Canada

The 15q13.3 microdeletion syndrome is a highly penetrant copy number variant (CNV) associated with epilepsy, autism spectrum disorder, schizophrenia, and intellectual disability. Within the deletion are seven protein coding genes, including the deubiquitinase and driver gene, OTUD7A. We used a proximity proteomics assay in neurons to determine the binding partners to OTUD7A and found enrichment in proteins localized to the axon and axon initial segment (AIS). Using neurons generated from our cohort of 15q13.3 patient-derived induced pluripotent stem cells (iPSCs), we sought to understand the role of OTUD7A at the axon in 2D and 3D models of the CNV. 15q13.3 and mutant OTUD7A patient-derived neurons were generated from iPSCs and assayed for early growth and axonal properties using immunofluorescence techniques. We found that OTUD7A interacts with the axon growth protein, Ankyrin B, and AIS regulator, Ankyrin G. We examined 15q13.3 neurons at an early timepoint during axon outgrowth and found an immature phenotype among three 15q13.3 probands. We then looked at a mature timepoint to find aberrant localization of Ankyrin G at the AIS. To better profile the developmental trajectory of the 15q13.3 CNV, we are examining cerebral organoids at various timepoints to investigate neural progenitor proliferation, cellular migration, and maturation in a 3D system across development.

CAN-ACN 2021

Investigating neurodevelopmental and axonal defects in human models of the 15q13.3 microdeletion disorder

autism spectrum disorder

bioid2

copy number variants

neurodevelopmental disorders

axon

ipsc

axon initial segment

organoids

proteomics

epilepsy

Colleagues and Friends:

I am very happy to welcome you to the 14th annual Canadian Neuroscience Meeting, and to the first virtual edition of our meeting. I want to highlight the important work the CAN meeting chair, **Roger Thompson** and co-chair, **Alyson Fournier**, did to put together the great program we are offering this year. Moving to a virtual format required a complete re-design of the program, and both Roger and Alyson did a splendid job in providing us with an appealing and well adapted program for our first virtual event. I wish to thank them for this very important effort.

Highlights of this year’s scientific meeting include invited speakers **Michael Hausser, Franck Polleux, Gwyneth Card, Magdalena Götz**and Brain Prize winner **Huda Zoghbi**. Plenary and parallel symposia, proposed by our members complete our diverse [scientific program.](https://can-acn.org/meeting-2021/program-2021/)

The 2021 Advocacy session, chaired and organized by CAN advocacy committee chair **Karun Singh**, will feature a special lecture and question period with Member of Parliament and past Science Minister **Kirsty Duncan**. This will be followed by a short overview of CAN’s advocacy activities over the last year. We invite you to participate in this session for insight from Dr. Duncan on the workings of Parliament Hill, and to learn more about what CAN has been doing to promote neuroscience research in Canada.

We are also excited to host **Lisa Willis** in the Equity Diversity and Inclusion session, organized by EDI committee chair **Jibran Khokhar**.

A special highlight of our meeting will be the lectures by award-winning young neuroscientists. We are please to host the CAN 2020 Young Investigator Award winner **Mihaela Iordanova**, and CAN 2021 Young Investigator Award winners **Aaron Phillips** and **Tabrez Siddiqui**. Highlights will also include presentation by the top three winners of the CAN-CIHR-INMHA Brain Star Award winners, **Claire Gizowski, Nuria Daviu Abant** and **Lukas Tamayo-Orrego**.

Independently organized satellite symposia complement and complete this exciting program, check them out here: https://can-acn.org/meeting-2021/2021-satellite-symposia/

Some of the most engaging sessions are always the poster sessions featuring the work of our trainees, so make sure to log in to Gather Town during the poster sessions to chat with the presenters.
We hope you enjoy this program, and look forward to welcoming you, virtually.

Shernaz Bamji

President of the Canadian Association for Neuroscience

![](https://assets.underline.io/uploads/markdown_image/1/image/b9aea25343a87d5aac3b9367dc780974.jpg)



Please head to https://can-acn.org/meeting-2021/registration-2021/ to register. 

