Doctor of Philosophy, University of South Florida (2013)
Bachelor of Science, University of South Florida (2009)
Thomas Sudhof, Postdoctoral Faculty Sponsor
Angelman Syndrome (AS) is a devastating neurological disorder caused by disruption of the maternal UBE3A gene. Ube3a protein is identified as an E3 ubiquitin ligase that shows neuron-specific imprinting. Despite extensive research evaluating the localization and basal expression profiles of Ube3a in mouse models, the molecular mechanisms whereby Ube3a deficiency results in AS are enigmatic. Using in vitro and in vivo systems we show dramatic changes in the expression of Ube3a following synaptic activation. In primary neuronal culture, neuronal depolarization was found to increase both nuclear and cytoplasmic Ube3a levels. Analogous up-regulation in maternal and paternal Ube3a expression was observed in Ube3a-YFP reporter mice following fear conditioning. Absence of Ube3a led to deficits in the activity-dependent increases in ERK1/2 phosphorylation, which may contribute to reported deficits in synaptic plasticity and cognitive function in AS mice. Taken together, our findings provide novel insight into the regulation of Ube3a by synaptic activity and its potential role in kinase regulation.
View details for DOI 10.1101/lm.032375.113
View details for Web of Science ID 000331396000006
View details for PubMedID 24434871
The secreted glycoprotein reelin plays an indispensable role in neuronal migration during development and in regulating adult synaptic functions. The upstream mechanisms responsible for initiating and regulating the duration and magnitude of reelin signaling are largely unknown. Here we report that reelin is cleaved between EGF-like repeats 6-7 (R6-7) by tissue plasminogen activator (tPA) under cell-free conditions. No changes were detected in the level of reelin and its fragments in the brains of tPA knockouts, implying that other unknown proteases are responsible for generating reelin fragments found constitutively in the adult brain. Induction of NMDAR-independent long-term potentiation with the potassium channel blocker tetraethylammonium chloride (TEA-Cl) led to a specific up-regulation of reelin processing at R6-7 in wild-type mice. In contrast, no changes in reelin expression and processing were observed in tPA knockouts following TEA-Cl treatment. These results demonstrate that synaptic potentiation results in tPA-dependent reelin processing and suggest that extracellular proteolysis of reelin may regulate reelin signaling in the adult brain.
View details for DOI 10.1016/j.neuroscience.2014.05.046
View details for PubMedID 24892761
Disabled-1 (Dab1) is an adaptor protein that is an obligate effector of the Reelin signaling pathway, and is critical for neuronal migration and dendrite outgrowth during development. Components of the Reelin pathway are highly expressed during development, but also continue to be expressed in the adult brain. Here we investigated in detail the expression pattern of Dab1 in the postnatal and adult forebrain, and determined that it is expressed in excitatory as well as inhibitory neurons. Dab1 was found to be localized in different cellular compartments, including the soma, dendrites, presynaptic and postsynaptic structures. Mice that are deficient in Dab1, Reelin, or the Reelin receptors ApoER2 and VLDLR exhibit severely perturbed brain cytoarchitecture, limiting the utility of these mice for investigating the role of this signaling pathway in the adult brain. In this study, we developed an adult forebrain-specific and excitatory neuron-specific conditional knock-out mouse line, and demonstrated that Dab1 is a critical regulator of synaptic function and hippocampal-dependent associative and spatial learning. These dramatic abnormalities were accompanied by a reduction in dendritic spine size, and defects in basal and plasticity-induced Akt and ERK1/2 signaling. Deletion of Dab1 led to no obvious changes in neuronal positioning, dendrite morphology, spine density, or synaptic composition. Collectively, these data conclusively demonstrate an important role for Reelin-Dab1 signaling in the adult forebrain, and underscore the importance of this pathway in learning and memory.
View details for DOI 10.1523/JNEUROSCI.2010-13.2013
View details for Web of Science ID 000324912500034
View details for PubMedID 24068831
The lipoprotein receptor ligand Reelin is important for the processes of normal synaptic plasticity, dendritic morphogenesis, and learning and memory. Heterozygous reeler mice (HRM) show many neuroanatomical, biochemical, and behavioral features that are associated with schizophrenia. HRM show subtle morphological defects including reductions in dendritic spine density, altered synaptic plasticity and behavioral deficits in associative learning and memory and pre-pulse inhibition. The present studies test the hypothesis that in vivo elevation of Reelin levels can rescue synaptic and behavioral phenotypes associated with HRM. We demonstrate that a single in vivo injection of Reelin increases GAD67 expression and alters dendritic spine morphology. In parallel we observed enhancement of hippocampal synaptic function and associative learning and memory. Reelin supplementation also increases pre-pulse inhibition. These results suggest that characteristics of HRM, similar to those observed in schizophrenia, are sensitive to Reelin levels and can be modified with Reelin supplementation in male and female adults.
