Résumés / Abstracts 2023

Authors: Morgan Bérard1,2, Laura Martinez-Drudis1,2, Razan Sheta, PhD1,2, Omar M A El-Agnaf3, Abid Oueslati, PhD1,2.

Affiliations : 1.CHU de Quebec Research Center, Axe Neurosciences. 2.Department of Molecular Medicine, Faculty of Medicine, Université Laval, Quebec City, Canada. 3. Neurological Disorder Research Center, Qatar Biomedical Research Institute (QBRI), Hamad Bin Khalifa University (HBKU), Qatar Foundation, Doha, Qatar.

Alpha-synuclein (aSyn) aggregation into proteinaceous intraneuronal inclusions, known as Lewy bodies, is one of the major neuropathological hallmarks of Parkinson’s disease (PD) and related synucleinopathies. However, the exact role of aSyn inclusions in the pathogenesis of this diseases remains elusive. Currently, none of transgenic models exhibited significant early dopaminergic neuronal loss. An alternative approach using Adeno-associated viral (AAV) allowed for a local aSyn accumulation and a significant dopaminergic neuronal loss. However, the viral delivery requires specific equipment and technical training. Here, we describe a novel approach for a non-invasive systemic delivery of viral particles overexpressing human aSyn allowing for a large-scale overexpression in mouse brain. We cloned plasmid for human aSyn expression under ubiquitous CAG promotor to produce the recombinant AAV2 with specific capsid called “PHP.eB”. Mice received retro-orbital injection of AAV-PHP.eB-CAG-human_aSyn. Examination of human aSyn overexpression in mouse brains was performed 2 weeks post-injection. Subsequently, aSyn overexpression impact was also assessed using behavioral tasks several months post-injection. Using this model, we report that the widespread human aSyn overexpression in mouse brains induced the selective dopaminergic neurons degeneration in the substantia nigra. This neuronal degeneration was associated with a progressive manifestation of PD-like motor impairment, without affecting cognitive performance. Our data demonstrate that this non-invasive novel in vivo delivery of aSyn represents a viable strategy for modeling PD to study the selective vulnerability of the nigral dopaminergic neurons that can essentially help to decorticate the role of aSyn in PD pathogenesis.

EVERARD GAUTHIER, Lejeune Thierry, Batcho Charles, Centre interdisciplinaire de recherche en réadaptation et intégration sociale

OBJECTIF – OBJECTIVE

This study aimed to evaluate the effect of age, visual feedback and movement repetition on the course of upper limb (UL) precision and kinematics.

MÉTHODES – METHODS

Fifty-one healthy participants were asked to perform 25 trials of a reaching task in immersive virtual reality (VR) with and without benefiting from visual feedback of their tested hand. They were instructed to place, as accurately as possible, a controller in the centre of a virtual red cube. For each trial, the error distance (distance between the controller and the cube), a coefficient of linearity (CL), the movement time (MT), and the SPARC (a movement smoothness index) were calculated. Correlations and multivariate analyses of variance were conducted to assess the influence of age, movement repetition and visual feedback on the course of the error distance, SPARC, CL and MT throughout the 25 trials

RÉSULTATS – RESULTS

Medium correlations were observed between age and mean error distance (r=0.40; p=0.001) (r=0.354; p=0.01), and age and mean CL (r=-0.49; p=0.005) (r=-0.4; p=0.004) in presence and absence of visual feedback. The error distance evolvement was significantly influenced by age (p=0.027) and visual feedback (p<0.001); CL by movement repetition (p<0.001) and age (p=0.013); SPARC by movement repetition (p<0.001), visual feedback (p<0.001) and age (p=0.015); and MT by visual feedback (p=0.001) and movement repetition (p=0.001) but not by age (p=0.671).

CONCLUSION

Results of this study demonstrated that age and visual feedback of the hand have a significant influence on the course of UL precision and movement smoothness in immersive VR.

IDI WALID, Bérard Morgan, Teixeira Maxime, Sheta Razan and Oueslati Abid

CHU de Quebec Research Center, Axe Neuroscience and Department of Molecular Medicine, Université Laval, Quebec, QC G1V 4G2, Canada​

OBJECTIVE

Parkinson’s Disease (PD) is the second most common neurodegenerative disease in the world. One of the major pathological hallmarks of the disease is the aggregation of a misfolded protein called alpha-synuclein (a-syn), which will lead to the formation of cellular inclusion known as Lewy bodies (Spillantini et al., 1997). However, how these aggregates disturb neuronal homeostasis leading to neurodegeneration remains elusive. Several studies showed a correlation between alterations of the degradation systems (autophagic or proteasomal), implicated in protein quality control, and a-syn aggregation (Lehtonen et al., 2019). Nevertheless, it is not clear yet how the degradation is impaired.

Our aim is to precisely understand how an alteration of the degradation systems is involved in the pathogenesis of PD.

METHODS

To study the effect of a-syn aggregation, the LIPA (Light-Inducible Protein Aggregation) system recently developed by our laboratory is used (Bérard et al., 2022). This system nicely mimics key features of Lewy bodies and allows us to optogenetically control and observe in real-time the aggregation of a-syn.

RESULTS

Using this model, we were able to observe for the first time the effect of LIPA-induced aggregates on the proteasome and autophagy systems by using specific markers (Larsen et al., 2010; Menéndez-Benito et al., 2005). Moreover, we also get interested in the inhibition of these systems and the effect on aggregation. The results obtained show us the capacity of our LIPA system to mimic the potential effects of a-syn on the degradation systems.

CONCLUSION

Taken together, our observations reveal the impact of autophagy and proteasome dysfunctions in PD pathogenesis. Interestingly, we found that both systems seem involved but in a different manner and with a different kinetic.

Anna A. Chami1, Silvia Pozzi1, Claude Gravel 1,2, Daniel Phaneuf 1, Jean-Pierre Julien 1,2

1CERVO Brain Research Centre, Québec, Québec, Canada.

2Department of Psychiatry and Neuroscience, Université Laval, Québec City, Québec, Canada.

Abnormal cytoplasmic aggregates of TDP-43 (TAR DNA-binding protein 43) are a pathological hallmark of degenerating neurons in many neurodegenerative disorders, including Amyotrophic Lateral Sclerosis (ALS), Frontotemporal Dementia (FTD), Alzheimer’s disease, Parkinson’s disease. In order to target TDP-43 and reduce its pathology, we developed an AAV (Adeno-Associated Virus) vector encoding a single chain antibody (scFv) against the RRM1 (RNA Recognition Motif 1) of TDP-43. Previously, our lab (Pozzi et al.,2019) reported that this AAV-mediated delivery intrathecal of the scFv can be used to mitigate TDP-43 pathology in transgenic mice expressing mutant TDP-43. Here, we propose to further validate the antibody approach in mitigating pathology and cognitive defects in new mouse models with robust TDP-43 pathology and the use of scFv-encoding AAV vector bearing a recombinant capsid designed to achieve efficient neuronal transduction after injection directly into blood circulation. With this improved AAV vector and an injection directly into blood circulation, it is possible to reach and transduced neurons over a large portion of the nervous system. It is our hope that such AAV-delivery of scFv antibodies will succeed in TDP-43 proteinopathy. A single intravenous administration of AAV vector to achieve sustained production in neurons of therapeutic antibody mitigating TDP-43 proteinopathy is an appealing strategy for treatment of neurodegenerative diseases.

Keywords : ALS, intrabodies, scFv, TDP-43, in vivo animal models

RODRIGUEZ-ALLER RAQUEL1,2,3, Malvaut Sarah3,4, Bérard Morgan1,2, Sheta Razan1,2, Saghatelyan Armen 3,4, Oueslati Abid 1,2 

1CHU de Quebec Research Center, Axe Neurosciences. 2Department of Molecular Medicine, Faculty of medicine, Université Laval, Quebec City, Canada.3CERVO Brain Research Centre. 4Department of Psychiatry and Neurosciences, Faculty of medicine, Université Laval, Quebec City. 

OBJECTIF – OBJECTIVE

Intracellular deposits of misfolded a-synuclein (a-syn) constitute one of the main pathological hallmarks that have been attributed to Parkinson’s disease (PD). While it has been proposed that synaptic aggregation of a-syn represents one of the first events in the pathogenesis of PD, its exact role in synaptic function and subsequent progressive neurodegeneration remains elusive.

MÉTHODES – METHODS

One of the main hurdles in studying the early mechanisms inducing synaptopathy in PD is the limited spatial and temporal resolution of current PD models. However, our laboratory has developed an in vitro and in vivo model that mimics the histopathological features of PD, and is based on the use of optogenetics technology named LIPA (light-inducible protein aggregation) that allows for inducing the aggregation of a-syn in a spatio-temporal manner under the control of blue light.

In order to examine what role LIPA-a-syn aggregation may play in impairing synaptic function we first assessed the activity of striatal cells combining the LIPA model with the in vivo Ca2+ imaging through mini-endoscopes. Subsequently, we evaluated the dopamine content, an important neurotransmitter involved in the circuitry behind movement control, in the striatum of mice using High Performance Liquid Chromatography (HPLC).

RÉSULTATS – RESULTS

Our results revealed changes in the striatal neuronal network after inducing the aggregation of a-syn in the substantia nigra pars compacta (SNc). We reported a significant decrease in the synchronization and frequency of Ca2+ events in striatal neurons shortly after the onset of stimulation. In addition, our data evaluating the dopamine levels in the striatum of mice has shown an increase in the dopamine levels caused by the aggregation of a-syn.

CONCLUSION

Altogether, our data highlight the synaptic function as an initial component to be affected in the progression of PD. Now, we seek to further assess the underlying mechanisms by which LIPA a-syn aggregates alter the synaptic homeostasis.

Amélie Poulin-Brière, Silvia Pozzi, Jean-Pierre Julien

The majority of amyotrophic lateral sclerosis (ALS) cases (~90%) are considered sporadic (sALS) and their etiology still remains unknown. However, a TDP-43 proteinopathy is observed in more than 95% of ALS cases, suggesting that TDP-43 could play a role in neurodegeneration.

Our hypothesis is that the pathogenic effect of sALS patients CSF, previously observed, is in part due to the presence of misfolded TDP-43 and SOD1, which can spread prion-like proteinopathy to healthy cells.

OBJECTIF – OBJECTIVE

The objective is to study the effect of a therapy based on an antibody directed against RRM1 domain of TDP-43 on motor and cognitive performances as well as on pathological changes in tissues of mice models of ALS induced by infusion of CSF from sporadic ALS patients.

MÉTHODES – METHODS

Human TDP-43 WT transgenic mice (hTDP-43WT) are infused intracerebroventricularly (ICV) with sALS patients CSF.  Mice are treated with a full-length antibody directed against the RRM1 domain of TDP-43, named E6, administered by intrathecal (IT) injection or ICV infusion. Effects on cognitive and motor functions as well as on pathological changes in tissues are analysed.

RÉSULTATS – RESULTS

E6 immunotherapy decreased TDP-43 mislocalisation and improved motor and cognitive functions. Immunofluorescence analysis showed an increased nuclear/cytoplasmic ratio of TDP-43 in lumbar motor neurons of IT E6 treated mice as well as in motor cortex of ICV E6 treated mice compared to control. Gait analysis revealed an increased stride length in both IT and ICV E6 treated mice compared to control. Novel object recognition test showed an increased interaction time with the novel object in ICV E6 treated mice compared to controls.

CONCLUSION

Immunotherapy against TDP-43 provided promising results. We now propose to study the long-term effects of CSF infusion on disease progression after its delivery. These studies will provide new insights on disease mechanisms and will serve to advance the development of new treatments to halt the disease propagation.

SROUR NADER1,2, Khouma Axelle1,2, Minbashi Moeini Moein1,2, Michael Natalie Jane1,2 & Caron Alexandre1,2

1Faculté de Pharmacie, Université Laval; 2Institut Universitaire de Cardiologie et de Pneumologie de Québec

Introduction: A prevalent model in the field of neurometabolism is that leptin directly activates pro-opiomelanocortin (POMC) neurons of the arcuate nucleus of the hypothalamus (ARC) to suppress feeding. However, evidence reveals that POMC neurons are highly heterogenous. Furthermore, POMC neurons are also expressed in the retrochiasmatic area (RCA), and little is known about the effects of leptin on POMC neurons in females. Therefore, there is a need to better define the effects of leptin on hypothalamic POMC neurons in both males and females.

Objectives: 1) Determine the differences in leptin responsiveness of POMC neurons in the RCA and ARC 2) Determine if leptin responsiveness of these neurons differs by sex.

Methods: We used a mouse model allowing fluorescent labeling of adult POMC neurons (Pomc-CreERT2::tdTomato). Male and female mice (8-12 wk) were euthanized, and brains were rapidly removed and sectioned for whole-cell patch-clamp electrophysiology experiments. Current-clamp recordings were established from POMCARC and POMCRCA neurons and slices were bath exposed to leptin (100nM). Changes in neuronal electrical excitability were assessed.

Results: In males, leptin excited 32% of POMCARC neurons and inhibited a smaller population of these cells. However, 26% of POMCRCA neurons were inhibited by leptin with only a small proportion being excited. In contrast, most POMC neurons were nonresponsive to leptin in females (70-80%). Of the female leptin-responsive POMC neurons, similar percentages of excitatory or inhibitory effects were observed between the regions examined.

Conclusion: Our data demonstrate that POMCARC and POMCRCA respond differently to leptin in males; POMCARC are mainly excited while POMCRCA are mainly inhibited. We also show that the majority of POMC(ARC or RCA) neurons are nonresponsive to leptin in females. Unlike males, leptin-induced inhibition of POMC neuron activity is virtually absent in females. This suggests that anatomical and sexual differences exist in the hypothalamic response to leptin.

* CANIVET CORALINE 1, Boilard Éric 2,3, Merette Chantal 3,4, C.M. Morin Charles.M 3 and Deslauriers Jessica 1,3

1Neurosciences, CRCHU de Québec-ULaval, Québec, QC, Canada, 2Infectious and Inflammatory Diseases, CRCHU de Québec-ULaval, Québec, QC, Canada,3Univ. Laval, Québec, QC, Canada; 4 Neuroscience Biostatistics Platform, Brain Research Center, Québec (QC), CANADA

Background: How the perception of the measures implemented during COVID-19 pandemic, affect the psychological health and stress of healthcare workers remains unknown. Moreover, numerous studies show a relationship between inflammatory status and work-related distress. Brain-derived exosomes (BDEs) are extracellular vesicles that may be produced by brain cells, cross the blood-brain barrier (BBB), then passively release cytokines in the blood. These BDEs may be potential targets to blood identify brain-specific biomarkers. We hypothesized that specific measures implemented are critical to prevent distress in healthcare workers, and that inflammation from BDEs are associated with risk to develop psychiatric symptoms.

Methods: Fifteen healthcare workers were recruited, and completed four self-report mental health surveys three times (1st visit, 3 and 6 months later): Blood sample was drawn for enrichment of BDEs and quantification of immune cargo from BDEs. We assigned the participants according to their K10 score on their 3rd visit: no/low vs moderate/severe psychological distress. Welch-corrected t test was used for statistical analyses.

Results: Participants with moderate/severe psychological distress on their 3rd visit were already at elevated risk for exhaustion (p≤0.01) and hyperarousal (p≤0.05) early on vs participants with low distress. On the 3rd visit, moderate/severe distress in healthcare workers was associated with higher job-related stress (p≤0.05) compared to the low distress group.

Conclusion: Altogether these findings show that healthcare workers with a moderate/severe psychological distress exhibit increased intrusion and hyperarousal symptoms, and are at high risk for emotional burnout. Moderate or severe psychological distress is associated with high job-related stress and negative perception of the implemented measures. Complementary analyses, and measurements of circulating levels of inflammatory markers from BDEs will also be presented.

THIBAULT NICOLA, D’Amours Stéphanie, Albouy Philippe, Grondin Simon, Centre de recherche CERVO.

