Yu Tian Wang

The full abstract for this thesis is available in the body of the thesis, and will be available when the embargo expires.

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Both N-methyl-D-aspartate (NMDA) receptor, one of the subtypes of ionotropic glutamatergic receptors at the vast majority of excitatory synapses, and the type A γ-aminobutyric acid receptor (GABAAR), the principal ionotropic GABARs at the inhibitory GABAergic synapses play essential roles in regulating neuronal activities in the mammalian central nervous system (CNS). Their dysfunctions contribute to the pathogenesis of many neurological disorders. Overactivation of NMDAR-mediated excitotoxicity is a common cause of many neurodegenerative diseases including stroke. There are two prevalent theories, the ‘NMDAR subtype hypothesis’ that proposes that activating GluN2A-containing NMDAR promotes neuronal survival, whereas activating GluN2B-containing NMDARs leads to neuronal death, and the ‘NMDAR location hypothesis’ that suggests that activating synaptic, primarily GluN2A-containing NMDARs favors neuronal survival while activating extrasynaptic, predominantly GluN2B-containing NMDARs induces neuronal death. Since both hypotheses support allosteric modulators that potentiate GluN2A function and inhibit GluN2B function may have promising potentials as a new and more effective class of neuroprotective therapy for stroke, we performed computer-aided virtual screening and in-silico drug design to discover a lead compound 813 that functions as an NMDAR dual allosteric modulator (Ndam) that potentiates GluN1/GluN2A and at the same time inhibits GluN1/GluN2B. We further optimized Ndam813 and thereby developed two more efficacious compounds, Ndam830 and Ndam844. Ndam830 protected cortical neurons from NMDA-induced excitotoxicity and H2O2-induced oxidative stress, and also reduced ischemia-induced infarct volume, and promoted behavioral recoveries in rat MCAo models. Thus, our results strongly suggest that Ndam830 by promoting synaptic/GluN2A-containing NMDAR mediated cell survival signaling and inhibiting extrasynaptic/GluN2B-containing receptor mediated cell death signaling, is a novel neuroprotective stroke therapeutic.Ndam844 is a potent pan-NMDAR potentiator that can potentially treat NMDAR hypofunction related disorders. Apart from NMDARs, dysfunction of GABAARs is also implicated in various neurological conditions. Using electrophysiological and biochemical methods, we characterized the functional alterations of two de novo GABAAR variants T292S and T292I identified in patients with epileptic encephalopathy (EE) and developmental delay. Moreover, we found clinically approved allosteric modulators of GABAAR that may treat the patients carrying the variants. In summary, allosteric modulators of both NMDARs and GABAAR showed great therapeutic potential for neurological disorders.

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Accumulating evidence implicates dysfunction within the glutamatergic system anddysregulation of synaptic plasticity in the pathophysiology of depression, particularly in thehippocampus (HPC). Ketamine has rapid and sustained antidepressant activity in treatmentresistant depression and various animal models; however, its effects on synaptic plasticity, as well as their contribution to ketamine’s antidepressant action, are still unclear. To address this, we utilized the Wistar-Kyoto (WKY) model of endogenous stress susceptibility and depression. Consistent with the literature, WKY rats exhibited various depressive-like phenotypes compared to Wistar controls. In addition, we revealed that while in vivo hippocampal long-term depression (LTD) at the Schaffer collateral–CA1 (SC-CA1) synapse was not facilitated in the WKY strain, both early and late long-term potentiation (LTP) were significantly impaired. Importantly, both ketamine (5mg/kg, ip), as well as its metabolite (2R,6R)-HNK (5mg/kg, ip), acutely rescued the LTP deficit in WKYs at 3.5h following injection. Consistent with a sustained LTP-like effect,ketamine also increased SC-CA1 basal synaptic transmission at 24h in these rats. Importantly,ketamine, but not (2R,6R)-HNK, was found to have rapid and sustained antidepressant effects inWKY rats in the FST, leading to a dissociation between FST antidepressant-like activity anddorsal HPC synaptic plasticity. However, consistent with the observed SC-CA1 L-LTP deficitand corresponding effects of drug treatment, WKY rats exhibited impaired hippocampaldependent long-term spatial memory compared to Wistar controls (as measured by the novelobject location recognition test at a delay of 24h), which was effectively restored by bothketamine and (2R,6R)-HNK. We propose that, in the WKY rat model, restoring dorsal HPC LTPdoes not underlie ketamine’s antidepressant effects in FST, but may instead mediate reversal ofhippocampal-dependent cognitive deficits, which are also key features of clinical depression.ivThis work supports the theory that ketamine may reverse the stress-induced loss of connectivityin key neural circuits by engaging synaptic plasticity processes to “reset the system”, andhighlights the importance of deconstructing depression-like phenotypes and identifying theneural circuits that mediate them more precisely. Based on our results, the existing hypothesisthat ketamine’s antidepressant effects are solely due to the actions of its metabolite (2R,6R)-HNK is effectively challenged.

