynchronous release, or short-term synaptic plasticity of release of neurotransmitters. Thus, our results show that Ca(2+) binding to the synaptotagmin-7 C(2) domains is physiologically important for stabilizing synaptotagmin-7, but that Ca(2+) binding by synaptotagmin-7 likely does not regulate SV exocytosis, consistent with a role for synaptotagmin-7 in other forms of Ca(2+)-dependent synaptic exocytosis.

    View details for DOI 10.1073/pnas.0712372105

    View details for Web of Science ID 000253930600059

    View details for PubMedID 18308933

  • Impaired insulin secretion and glucose intolerance in synaptotagmin-7 null mutant mice PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Gustavsson, N., Lao, Y., Maximov, A., Chuang, J., Kostromina, E., Repa, J. J., Li, C., Radda, G. K., Suedhof, T. C., Han, W. 2008; 105 (10): 3992-3997

    Abstract

    Vertebrates express at least 15 different synaptotagmins with the same domain structure but diverse localizations and tissue distributions. Synaptotagmin-1,-2, and -9 act as calcium sensors for the fast phrase of neurotransmitter release, and synaptotagmin-12 acts as a calcium-independent modulator of release. The exact functions of the remaining 11 synaptotagmins, however, have not been established. By analogy to the role of synaptotagmin-1, -2, and -9 in neurotransmission, these other synaptotagmins may serve as Ca(2+) transducers regulating other Ca(2+)-dependent membrane processes, such as insulin secretion in pancreatic beta-cells. Of these other synaptotagmins, synaptotagmin-7 is one of the most abundant and is present in pancreatic beta-cells. To determine whether synaptotagmin-7 regulates Ca(2+)-dependent insulin secretion, we analyzed synaptotagmin-7 null mutant mice for glucose tolerance and insulin release. Here, we show that synaptotagmin-7 is required for the maintenance of systemic glucose tolerance and glucose-stimulated insulin secretion. Mutant mice have normal insulin sensitivity, insulin production, islet architecture and ultrastructural organization, and metabolic and calcium responses but exhibit impaired glucose-induced insulin secretion, indicating a calcium-sensing defect during insulin-containing secretory granule exocytosis. Taken together, our findings show that synaptotagmin-7 functions as a positive regulator of insulin secretion and may serve as a calcium sensor controlling insulin secretion in pancreatic beta cells.

    View details for DOI 10.1073/pnas.0711700105

    View details for Web of Science ID 000253930600060

    View details for PubMedID 18308938

  • A molecular pathway of neurodegeneration linking alpha-synuclein to ApoE and A beta peptides NATURE NEUROSCIENCE Gallardo, G., Schlueter, O. M., Suedhof, T. C. 2008; 11 (3): 301-308

    Abstract

    Pathogenic aggregates of alpha-synuclein are thought to contribute to the development of Parkinson's disease. Inclusion bodies containing alpha-synuclein are present in Parkinson's disease and other neurodegenerative diseases, including Alzheimer's disease. Moreover, alpha-synuclein mutations are found in cases of familial Parkinson's disease, and transgenic overexpression of alpha-synuclein causes neurodegeneration in mice. The molecular mechanisms involved, however, remain incompletely understood. Here we show that, in transgenic mice, alpha-synuclein induced neurodegeneration involves activation of the ubiquitin/proteasome system, a massive increase in apolipoprotein E (ApoE) levels and accumulation of insoluble mouse Abeta. ApoE was not protective, but was injurious, as deletion of ApoE delayed the neurodegeneration caused by alpha-synuclein and suppressed the accumulation of Abeta. Our data reveal a molecular link between central pathogenic mechanisms implicated in Parkinson's disease and Alzheimer's disease and suggest that intracellular alpha-synuclein is pathogenic, at least in part, by activation of extracellular signaling pathways involving ApoE.

    View details for DOI 10.1038/nn2058

    View details for Web of Science ID 000253548300013

    View details for PubMedID 18297066

  • Structures of neuroligin-1 and the Neuroligin-l/Neurexin-1 beta complex reveal specificprotein-protein and protein-Ca2+ interactions NEURON Arac, D., Boucard, A. A., Ozkan, E., Strop, P., Newell, E., Sudhof, T. C., Brunger, A. T. 2007; 56 (6): 992-1003

    Abstract

    Neurexins and neuroligins provide trans-synaptic connectivity by the Ca2+-dependent interaction of their alternatively spliced extracellular domains. Neuroligins specify synapses in an activity-dependent manner, presumably by binding to neurexins. Here, we present the crystal structures of neuroligin-1 in isolation and in complex with neurexin-1 beta. Neuroligin-1 forms a constitutive dimer, and two neurexin-1 beta monomers bind to two identical surfaces on the opposite faces of the neuroligin-1 dimer to form a heterotetramer. The neuroligin-1/neurexin-1 beta complex exhibits a nanomolar affinity and includes a large binding interface that contains bound Ca2+. Alternatively spliced sites in neurexin-1 beta and in neuroligin-1 are positioned nearby the binding interface, explaining how they regulate the interaction. Structure-based mutations of neuroligin-1 at the interface disrupt binding to neurexin-1 beta, but not the folding of neuroligin-1 and confirm the validity of the binding interface of the neuroligin-1/neurexin-1 beta complex. Our results provide molecular insights for understanding the role of cell-adhesion proteins in synapse function.

    View details for DOI 10.1016/j.neuron.2007.12.002

    View details for Web of Science ID 000252138000009

    View details for PubMedID 18093522

  • A dual-Ca2+-sensor model for neurotransmitter release in a central synapse NATURE Sun, J., Pang, Z. P., Qin, D., Fahim, A. T., Adachi, R., Suedhof, T. C. 2007; 450 (7170): 676-U4

    Abstract

    Ca2+-triggered synchronous neurotransmitter release is well described, but asynchronous release-in fact, its very existence-remains enigmatic. Here we report a quantitative description of asynchronous neurotransmitter release in calyx-of-Held synapses. We show that deletion of synaptotagmin 2 (Syt2) in mice selectively abolishes synchronous release, allowing us to study pure asynchronous release in isolation. Using photolysis experiments of caged Ca2+, we demonstrate that asynchronous release displays a Ca2+ cooperativity of approximately 2 with a Ca2+ affinity of approximately 44 microM, in contrast to synchronous release, which exhibits a Ca2+ cooperativity of approximately 5 with a Ca2+ affinity of approximately 38 muM. Our results reveal that release triggered in wild-type synapses at low Ca2+ concentrations is physiologically asynchronous, and that asynchronous release completely empties the readily releasable pool of vesicles during sustained elevations of Ca2+. We propose a dual-Ca2+-sensor model of release that quantitatively describes the contributions of synchronous and asynchronous release under conditions of different presynaptic Ca2+ dynamics.

    View details for DOI 10.1038/nature06308

    View details for Web of Science ID 000251209700046

    View details for PubMedID 18046404

  • Dual modes of Munc18-1/SNARE interactions are coupled by functionally critical binding to syntaxin-1 n terminus JOURNAL OF NEUROSCIENCE Khvotchev, M., Dulubova, I., Sun, J., Dai, H., Rizo, J., Suedhof, T. C. 2007; 27 (45): 12147-12155

    Abstract

    The SM (Sec1/Munc18-like) protein Munc18-1 and the soluble N-ethylmaleimide-sensitive factor attachment protein (SNAP) receptor (SNARE) proteins syntaxin-1, SNAP-25, and synaptobrevin/VAMP (vesicle-associated membrane protein) constitute the core fusion machinery for synaptic vesicle exocytosis. Strikingly, Munc18-1 interacts with neuronal SNARE proteins in two distinct modes (i.e., with isolated syntaxin-1 alone in a "closed" conformation and with assembled SNARE complexes containing syntaxin-1 in an "open" conformation). However, it is unclear whether the two modes of Munc18/SNARE interactions are linked. We now show that both Munc18/SNARE interaction modes involve the same low-affinity binding of the extreme syntaxin-1 N terminus to Munc18-1, suggesting that this binding connects the two Munc18/SNARE interaction modes to each other. Using transfected cells as an in vitro assay system, we demonstrate that truncated syntaxins lacking a transmembrane region universally block exocytosis, but only if they contain a free intact N terminus. This block is enhanced by coexpression of either Munc18-1 or SNAP-25, suggesting that truncated syntaxins block exocytosis by forming an untethered inhibitory SNARE complex/Munc18-1 assembly in which the N-terminal syntaxin/Munc18 interaction is essential. Introduction of an N-terminal syntaxin peptide that disrupts this assembly blocks neurotransmitter release in the calyx of Held synapse, whereas a mutant peptide that does not disrupt the SNARE complex/Munc18 assembly has no effect. Viewed together, our data indicate that binding of Munc18 to the syntaxin N terminus unites different modes of Munc18/SNARE interactions and is essential for exocytic membrane fusion.

    View details for DOI 10.1523/JNEUROSCI.3655-07.2007

    View details for Web of Science ID 000250758600006

    View details for PubMedID 17989281

  • Neuroligin-3 mutation implicated in autism increases inhibitory synaptic transmission in mice SCIENCE Tabuchi, K., Blundell, J., Etherton, M. R., Hammer, R. E., Liu, X., Powell, C. M., Suedhof, T. C. 2007; 318 (5847): 71-76

    Abstract

    Autism spectrum disorders (ASDs) are characterized by impairments in social behaviors that are sometimes coupled to specialized cognitive abilities. A small percentage of ASD patients carry mutations in genes encoding neuroligins, which are postsynaptic cell-adhesion molecules. We introduced one of these mutations into mice: the Arg451-->Cys451 (R451C) substitution in neuroligin-3. R451C mutant mice showed impaired social interactions but enhanced spatial learning abilities. Unexpectedly, these behavioral changes were accompanied by an increase in inhibitory synaptic transmission with no apparent effect on excitatory synapses. Deletion of neuroligin-3, in contrast, did not cause such changes, indicating that the R451C substitution represents a gain-of-function mutation. These data suggest that increased inhibitory synaptic transmission may contribute to human ASDs and that the R451C knockin mice may be a useful model for studying autism-related behaviors.

    View details for DOI 10.1126/science.1146221

    View details for Web of Science ID 000249915400041

    View details for PubMedID 17823315

  • Membrane fusion as a team effort PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Suedhof, T. C. 2007; 104 (34): 13541-13542

    View details for Web of Science ID 000249064700004

    View details for PubMedID 17699625

  • Integrative genomic and functional analyses reveal neuronal subtype differentiation bias in human embryonic stem cell lines PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Wu, H., Xu, J., Pang, Z. P., Ge, W., Kim, K. J., Blanchi, B., Chen, C., Suedhof, T. C., Sun, Y. E. 2007; 104 (34): 13821-13826

    Abstract

    The self-renewal and differentiation potential of human embryonic stem cells (hESCs) suggests that hESCs could be used for regenerative medicine, especially for restoring neuronal functions in brain diseases. However, the functional properties of neurons derived from hESC are largely unknown. Moreover, because hESCs were derived under diverse conditions, the possibility arises that neurons derived from different hESC lines exhibit distinct properties, but this possibility remains unexplored. To address these issues, we developed a protocol that allows stepwise generation from hESCs of cultures composed of approximately 70-80% human neurons that exhibit spontaneous synaptic network activity. Comparison of neurons derived from the well characterized HSF1 and HSF6 hESC lines revealed that HSF1- but not HSF6-derived neurons exhibit forebrain properties. Accordingly, HSF1-derived neurons initially form primarily GABAergic synaptic networks, whereas HSF6-derived neurons initially form glutamatergic networks. microRNA profiling revealed significant expression differences between the two hESC lines, suggesting that microRNAs may influence their distinct differentiation properties. These observations indicate that although both HSF1 and HSF6 hESCs differentiate into functional neurons, the two hESC lines exhibit distinct differentiation potentials, suggesting that they are preprogrammed. Information on hESC line-specific differentiation biases is crucial for neural stem cell therapy and establishment of novel disease models using hESCs.

    View details for DOI 10.1073/pnas.0706199104

    View details for Web of Science ID 000249064700054

    View details for PubMedID 17693548

  • Crystal structure of the RIM1 alpha C2B domain at 1.7 angstrom resolution BIOCHEMISTRY Guan, R., Dai, H., Tomchick, D. R., Dulubova, I., Machius, M., Suedhof, T. C., Rizo, J. 2007; 46 (31): 8988-8998

    Abstract

    RIM proteins play critical roles in synaptic vesicle priming and diverse forms of presynaptic plasticity. The C-terminal C2B domain is the only module that is common to all RIMs but is only distantly related to well-studied C2 domains, and its three-dimensional structure and interactions have not been characterized in detail. Using NMR spectroscopy, we now show that N- and C-terminal extensions beyond the predicted C2B domain core sequence are necessary to form a folded, stable RIM1alpha C2B domain. We also find that the isolated RIM1alpha C2B domain is not sufficient for previously described protein-protein interactions involving the RIM1alpha C-terminus, suggesting that additional sequences adjacent to the C2B domain might be required for these interactions. However, analytical ultracentrifugation shows that the RIM1alpha C2B domain forms weak dimers in solution. The crystal structure of the RIM1alpha C2B domain dimer at 1.7 A resolution reveals that it forms a beta-sandwich characteristic of C2 domains and that the unique N- and C-terminal extensions form a small subdomain that packs against the beta-sandwich and mediates dimerization. Our results provide a structural basis to understand the function of RIM C2B domains and suggest that dimerization may be a crucial aspect of RIM function.

