Canada

Multiple sclerosis (MS) is a neurodegenerative disease which is largely associated with progressive disability. It is characterized by multiple focal demyelinating lesions where the extent of myelin loss correlates with the advancement of disease and also age. Substantial work has focused on preventing demyelination from occurring, but the alleviation of MS symptoms through regeneration of myelin, or remyelination, remains an under-researched area. Remyelination is associated with less clinical disability and linearly declines with age. This process is dependent upon microglia, the resident immune mediators of the central nervous system (CNS). Microglia are a heterogeneous phenotype, forming subpopulations that vary across development, anatomical regions and disease. Using advanced analytical tools and bioinformatic techniques, we show that a subpopulation of microglia arises at the onset of remyelination in a mouse model of MS which presents acute demyelination followed by spontaneous, but robust, remyelination. These remyelination-associated microglia (RAM) change their transcriptional signature throughout remyelination, moving from an Igf1/Irf7 dominated phenotype to one that is characterized by genes responsible for regeneration, such as Meg3. We have validated this by showing the expression of Igf1 and Irf7 within mouse lesions using RNA scope. We are currently sequencing microglia from aged, lesioned environments to assess how microglia responses differ through ageing

CAN-ACN 2021

Phenotypic profiling of microglia across remyelination and ageing

single cell sequencing

remyelination

heterogeneity

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

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.

Mapping and characterizing ALS-linked TDP-43 protein-protein interactions

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.

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

Lysosomes are acidic organelles that contain a myriad of hydrolytic enzymes. In neurons, lysosomes traffic throughout the axons and dendrites to deliver catabolic enzymes to distal regions of the cell and maintain local degradative demands. Of the various lysosomal enzymes, glucocerebrosidase (GBA) is of particular interest due to its relationship with synucleinopathies such as Parkinson's Disease and Dementia with Lewy Bodies. Mutations in GBA are the most common genetic risk factor for synucleinopathies, but the mechanistic relationship is unclear. GBA cleaves the glycolipid glucosylceramide (GC) into glucose and ceramide, but mutations in GBA, or its catalytic inhibition, can disrupt the composition of the lysosomal membrane. The lysosomal membrane serves as the platform to which trafficking complexes are recruited and activated. Thus, we investigated if the prolonged catalytic inhibition of GBA by Conduritol B Epoxide (CBE) could interfere with lysosomal trafficking dynamics in axons. In human iPSC-derived cortical neurons treated with CBE for 10 days, we did not observe a change in the motile fraction, velocity, or run length of LAMP1-GFP vesicles. Additionally, we did not find any alterations in lysosomal size or density in the axon, dendrites, or neuronal cell bodies, despite complete inhibition of enzymatic activity. These results suggest that CBE-mediated inhibition of GBA in neurons does not directly impact lysosomal dynamics or size in the time course of our experiments.

Lysosomal transport dynamics in GBA-inhibited iPSC-derived human neurons

The previous study from our group has identified that after peripheral nerve injury there is an increase of kynurenine in the plasma, which seems to be involved in the maintenance of neuropathic pain. However, the mechanisms which peripheral kynurenine (Kyn) mediates neuropathic pain is unknown. Therefore, the aim of the present study was to test the hypothesis that peripheral Kyn reaches the spinal cord and maintain neuropathic pain by KMO metabolism, leading to downstream nociceptive metabolites. Methods: Spared Nerve Injury (SNI) model of neuropathic pain was induced in C57BL/6 mice and the following test and methods were used: von Frey filaments nociceptive test, Real-time PCR and western blotting, inhibition of KMO activity. This study was approved by the Local Ethical Commission in Animal Research (045/2013). Results: SNI induced mechanical allodynia in a time-dependent manner, which peaked from 7 up to 21 days. SNI- induced mechanical allodynia was associated with an increase in the expression of KMO in the spinal cord, mainly at day 10 and 14 after injury. KMO expression was restricted to spinal cord astrocytes. Functionally, pharmacological inhibitor against KMO injected intrathecally after SNI, reduced mechanical allodynia. Also, kyn injected systemically (i.v) promoted mechanical allodynia, which was educed when KMO was pharmacologically inhibited. In summary, these results indicated that after peripheral nerve injury spinal astrocytes-expressing KMO plays a critical role in the development of neuropathic pain. In conclusion, the kynurenine metabolic pathway is a critical link in the neuroimmune response after peripheral nerve injury in the maintenance of neuropathy.

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

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