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  • Neuromodulator Signaling Bidirectionally Controls Vesicle Numbers in Human Synapses. Cell Patzke, C., Brockmann, M. M., Dai, J., Gan, K. J., Grauel, M. K., Fenske, P., Liu, Y., Acuna, C., Rosenmund, C., Südhof, T. C. 2019; 179 (2): 498–513.e22

    Abstract

    Neuromodulators bind to pre- and postsynaptic G protein-coupled receptors (GPCRs), are able to quickly change intracellular cyclic AMP (cAMP) and Ca2+ levels, and are thought to play important roles in neuropsychiatric and neurodegenerative diseases. Here, we discovered in human neurons an unanticipated presynaptic mechanism that acutely changes synaptic ultrastructure and regulates synaptic communication. Activation of neuromodulator receptors bidirectionally controlled synaptic vesicle numbers within nerve terminals. This control correlated with changes in the levels of cAMP-dependent protein kinase A-mediated phosphorylation of synapsin-1. Using a conditional deletion approach, we reveal that the neuromodulator-induced control of synaptic vesicle numbers was largely dependent on synapsin-1. We propose a mechanism whereby non-phosphorylated synapsin-1 "latches" synaptic vesicles to presynaptic clusters at the active zone. cAMP-dependent phosphorylation of synapsin-1 then removes the vesicles. cAMP-independent dephosphorylation of synapsin-1 in turn recruits vesicles. Synapsin-1 thereby bidirectionally regulates synaptic vesicle numbers and modifies presynaptic neurotransmitter release as an effector of neuromodulator signaling in human neurons.

    View details for DOI 10.1016/j.cell.2019.09.011

    View details for PubMedID 31585084

  • The fragile X mutation impairs homeostatic plasticity in human neurons by blocking synaptic retinoic acid signaling. Science translational medicine Zhang, Z., Marro, S. G., Zhang, Y., Arendt, K. L., Patzke, C., Zhou, B., Fair, T., Yang, N., Sudhof, T. C., Wernig, M., Chen, L. 2018; 10 (452)

    Abstract

    Fragile X syndrome (FXS) is an X chromosome-linked disease leading to severe intellectual disabilities. FXS is caused by inactivation of the fragile X mental retardation 1 (FMR1) gene, but how FMR1 inactivation induces FXS remains unclear. Using human neurons generated from control and FXS patient-derived induced pluripotent stem (iPS) cells or from embryonic stem cells carrying conditional FMR1 mutations, we show here that loss of FMR1 function specifically abolished homeostatic synaptic plasticity without affecting basal synaptic transmission. We demonstrated that, in human neurons, homeostatic plasticity induced by synaptic silencing was mediated by retinoic acid, which regulated both excitatory and inhibitory synaptic strength. FMR1 inactivation impaired homeostatic plasticity by blocking retinoic acid-mediated regulation of synaptic strength. Repairing the genetic mutation in the FMR1 gene in an FXS patient cell line restored fragile X mental retardation protein (FMRP) expression and fully rescued synaptic retinoic acid signaling. Thus, our study reveals a robust functional impairment caused by FMR1 mutations that might contribute to neuronal dysfunction in FXS. In addition, our results suggest that FXS patient iPS cell-derived neurons might be useful for studying the mechanisms mediating functional abnormalities in FXS.

    View details for PubMedID 30068571

  • Autism-associated SHANK3 haploinsufficiency causes I-h channelopathy in human neurons SCIENCE Yi, F., Danko, T., Botelho, S. C., Patzke, C., Pak, C., Wernig, M., Sudhof, T. C. 2016; 352 (6286): 672-?

    Abstract

    Heterozygous SHANK3 mutations are associated with idiopathic autism and Phelan-McDermid syndrome. SHANK3 is a ubiquitously expressed scaffolding protein that is enriched in postsynaptic excitatory synapses. Here, we used engineered conditional mutations in human neurons and found that heterozygous and homozygous SHANK3 mutations severely and specifically impaired Ih channels. SHANK3 mutations caused alterations in neuronal morphology and synaptic connectivity; chronic pharmacological blockage of Ih channels reproduced these phenotypes, suggesting that they may be secondary to Ih-channel impairment. Moreover, mouse Shank3-deficient neurons also exhibited severe decreases in Ih currents. SHANK3 protein interacted with hyperpolarization-activated cyclic nucleotide-gated channel proteins (HCN proteins) forming Ih channels, indicating that SHANK3 functions to organize HCN channels. Our data suggest SHANK3 mutations predispose to autism, at least partially, by inducing an Ih channelopathy that may be amenable to pharmacological intervention.

    View details for DOI 10.1126/science.aaf2669

    View details for PubMedID 26966193

  • Conditional deletion of L1CAM in human neurons impairs both axonal and dendritic arborization and action potential generation JOURNAL OF EXPERIMENTAL MEDICINE Patzke, C., Acuna, C., Giam, L. R., Wernig, M., Suedhof, T. C. 2016; 213 (4): 499-515

    Abstract

    Hundreds ofL1CAMgene mutations have been shown to be associated with congenital hydrocephalus, severe intellectual disability, aphasia, and motor symptoms. How such mutations impair neuronal function, however, remains unclear. Here, we generated human embryonic stem (ES) cells carrying a conditionalL1CAMloss-of-function mutation and produced precisely matching control andL1CAM-deficient neurons from these ES cells. In analyzing two independent conditionally mutant ES cell clones, we found that deletion ofL1CAMdramatically impaired axonal elongation and, to a lesser extent, dendritic arborization. Unexpectedly, we also detected an ∼20-50% and ∼20-30% decrease, respectively, in the levels of ankyrinG and ankyrinB protein, and observed that the size and intensity of ankyrinG staining in the axon initial segment was significantly reduced. Overexpression of wild-type L1CAM, but not of the L1CAM point mutants R1166X and S1224L, rescued the decrease in ankyrin levels. Importantly, we found that theL1CAMmutation selectively decreased activity-dependent Na(+)-currents, altered neuronal excitability, and caused impairments in action potential (AP) generation. Thus, our results suggest that the clinical presentations ofL1CAMmutations in human patients could be accounted for, at least in part, by cell-autonomous changes in the functional development of neurons, such that neurons are unable to develop normal axons and dendrites and to generate normal APs.