View details for DOI 10.1177/0269881112463468
View details for Web of Science ID 000316907600008
View details for PubMedID 23104248
The cellular and molecular mechanisms responsible for the development of inner retinal circuitry are poorly understood. Reelin and apolipoprotein E (apoE), ligands of apoE receptor 2 (ApoER2), are involved in retinal development and degeneration, respectively. Here we describe the function of ApoER2 in the developing and adult retina. ApoER2 expression was highest during postnatal inner retinal synaptic development and was considerably lower in the mature retina. Both during development and in the adult, ApoER2 was expressed by A-II amacrine cells. ApoER2 knock-out (KO) mice had rod bipolar morphogenic defects, altered A-II amacrine dendritic development, and impaired rod-driven retinal responses. The presence of an intact ApoER2 NPxY motif, necessary for binding Disabled-1 and transducing the Reelin signal, was also necessary for development of the rod bipolar pathway, while the alternatively spliced exon 19 was not. Mice deficient in another Reelin receptor, very low-density lipoprotein receptor (VLDLR), had normal rod bipolar morphology but altered A-II amacrine dendritic development. VLDLR KO mice also had reductions in oscillatory potentials and delayed synaptic response intervals. Interestingly, age-related reductions in rod and cone function were observed in both ApoER2 and VLDLR KOs. These results support a pivotal role for ApoER2 in the establishment and maintenance of normal retinal synaptic connectivity.
View details for DOI 10.1523/JNEUROSCI.3135-11.2011
View details for Web of Science ID 000295805500038
View details for PubMedID 21976526
Emerging concepts in developmental biology, such as facilitated variation and dynamical patterning modules, address a major shortcoming of the Modern Synthesis in Biology: how genotypic variation is transduced into functional yet diverse phenotypic variation. Still, we lack a theory to explain how variation at the cellular and tissue level is coordinated into variation at the whole-organism level, especially as priority of cellular and tissue functions change over an individual's lifetime and are influenced by environmental variation. Here, we propose that interactions among a limited subset of physiological factors that we call, integrators, regulate most phenotypic variation at the organismal level. Integrators are unique among physiological factors in that they have the propensity to coordinate the expression of conserved gene modules of most types of tissues because they participate as nodes in a hierarchical network. In other words, integrator networks impose physiological epistasis, meaning that whole-organism phenotypic responses will be influenced by previous experiences, current environmental conditions, and fitness priorities as encoded by individual integrators. Below, we provide examples of how integrator networks are responsible for both profound and irreversible phenotypic changes (i.e., metamorphosis, sexual differentiation) as well as subtler, transient (e.g., pelage color, seasonal fluctuations in lymphoid and reproductive tissues) variation. The goal of this article is not to describe completely how integrator networks function, but to stimulate discussion about the role of physiology in linking genetic to phenotypic variation. To generate useful data sets for understanding integrator networks and to inform whole-organism physiology generally, we describe several useful tools including vector-field editing, response-surface regression, and experiments of life-table responses. We then close by highlighting some implications of integrator networks for conservation and biomedicine.
View details for DOI 10.1093/icb/icr049
View details for Web of Science ID 000295172700002
View details for PubMedID 21705794
Apolipoprotein receptors belong to an evolutionarily conserved surface receptor family that has intimate roles in the modulation of synaptic plasticity and is necessary for proper hippocampal-dependent memory formation. The known lipoprotein receptor ligand Reelin is important for normal synaptic plasticity, dendritic morphology, and cognitive function; however, the in vivo effect of enhanced Reelin signaling on cognitive function and synaptic plasticity in wild-type mice is unknown. The present studies test the hypothesis that in vivo enhancement of Reelin signaling can alter synaptic plasticity and ultimately influence processes of learning and memory. Purified recombinant Reelin was injected bilaterally into the ventricles of wild-type mice. We demonstrate that a single in vivo injection of Reelin increased activation of adaptor protein Disabled-1 and cAMP-response element binding protein after 15 min. These changes correlated with increased dendritic spine density, increased hippocampal CA1 long-term potentiation (LTP), and enhanced performance in associative and spatial learning and memory. The present study suggests that an acute elevation of in vivo Reelin can have long-term effects on synaptic function and cognitive ability in wild-type mice.