PROBLÉMATIQUE ET OBJECTIFS

Différentes études de neuroimagerie (imagerie par résonance magnétique fonctionnelle, IRMf) et d’électrophysiologie (électroencéphalographie, EEG) offrent des résultats contradictoires concernant le rôle de différentes régions cérébrales dans la perception temporelle. On trouve cependant un certain consensus relatif à l’effet de la longueur des intervalles sur les processus de perception temporelle (sous une seconde ou au-delà d’une seconde). L’objectif de cette étude est d’identifier les réseaux cérébraux associés à la perception passive d’intervalles temporels en fonction de la longueur des intervalles présentés (sous une seconde ou au-delà d’une seconde). Pour ce faire nous utilisons des approches de reconstruction de sources des signaux EEG enregistrés lors d’une tâche auditive d’oddball. Puisqu’il est également connu que les musiciens sont plus efficaces dans la discrimination de stimuli temporels que les non-musiciens, un objectif secondaire est d’observer si les musiciens et non-musiciens recrutent des réseaux cérébraux distincts et ce, autant pour le traitement de courts que pour celui de longs intervalles temporels.

MÉTHODES

L’échantillon est composé de 35 participants, dont 15 musiciens et 20 non-musiciens. Le paradigme oddball basé sur les intervalles temporels est réparti en six blocs de 40 essais séparés également entre les courts (800 ms) et longs (1600 ms) intervalles. Chaque essai comprend dix intervalles de 800 ou 1600 ms, dont un survenant à un moment discordant.

RÉSULTATS

Des comparaisons déviants-standards sur les réponses évoquées sont effectuées pour les intervalles courts et longs et les réponses évoquées sont comparées entre les deux conditions. Des tests-t sur les reconstructions de sources corrigés avec 1000 permutations de cluster sont effectués pour comparer les standards et déviants pour chacun des groupes. Les mêmes analyses sont reprises pour comparer les musiciens et non-musiciens. Pour les courts intervalles, c’est l’aire supplémentaire motrice et le cortex auditif gauche qui s’activent alors que pour les intervalles longs, ce sont le sulcus intrapariétal gauche et le cortex moteur qui s’activent. Les analyses des données du groupe de musiciens sont en cours, mais nous faisons l’hypothèse que les musiciens recruteront les mêmes aires corticales que les non-musiciens avec une plus grande amplitude, autant pour les courts que les longs intervalles.

CONCLUSION

Cette étude permet donc de distinguer les fondements neurophysiologiques de la perception temporelle de courts et de longs intervalles temporels et de vérifier si ces fondements neurophysiologiques diffèrent selon l’expertise musicale.

P LEVESQUE1,2, C Canivet1,2, and J Deslauriers1,2

1 Neuroscience axis, CRCHU de Québec-UL; 2 Faculté de Pharmacie, Université Laval

Background: Posttraumatic stress disorder (PTSD) affects over 9% of the population. The only FDA-approved drugs for treatment of PTSD have limited efficacy. It is therefore urgent to better understand the pathophysiological mechanisms of PTSD to developing new therapeutic avenues. Studies support a role of angiotensin II (Ang II) in the response to psychological trauma, leading to higher risk for PTSD. Still, the mechanisms underlying this relationship remain poorly understood. We hypothesized that following psychological trauma, Ang II binding to endothelial angiotensin type 1a receptors (AT1aRs) leads to BBB disruption and infiltration of inflammatory molecules into the brain, contributing to the onset of PTSD symptoms. Methods: Male and female wild-type (WT) mice and mice deficient for AT1aR in endothelial cells (eAT1aR-/-) were exposed to predator stress. A week later, anxiety-like and avoidance behaviors were evaluated using the open field (OF), light-dark box (LD), and trauma-reminder (TR) tests. Blood pressure (BP) was monitored by plethysmography at baseline, as well as 1 and 10 days after stress. Results: Across all three tests, predator stress increased anxiety-like and avoidance behaviors independently of eAT1aR phenotype in female mice (p < 0.05 or p < 0.001). In the LD test, eAT1aR -/- phenotype reduced overall anxiety-like behavior in females regardless of stress exposure (p < 0.05). In males, the TR test, stress exposure increased avoidance behavior (p < 0.0001). Only in males, the eAT1aR-/- phenotype reduced anxiety-like and avoidance behavior in the LD (p < 0.05) and TR tests (p < 0.05). Surprisingly, eAT1aR-/- mice had higher systolic and diastolic BP vs WT mice. This effect was observed in both sexes, with a stronger effect in males. Conclusion: These findings suggest that peripheral Ang II signaling through endothelial AT1aRs is implicated in the development of overall anxiety and avoidance of trauma-reminder cues, and tends to promote resilience following psychological trauma in male mice. Brain levels of inflammatory markers and BBB integrity ​​will also be presented to provide additional key insights on the mechanisms underlying the association between Ang II and PTSD risk.

CHAUVETTE LOONAN, Whittom Alexis, Sharp Andréanne

OBJECTIVE

The study investigates the interaction of two auditory scene analysis abilities in professional musicians: missing fundamental perception and mistuned harmonic detection. The missing fundamental phenomenon refers to the brain’s ability to reconstruct and accurately perceive the pitch of complex tones even when the fundamental frequency (F0) is missing. The mistuned harmonic detection threshold (MHDT) is a measure of harmonic analysis, which underpins sound fusion and pitch perception. The objective of this study was to investigate the effect of missing fundamentals on the MHDT of professional musicians to understand the relationship between musical training and auditory scene analysis.

METHODS

We used the novel adaptive method introduced in our prior study to determine the MHDT in complex tones with present or missing fundamentals. Adult musicians (N=10) were asked to untune the 2nd harmonic of complex tones containing 6 harmonics until they perceived 2 distinct sounds. This task was repeated 5 times to estimate a MHDT. Three tone frequency conditions were used (125, 200, 440 Hz) and MHDTs were obtained with the F0 present and with the F0 missing. These preliminary results were compared to those of non-musician participants from a previous study.

RESULTS

The MHDTs did not vary significantly between the conditions where the F0 was present and where the F0 was missing for the 125, 200, and 400 Hz complex tones. The MHDT of musicians were comparable to the MHDT with present F0 of non-musicians of previous studies.

CONCLUSION

Our results suggest that the MHDT of musicians are not affected by missing fundamentals. Previous research had suggested that missing fundamentals may impair auditory scene analysis in non-musicians, but the present results suggest that this may not be the case for musicians. This suggests that musical training may enhance central auditory processing in situations where the F0 is removed (e.g., on the telephone) or masked (e.g., by ambient noise), which could have implications for the development music training interventions for people with central or peripheral hearing difficulties.

Lida Sanchez & Jorge Soliz

Laval University, Centre de Recherche de l’Institute Universitaire de Cardiologie et de Pneumologie de Québec (IUCPQ), Québec, Canada

Mitochondria are small organelles within cells that generate almost all the energy we need throughout life. The human brain, being the most energy-demanding organ, consumes between 50% (in postnatal age) and 20% (in adulthood) of this energy. There are around 2 million mitochondria per neural cell orchestrating brain functions such as synapsis formation and axonal growth. Erythropoietin (EPO) is a hormone endogenously produced by several nervous cells, including neurons, astrocytes, and microglia. When the brain is injured, this endogenous production of EPO activates anti-oxidative, anti-apoptotic, and anti-inflammatory mechanisms. Animals injected with EPO or overexpressing this hormone exclusively in the brain, have been shown to increase the number of mitochondria and the number of vesicles in the synapses. These beneficial effects of EPO could be used to treat brain regions affected by stress and its long-term consequences. Stress caused at early life stages, particularly in preterm babies, in which the brain is developing, could cause a malfunctioning of the proliferating synapses and cause respiratory disorders in adulthood. In our laboratory (using animal models), we have shown that stress experienced in early life (such as that induced in cases of maternal isolation or conditions that interfere with mother-child interactions), leads to the development of respiratory pathologies, such as sleep apnea. Oxidative stress, apoptosis, and inflammation are cellular features of sleep apnea, which all have a common factor: the mitochondria. Finding a treatment that could aid to keep healthy mitochondria may assist decrease the effects of sleep apnea. EPO could be one of those treatments, potentiating protective mechanisms and cellular dynamics. In this work, we will focus on the effects of EPO on the mitochondria in two brain regions: the paraventricular nucleus of the hypothalamus (PVH) and the nucleus of the solitary tract (NTS), involved in the control of stress and respiration respectively. We will evaluate mitochondrial activity, and oxidative stress, as well as the structure and the distribution of mitochondria in the synaptosomes of these regions. This project will lead us to define the role of mitochondria in the process in which neonatal stress leads to respiratory dysfunction in adulthood.

WHITTOM, ALEXIS; Chauvette, Loonan; Sharp, Andréanne; Centre de recherche CERVO

OBJECTIF

Les illusions auditives sont de précieux outils permettant d’explorer les limites de la perception auditive. L’ «Octave Illusion» est un exemple de ce type de phénomène auditif. Celle-ci est perçue lorsque deux sons séparés par une octave sont joués de manière répétée en alternance entre les deux oreilles. Jusqu’à maintenant, peu d’études ont exploré l’impact de l’entraînement musical sur la perception de cette illusion qui génère une réorganisation corticale dans les aires associées aux modalités sollicitées. Au niveau comportemental, les musiciens présentent d’ailleurs de meilleures performances dans des tâches impliquant le traitement auditif de la musique. Cependant, il a été rapporté que même parmi les musiciens les plus entraînés, seule une petite minorité percevait le véritable stimulus physique au lieu d’un percept illusoire. Toutefois, les études précédentes n’ont utilisé que les fréquences centrales du spectre musical pour provoquer l’illusion. Une étude récente de notre laboratoire suggère que l’utilisation de paires de fréquences inférieures ou supérieures aux expérimentations antérieures peut moduler l’illusion de l’octave chez les non-musiciens. Il reste à déterminer si cette modulation est identique chez les musiciens. La présente étude avait pour but d’examiner l’impact d’un entraînement musical sur la distribution des percepts sur une gamme fréquentielle plus large.

MÉTHODES

7 paires de fréquences séparées par une octave allant de 40-80 à 2000-4000 Hz ont été présentées à 30 non-musiciens et 9 musiciens entre 18 et 40 ans. Les participants devaient sélectionner un choix de réponse correspondant à leur perception : 1) Un son aigu à droite alternant avec un son grave à gauche; 2) Un son aigu à gauche alternant avec un son grave à droite; 3) Un son passant d’une oreille à l’autre sans changement de hauteur; 4) Aucune de ses réponses. Les réponses ont été divisées en 3 catégories : octave (réponse 1 et 2), simple (réponse 3) et complexe (réponse 4).

RÉSULTATS

Les résultats suggèrent des différences dans la distribution des percepts rapportés en fonction des paires de fréquences utilisées pour susciter l’illusion entre les non-musiciens et les musiciens.

CONCLUSION

Ces données suggèrent que l’entraînement musical a un impact sur la perception de l’illusion et contribue à mieux comprendre l’impact de la réorganisation corticale liée à l’entraînement musical sur la perception auditive lors de la présentation de stimuli ambigu.

Antoine Frasie, Mathieu Bielmann, Nicolas Gauthier, Mourad Roudjane, Hugo Massé-Alarie, Isabelle Pagé, Benoit Gosselin, Jean-Sébastien Roy, Younes Messaddeq et Laurent J. Bouyer . 

CONTEXTE : La lombalgie constitue une charge socio-économique importante et génère le plus grand nombre de demandes d’indemnisation liées à des accidents du travail. Cette affection peut être associée à des modifications de la biomécanique de la colonne vertébrale et à une altération du contrôle moteur, et ces modifications pourraient être secondaires à la fatigue musculaire des muscles stabilisateurs tels que l’erector spinae. De nombreuses études sur ce sujet ont été réalisées dans des environnements contrôlés. Cependant, l’émergence de nouveaux tissus, la miniaturisation des technologies sans fil permettront bientôt de mener des recherches dans des environnements réels.

OBJECTIF : L’objectif de cette étude était de tester si un nouveau type d’électrodes en fibre polymère multi-matériaux et intégrables dans une chemise intelligente était suffisamment sensible pour mesurer la fatigue des muscles erector spinae.

MÉTHODES : deux paires de fibres polymères multi-matériaux ont été placées sur les muscles erector spinae aux niveaux T12-L1 et L4-L5, avec des électrodes commerciales Delsys Trigno. Le test de Biering-Sorensen, permet d’évaluer l’endurance musculaire des muscles extenseurs lombaires lors d’un maintien du tronc horizontal en décubitus ventral contre la gravité jusqu’à épuisement. Une version modifiée avec une contraction correspondant à 40% de la force maximale volontaire (MVC) a été utilisé pour fatiguer les muscles du bas du dos. La MVC a été déterminé à la suite de trois extensions du tronc relié à un dynamomètre fixé au sol. Douze jeunes adultes en santé ont participé à l’étude. La fréquence médiane du spectre de puissance EMG (MF) été mesurée toutes les deux secondes.

RÉSULTATS : Une baisse de MF dans les muscles erector spinae a été observée chez l’ensemble des participants avec des enregistrements utilisables (n=7/12) avec les deux systèmes (p<0.01) sans différence significative entre les systèmes (p>0.05).

CONCLUSION : Les électrodes en fibre polymère multi-matériaux à faible coût et faible puissance sont une source potentielle d’électrodes pour la conception de vêtements intelligents capables de mesurer l’apparition de fatigue dans les muscles du bas du dos. Cela ouvre de nouvelles possibilités de mesure en environnement réel et est potentiellement important pour la prévention des blessures musculosquelettiques et le développement de douleur chronique.

BOURBONNAIS JULIEN¹, Keramidis Iason^1,2, Khademullah Sahara¹, Barbeau Annie¹, De Koninck Yves^1,2 et Godin Antoine^1,2

1. CERVO Brain Research Centre, Quebec Mental Health Institute, Québec City, QC, Canada
2. Department of Psychiatry & Neuroscience, Laval University, Québec, QC, Canada

OBJECTIF – OBJECTIVE

Intracellular chloride regulates the inhibitory response of the γ -aminobutyric acid type-A receptor (GABAAR). In mature neurons, the potassium-chloride co-transporter KCC2 is the principal chloride extruder which maintains low intracellular chloride concentration allowing for an inhibitory response to GABA release. Loss of KCC2 activity has been associated with many neurological disorders such as epilepsy, anxiety, schizophrenia, and chronic pain. We previously showed that membrane KCC2 levels are reduced in medial prefrontal cortex (mPFC) and hippocampal neurons of Alzheimer’s disease (AD) transgenic mice and the motor cortex of ALS mouse models. This study aims to determine whether the KCC2 downregulation observed in AD and ALS mouse models is also associated with a decrease in its function.

MÉTHODES – METHODS

We used a chloride sensor combined with fluorescence lifetime imaging microscopy (FLIM) to monitor intracellular chloride in neurons. This sensor called SuperClomeleon is composed of a pair of fluorescent proteins linked together: Cerulean and YFP chloride sensitive. The lifetime of the donor (Cerulean) decreases when its energy is transferred (FRET) to an acceptor (YFP) and this transfer was shown to depend on local chloride concentrations. Thus, we virally transduced SuperClomeleon in the mPFC and hippocampus of the AD mice and the motor cortex of ALS mice. We then monitored the neuronal intracellular chloride in brain slices while increasing the extracellular KCl concentration to reverse KCC2 transporter function.

RÉSULTATS – RESULTS

We found that, the rate of chloride transport in AD mice was diminished by ~20%. Similarly, a ~25% decrease in the rate of chloride transport was also observed in ALS mice.

CONCLUSION

Our findings confirm that KCC2 downregulation is associated with a decrease of chloride transport and that it plays an important role in the abnormal chloride homeostasis observed in AD and ALS mouse models. This suggests that KCC2 could be a promising pharmacological target to treat neurodegenerative diseases.