14th Canadian Neuroscience Meeting from August 23rd-25th

poster

Ischemic stroke is a devastating neurological disease caused by a reduction in blood flow to the brain that results in neuronal cell death. Subsequent neuroinflammation is mediated by central nervous system resident immune cells and peripheral immune cells. Evidence suggests that stroke-induced gut dysbiosis results in priming of pro-inflammatory immune cells in the intestine that migrate to the stroke lesion and contribute to neuroinflammation. Currently, it is unknown whether gut dysbiosis and the migration of gut-derived immune cells to the stroke lesion persist in the chronic phase following injury. In this study, we aimed to characterize; (I) stroke induced gut dysbiosis and (II) the contribution of gut-derived immune cells to neuroinflammation, over time in a rodent model of ischemic stroke. Using 16s rRNA sequencing we observed that gut dysbiosis is observed at D4 following injury in both males and female animals. Interestingly, females exhibit a return to baseline microbial composition at D20 while males continue to exhibit gut dysbiosis. In addition, we injected a lipophilic cell-tracking dye (Cm-DiI) into peyer's patches at various timepoints following injury to observe whether gut derived immune cells migrate to the brain beyond D3. Using immunohistochemical analyses, we observed Cm-DiI+/CD45+/DAPI+ cells in the stroke injured hemisphere at D4 and D9 following ischemic stroke induction. These findings suggest that stroke induced gut dysbiosis and the migration of gut-derived immune cells to the stroke lesion are phenomena that persist beyond the acute phase of injury.

Characterizing gut dysbiosis and the gut derived immune response to ischemic stroke

Alzheimer's Disease (AD) remains the major form of Dementia. Progressive neuronal loss is the major cause for the disease; however, the etiology of AD remains a controversial topic. Excessive oxidative stress, Tau hyperphosphorylation and formation of intracellular neurofibrillary tangles, as well as accumulation of extracellular Amyloid beta plaques represent the main targets for finding a cure. There is currently no effective treatment available for AD. Loss of cellular antioxidants including Thioredixin-1 (Trx1) is observed in AD tissue samples. Trx1 is a small redox reductase protein responsible for maintaining the oxidized proteins. Examining the importance of Trx1 depletion in pathology of AD, we have previously shown that pharmacological and genetic inhibition of Trx1 impairs autophagy, a vital housekeeping process responsible for protein homeostasis in neurons. We also have identified that loss of Trx1 is an important upstream event in induction of neuronal nuclear lamina invagination. Nuclear lamina damage or laminopathy is a newly identified mechanism in pathophysiology of AD. In the current studies we use genetic inhibition of Trx1 in human neuroblastoma cells and examine the effect on nuclear events. Our results indicate that downregulation of Trx1 is sufficient for induction of NL damage. These results lead to histone modification and changes in nuclear chromatin methylation. These studies were also complemented in hippocampi tissues from a mouse model of neuronal specific Trx1 knockout. Downregulation of Trx1 in these mice was associated with nuclear lamina damage and accumulation of amyloid beta and Tau hyperphosphorylation. Our studies provide a mechanistic link between oxidative stress and the key downstream events observed in AD pathology.