    View details for DOI 10.1021/bi700698a

    View details for Web of Science ID 000248439000009

    View details for PubMedID 17630786

  • Differential effects of SNAP-25 deletion on Ca2+-dependent and Ca2+-independent neurotransmission JOURNAL OF NEUROPHYSIOLOGY Bronk, P., Deak, F., Wilson, M. C., Liu, X., Sudhof, T. C., Kavalali, E. T. 2007; 98 (2): 794-806

    Abstract

    At the synapse, SNAP-25, along with syntaxin/HPC-1 and synaptobrevin/VAMP, forms SNARE N-ethylmaleimide-sensitive factor [soluble (NSF) attachment protein receptor] complexes that are thought to catalyze membrane fusion. Results from neuronal cultures of synaptobrevin-2 knockout (KO) mice showed that loss of synaptobrevin has a more severe effect on calcium-evoked release than on spontaneous release or on release evoked by hypertonicity. In this study, we recorded neurotransmitter release from neuronal cultures of SNAP-25 KO mice to determine whether they share this property. In neurons lacking SNAP-25, as those deficient in synaptobrevin-2, we found that approximately 10-12% of calcium-independent excitatory and inhibitory neurotransmitter release persisted. However, in contrast to synaptobrevin-2 knockouts, this remaining readily releasable pool in SNAP-25-deficient synapses was virtually insensitive to calcium-dependent-evoked stimulation. Although field stimulation reliably evoked neurotransmitter release in synaptobrevin-2 KO neurons, responses were rare in neurons lacking SNAP-25, and unlike synaptobrevin-2-deficient synapses, SNAP-25-deficient synapses did not exhibit facilitation of release during high-frequency stimulation. This severe loss of evoked exocytosis was matched by a reduction, but not a complete loss, of endocytosis during evoked stimulation. Moreover, synaptic vesicle turnover probed by FM-dye uptake and release during hypertonic stimulation was relatively unaffected by the absence of SNAP-25. This last difference indicates that in contrast to synaptobrevin, SNAP-25 does not directly function in endocytosis. Together, these results suggest that SNAP-25 has a more significant role in calcium-secretion coupling than synaptobrevin-2.

    View details for DOI 10.1152/jn.00226.2007

    View details for Web of Science ID 000248601100021

    View details for PubMedID 17553942

  • Activity-dependent validation of excitatory versus inhibitory synapses by neuroligin-1 versus neuroligin-2 NEURON Chubykin, A. A., Atasoy, D., Etherton, M. R., Brose, N., Kavalali, E. T., Gibson, J. R., Suedhof, T. C. 2007; 54 (6): 919-931

    Abstract

    Neuroligins enhance synapse formation in vitro, but surprisingly are not required for the generation of synapses in vivo. We now show that in cultured neurons, neuroligin-1 overexpression increases excitatory, but not inhibitory, synaptic responses, and potentiates synaptic NMDAR/AMPAR ratios. In contrast, neuroligin-2 overexpression increases inhibitory, but not excitatory, synaptic responses. Accordingly, deletion of neuroligin-1 in knockout mice selectively decreases the NMDAR/AMPAR ratio, whereas deletion of neuroligin-2 selectively decreases inhibitory synaptic responses. Strikingly, chronic inhibition of NMDARs or CaM-Kinase II, which signals downstream of NMDARs, suppresses the synapse-boosting activity of neuroligin-1, whereas chronic inhibition of general synaptic activity suppresses the synapse-boosting activity of neuroligin-2. Taken together, these data indicate that neuroligins do not establish, but specify and validate, synapses via an activity-dependent mechanism, with different neuroligins acting on distinct types of synapses. This hypothesis reconciles the overexpression and knockout phenotypes and suggests that neuroligins contribute to the use-dependent formation of neural circuits.

    View details for DOI 10.1016/j.neuron.2007.05.029

    View details for Web of Science ID 000247645600011

    View details for PubMedID 17582332

  • Endocannabinoid-mediated long-term plasticity requires cAMP/PKA signaling and RIM1 alpha NEURON Chevaleyre, V., Heifets, B. D., Kaeser, P. S., Sudhof, T. C., Purpura, D. P., Castillo, P. E. 2007; 54 (5): 801-812

    Abstract

    Endocannabinoids (eCBs) have emerged as key activity-dependent signals that, by activating presynaptic cannabinoid receptors (i.e., CB1) coupled to G(i/o) protein, can mediate short-term and long-term synaptic depression (LTD). While the presynaptic mechanisms underlying eCB-dependent short-term depression have been identified, the molecular events linking CB1 receptors to LTD are unknown. Here we show in the hippocampus that long-term, but not short-term, eCB-dependent depression of inhibitory transmission requires presynaptic cAMP/PKA signaling. We further identify the active zone protein RIM1alpha as a key mediator of both CB1 receptor effects on the release machinery and eCB-dependent LTD in the hippocampus. Moreover, we show that eCB-dependent LTD in the amygdala and hippocampus shares major mechanistic features. These findings reveal the signaling pathway by which CB1 receptors mediate long-term effects of eCBs in two crucial brain structures. Furthermore, our results highlight a conserved mechanism of presynaptic plasticity in the brain.

    View details for DOI 10.1016/j.neuron.2007.05.020

    View details for Web of Science ID 000247329900012

    View details for PubMedID 17553427

  • Synaptotagmin-1,-2, and-9: Ca2+ sensors for fast release that specify distinct presynaptic properties in subsets of neurons NEURON Xu, J., Mashimo, T., Sudhof, T. C. 2007; 54 (4): 567-581

    Abstract

    Synaptotagmin-1 and -2 are known Ca(2+) sensors for fast synchronous neurotransmitter release, but the potential Ca(2+)-sensor functions of other synaptotagmins in release remain uncharacterized. We now show that besides synaptotagmin-1 and -2, only synaptotagmin-9 (also called synaptotagmin-5) mediates fast Ca(2+) triggering of release. Release induced by the three different synaptotagmin Ca(2+) sensors exhibits distinct kinetics and apparent Ca(2+) sensitivities, suggesting that the synaptotagmin isoform expressed by a neuron determines the release properties of its synapses. Conditional knockout mice producing GFP-tagged synaptotagmin-9 revealed that synaptotagmin-9 is primarily expressed in the limbic system and striatum. Acute deletion of synaptotagmin-9 in striatal neurons severely impaired fast synchronous release without changing the size of the readily-releasable vesicle pool. These data show that in mammalian brain, only synaptotagmin-1, -2, and -9 function as Ca(2+) sensors for fast release, and that these synaptotagmins are differentially expressed to confer distinct release properties onto synapses formed by defined subsets of neurons.

    View details for DOI 10.1016/j.neuron.2007.05.004

    View details for Web of Science ID 000246970800012

    View details for PubMedID 17521570

  • Deletion of alpha-neurexins does not cause a major impairment of axonal pathfinding or synapse formation JOURNAL OF COMPARATIVE NEUROLOGY Dudanova, I., Tabuchi, K., Rohlmann, A., Suedhof, T. C., Missler, M. 2007; 502 (2): 261-274

    Abstract

    Alpha-neurexins are synaptic cell-surface molecules that are required for Ca(2+)-triggered exocytosis. Mice lacking all three alpha-neurexins show drastically reduced neurotransmitter release at excitatory and inhibitory synapses and die early postnatally. Although previous histological analysis of newborn alpha-neurexin triple mutants revealed only a moderate reduction in the density of type II synapses in the brainstem, cell culture studies proposed that neurexins are prominently involved in synapse formation. To assess the contribution of alpha-neurexins to the formation and structural properties of synapses in vivo, we performed a detailed morphological analysis of the brains from surviving adult double knockout mice lacking two of the three alpha-neurexins. Despite their impaired neurotransmission, we did not observe any gross anatomical defects or changes in the distribution of synaptic proteins in adult mutants. Only mild structural alterations were found: a approximately 20% reduction of neuropil area in many brain regions, resulting predominantly from shortened distal dendritic branches and fewer spines, as demonstrated by Golgi impregnation of pyramidal neurons. Quantitative electron microscopy revealed ultrastructurally normal type I and II terminals and a approximately 30% decrease in the density of type II synapses in the neocortex. To exclude errors in pathfinding, we investigated axonal projections in the olfactory bulb of newborn knockouts and did not observe any changes. Therefore, alpha-neurexins are not essential for the formation of the vast majority of synapses in vivo but rather regulate the function of these synapses.

    View details for DOI 10.1002/cne.21305

    View details for Web of Science ID 000245688600007

    View details for PubMedID 17347997

  • Monitoring synaptic transmission in primary neuronal cultures using local extracellular stimulation JOURNAL OF NEUROSCIENCE METHODS Maximov, A., Pang, Z. P., Tervo, D. G., Sudhof, T. C. 2007; 161 (1): 75-87

    Abstract

    Various techniques have been applied for the functional analysis of synaptic transmission in cultured neurons. Here, we describe a method of studying synaptic transmission in neurons cultured at high-density from different brain regions such as the cortex, striatum and spinal cord. We use postsynaptic whole-cell recordings to monitor synaptic currents triggered by presynaptic action potentials that are induced by brief stimulations with a nearby extracellular bipolar electrode. Pharmacologically isolated excitatory or inhibitory postsynaptic currents can be reliably induced, with amplitudes, synaptic charge transfers, and short-term plasticity properties that are reproducible from culture to culture. We show that the size and kinetics of pharmacologically isolated inhibitory postsynaptic currents triggered by single action potentials or stimulus trains depend on the Ca2+ concentration, temperature and stimulation frequency. This method can be applied to study synaptic transmission in wildtype neurons infected with lentiviruses encoding various components of presynaptic release machinery, or in neurons from genetically modified mice, for example neurons carrying floxed genes in which gene expression can be acutely ablated by expression of Cre recombinase. The preparation described in this paper should be useful for analysis of synaptic transmission in inter-neuronal synapses formed by different types of neurons.

    View details for DOI 10.1016/j.jneumeth.2006.10.009

    View details for Web of Science ID 000245490900010

    View details for PubMedID 17118459

  • E-Syts, a family of membranous Ca2+-sensor proteins with multiple C-2 domains PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Min, S., Chang, W., Sudhof, T. C. 2007; 104 (10): 3823-3828

    Abstract

    C(2) domains are autonomously folded protein modules that generally act as Ca(2+)- and phospholipid-binding domains and/or as protein-protein interaction domains. We now report the primary structures and biochemical properties of a family of evolutionarily conserved mammalian proteins, referred to as E-Syts, for extended synaptotagmin-like proteins. E-Syts contain an N-terminal transmembrane region, a central juxtamembranous domain that is conserved from yeast to human, and five (E-Syt1) or three (E-Syt2 and E-Syt3) C-terminal C(2) domains. Only the first E-Syt C(2) domain, the C(2)A domain, includes the complete sequence motif that is required for Ca(2+) binding in C(2) domains. Recombinant protein fragments of E-Syt2 that include the first C(2) domain are capable of Ca(2+)-dependent phospholipid binding at micromolar concentrations of free Ca(2+), suggesting that E-Syts bind Ca(2+) through their first C(2) domain in a phospholipid complex. E-Syts are ubiquitously expressed, but enriched in brain. Expression of myc-tagged E-Syt proteins in transfected cells demonstrated localization to intracellular membranes for E-Syt1 and to plasma membranes for E-Syt2 and E-Syt3. Structure/function studies showed that the plasma-membrane localization of E-Syt2 and E-Syt3 was directed by their C-terminal C(2)C domains. This result reveals an unexpected mechanism by which the C(2)C domains of E-Syt2 and E-Syt3 functions as a targeting motif that localizes these proteins into the plasma membrane independent of their transmembrane region. Viewed together, our findings suggest that E-Syts function as Ca(2+)-regulated intrinsic membrane proteins with multiple C(2) domains, expanding the repertoire of such proteins to a fourth class beyond synaptotagmins, ferlins, and MCTPs (multiple C(2) domain and transmembrane region proteins).