Yu Zhang, Louis-Charles Béland, Catherine Bélanger, Noopur Singh, Luc Vallières
Neuroscience Unit, University Hospital Center of Quebec – Laval University, Quebec City, Canada

Introduction
Multiple sclerosis (MS) is an autoimmune disease characterized by demyelination and
neurodegeneration in the central nervous system (CNS). It remains incurable. Experimental
autoimmune encephalomyelitis (EAE) is the most studied mouse model of MS. EAE is mediated
primarily by T cells, which depend on antigen-presenting cells (APCs) to access processed CNS
antigens. Dendritic cells (DCs) are considered the most powerful and professionally equipped APCs.
However, no DC-specific markers have been discovered. This limits our ability to identify DC
subsets and target them for the treatment of MS.
Methods and results
In this study, we used single cell-RNA sequencing (scRNAseq) to compare the transcriptome of
lymph node CD11c+ cells from mice developing EAE. We identified five subsets of DCs, namely
plasmacytoid DCs (pDCs), monocyte-derived DCs (mDC1 and mDC2), and conventional DCs
(cDC1 and cDC2). Using mass cytometry, we validated the identification of novel DCs markers.
Among these cells, only cDC2s expressed the full range of molecules required for priming naïve T
helper cells. To better understand the unique properties of cDC2s, we followed up on Mir155hg,
one of the top 10 mRNAs specifically expressed in cDC2s. To identify potential targets of miR155,
we performed scRNAseq of cDC2 cells from miR155 knockout and wild-type EAE mice.
Surprisingly, we found only one differentially expressed mRNA, called here mRNAX. The capacity
of miR155 to downregulate mRNAX was confirmed in vitro and in vivo.
Conclusion
This study provides new markers to study DC subsets and reveals cDC2s as the main antigen
presenting cells in EAE

HAMEL, JUSTIN P.1.2., Froux, Lionel1., Godin, Antoine G.1.3.

1. Centre de recherche CERVO 2. Département de biochimie, microbiologie et bio-informatique, Université Laval 3. Département de psychiatrie et neurosciences, Université Laval

OBJECTIF – OBJECTIVE

In this project, we aim at developing an intelligent analytical approach to optimize the spatiotemporal binning as well as modulating laser line scanning frequency to better reveal the fast biological processes studied in the central nervous system, in the context of the shift in current polarity of the GABAA receptor during neurodevelopment in primary neuronal cultures.
MÉTHODES – METHODS

Using Fluorescence Lifetime Imaging Microscopy (FLIM), video-rate two-photon laser scanning microscopy and the MQAE chloride indicator, the shift in current polarity of the GABAA receptor during neurodevelopment in primary neuronal cultures is studied by monitoring Cl- concentration upon GABA stimulations.
RÉSULTATS – RESULTS

The number of photons detected is mainly limited by the photophysical properties of the indicator (photobleaching, quantum yield, dynamic range), the expression of the marker, the laser intensity and the timescale of the biological process of interest. Increasing the laser power to detect more photons is limited as it induces photobleaching and phototoxicity.  The accuracy of a FLIM measurement highly depends on the number of photons detected which can be increased by using spatial and temporal binning. Nevertheless, for a given number of photons per analysis, the spatial resolution will be directly linked to chosen temporal resolution chosen and both cannot be optimized concurrently.
CONCLUSION

The strategy developed to optimize spatiotemporal resolution in FLIM is used with MQAE, a Cl- indicator, to study the shift in current polarity of the GABAA receptor with TCSPC, but can easily be transposed to study many other phenomena, other fluorescent indicators and using other FLIM analytical modalities like the phasor approach.

AKBAR IRSHAD, (1), Prenitha Mercy Ignatius Arokia Doss (1), Baillargeon Joanie (1), Mohmed Reda Fazazi (1), Muhammad Umair (1),  Rangachari Manu (1,2)

1. Department of Neurosciences, Centre de recherche du CHU de Québec – Université Laval, Quebec City, QC, Canada; 2. Department of Molecular Medicine, Faculty of Medicine, Université Laval, Quebec City, QC, Canada.

OBJECTIF – OBJECTIVE

Both CD4+ and CD8+ T cells play critical roles in the immunopathogenesis of MS. 1C6 T cell receptor transgenic (TcR-Tg) mice on the nonobese diabetic (NOD) background have a MOG[35-55]-specific, MHC class II-restricted, TcR that selects for both CD4+ and CD8+ T cells. We recently demonstrated that in vitro-differentiated 1C6 CD4 Th1 and Th17 cells can induce a progressive form of experimental autoimmune encephalomyelitis (EAE) upon adoptive transfer to NOD.Scid recipient mice. The objective of this study was to assess whether the same is true for 1C6 CD8+ T cells.

MÉTHODES – METHODS

Naive CD8+ T cells were isolated from 1C6 mice, by MACS column isolation and high-speed cell sorting. Cells were cultured under T cell culture conditions for 5 days, and on day 5, 5×106 cells per mouse were adoptively transferred to NOD. Scid mice. After the adoptive transfer of cells, the mice were monitored for signs of EAE until day 70.

RÉSULTATS – RESULTS

1C6 CD8 T cells differentiated and activated in response to plate bound anti-CD3/CD28 stimulus and also to the MOG35-55-antigen+APCs. Upon adoptive transfer 1C6 Tc1 and Tc17 cells (5×106 per recipient) induced progressive EAE. However, a disease of increased severity and a higher number of CD8+ T cells was seen in the CNS upon co-transfer of Th1+Tc1 (2.5×106 of each per recipient). Intriguingly, ex vivo analysis of the spleens and CNS of Tc1-alone or Tc17-alone recipients revealed the presence of CD4+ T cells. This was observed even in mice receiving CD8+ T cells that were purified by high-speed cell sorting upon initial isolation from 1C6 mice; after in vitro culture but just prior to injection; and also at both time points. In vivo blockade of CD4 reduced not only the presence of CD4+ T cells in the CNS of Tc17 alone recipients but also the presence of CD8+ T cells; the frequency of splenic CD8+ T cells was either increased or not affected. This suggests that CD4/CD8 cooperation is required for the infiltration of the target organ by the latter.

CONCLUSION

Together, our data indicate that the presence of both CD4+ and CD8+ T cells specific for the same CNS antigen is required for optimal CD8+ T cell pathogenicity in an adoptive transfer EAE model.

DIADHIOU SANDRA1,2, Calon Frederic1,2, Darweesh Sirwan3, De Rus Jacquet Aurelie1,4

1Axe Neurosciences, Centre de Recherche du CHU de Québec-Université Laval

2Faculté de Pharmacie, Université Laval

 3Radboud University Medical Centre, Donders Institute for Brain, Cognition and Behaviour, Department of Neurology, The Netherlands.

4 Département de Psychiatrie & Neurosciences, Faculté de Médecine, Université Laval

OBJECTIF – OBJECTIVE

Parkinson’s disease (PD) is a neurodegenerative disorder that causes a selective decline in populations of dopaminergic neurons in the substantia nigra. PD is associated with motor symptoms, but patients also suffer from non-motor symptoms. Current PD medication includes dopamine replacement therapy, but complementary approaches using natural health products could offer promising strategies. Various plant-derived supplements are available over the counter, but there is a lack of insight into PD patients’ interest in using these complementary products. The purpose of this study is to evaluate the prevalence, interest, and determinants associated with the use of natural products to alleviate PD-related symptoms.

MÉTHODES – METHODS

This study is a cross-sectional survey embedded in the ongoing prospective PRIME-NL cohort (Radboud University, the Netherlands), and participants were administered a questionnaire to assess potential determinants of herbal natural product use, interest, and awareness of plant-drug interactions. A number of plant-based products have previously been investigated for their pro-health benefits, and this study included chamomile, guarana, passionflower, St. John’s wort, turmeric, and velvet bean. We also focused on curcumin and resveratrol, two natural products with known in vivo and in vitro neuroprotective properties.

RÉSULTATS – RESULTS

This analysis revealed that 25.5 % of the participants reported using natural health products to mitigate PD-related symptoms. We did not observe a gender effect in the consumption of herbal supplements, however, a greater number of women appeared interested in learning more about the use of herbs to treat PD-related symptoms compared to men (women, 64.4 %; men 41.9 %). Of those who report using herbal supplements, 34.6 % reported being aware of possible herb-drug interactions, and 40.4 % mentioned their use of natural health products to their PD specialists.

CONCLUSION

This study suggests that a subset of patients use natural health products to attenuate PD-related symptoms, and a significant proportion of participants appears interested in learning more about possible herbal supplements to complement their medication. Future studies could use a larger cohort, extend the survey to other countries, and identify which additional supplements are used by PD patients.

Reda Fazazi1, Prenitha Doss1, Resel Pereira2, Neva Fudge3, Irshad Akbar1, Joanie Baillargeon1, Craig S. Moore3, Olga L. Rojas2, Manu Rangachari1.

1Department of Molecular Medicine, Faculty of Medicine, Laval University, Quebec City, CANADA;

2Department of Immunology, University of Toronto, Toronto, ON, CANADA, M5S 1A1; 

3Department of Neurology, Memorial University of Newfoundland, St. John’s, CANADA.

OBJECTIF – OBJECTIVE

Multiple sclerosis (MS) is an autoimmune disorder of the central nervous system (CNS) that has traditionally been considered a T cell-mediated disease. However, accumulating evidence points to a crucial role for B cells in disease processes. Experimental autoimmune encephalomyelitis (EAE) is a well-established mouse model for studying the immune aspects of CNS autoimmunity.

MÉTHODES – METHODS

In order to study the paramount interaction of B and T lymphocytes in disease processes, we immunized transgenic IgH[MOG] mice with the peptide MOG35-55. IgH[MOG] mice possess a transgenic immunoglobulin heavy chain derived from a monoclonal antibody specific to myelin oligodendrocyte glycoprotein (MOG), a key target for autoimmune responses. This mouse model associated with MOG[35-55] autoantigen that drives an mandatory CD4+ T cell-driven response is a useful model to study B and T cell interactions.

RÉSULTATS – RESULTS

We found that immunized IgH[MOG] mice rapidly developed severe EAE. While the frequency and absolute number of CNS-infiltrating B cells was similar between WT and IgH[MOG] mice, a greater frequency of class-switched and inflammatory cytokine-positive B cells were seen in the IgH[MOG] CNS. We observed an increased presence of class-switched and inflammatory cytokine-positive B cells in the IgH[MOG] CNS, as well as a greater frequency of  IL-17- and GM-CSF-producing CD4+ T cells. Production of the Th17 maintenance factor IL-23 was increased from IgH[MOG]CNS-infiltrating B cells, and in vivo blockade of IL-23p19 strongly attenuated disease severity in IgH[MOG] mice. Strikingly, we observed an increased frequency of PD-1+CXCR5- T peripheral helper (Tph)-like cells in the CNS of IgH[MOG] mice and we also found that the meninges of immunized IgH[MOG] mice were characterized by an accumulation of tertiary lymphoid organs. Both Tph accumulation in the CNS, as well as meningeal inflammation, were again sharply reduced upon IL-23p19 blockade in vivo. Intriguingly, the expression of IL23a transcript in the cerebrospinal fluid of MS-affected individuals was positively correlated with the frequency of B cells.

CONCLUSION

Altogether, these data show that MOG-specific B cells contribute to severe CD4+ T cell-driven EAE by promoting CNS accumulation of Th17 and Tph cells, as well as tertiary lymphoid organs in the CNS meninges, in a IL-23 dependent manner.

Maxime Bernard, Romain Menet, Sarah Lecordier, Ayman ElAli

Problems and objectives: Stroke is one of the leading causes of death and disability in the world. Unfortunately, there is currently no cure. Cerebral pericytes are specialized cells that are critically involved in generating neurovascular functions that include maintenance of the blood-brain barrier, stabilization of the vascular system, and immunomodulation. Because of their multifunctional properties, pericytes are a promising target for the development of novel therapeutic interventions for stroke. Platelet-derived growth factor (PDGF)-D (an isoform of PDGF), which specifically activates PDGFRβ in pericytes, could thus modulate vascular system activity. We postulate that PDGF-D plays an important role in regulating pericyte function to mitigate stroke-associated damage.

Methods: Ischemic stroke was induced in C57BL6/J mice by middle cerebral artery occlusion. Mice were treated subacutely intranasally with siRNA or recombinant active peptide of PDGF-D. In addition, primary pericytes harvested from the human brain were exposed to ischemic-like conditions in vitro to identify mechanisms underlying PDGF-D stimulation.

Results: Our results indicate that endogenous PDGF-D is transiently increased in the brain after stroke. Reduced PDGF-D expression exacerbated brain damage after stroke, whereas PDGF-D administration reduced structural damage, promoted by pericyte recruitment associated with improved vascularization of injured tissue, mediated by angiogenic factor release. These changes were accompanied by an improvement in motor recovery.

Conclusion: Our study highlighted that PDGF-D plays a key role in the modulation of pericyte function. Our results indicate that PDGF-D is primarily expressed in vascular structures. Early depletion of PDGF-D exacerbated subacute brain injury. Intranasal administration of PDGF-D improved neurological recovery by promoting stable new vessel formation. PDGF-D promoted the survival of pericytes exposed to ischemic stressors. Our results demonstrate that PDGF-D stimulates neovascularization of ischemic tissue. Therefore, administration of PDGF-D in the brain would be an attractive means of therapeutic angiogenesis after stroke.

MENET ROMAIN1,2, Bernard Maxime1,2, Lecordier Sarah1,2, Aldib Natija1,2, Allain Anne-Sophie1,2, and ElAli Ayman1,2

1,2Department of Psychiatry and Neuroscience, Faculty of Medicine, Neuroscience Axis, Research Center of CHU de Québec – Université Laval, Quebec City, QC, Canada

OBJECTIF – OBJECTIVE

Ischemic stroke constitutes a major cause of death and disability of the adult in Canada. Despite efforts, still no disease-modifying therapy exists. We have previously demonstrated that ischemic stroke deregulates canonical Wnt pathway activity, which plays key roles in regulating neurovascular functions. Interestingly, the levels of Dickkopf-1 (DKK1), an endogenous inhibitor of the pathway, has been shown to be elevated in the blood circulation of stroke patients, correlating with poor neurological outcomes. Yet, how DKK1 affects brain injury and subsequent repair after stroke remains unknown.

MÉTHODES – METHODS

The current study aims to elucidate the role of DKK1 in stroke pathobiology and therapy. For this purpose, conditional mice allowing temporal conditional widespread induction of DKK1 subjected to ischemic stroke via middle cerebral artery occlusion (MCAo) were used to investigate the impact on neurovascular functions. Furthermore, pharmacological approaches were used to neutralize DKK1’s biological activity as a novel approach to stimulate repair and recovery.

RÉSULTATS – RESULTS

Our findings indicate that DKK1 was mainly produced by bone marrow derived cells and its induction prior to stroke exacerbated infarct and oedema sizes, while aggravating motor deficits after stroke. Moreover, DKK1 widespread induction increased neuronal degeneration and synaptic dysfunction, as well as altered neurogenesis and angiogenesis. Interestingly, DKK1 prolonged widespread induction attenuated long-term structural restorative processes and functional recovery, associated with emergence of anxiety-like behavior. These changes were associated with an impaired astroglial scar organization and a chronic neuroinflammatory response. Finally, pharmacological neutralization of DKK1’s biological activity improved structural and functional recovery after stroke.

CONCLUSION

Our results indicate that DKK1 plays a central role in stroke pathobiology and its neutralization constitutes a clinically relevant approach to improve structural repair and neurological recovery after stroke.

Desmeules C1-3, Anderson E2,4, Huot-Lavoie M1,2, Corbeil O2,5,6, Béchard L2,5,6, Essiambre A-M2,4, Claveau C2, Thériault C2, Lehoux C2,4,7, Deslauriers J3,5, Roy M-A1,2,6, Demers M-F2,5,6

1. Faculté de médecine, Université Laval;
2. Centre de recherche CERVO;
3. Centre de recherche du CHU de Québec, Université Laval;
4. Faculté des sciences sociales, Université Laval;
5. Faculté de pharmacie, Université Laval;
6. Institut universitaire en santé mentale de Québec, CIUSSS-CN ;
7. Hotel-Dieu de Levis, CISSS Chaudière-Appalaches

Introduction : Le trauma psychologique est un facteur de risque environnemental connu des troubles psychotiques (TP). La neuro-inflammation est un phénomène observé tant dans la pathophysiologie des TP que dans la réponse au trauma. Cependant, les mécanismes qui sous-tendent ces associations, et comment l’historique de trauma interagis avec les fonctions immunitaires dans les TP restent incompris. Récemment, un intérêt grandissant a été porté sur les exosomes dérivés du cerveau (EDC), qui sont des vésicules qui transportent du cargo inflammatoire et qui traversent la barrière hématoencéphalique pour atteindre le sang. Facilement accessibles par une prise de sang, les EDC sont des outils puissants pour identifier des biomarqueurs spécifiques aux troubles psychiatriques.

Objectifs : Les objectifs de l’étude sont d’évaluer l’association entre les marqueurs inflammatoires des exosomes dérivés du cerveau et : (1) la sévérité des symptômes psychotiques; et (2) l’historique de trauma.

