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Graduate Student Supervision
Doctoral Student Supervision (Jan 2008 - Nov 2020)
No abstract available.
Ocular dominance plasticity (ODP) is a well-characterized example of experience-dependentplasticity. Multiple molecular mechanisms have been implicated in the studying of ODP. Wehave previously demonstrated a temporal correlation between long-term depression (LTD) andocular dominance plasticity. It has also been shown that blockade of LTD abolishes oculardominance shift during the critical period, suggesting that LTD is necessary for oculardominance plasticity. Here I go on to explore if LTD is sufficient for ocular dominance plasticityby augmenting it in adulthood. By administering D-serine, an NMDAR co-agonist thatselectively enhances LTD in adult visual cortex, I am able to enhance ocular dominanceplasticity in adulthood, as evidenced by data collected from single-unit recordings. D-serineoperates via an LTD-like mechanism as its effect could be abolished by GluR23Y peptide, aselective LTD blocker. I therefore argue that LTD plays a key regulatory role in both juvenileand adult ocular dominance plasticity. In addition, D-serine helps facilitate recovery of visualinput in long-term monocularly deprived adult mice, suggestive of therapeutic potentials. In addition, I have examined the functional consequences of monocular deprivation onthe rest of primary visual cortex (V1) and cerebral cortex. This is achieved with help of in vivo imaging of intrinsic optic signals and calcium imaging. Within the visual cortex, monoculardeprivation decreases the correlation between the contralateral monocular zone with the rest ofV1. I have also observed transient changes in global functional connectivity correlating with theduration of lid suture during the critical period, in keeping with cross-modal plasticity. However,this change in functional connectivity is not observed in adulthood, suggesting a sensory periodfor cross-modal connectivity.
The visual cortex of the brain is one of the fundamental preparations to study critical periods andactivity dependent changes in the brain. During development, when sensory input from one eyeis prevented, visual acuity and brain connectivity is lost in favour of inputs from the active eye.Because of the brain’s complexity, it is difficult to perform thorough analyses of synapticmechanisms that exist during development. Therefore, the development of simpler in vitromodels would be advantageous. In our studies, we used a 3-compartment microfluidic deviceand created a new model for dual input in vitro activity dependent synaptic plasticity.Microfluidics offered the advantage of being able to physically and chemically isolate neurons indistinct environments.In chapter 2, we optimized previously developed microfluidic devices for use in our cell cultureexperiments and demonstrated that their application can create a dual input activity dependentsystem. Using a 3-compartment microfluidic device, the activity of one neuronal group wasreduced by application of tetrodotoxin or the GABA agonist, muscimol. Treatment caused theformation of a greater number of synaptic contacts between the target ‘postsynaptic’ neurons andthe ‘presynaptic’ inputs at normal working activity levels compared to the opposing‘presynaptic’ inputs with reduced activity.In chapter 3, we established that ‘critical periods’ exist in our in vitro model by varying the agesat which we reduced neuronal input activity. Muscimol treatment had an earlier time window toinduce activity dependent synaptic changes compared to inhibition by tetrodotoxin. By thefourth week in culture, neither treatment induced any synaptic difference between inputs.In chapter 4, we examined the mechanisms involved in our model. We manipulated NMDARactivity, CamKII activity, or GluR2 internalization postynaptically under the same conditionsthat we previously established. In our model, both treatments were NMDAR activity dependentwhile the requirement for CamKII activity and GluR2 internalization was dependent on theapplication of either muscimol or tetrodotoxin respectively.Taken together, we showed the ability to create a new in vitro model for activity dependentsynaptic plasticity and that even in a simple system multiple mechanisms can exist.