    View details for DOI 10.1073/pnas.0611725104

    View details for Web of Science ID 000244972400030

    View details for PubMedID 17360437

  • cAMP oscillations and retinal activity are permissive for ephrin signaling during the establishment of the retinotopic map NATURE NEUROSCIENCE Nicol, X., Voyatzis, S., Muzerelle, A., Narboux-Neme, N., Sudhof, T. C., Miles, R., Gaspar, P. 2007; 10 (3): 340-347

    Abstract

    Spontaneous activity generated in the retina is necessary to establish a precise retinotopic map, but the underlying mechanisms are poorly understood. We demonstrate here that neural activity controls ephrin-A-mediated responses. In the mouse retinotectal system, we show that spontaneous activity of the retinal ganglion cells (RGCs) is needed, independently of synaptic transmission, for the ordering of the retinotopic map and the elimination of exuberant retinal axons. Activity blockade suppressed the repellent action of ephrin-A on RGC growth cones by cyclic AMP (cAMP)-dependent pathways. Unexpectedly, the ephrin-A5-induced retraction required cAMP oscillations rather than sustained increases in intracellular cAMP concentrations. Periodic photo-induced release of caged cAMP in growth cones rescued the response to ephrin-A5 when activity was blocked. These results provide a direct molecular link between spontaneous neural activity and axon guidance mechanisms during the refinement of neural maps.

    View details for DOI 10.1038/nn1842

    View details for Web of Science ID 000244480300016

    View details for PubMedID 17259982

  • Munc18-1 binds directly to the neuronal SNARE complex PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Dulubova, I., Khvotchev, M., Liu, S., Huryeva, I., Sudhof, T. C., Rizo, J. 2007; 104 (8): 2697-2702

    Abstract

    Both SM proteins (for Sec1/Munc18-like proteins) and SNARE proteins (for soluble NSF-attachment protein receptors) are essential for intracellular membrane fusion, but the general mechanism of coupling between their functions is unclear, in part because diverse SM protein/SNARE binding modes have been described. During synaptic vesicle exocytosis, the SM protein Munc18-1 is known to bind tightly to the SNARE protein syntaxin-1, but only when syntaxin-1 is in a closed conformation that is incompatible with SNARE complex formation. We now show that Munc18-1 also binds tightly to assembled SNARE complexes containing syntaxin-1. The newly discovered Munc18-1/SNARE complex interaction involves contacts of Munc18-1 with the N-terminal H(abc) domain of syntaxin-1 and the four-helical bundle of the assembled SNARE complex. Together with earlier studies, our results suggest that binding of Munc18-1 to closed syntaxin-1 is a specialization that evolved to meet the strict regulatory requirements of neuronal exocytosis, whereas binding of Munc18-1 to assembled SNARE complexes reflects a general function of SM proteins involved in executing membrane fusion.

    View details for DOI 10.1073/pnas.0611318104

    View details for Web of Science ID 000244511200026

    View details for PubMedID 17301226

  • Deletion of CASK in mice is lethal and impairs synaptic function PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Atasoy, D., Schoch, S., Ho, A., Nadasy, K. A., Liu, X., Zhang, W., Mukherjee, K., Nosyreva, E. D., Fernandez-Chacon, R., Missler, M., Kavalali, E. T., Suedhof, T. C. 2007; 104 (7): 2525-2530

    Abstract

    CASK is an evolutionarily conserved multidomain protein composed of an N-terminal Ca2+/calmodulin-kinase domain, central PDZ and SH3 domains, and a C-terminal guanylate kinase domain. Many potential activities for CASK have been suggested, including functions in scaffolding the synapse, in organizing ion channels, and in regulating neuronal gene transcription. To better define the physiological importance of CASK, we have now analyzed CASK "knockdown" mice in which CASK expression was suppressed by approximately 70%, and CASK knockout (KO) mice, in which CASK expression was abolished. CASK knockdown mice are viable but smaller than WT mice, whereas CASK KO mice die at first day after birth. CASK KO mice exhibit no major developmental abnormalities apart from a partially penetrant cleft palate syndrome. In CASK-deficient neurons, the levels of the CASK-interacting proteins Mints, Veli/Mals, and neurexins are decreased, whereas the level of neuroligin 1 (which binds to neurexins that in turn bind to CASK) is increased. Neurons lacking CASK display overall normal electrical properties and form ultrastructurally normal synapses. However, glutamatergic spontaneous synaptic release events are increased, and GABAergic synaptic release events are decreased in CASK-deficient neurons. In contrast to spontaneous neurotransmitter release, evoked release exhibited no major changes. Our data suggest that CASK, the only member of the membrane-associated guanylate kinase protein family that contains a Ca2+/calmodulin-dependent kinase domain, is required for mouse survival and performs a selectively essential function without being in itself required for core activities of neurons, such as membrane excitability, Ca2+-triggered presynaptic release, or postsynaptic receptor functions.

    View details for DOI 10.1073/pnas.0611003104

    View details for Web of Science ID 000244438500086

    View details for PubMedID 17287346

  • Synaptotagmin-12, a synaptic vesicle phosphoprotein that modulates spontaneous neurotransmitter release JOURNAL OF CELL BIOLOGY Maximov, A., Shin, O., Liu, X., Sudhof, T. C. 2007; 176 (1): 113-124

    Abstract

    Central synapses exhibit spontaneous neurotransmitter release that is selectively regulated by cAMP-dependent protein kinase A (PKA). We now show that synaptic vesicles contain synaptotagmin-12, a synaptotagmin isoform that differs from classical synaptotagmins in that it does not bind Ca(2+). In synaptic vesicles, synaptotagmin-12 forms a complex with synaptotagmin-1 that prevents synaptotagmin-1 from interacting with SNARE complexes. We demonstrate that synaptotagmin-12 is phosphorylated by cAMP-dependent PKA on serine(97), and show that expression of synaptotagmin-12 in neurons increases spontaneous neurotransmitter release by approximately threefold, but has no effect on evoked release. Replacing serine(97) by alanine abolishes synaptotagmin-12 phosphorylation and blocks its effect on spontaneous release. Our data suggest that spontaneous synaptic-vesicle exocytosis is selectively modulated by a Ca(2+)-independent synaptotagmin isoform, synaptotagmin-12, which is controlled by cAMP-dependent phosphorylation.

    View details for DOI 10.1083/jcb.200607021

    View details for Web of Science ID 000243581600012

    View details for PubMedID 17190793

  • Synaptotagmin-2 is essential for survival and contributes to Ca2+ triggering of neurotransmitter release in central and neuromuscular synapses JOURNAL OF NEUROSCIENCE Pang, Z. P., Melicoff, E., Padgett, D., Liu, Y., Teich, A. F., Dickey, B. F., Lin, W., Adachi, R., Sudhof, T. C. 2006; 26 (52): 13493-13504

    Abstract

    Biochemical and genetic data suggest that synaptotagmin-2 functions as a Ca2+ sensor for fast neurotransmitter release in caudal brain regions, but animals and/or synapses lacking synaptotagmin-2 have not been examined. We have now generated mice in which the 5' end of the synaptotagmin-2 gene was replaced by lacZ. Using beta-galactosidase as a marker, we show that, consistent with previous studies, synaptotagmin-2 is widely expressed in spinal cord, brainstem, and cerebellum, but is additionally present in selected forebrain neurons, including most striatal neurons and some hypothalamic, cortical, and hippocampal neurons. Synaptotagmin-2-deficient mice were indistinguishable from wild-type littermates at birth, but subsequently developed severe motor dysfunction, and perished at approximately 3 weeks of age. Electrophysiological studies in cultured striatal neurons revealed that the synaptotagmin-2 deletion slowed the kinetics of evoked neurotransmitter release without altering the total amount of release. In contrast, synaptotagmin-2-deficient neuromuscular junctions (NMJs) suffered from a large reduction in evoked release and changes in short-term synaptic plasticity. Furthermore, in mutant NMJs, the frequency of spontaneous miniature release events was increased both at rest and during stimulus trains. Viewed together, our results demonstrate that the synaptotagmin-2 deficiency causes a lethal impairment in synaptic transmission in selected synapses. This impairment, however, is less severe than that produced in forebrain neurons by deletion of synaptotagmin-1, presumably because at least in NMJs, synaptotagmin-1 is coexpressed with synaptotagmin-2, and both together mediate fast Ca2+-triggered release. Thus, synaptotagmin-2 is an essential synaptotagmin isoform that functions in concert with other synaptotagmins in the Ca2+ triggering of neurotransmitter release.

    View details for DOI 10.1523/JNEUROSCI.3519-06.2006

    View details for Web of Science ID 000243277800010

    View details for PubMedID 17192432

  • Redundant functions of RIM1 alpha and RIM2 alpha in Ca2+-triggered neurotransmitter release EMBO JOURNAL Schoch, S., Mittelstaedt, T., Kaeser, P. S., Padgett, D., Feldmann, N., Chevaleyre, V., Castillo, P. E., Hammer, R. E., Han, W., Schmitz, F., Lin, W., Sudhof, T. C. 2006; 25 (24): 5852-5863

    Abstract

    Alpha-RIMs (RIM1alpha and RIM2alpha) are multidomain active zone proteins of presynaptic terminals. Alpha-RIMs bind to Rab3 on synaptic vesicles and to Munc13 on the active zone via their N-terminal region, and interact with other synaptic proteins via their central and C-terminal regions. Although RIM1alpha has been well characterized, nothing is known about the function of RIM2alpha. We now show that RIM1alpha and RIM2alpha are expressed in overlapping but distinct patterns throughout the brain. To examine and compare their functions, we generated knockout mice lacking RIM2alpha, and crossed them with previously produced RIM1alpha knockout mice. We found that deletion of either RIM1alpha or RIM2alpha is not lethal, but ablation of both alpha-RIMs causes postnatal death. This lethality is not due to a loss of synapse structure or a developmental change, but to a defect in neurotransmitter release. Synapses without alpha-RIMs still contain active zones and release neurotransmitters, but are unable to mediate normal Ca(2+)-triggered release. Our data thus demonstrate that alpha-RIMs are not essential for synapse formation or synaptic exocytosis, but are required for normal Ca(2+)-triggering of exocytosis.

    View details for DOI 10.1038/sj.emboj.7601425

    View details for Web of Science ID 000242891100021

    View details for PubMedID 17124501

  • Genetic analysis of Mint/X11 proteins: Essential presynaptic functions of a neuronal adaptor protein family JOURNAL OF NEUROSCIENCE Ho, A., Morishita, W., Atasoy, D., Liu, X., Tabuchi, K., Hammer, R. E., Malenka, R. C., Sudhof, T. C. 2006; 26 (50): 13089-13101

    Abstract

    Mints/X11s are adaptor proteins composed of three isoforms: neuron-specific Mints 1 and 2, and the ubiquitously expressed Mint 3. We have now analyzed constitutive and conditional knock-out mice for all three Mints/X11s. We found that approximately 80% of mice lacking both neuron-specific Mint isoforms (Mints 1 and 2) die at birth, whereas mice lacking any other combination of Mint isoforms survive normally. The approximately 20% surviving Mint 1/2 double knock-out mice exhibit a decrease in weight and deficits in motor behaviors. Hippocampal slice electrophysiology uncovered a decline in spontaneous neurotransmitter release, lowered synaptic strength, and enhanced paired-pulse facilitation in Mint-deficient mice, suggesting a decreased presynaptic release probability. Acute ablation of Mint expression in cultured neurons from conditional Mint 1/2/3 triple knock-in mice also revealed a decline in spontaneous release, confirming that deletion of Mints impair presynaptic function. Quantitation of synaptic proteins showed that acute deletion of Mints caused a selective increase in Munc18-1 and Fe65 proteins, and overexpression of Munc18-1 in wild-type neurons also produced a decrease in spontaneous release, suggesting that the interaction of Mints with Munc18-1 may contribute to the presynaptic phenotype observed in Mint-deficient mice. Our studies thus indicate that Mints are important regulators of presynaptic neurotransmitter release that are essential for mouse survival.

    View details for DOI 10.1523/JNEUROSCI.2855-06.2006

    View details for Web of Science ID 000242996200024

    View details for PubMedID 17167098

  • Differential expression of active zone proteins in neuromuscular junctions suggests functional diversification EUROPEAN JOURNAL OF NEUROSCIENCE Juranek, J., Mukherjee, K., Rickmann, M., Martens, H., Calka, J., Suedhof, T. C., Jahn, R. 2006; 24 (11): 3043-3052

    Abstract

    Nerve terminals of the central nervous system (CNS) contain specialized release sites for synaptic vesicles, referred to as active zones. They are characterized by electron-dense structures that are tightly associated with the presynaptic plasma membrane and organize vesicle docking and priming sites. Recently, major protein constituents of active zones have been identified, including the proteins Piccolo, Bassoon, RIM, Munc13, ERCs/ELKs/CASTs and liprins. While it is becoming apparent that each of these proteins is essential for synaptic function in the CNS, it is not known to what extent these proteins are involved in synaptic function of the peripheral nervous system. Somatic neuromuscular junctions contain morphologically and functionally defined active zones with similarities to CNS synapses. In contrast, sympathetic neuromuscular varicosities lack active zone-like morphological specializations. Using immunocytochemistry at the light and electron microscopic level we have now performed a systematic investigation of all five major classes of active zone proteins in peripheral neuromuscular junctions. Our results show that somatic neuromuscular endplates contain a full complement of all active zone proteins. In contrast, varicosities of the vas deferens contain a subset of active zone proteins including Bassoon and ELKS2, with the other four components being absent. We conclude that Bassoon and ELKS2 perform independent and specialized functions in synaptic transmission of autonomic synapses.

