Lynn Raymond

Preclinical drug discovery relies on the identification of appropriate target compounds and usage of effective animal model screening tests. In this work excitability across systems as well as spatial and temporal scales were evaluated to understand long QT syndrome (LQTS) therapeutic targets and develop a behavioral assay that could sensitively characterize motor phenotype onset in a Huntington Disease (HD) mouse model. Case I: The pairing of the tetrameric voltage-gated potassium channel, KCNQ1, with an accessory β-subunit, KCNE1, gives rise to the slow delayed cardiac rectifier current (IKs), known to play an important role in the physiological shortening of the cardiac action potential. With loss-of-function mutations in both subunits found to be associated with LQTS, enhancing IKs has been identified as a therapeutic approach. Here, the NSAID, mefenamic acid, was found to dose- and rate-dependently activate IKs. More KCNE1-saturated IKs channel complexes had a greater response to mefenamic acid treatment. KCNE1 residue K41 was identified as critical for mefenamic acid action, suggesting a potential binding site. Case II: HD is a dominantly inherited neurodegenerative disease with characteristic motor symptoms. Animal models with increasingly better face and construct validity have been developed to understand HD pathophysiology. Although HD gross motor defects have been extensively characterized, less is known about forelimb motor deficits. Using a high-throughput alternating reward/non-reward water-reaching task, HD forelimb movement defects and associated aberrant cortical activity were examined. HD heterozygous-zQ175 mice displayed an event sequence defect at ~5.5 months with progressive forelimb deficits starting at ~6 months. Cortical activity associated with water-reaching increased over time in HD but not wildtype mice. Gross motor defects characterized using the tapered beam and rotarod tasks, as well as post-hoc striatal immunostaining, confirmed HD pathology at ~8 months. Overall, at the nanoscale level, a biophysical and pharmacological characterization of mefenamic acid’s effect on IKs highlighted a binding site and the potential of the NSAID to act as a precursor compound for LQTS therapeutic development. At the mesoscale level, a water-reaching task was developed and used to characterize HD phenotype demonstrating the potential of the behavioral task to examine therapeutic efficacy and intervention windows.

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Huntington disease (HD) is a progressive neurodegenerative disorder with no disease-modifying treatments. Patients experience motor, cognitive, and psychiatric disturbances, and the dorsal striatum is the main target of neurodegeneration. Mouse models of HD show altered striatal synaptic signaling in vitro, including changes to cortico-striatal glutamate signaling. Previous studies demonstrate altered glutamate receptor distribution and signaling at cortico-striatal synapses in HD mice, and some studies suggest that glutamate release may be altered, but the presynaptic mechanisms underlying aberrant glutamate release in HD are unknown. Additionally, although changes to striatal signaling have been studied extensively in vitro, it is unclear how these changes correlate with behavioural impairments in vivo. Here, we utilize optogenetic sensors to explore cortico-striatal signaling in HD mice. In acute brain slice, we used iGluSnFR, a modified green fluorescent protein reporter for real-time imaging of glutamate dynamics, to study the presynaptic modulation of glutamate release. We validated iGluSnFR as a valuable tool to accurately measure short- and long-term changes in glutamate release caused by changes to extracellular calcium levels, modulation by presynaptic receptors, and plasticity-inducing stimulation protocols. We confirmed a deficit in HFS-LTD and found changes to D2-receptor-mediated inhibition of glutamate release in YAC128 HD mice. In vivo, we used GCaMP7f, a calcium-sensing fluorescent reporter, to image striatal activity during motor learning on the accelerating rotarod and open field exploration. Mice showed increased neuronal activity on the rotarod, which diminished by late stages of learning. 2–3-month-old YAC128 mice did not show a deficit in latency to fall, but did display significant deficits in paw kinematics, including increased frequency of paw slips. These mice also exhibited aberrant striatal activity during rotarod performance, including a weaker correlation between striatal activity and behaviour. 6–7-month-old YAC128 mice displayed severe rotarod deficits, and elevated striatal activity while on the rotarod. In the open field, YAC128 mice showed increased neuronal activity at rest. Overall, this thesis presents new insights into the mechanisms underlying cortico-striatal glutamate transmission, and alterations to striatal activity associated with behavioural impairments, in mouse models of HD.