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The NMDA receptor is a glutamatergic ionotropic receptor key in mediating neuronalplasticity across virtually all synaptic circuits in the brain. An increasing list of neurologicaldisorders have implicated NMDA receptor hypofunction as an integral part of pathogenesis,necessitating the production of NMDA receptor potentiators as therapeutics. To date, most ofthese attempts have used increased co-agonism at the glycine binding site of NMDA receptors,but this strategy has been plagued by low specificity and efficacy. Specific allosteric modulationof NMDA receptors is an ideal solution, but until recently, no known drugs were capable ofdoing so. Building off previous work in our lab that discovered a novel family of compoundscapable of modulating NMDA receptor activity through its apical N-terminal domain, weidentified and characterized a drug candidate, Npam59, predicted to potentiate both GluN2A- and 2B-containing NMDA receptors. Npam59 was shown to potentiate NMDA currentsmediated by both subtypes with EC50 in the low-micromolar range. Npam59 also potentiated d-amphetamine-induced dopamine release in the ventral striatum in an NMDA receptor-dependent manner, but had no observable effect when administered alone. Finally, Npam59 potentiated d-amphetamine-induced hyperlocomotion in Sprague-Dawley rats. These results demonstrate that Npam59 can potentiate the function of NMDA receptors, including both GluN2A- and 2B-containing ones, suggesting its potential as a research tool and drug candidate for further development. Npam59 is the first known NMDA receptor allosteric potentiator with specificity for both GluN2A and GluN2B. Its characterization provides the foundation for therapeutic development and novel insights into the interaction of dopamine-glutamate signaling in the ventral striatum.

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The exploration and encoding of a novel environment is a fundamental learning process that occurs on a short time scale, and is a useful model for studying how the hippocampus encodes and represents complex and arbitrary associations, as is required for episodic memory. Novelty exploration has been demonstrated to promote long-term depression (LTD) induction in area CA1 of the hippocampus, but it is unclear what role this LTD plays as the novel space becomes familiarized and encoded in the hippocampus.In order to determine whether de novo LTD occurs during novelty exploration, we utilized multi-array electrophysiological recording in freely moving rats and demonstrated an AMPA receptor endocytosis-dependent LTD in CA1 in the absence of a paired LTD induction protocol. To determine what role this LTD played in forming spatial representations, we recorded the spiking activity of multiple single units in hippocampal CA1 using multi-tetrode electrophysiological recordings to observe the development of place field firing in a novel environment, and the effect of LTD blockade using an inhibitor of AMPAR endocytosis. Place fields formed in the presence of LTD blockade, however the maintenance of place field firing location between the novel environmental exposure and re-exposure one day later was impaired by inhibition of LTD.To investigate the dynamics of place field formation over the first several minutes of exposure, hippocampal neurons were recorded during exposure to a novel linear environment. While fields developed and stabilized over several minutes in control rats, we found that LTD blockade produced a rapid establishment of stable place fields after a single lap, suggesting the dynamics of field formation are altered with LTD blockade.To test the role of this LTD in novel spatial learning, the effect of LTD blockade on contextual fear was assessed using a modification of inhibitory avoidance training that separated the acquisition of contextual information from the pairing with an aversive stimulus. This demonstrated that LTD is required on the first exposure to a novel context.These results place the activity dependent weakening of synapses as a central process in the rapid acquisition of novel spatial information in the hippocampus.