    View details for DOI 10.1111/j.1460-9568.2006.05183.x

    View details for Web of Science ID 000243361700008

    View details for PubMedID 17156365

  • A gain-of-function mutation in synaptotagmin-1 reveals a critical role of Ca2+-dependent soluble N-ethylmaleimide-sensitive factor attachment protein receptor complex binding in synaptic exocytosis JOURNAL OF NEUROSCIENCE Pang, Z. P., Shin, O., Meyer, A. C., Rosenmund, C., Suedhof, T. C. 2006; 26 (48): 12556-12565

    Abstract

    Synaptotagmin-1, the Ca2+ sensor for fast neurotransmitter release, was proposed to function by Ca2+-dependent phospholipid binding and/or by Ca2+-dependent soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) complex binding. Extensive in vivo data support the first hypothesis, but testing the second hypothesis has been difficult because no synaptotagmin-1 mutation is known that selectively interferes with SNARE complex binding. Using knock-in mice that carry aspartate-to-asparagine substitutions in a Ca2+-binding site of synaptotagmin-1 (the D232N or D238N substitutions), we now show that the D232N mutation dramatically increases Ca2+-dependent SNARE complex binding by native synaptotagmin-1, but leaves phospholipid binding unchanged. In contrast, the adjacent D238N mutation does not significantly affect SNARE complex binding, but decreases phospholipid binding. Electrophysiological recordings revealed that the D232N mutation increased Ca2+-triggered release, whereas the D238N mutation decreased release. These data establish that fast vesicle exocytosis is driven by a dual Ca2+-dependent activity of synaptotagmin-1, namely Ca2+-dependent binding both to SNARE complexes and to phospholipids.

    View details for DOI 10.1523/JNEUROSCI.3804-06.2006

    View details for Web of Science ID 000242387900019

    View details for PubMedID 17135417

  • Synaptic vesicles: An organelle comes of age CELL Sudhof, T. C. 2006; 127 (4): 671-673

    Abstract

    Synaptic vesicles mediate the release of neurotransmitters at nerve terminals. In this issue of Cell, Takamori et al. (2006) analyze the lipid and protein components of synaptic vesicles, providing the most comprehensive description of synaptic vesicles to date.

    View details for DOI 10.1016/j.cell.2006.10.033

    View details for Web of Science ID 000242330600013

    View details for PubMedID 17110326

  • Gene selection, alternative splicing, and post-translational processing regulate neuroligin selectivity for beta-neurexins BIOCHEMISTRY Comoletti, D., Flynn, R. E., Boucard, A. A., Demeler, B., Schirf, V., Shi, J., Jennings, L. L., Newlin, H. R., Sudhof, T. C., Taylor, P. 2006; 45 (42): 12816-12827

    Abstract

    Neuroligins 1-4 are postsynaptic transmembrane proteins capable of initiating presynaptic maturation via interactions with beta-neurexin. Both neuroligins and beta-neurexins have alternatively spliced inserts in their extracellular domains. Using analytical ultracentrifugation, we determined that the extracellular domains of the neuroligins sediment as dimers, whereas the extracellular domains of the beta-neurexins appear monomeric. Sedimentation velocity experiments of titrated stoichiometry ratios of beta-neurexin and neuroligin suggested a 2:2 complex formation. The recognition properties of individual neuroligins toward beta-neurexin-1 (NX1beta), along with the influence of their splice inserts, were explored by surface plasmon resonance and affinity chromatography. Different neuroligins display a range of NX1beta affinities spanning more than 2 orders of magnitude. Whereas splice insert 4 in beta-neurexin appears to act only as a modulator of the neuroligin/beta-neurexin association, splice insert B in neuroligin-1 (NL1) is the key element regulating the NL1/NX1beta binding. Our data indicate that gene selection, mRNA splicing, and post-translational modifications combine to give rise to a controlled neuroligin recognition code with a rank ordering of affinities for particular neurexins that is conserved for the neuroligins across mammalian species.

    View details for DOI 10.1021/bi0614131

    View details for Web of Science ID 000241325700021

    View details for PubMedID 17042500

  • A complexin/synaptotagmin 1 switch controls fast synaptic vesicle exocytosis CELL Tang, J., Maximov, A., Shin, O., Dai, H., Rizo, J., Sudhof, T. C. 2006; 126 (6): 1175-1187

    Abstract

    Ca(2+) binding to synaptotagmin 1 triggers fast exocytosis of synaptic vesicles that have been primed for release by SNARE-complex assembly. Besides synaptotagmin 1, fast Ca(2+)-triggered exocytosis requires complexins. Synaptotagmin 1 and complexins both bind to assembled SNARE complexes, but it is unclear how their functions are coupled. Here we propose that complexin binding activates SNARE complexes into a metastable state and that Ca(2+) binding to synaptotagmin 1 triggers fast exocytosis by displacing complexin from metastable SNARE complexes. Specifically, we demonstrate that, biochemically, synaptotagmin 1 competes with complexin for SNARE-complex binding, thereby dislodging complexin from SNARE complexes in a Ca(2+)-dependent manner. Physiologically, increasing the local concentration of complexin selectively impairs fast Ca(2+)-triggered exocytosis but retains other forms of SNARE-dependent fusion. The hypothesis that Ca(2+)-induced displacement of complexins from SNARE complexes triggers fast exocytosis accounts for the loss-of-function and gain-of-function phenotypes of complexins and provides a molecular explanation for the high speed and synchronicity of fast Ca(2+)-triggered neurotransmitter release.

    View details for DOI 10.1016/j.cell.2006.08.030

    View details for Web of Science ID 000240897600022

    View details for PubMedID 16990140

  • Neuroligins determine synapse maturation and function NEURON Varoqueaux, F., Aramuni, G., Rawson, R. L., Mohrmann, R., Missler, M., Gottmann, K., Zhang, W., Suedhof, T. C., Brose, N. 2006; 51 (6): 741-754

    Abstract

    Synaptogenesis, the generation and maturation of functional synapses between nerve cells, is an essential step in the development of neuronal networks in the brain. It is thought to be triggered by members of the neuroligin family of postsynaptic cell adhesion proteins, which may form transsynaptic contacts with presynaptic alpha- and beta-neurexins and have been implicated in the etiology of autism. We show that deletion mutant mice lacking neuroligin expression die shortly after birth due to respiratory failure. This respiratory failure is a consequence of reduced GABAergic/glycinergic and glutamatergic synaptic transmission and network activity in brainstem centers that control respiration. However, the density of synaptic contacts is not altered in neuroligin-deficient brains and cultured neurons. Our data show that neuroligins are required for proper synapse maturation and brain function, but not for the initial formation of synaptic contacts.

    View details for DOI 10.1016/j.neuron.2006.09.003

    View details for Web of Science ID 000240997900013

    View details for PubMedID 16982420

  • Crystal structure of the second LNS/LG domain from neurexin 1 alpha - Ca2+ binding and the effects of alternative splicing JOURNAL OF BIOLOGICAL CHEMISTRY Sheckler, L. R., Henry, L., Sugita, S., Sudhof, T. C., Rudenko, G. 2006; 281 (32): 22896-22905

    Abstract

    Neurexins mediate protein interactions at the synapse, playing an essential role in synaptic function. Extracellular domains of neurexins, and their fragments, bind a distinct profile of different proteins regulated by alternative splicing and Ca2+. The crystal structure of n1alpha_LNS#2 (the second LNS/LG domain of bovine neurexin 1alpha) reveals large structural differences compared with n1alpha_LNS#6 (or n1beta_LNS), the only other LNS/LG domain for which a structure has been determined. The differences overlap the so-called hyper-variable surface, the putative protein interaction surface that is reshaped as a result of alternative splicing. A Ca2+-binding site is revealed at the center of the hyper-variable surface next to splice insertion sites. Isothermal titration calorimetry indicates that the Ca2+-binding site in n1alpha_LNS#2 has low affinity (Kd approximately 400 microm). Ca2+ binding ceases to be measurable when an 8- or 15-residue splice insert is present at the splice site SS#2 indicating that alternative splicing can affect Ca2+-binding sites of neurexin LNS/LG domains. Our studies initiate a framework for the putative protein interaction sites of neurexin LNS/LG domains. This framework is essential to understand how incorporation of alternative splice inserts expands the information from a limited set of neurexin genes to produce a large array of synaptic adhesion molecules with potentially very different synaptic function.

    View details for DOI 10.1074/jbc.M603464200

    View details for Web of Science ID 000239542600052

    View details for PubMedID 16772286

  • Structural basis for a Munc13-1 homodimer to Munc13-1/RIM heterodimer switch PLOS BIOLOGY Lu, J., Machius, M., Dulubova, I., Dai, H., Sudhof, T. C., Tomchick, D. R., Rizo, J. 2006; 4 (7): 1159-1172

    Abstract

    C(2) domains are well characterized as Ca(2+)/phospholipid-binding modules, but little is known about how they mediate protein-protein interactions. In neurons, a Munc13-1 C(2)A-domain/RIM zinc-finger domain (ZF) heterodimer couples synaptic vesicle priming to presynaptic plasticity. We now show that the Munc13-1 C(2)A domain homodimerizes, and that homodimerization competes with Munc13-1/RIM heterodimerization. X-ray diffraction studies guided by nuclear magnetic resonance (NMR) experiments reveal the crystal structures of the Munc13-1 C(2)A-domain homodimer and the Munc13-1 C(2)A-domain/RIM ZF heterodimer at 1.44 A and 1.78 A resolution, respectively. The C(2)A domain adopts a beta-sandwich structure with a four-stranded concave side that mediates homodimerization, leading to the formation of an eight-stranded beta-barrel. In contrast, heterodimerization involves the bottom tip of the C(2)A-domain beta-sandwich and a C-terminal alpha-helical extension, which wrap around the RIM ZF domain. Our results describe the structural basis for a Munc13-1 homodimer-Munc13-1/RIM heterodimer switch that may be crucial for vesicle priming and presynaptic plasticity, uncovering at the same time an unexpected versatility of C(2) domains as protein-protein interaction modules, and illustrating the power of combining NMR spectroscopy and X-ray crystallography to study protein complexes.

    View details for DOI 10.1371/journal.pbio.0040192

    View details for Web of Science ID 000238974300009

    View details for PubMedID 16732694

  • Structural determinants of synaptobrevin 2 function in synaptic vesicle fusion JOURNAL OF NEUROSCIENCE Deak, F., Shin, O. H., Kavalali, E. T., Sudhof, T. C. 2006; 26 (25): 6668-6676

    Abstract

    Deletion of synaptobrevin/vesicle-associated membrane protein, the major synaptic vesicle soluble N-ethylmaleimide-sensitive factor attachment protein receptor (R-SNARE), severely decreases but does not abolish spontaneous and evoked synaptic vesicle exocytosis. We now show that the closely related R-SNARE protein cellubrevin rescues synaptic transmission in synaptobrevin-deficient neurons but that deletion of both cellubrevin and synaptobrevin does not cause a more severe decrease in exocytosis than deletion of synaptobrevin alone. We then examined the structural requirements for synaptobrevin to function in exocytosis. We found that substituting glutamine for arginine in the zero-layer of the SNARE motif did not significantly impair synaptobrevin-dependent exocytosis, whereas insertion of 12 or 24 residues between the SNARE motif and transmembrane region abolished the ability of synaptobrevin to mediate Ca2+-evoked exocytosis. Surprisingly, however, synaptobrevin with the 12-residue but not the 24-residue insertion restored spontaneous release in synaptobrevin-deficient neurons. Our data suggest that synaptobrevin mediates Ca2+-triggered exocytosis by tight coupling of the SNARE motif to the transmembrane region and hence forcing the membranes into close proximity for fusion. Furthermore, the fusion reactions underlying evoked and spontaneous release differ mechanistically.

    View details for DOI 10.1523/JNEUROSCI.5272-05.2006

    View details for Web of Science ID 000238473600004

    View details for PubMedID 16793874

  • Rabphilin regulates SNARE-dependent re-priming of synaptic vesicles for fusion EMBO JOURNAL Deak, F., Shin, O., Tang, J., Hanson, P., Ubach, J., Jahn, R., Rizo, J., Kavalali, E. T., Sudhof, T. C. 2006; 25 (12): 2856-2866

    Abstract

    Synaptic vesicle fusion is catalyzed by assembly of synaptic SNARE complexes, and is regulated by the synaptic vesicle GTP-binding protein Rab3 that binds to RIM and to rabphilin. RIM is a known physiological regulator of fusion, but the role of rabphilin remains obscure. We now show that rabphilin regulates recovery of synaptic vesicles from use-dependent depression, probably by a direct interaction with the SNARE protein SNAP-25. Deletion of rabphilin dramatically accelerates recovery of depressed synaptic responses; this phenotype is rescued by viral expression of wild-type rabphilin, but not of mutant rabphilin lacking the second rabphilin C2 domain that binds to SNAP-25. Moreover, deletion of rabphilin also increases the size of synaptic responses in synapses lacking the vesicular SNARE protein synaptobrevin in which synaptic responses are severely depressed. Our data suggest that binding of rabphilin to SNAP-25 regulates exocytosis of synaptic vesicles after the readily releasable pool has either been physiologically exhausted by use-dependent depression, or has been artificially depleted by deletion of synaptobrevin.