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Rodent genetic models are a critical tool for understanding the pathogenesis of neurological disorders, and for evaluating the efficacy and safety of novel therapeutics. Unfortunately, behavioural studies of rodents can be vulnerable to false positives or negatives, as many behaviours have substantial inter-animal variability and are sensitive to environmental stressors (which in turn vary between facilities and experimenters). Developing tools to decrease the impact of these stressors and increase the throughput of pre-clinical research is an important area of focus to help deal with this problem. To this end, my thesis project is focused on the development and testing of two automated, self-directed behavioural testing systems that are accessible to mice from their home-cage and can be accessed at will, 24 hours per day. Animals are individually identified through radio-frequency identification (RFID) tagging, allowing for mice to be group-housed and tested alongside their littermates. This design eliminates the need for the animal to be exposed to novel environments, and minimizes experimenter interaction, significantly reducing two of the largest stressors associated with animal behaviour. These two systems can be used, respectively, to assess motor phenotypes via a forelimb lever-positioning task (PiPaw), and to treat animals with drug through their drinking water (PiDose). I applied these home-cage tools to two mouse models of Huntington’s disease (HD), a genetic neurodegenerative disorder that causes debilitating motor dysfunction, in addition to cognitive and psychiatric symptoms. Using the PiPaw system, I found that young HD mice had impairments on a task that required them to hold a lever within a rewarded position range, but not when they had to make a short-duration pull to a defined target. Deficits in older HD mice were dependent on the specific genetic model, with the transgenic YAC128 model showing little to no impairment on the task but knock-in Q175-FDN mice showing substantial motor deficits. We also observed altered patterns of task engagement and changes in the circadian activity patterns of both HD mouse models. These two home-cage systems are open-source, low-cost and built with easily obtainable parts, and should prove useful for experimenters performing basic and translational rodent research.

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Huntington disease (HD) is an inherited neurodegenerative disorder caused by expansion of the CAG repeat region of the huntingtin (Htt) gene. Early in the disease neuronal degeneration is preceded by synaptic dysfunction and changes in cellular signaling. This includes reduced BDNF signaling and altered calcium homeostasis, which could interfere with the group of processes known as homeostatic plasticity which alter neuronal connectivity and excitability to maintain neuronal network stability. We compared neurons cultured from normal (wild-type) mice with those from mice expressing the human genomic DNA for mutant huntingtin (YAC128). We focused on synaptic scaling, the process whereby the strength of synapses onto a neuron changes based on its level of activity. This is typically measured using the amplitude and frequency of miniature excitatory postsynaptic currents (mEPSCs), which represent the response to neurotransmitter release from individual synaptic vesicles. We attempted to induce scaling at excitatory glutamatergic synapses in striatal projection neurons (SPNs) in cortico-striatal co-cultures, and in cortical pyramidal neurons (CPNs) in cortical mono-cultures, by suppressing activity with tetrodotoxin (TTX) or disinhibiting activity with bicuculline (BIC) over 48 hours. This failed to induce homeostatic plasticity in either wild-type or YAC128 SPNs; however, TTX did induce an increase in synaptic AMPA receptor content and glutamatergic synapse density in wild-type (WT) CPNs, which was reflected in increased mEPSC amplitude and frequency. In CPNs from YAC128 HD mice this occurred only after pre-treatment with pridopidine – a drug previously tested in HD clinical trials – or the sigma-1 receptor (S1R) agonist 3-PPP. These data, combined with the results of manipulating culture medium BDNF concentration in WT CPN cultures, led us to conclude that impairment and restoration of homeostatic plasticity in YAC128 CPNs depends on changes to multiple signaling pathways modulated by S1R, including BDNF signaling. These results suggest that cortical homeostatic plasticity at glutamatergic cortical synapses is disrupted early in HD and may play a role in the disease’s early cognitive and psychiatric symptoms. They also indicate that S1R agonists can ameliorate this disruption, adding to the evidence that drugs of this class may be of use in treating the early symptoms of HD.

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Huntington’s disease (HD) is a genetic neurodegenerative disorder caused by expansion of a CAG repeat in exon 1 of the HTT gene, encoding an elongated poly-glutamine repeat in the N-terminal region of the protein huntingtin (mutant huntingtin; mHtt). The average age of onset is 38, and the disease is characterized by psychiatric disorders and cognitive deficits that, in general, gradually develop over 10 years before the overt onset of the disease phenotype – difficulties in movement control. In the past two decades, many studies have focused on cell death that is obvious in mid to late stage of the disease when the overt disease symptoms become irreversible, despite the fact that altered neuronal/synaptic functions may underlie the mood/cognitive disorders that precede a motor diagnosis.In order to uncover the potentially preventable and/or reversible changes in cortico-striatal (C-S) connections in pre- and early stages of HD, we first studied the C-S coculture platform that represents its in vivo counterparts in order to investigate the role of wild-type huntingtin (wtHtt) protein in cell-death and C-S synaptic malfunctions in HD. I found that coculture with low cortical-to-striatal neuronal plating ratio (1:3 plating ratio) is a closer replica of its in vivo origin with slight differences in membrane properties, but with a significant increase in extrasynaptic NMDA receptor portion and a decrease in cell-survival signaling compared with the control (1:1). On the other hand, we found that wtHtt provides neuroprotective effects to striatal, cortical and hippocampal neurons, in a phospho-CREB-independent way in the case of the latter two neuronal types. Finally, using the C-S coculture and acute brain slice to study C-S synapse development and functions, I found that mHtt impairs the connection not only via suppressing striatal dendritic tree development but also by altering excitatory presynaptic vesicle release and recovery of the glutamate pool.In summary, this work is a further proof of HD as synaptopathy, and is a foundation for future research of drug discovery for HD targeting synaptic malfunctions at the pre-symptomatic stage.