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Accumulating evidence supports the premise that reducing α-synuclein levels may be an effective and specific therapy for Parkinson’s disease. To date, a clinically applicable α-synuclein reducing therapeutic strategy has yet to be developed. To remedy this, I developed a blood brain barrier and plasma membrane-permeable α-synuclein knockdown peptide that may have therapeutic potentials. By modifying a peptide-based method that was recently developed in our lab to rapidly and reversibly decrease the levels of endogenous proteins, I developed an α-synuclein knockdown peptide, Tat-βsyn-degron, and tested its specificity and efficacy in reducing the level of α-synuclein and its neuroprotective efficacy in well-characterized cellular and animal models of Parkinson’s disease. I found that the peptide effectively reduced the level of α-synuclein via proteasomal degradation both in cell cultures and in freely moving animals. More importantly, the peptide-induced α-synuclein reduction was associated with a significant decrease in parkinsonian toxin-induced neuronal damage and motor impairment in an animal model of Parkinson’s disease. These results suggest that targeted degradation of α-synuclein using the Tat-βsyn-degron peptide represents a novel, specific and effective therapeutic strategy for reversing a core cellular mechanism contributing to Parkinson’s disease.

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N-methyl-d-aspartate glutamate receptors (NMDARs) are fundamental to many normal brain functions such as cognition and memory; however, NMDAR over-activation can cause neuronal death as a result of excitotoxicity. Although the mechanisms underlying these paradoxical roles of NMDARs remain unclear, accumulating evidence from both in-vitro and in-vivo studies suggested that GluN1/GluN2B-NMDAR subtypes mediated signaling may contribute cell death, while GluN1/GluN2A-NMDARs signaling promote pro-survival outcomes. Employing an extensive drug discovery pipeline process, we identified and characterized a class of novel small molecules that specifically potentiate GluN1/GluN2A NMDARs in an allosteric manner. This new class of molecules is referred to as NMDAR positive allosteric modulators (Npams) with the Npam43 being the lead compound. Mutational analysis demonstrates that Npam43 binds to a novel binding site on the N-terminal domain (NTD) at the interface between the GluN1 and GluN2A subunits. Functional characterizations in in-vitro show that Npam43 activates cell survival signaling, increasing phosphorylated CREB levels, and thereby protects neurons against NMDAR-mediated excitotoxicity and NMDA-independent H₂O₂ oxidative stress. Moreover, Npam43 potentiates GluN1/GluN2A-mediated synaptic currents, and facilitates the induction of long-term potentiation (LTP) in hippocampal slices acutely prepared from mouse brain. Using a rat focal ischemia model of stroke in-vivo, we show that systemic administration of Npam43 not only modulates GluN1/GluN2A containing NMDARs but also substantially reduces neuronal damage and improves behavioral outcomes. Together, our study not only develops a novel class of Npams for GluN1/GluN2A NMDARs, but also demonstrates their therapeutic potential as novel neuroprotectants for stroke. In addition, the present work also provides strong evidence supporting a critical role of GluN1/GluN2A subtype of NMDARs in promoting cell survival.

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The A type γ-aminobutyric acid receptor (GABAAR) mediates major inhibition to counteract glutamate receptor-mediated excitation in the Central Nervous System (CNS). However, work in this dissertation identified a novel glutamate-binding site at the α+β- interface of the GABAAR. Activation of this glutamate binding site by glutamate and analogues can potentiate both the synaptic GABAAR-mediated phasic responses and the extrasynaptic GABAAR-mediated tonic inhibition. Using systematic mutagenesis analysis, we identified a conserved group of charged amino acid residues including α1K104, α1K155, α1E137 and β2E181, that form this glutamate binding site at the extracellular domain of the GABAAR. Spatial and electrostatic accessibility are both crucial for glutamate binding on this site. Furthermore, through in-silicon and electrophysiological screening, we identified that ampicillin, an antibiotic, and BRC640 as novel compounds that can target this newly identified glutamate-binding site, leading to an enhancement of the GABAAR function. Comparing with traditional benzodiazepine drugs, these two compounds were able to regulate both synaptic and extrasynaptic GABAARs. In the cerebellum, depolarization of the Purkinje cells induces both dendritic glutamate release and a rebound potentiation of GABA responsiveness. Application of ampicillin occluded the early phase of rebound potentiation, presumably by saturating the glutamate-binding site on the GABAARs and preventing the further potentiation induced by dendritically released glutamate. Taken together, our present study demonstrated a novel glutamate-binding site on the GABAAR that might lead to future development of novel GABAAR-based therapeutics. This type of excitation/inhibition crosstalk may play an essential role in Purkinje cell inhibitory plasticity.