    View details for DOI 10.1038/sj.embjol.7601165

    View details for Web of Science ID 000238709800018

    View details for PubMedID 16763567

  • Phosphatidylinositol phosphates as co-activators of Ca2+ binding to C-2 domains of synaptotagmin 1 JOURNAL OF BIOLOGICAL CHEMISTRY Li, L., Shin, O., Rhee, J., Arac, D., Rah, J., Rizo, J., Sudhof, T., Rosenmund, C. 2006; 281 (23): 15845-15852

    Abstract

    Ca2+-dependent phospholipid binding to the C2A and C2B domains of synaptotagmin 1 is thought to trigger fast neurotransmitter release, but only Ca2+ binding to the C2B domain is essential for release. To investigate the underlying mechanism, we have compared the role of basic residues in Ca2+/phospholipid binding and in release. Mutations in a polybasic sequence on the side of the C2B domain beta-sandwich or in a basic residue in a top Ca2+-binding loop of the C2A domain (R233) cause comparable decreases in the apparent Ca2+ affinity of synaptotagmin 1 and the Ca2+ sensitivity of release, whereas mutation of the residue homologous to Arg233 in the C2B domain (Lys366) has no effect. Phosphatidylinositol polyphosphates co-activate Ca2+-dependent and -independent phospholipid binding to synaptotagmin 1, but the effects of these mutations on release only correlate with their effects on the Ca2+-dependent component. These results reveal clear distinctions in the Ca2+-dependent phospholipid binding modes of the synaptotagmin 1 C2 domains that may underlie their functional asymmetry and suggest that phosphatidylinositol polyphosphates may serve as physiological modulators of Ca2+ affinity of synaptotagmin 1 in vivo.

    View details for DOI 10.1074/jbc.M600888200

    View details for Web of Science ID 000237996000033

    View details for PubMedID 16595652

  • Genetic analysis of synaptotagmin 2 in spontaneous and Ca2+-triggered neurotransmitter release EMBO JOURNAL Pang, Z. P., Sun, J., Rizo, J., Maximov, A., Sudhof, T. C. 2006; 25 (10): 2039-2050

    Abstract

    Synaptotagmin 2 resembles synaptotagmin 1, the Ca2+ sensor for fast neurotransmitter release in forebrain synapses, but little is known about synaptotagmin 2 function. Here, we describe a severely ataxic mouse strain that harbors a single, destabilizing amino-acid substitution (I377N) in synaptotagmin 2. In Calyx of Held synapses, this mutation causes a delay and a decrease in Ca2+-induced but not in hypertonic sucrose-induced release, suggesting that synaptotagmin 2 mediates Ca2+ triggering of evoked release in brainstem synapses. Unexpectedly, we additionally observed in synaptotagmin 2 mutant synapses a dramatic increase in spontaneous release. Synaptotagmin 1-deficient excitatory and inhibitory cortical synapses also displayed a large increase in spontaneous release, demonstrating that this effect was shared among synaptotagmins 1 and 2. Our data suggest that synaptotagmin 1 and 2 perform equivalent functions in the Ca2+ triggering of action potential-induced release and in the restriction of spontaneous release, consistent with a general role of synaptotagmins in controlling 'release slots' for synaptic vesicles at the active zone.

    View details for DOI 10.1038/sj.emboj.7601103

    View details for Web of Science ID 000237590000001

    View details for PubMedID 16642042

  • Close membrane-membrane proximity induced by Ca2+-dependent multivalent binding of synaptotagmin-1 to phospholipids NATURE STRUCTURAL & MOLECULAR BIOLOGY Arac, D., Chen, X. C., Khant, H. A., Ubach, J., Ludtke, S. J., Kikkawa, M., Johnson, A. E., Chiu, W., Sudhof, T. C., Rizo, J. 2006; 13 (3): 209-217

    Abstract

    Synaptotagmin acts as a Ca(2+) sensor in neurotransmitter release through its two C(2) domains. Ca(2+)-dependent phospholipid binding is key for synaptotagmin function, but it is unclear how this activity cooperates with the SNARE complex involved in release or why Ca(2+) binding to the C(2)B domain is more crucial for release than Ca(2+) binding to the C(2)A domain. Here we show that Ca(2+) induces high-affinity simultaneous binding of synaptotagmin to two membranes, bringing them into close proximity. The synaptotagmin C(2)B domain is sufficient for this ability, which arises from the abundance of basic residues around its surface. We propose a model wherein synaptotagmin cooperates with the SNAREs in bringing the synaptic vesicle and plasma membranes together and accelerates membrane fusion through the highly positive electrostatic potential of its C(2)B domain.

    View details for DOI 10.1038/nsmb1056

    View details for Web of Science ID 000235776900011

    View details for PubMedID 16491093

  • Synapsins regulate use-dependent synaptic plasticity in the calyx of Held by a Ca2+/calmodulin-dependent pathway PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Sun, J. Y., Bronk, P., Liu, X. R., Han, W. P., Sudhof, T. C. 2006; 103 (8): 2880-2885

    Abstract

    Synapsins are abundant synaptic-vesicle phosphoproteins that are known to regulate neurotransmitter release but whose precise function has been difficult to pinpoint. Here, we use knockout mice to analyze the role of synapsins 1 and 2 in the calyx of Held synapse, allowing precise measurements of neurotransmitter release. We find that deletion of synapsins did not induce significant changes in spontaneous release or release evoked by isolated action potentials (APs) and did not alter the size of the readily releasable vesicle pool (RRP), the kinetics of RRP depletion, or the rate of recovery of the RRP after depletion. Deletion of synapsins, however, did increase use-dependent synaptic depression induced by a high-frequency stimulus train (> or = 50 Hz). The increased depression was due to a decrease in the fraction of the RRP, whose release was evoked by APs late in the stimulus train. The effect of synapsin deletions was occluded by intracellular application of the Ca2+-chelator EGTA or of a calmodulin inhibitor. Our results show that synapsins boost the release probability during high-frequency stimulation and suggest that this effect involves Ca2+/calmodulin-dependent phosphorylation of synapsins.

    View details for Web of Science ID 000235554900072

    View details for PubMedID 16481620

  • CSP alpha-deficiency causes massive and rapid photoreceptor degeneration PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Schmitz, F., Tabares, L., Khimich, D., Strenzke, N., de la Villa-Polo, P., Castellano-Munoz, M., Bulankina, A., Moser, T., Fernandez-Chacon, R., Sudhof, T. C. 2006; 103 (8): 2926-2931

    Abstract

    Cysteine string protein (CSP) alpha is an abundant synaptic vesicle protein that contains a DNA-J domain characteristic of Hsp40-type cochaperones. Previous studies showed that deletion of CSPalpha in mice leads to massive lethal neurodegeneration but did not clarify how the neurodegeneration affects specific subpopulations of neurons. Here, we analyzed the effects of the CSPalpha deficiency on tonically active ribbon synapses of the retina and the inner ear. We show that CSPalpha-deficient photoreceptor terminals undergo dramatic and rapidly progressive neurodegeneration that starts before eye opening and initially does not affect other retinal synapses. These changes are associated with progressive blindness. In contrast, ribbon synapses of auditory hair cells did not exhibit presynaptic impairments in CSPalpha-deficient mice. Hair cells, but not photoreceptor cells or central neurons, express CSPbeta, thereby accounting for the lack of a hair-cell phenotype in CSPalpha knockout mice. Our data demonstrate that tonically active ribbon synapses in retina are particularly sensitive to the deletion of CSPalpha and that expression of at least one CSP isoform is essential to protect such tonically active synapses from neurodegeneration.

    View details for DOI 10.1073/pnas.0510060103

    View details for Web of Science ID 000235554900080

    View details for PubMedID 16477021

  • Rab3 superprimes synaptic vesicles for release: Implications for short-term synaptic plasticity JOURNAL OF NEUROSCIENCE Schluter, O. M., Basu, J., Sudhof, T. C., Rosenmund, C. 2006; 26 (4): 1239-1246

    Abstract

    Presynaptic vesicle trafficking and priming are important steps in regulating synaptic transmission and plasticity. The four closely related small GTP-binding proteins Rab3A, Rab3B, Rab3C, and Rab3D are believed to be important for these steps. In mice, the complete absence of all Rab3s leads to perinatal lethality accompanied by a 30% reduction of probability of Ca2+-triggered synaptic release. This study examines the role of Rab3 during Ca2+-triggered release in more detail and identifies its impact on short-term plasticity. Using patch-clamp electrophysiology of autaptic neuronal cultures from Rab3-deficient mouse hippocampus, we show that excitatory Rab3-deficient neurons display unique time- and frequency-dependent short-term plasticity characteristics in response to spike trains. Analysis of vesicle release and repriming kinetics as well as Ca2+ sensitivity of release indicate that Rab3 acts on a subset of primed, fusion competent vesicles. They lower the amount of Ca2+ required for action potential-triggered release, which leads to a boosting of release probability, but their action also introduces a significant delay in the supply of these modified vesicles. As a result, Rab3-induced modifications to primed vesicles causes a transient increase in the transduction efficacy of synaptic action potential trains and optimizes the encoding of synaptic information at an intermediate spike frequency range.

    View details for DOI 10.1523/JNEUROSCI.3553-05.2006

    View details for Web of Science ID 000234896200022

    View details for PubMedID 16436611

  • Different effects on fast exocytosis induced by synaptotagmin 1 and 2 isoforms and abundance but not by phosphorylation JOURNAL OF NEUROSCIENCE Nagy, G., Kim, J. H., Pang, Z. P., Matti, U., Rettig, J., Sudhof, T. C., Sorensen, J. B. 2006; 26 (2): 632-643

    Abstract

    Synaptotagmins comprise a large protein family, of which synaptotagmin 1 (Syt1) is a Ca2+ sensor for fast exocytosis, and its close relative, synaptotagmin 2 (Syt2), is assumed to serve similar functions. Chromaffin cells express Syt1 but not Syt2. We compared secretion from chromaffin cells from Syt1 null mice overexpressing either Syt isoform. High time-resolution capacitance measurement showed that Syt1 null cells lack the exocytotic phase corresponding to the readily-releasable pool (RRP) of vesicles. Comparison with the amperometric signal confirmed that the missing phase of exocytosis consists of catecholamine-containing vesicles. Overexpression of Syt1 rescued the RRP and increased its size above wild-type values, whereas the size of the slowly releasable pool decreased, indicating that the availability of Syt1 regulates the relative size of the two releasable pools. The RRP was also rescued by Syt2 overexpression, but the kinetics of fusion was slightly slower than in cells expressing Syt1. Biochemical experiments showed that Syt2 has a slightly lower Ca2+ affinity for phospholipid binding than Syt1 because of a difference in the C2A domain. These data constitute evidence for the function of Syt1 and Syt2 as alternative, but not identical, calcium-sensors for RRP fusion. By overexpression of Syt1 mutated in the shared PKC/calcium/calmodulin-dependent kinase phosphorylation site, we show that phorbol esters act independently and upstream of Syt1 to regulate the size of the releasable pools. We conclude that exocytosis from mouse chromaffin cells can be modified by the differential expression of Syt isoforms and by Syt abundance but not by phosphorylation of Syt1.

    View details for DOI 10.1523/JNEUROSCI.2589-05.2006

    View details for Web of Science ID 000234556200032

    View details for PubMedID 16407561

  • Augmenting neurotransmitter release by enhancing the apparent Ca2+ affinity of synaptotagmin 1 PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Rhee, J. S., Li, L. Y., Shin, O. H., Rah, J. C., Rizo, J., Sudhof, T. C., Rosenmund, C. 2005; 102 (51): 18664-18669

    Abstract

    Synaptotagmin 1 likely acts as a Ca2+ sensor in neurotransmitter release by Ca2+-binding to its two C2 domains. This notion was strongly supported by the observation that a mutation in the C2A domain causes parallel decreases in the apparent Ca2+ affinity of synaptotagmin 1 and in the Ca2+ sensitivity of release. However, this study was based on a single loss-of-function mutation. We now show that tryptophan substitutions in the synaptotagmin 1 C2 domains act as gain-of-function mutations to increase the apparent Ca2+ affinity of synaptotagmin 1. The same substitutions, when introduced into synaptotagmin 1 expressed in neurons, enhance the Ca2+ sensitivity of release. Mutations in the two C2 domains lead to comparable and additive effects in release. Our results thus show that the apparent Ca2+ sensitivity of release is dictated by the apparent Ca2+ affinity of synaptotagmin 1 in both directions, and that Ca2+ binding to both C2 domains contributes to Ca2+ triggering of release.