Méthode : 50 participants sains et 50 participants atteints de premier épisode psychotique seront recrutés. Les participants seront divisés en groupe avec et sans historique de trauma. Les participants seront évalués à deux reprises avec 6 mois d’intervalle. Des données sociodémographiques, psychiatriques et d’historique de traitements, de sévérité de la symptomatologie et d’historique de trauma seront recueillies en plus d’un prélèvement sanguin pour l’analyse des exosomes.

Retombées potentielles : En utilisant une technique innovante et non invasive, l’étude présente une opportunité unique pour l’évaluation longitudinale de marqueurs inflammatoires spécifiques aux changements du cerveau en association avec la pathophysiologie des TP. En considérant l’effet de l’historique de trauma, cette étude est cruciale pour permettre un diagnostic et un traitement plus précis des troubles psychotiques.

NASRALLAH LEILA 1,2 , Bernard Maxime 1,2, Menet Romain 1,2, ElAli Ayman 1,2

1 Neuroscience Axis, Research Center of CHU de Québec – Université Laval, 2705 Laurier Boulevard, Quebec City, QC G1V 4G2, Canada

2 Department of Psychiatry and Neuroscience, Faculty of Medicine, Université Laval, Québec City, QC, Canada

OBJECTIF – OBJECTIVE

Ischemic stroke constitutes a major cause of death and disability of the adult in Canada. Upon stroke, pro-angiogenic responses are activated as an attempt to promote tissue vascularization and subsequent repair. Elevated vascular endothelial growth factor (VEGF) levels prior to stroke are associated with attenuated infarct progression due to the formation of collateral vasculature. This suggests that neo-vascularization constitutes a promising therapeutic strategy. Nonetheless, administration of the potent angiogenic factor VEGF-A increases the risk of vascular destabilization. VEGF-E, a VEGF-A-like isoform encoded by Orf virus, has been shown to stimulate the formation of stable cutaneous vasculature network via activation of divergent signaling pathways in comparison to VEGF-A.

MÉTHODES – METHODS

We aimed to evaluate the therapeutic potential of VEGF-E in stroke. For this purpose, C57BL6/J mice subjected to ischemic stroke via transient middle cerebral artery occlusion (MCAo) were intranasally infused with VEGF-E 24 and 72 hours after stroke onset and euthanized 24 hours after last infusion.

RÉSULTATS – RESULTS

VEGF-E reduced infarct size and neuronal degeneration as well as improved neurological recovery. These changes were associated with an increased microvascular density without compromising permeability. Interestingly, VEGF-E enhanced the density of pericytes localized at the vasculature within the lesion site. The attenuated neuronal damage upon VEGF-E infusion was accompanied with a reduced reactivity of astrocytes in the peri-infarct region.

CONCLUSION

Our results suggest that VEGF-E infusion constitutes a promising approach to reduce sub-acute damage after stroke via promotion of angiogenesis while conserving microvascular integrity.

Laila Blanc Árabe; Benoit Labonté

The cortico-accumbal and cortico-tegmental pathways are involved in the expression of anxiety- and depressive-like behaviors induced by chronic stress. Previous studies indicate that chronic variable stress (CVS) induces morphological and functional changes in a pathway- and sex-specific fashion. Here, we investigated the upstream neural circuitry of both pathways to understand which inputs are affected by stress and may contribute to those effects observed on cortico-accumbal and tegmental pathways in males and females.

We used a 21-day CVS model to stress female mice. Inputs targeting specifically neurons from the medial prefrontal cortex (mPFC) projecting either to the nucleus accumbens (NAc) or ventral tegmental area (VTA) were identified through a trans-sectional viral approach combined with rabies trans-synaptic tracing. Neural activity patterns in stressed and control mice were assessed via c-Fos immunohistochemistry. We used whole brain light sheet microscopy to unbiasedly map every neuronal input projecting to either NAc- or VTA-projecting cells along with their activity patterns.

Our results show that the largest proportion of inputs to cortico-accumbal and tegmental pathways come from cortical areas. For instance, cortico-tegmental neurons are targeted mainly by inputs from the insular cortex, whereas the orbital area projects more densely to NAc-projecting neurons. Limbic and brain stem areas such as the entorhinal area, periaqueductal grey, and amygdala (AMY) also project densely to both pathways. We also identified retro-projections from the VTA and NAc to the cortico-tegmental and accumbal pathways respectively. Our analysis of c-Fos expression in stressed mice showed pathway-specific activation patterns. For instance,c-Fos expression was lower in neurons from the insular cortex and higher in neurons from the basolateral AMY projecting to cortico-tegmental neurons in stressed female mice. In contrast, higher c-Fos expression was found in neurons from the orbito-frontal area projecting on cortico-accumbal neurons.

Overall, our findings map the neuronal circuits projecting on cortico-accumbal and tegmental pathways. Importantly, our analyses also revealed how stress impacts the activity of these circuits which may contribute to the morphological and functional changes affecting mPFC neurons projecting either to the NAc or the VTA and ultimately to the elaboration of stress responses

MICAËL CARRIER1,2,3, Chin W. Hui3, Valerie Watters3, Eva Simoncicova2, Katherine Picard2,3,4, Fernando Gonzalez Ibanez2,3,4, Nathalie Vernoux3, Arnaud Droit3,4, Michèle Desjardin5,6 and Marie-Ève Tremblay2,3,4,7,8

1 Department of Psychiatry and Neuroscience, Faculty of Medicine, Université Laval, Quebec, QC, Canada

2 Division of Medical Sciences, University of Victoria, Victoria, BC, Canada.

3 Centre de recherche du CHU de Québec-Université Laval, Québec, QC, Canada.

4 Département de médecine moléculaire, Faculté de médecine, Université Laval, Québec, QC, Canada.

5Department of Physics, Physical Engineering and Optics, Université Laval, Québec City, QC, Canada

6Oncology Axis, Centre de Recherche du CHU de Québec, Université Laval, Québec City, QC, Canada

7 Department of Biochemistry and Molecular Biology, The University of British Columbia, Vancouver, BC, Canada.

8 Department of Neurology and Neurosurgery, McGill University, Montréal, QC, Canada.

OBJECTIF – OBJECTIVE

Considering that genetic variants affecting the function of fractalkine signalling between neurons and microglia were linked to schizophrenia, we compared the outcomes of a well characterized model of maternal immune activation using the viral mimetic polyinosinic:polycytidylic acid (Poly I:C) in wild-type versus fractalkine receptor knockout mice.

MÉTHODES – METHODS

Possible behavioral and immune alterations were assessed in male and female offspring at adulthood. Considering the role of the hippocampus in schizophrenia, microglial analyses and bulk RNA sequencing were performed among the hippocampus to assess the neuroimmune dynamics at play.

RÉSULTATS – RESULTS

Offspring exposed to the dual challenge paradigm exhibited many positive symptoms relevant to schizophrenia and mood disorders which differed between males and females. Males displayed social disconnection and cognitive deficits while females mainly presented anxiety-like behaviors. Hippocampal microglia in the females exposed to the dual challenge were hyper-ramified while those in males showed on the other end of the morphological spectrum hypertrophic morphologies indicative of increased surveillance and metabolic activity. Hippocampal bulk-RNA sequencing further revealed downregulation in females of genes related to GABAergic transmission, which represents one of the main proposed causes of mood disorders.

CONCLUSION

Building on previous results, we identify in the current study specific behavioral phenotypes in female mice experiencing a dual genetic and environmental challenge, thus revealing a novel model of mood disorder. This opens the field to new investigations into the susceptibility to stress using a model based on genetic and immune vulnerability as presented here.

Marco D’Angelo and Benoit Labonté

CERVO brain research center

The medial prefrontal cortex (mPFC) is a major hub in the control and elaboration of stress responses. It does so by integrating and processing information from different brain regions before redirecting it to limbic structures controlling different aspects of emotional behaviors. Somatostatin (SST) interneurons and parvalbumin (PV) maintain a homeostatic balanced activity in the mPFC by controlling the inputs and the output of pyramidal cells, respectively. Work from our group suggests that chronic stress impairs the excitatory and inhibitory (E/I) balance in the mPFC. Here, we tested whether chronic stress changes with the morphological and functional properties of SST and PV GABAergic interneurons to disrupt the E/I balance in the mPFC of male and female mice.

We used 21 days of chronic variable stress (CVS) to induce an emotional stress response in PV-cre and SST-flpo transgenic mice. A trans-sectional viral approach was used to label SST and PV cells and assess neuronal complexity and functional alterations in stressed male and female mice. Connectivity and functional properties of both SST and PV GABAergic interneurons in male and female mice were addressed using the mGRASP viral approach.

Our results show a reduction in the complexity of SST interneurons in both male and female stressed mice compared to controls. On the other hand, we observed an increase in the number and complexity of PV interneurons in stressed female mice compared to controls, while stressed males exhibited reduced complexity compared to controls. Interestingly, this was accompanied by a concomitant increase in the dendritic arborization of PV interneurons in stressed female mice, while stressed male mice showed reduced dendritic arborization. SST interneurons showed a reduction of the dendritic arborization in stressed males and females compared to controls.

Our results provide a better understanding on how chronic stress might affect morphological and functional properties of SST and PV interneurons and their relationship with excitatory pyramidal neurons in modulating behavioral stress responses in a sex-specific way.

ANNE-MARIE CASTONGUAY1, Béatrice Morin1, Thomas Durcan2, Claude Gravel1 & Martin Lévesque1.

1 CERVO Brain Research Center, Laval University, Québec, Qc, Canada

2 The Neuro, McGill University, Montréal, Qc, Canada

Parkinson’s disease (PD) in mainly characterized by the degeneration of dopaminergic neurons from the substantia nigra. Degenerating neurons present intracytoplasmic inclusions called Lewy bodies, that are mainly composed of the protein alpha-synuclein (aSyn). In those insoluble aggregates, aSyn is found hyperphosphorylated and misfolded. Evidence in various animal and cell models suggest that pathological alpha-synuclein (aSyn) can be transmitted from cell-to-cell in a prion-like manner. Blocking either the aggregation process or the transmission of the pathological protein across the brain could therefore be effective therapeutic strategies. We generated small antibodies in the form of secreted single-chain variable fragments (scFv) and intracellular antigen-binding fragments (Fab) that bind to aSyn. We also generated a control antibody that targets GFP. We validated the specificity of our antibodies with dot blots and immunoprecipitation and identified one scFv specific for phosphorylated aSyn, and one recognizing both normal and phosphorylated aSyn. We also verified the efficiency of our intracellular Fabs to bind and degrade aSyn in vitro using HEK cells co-transfected with aSyn and by measuring the aSyn protein level. Our Fabs significantly reduced aSyn protein level compared to the control antibody. We then encoded the single-chain antibodies in an adeno-associated viral vector (AAV) of a serotype that can cross the blood-brain barrier when injected intravenously (IV). After IV injection, we verified the expression of the antibodies in the brain with immunohistochemistry for the Myc tag expressed by the antibodies. To model aSyn pathology spreading, we injected human pre-formed fibrils (PFF) of aSyn in the dorsal striatum of transgenic mice overexpressing human A53T-aSyn (M83) and 7 days later we injected IV the AAV encoding the aSyn scFvs or control scFv. Control groups were also injected with PBS instead of PFF. We closely monitored the motor impairments arising after the injections with a scoring system. Results show a strong protective function of the scFvs against motor impairments and aSyn brain pathology. In conclusion, we developed scFvs and Fabs targeting phospho- or total aSyn and proved that they engage their target. Our scFvs also show a great potential in reducing aSyn pathology in vivo in an aggressive PD mouse model.

MARROQUIN Arturo – CERVO brain research center

Droit Arnaud – Research center of CHU de Québec Université Laval

Labonté Benoit – CERVO brain research center

Objectif

To cope with external harmful stimuli, the immune system responds via inflammation. Although, the latter is mostly a protective feature, it also plays a role in the occurrence of disease across organs, including the brain. One cell-type implicated in chronic neuroinflammation is microglia. These cells can have pro or anti-inflammatory activity, phenotypes partly induced by epigenetic mechanisms, however, little is known about this process. To give insights into the role of histone modifications (HM) in the regulation of the neuro-inflammatory response and, specifically, in microglial programming, we aimed to explore the dynamics of the epigenetic landscape during inflammation and its relationship with the inflammatory transcriptomic profile.

Methods

We exposed cells to lipopolysaccharide (LPS) and, through a CUT&RUN methodology, profiled four genome-wide HMs (H3K4me1, H3K4me3, H3K27ac and H3K27me3). The distributions were then compared to control cells exposed solely to saline solution. Finally, the inflammatory transcriptomic profile was determined through RNA-seq and differentially expressed genes were identified and contrasted with the epigenetic landscapes. Other downstream analyses were also included in this study (MA, chromHMM, GSEA, etc.).

Results

We found the combination of H3K4me1, H3K27Ac and H3K4me3 to be the most common state across the genome, whereas only the two latter marks constitute the state most frequently seen in promoters (PR). Regarding epigenetic configuration induced by LPS, we found that the differentially bound “active” regions were strongly described by immunological-related ontology terms (“defense to”, “response to”). Additionally, the association between differentially expressed genes involved in immune response and chromatin organization and HM patterns was assessed. Strong correlation between repressive H3K27me3 and lower gene expression and active marks and higher expression was observed. Conversely, H3K4me3 and H3K27ac were frequently observed in PR of expressed genes.

Conclusion

This study exhibits important differences in the distribution of HM in BV2 cells, constituting an epigenetic reconfiguration that leads to the inflammatory response. Also, it highlights the importance of HM regulatory role in gene expression and provides possible targets for further studies in the context of inflammation.

Dovonou Axelle, Yves De Koninck, Emmanouil Metzakopian, et Lévesque Martin.

Centre de recherche CERVO , Département de psychiatrie et neuroscience, Faculté de médecine, Université Laval.

La maladie de Parkinson est l’un des désordres neurodégénératifs les plus fréquent, touchant environ 1% de la population de 60 ans et plus.  Elle est caractérisée par la mort des neurones dopaminergiques (nDA) de la substance noire compacte (SNc) entrainant la majorité des symptômes de la maladie. Il est à présent connu que le stress oxydatif, des défauts mitochondriaux ou encore la toxicité d’agrégats protéiques, en particulier l’alpha-synucléine (α-syn), sont associés à la mort des nDA.  Cependant, les mécanismes sous-jacents de cette mort neuronale restent nébuleux et il n’existe encore aucun remède. Il est donc urgent de développer un traitement curatif qui contrera la maladie.

Notre objectif est donc d’identifier de nouvelles cibles thérapeutiques contre la maladie de Parkinson. Dans ce but, nous avons procédé à un criblage génétique en appliquant une librairie CRISPR-CAS9. La survie de nDA à la neurotoxine Roténone nous a permis d’identifier de nouveaux gènes cibles afin de protéger contre le stress oxydatif et le dysfonctionnement mitochondrial. L’un de ces candidats fait partie des acteurs de la réparation de l’ADN, la ligase à ADN IV (Lig4). La suppression de Lig4 abolit la toxicité mortelle induite par la Roténone dans les nDA in vitro. Afin de valider Lig4 comme cible pour la neuroprotection des nDA, nous avons supprimé Lig4 in vivo chez la souris adulte. Nous injectons un vecteur AAV2 encodant pour deux ARN guides dirigés contre Lig4, dans une souris exprimant Cas9 spécifiquement dans les nDA du mésencéphale. À la suite de la suppression de Lig4, nous induisons un phénotype parkinsonien grâce à la 6-hydroxydopamine (6-OHDA) ou par la surexpression virale de la forme mutée de l’α-syn (A53T). Une série de tests comportementaux et une analyse stéréologique nous ont permis de confirmer les résultats préalablement observés in vitro. Les nDA présentent une plus grande survie à l’exposition au 6-OHDA et à l’a-syn A53T lorsque Lig4 est absente. De plus, nous observons une réduction des niveaux d’α-syn pathologique et des dommages à l’ADN. Nos résultats suggèrent que la suppression de Lig4, à la foi in vitro et in vivo, semble avoir un effet protecteur dans les nDA. L’inactivation de Lig4 pourrait donc constituer un nouveau traitement neuroprotecteur contre la maladie de Parkinson.