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Huntington’s disease (HD) is an inherited neurodegenerative disease with progressive striatal loss. No treatments exist, though availability of predictive testing offers possibility for early intervention. Peptides represent an exciting way to interact with molecular signaling. My thesis investigates three potential early targets for preventative HD therapies: NMDAR-mediated PTEN nuclear translocation, caspase-6 activation, and peptide-mediated mutant huntingtin (mHTT) knockdown. Excitotoxicity via N-methyl-D-aspartate receptor (NMDAR) over-activation has a role in HD pathogenesis. We recently demonstrated that nuclear translocation of phosphatase and tensin homolog deleted on chromosome ten (PTEN) is a critical step in NMDAR-mediated excitotoxicity, and blocking PTEN nuclear translocation with peptide Tat-K13 prevents excitotoxic neuronal death. Given the role of NMDAR-mediated excitoxicity in HD, PTEN nuclear translocation may have a role in excitotoxic neuron death in HD. Here, PTEN nuclear translocation was associated with NMDAR-mediated death in cultured HD neurons. I also detected small increases in nuclear PTEN in HD transgenic mouse brains vs control. Interestingly, Tat-K13 effectively blocked PTEN translocation and prevented excitotoxicity in cortex/hippocampus, but not striatum, in vitro and in vivo, suggesting differential mechanisms of PTEN nuclear translocation. Caspase activation downstream of NMDARs may be critically involved in excitotoxicity. Caspase-6 (casp6) particularly, has roles in pathogenesis of HD and other conditions. Using NMDA-induced excitotoxicity in cultured neurons, I demonstrated early increase in caspase profiles via mRNA, protein and activity. Casp6 is elevated and activated first, followed by caspase-8 and caspase-3. Casp6 substrate huntingtin, and novel casp6 substrates STK3 and DAXX, are cleaved in similar temporal patterns post-NMDA, pointing to casp6 is an initiator caspase in NMDA-mediated apoptotic cascades and a potential therapeutic target. Many studies suggest that reducing mHTT protein may be an effective HD therapy. Our lab recently developed a technique for targeted protein degradation using peptides that signal proteins for lysosome- or proteasome-mediated degradation. Utilizing mHTT-binding domains paired with degradation sequences, I designed and tested mHTT-degradation peptides. In cultured neurons from HD transgenic mice, mHTT-degradation peptides led to robust mHTT knockdown, at least in part due to degradation. mHTT degradation peptides were ineffective in vivo, but pointed to potential for development of peptide-mediated mHTT-lowering therapies.

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Rapid and reversible methods for altering the function of endogenous proteins are not only indispensable tools for probing complex biological systems, but may potentially drive the development of new therapeutics for the treatment of human diseases. Genetic approaches have provided insights into protein function, but are limited in speed, reversibility and spatiotemporal control. To overcome these limitations, we have developed a peptide-based method to degrade a given endogenous protein at the post-translational level by harnessing chaperone-mediated autophagy, a major intracellular protein degradation pathway mediated by the lysosome. This thesis presents the design and validation of SNIPER (Selective Native Protein ERadication) for use in cultured cells as well as in intact animals. Specifically, we demonstrate the specificity, efficacy and generalizability of SNIPER by showing efficient knockdown of various proteins, including death-associated protein kinase 1 (160 kDa), scaffolding protein PSD-95 (95 kDa) and α-synuclein (19 kDa), with their respective SNIPER peptides in cell lines and rat neuronal cultures. Moreover, we examine the characteristics of SNIPER-mediated protein knockdown and found that the level of protein knockdown is dose-, time- and lysosome-dependent. Furthermore, we demonstrate that SNIPER efficiently knocked down a dephosphorylated subpopulation of a given protein while sparing its phosphorylated form, attesting to the specificity of the method. Finally, using a rat model of focal ischemic stroke, we show that a single intravenous injection of a SNIPER peptide efficiently knocked down a death-promoting protein in the brains of freely moving rats and protected the rat brain from ischemic injury. Taken together, SNIPER is a robust and convenient research tool for manipulating the levels of endogenous proteins in situ, and may also lead to the development of novel protein knockdown–based therapeutics for treating human diseases.