    View details for DOI 10.1073/pnas.0509153102

    View details for Web of Science ID 000234174300075

    View details for PubMedID 16352718

  • RIM function in short- and long-term synaptic plasticity BIOCHEMICAL SOCIETY TRANSACTIONS Kaeser, P. S., Sudhof, T. C. 2005; 33: 1345-1349

    Abstract

    RIM1alpha (Rab3-interacting molecule 1alpha) is a large multidomain protein that is localized to presynaptic active zones [Wang, Okamoto, Schmitz, Hofmann and Südhof (1997) Nature (London) 388, 593-598] and is the founding member of the RIM protein family that also includes RIM2alpha, 2beta, 2gamma, 3gamma and 4gamma [Wang and Südhof (2003) Genomics 81, 126-137]. In presynaptic nerve termini, RIM1alpha interacts with a series of presynaptic proteins, including the synaptic vesicle GTPase Rab3 and the active zone proteins Munc13, liprins and ELKS (a protein rich in glutamate, leucine, lysine and serine). Mouse KOs (knockouts) revealed that, in different types of synapses, RIM1alpha is essential for different forms of synaptic plasticity. In CA1-region Schaffer-collateral excitatory synapses and in GABAergic synapses (where GABA is gamma-aminobutyric acid), RIM1alpha is required for maintaining normal neurotransmitter release and short-term synaptic plasticity. In contrast, in excitatory CA3-region mossy fibre synapses and cerebellar parallel fibre synapses, RIM1alpha is necessary for presynaptic long-term, but not short-term, synaptic plasticity. In these synapses, the function of RIM1alpha in presynaptic long-term plasticity depends, at least in part, on phosphorylation of RIM1alpha at a single site, suggesting that RIM1alpha constitutes a 'phosphoswitch' that determines synaptic strength. However, in spite of the progress in understanding RIM1alpha function, the mechanisms by which RIM1alpha acts remain unknown. For example, how does phosphorylation regulate RIM1alpha, what is the relationship of the function of RIM1alpha in basic release to synaptic plasticity and what is the physiological significance of different forms of RIM-dependent plasticity? Moreover, the roles of other RIM isoforms are unclear. Addressing these important questions will contribute to our view of how neurotransmitter release is regulated at the presynaptic active zone.

    View details for Web of Science ID 000233930800030

    View details for PubMedID 16246115

  • Autonomous function of synaptotagmin 1 in triggering synchronous release independent of asynchronous release NEURON Maximov, A., Sudhof, T. C. 2005; 48 (4): 547-554

    Abstract

    Ca(2+) triggers neurotransmitter release in at least two principal modes, synchronous and asynchronous release. Synaptotagmin 1 functions as a Ca(2+) sensor for synchronous release, but its role in asynchronous release remains unclear. We now show that in cultured cortical neurons stimulated at low frequency (or Hz), deletion of synaptotagmin 1 also alters only synchronous, not asynchronous, release during the stimulus train, but dramatically enhances "delayed asynchronous release" following the stimulus train. Thus synaptotagmin 1 functions as an autonomous Ca(2+) sensor independent of asynchronous release during isolated action potentials and action potential trains, but restricts asynchronous release induced by residual Ca(2+) after action potential trains. We propose that synaptotagmin 1 occupies release "slots" at the active zone, possibly in a Ca(2+)-independent complex with SNARE proteins that are freed when action potential-induced Ca(2+) influx activates synaptotagmin 1.

    View details for DOI 10.1016/j.neuron.2005.09.006

    View details for Web of Science ID 000233677300009

    View details for PubMedID 16301172

  • alpha-synuclein cooperates with CSP alpha in preventing neurodegeneration CELL Chandra, S., Gallardo, G., Fernandez-Chacon, R., Schluter, O. M., Sudhof, T. C. 2005; 123 (3): 383-396

    Abstract

    Alpha-synuclein and cysteine-string protein-alpha (CSPalpha) are abundant synaptic vesicle proteins independently linked to neurodegeneration. Dominantly inherited mutations in alpha-synuclein cause Parkinson's disease, but the physiological role of alpha-synuclein remains unknown. Deletion of CSPalpha produces rapidly progressive neurodegeneration in mice, presumably because the cochaperone function of CSPalpha is essential for neuronal survival. Here, we report the surprising finding that transgenic expression of alpha-synuclein abolishes the lethality and neurodegeneration caused by deletion of CSPalpha. Conversely, ablation of endogenous synucleins exacerbates these phenotypes. Deletion of CSPalpha inhibits SNARE complex assembly; transgenic alpha-synuclein ameliorates this inhibition. In preventing neurodegeneration in CSPalpha-deficient mice, alpha-synuclein does not simply substitute for CSPalpha but acts by a downstream mechanism that requires phospholipid binding by alpha-synuclein. These observations reveal a powerful in vivo activity of alpha-synuclein in protecting nerve terminals against injury and suggest that this activity operates in conjunction with CSPalpha and SNARE proteins on the presynaptic membrane interface.

    View details for DOI 10.1016/j.cell.2005.09.028

    View details for Web of Science ID 000233264300008

    View details for PubMedID 16269331

  • A splice code for trans-synaptic cell adhesion mediated by binding of neuroligin 1 to alpha- and beta-neurexins NEURON Boucard, A. A., Chubykin, A. A., Comoletti, D., Taylor, P., Sudhof, T. C. 2005; 48 (2): 229-236

    Abstract

    Previous studies suggested that postsynaptic neuroligins form a trans-synaptic complex with presynaptic beta-neurexins, but not with presynaptic alpha-neurexins. Unexpectedly, we now find that neuroligins also bind alpha-neurexins and that alpha- and beta-neurexin binding by neuroligin 1 is regulated by alternative splicing of neuroligin 1 (at splice site B) and of neurexins (at splice site 4). In neuroligin 1, splice site B is a master switch that determines alpha-neurexin binding but leaves beta-neurexin binding largely unaffected, whereas alternative splicing of neurexins modulates neuroligin binding. Moreover, neuroligin 1 splice variants with distinct neurexin binding properties differentially regulate synaptogenesis: neuroligin 1 that binds only beta-neurexins potently stimulates synapse formation, whereas neuroligin 1 that binds to both alpha- and beta-neurexins more effectively promotes synapse expansion. These findings suggest that neuroligin binding to alpha- and beta-neurexins mediates trans-synaptic cell adhesion but has distinct effects on synapse formation, indicating that expression of different neuroligin and neurexin isoforms specifies a trans-synaptic signaling code.

    View details for DOI 10.1016/j.neuron.2005.08.026

    View details for Web of Science ID 000232838700013

    View details for PubMedID 16242404

  • Crystal structure of the RIM2 C(2)A-domain at 1.4 angstrom resolution BIOCHEMISTRY Dai, H., Tomchick, D. R., Garcia, J., Sudhof, T. C., Machius, M., Rizo, J. 2005; 44 (41): 13533-13542

    Abstract

    RIMs are large proteins that contain two C2-domains and are localized at presynaptic active zones, where neurotransmitters are released. RIMs play key roles in synaptic vesicle priming and regulation of presynaptic plasticity. A mutation in the RIM1 C2A-domain has been implicated in autosomal dominant cone-rod dystrophy (CORD7). The RIM C2A-domain does not contain the full complement of aspartate residues that commonly mediate Ca2+ binding at the top loops of C2-domains, and has been reported to interact with SNAP-25 and synaptotagmin 1, two proteins from the Ca2+-dependent membrane fusion machinery. Here we have used NMR spectroscopy and X-ray crystallography to analyze the structure and biochemical properties of the RIM2 C2A-domain, which is closely related to the RIM1 C2A-domain. We find that the RIM2 C2A-domain does not bind Ca2+. Moreover, little binding of the RIM2 C2A-domain to SNAP-25 and to the C2-domains of synaptotagmin 1 was detected by NMR experiments, suggesting that as yet unidentified interactions of the RIM C2A-domain mediate its function. The crystal structure of the RIM2 C2A-domain using data to 1.4 A resolution reveals a beta-sandwich that resembles those observed for other C2-domains, but exhibits a unique dipolar distribution of electrostatic charges whereby one edge of the beta-sandwich is highly positive and the other edge is highly negative. The location of the mutation site implicated in CORD7 at the bottom of the domain and the pattern of sequence conservation suggest that, in contrast to most C2-domains, the RIM C2A-domains may function through Ca2+-independent interactions involving their bottom face.

    View details for DOI 10.1021/bi0513608

    View details for Web of Science ID 000232632100011

    View details for PubMedID 16216076

  • Solution structure of the RIM1 alpha PDZ domain in complex with an ELKS1 b C-terminal peptide JOURNAL OF MOLECULAR BIOLOGY Lu, J., Li, H. M., Wang, Y., Sudhof, T. C., Rizo, J. 2005; 352 (2): 455-466

    Abstract

    PDZ domains are widespread protein modules that commonly recognize C-terminal sequences of target proteins and help to organize macromolecular signaling complexes. These sequences usually bind in an extended conformation to relatively shallow grooves formed between a beta-strand and an alpha-helix in the corresponding PDZ domains. Because of this binding mode, many PDZ domains recognize primarily the C-terminal and the antepenultimate side-chains of the target protein, which commonly conform to motifs that have been categorized into different classes. However, an increasing number of PDZ domains have been found to exhibit unusual specificities. These include the PDZ domain of RIMs, which are large multidomain proteins that regulate neurotransmitter release and help to organize presynaptic active zones. The RIM PDZ domain binds to the C-terminal sequence of ELKS with a unique specificity that involves each of the four ELKS C-terminal residues. To elucidate the structural basis for this specificity, we have determined the 3D structure in solution of an RIM/ELKS C-terminal peptide complex using NMR spectroscopy. The structure shows that the RIM PDZ domain contains an unusually deep and narrow peptide-binding groove with an exquisite shape complementarity to the four ELKS C-terminal residues in their bound conformation. This groove is formed, in part, by a set of side-chains that is conserved selectively in RIM PDZ domains and that hence determines, at least in part, their unique specificity.

    View details for DOI 10.1016/j.jmb.2005.07.047

    View details for Web of Science ID 000231921700018

    View details for PubMedID 16095618

  • A Munc13/RIM/Rab3 tripartite complex: from priming to plasticity? EMBO JOURNAL Dulubova, I., Lou, X. L., Lu, J., Huryeva, I., Alam, A., Schneggenburger, R., Sudhof, T. C., Rizo, J. 2005; 24 (16): 2839-2850

    Abstract

    alpha-RIMs and Munc13s are active zone proteins that control priming of synaptic vesicles to a readily releasable state, and interact with each other via their N-terminal sequences. The alpha-RIM N-terminal sequence also binds to Rab3s (small synaptic vesicle GTPases), an interaction that regulates presynaptic plasticity. We now demonstrate that alpha-RIMs contain adjacent but separate Munc13- and Rab3-binding sites, allowing formation of a tripartite Rab3/RIM/Munc13 complex. Munc13 binding is mediated by the alpha-RIM zinc-finger domain. Elucidation of the three-dimensional structure of this domain by NMR spectroscopy facilitated the design of a mutation that abolishes alpha-RIM/Munc13 binding. Selective disruption of this interaction in the calyx of Held synapse decreased the size of the readily releasable vesicle pool. Our data suggest that the ternary Rab3/RIM/Munc13 interaction approximates synaptic vesicles to the priming machinery, providing a substrate for presynaptic plasticity. The modular architecture of alpha-RIMs, with nested binding sites for Rab3 and other targets, may be a general feature of Rab effectors that share homology with the alpha-RIM N-terminal sequence.

    View details for DOI 10.1038/sj.emboj.7600753

    View details for Web of Science ID 000231789300001

    View details for PubMedID 16052212

  • Nicastrin functions as a gamma-secretase-substrate receptor CELL Shah, S., Lee, S. F., Tabuchi, K., Hao, Y. H., Yu, C., LaPlant, Q., Ball, H., Dann, C. E., Sudhof, T., Yu, G. 2005; 122 (3): 435-447

    Abstract

    gamma-secretase catalyzes the intramembrane cleavage of amyloid precursor protein (APP) and Notch after their extracellular domains are shed by site-specific proteolysis. Nicastrin is an essential glycoprotein component of the gamma-secretase complex but has no known function. We now show that the ectodomain of nicastrin binds the new amino terminus that is generated upon proteolysis of the extracellular APP and Notch domains, thereby recruiting the APP and Notch substrates into the gamma-secretase complex. Chemical- or antibody-mediated blocking of the free amino terminus, addition of purified nicastrin ectodomain, or mutations in the ectodomain markedly reduce the binding and cleavage of substrate by gamma-secretase. These results indicate that nicastrin is a receptor for the amino-terminal stubs that are generated by ectodomain shedding of type I transmembrane proteins. Our data are consistent with a model where nicastrin presents these substrates to gamma-secretase and thereby facilitates their cleavage via intramembrane proteolysis.