MOREIRA PESSONI, ANDRE – Cervo Brain Research Centre
Mansouri, Samaneh – Cervo Brain Research Centre
Zhao, Chenqi – Cervo Brain Research Centre
Geoffroy, Olivier – Cervo Brain Research Centre
Marroquin Rivera, Arturo – Cervo Brain Research Centre
Droit, Arnaud – Research Centre of CHU de Québec-Université Laval
Labonté, Benoit – Cervo Brain Research Centre

OBJECTIF – OBJECTIVE

The transcriptional organization of gene networks is affected differently in males and females with major depressive disorder (MDD), and alternative splicing (AS) produces distinct mRNA molecules encoding proteins with different functional properties. Alternatively spliced variants could contribute to the functional diversity of genes involved in different fundamental processes across several tissues, including brain regions in males and females. Here, we quantified the exon expression and characterized the alternative splicing events across brain regions of males and females with MDD.

MÉTHODES – METHODS

A genome-wide gene expression dataset from 6 brain regions that included more than 600 human post-mortem samples from males and females with and without MDD was processed, and exon-based (Limma package) and event-based approaches (rMATS) were used to quantify the differential expression of exons and several AS features, respectively. qPCR was used to quantify the expression of a few differentially expressed exons to validate the preliminary findings.

RÉSULTATS – RESULTS

Our results suggest that the representation of exons differs significantly between males and females across all 6 brain regions. In the dorsolateral prefrontal cortex (dlPFC), we identified 1650 downregulated and 1584 upregulated exons in females with MDD, and 841 downregulated and 467 upregulated exons in males with MDD. We confirmed via qPCR that the exon 2 from gene EGR4 was downregulated in both sexes in this brain region. We also identified striking differences between AS events, in which females with MDD showed an increase of total events when compared to males with MDD. In dlPFC, increased exon skipping, alternative splice site and intron retention were found in females with MDD (i.e., SCN1A-AS1 and CTSL), while males with MDD showed larger amount of mutually exclusive exons.

CONCLUSION

Our analysis suggests that the representation of exons and splicing events differs significantly in both males and females across all studied brain regions, indicating the existence of important sex differences. This understanding could elucidate the molecular mechanisms underlying the expression of depression and its related symptoms in a sex-specific fashion, to ultimately improve treatment efficacy.

MORIN, BÉATRICE1, Castonguay, Anne-Marie1, Obergasteiger, Julia1, Cardoso, Tiago1, Watters, Valérie2, Gravel, Claude1, Hussein, Samer2, Durcan, Thomas3, Lévesque, Martin1

1. CERVO Brain Research Center, 2601, chemin de la Canardière, Québec, QC, Canada
2. St. Patrick Research Group in Basic Oncology, Oncology Division of CHU de Québec-Université Laval Research Center, 9, Rue McMahon, Québec, QC, Canada
3. The Neuro – Montreal Neurological Institute, 3801, Rue University, Montréal, QC, Canada

PD is a chronic neurodegenerative disease characterized by the extensive loss of dopamine (DA) neurons in the substantia nigra pars compacta through the accumulation of intraneuronal Lewy bodies containing misfolded fibrillar alpha-synuclein (aSyn). These aggregates are toxic and can spread the pathology to neighboring cells, contributing to disease progression. Neuroprotective therapy based on mini antibodies, such as single-chain variable fragment (scFv) is a promising treatment. They are efficient at an early stage of the disease when neurodegeneration is not too advanced. Cell replacement therapy is then an attractive option for more advanced PD to restore dopaminergic innervation. Although recent advances facilitate cell replacement therapy for PD, there are still hurdles to overcome. One major challenge is the survival of the grafted neurons in a brain environment containing toxic protein aggregates. Analysis of post-mortem brains from PD patients revealed that the grafted neurons acquire aSyn pathology, thus limiting their efficacy and survival, especially in the long term. OBJECTIVE Our main objective is to develop strategies to promote survival of transplanted DA neurons to efficiently restore the DA deficiencies. Here, we tested the efficacy of secreted and non-secreted scFvs against aSyn to protect human induced pluripotent stem cell-derived neurons from the spread of pS129aSyn pathology. METHODS To model PD, we used unilateral striatal injection of aSyn preformed fibrils (PFF). To control the production of our scFvs, we used the TeT-ON system. This system allows the expression of the scFv only in the presence of tetracycline or one of its analogues, such as doxycycline (DOX). To evaluate the efficiency of scFv in protecting grafted cells, we transplanted differentiated DA neurons into mice previously injected with PFF. DOX was then added in water to activate scFv transcription. RESULTS Our preliminary data suggest that our scFvs can protect the graft from aSyn pathology. CONCLUSION We believe that this neuroregenerative approach will lead to a significant advance in cell replacement therapies for PD.

MAROIS ÉLODIE (1, 4), Belleville Sylvie (2, 3), Potvin Olivier (4), Macoir Joël (4, 5), Hudon Carol (1, 4)

1 École de psychologie, Université Laval, Québec, Canada
2 Centre de recherche de l’Institut universitaire de gériatrie de Montréal, Montréal, Canada
3 Département de psychologie, Université de Montréal, Montréal, Canada
4 Centre de recherche CERVO, Québec, Canada
5 Département de réadaptation, Université Laval, Québec, Canada

OBJECTIF :

Le sous-test de rappel d’une histoire de la Batterie d’Efficience Mnésique (BEM-144) est un outil permettant d’évaluer la mémoire épisodique verbale chez les adultes et les personnes âgées. Il n’existe cependant pas de données normatives adaptées à la population franco-québécoise. L’objectif de cette étude était donc de développer des normes pour le sous-test de rappel d’histoire de la BEM-144 pour les Québécois francophones de 50 ans et plus.

MÉTHODES :

L’échantillon normatif comportait 260 participants franco-québécois en bonne santé âgés de 50 à 90 ans. Des analyses ont été effectuées pour estimer l’influence des caractéristiques sociodémographiques, soit l’âge, le sexe et le niveau de scolarité, sur les scores aux rappels immédiat et différé.

RÉSULTATS :

L’âge, le sexe et le niveau de scolarité étaient significativement associés à la performance aux rappels immédiat et différé de l’histoire. Des équations de régression pour le calcul des scores Z ont été créées pour les scores aux deux types de rappels.

CONCLUSION :

Les données normatives de ce sous-test seront utiles aux cliniciens afin de mieux évaluer la mémoire épisodique chez la population franco-québécoise de 50 ans et plus, favorisant ainsi une détection plus précoce des troubles cognitifs pour encourager une prise en charge plus rapide des patients.

MUNRO JONATHAN, Desmeules Francis, Cottin Sylvine Carrondo, Noecker Angela, Tremblay Marie-Ève, Gould Peter V,
Saikali Stephan, Langlois Mélanie, McIntyre Cameron C, Prud’homme Michel, Cantin Léo and Parent Martin. CERVO brain research centre.

OBJECTIF – OBJECTIVE

Deep brain stimulation (DBS) of the subthalamic nucleus (STN) is an effective surgical treatment for Parkinson’s disease (PD), alleviating motor symptoms and restoring patients’ quality of life. However, research into the long term effects of DBS on the human brain is lacking. This study aims to investigate the neuroanatomical and neurochemical alterations induced by chronic stimulations of the STN, and to correlate these changes with clinical outcomes and estimated electrical current delivered in the brain parenchyma.

MÉTHODES – METHODS

Brains of PD patients who had received more than 9 years of DBS treatment in the STN were used. For each brain, 3D graphical representations of the basal ganglia (BG) and DBS electrode were produced to determine the electrical current propagation using a patient-specific computational model (StimVision2). Immunofluorescence and confocal microscopy were used to determine the immunoreactivity of various proteins within the basal ganglia. Stereological quantification and morphological analyses of different cell types and blood vessels were performed.

RÉSULTATS – RESULTS

Near active contacts, elevated levels of GLUT1 were observed, indicating increased blood vessel growth. Additionally, IBA1+ microglia count was unchanged while CD68 expression was reduced suggesting no inflammatory response. However, detailed morphological analysis of microglia indicates a more active state near the stimulated area of the STN.

CONCLUSION

Our post-mortem analysis indicates changes induced by long-term DBS of the STN involving glial cells and blood vessels a which do not appear to be detrimental to the patient.

Alice Cadoret, Laurence Dion-Albert, Sara Amrani, Laurianne Caron, Mathilde Théberge, Audrey Turmel, Manon Lebel, Caroline Menard

Experiences are linked to emotions impacting memory consolidation and associated brain neuronal circuits. Posttraumatic stress disorder is an example of strong negative emotions affecting memory processes by flashbacks of past traumas. Stress-related memory deficits are also observed in major depressive disorder (MDD). We recently highlighted that sex-specific blood-brain barrier (BBB) alterations underlie stress responses in mice and human depression. However, little is known about the relationship between emotional valence, memory encoding and BBB function. Here, we investigated the effects of novel object recognition (NOR) test, an experience considered of neutral emotional valence, on BBB properties in dorsal vs ventral hippocampus in the context of various environmental conditions (arena size, handling, age). The hippocampus is a brain area central for learning and memory processes with the dorsal and ventral subregions being associated with working memory vs reference memory retrieval, respectively. Expression of genes related to BBB integrity are altered in line with learning and memory processes in a region- and sex-specific manner. We observed correlations between poor learning, anxiety, stress-induced corticosterone release and changes in BBB-associated gene expression. Comparison of BBB transcriptomes between sexes also revealed profound differences at baseline in both ventral and dorsal hippocampus. Finally, we identified circulating vascular biomarkers, such as sE-selectin and Mmp-9, altered following NOR exposure supporting that recognition memory formation has an impact on the neurovasculature. Although deemed as a neutral valence test, NOR experimental conditions impact performance, highlighting the need to minimize anxiety when performing this commonly used test in mice.

SAIDI LYDIA1, Rajan Sandeep Sundara2, Pozzi Silvia1, Metzakopian Emmanouil2, Proulx Christophe1, Parent Martin1*

1 CERVO Brain Research Center, Université Laval, QC
2 UK Dementia Research Institute, University of Cambridge, UK

OBJECTIF – OBJECTIVE Parkinson’s disease is mainly characterized by the progressive loss of midbrain dopaminergic neurons that innervate the striatum. The dopamine precursor L-3,4-dihydroxyphenylalanine (L-Dopa) is the most effective pharmacotherapy but its chronic use is hampered by side effects such as abnormal involuntary movements (AIMs), also termed L-Dopa-induced dyskinesia (LID). Studies have shown the crucial role of serotonin (5-HT) neurons in the conversion of exogenous L-Dopa and LID expression. Through this study, we specifically addressed the functional role of glutamate co-transmission by 5-HT neurons of the dorsal raphe nucleus (DRN) in the regulation of motor behavior and LID expression.

MÉTHODES – METHODS We used CRIPSR-Cas9 technology and viral injections to knock out or overexpress the atypical vesicular glutamate transporter 3 (VGluT3), specifically in the DRN 5-HT neurons of adult mice. Two weeks later, mice were injected with 6-OHDA in the medial forebrain bundle to selectively damage dopaminergic axons and then treated with L-Dopa to induce AIMs.

RÉSULTATS – RESULTS Post-mortem analysis confirmed the depletion or overexpression of VGluT3 in AAV-infected 5-HT neurons of the DRN as well as severe dopamine denervation. After dopamine lesion and L-Dopa administration, VGluT3-depleted mice show exacerbated AIMs following the administration of a low dose of L-Dopa (1mg/kg), compared to controls and transgenic mice overexpressing VGluT3. At higher L-Dopa doses (3, 6, 12 mg/kg), mice overexpressing VGluT3 show higher severity of the orofacial AIMs subtype.

CONCLUSION Glutamate that is co-released by 5-HT neurons of the DRN appears to be involved in the expression of LID.

Hayatte-Dounia Mir1*, Alexandre Milman2, Magali Monnoye1, Véronique Douard1, Catherine Philippe1, Agnès Aubert1, Nathalie Castanon3, Sylvie Vancassel3, Nathalie C. Guérineau2, Laurent Naudon1,4, Sylvie Rabot1

1 Université Paris-Saclay, INRAE, AgroParisTech, Micalis Institute, Jouy-en-Josas, France
* Université Laval, Département de Médecine, CRIUCPQ, Québec, Canada
2 IGF, Univ. Montpellier, CNRS, INSERM, Montpellier, France
3 Université de Bordeaux, INRAE, UMR NutriNeurO, Bordeaux, France
4 CNRS

hdmir@ulaval.ca

OBJECTIVE. Several studies have brought forward that the intestinal microbiota could be involved in Major depressive disorder (MDD). Different pathways between the central nervous system and the gut have been identified and used by either bacterial components or bacterial metabolites. Recently, we have shown that an overproduction of indole, a bacterial metabolite produced in the gut from dietary tryptophan, induces anxiety-like and depressive-like behaviors. We aim to better understand the implication of indole in the pathophysiology of MDD and elucidate whether it triggers and/or aggravates this psychiatric condition.

METHODS. We colonized germ-free mice with a wild-type indole-producing Escherichia coli strain, or with the non-indole producing mutant. Gnotobiotic mice were subjected to an unpredictable chronic mild stress (UCMS) procedure, then to a set of tests aimed at assessing anxiety-like and depression-like behaviors. We also carried out biochemical and molecular analyses in gut mucosa, plasma, brain and adrenal glands, to examine biological correlates that are usually associated with stress, anxiety and MDD.

RESULTS. Chronic indole production by the gut microbiota increased the emotional response of mice to the adverse effects of chronic mild stress. On the same mice, indole induced a dramatic increase of the expression of the adrenomedullary Pnmt gene, which is involved in catecholamine biosynthesis. Interestingly, this occurred without any low-grade inflammation or any change in the host tryptophan metabolism pathways.

CONCLUSION. Chronic indole production by the gut microbiota increased the vulnerability of mice to the adverse effects of chronic mild stress on emotional behaviors. It also targeted catecholamine biosynthetic pathway of the adrenal medulla, which plays a pivotal role in body’s physiological adaptation to stressful events.

PATON SAM1, Richer Émanuelle1, Coulombe-Rozon François1, Dion-Albert Laurence1, Lebel Manon1, & Menard Caroline1

1. Department of Psychiatry and Neuroscience, Faculty of Medicine and CERVO Brain Research Center, Université Laval, Quebec City, QC, Canada

OBJECTIF – OBJECTIVE

Major depressive disorder (MDD) is a leading cause of disability, affecting more than 300 million people worldwide. However, current antidepressants are largely ineffective, with 30-50% of patients experiencing treatment resistance. Further, the symptoms and underlying biology of MDD are strikingly different in males and females, highlighting the need for sex-specific antidepressant approaches. Alternate therapeutic strategies could potentially target the blood brain barrier (BBB), which is damaged in people with MDD by elevated peripheral inflammation associated with chronic stress. The BBB, a highly selective membrane consisting of specialized tight junctions between endothelial cells lining blood vessels in the brain, is damaged in different brain regions in males and females with depression, representing a sex-specific antidepressant target. In humans, socioeconomic status is negatively correlated with depression risk, while in mice access to a nest, house, and chew toy (enriched environment, EE) promotes resilience to chronic stress; however, it is unclear whether these approaches improve underlying BBB damage. Here, we show that access to EE during appropriate chronic stress models for male and female mice prevents onset of behavioural and BBB deficits.

MÉTHODES – METHODS

To address this question, we employ two mouse models of stress induced depression: chronic social defeat stress (CSDS, males) and chronic variable stress (CVS, females). Behavioural tests, gene expression analysis, and immunofluorescent staining was used to characterize stress-induced changes in the brain.

RÉSULTATS – RESULTS

Once we confirmed protective effects of EE against stress-induced behavioural deficits in both sexes, gene expression analysis revealed that EE substantially modifies stress-induced transcription in the BBB. Importantly, EE rescues stress-induced downregulation of Cldn5, an important tight junction protein, in a sex- and region-dependent manner. Immunofluorescent staining showed discrepancies between gene and protein expression of Cldn5 suggesting that EE protects against some, but not all, pathways of stress-related BBB damage.

CONCLUSION

Future work will delve into molecular signalling pathways underlying these pro-resilient effects, in order to identify pharmacological targets.