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Huntington disease (HD) is an inherited neurodegenerative disease lacking effective treatment, characterized by involuntary movements, psychiatric disorders, and cognitive symptoms. Pathology shows prominent degeneration of γ-aminobutyric acid (GABA)-ergic medium-sized spiny neurons (MSNs) of the striatum and certain cortical layers (Vonsattel and DiFiglia, 1998). HD is caused by a dominant mutation in the HD gene that leads to >35 glutamine repeats (polyQ) near the N-terminus of the protein huntingtin (htt) (The Huntington’s Disease Collaborative Research Group, 1993). Increasing evidence suggests that the N-methyl-D-aspartate (NMDA)-type glutamate receptor (NMDAR) plays a role in mediating death of MSNs observed in HD (Fan and Raymond, 2007). Previous results from our laboratory demonstrate that NMDAR-mediated current and toxicity are increased in MSNs from the Yeast Artificial Chromosome (YAC) transgenic mouse model expressing polyglutamine-expanded full-length human htt (Shehadeh et al., 2006; Zeron et al., 2002). However, the mechanism underlying altered function and enhanced toxicity of NMDAR in HD remains unknown. Previous studies have shown that membrane-associated guanylate kinases (MAGUKs), such as postsynaptic density protein 95 (PSD-95) modulate NMDAR surface expression and excitotoxicity in rat hippocampal and cortical neurons (Aarts et al., 2002; Roche et al., 2001), and that htt interacts with PSD-95 in a polyglutamine dependent manner (Sun et al., 2001). Here, I tested the hypothesis that an altered association and/or regulation between PSD-95 and NMDARs in mutant htt-expressing cells contributes to increased susceptibility to excitotoxicity and investigated mechanism by which this occurs. Specifically, I investigated the association of PSD-95 with htt and the NMDAR GluN2 subunits; signaling downstream of activation of the NMDAR/PSD-95 complex; and NMDA-induced cell death. My results suggest that at the presymptomatic stage of HD, the enhanced interaction of PSD-95 with GluN2B, and its signaling through p38 mitogen-activated protein kinase (MAPK) but not neuronal nitric oxide synthase (nNOS) activation, contributes to mutant htt-mediated sensitivity to NMDAR-dependent excitotoxicity in YAC128 striatal neurons. This work contributes to the understanding of both NMDAR-dependent neuronal death mechanisms in striatal neurons and early synaptic changes in HD pathogenesis, as well as providing potential drug candidates for future HD treatment.

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Regulation of NMDAR activity by desensitization is important in physiological and pathological states. We previously reported that desensitization decreases during hippocampal neuronal development, correlating with NMDAR composition, synaptic localization and association with PSD-95. To determine if PSD-95-induced changes in NMDAR desensitization occur because of direct binding to NR2 subunits or due to recruitment of regulatory proteins, we tested the effects of various PSD-95 constructs on NMDAR currents in HEK293 cells and neurons. In HEK cells, wt PSD-95 significantly reduced wt NMDAR desensitization without altering currents of NMDARs containing NR2A-S1462A, a mutation that abolishes PSD-95 binding. Moreover, PDZ1-2 domain was sufficient for this effect in neurons with low endogenous PSD-95 levels. Moreover, other PSD-95 family members with highly homologous PDZ1-2 domains significantly reduced NMDAR desensitization. In mature neurons, disruption of PSD-95/NMDAR interaction through PKC activation, or through interference peptides, increased desensitization to levels found in immature neurons. We conclude that direct binding of PSD-95 increases stability of NMDAR responses to agonist exposure.Desensitization is a property that shapes synaptic responses, and modulates the calcium signal mediated by the two predominant NMDARs subtypes in hippocampus, with possible consequences for their functioning. Further, we examined the involvement of NR2 subtypes in synaptic plasticity in hippocampal dentate gyrus of juvenile mice. Exercise was used as a means to alter expression of the NR2 subunits in this region. We compared two groups of animals: Controls, which were housed in conditions of minimal enrichment, andiiiRunners, which had access to an exercise wheel. NMDAR-dependent LTP expression was significantly greater in Runners than in Controls; in the presence of NR2B subunit antagonists, it was significantly reduced in both groups. NR2A subunit antagonist blocked LTP in slices from Runners and produced a slight depression in Control animals. LTD could not be prevented by either of the NR2B specific antagonists. Strikingly, eliminating NR2A subunit-containing receptor activity prevented LTD in Runners, but not in Control animals. Overall, these results indicate that interplay between subtype, subcellular localization and size of NMDAR subpopulations accounts for their diverse role in synaptic plasticity induction, and that exercise increases the contribution of NR2A to plasticity.

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