    View details for DOI 10.1016/j.cell.2005.05.022

    View details for Web of Science ID 000231254400014

    View details for PubMedID 16096062

  • Genetic evidence for a protein-kinase-A-mediated presynaptic component in NMDA-receptor-dependent forms of long-term synaptic potentiation PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Huang, Y. Y., Zakharenko, S. S., Schoch, S., Kaeser, P. S., Janz, R., Sudhof, T. C., Siegelbaum, S. A., Kandel, E. R. 2005; 102 (26): 9365-9370

    Abstract

    The synaptic vesicle protein Rab3A is a small GTP-binding protein that interacts with rabphilin and RIM1alpha, two presynaptic substrates of protein kinase A (PKA). Mice lacking RIM1alpha and Rab3A have a defect in PKA-dependent and NMDA receptor (NMDAR)-independent presynaptic long-term potentiation (LTP) at hippocampal mossy-fiber and cerebellar parallel-fiber synapses. In contrast, the NMDAR-dependent and PKA-independent early phase of LTP at hippocampal CA3-CA1 synapses does not require these presynaptic proteins. Here, we ask whether Rab3A and RIM1alpha participate in forms of LTP that require both PKA and NMDAR activation. We find that Rab3A is necessary for corticoamygdala LTP and late-phase LTP at CA3-CA1 synapses, two forms of LTP that require NMDAR and PKA activation. The latter form of LTP also requires RIM1alpha. These results provide genetic evidence that presynaptic proteins are required in LTP induced through the postsynaptic activation of NMDARs. Thus Rab3A and its effectors are general modules for four distinct types of PKA-dependent LTP in the brain.

    View details for DOI 10.1073/pnas.0503777102

    View details for Web of Science ID 000230191400050

    View details for PubMedID 15967982

  • v-SNAREs control exocytosis of vesicles from priming to fusion EMBO JOURNAL Borisovska, M., Zhao, Y., Tsytsyura, Y., Glyvuk, N., Takamori, S., Matti, U., Rettig, J., Sudhof, T., Bruns, D. 2005; 24 (12): 2114-2126

    Abstract

    SNARE proteins (soluble NSF-attachment protein receptors) are thought to be central components of the exocytotic mechanism in neurosecretory cells, but their precise function remained unclear. Here, we show that each of the vesicle-associated SNARE proteins (v-SNARE) of a chromaffin granule, synaptobrevin II or cellubrevin, is sufficient to support Ca(2+)-dependent exocytosis and to establish a pool of primed, readily releasable vesicles. In the absence of both proteins, secretion is abolished, without affecting biogenesis or docking of granules indicating that v-SNAREs are absolutely required for granule exocytosis. We find that synaptobrevin II and cellubrevin differentially control the pool of readily releasable vesicles and show that the v-SNARE's amino terminus regulates the vesicle's primed state. We demonstrate that dynamics of fusion pore dilation are regulated by v-SNAREs, indicating their action throughout exocytosis from priming to fusion of vesicles.

    View details for DOI 10.1038/sj.emboj.7600696

    View details for Web of Science ID 000230511000007

    View details for PubMedID 15920476

  • Dissection of synapse induction by neuroligins - Effect of a neuroligin mutation associated with autism JOURNAL OF BIOLOGICAL CHEMISTRY Chubykin, A. A., Liu, X. R., Comoletti, D., Tsigelny, I., Taylor, P., Sudhof, T. C. 2005; 280 (23): 22365-22374

    Abstract

    To study synapse formation by neuroligins, we co-cultured hippocampal neurons with COS cells expressing wild type and mutant neuroligins. The large size of COS cells makes it possible to test the effect of neuroligins presented over an extended surface area. We found that a uniform lawn of wild type neuroligins displayed on the cell surface triggers the formation of hundreds of uniformly sized, individual synaptic contacts that are labeled with neurexin antibodies. Electron microscopy revealed that these artificial synapses contain a presynaptic active zone with docked vesicles and often feature a postsynaptic density. Neuroligins 1, 2, and 3 were active in this assay. Mutations in two surface loops of neuroligin 1 abolished neuroligin binding to neurexin 1beta, a presumptive presynaptic binding partner for postsynaptic neuroligins, and blocked synapse formation. An analysis of mutant neuroligins with an amino acid substitution that corresponds to a mutation described in patients with an autistic syndrome confirmed previous reports that these mutant neuroligins have a compromised capacity to be transported to the cell surface. Nevertheless, the small percentage of mutant neuroligins that reached the cell surface still induced synapse formation. Viewed together, our data suggest that neuroligins generally promote artificial synapse formation in a manner that is associated with beta-neurexin binding and results in morphologically well differentiated synapses and that a neuroligin mutation found in autism spectrum disorders impairs cell-surface transport but does not completely abolish synapse formation activity.

    View details for Web of Science ID 000229557900082

    View details for PubMedID 15797875

  • Munc18-1 stabilizes syntaxin 1, but is not essential for syntaxin 1 targeting and SNARE complex formation JOURNAL OF NEUROCHEMISTRY Toonen, R. F., de Vries, K. J., Zalm, R., Sudhof, T. C., Verhage, M. 2005; 93 (6): 1393-1400

    Abstract

    Munc18-1, a member of the Sec1/Munc18 (SM) protein family, is essential for synaptic vesicle exocytosis. Munc18-1 binds tightly to the SNARE protein syntaxin 1, but the physiological significance and functional role of this interaction remain unclear. Here we show that syntaxin 1 levels are reduced by 70% in munc18-1 knockout mice. Pulse-chase analysis in transfected HEK293 cells revealed that Munc18-1 directly promotes the stability of syntaxin 1, consistent with a chaperone function. However, the residual syntaxin 1 in munc18-1 knockout mice is still correctly targeted to synapses and efficiently forms SDS-resistant SNARE complexes, demonstrating that Munc18-1 is not required for syntaxin 1 function as such. These data demonstrate that the Munc18-1 interaction with syntaxin 1 is physiologically important, but does not represent a classical chaperone-substrate relationship. Instead, the presence of SNARE complexes in the absence of membrane fusion in munc18-1 knockout mice indicates that Munc18-1 either controls the spatially correct assembly of core complexes for SNARE-dependent fusion, or acts as a direct component of the fusion machinery itself.

    View details for DOI 10.1111/j.1471-4159.2005.03128.x

    View details for Web of Science ID 000229425800004

    View details for PubMedID 15935055

  • Extracellular domains of alpha-neurexins participate in regulating synaptic transmission by selectively affecting N- and P/Q-type Ca2+ channels JOURNAL OF NEUROSCIENCE Zhang, W. Q., Rohlmann, A., Sargsyan, V., Aramuni, G., Hammer, R. E., Sudhof, T. C., Missler, M. 2005; 25 (17): 4330-4342

    Abstract

    Neurexins constitute a large family of highly variable cell-surface molecules that may function in synaptic transmission and/or synapse formation. Each of the three known neurexin genes encodes two major neurexin variants, alpha- and beta-neurexins, that are composed of distinct extracellular domains linked to identical intracellular sequences. Deletions of one, two, or all three alpha-neurexins in mice recently demonstrated their essential role at synapses. In multiple alpha-neurexin knock-outs, neurotransmitter release from excitatory and inhibitory synapses was severely reduced, primarily probably because voltage-dependent Ca2+ channels were impaired. It remained unclear, however, which neurexin variants actually influence exocytosis and Ca2+ channels, which domain of neurexins is required for this function, and which Ca2+-channel subtypes are regulated. Here, we show by electrophysiological recordings that transgenic neurexin 1alpha rescues the release and Ca2+-current phenotypes, whereas transgenic neurexin 1beta has no effect, indicating the importance of the extracellular sequences for the function of neurexins. Because neurexin 1alpha rescued the knock-out phenotype independent of the alpha-neurexin gene deleted, these data are consistent with a redundant function among different alpha-neurexins. In both knock-out and transgenically rescued mice, alpha-neurexins selectively affected the component of neurotransmitter release that depended on activation of N- and P/Q-type Ca2+ channels, but left L-type Ca2+ channels unscathed. Our findings indicate that alpha-neurexins represent organizer molecules in neurotransmission that regulate N- and P/Q-type Ca2+ channels, constituting an essential role at synapses that critically involves the extracellular domains of neurexins.

    View details for DOI 10.1523/JNEUROSCI.0497-05.2005

    View details for Web of Science ID 000228702900014

    View details for PubMedID 15858059

  • CAPS in search of a lost function NEURON Sudhof, T. C. 2005; 46 (1): 2-4

    Abstract

    Ca2+-dependent activator protein for secretion (CAPS) is an evolutionarily conserved secretory protein that was previously thought to mediate Ca2+-triggered fusion of dense-core vesicles. In an elegant study of CAPS1-deficient mice, Speidel et al. (this issue of Neuron) now show that CAPS function may have been misunderstood. CAPS appears to act upstream of fusion in the biogenesis or maintenance of mature secretory vesicles, raising the possibility of a completely new type of function for an essential component of the secretory machinery.

    View details for DOI 10.1016/j.neuron.2005.03.017

    View details for Web of Science ID 000228228600002

    View details for PubMedID 15820687

  • Are neuronal SNARE proteins Ca2+ sensors? JOURNAL OF MOLECULAR BIOLOGY Chen, X. C., Tang, J., Sudhof, T. C., Rizo, J. 2005; 347 (1): 145-158

    Abstract

    The neuronal SNARE complex formed by synaptobrevin, syntaxin and SNAP-25 plays a central role in Ca2+-triggered neurotransmitter release. The SNARE complex contains several potential Ca2+-binding sites on the surface, suggesting that the SNAREs may be involved directly in Ca2+-binding during release. Indeed, overexpression of SNAP-25 bearing mutations in two putative Ca2+ ligands (E170A/Q177A) causes a decrease in the Ca2+-cooperativity of exocytosis in chromaffin cells. To test whether the SNARE complex might function in Ca2+-sensing, we analyzed its Ca2+-binding properties using transverse relaxation optimized spectroscopy (TROSY)-based NMR methods. Several Ca2+-binding sites are found on the surface of the SNARE complex, but most of them are not specific for Ca2+ and all have very low affinity. Moreover, we find that the E170A/Q177A SNAP-25 mutation does not alter interactions between the SNAREs and the Ca2+ sensor synaptotagmin 1, but severely impairs SNARE complex assembly. These results suggest that the SNAREs do not act directly as Ca2+ receptors but SNARE complex assembly is coupled tightly to Ca2+-sensing during neurotransmitter release.

    View details for DOI 10.1016/j.jmb.2005.01.024

    View details for Web of Science ID 000227505200012

    View details for PubMedID 15733924

  • Parkinson-like syndrome induced by continuous MPTP infusion: Convergent roles of the ubiquitin-proteasome system and alpha-synuclein PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Fornai, F., Schluter, O. M., Lenzi, P., Gesi, M., Ruffoli, R., Ferrucci, M., Lazzeri, G., Busceti, C. L., Pontarelli, F., Battaglia, G., Pellegrini, A., Nicoletti, F., Ruggieri, S., Paparelli, A., Sudhof, T. C. 2005; 102 (9): 3413-3418

    Abstract

    In animals, sporadic injections of the mitochondrial toxin 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) selectively damage dopaminergic neurons but do not fully reproduce the features of human Parkinson's disease. We have now developed a mouse Parkinson's disease model that is based on continuous MPTP administration with an osmotic minipump and mimics many features of the human disease. Although both sporadic and continuous MPTP administration led to severe striatal dopamine depletion and nigral cell loss, we find that only continuous administration of MPTP produced progressive behavioral changes and triggered formation of nigral inclusions immunoreactive for ubiquitin and alpha-synuclein. Moreover, only continuous MPTP infusions caused long-lasting activation of glucose uptake and inhibition of the ubiquitin-proteasome system. In mice lacking alpha-synuclein, continuous MPTP delivery still induced metabolic activation, but induction of behavioral symptoms and neuronal cell death were almost completely alleviated. Furthermore, the inhibition of the ubiquitinproteasome system and the production of inclusion bodies were reduced. These data suggest that continuous low-level exposure of mice to MPTP causes a Parkinson-like syndrome in an alpha-synuclein-dependent manner.