Studying the mechanisms underlying glutamate excitotoxicity and inflammatory responses provides hints to the pathology of neurological diseases such as epilepsy. In this dissertation I investigated the expression and function of Krüppel-like factor 4 (KLF4) in glutamate excitotoxicity. I also studied the distribution and the role of progranulin (PGRN) in inflammatory stimulation, in epilepsy and in astrocytes subjected to glutamate excitotoxicity. First, I studied the role of KLF4 and found that NMDA induced KLF4 expression in cultured neurons and in brain slices. Overexpression of KLF4 upregulated cyclin D1 and downregulated p21Waf1/Cip1, suggesting the neuron’s progression into cell cycle. KLF4 expression also induced the cleavage of caspase-3 under conditions of a subtoxic dose of NMDA. Thus our work suggests that KLF4 might play a role in NMDA-induced apoptosis. Second, I studied the function of PGRN and observed that PGRN was enhanced in activated microglia after pilocarpine-induced epilepsy. In mixed cultures, lipopolysaccharide (LPS) also induced PGRN expression. Recombinant PGRN protein promoted microglial activation in the dentate gyrus after epilepsy and in purified microglial cell culture. PGRN was also required for LPS-induced microglial migration. Our work suggests that PGRN may contribute to microglial activation after epileptic and inflammatory insults.Third, I performed a preliminary study on the role of PGRN in purified culture of astrocytes. I found that our cultured astrocytes express PGRN, and PGRN was required for glutamate-induced lactate release. PGRN was also involved in glutamate-induced glucose uptake and participated in the regulation of monocarboxylate transporter 1 (MCT1) expression in excitotoxic conditions. Our findings suggest that PGRN may be involved in glutamate-evoked increase of glycolysis in cultured astrocytes. In conclusion, our findings provide insights into factors involved in glutamate excitotoxicity, inflammation, and epilepsy.
Protein palmitoylation is an important post-translational lipid modification. While hundreds of palmitoyl proteins have been identified in neurons, little is known about how palmitoylation regulates these neuronal proteins and how it contributes to neuronal development and function. A special group of palmitoyl proteins, 23 mammalian zinc-finger DHHC-type containing (zD) proteins are potent palmitoyl acyltransferases (PATs) that catalyze protein palmitoylation. However, the physiopathological roles of these PATs in brain function are largely elusive. AMPA receptor (AMPAR) subunit GluR1 and GluR2 are palmitoyl proteins. In this thesis, I have found that GluR1 and GluR2 show different palmitoylation properties in neurons. Palmitoylation regulate AMPAR stability in a subunit-selective manner in response to synaptic stimulations. In addition, c-jun N-terminal kinase 3 (JNK3), but not other JNK isoforms, has been identified in this thesis as a novel palmitoyl protein. Without palmitoylation, JNK3 is associated more strongly with the cytoskeleton and promotes axonal branching. This suggests a potential role of palmitoylation in modulating axonal development via isoform-specific regulation of JNK3. I have further revealed that zD17 mediates neuronal responses in acute ischemic brain injury via a mechanism independent of its PAT activity. ZD17 directly interacts with JNK to form a signaling module for JNK activation. Pathological stressors induce the zD17-JNK interaction which promotes downstream neuronal cell death signals. I have developed novel peptides targeting the JNK-interacting motif on zD17 to selectively block the enhancement of the zD17-JNK interaction and the activation of JNK isoforms 2 and 3. Application of these peptides successfully blocks JNK activation and neuronal cell death pathways, protects cultured neurons from excitotoxicity, and dramatically reduces brain damage and behavioural deficits in a rat model of focal ischemic stroke. These findings indicate PAT zD17 as a key player in ischemic stroke, and suggest the potential therapeutic value of targeting palmitoyl proteins for neuroprotection.