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Ischemic stroke is a major cause of death and disability in developed countries, and a major economic burden in the world. The mechanisms mediating stroke damage are likely multifactorial, with N- methyl-D-aspartate receptor (NMDAR) mediated excitotoxicity being an important factor. But NMDAR blockers are not clinically feasible due to their side effects and short therapeutic window. This doctoral dissertation discusses an ongoing effort to develop novel anti-excitotoxic therapeutics that can overcome these limitations. Our strategies are largely based on the premises that NR2A- subunit containing NMDARs (NR2ARs) are pro-survival whereas NR2B-subunit containing NMDARs (NR2BRs) are pro-death, and NR2BRs mediate neuronal death in large by activating a set of neuronal death signaling proteins.Specific Findings: 1, we investigated whether stimulation of NR2ARs by the NMDAR co- agonist glycine rather than blocking NR2BRs would confer a wider therapeutic time window in an in vivo rat model of focal and global ischemia. Because we stimulated neuronal survival rather than blocked neuronal death, our therapeutic remained efficacious up to 6 h post-ictus - a time point when many known death signaling proteins downstream of NR2BRs were already activated. 2, we studied the death-signaling pathway of SREBP1 (sterol response element binding protien 1), a transcription factor downstream of NR2BR, in an in vivo rat model of focal ischemia. In the ischemic brain, we found SREBP1 activation and nuclear translocation due to the ubiquitination and degradation of its inhibitory partner Insig1 (protein encoded by insulin signaling gene1). Notably, the new therapeutic peptide Indip (Insig1 degradation inhibiting peptide) prevented neuronal death when administered 1 h pre- and 2 h post-ictus. Because we targeted a death-signaling protein rather than all signaling proteins downstream of NMDARs, our treatment would have fewer side effects than NMDAR blockers. 3, we developed a novel method to selectively knockdown death-signaling proteins downstream of NR2BRs by means of ubiquitin-tagged peptides. Because interference peptides in the past were limited to disrupting protein-protein interactions and post-translational modifications, this opened a new avenue to develop therapeutics for excitotoxicity following stroke.

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Transplantation of neural progenitor cells (NPC) constitutes a putative therapeutic maneuver for use in treatment of neurodegenerative diseases. At present, effects of NPC transplantation in the Alzheimer’s disease (AD) brain are largely unknown and a primary objective of this work is to demonstrate possible efficacy of NPC administration in an AD animal model. The benefits of transplantation could involve a spectrum of effects including replacement of endogenous neurons, conferring neuroprotection with enhancement of neurotrophic factors, and diminishing levels of neurotoxic agents. Additionally, since chronic inflammation is a characteristic property of the AD brain, I considered NPC transplantation could have a particular utility in inhibiting ongoing inflammatory reactivity. Accordingly, intra-hippocampal transplantation of NPC has been examined for efficacy in attenuating inflammatory responses and conferring neuroprotection in the hippocampus. These findings indicate efficacy for NPC transplantation with effects consistent with cellular actions to attenuate inflammatory reactivity. Synaptic plasticity, such as long-term potentiation (LTP), is thought to play a critical role in modification of neuronal circuitry in learning and memory, but the role in neurogenesis is not well known. A critical aspect of my study was to examine potential roles of N-methyl-D-aspartate receptor (NMDAR)-dependent LTP in promoting neurogenesis by facilitating proliferation/survival and neuronal differentiation of endogenous NPCs in the dentate gyrus (DG) and exogenously transplanted neural stem cells (NSCs) in the CA1. I found that LTP induction significantly facilitates proliferation/survival and neuronal differentiation of endogenous NPCs and exogenously transplanted NSCs in the hippocampus. These effects were eliminated by a NMDAR competitive antagonist, CPP. Accordingly, chemical LTP stimulation reproduced enhanced proliferation/survival and neuronal differentiation of NSCs when co-cultured with hippocampal neurons. These effects were eliminated by a NMDAR competitive antagonist, D-APV and inhibited by the tyrosine kinase inhibitor, K252a. ELISA and biotinylation results revealed that NMDAR-mediated LTP facilitates the release of a neurotrophic factor, BDNF. The conditioned media from cLTP-induced hippocampal neurons were sufficient to activate the BDNF receptor, TrkB. Overall, my results suggest that NMDAR-dependent LTP plays a critical role in neurogenesis and may contribute to the utility of NSC transplantation as an effective cell therapy for a variety of neurodegenerative diseases.