    View details for Web of Science ID 000373394100005

    View details for PubMedCentralID PMC4821644

  • The conditional KO approach: Cre/Lox technology in human neurons. Rare diseases (Austin, Tex.) Patzke, C., Südhof, T. C. 2016; 4 (1)

    Abstract

    The use of human pluripotent stem cells to model human diseases has become a new standard in biomedical sciences. To this end, patient-derived somatic cells are studied in vitro to mimic human pathological conditions. Here, we describe an alternative experimental strategy, the 'conditional KO approach', which allows engineering disease-relevant mutations in pluripotent stem cells from healthy donors. In combination with the Cre/Lox technology, this strategy enables us to study the molecular causes of human diseases independent of the genetic background or of genetic alterations induced by clonal selection. As a proof-of-principle, we generated pluripotent stem cells with conditional loss-of-function mutations in the human STXBP1 gene that encodes Munc18-1. Using neurons derived from these cells, we show that heterozygous disruption of STXBP1 produces a specific and selective impairment in synaptic transmission that may account for the severe neurological disease caused by such mutations in human patients.

    View details for DOI 10.1080/21675511.2015.1131884

    View details for PubMedID 27141410

  • Analysis of conditional heterozygous STXBP1 mutations in human neurons JOURNAL OF CLINICAL INVESTIGATION Patzke, C., Han, Y., Covy, J., Yi, F., Maxeiner, S., Wernig, M., Suedhof, T. C. 2015; 125 (9): 3560-3571

    Abstract

    Heterozygous mutations in the syntaxin-binding protein 1 (STXBP1) gene, which encodes Munc18-1, a core component of the presynaptic membrane-fusion machinery, cause infantile early epileptic encephalopathy (Ohtahara syndrome), but it is unclear how a partial loss of Munc18-1 produces this severe clinical presentation. Here, we generated human ES cells designed to conditionally express heterozygous and homozygous STXBP1 loss-of-function mutations and studied isogenic WT and STXBP1-mutant human neurons derived from these conditionally mutant ES cells. We demonstrated that heterozygous STXBP1 mutations lower the levels of Munc18-1 protein and its binding partner, the t-SNARE-protein Syntaxin-1, by approximately 30% and decrease spontaneous and evoked neurotransmitter release by nearly 50%. Thus, our results confirm that using engineered human embryonic stem (ES) cells is a viable approach to studying disease-associated mutations in human neurons on a controlled genetic background, demonstrate that partial STXBP1 loss of function robustly impairs neurotransmitter release in human neurons, and suggest that heterozygous STXBP1 mutations cause early epileptic encephalopathy specifically through a presynaptic impairment.

    View details for DOI 10.1172/JCI78612

    View details for Web of Science ID 000362303600030

    View details for PubMedCentralID PMC4588304

  • 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

  • The Coxsackievirus-Adenovirus Receptor Reveals Complex Homophilic and Heterophilic Interactions on Neural Cells JOURNAL OF NEUROSCIENCE Patzke, C., Max, K. E., Behlke, J., Schreiber, J., Schmidt, H., Dorner, A. A., Kroeger, S., Henning, M., Otto, A., Heinemann, U., Rathjen, F. G. 2010; 30 (8): 2897-2910

    Abstract

    The coxsackievirus-adenovirus receptor (CAR) is a member of the Ig superfamily strongly expressed in the developing nervous system. Our histological investigations during development reveal an initial uniform distribution of CAR on all neural cells with a concentration on membranes that face the margins of the nervous system (e.g., the basal laminae and the ventricular side). At more advanced stages, CAR becomes downregulated and restricted to specific regions including areas rich in axonal and dendritic surfaces. To study the function of CAR on neural cells, we used the fiber knob of the adenovirus, extracellular CAR domains, blocking antibodies to CAR, as well as CAR-deficient neural cells. Blocking antibodies were found to inhibit neurite extension in retina organ and retinal explant cultures, whereas the application of the recombinant fiber knob of the adenovirus subtype Ad2 or extracellular CAR domains promoted neurite extension and adhesion to extracellular matrices. We observed a promiscuous interaction of CAR with extracellular matrix glycoproteins, which was deduced from analytical ultracentrifugation experiments, affinity chromatography, and adhesion assays. The membrane proximal Ig domain of CAR, termed D2, was found to bind to a fibronectin fragment, including the heparin-binding domain 2, which promotes neurite extension of wild type, but not of CAR-deficient neural cells. In contrast to heterophilic interactions, homophilic association of CAR involves both Ig domains, as was revealed by ultracentrifugation, chemical cross-linking, and adhesion studies. The results of these functional and binding studies are correlated to a U-shaped homodimer of the complete extracellular domains of CAR detected by x-ray crystallography.

    View details for DOI 10.1523/JNEUROSCI.5725-09.2010

    View details for Web of Science ID 000274930500011

    View details for PubMedID 20181587