Master's Student Supervision (2010 - 2018)
Aberrant cellular processing and targeting of TDP-43 has been implicated in a wide variety of neurological diseases such as frontotemporal lobar degeneration with ubiquitinated inclusions (FTLD-U) and amyotrophic lateral sclerosis (ALS). These diseases are characterized by the sequestration of TDP-43 into the cytoplasm of afflicted neurons, leading to the formation of ubiquitinated, cytoplasmic inclusions and an increased susceptibility to cellular insults. While the underlying causes of TDP-43 proteinopathy are unknown, we are investigating the role of a protein family known as the karyopherins in the nuclear targeting of TDP-43. Using co-immunoprecipitation in SH-SY5Y cells we determined that a major binding partner of TDP-43 is karyopherin-alpha 2 (KPNA2). Next, utilizing a high-density peptide array comprised of overlapping peptide sequences derived from TDP-43 we identified six regions where KPNA2 may directly interact with TDP-43. From these regions we developed six small, cell-penetrating peptides designed to specifically inhibit the interaction between KPNA2 and TDP-43. Through the use of these synthetic peptides, we were able to interfere with the binding of KPNA2 to TDP-43 in vitro. We found that the disruption of this specific protein-protein interaction was not sufficient to induce TDP-43 cytoplasmic sequestration, as determined by co-immunoprecipitation and subcellular fractionation assays. As our research focused primarily on healthy SH-SY5Y cells, future studies will focus on investigating the effects of peptide-mediated TDP-43 nuclear import impairment paired alongside oxidative insult. We will also investigate whether compensatory mechanisms within SH-SY5Y cells are responsible for the nuclear localization of TDP-43 in the absence of KPNA2-mediated nuclear import.
Excitotoxicity is caused by prolonged stimulation of N-methyl-D-aspartate type glutamate receptors (NMDARs) resulting in internalization of α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid type glutamate receptors (AMPARs) and long-term depression (LTD) of post-synaptic response, and this process has been causally linked to neuronal cell death. Indeed, NMDAR/AMPAR excitotoxicity is believed to underlie neurodegenerative diseases including Alzheimer’s disease, Parkinson’s disease and ischaemic brain injury. Neuronal activity regulated pentraxin (NARP) is a secreted immediate early gene product that binds to and clusters AMPAR subunits GluR1-4 in response to normal and pathological synaptic activity. To test whether NARP could potentiate excitotoxic neuronal death by clustering AMPARs at cortical synapses, we used peptide arrays to develop four peptides that mimic sites on GluR1 that might bind NARP. Three of the four peptides correspond to sites on the N-terminal domain of GluR1, a region previously implicated in facilitating NARP-mediated GluR1 clustering at synapses. We show that NARP is up-regulated 4-8 hours after excitotoxic NMDA treatment of primary cortical neurons. We found that a mixture of all four peptides inhibited NMDA-induced GluR1 internalization and was neuroprotective in a dose-dependent manner. Also, three of peptides were individually neuroprotective. We conclude that the peptides inhibit NARP’s ability to form synaptic GluR1 clusters which may be required for coordinated and sustained GluR1 internalization 4-8 hours after NMDA stimulation. This is an essential step in NMDA-induced cell death since blocking it is neuroprotective. These peptides suggest new approaches to treatment of neurodegenerative diseases caused by excitotoxicity.
Bisphenol A (BPA) is an ubiquitous environmental xenoestrogen excreted in the urine of 95-99\% of humans studied. In this investigation, we examine the effects of 1mM to 10fM concentrations of BPA on the viability of rat cortical neurons under acute (5h) and chronic (DIV3-9, DIV3-15) exposure conditions. Post-exposure, we also challenged the cultures with an oxidative or excitotoxic stress to determine whether BPA conferred neuroprotection, susceptibility or neither to the challenged cultures. Finally, we studied the effects of different concentrations of BPA on the activation of SREBP~1, a transcription factor that regulates many genes with important effects on neuron function. We found that 1mM and 500uM BPA are neurotoxic under chronic exposure, but only 1mM BPA decreased cell viability after 5h. Acute and chronic exposure to BPA conferred neither neuroprotection nor susceptibility to oxidative or excitotoxic stress in the neuron cultures. Five hours exposure to 10pM and 1pM BPA increased SREBP~1 activation two-fold. In DIV9 cultures, 10pM BPA stimulated a maximal three-fold SREBP~1 activation at 8h post exposure, while at DIV15, 10pM BPA stimulated a maximal two-fold SREBP~1 activation at 5h post exposure. High, physiologically irrelevant concentrations of BPA induce neuronal cell death, and while sublethal concentrations do not predispose cultures to oxidative or excitotoxic stress, they also do not confer neuroprotection as a true estrogen, estradiol, would. Sublethal concentrations of BPA activate SREBP~1 in a hormetic manner, resulting in a non-monotonic dose response curve.