MARTINIE OPHELIE1,2, Karan Philippe3, Mercier Catherine1,2, Descoteaux Maxime3, Robert T Maxime1,2 

1 Centre Interdisciplinaire de recherche en réadaptation et intégration sociale (Cirris), Québec, QC, CAN

2 Département de réadaptation, Université Laval, Québec, QC, CAN

3 Département d’informatique, Université de Sherbrooke, QC, CAN

INTRODUCTION : La paralysie cérébrale (PC) est un trouble neuromoteur caractérisé par des lésions neurologiques chez le fœtus ou à la naissance. Ces lésions neurologiques altèrent différents réseaux, notamment ceux impliqués dans le contrôle moteur. Peu d’études décrivent les réseaux de matière blanche tels que le système cortico-cérébelleux avec une méthodologie rigoureuse permettant une correction et un pré-traitement avancé des données de neuroimagerie, en paralysie cérébrale.

OBJECTIF : L’objectif est de tester une méthodologie robuste pour identifier et analyser le système cortico-cérébelleux chez des enfants avec la PC.

MÉTHODES : Six enfants avec la PC âgés de 8 à 12 ans ont passé une imagerie par résonance magnétique de diffusion (Philipps, 3T). Les images ont été corrigées et analysées grâce à Tractoflow, un outil de pointe dans l’analyse et le prétraitement de données de neuroimagerie. Le réseau cortico-cérébelleux a été tractographié grâce à des outils standardisés comme ANTs et la libraire Python Scilpy.

RÉSULTATS : Les résultats montrent qu’il est possible de reconstruire le système cortico-cérébelleux chez les enfants avec la PC avec une approche rigoureuse et d’en extraire différentes variables d’intégrité.

CONCLUSION : Les résultats contribueront à mieux décrire les réseaux de matière blanche dans la PC pour mieux comprendre les symptômes sensorimoteurs. Ces résultats pourront être corrélés avec des mesures comportementales dans l’ultime but d’améliorer les interventions.

GELON PAULINE, sephton chantelle, CERVO

BACKGROUND

ALS/FTD is a multisystem neurodegenerative disease that causes loss of motor function and lethality. In 10% of familial ALS cases, mutations in the gene coding for the DNA/RNA-binding protein fused in sarcoma (FUS) are linked with the disease. A pathological feature of ALS-FUS mutations is the aberrant cytoplasmic localization of FUS and its aggregation. FUS is ubiquitously expressed and regulates several aspects of RNA metabolism, including translation. Recently, we demonstrated that FUS can regulate RNA translation at polyribosomes in response to inhibition of the mechanistic target of the rapamycin (mTOR) signaling pathway, a major pathway responsible for translation regulation. We also demonstrated that the ALS-linked FUSR521G mutant aberrantly associates with polyribosomes and promotes global translation inhibition. The mTOR pathway is regulated by dietary intake (i.e. nutrient and growth factor availability), which is also shown to be disrupted in patients with ALS. Moreover, there is evidence that dietary intake, especially a high fat diet, can affect disease progression and quality of life in patients with ALS. Our preliminary data show that ALS-FUSR521G mice have defects in lipid metabolism, which suggests their ability to use dietary fats is defective. However, we do not understand how defects in lipid metabolism occur in ALS-FUSR521G mice or how dietary intake would impact our model of ALS.

HYPOTHESIS

Our hypothesis is that certain dietary inputs can promote or restrict protein translation and that ALS-link FUS mutations further exacerbate defects in translation in response to dietary intake.

METHODS

Both presymptomatic ALS-FUS mice and littermate controls (WT) will be placed on a standard or ketogenic diet (high-fat diet) for 4 weeks, or starved for 24 hours. Behavior tests will be performed before and after the dietary treatments (except for starvation groups). Brains and spinal cord tissues will be harvested and biochemically fractionated to isolate cytosolic fraction. Polyribosomes will be extracted using a continuous sucrose gradient (10%-50%) and Brandel Density Gradient fractionation system and protein and RNA levels will be measured.

RESULTS

Behavioral tests have shown that the ketogenic diet significantly improves cognitive behaviors in ALS-FUSR521G mice and modestly improves motor function in these mice. We examined the translation state of mice on different diets and found a differential effect of diet on the translation state in the ALS-FUSR521G mice.

Letícia Priscila Pacheco  

Lilian Cristine Hübner 

Maximiliano A. Wilson 

The scripts of different languages vary in the consistency between orthography and phonology. Brazilian Portuguese (BP) has a highly consistent script. This means that most of its letters have only one possible pronunciation. However, some letters have up to four possible pronunciations independent of context, causing inconsistencies in the language.  In French it has been shown that people improve at reading inconsistent words during aging. These inconsistencies and their change during aging have not yet been studied in BP. This study investigates the influence of word frequency and consistency in word reading in young and older adults in BP. METHOD. Thirty young adults and 30 older adults, matched by sex, education, and socioeconomic status, participated in the study. TASK. Word reading aloud task with 4 groups of words varying their frequency (high and low) and their consistency (high and low). Stimuli were matched by critical psycholinguistic variables such as imageability, concreteness, orthographic neighborhood, initial phoneme, number of letters, phonemes, and syllables. We used linear mixed effects models with age group (young and older adults), frequency (high and low) and consistency (consistent and inconsistent) as fixed factors and response times (RT) as dependent variable. We used logit mixed effects models for accuracy. Participants and items were entered as random effects. RESULTS. Young adults were significantly faster and more accurate than older adults. High frequency words were read faster and more accurately than low frequency words. Interestingly, consistency affected accuracy but not RTs. Consistent words were read more accurately than inconsistent words. The triple interaction group x frequency x consistency was significant for RTs. Simple effects showed that the effect of frequency was larger for young adults. None of the other effects nor interactions achieved significance. CONCLUSION. Even in a highly consistent script as BP, these rare inconsistencies make it more difficult to read. These results have an impact on the way children learn to read. Thus, reading ability in BP may present some challenges during its acquisition and aging. This is important for rehabilitation and teaching strategies.

GLASSON BASTIEN, Yousri Benchaar, Valerie Clavet-Fournier Flavie Lavoie-Cardinal and Chantelle F Sephton

BACKGROUND

Mutations in the gene encoding the RNA-binding protein fused in sarcoma (FUS), are associated with amyotrophic lateral sclerosis (ALS) familial forms. Mutated FUS is predominantly present in the cytoplasm, which correlates with decrease in protein synthesis. mTOR signalling pathway is essential for regulation of local protein synthesis and synaptic plasticity. Recently, we showed that the mTORC2 signaling pathway affects the cytoplasmic localization of FUS and increased his association with stalled polyribosomes and inhibits protein synthesis in HEK293T cells.

OBJECTIVE

Investigate the regulatory role of mTORC2 in FUS function in neurons and whether defects in mTORC2 signaling can further exacerbate cytoplasmic toxicity caused by ALS-linked FUS mutations

METHODS

Primary cortical neurons were treated with Torin1, which inhibits the mTORC1 and mTORC2 pathways or Rapamycin, which inhibits the mTORC1 pathway. STimulated Emission Depletion (STED) microscopy provided images of FUS protein, ribosomes (60s ribosomal subunit) and vesicular glutamate transporter 1 VGLUT1. Acute slices were treated with DMSO, Torin1, Rapamycin. We isolated synaptoneurosomes (SN) from these slices.

RESULTS

After mTOR pathway inhibition, FUS is recruited more abundantly to the cytoplasm. Inhibition of mTORC1 pathway is not sufficient to induce a significant change in FUS localization. We also investigate ALS-linked FUS mutants R521G and there are localized more predominantly to the cytoplasm. With Torin1 acute slices treatment, there is a decrease in the downstream effectors of mTORC1 and mTORC2, respectively pS6 and pAKT. Treatment with rapamycin causes a decrease only for pS6. Preliminary results show an increase in FUS expression with Torin1 treatment in the SN fraction. Next, we investigated the inhibition impact of translational activity on the interaction between FUS and ribosomes localized at synapses. STED microscopy show that with Torin 1 treatment, FUS localized near the 60S ribosomal subunit at the presynaptic region.

CONCLUSION

These results suggests a potential role of FUS in the regulation of local translation in the context of translation inhibition. Future studies will examine the effects of mTORC2 on ALS-linked FUS mutations and identify the mRNA targets translationally repressed by FUS.

Manon Spigarelli  

Maximiliano A. Wilson 

OBJECTIF – OBJECTIVE

Le manque du mot, plus marqué pour les verbes d’action que pour les objets, est la plainte cognitive la plus importante chez les personnes aînées. La Stimulation Magnétique Transcrânienne répétitive (rTMS) est une méthode de stimulation non invasive du cerveau qui permet de moduler l’excitabilité de la zone visée et modifier son fonctionnement. La rTMS appliquée sur le cortex préfrontal dorsolatéral gauche réduit la latence chez l’adulte pour nommer une action. Aucune étude n’a été réalisée chez les personnes saines aînées. La dénomination d’actions est meilleure avec des vidéos plutôt que des images. L’objectif est de déterminer l’effet de la rTMS sur la performance de dénomination d’actions avec vidéos chez des personnes aînées en santé.

MÉTHODES – METHODS

Nous recruterons 30 participants en santé. Un protocole rTMS de facilitation sera appliqué sur 3 aires cérébrales : 2 connues pour moduler la dénomination d’actions et une aire contrôle. Pendant la stimulation rTMS de chaque région, les participants dénommeront des actions.

RÉSULTATS – RESULTS

Les résultats préliminaires prédisent que la rTMS aux aires cérébrales liées à la tâche réduit le temps de réponse pour la dénomination d’actions.

CONCLUSION

Étudier l’effet de la rTMS sur la dénomination d’actions chez des personnes aînées en santé permettra de mieux connaître les aires cérébrales spécifiques aux actions. Ceci guidera le développement de protocoles de rééducation chez des populations pathologiques.

Mari Carmen Pelaez, Pauline Gelon, Alicia Rodgers, Myriam Sévigni, Daniel Phaneuf, Silvia Pozzi, Paul A. Dutchak, Jean-Pierre Julien, Chantelle F.  Sephton

Background: There is mounting evidence that suggests dendritic attrition and synaptic loss are important contributors to amyotrophic lateral sclerosis (ALS). Post-mortem histological analysis of ALS patients’ brain and spinal cord tissues showed neuromorphological alterations in upper (UMNs) and lower motor neurons (LMNs). These structural changes are proposed to drive cognitive and motor impairments in ALS. We previously demonstrated that global expression of the ALS-linked FUSR521G variant in mice caused dendritic attrition and spine loss in motor-neurons (MNs), coinciding with cognitive and motor impairments. However, many questions remain concerning the contribution of neuromorphological alterations on the behavioral impairments observed in our model; the influence of cell-autonomous versus non-cell autonomous mechanisms that contribute to these changes; and whether therapies that stabilize dendritic structures can attenuate ALS-associated phenotypes.

Objectives: In this study, we aimed to generate a neuron-specific mouse model expressing the FUSR521G variant to determine the effects of FUSR521G expression on neuromorphology and behavior phenotypes. Moreover, we aimed to assess the therapeutic potential of an NFkb inhibitor, IMS-088, in attenuating these changes in this mouse model.

Methods and results: A neuron-specific mouse model expressing the FUSR521G variant was generated by crossing the CAG-Z-FUSR521G-IRES-EGFP mice with Syn1Cre mice, hereafter referred to as FUSR521G/Syn1. FUSR521G/Syn1 and littermate control mice were analyzed for cognitive and motor function at different timepoints. In parallel, the dendritic branching and spine density of MNs were analyzed. We found that 1-month-old FUSR521G/Syn1 mice presented dendritic attrition without loss of spine density in their UMNs; corresponding with cognitive impairments in these mice. Importantly, we observed a progressive loss of spine density of UMNs, a loss of dendritic branching of LMNs and glial activation in 6-month-old mice, which preceded the motor impairments observed in 8-month-old mice. We then assessed the therapeutic potential of IMS-088 on attenuating dendritic branching and synaptic loss in FUSR521G/Syn1 mice. We found that IMS-088 treatment led to a corresponding decrease in glial activation, enhancement of dendritic structures and synapses, and cognitive and motor improvement.

Conclusion: Our study shows that dendritic and spine defects that occur in MNs are due to the intrinsic mechanisms caused by FUSR521G. Moreover, these changes are progressive and sufficient to cause behavioral impairments. Importantly, treatment of FUSR521G/Syn1 mice with IMS-088 greatly improves the neuronal morphology and synaptic structures in these mice. These findings indicate that therapeutic strategies that stabilize dendritic structures of MNs should be considered in the treatment of ALS.

Thiéry De Serres-Bérard, Jack Puymirat and Mohamed Chahine

OBJECTIF – OBJECTIVE

Myotonic dystrophy type 1 (DM1) is caused by the expansion of CTG repeats in the DMPK gene. Mutated RNAs form toxic nuclear aggregates called foci and disrupt the activity of RNA-binding proteins of the MBNL and CELF families. Patients with more than 1000 repeats and hypermethylation patterns upstream the mutation often manifest a severe congenital form (CDM1) characterized by mental retardation and brain malformations of unknown cause. We hypothesize that impaired CELF and/or MBNL activities in neural stem cells affect embryonic brain development in patients with CDM1. The objective of the project is to develop a forebrain organoid model to identify the cellular mechanisms causing prenatal neurodevelopmental defects in this disease.

MÉTHODES – METHODS

We have reprogrammed somatic cells derived from 3 healthy individuals or 3 patients with CDM1 into induced pluripotent stem cells (hiPSCs) using the Sendai virus. These iPSCs were differentiated into forebrain organoids using the Lancaster method and then analyzed by immunofluorescence and fluorescence in situ hybridization (FISH) at multiple time points. In addition, the CTG repeats of the CDM1 hiPSCs were excised by the CRISPR-Cas9 technique to create isogenic controls.

RÉSULTATS – RESULTS

Forebrain organoids recapitulated the formation of ventricular and subventricular zones containing proliferating neural progenitors as well as rudimentary organized cortical layers containing mature neurons. Immunostaining of the ventricular zones of organoids showed strong nuclear expression of CELF1 and western immunoblotting showed stronger expression of the protein in neural stem cells. Furthermore, ventricular zones of CDM1 manifested the presence of numerous intranuclear CUG-expanded foci, suggesting that RNA toxicity may affect neural stem cells. Deletion of CTG repeats corrected foci formation in all three CDM1 hiPSC lines as well as aberrant methylation upstream the mutation, confirming the establishment of isogenic controls to validate the effect of the mutation in organoids.

CONCLUSION

We have confirmed the relevance of forebrain organoids for modeling neurological disorders in DM1 by assessing its ability to recapitulate key events of embryonic brain development and molecular phenotypes characteristic of DM1. Further investigations will allow to determine the functional consequence of RNA toxicity caused by the mutation in neural stem cells.

A. Jochmans-Lemoine, E. Hudon, J. Boudreault, S. Caparos, G. Belleville, I. Blanchette.

Une majorité d’individus sera exposée à au moins un événement potentiellement traumatique (EPT) au cours de sa vie. Seule une minorité développera des réactions psychopathologiques. Ces événements peuvent affecter le fonctionnement affectif et cognitif, mais leur impact causal reste à déterminer. L’objectif principal de cette étude est de déterminer si l’exposition traumatique cause des altérations dans le fonctionnement cognitif, particulièrement la mémoire de travail. Nous examinerons également les mécanismes qui pourraient l’expliquer. Nous postulons que vivre un EPT entrainera une activation en mémoire dudit EPT mis en évidence par la présence d’intrusions et de ruminations. Ceci pourrait diminuer les capacités de la mémoire de travail.

Nous avons conduit une étude prospective sur 4 ans chez une cohorte d’étudiants. Les participants remplissaient des questionnaires sur l’exposition aux EPT ainsi qu’un inventaire des ruminations et intrusions et divers questionnaires visant à établir la présence de symptômes liés au TSPT, à l’anxiété, la dépression et la consommation de substance. La mémoire de travail était mesurée par une tâche d’empan à l’endroit et à l’envers.

L’analyse des résultats des 3 premières années établit que l’exposition traumatique impacte la mémoire de travail. Les participants exposés à au moins un événement potentiellement traumatique durant le cours de l’étude avaient une moins bonne performance à la tâche de mémoire de travail à l’an 3 que les participants ne rapportant aucun tel événement, même en contrôlant pour le niveau de performance initial, à l’an 1. Cependant un meilleur contrôle attentionnel réduisait cet impact du trauma sur la mémoire de travail. Ces impacts n’étaient pas expliqués par une augmentation des intrusions/ruminations.