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Stroke is a leading cause of death and disability in developed countries. About 87% of stroke cases results from blood vessel(s) occlusion in the brain, leading toneuronal death and neurological impairment (Lloyd-Jones et al., 2010). The ischemic progression likely involves multiple events, and increasing evidence showed that the ischemic neuronal death is caused, at least in part, by over-activation of N-methyl-Daspartate subtype glutamate receptors (NMDARs) (Rothman, 1983, Rothman, 1984, Simon et al., 1984). A large number of pre-clinical studies showed that NMDAR antagonists have strong neuroprotective effects against ischemic insults (Park et al.,1988b, Bullock et al., 1990). However, none of the following human clinical trials have succeeded yet (Muir, 2006). Several explanations have been suggested (Gladstone et al., 2002), including the following two major reasons that may beovercome by the novel therapeutics proposed here. First, the stroke patient inclusion periods (>6 hours) used by most clinical trials are beyond the therapeutic time window (
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Epilepsy is a common neurological disorder with a strong hereditary component. A mutation in the α1 subunit (A322D) of the GABA-A receptor is responsible for juvenile myoclonic epilepsy in a large Canadian family. Previous work has identified that this mutation affects the function of GABA-A receptors, expressed in HEK293 cells. Here I have examined the underlying mechanisms of this dysfunction and shown that the mutation reduces the cell surface expression of the GABA-A receptor, promotes association with the endoplasmic reticulum chaperone calnexin, enhances degradation and accelerates the degradation rate of the subunits approximately 2.5 fold. I have also found that the mutation causes the receptor to be degraded by a lysosomal-dependent process. Furthermore, I find that the mutation results in receptors that are inserted into the plasma membrane but are more rapidly endocytosed by a dynamin and caveolin-dependent mechanism. These results suggest that the mutant subunit can form trafficking-competent receptors that have a shorter lifetime on the plasma membrane. Collectively, my results strongly implicate defects in both the biogenesis and trafficking of the GABA-A receptors, as part of the mechanistic basis for the epileptic phenotype observed.

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The α-amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA) subtype of glutamate receptors mediates the fast excitatory synaptic transmission in the mammalian brain. Increasing evidence suggests that rapid movement of AMPARs into and out of synapses is the major mechanism for changes in synaptic strength, which is thought to underlie learning and memory. Although the bidirectional hippocampal synaptic plasticity, particularly long-term potentiation (LTP) and long-term depression (LTD), has been regarded as a candidate mechanism for long-term spatial memory (LSM), the precise contribution of LTP and LTD to LSM remains poorly understood. Using antagonists that target NMDARs carrying specific NR2 subunits, we found that LTP and LTD in freely moving rats can be selectively abolished by NR2A and NR2B antagonists, respectively. Using the Morris water maze, we found that only the NR2B antagonist disturbed the consolidation of LSM. In addition, a similar LSM deficit was observed when the expression of LTD was prevented by inhibiting regulated AMPAR endocytosis. Thus, these findings support a functional requirement of hippocampal LTD in the consolidation of LSM. Blocking LTP by NR2A-preferential antagonist had no effects on LSM. However, another structurally and mechanistically different LTP-specific inhibitor is still lacking. Since the expression of LTP is thought to be mediated by the facilitated exocytosis of AMPARs, we therefore attempted to identify novel AMPAR binding partner(s) using co-immunoprecipitatoin with anti-GluR1 or anti-GluR2 antibody followed by mass spectrometric analysis. We found that p97, also called valosin-containing protein (VCP), specifically interacts with and modulates trafficking of homomeric GluR1 receptors. Using various truncated and deleted constructs of GluR1, we found that p97 interacts with N-terminal of GluR1, but not GluR2, resulting in facilitated formation of homomeric GluR1 receptors by decreasing GluR1/GluR2 heteromeric receptors formation. Moreover, we found that under basal conditions, p97 retained homomeric GluR1 AMPARs in the intracellular pool, but immediately after the induction of LTP, it disassociated from GluR1 and hence, allowed these homomeric AMPARs insert into the postsynaptic membrane, thereby contributing to LTP expression. Thus, our results highlight a previously unknown molecular mechanism by which p97 regulates formation and trafficking of homomeric GluR1 AMPARs, and thereby plays a critical role in LTP expression.