View details for DOI 10.1101/lm.2153511
View details for Web of Science ID 000295080700003
View details for PubMedID 21852430
The microtubule-associated protein tau, which becomes hyperphosphorylated and pathologically aggregates in a number of these diseases, is extremely sensitive to manipulations of chaperone signaling. For example, Hsp90 inhibitors can reduce the levels of tau in transgenic mouse models of tauopathy. Because of this, we hypothesized that a number of Hsp90 accessory proteins, termed co-chaperones, could also affect tau stability. Perhaps by identifying these co-chaperones, new therapeutics could be designed to specifically target these proteins and facilitate tau clearance. Here, we report that the co-chaperone Cdc37 can regulate aspects of tau pathogenesis. We found that suppression of Cdc37 destabilized tau, leading to its clearance, whereas Cdc37 overexpression preserved tau. Cdc37 was found to co-localize with tau in neuronal cells and to physically interact with tau from human brain. Moreover, Cdc37 levels significantly increased with age. Cdc37 knockdown altered the phosphorylation profile of tau, an effect that was due in part to reduced tau kinase stability, specifically Cdk5 and Akt. Conversely, GSK3β and Mark2 were unaffected by Cdc37 modulation. Cdc37 overexpression prevented whereas Cdc37 suppression potentiated tau clearance following Hsp90 inhibition. Thus, Cdc37 can regulate tau in two ways: by directly stabilizing it via Hsp90 and by regulating the stability of distinct tau kinases. We propose that changes in the neuronal levels or activity of Cdc37 could dramatically alter the kinome, leading to profound changes in the tau phosphorylation signature, altering its proteotoxicity and stability.
View details for DOI 10.1074/jbc.M110.182493
View details for Web of Science ID 000290301900047
View details for PubMedID 21367866
Apolipoprotein E receptor 2 (ApoEr2) is a postsynaptic protein involved in long-term potentiation (LTP), learning, and memory through unknown mechanisms. We examined the biological effects of ApoEr2 on synapse and dendritic spine formation-processes critical for learning and memory.In a heterologous co-culture synapse assay, overexpression of ApoEr2 in COS7 cells significantly increased colocalization with synaptophysin in primary hippocampal neurons, suggesting that ApoEr2 promotes interaction with presynaptic structures. In primary neuronal cultures, overexpression of ApoEr2 increased dendritic spine density. Consistent with our in vitro findings, ApoEr2 knockout mice had decreased dendritic spine density in cortical layers II/III at 1 month of age. We also tested whether the interaction between ApoEr2 and its cytoplasmic adaptor proteins, specifically X11α and PSD-95, affected synapse and dendritic spine formation. X11α decreased cell surface levels of ApoEr2 along with synapse and dendritic spine density. In contrast, PSD-95 increased cell surface levels of ApoEr2 as well as synapse and dendritic spine density.These results suggest that ApoEr2 plays important roles in structure and function of CNS synapses and dendritic spines, and that these roles are modulated by cytoplasmic adaptor proteins X11α and PSD-95.
View details for DOI 10.1371/journal.pone.0017203
View details for Web of Science ID 000287369200029
View details for PubMedID 21347244
Target-based drug discovery for Alzheimer's disease (AD) centered on modulation of the amyloid β peptide has met with limited success. Therefore, recent efforts have focused on targeting the microtubule-associated protein tau. Tau pathologically accumulates in more than 15 neurodegenerative diseases and is most closely linked with postsymptomatic progression in AD. We endeavored to identify compounds that decrease tau stability rather than prevent its aggregation. An extract from Myrica cerifera (bayberry/southern wax myrtle) potently reduced both endogenous and overexpressed tau protein levels in cells and murine brain slices. The bayberry flavonoids myricetin and myricitrin were confirmed to contribute to this potency, but a diarylheptanoid, myricanol, was the most effective anti-tau component in the extract, with potency approaching the best targeted lead therapies. (+)-aR,11S-Myricanol, isolated from M. cerifera and reported here for the first time as the naturally occurring aglycone, was significantly more potent than commercially available (±)-myricanol. Myricanol may represent a novel scaffold for drug development efforts targeting tau turnover in AD.
View details for DOI 10.1021/np100572z
View details for Web of Science ID 000286577800008
View details for PubMedID 21141876
View details for DOI 10.2217/fnl.10.80
The vast majority of Alzheimer's disease (AD) cases are late onset with progressive synapse loss and neurodegeneration. Although the amyloid hypothesis has generated great insights into the disease mechanism, several lines of evidence indicate that other risk factors might precondition the brain to amyloid toxicity. Here, we show that the deletion of a major lipoprotein receptor, low-density lipoprotein receptor-related protein 1 (LRP1), in forebrain neurons in mice leads to a global defect in brain lipid metabolism characterized by decreased brain levels of cholesterol, sulfatide, galactosylceramide, and triglyceride. These lipid deficits correlate with progressive, age-dependent dendritic spine degeneration, synapse loss, neuroinflammation, memory loss, and eventual neurodegeneration. We further show that the levels of glutamate receptor subunits NMDA receptor 1 and Glu receptor 1 are selectively reduced in LRP1 forebrain knock-out mice and in LRP1 knockdown neurons, which is partially rescued by restoring neuronal cholesterol. Together, these studies support a critical role for LRP1 in maintaining brain lipid homeostasis and associated synaptic and neuronal integrity, and provide important insights into the pathophysiological mechanisms in AD.