Support et financement :

Financé par le CRSH

VÉZINA, ALEXANDRE (1,2), Boutej, Hejer (1), Picher-Martel, Vincent (1), Dupré, Nicolas (2), Kriz, Jasna (1)

1. Département de psychiatrie et de neuroscience, Faculté de médecine, Centre de recherche CERVO , 2601 Chemin de la Canardière, Québec, QC, G1J 2G3, Canada
2. CHU de Québec, Axe Neurosciences, HEJ, Université Laval, 1401 18e Rue, Québec, QC, G1J 1Z4, Canada

CONTEXTE – BACKGROUND

Un hypermétabolisme surviendrait chez plusieurs patients atteints de la sclérose latérale amyotrophique (SLA), étant potentiellement lié à un pronostique défavorable. L’AMP-activated protein kinase (AMPK), régulateur métabolique majeur, est souvent hyperactivé, mais son influence dans l’axe immun-métabolique en SLA demeure incertain. Des travaux ont démontré une potentielle signature immune-métabolique spécifique caractérisée par des niveaux plasmatiques diminués en leptine et augmentés en soluble tumor necrosis factor receptor II (sTNF-RII) et CC-motif chemokine ligand 16 (CCL16).

OBJECTIF – OBJECTIVE

L’objectif principal du projet est de concevoir un modèle in vitro humanisé valide afin d’examiner la réponse immun-métabolique des adipocytes en réponse au plasma de patients atteints de la SLA. L’hypothèse est qu’une dérégulation immunitaire chez les patients SLA à progression rapide résulterait en hyperactivation de l’AMPK au sein des adipocytes et en réduction de leur sécrétion en leptine.

MÉTHODES – METHODS

1. Caractériser le sécrétome des adipocytes humains après une stimulation inflammatoire contrôlée à l’aide d’un traitement au LPS.
2. Étudier la signature moléculaire des adipocytes à la suite d’un conditionnement au plasma de patients SLA.
3. Identifier des prédicteurs moléculaires spécifiques à la SLA à progression rapide (en plus de la leptine).

RÉSULTATS – RESULTS

1. En réponse à une stimulation inflammatoire contrôlée, la sécrétion de la leptine et de plusieurs autres marqueurs immuns-métaboliques est diminuée de façon dose-dépendante.
2. À la suite d’une exposition au plasma de patients SLA, une diminution de la sécrétion de la leptine et de plusieurs autres marqueurs immuns-métaboliques par les adipocytes humains est observée, spécialement lors d’une exposition au plasma de patients ayant la forme rapide de la maladie.
3. (Résultats non disponibles à l’heure actuelle).

CONCLUSION

Notre modèle in vitro humanisé conçu à partir d’adipocytes humains offre la possibilité de mieux comprendre le rôle de l’axe immun-métabolique au sein des adipocytes en SLA.  L’étude de la réponse adipocytaire spécifique au plasma de patients SLA pourrait mener au développement de nouveaux outils diagnostiques, pronostiques et thérapeutiques.

L. Pancotti – É. Dumas – C. Proulx – B. Labonté

OBJECTIF – OBJECTIVE

Several lines of evidence suggest that cortically driven dysregulation of subcortical circuits triggers depressive-like behaviours in humans and animals. Amongst these circuits, the medial prefrontal cortex (mPFC) sends dense projections to the ventral tegmental area (VTA). Recent findings from our lab showed that chronic stress induces functional and morphological changes affecting the activity of the cortico-tegmental pathway in a sex-dependent fashion. These results raise an important question: how do these stress-driven changes that occur in the mPFC affect synaptic transmission in downstream output structures in males and females?

MÉTHODES – METHODS

Chronic variable stress (CVS) was used to produce a persistent depressive-like phenotype in males and females. A trans-sectional viral strategy was used to expressed channelrhodopsin in the cortico-tegmental pathway and to label downstream neurons in the VTA. Optogenetic stimulation on live VTA slices was used to activate the cortical projections in naïve and stressed animals. Whole-cell patch-clamp experiments were performed to measure optogenetically-evoked responses (oEPSCs) in VTA neurons and detect pre-and post-synaptic modifications.

RÉSULTATS – RESULTS

Stimulation of cortical axons in the VTA region induced a significant short-term synaptic potentiation of glutamatergic transmission. Interestingly, mPFC-VTA plasticity was significantly impaired after CVS in both male and female mice.  The potentiation was not affected by NMDARs blockade, which excludes a main postsynaptic involvement. In turn, the presynaptic chelation of intracellular Ca2+, obtained by EGTA-AM administration, strongly reduced the short-term facilitation. Evidence from pathway-specific RNAseq from naïve and stressed animals supports our pharmacological results: CVS changed the expression of Ca2+-interacting proteins in VTA-projecting cortical neurons, which could represent a maladaptive substrate for the loss of potentiation observed in this pathway.

CONCLUSION

We characterized a novel form of presynaptic plasticity at mPFC-VTA synapses, which is likely involved in the emotional processing of male and female mice. Chronic stress disrupts such signalling by dysregulating Ca2+-binding proteins at the presynaptic site. Investigating this pathway will provide novel insights into the neuronal mechanisms underlying adaptations to stress in males and females.

NDONGO SONFACK DAVAINE JOEL1, 2, 3, 4 ; Touzel Deschênes Lydia2, 3 ; Berthod François1, 2, 3 ; Gros-Louis François1, 2, 3 ; Champagne Pierre Olivier1, 2, 3, 4

1 Faculté de Médecine de l’Université Laval, Département de chirurgie 

2 Centre de recherche du CHU de Québec-Université Laval 

3 Laboratoire d’organogénèse expérimentale de l’université Laval (LOEX) 

4 Laboratoire d’Innovation en Neurochirurgie de l’Université Laval (LINUL)

OBJECTIF – OBJECTIVE

La chirurgie endoscopique par voie endonasale (CEE) est une procédure chirurgicale de choix pour la résection de certaines tumeurs de la base du crâne grâce à une caméra insérée par les narines. La complication la plus redoutée de la CEE est une fuite de liquide céphalo-rachidien (LCR). Le LCR est le liquide dans lequel baigne le cerveau. Une fuite de LCR indique la présence d’un passage entre le cerveau et les cavités nasales, mettant le patient à risque d’infections potentiellement mortelles. L’utilisation d’un lambeau de muqueuse nasale pour fermer l’ouverture créée à la base du crâne est une des méthodes ayant permis de réduire ce risque. Ce dernier est une forme de « pansement » de muqueuse du nez, venant du patient, mais dans plusieurs situations cette muqueuse n’est pas toujours disponible.  L’objectif est de fabriquer par génie tissulaire de la muqueuse nasale à partir des cellules du patient pour remplacer celle du patient lors de la reconstruction endoscopique endonasale des chirurgies de la base du crâne.

MÉTHODES – METHODS

Pour y parvenir, nous procéderons d’abord au prélèvement de muqueuse nasale saine, chez des patients opérés par CEE au CHU de Québec – Université Laval.  Ensuite, nous procéderons à l’extraction des cellules des tissus recueillis en comparant plusieurs protocoles de digestion enzymatique. Par la suite, des approches de génie tissulaire seront utilisées pour reconstruire une muqueuse nasale tridimensionnelle permettant la croissance et la différenciation de nos cellules. Finalement, afin de comparer le nouveau biomatériau à la muqueuse originale, nous effectuerons des tests de qualité tels que la mesure de la résistance électrique transépithéliale (TEER).

RÉSULTATS – RESULTS

Les résultats préliminaires démontrent une extraction réussie des cellules.

CONCLUSION

Les applications de cette muqueuse sont nombreuses autant pour la recherche pharmaceutique, toxicologique et chirurgicale.

IHIDOYPE MARINA, Derwel Cléo, Martianova Ekaterina & Proulx Christophe.

CERVO brain research center.

INTRODUCTION

Learning to adapt behavioral responses to threatening or unpleasant events is essential to maximize survival. The lateral habenula (LHb) is a small nucleus of the epithalamus receiving neural inputs from the basal ganglia and limbic system, and in turn sends neural projections to the dopaminergic ventral tegmental area (VTA). It is then ideally positioned to integrate affective signals into the selection of action.

OBJECTIF – OBJECTIVE

In this project, we test the hypothesis that VTA-projecting LHb neurons encode aversive signals involved in associative learning to promote escape behavior.

MÉTHODES – METHODS

Specifically, we use an intersectional viral approach to direct expression of the genetically encoded calcium sensor GCaMP6s specifically in VTA-projecting LHb neurons to monitor their activity using fiber photometry calcium imaging in freely moving mice during an active avoidance task. In this protocol, mice learn to associate a neutral stimulus (tone) with an upcoming mild foot shock, which is required to engage either escape or avoidance responses.

RÉSULTATS – RESULTS

We have observed that neuronal activity of VTA-projecting LHb neurons increases when mice are presented with an aversive airpuff or mild foot shocks, confirming their role in encoding aversive signals. Moreover, we found that an auditory cue (tone) paired with a foot shock progressively causes cue-driven activity in VTA-projecting LHb neurons during avoidance learning, which is not observed when tone is not contingent to foot shock (unpaired).

CONCLUSION

Taken together, these findings suggest that VTA-projecting LHb neurons encode aversive signals and cues predicting them, and that they may be involved in the associative learning to promote defensive escape behaviors.

Marieva Vlachoua, Juliette Legrosa, Cécile Sellina, Martin Simoneaub,c, Laurence Mouchninoa,d, Jean Blouina

aLaboratoire de Neurosciences Cognitives,Aix-Marseille Université/CNRS.Marseille

bCentre interdisciplinaire de recherche en réadaptation et intégration sociale du CIUSSS de la Capitale-Nationale, Québec,Québec

cFaculté de médecine,Université Laval,Québec

dInstitut Universitaire de France,Paris

The sense of touch does not only provide information about the external environment but also acts as a crucial auxiliary proprioceptive cue. Activation of cutaneous mechanoreceptors on the fingertips has a key role in constructing a representation of hand position and movement. However, most of the studies have focused on the role of tactile information in grasping and reaching tasks and little is known about its role in the control of spatially oriented movements. Our aim was to determine if cutaneous touch provides information for the control of finger slide movement. To assess this, we used an established paradigm, during which the performance of a visually-guided motor task is perturbed by conflicting visual and somatosensory inputs. In this context, previous work demonstrated that the greater the weight given to somatosensory input, the worse the tracing performance. Participants traced the outline of an irregular polygon on a surface with their index finger (Cutaneous group,N=16) while looking both at the polygon and their hand directly (NoConflict) and through an inclined mirror (Conflict). The same tasks were performed by a control group (NoCutaneous group,N=15) wearing a finger splint to eliminate tactile stimulation from the surface. Tracing performance was evaluated by calculating jerk as smoothness metric. If the Cutaneous group exhibits a jerkier movement profile than the NoCutaneous group, our hypothesis of a pivotal contribution of tactile information in movement control would be supported. The analysis revealed that under the sensory conflict, the sum of the absolute jerk was significantly greater in the Cutaneous compared to the NoCutaneous group (permutation test: p=0.01). Accordingly, the power spectrum density of the jerk showed higher power in frequency between 0.1 and 3Hz for the Cutaneous compared to the NoCutaneous group. These results support the hypothesis that cutaneous feedback contributes to motion control and shed new light on the interplay between proprioception and touch in active tasks.

SADRETDINOVA RENATA, Benmammar Zakaria , Proulx Christophe D.

OBJECTIF – OBJECTIVE

The dorsal raphe nucleus (DRN) regulates affective behaviors and energy balance. Positive and negative emotional states might be encoded in DRN by the neural inputs from numerous brain regions. Although one of the major reciprocal synaptic partners of DRN is the lateral hypothalamic area (LHA), the circuitry between LHA and DRN was not characterized yet.

MÉTHODES – METHODS

In the current study, we use anterograde and retrograde viral strategies to investigate the anatomical and functional reciprocal circuitry between the LHA and the DRN. Specifically, we have injected an anterograde virus encoding the recombinase cre (AAV1-cre) along with a retrograde virus encoding the recombinase flippase (retroAAV-FlpO) into LHA to target DRN cells receiving input from LHA (DRN input) and DRN cells projecting on LHA (DRN output). The same mice were injected in the DRN with a cre- and a FlpO-dependent AAVs encoding eGFP and mCherry respectively (AAV5-DIO-eGFP and AAV5-fDIO-mCherry). A similar viral approach was used to drive the expression of genetically-encoded calcium indicators (GECIs) and opsins in DRN neurons to examine their functions in freely moving animals. Post-hoc identification of serotonin cells in the DRN was done using IHC for the tryptophan hydroxylase.

RÉSULTATS – RESULTS

Our results show that DRN input and DRN output neurons are largely two distinct neuronal populations that show differential spatial distribution. LHA makes synaptic contact with 5-HT and non-5-HT neurons in the DRN, which is spatially biased to the ventromedial part and dorsolateral parts of DRN, respectively. DRN output neurons appeared to be mostly non-serotonergic indicating the bias of the retroAAV-FlpO used. Both DRN input and DRN output neurons target a stereotypical set of subcortical regions while there were no terminals detected in cortical areas. Neural activity of studied populations was increased in aversive stressful contexts such as air puff and tail suspension.

CONCLUSION

Overall, these results show that 1) anterograde transsynaptic tracing can be applied to identify DRN neurons innervated by the LHA, whereas retroAAV failed to infect serotonergic cells in DRN 2) LHA targets specific DRN neuron populations with a spatial bias, 3) DRN input neurons projects mostly to subcortical limbic regions 4) DRN input and DRN output neurons are involved in encoding aversive signals.

SICARD, ALEXANDRE1,2 et Tremblay, Pascale1,2

1. Centre de recherche CERVO, Québec, Canada

2. Université Laval, Faculté de Médecine, Département de réadaptation, Québec, Canada

OBECTIFS

(1) Examiner la relation entre la matière grise corticale et les fonctions cognitives au cours du vieillissement et (2) identifier des différences potentielles entre les chanteurs et les musiciens amateurs.

MÉTHODES

105 participants ont été recrutés (20-88 ans, 50 ♀), séparés en trois groupes (33 chanteurs, 35 musiciens et 37 contrôles actifs) et appariés pour l’âge, l’éducation, le niveau de santé et la cognition. Les participants ont effectué une batterie de tests, incluant une tâche d’attention soutenue auditive (TAiL), en plus d’une séance d’IRM où des images anatomiques (1 mm3) ont été acquises. Un modèle linéaire mixte a été utilisé pour les analyses. L’épaisseur corticale moyenne, des régions d’intérêt sélectionnées, a été utilisée comme variable dépendante et les variables indépendantes étaient le groupe, l’âge et les scores d’attention (résolution de conflit et distractibilité). Les covariables étaient le sexe, l’audition, l’expérience musicale et le risque de démence.

RÉSULTATS

Des résultats préliminaires basés sur 30 participants (10/groupe) sont présentés dans ce résumé pour la résolution de conflit (CR) exprimée en coût en exactitude. Dû à leur rôle clé dans les fonctions exécutives, les cortex préfrontal et cingulaire antérieur ont d’abord été analysés. Les chanteurs avec un meilleur CR ont un IFG orbital gauche plus épais que ceux avec un faible CR (p=0.006). Les chanteurs âgés avec un meilleur CR ont un IFG triangulaire gauche plus épais comparé aux jeunes chanteurs; l’inverse est observé pour ceux avec un faible CR (p=0.025). Les musiciens avec un meilleur CR ont un ACC droit moins épais que ceux avec un faible CR (p=0.001). Les jeunes musiciens avec un meilleur CR ont un sulcus frontal moyen gauche plus épais que les plus âgés; l’inverse est observé pour ceux avec un faible CR (p=0.019).

CONCLUSION

Nos résultats suggèrent une relation complexe entre la cognition et l’anatomie cérébrale. Dans certains cas, une plus grande épaisseur corticale est liée à de meilleures performances, alors que dans d’autres, l’inverse est trouvé. Trouver de nouvelles façons de réduire le vieillissement cérébral est essentiel dans nos sociétés vieillissantes pour maintenir la qualité de vie des aînés.