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Basal level neuronal excitability in the mammalian brain is fundamental for physiological brain functions. It is primarily maintained by a fine balance between two types of synaptic transmission: the excitatory transmission mediated by glutamate-gated ion channels including α-amino-3-hydroxyl-5-methyl-4-isoxazole-propionate receptors (AMPARs) and N-methyl-D-aspartic acid receptors (NMDARs), and the inhibitory transmission mediated by chloride channels including γ-Aminobutyric acid receptors (GABAARs) found in the brain and glycine receptors (GlyRs) orginating in the spinal cord and brainstem. Therefore, understanding mechanisms by which these ligand-gated ion channels (LIGCs) are regulated is critical for our understanding of both brain functions and dysfunctions, and is the major focus of this thesis. In particular, this research project investigates: 1) how rapid alteration of AMPAR trafficking results in changes in strength of synaptic transmission, with a particular emphasis on its contribution to amygdala long-term potentiation (LTP) and depression (LTD), the two most well-characterized forms of synaptic plasticity, and 2) how excitatory transmitter glutamate modulates functions of the inhibitory GABAA and Gly receptors. In chaper 2 and 3, we show in lateral amygdale (LA) slices that the induction of LTP requires NR2A-containing NMDAR activation, while the expression of LTP requires AMPARs insertion (sensitive to TeTx or GluR1-derived peptide). On the contrary, the induction of LTD involves activation of NR2B-containing NMDARs and the expression of LTD involves AMPARs endocytosis (sensitive to GluR2-3Y peptide). The inhibitory receptors GABAARs and GlyRs are respectively activated by binding with their respective transmitters, GABA and glycine. In chapter 4 and 5, we show novel and unexpected findings where glutamate potentiates currents mediated by either GABAARs or GlyRs in neurons and in HEK cells over-expressing recombinant GABAARs and GlyRs. This potentiation was not dependent on activation of any known ionotropic or metabotropic glutamate receptors. Thus, our results strongly suggest that glutamate can allosterically potentiate the function of GABAARs and GlyRs, thereby blurring the traditional distinction between excitatory and inhibitory transmitters. Such a rapid homeostatic regulatory mechanism may have a significant role in tuning functional balance between synaptic excitation and inhibition in the central nervous system (CNS).

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At post synaptic sites, the neuroligin (NL) family of proteins is thought to play an important role in synapse maturation, and regulation of excitatory and inhibitory synapses. Being selectively enriched at either excitatory (NL1,3) or inhibitory (NL2) synapses, NL’s have been shown to regulate the ratio of excitation to inhibition (E/I ratio), a process critical for normal brain development. In addition, NLs have been linked to neurodevelopmental disorders through genetic studies. To advance our understanding of synaptic regulation by NLs, and their potential role in synaptic dysfunciton in neurodevelopmental disorders, we have developed strains of transgenic mice which overexpress either HA tagged-NL1, or -NL2 under control of the Thy1 promoter. Detailed behavioural analysis of TgNL2 mice revealed anxiety, stereotyped jumping behaviour, and impairments in social approach and reciprocal social interactions. These animals also displayed fronto-parietal seizure activity as shown by chronic in vivo EEG recording. Synapse analysis in TgNL2 frontal cortex revealed changes in the number and morphology of synapses compared to wildtype littermates. A small change in NL2 expression results in enlarged synaptic contact size and vesicle reserve pool and an overall reduction in the E/I ratio. In addition, the frequency of miniature inhibitory synaptic currents was also found to be increased in the frontal cortex of TgNL2 mice. Behavioural assessment of TgNL1 mice revealed deficits in memory acquisition and retrieval in water maze paradigms. Golgi and electron microscopy analysis revealed changes in synapse morphology indicative of increased maturation of excitatory synapses. In parallel, electrophysiological examination indicated a shift in the E/I ratio towards increased excitation. Further experiments revealed impairment in the induction of long term potentiation.These data demonstrate that altered expression of members of the NL family in vivo leads to altered synapse number and morphology, which potentially underlies the profound behavioural changes. We also observed a predominant effect of NL2 expression on inhibitory synapses, with NL1 primarily influencing excitatory synapses, supporting the idea that NL’s may act to regulate the E/I ratio. In addition this data may provide insight into the pathology and symptoms of neurodevelopmental disorders such as autism thought be be caused by synaptic abnormalities.

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