    View details for DOI 10.1073/pnas.0409713102

    View details for Web of Science ID 000227423700045

    View details for PubMedID 15716361

  • C-terminal ECFP fusion impairs synaptotagmin 1 function - Crowding out synaptotagmin 1 JOURNAL OF BIOLOGICAL CHEMISTRY Han, W. P., Rhee, J. S., Maximov, A., Lin, W. C., Hammer, R. E., Rosenmund, C., Sudhof, T. C. 2005; 280 (6): 5089-5100

    Abstract

    To allow the monitoring of synaptotagmin 1 trafficking in vivo, we generated transgenic mice expressing a synaptotagmin 1-enhanced cyan fluorescent protein (ECFP) fusion protein under control of the Thy1 promoter. Transgenic synaptotagmin 1-ECFP is expressed throughout the brain where it localizes to synapses and marks synapses in vivo. However, when we crossed transgenic synaptotagmin 1-ECFP mice with synaptotagmin 1 knock-out mice, we detected no rescue of survival or function. Furthermore, viral overexpression of synaptotagmin 1-ECFP in synaptotagmin 1-deficient neurons failed to restore normal Ca2+-triggered release, whereas overexpression of wild type synaptotagmin 1 did so efficiently. To determine whether synaptotagmin 1-ECFP is non-functional because the ECFP-fusion interferes with its biochemical activities, we measured Ca2+-independent binding of synaptotagmin 1-ECFP to SNARE complexes, and Ca2+-dependent binding of synaptotagmin 1-ECFP to phospholipids and to itself. Although the apparent Ca2+ affinity of synaptotagmin 1-ECFP was decreased compared with wild type synaptotagmin 1, we observed no major changes in Ca2+-dependent or -independent activities, indicating that the non-functionality of the synaptotagmin 1-ECFP fusion protein was not because of inactivation of its biochemical properties. These data suggest that synaptotagmin 1-ECFP is suitable for monitoring synaptic vesicle traffic in vivo because the synaptotagmin 1-ECFP marks synaptic vesicles without participating in exocytosis. In addition, the data demonstrate that synaptotagmin 1 function requires a free C terminus, possibly because of spatial constraints at the release sites.

    View details for DOI 10.1074/jbc.M408757200

    View details for Web of Science ID 000227096600131

    View details for PubMedID 15561725

  • Evolutionarily conserved multiple C-2 domain proteins with two transmembrane regions (MCTPs) and unusual Ca2+ binding properties JOURNAL OF BIOLOGICAL CHEMISTRY Shin, O. H., Han, W. P., Wang, Y., Sudhof, T. C. 2005; 280 (2): 1641-1651

    Abstract

    C2 domains are primarily found in signal transduction proteins such as protein kinase C, which generally contain a single C2 domain, and in membrane trafficking proteins such as synaptotagmins, which generally contain multiple C2 domains. In both classes of proteins, C2 domains usually regulate the respective protein's function by forming Ca(2+)-dependent or Ca(2+)-independent phospholipid complexes. We now describe MCTPs (multiple C2 domain and transmembrane region proteins), a novel family of evolutionarily conserved C2 domain proteins with unusual Ca(2+)-dependent properties. MCTPs are composed of a variable N-terminal sequence, three C2 domains, two transmembrane regions, and a short C-terminal sequence. The invertebrate organisms Caenorhabditis elegans and Drosophila melanogaster express a single MCTP gene, whereas vertebrates express two MCTP genes (MCTP1 and MCTP2) whose primary transcripts are extensively alternatively spliced. Most of the MCTP sequences, in particular the C2 domains, are highly conserved. All MCTP C2 domains except for the second C2 domain of MCTP2 include a perfect Ca2+/phospholipid-binding consensus sequence. To determine whether the C2 domains of MCTPs actually function as Ca2+/phospholipid-binding modules, we analyzed their Ca2+ and phospholipid binding properties. Surprisingly, we found that none of the three MCTP1 C2 domains interacted with negatively charged or neutral phospholipids in the presence or absence of Ca2+. However, Ca2+ titrations monitored via intrinsic tryptophan fluorescence revealed that all three C2 domains bound Ca2+ in the absence of phospholipids with a high apparent affinity (EC50 of approximately 1.3-2.3 microM). Our data thus reveal that MCTPs are evolutionarily conserved C2 domain proteins that are unusual in that the C2 domains are anchored in the membrane by two closely spaced transmembrane regions and represent Ca(2+)-binding but not phospholipid-binding modules.

    View details for DOI 10.1074/jbc.M407305200

    View details for Web of Science ID 000226195200095

    View details for PubMedID 15528213

  • Selective capability of SynCAM and neuroligin for functional synapse assembly JOURNAL OF NEUROSCIENCE Sara, Y., Biederer, T., Atasoy, D., Chubykin, A., Mozhayeva, M. G., Sudhof, T. C., Kavalali, E. T. 2005; 25 (1): 260-270

    Abstract

    Synaptic cell adhesion is central for synapse formation and function. Recently, the synaptic cell adhesion molecules neuroligin 1 (NL1) and SynCAM were shown to induce presynaptic differentiation in cocultured neurons when expressed in a non-neuronal cell. However, it is uncertain how similar the resulting artificial synapses are to regular synapses. Are these molecules isofunctional, or do all neuronal cell adhesion molecules nonspecifically activate synapse formation? To address these questions, we analyzed the properties of artificial synapses induced by NL1 and SynCAM, compared the actions of these molecules with those of other neuronal cell adhesion molecules, and examined the functional effects of NL1 and SynCAM overexpression in neurons. We found that only NL1 and SynCAM specifically induced presynaptic differentiation in cocultured neurons. The induced nerve terminals were capable of both spontaneous and evoked neurotransmitter release, suggesting that a full secretory apparatus was assembled. By all measures, SynCAM- and NL1-induced artificial synapses were identical. Overexpression in neurons demonstrated that only SynCAM, but not NL1, increased synaptic function in immature developing excitatory neurons after 8 d in vitro. Tests of chimeric molecules revealed that the dominant-positive effect of SynCAM on synaptic function in developing neurons was mediated by its intracellular cytoplasmic tail. Interestingly, morphological analysis of neurons overexpressing SynCAM or NL1 showed the opposite of the predictions from electrophysiological results. In this case, only NL1 increased the synapse number, suggesting a role for NL1 in morphological synapse induction. These results suggest that both NL1 and SynCAM act similarly and specifically in artificial synapse induction but that this process does not reflect a shared physiological function of these molecules.

    View details for DOI 10.1523/JNEUROSCI.3165-04.2005

    View details for Web of Science ID 000226130200032

    View details for PubMedID 15634790

  • Multiple roles for the active zone protein RIM1 alpha in late stages of neurotransmitter release NEURON Calakos, N., Schoch, S., Sudhof, T. C., Malenka, R. C. 2004; 42 (6): 889-896

    Abstract

    The active zone protein RIM1alpha interacts with multiple active zone and synaptic vesicle proteins and is implicated in short- and long-term synaptic plasticity, but it is unclear how RIM1alpha's biochemical interactions translate into physiological functions. To address this question, we analyzed synaptic transmission in autaptic neurons cultured from RIM1alpha-/- mice. Deletion of RIM1alpha causes a large reduction in the readily releasable pool of vesicles, alters short-term plasticity, and changes the properties of evoked asynchronous release. Lack of RIM1alpha, however, had no effect on synapse formation, spontaneous release, overall Ca2+ sensitivity of release, or synaptic vesicle recycling. These results suggest that RIM1alpha modulates sequential steps in synaptic vesicle exocytosis through serial protein-protein interactions and that this modulation is the basis for RIM1alpha's role in synaptic plasticity.

    View details for Web of Science ID 000222329300005

    View details for PubMedID 15207234

  • Role of alpha-synuclein in 1-methyl-4-phenyl-1,2,3,6-tetra-hydropyridine-induced parkinsonism in mice NEUROSCIENCE Schluter, O. M., Fornai, F., Alessandri, M. G., Takamori, S., Geppert, M., Jahn, R., Sudhof, T. C. 2003; 118 (4): 985-1002

    Abstract

    In humans, mutations in the alpha-synuclein gene or exposure to the neurotoxin 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) produce Parkinson's disease with loss of dopaminergic neurons and depletion of nigrostriatal dopamine. alpha-Synuclein is a vertebrate-specific component of presynaptic nerve terminals that may function in modulating synaptic transmission. To test whether MPTP toxicity involves alpha-synuclein, we generated alpha-synuclein-deficient mice by homologous recombination, and analyzed the effect of deleting alpha-synuclein on MPTP toxicity using these knockout mice. In addition, we examined commercially available mice that contain a spontaneous loss of the alpha-synuclein gene. As described previously, deletion of alpha-synuclein had no significant effects on brain structure or composition. In particular, the levels of synaptic proteins were not altered, and the concentrations of dopamine, dopamine metabolites, and dopaminergic proteins were unchanged. Upon acute MPTP challenge, alpha-synuclein knockout mice were partly protected from chronic depletion of nigrostriatal dopamine when compared with littermates of the same genetic background, whereas mice carrying the spontaneous deletion of the alpha-synuclein gene exhibited no protection. Furthermore, alpha-synuclein knockout mice but not the mice with the alpha-synuclein gene deletion were slightly more sensitive to methamphetamine than littermate control mice. These results demonstrate that alpha-synuclein is not obligatorily coupled to MPTP sensitivity, but can influence MPTP toxicity on some genetic backgrounds, and illustrate the need for extensive controls in studies aimed at describing the effects of mouse knockouts on MPTP sensitivity.

    View details for DOI 10.1016/S0306-4522(03)00036-8

    View details for Web of Science ID 000182926900011

    View details for PubMedID 12732244

  • RIM1 alpha is required for presynaptic long-term potentiation NATURE Castillo, P. E., Schoch, S., Schmitz, F., Sudhof, T. C., Malenka, R. C. 2002; 415 (6869): 327-330

    Abstract

    Two main forms of long-term potentiation (LTP)-a prominent model for the cellular mechanism of learning and memory-have been distinguished in the mammalian brain. One requires activation of postsynaptic NMDA (N-methyl d-aspartate) receptors, whereas the other, called mossy fibre LTP, has a principal presynaptic component. Mossy fibre LTP is expressed in hippocampal mossy fibre synapses, cerebellar parallel fibre synapses and corticothalamic synapses, where it apparently operates by a mechanism that requires activation of protein kinase A. Thus, presynaptic substrates of protein kinase A are probably essential in mediating this form of long-term synaptic plasticity. Studies of knockout mice have shown that the synaptic vesicle protein Rab3A is required for mossy fibre LTP, but the protein kinase A substrates rabphilin, synapsin I and synapsin II are dispensable. Here we report that mossy fibre LTP in the hippocampus and the cerebellum is abolished in mice lacking RIM1alpha, an active zone protein that binds to Rab3A and that is also a protein kinase A substrate. Our results indicate that the long-term increase in neurotransmitter release during mossy fibre LTP may be mediated by a unitary mechanism that involves the GTP-dependent interaction of Rab3A with RIM1alpha at the interface of synaptic vesicles and the active zone.

    View details for Web of Science ID 000173293500045

    View details for PubMedID 11797010

  • MAPPING OF SYNAPSIN-II (SYN2) GENES TO HUMAN-CHROMOSOME-3P AND MOUSE CHROMOSOME-6 BAND-F CYTOGENETICS AND CELL GENETICS Li, X., Rosahl, T. W., Sudhof, T. C., FRANCKE, U. 1995; 71 (3): 301-305

    Abstract

    Synapsins are neuron-specific phosphoproteins of small synaptic vesicles encoded by two different genes. While the gene for synapsin I (SYN1) is on the X chromosome, we have now assigned the human and mouse synapsin II (SYN2) genes to autosomes. By using PCR primers derived from rat synapsin II cDNA sequences we were able to amplify homologous sequences of the 3'-untranslated regions and to localize the human SYN2 gene to 3p and the mouse Syn2 gene to mouse chromosome 6 by single strand conformation analysis of PCR products from panels of somatic hybrid cell lines. The mouse gene was further mapped by FISH to chromosome 6 band F in a region of known conserved synteny with human 3p. Genotyping of a M. musculus x M. spretus backcross panel placed Syn2 close to a cluster of previously mapped loci on chromosome 6 in an interval between interleukin 5 receptor alpha (Il5ra) and hematopoietic cell phosphatase 1C (Hcph). Both physical and genetic mapping data indicate that Syn2 is near two mutant loci defined by neuromuscular disorders, opisthotonus (opt) and deaf waddler (dfw).

    View details for Web of Science ID A1995TD38500023

    View details for PubMedID 7587399

  • SYNAPTOPHYSIN - STRUCTURE OF THE HUMAN GENE AND ASSIGNMENT TO THE X-CHROMOSOME IN MAN AND MOUSE AMERICAN JOURNAL OF HUMAN GENETICS Ozcelik, T., Lafreniere, R. G., ARCHER, B. T., Johnston, P. A., WILLARD, H. F., FRANCKE, U., Sudhof, T. C. 1990; 47 (3): 551-561

    Abstract

    Synaptophysin is an integral membrane protein of small synaptic vesicles in brain and endocrine cells. We have determined the structure and organization of the human synaptophysin gene and have established the chromosome localizations in man and mouse. Analysis of a cosmid clone containing the human synaptophysin gene (SYP) revealed seven exons distributed over approximately 20 kb, when compared with the previously published cDNA sequence. The exon-intron boundaries have been identified and do not correlate with functional domains. One intron interrupts the 3' untranslated region. Chromosomal localization of the human and murine genes for synaptophysin established the human SYP locus on the X chromosome in subbands Xp11.22-p11.23 and the mouse synaptophysin gene locus (Syp) on the X chromosome in region A-D. In addition, an Eco0109 RFLP has been identified and used in genetic mapping of the human SYP locus and supports the order TIMP-SYP-DXS14 within a span of approximately 4-7 centimorgans.

    View details for Web of Science ID A1990DX22300020

    View details for PubMedID 1975480

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