View details for DOI 10.1523/JNEUROSCI.4067-10.2010
View details for Web of Science ID 000285342300032
View details for PubMedID 21159977
The three human alleles of apolipoprotein E (APOE) differentially influence outcome after CNS injury and affect one's risk of developing Alzheimer's disease (AD). It remains unclear how ApoE isoforms contribute to various AD-related pathological changes (e.g., amyloid plaques and synaptic and neuron loss). Here, we systematically examined whether apoE isoforms (E2, E3, E4) exhibit differential effects on dendritic spine density and morphology in APOE targeted replacement (TR) mice, which lack AD pathological changes. Using Golgi staining, we found age-dependent effects of APOE4 on spine density in the cortex. The APOE4 TR mice had significantly reduced spine density at three independent time points (4 weeks, 3 months, and 1 year, 27.7% +/- 7.4%, 24.4% +/- 8.6%, and 55.6% +/- 10.5%, respectively) compared with APOE3 TR mice and APOE2 TR mice. Additionally, in APOE4 TR mice, shorter spines were evident compared with other APOE TR mice at 1 year. APOE2 TR mice exhibited longer spines as well as significantly increased apical dendritic arborization in the cortex compared with APOE4 and APOE3 TR mice at 4 weeks. However, there were no differences in spine density across APOE genotypes in hippocampus. These findings demonstrate that apoE isoforms differentially affect dendritic complexity and spine formation, suggesting a role for APOE genotypes not only in acute and chronic brain injuries including AD, but also in normal brain functions.
View details for DOI 10.1523/JNEUROSCI.4026-09.2009
View details for Web of Science ID 000272361700032
View details for PubMedID 19955384
Alzheimer's disease and other tauopathies have recently been clustered with a group of nervous system disorders termed protein misfolding diseases. The common element established between these disorders is their requirement for processing by the chaperone complex. It is now clear that the individual components of the chaperone system, such as Hsp70 and Hsp90, exist in an intricate signaling network that exerts pleiotropic effects on a host of substrates. Therefore, we have endeavored to identify new compounds that can specifically regulate individual components of the chaperone family. Here, we hypothesized that chemical manipulation of Hsp70 ATPase activity, a target that has not previously been pursued, could illuminate a new pathway toward chaperone-based therapies. Using a newly developed high-throughput screening system, we identified inhibitors and activators of Hsp70 enzymatic activity. Inhibitors led to rapid proteasome-dependent tau degradation in a cell-based model. Conversely, Hsp70 activators preserved tau levels in the same system. Hsp70 inhibition did not result in general protein degradation, nor did it induce a heat shock response. We also found that inhibiting Hsp70 ATPase activity after increasing its expression levels facilitated tau degradation at lower doses, suggesting that we can combine genetic and pharmacologic manipulation of Hsp70 to control the fate of bound substrates. Disease relevance of this strategy was further established when tau levels were rapidly and substantially reduced in brain tissue from tau transgenic mice. These findings reveal an entirely novel path toward therapeutic intervention of tauopathies by inhibition of the previously untargeted ATPase activity of Hsp70.
View details for DOI 10.1523/JNEUROSCI.3345-09.2009
View details for Web of Science ID 000270363300007
View details for PubMedID 19793966
The lipoprotein receptor system in the hippocampus is intimately involved in the modulation of synaptic transmission and plasticity. The association of specific apoE isoform expression with human neurodegenerative disorders has focused attention on the role of these apoE isoforms in lipoprotein receptor-dependent synaptic modulation. In the present study, we used the apoE2, apoE3 and apoE4 targeted replacement (TR) mice along with recombinant human apoE isoforms to determine the role of apoE isoforms in hippocampus area CA1 synaptic function. While synaptic transmission is unaffected by apoE isoform, long-term potentiation (LTP) is significantly enhanced in apoE4 TR mice versus apoE2 TR mice. ApoE isoform-dependent differences in LTP induction require NMDA-receptor function, and apoE isoform expression alters activation of both ERK and JNK signal transduction. Acute application of specific apoE isoforms also alters LTP induction while decreasing NMDA-receptor mediated field potentials. Furthermore, acute apoE isoform application does not have the same effects on ERK and JNK activation. These findings demonstrate specific, isoform-dependent effects of human apoE isoforms on adult hippocampus synaptic plasticity and highlight mechanistic differences between chronic apoE isoform expression and acute apoE isoform exposure.
View details for DOI 10.1186/1750-1326-4-21
View details for Web of Science ID 000267252500001
View details for PubMedID 19725929