Nuclear Laminopathy and oxidative stress in Alzheimer's Disease

Alzheimer's Disease (AD) is more common in women, while low free testosterone has been associated with increased AD risk in men. However, treatment with testosterone has yielded mixed results in improving AD-related cognitive decline. Previous in vitro studies have shown that a major neurometabolite of testosterone: 5α-androstane-3α,17β-diol (3α-diol) inhibited the prolonged extracellular signal-regulated kinase (ERK) phosphorylation associated with oxidative stress. The role of 3α-diol in vivo remains unknown. We therefore examined the effects of continuous 3α-diol treatment on protein markers associated with AD progression in a triple transgenic (3xTg) mouse model. 3-month-old wild-type and 3xTg mice received subcutaneous implants of a 1 cm long Silastic capsule containing either 3α-diol dipropionate, or a blank capsule. Brains were collected at 6 and 9 months of age to analyze changes in expression of hippocampal ERK, MAP Kinase Phosphatase 3 (MKP3), plus phosphorylated (Ser202) and total tau. 3α-diol had no significant effect on the ratio of phosphorylated: total tau, at either age. No significant differences in ERK phosphorylation were observed at 6 months, but at 9 months sex and genotype-dependent differences were observed, with 9-month 3xTg females exhibiting higher ERK phosphorylation as well as markedly higher MKP3 expression. Treatment with 3α-diol had no effect on ERK phosphorylation, but significantly reduced MKP3 at 9 months, to levels indistinguishable from those observed in 3xTg males. 3α-diol may contribute to sex differences in AD susceptibility.

Effect of 5α-androstane-3α,17β-diol on ERK and MKP3 in a mouse model of Alzheimer's Disease

Reduced ability of the salience network (SN) to control switching between the central executive and default mode network (DMN) is linked to deterioration of cognitive functioning in normal aging, mild cognitive impairment (MCI) and AD dementia (He et al., 2014). In the current study, we compared functional connectivity between SN and the anterior DMN amongst older adults with subjective cognitive decline (SCD), MCI, and normal cognition (NC). Participants were 72 older adults [39 females, mean age=71.5] who were classified as either NC (n=26), SCD (n=29), or MCI (n=17). Participants underwent a 6-minute resting state functional magnetic resonance imaging using gradient-echo EPI BOLD at 3T (TR=2000ms, TE=30ms). Seed-based analysis using CONN with an anterior cingulate cortex (ACC; [0, 22,35]) seed, a key node in the SN, was conducted to assess functional connectivity between the SN and the anterior DMN within each group. Groups did not differ in age, years of education, or sex distribution [F(2,69)=0.415, p=0.66; F(2,69)=2.20, p=0.12; X² (2, N=72)=1.89, p=0.39, respectively]. Significant functional connectivity was found between ACC and medial prefrontal cortex (mPFC) in SCD and MCI groups (T(28)=2.39, p-FDR=0.04; T(16)=2.89, p-FDR=0.02, respectively) but not in the NC group. These findings show support for increased functional connectivity in SCD and MCI between SN and anterior DMN compared to NC. This finding is consistent with evidence of increased functional connectivity between the posterior DMN and mPFC in individuals with MCI and AD, an alteration that is believed to be a compensatory response to decreased functional connectivity between the posterior DMN and other brain regions such as the medial temporal lobe, responsible for memory formation and processing.

Elucidating functional connectivity differences amongst older adults with mild cognitive impairment, subjective cognitive decline or normal cognition

Chemotherapy-induced neuropathic pain (CINP) is a treatment-limiting complication of several classes of anticancer agents, including the taxanes, platins, vinca alkaloids, and bortezomib. It negatively affects the quality of life of patients, and unfortunately, there is no effective treatment at present. The current treatment approaches utilize antidepressant drugs, which offer only mild improvement. Despite several research efforts in the past, the underlying mechanism behind CINP is not clear, which poses a major hurdle in developing effective therapies. In this study, we explored if neuroinflammation has any role in inducing CINP. We considered the frequently used chemotherapy agents paclitaxel and cisplatin in this study. Intraperitoneal administration of either paclitaxel or cisplatin demonstrated robust mechanical allodynia in male CD-1 mice. Co-administration of an anti-inflammatory agent, dexamethasone, significantly reversed mechanical allodynia in these models. We further investigated the impact of these drugs on neurite outgrowth of DRG cultures established from paclitaxel/cisplatin treated animals and did not observe any significant effect. However, these drugs induced hind paw denervation in these animals, and dexamethasone reversed the loss of the nerve extremities in the paws, as evident from increased PGP9.5 labeling of nerve fibers. Evaluation of macrophages' response to these drug treatments in the DRGs and nerve extremities is ongoing. Overall, our findings suggest that local inflammatory response at the nerve extremities contributes to CINP.

Neuroinflammation facilitates chemotherapy induced neuropathic pain

Astrocytes comprise a highly complex cell population with diverse structural and functional properties in both the healthy and diseased central nervous system. Astrocytic pathology has been implicated in numerous neurodegenerative diseases, however, their structural rearrangement in brain diseases such as Alzheimer's disease (AD) remain poorly understood. Here, we applied super-resolution imaging and 3-dimensional (3D) electron microscopy approaches to better understand the structural changes of astrocytes in AD model (APP/PS1) mice. Structured illumination microscopy (SIM) of genetically-labeled astrocytes allowed us to capture global modifications to individual astrocyte shape while resolving certain aspects of their subcellular organelle distribution. We observed perturbations to the complex branching pattern of whole astrocytes and additionally, mapped the organization of mitochondria throughout the cell in AD model samples. Results from SIM imaging were complemented with analysis using focused ion beam scanning electron microscopy (FIB-SEM) that enabled 3D serial reconstruction of astrocyte ultrastructure. Using FIB-SEM, we found dramatic restructuring of astrocyte subcompartments in neuropil and astrocytic endfeet, including major alterations of astrocytic process shape and the restructuring/redistribution of mitochondria. Quantitative analysis of SIM and FIB-SEM approaches allowed us to directly compare the astrocytic surface structure and the organelle distribution/morphology within astrocyte sub-compartments using custom MATLAB scripts, in healthy and AD model tissue. This multi-level structural analysis of astrocytes provides an important understanding of how astrocytes are constructed in the healthy brain and altered with brain disease.

Multi-scale analysis of astrocyte morphology and ultrastructure in healthy and Alzheimer's disease model mice

Huntington's disease (HD) is a neurodegenerative disorder characterized by motor, cognitive and psychiatric deficits. The dorsal striatum is the major site of neurodegeneration in HD, particularly the D2-expressing spiny projection neurons (D2-SPNs) early on, and the D1-expressing SPNs (D1-SPNs) late in the disease. Studies have shown aberrant cortico-striatal signalling in HD mice, including deficits in cortico-striatal plasticity. Although there has been extensive research on changes to cortico-striatal signalling in vitro, there has been relatively little research on how these changes correlate with behaviour in vivo. We combined the accelerating rotarod and open field tasks with GCaMP7f imaging using fiber photometry to correlate activity of striatal neurons with task performance and motor learning. Both WT and YAC128 HD mice showed increased striatal activity over baseline when they performed the rotarod, and this activity reduced over days of training. However, we found significant disruptions in the correlation between striatal activity and behaviour on the rotarod and in the open field in YAC128 HD mice. We also discovered changes to paw kinematics during rotarod in YAC128 mice, even when mice had no differences in latency to fall. We are also performing experiments measuring activity of D1-SPNs and D2-SPNs simultaneously, in YAC128 and Q175 HD mice. This work begins to bridge the gap between our understanding of cortico-striatal changes determined from in vitro studies and the behavioural deficits observed in vivo.

In vivo striatal neural activity during motor skill learning and spontaneous behaviour in Huntington's Disease mice

Sodium-dependent high affinity glutamate transporters are essential in mitigating the toxic effects of extracellular glutamate accumulation. Glutamate uptake is primarily mediated by astrocytes, although glial coverage is reported to be four times higher postsynaptically than presynaptically. The functional consequences of this synapse asymmetry in glial coverage is poorly understood, but it implies the presynapse is more vulnerable to glutamate uptake impairments than the postsynapse in brain diseases with compromised glutamate uptake, such as Alzheimer disease (AD). Here, we developed a novel approach to quantify the unique glutamate clearance dynamics in presynaptic and postsynaptic microenvironments in the hippocampus of the 3xTg mouse model of AD through a combination of intensity-based glutamate sensing fluorescent reporter (iGluSnFR) and two-photon microscopy. By 6-months, an age corresponding to the emergence of an AD-like phenotype in the 3xTg mouse, glutamate clearance in presynaptic microenviornments was impaired while clearance in postsynaptic microenvironments was unimpaired in 3xTg mice. This impairment is mediated by GLT-1 as confirmed by 2 observations: DHK application to acute hippocampal slices increases glutamate clearance time in presynaptic microenvironments, but the extent of this increase is lesser in 3xTg mice compared to control mice; and EAAT2 overexpression via ceftriaxone speeds up glutamate clearance in presynaptic microenvironments. This implies that glutamate transporters have different capabilities depending on the specific microenvironment within a given region, and in the context of AD, suggests that the emergence of presynaptic microenvironment glutamate clearance deficits may correlate with the emergence of AD-like synaptic pathology

Glutamate uptake asymmetry in a mouse model of alzheimer disease

One of the most vulnerable neuronal cell types in the mammalian brain are cerebellar Purkinje cells (PC). In many ataxias, certain PCs show remarkable resilience to disease, with only a subpopulation of PCs typically vulnerable to the same insult. Recently we have found that certain PCs located in the anterior region of the cerebellar vermis are more likely to die than those located in the posterior region in Christianson syndrome (CS) ataxia. We now wish to investigate what leads to PC vulnerabilities versus resilience in CS ataxia. CS is a rare X-linked neurodevelopmental and neurodegenerative disorder caused by loss of function mutations in NHE6, an endosomal Na+/H+ exchanger responsible for regulating pH in recycling endosomes of the endocytic pathway. Loss of NHE6 results in over acidification of endosomes leading to mistrafficking of cargo which could affect neuronal function. PCs have a unique ability to fire high frequency action potentials, even in the absence of synaptic input and motor control. Using electrophysiology techniques we investigated if PCs in the anterior region have alterations in intrinsic firing prior to cell death and motor deficits in CS. Using a mouse model of CS (NHE6KO), we observed a significant decrease in PC intrinsic firing ability in the anterior region at age P40, an age which precedes manifestation of PC death and motor impairment. Given these results, we are testing therapeutic approaches targeting intrinsic firing in vulnerable PCs with the goal of restoring firing output and prevent behavioral deficits.

Purkinje cell intrinsic firing deficits precede cell death and motor impairment in a mouse model of Christianson syndrome

Amyotrophic lateral sclerosis (ALS) is a moto-neuron disorder in which an RNA-binding protein, TDP-43, mislocalizes and pathologically accumulates from its normal nuclear locale to the cytosol. Given that the subcellular localization and expression of TDP-43 is tightly regulated and affected by its protein-protein interactions (PPIs), we posit that identifying novel interactors of wild-type and mutant TDP-43 could reveal insight into networks involved in driving ALS pathogenesis. Using CRISPR/Cas9, our lab has generated knockin cell lines expressing GFP-tagged wildtype (WT) and an ALS-causing mutant (Q331K) TDP-43, in the endogenous TARDBP locus (coding for TDP-43). We have shown that the Q331K mutation causes loss-of-function and mislocalization of TDP-43. We have performed immunoprecipitation-mass spectrometry (IP-MS) on these cell lines to elucidate interactors of WT- and Q331K, TDP-43. Our data has shown that there is an overall loss of interactors with the Q331K mutation. We have analyzed these data using bioinformatic approaches to shortlist and validate 14 candidates. From this, 4 interactors have shown robust interaction with TDP-43 via IP-western blot. We are using cellular and biochemical assays to assess the effects of knockdown and overexpression of these top 4 hits on TDP-43 localization and loss-of-function. Using this unbiased approach, we will identify TDP-43 PPIs and characterize their roles in cellular functions in the context of ALS, giving insight into pathways involved in driving neurodegeneration.

Investigating neurodevelopmental and axonal defects in human models of the 15q13.3 microdeletion disorder

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Characterizing gut dysbiosis and the gut derived immune response to ischemic stroke

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