Estelle Gouriou1,2,3, Thérèse Di Paolo2,4 and Cyril Schneider1,2,3,5

1 Noninvasive neurostimulation laboratory; 2 Research center of CHU de Québec – Université Laval, neuroscience division; 3 Faculty of medicine, Université Laval; 4 Faculty of pharmacy, Université Laval; 5 Dept rehabilitation of faculty of medicine, Université Laval

Parkinson’s disease (PD) is the second most frequent neurodegenerative illness worldwide. The loss of nigrostriatal dopamine (DA) neurons alters the basal ganglia-thalamus-cortical loops including connections from thalamus to primary motor cortex (M1), resulting in M1 hypoactivation with motor and non-motor symptoms. DA neurons denervation and L-Dopa medication led to inappropriate striato-cortical signaling and M1 hyperactivation resulting in the development of L-Dopa induced dyskinesia (LID). Pharmacology to reduce LID may not work in all patients and induce more debilitating side-effects. Therefore, non-pharmacological approaches are studied, such as transcranial direct current stimulation (tDCS) and repetitive peripheral magnetic stimulation (rPMS). These approaches influence M1 plasticity directly (tDCS of M1) or indirectly via the sensory thalamo-cortical pathways (rPMS of muscles). We tested the excitatory effects of tDCS alone and combined with rPMS on motor symptoms in patients (a-tDCS and i-rPMS) and the inhibitory tDCS influence on LID in a preclinical model (c-tDCS).

In patients (n=4), a-tDCS was applied over M1 contralateral to the most affected side (1mA, 20 min) and i-rPMS on leg, arm, abdominal and back muscles. As compared to sham, five sessions of tDCS alone and of the combo (tDCS+rPMS) improved the motor scores (MDS-UPDRS III score 4.5-point and 8.75-point decreases, respectively) leading to less bradykinesia and rigidity. These changes lasted until a one-month follow-up along with changes of M1 excitability (measured by transcranial magnetic stimulation).

In non-human primate (NHP) lesioned with MPTP and treated with L-Dopa (PD-LID model, n=1), c-tDCS of M1 aimed at reducing LID (ULaval dyskinesia scale) without reducing the anti-parkinsonian effects of L-Dopa (ULaval disability scale). L-Dopa was administered immediately after 5-min c-tDCS per each M1 (0.375 mA). As compared to sham, five tDCS sessions delayed LID appearance and shortened their duration.

These results warrant more studies on noninvasive neurostimulation to reduce motor symptoms and LID. M1 changes reflect brain plasticity at the origin of clinical improvement. LID decrease in NHP should be reproduced in larger samples. This shed new light on the non-pharmacological possible treatment to combine with L-Dopa.

BIENJONETTI ISABELLA1-2, Beaudet Marie-Josée2, Louit Aurélie1-2, Gros-Louis François1-2, Dupré Nicolas3

1Département de chirurgie, Faculté de médecine, Université Laval, Québec, QC, Canada; 2Axe Médecine régénératrice, Centre de recherche du CHU de Québec, Université Laval, Québec, QC, Canada; 3 Neurologie, CHU de Québec, Université Laval, Québec, QC, Canada.

OBJECTIF – OBJECTIVE

Les cellules souches pluripotentes induites (iPSC) sont des cellules souches génétiquement reprogrammées pouvant être générées à partir de cellules adultes somatiques. Les cellules iPSC permettent de produire en quantité illimitée des cellules du système nerveux central (SNC) difficilement accessibles, tels que les oligodendrocytes. Ces cellules sont responsables de la myélinisation des axones du SNC et sont impliquées dans diverses maladies neurologiques. Le but du projet est de générer des oligodendrocytes à partir d’iPSC dérivées de patients souffrant de diverses maladies neurologiques (Leucodystrophies, Ataxies, Sclérose Latérale Amyotrophique). Un modèle de co-culture des oligodendrocytes dérivés d’iPSC avec des neurones moteurs sera aussi développé.

MÉTHODES – METHODS

Des lignées d’iPSC dérivées de cellules de patients porteurs de mutations spécifiques connues seront produites et caractérisées par la plateforme de génération des iPSC du CRCHU de Québec. Les iPSC seront ensuite redifférenciées en oligodendrocytes. Des analyses par immunofluorescence (IF), RNA-seq et qPCR permettront d’évaluer le potentiel de différenciation terminale des oligodendrocytes à l’aide de différents marqueurs de différenciations (OLIG2, O4, O1, MOG et SOX10) et de caractériser leurs profils d’expression génique.

RÉSULTATS – RESULTS

Un protocole permettant la redifférenciation des iPSC en oligodendrocytes a été établit. Des analyses par IF et immunobuvardage de type Western ont montré que les oligodendrocytes générés expriment des marqueurs d’oligodendrocytes matures après seulement 25 jours de culture, en plus de favoriser la myélinisation des neurones moteurs dans un modèle de co-culture 3D issu du génie tissulaire.

CONCLUSION

La production d’oligodendrocytes dérivés d’iPSC de patients nous permettra d’une part de mieux comprendre les maladies à l’étude et facilitera l’utilisation de ces cellules pour le développement de thérapies cellulaires.

Valérie Gagné1, Rose Turgeon1, Valérie Jomphe 2*, Claude MH. Demers2 Marc Hébert 1,3

1 CERVO Centre de recherche, Centre Intégré Universitaire de Santé et des Services Sociaux de la Capitale Nationale, Quebec, QC, Canada 

2 École d’Architecture, Faculté d’aménagement, d’architecture, d’art et de design

3 Département d’ophtalmologie et ORL-chirurgie cervico-faciale- Faculté de Médecine, Université Laval

OBJECTIF: La lumière joue un rôle important dans l’horloge biologique, la cognition, la vigilance et la santé mentale. Ces effets non-visuels sont dus à la présence de lumière bleue dans la lumière du jour, perçue par les cellules de la rétine, appelées mélanopsine. Les personnes qui reçoivent peu de lumière bleue en hiver, comme les travailleurs ou les habitants du Grand Nord, risquent de subir des perturbations circadiennes qui peuvent être préjudiciables à leur bien-être. Récemment, des experts en photobiologie ont recommandé qu’une intensité lumineuse de 250 lux mélanopique (liée à la mélanopsine) devienne la norme pour une exposition diurne. Les LED émettant une forte proportion de bleu, il est possible d’atteindre cette norme, rarement atteinte par les fluorescents habituels. Cependant, ce type d’éclairage demeure nouveau. Notre étude vise à déterminer, dans un premier temps, si une courte exposition (50 minutes) à un éclairage LED pouvant atteindre 250 lux mélanopique (5000K), tout en produisant 600 lux photopique, a un impact sur la concentration et l’éveil par rapport à un éclairage standard (fluorescent) produisant le même niveau de 600 lux photopique, mais seulement 165 lux mélanopique (3500 K).

MÉTHODES: Les participants (n=30) ont été évalués dans les deux conditions d’éclairage (contrebalancées) au cours desquelles une évaluation de la somnolence subjective (KSS), de la concentration (test d2-R), de la performance visuelle (FrACT; Freiburg Acuity and Contrast Test), de l’appréciation générale (Échelles Visuelles Analogues), ainsi que des préférences et du confort (Office Lighting Survey-modifié) a été réalisée. Ainsi, les participants ont été soumis deux fois aux tests et questionnaires à un maximum d’un mois d’intervalle et ils ont été attribués aléatoirement à l’une ou l’autre des conditions en premier.

RÉSULTATS: Les résultats montrent que les deux lumières améliorent la concentration, mais que la lumière LED réduit de manière plus prononcée la somnolence sans nuire au confort ou à l’acuité visuelle. La sensibilité au contraste est réduite sous l’éclairage au LED et les participants ont préféré l’éclairage standard. Les participants ont eu tendance à considérer l’éclairage au LED plus clair et stimulant, mais moins confortable, joyeux et plaisant.

CONCLUSION: Ainsi, la lumière LED semble plus stimulante mais son appréciation est plus difficile. L’effet négatif sur la sensibilité aux contrastes demeure une préoccupation.

Alyssa Brodeur1,2, Vincent Roy1,2, Lydia Touzel-Deschênes1,2, Alexandre Winter3, Jean Ruel3 et François Gros-Louis1,2.

1 Département de chirurgie, Faculté de médecine, Université Laval, Québec, QC, Canada ;
2 Axe Médecine régénératrice, Centre de recherche du CHU de Québec-Université Laval, Québec, QC, Canada ;
3 Département de génie mécanique ; Faculté de sciences et génie, Université Laval, Québec, QC, Canada .

OBJECTIF – OBJECTIVE

Les anévrismes intracrâniens (AI) sont caractérisés par un ballonnement de la paroi des artères cérébrales, principalement aux embranchements du polygone de Willis. Le risque de rupture et d’hémorragie méningée subséquente, qui peut entrainer des conséquences graves chez les patients, sont présentement difficiles à évaluer et nécessite des méthodes d’imagerie cérébrales spécialisées. Notre objectif est d’étudier les AI par le biais d’un modèle de vaisseau sanguin humain de petit calibre à géométrie d’embranchement reconstruit par génie tissulaire (TEBV).

MÉTHODES – METHODS

Un système de culture rotatif sphérique, permettant l’ensemencement uniforme des cellules sur un modèle de TEBV à géométrie complexe, a été développé spécifiquement pour ce projet. Des mandrins en « Y » de polyéthylène téréphtalate glycol (PETG) et des cellules vasculaires humaines peuvent être placées à l’intérieur des chambres d’ensemencement faites sur mesure afin de permettre un attachement de ces dernières de façon plus uniforme et de stimuler la sécrétion de protéine de la matrice extracellulaire. Les mandrins sont ensuite retirés des chambres d’ensemencement et conservés en culture cellulaire pendant 21 jours. Plusieurs conditions d’ensemencement et de culture cellulaire ont été effectuées. Les mandrins avec la première couche de cellules maintenant intégrées dans un tissu/tube reconstruit sont réensemencés et placés en culture pour un autre 21 jours.  Des coupes histologiques des tissus ont été effectuées pour caractérisation de l’épaisseur et autres paramètres. La viabilité des tissus a aussi été mesurée en cytométrie en flux.

RÉSULTATS – RESULTS

Le système d’ensemencement sphérique permet la production de TEBV à embranchement sur PETG avec différents types cellulaires après 42 jours de culture. L’épaisseur du tissu est uniforme autant sur les branches du « Y » qu’à la jonction. La viabilité à l’ensemencement est supérieure avec ce système sphérique comparée à un ensemencement statique ou dynamique sur une plaque agitatrice.

CONCLUSION

Ce système novateur ouvre la porte à la production de vaisseaux sanguins à géométrie complexe pour l’étude notamment des AI et autres maladies vasculaires. Une compréhension supérieure de la pathologie et des étapes précoces de la pathogenèse permettrait une meilleure évaluation des risques, une meilleure planification des interventions chirurgicales et possiblement au développement de nouveaux traitements.

BELHAMITI NESRINE1,2, Fiola Stéphanie1, Distéfano-Gagné Félix1,2, Gosselin David1,2  

1. Centre de recherche du CHU Québec – Université laval, Axe Neurosciences, Québec.  2. Université Laval, Faculté de Médecine, Département de Médecine Moléculaire, Québec

Contexte : Les patients atteints de la sclérose en plaques développent éventuellement une forme progressive de SEP caractérisée par une aggravation irréversible des dommages de myéline. Parmi les modèles murins de lésions à la myéline, il y a le modèle Cuprizone (CPZ) qui consiste en l’ingestion de la neurotoxine CPZ, qui est toxique pour les oligodendrocytes et qui entraine une perte de myéline dans le cerveau. Nos expériences récentes indiquent que les microglies associées aux lésions dans le modèle CPZ expriment fortement des gènes inflammatoires antiviraux. De plus, l’analyse d’éléments régulateurs génomiques ont suggéré une forte activité des facteurs de transcription Interferon regulatory factors (Irf), ce concorde avec la signature génique « antivirale ». Ces données supportent l’hypothèse de mon projet, les Irfs coordonnent l’activité des microglies dans les lésions démyélinisantes.

OBJECTIF – OBJECTIVE : Étudier le rôle d’Irf3 dans la démyélinisation et la re-myélinisation subséquente ainsi que son effet sur l’activité inflammatoire dans le modèle CPZ.

MÉTHODES – METHODS​ : Afin de tester mon hypothèse, j’utilise des souris dont le gène codant pour Ir3 est invalidé, et des souris contrôles. Les souris sont soumises au protocole de CPZ, puis sacrifiées pour des analyses de lésions et de la réponse inflammatoire.

RÉSULTATS – RESULTS : Mes données indiquent que l’absence des Irf3 mène à une aggravation importante des dommages à la myéline. De plus, la réponse inflammatoire des microglies est également potentialisée. Ces observations ont été répliquées dans une seconde étude.

CONCLUSION​ et perspective : Ces données indiquent que la présence de Irf3 est nécessaire pour limiter une exacerbation destructrice de l’activité inflammatoire des microglies. Je procède actuellement à des analyses epigénomiques pour comprendre les mécanismes sous-jacents.

Alexandre Paquet1,2, Vincent Roy1,2, Hélène T. Khuong2, Nicolas Dupré2,3 et François Gros-Louis1,2

1 Département de chirurgie, Faculté de médecine, Université Laval, Québec, QC, Canada;
2 Axe de médecine régénératrice, Centre de recherche du CHU de Québec, Université Laval, Québec, QC, Canada;
3 Département de sciences neurologiques; Faculté de médecine, Université Laval, Québec, QC, Canada;

Problématique: La neurofibromatose de type 1 (NF1) est un désordre génétique rare causé par des mutations du gène de la neurofibromine (NF1). La NF1 est caractérisée par une grande hétérogénéité de manifestations cliniques, tel que le développement de neurofibromes. Les neurofibromes plexiformes sont des tumeurs très vascularisées qui peuvent évoluer vers une tumeur maligne. De plus, les cellules endothéliales haploinsuffisantes en NF1 (NF1-/+) ont une activité angiogénique plus élevée. L’hypothèse de l’étude est que les fibroblastes dermiques humains NF1-/+ sécrètent des vésicules extracellulaires, appelées exosomes, qui communiquent une signalisation pro-angiogénique et jouent un rôle crucial dans la modification du microenvironnement tumoral. Le projet consiste à étudier ces exosomes, sécrétés par des fibroblastes dermiques, et de valider leur effet sur les cellules endothéliales et l’angiogenèse.

Méthodes: Des peaux reconstruites par génie tissulaire ont été générées par la méthode d’auto-assemblage avec des cellules de patients NF1 et d’individus sains. Le modèle a été vascularisé par l’ajout de cellules endothéliales microvasculaires (MVEC). Les exosomes sécrétés par les feuillets de fibroblastes ont été isolés à l’aide d’un kit commercial et la concentration et la taille ont été évaluées. Le contenu exosomal a été analysé par spectrométrie de masse. L’influence directe des exosomes sur les MVEC a été évaluée par un test de formation de tubes sur Matrigel®.

Résultats: Les MVEC ensemencées dans les peaux NF1 ont formé un réseau micro-capillaire plus dense, comparativement aux contrôles. Aucune variation de la taille des exosomes et des concentrations de particules et de protéines n’a été mise en évidence entre les populations étudiées. L’analyse par spectrométrie de masse a également révélé que 99 protéines sont significativement modulées dans les exosomes dérivés des fibroblastes NF1-/+, dont 46 qui sont positivement modulées. Parmi celles-ci, on en retrouve plusieurs qui remplissent un rôle dans l’angiogenèse. De plus, les exosomes dérivés de fibroblastes NF1-/+ ont significativement augmenté la formation de tubes sur Matrigel® après 24h de co-culture avec des MVEC.

Conclusions: Notre étude suggère que l’haploinsuffisance du gène NF1 altère la fonction des fibroblastes dermiques et crée un signal pro-angiogénique, via la sécrétion d’exosomes, qui augmente la formation des micro-capillaires in vitro.

Marc Bazin1, Girish Shah1, Denis Soulet1

Centre de recherche du CHU de Québec – Université Laval

Contexte : L’échantillonage des données en recherche est encore la clé de voûte de l’analyse alors que l’instrumentation moderne permet de produire une grande quantité de données, souvent sous forme d’images à haute résolution. Néanmoins l’analyse massive de ces données représente un obstacle en terme de coût et de temps que les laboratoires contournent par des techniques de stéréologie. Ainsi, la majorité des précieuses données instrumentales est ignorée faute de moyens pour les explorer.

Problème : La limitation contrôlée des données soumises à l’analyse est souvent source de faiblesse ou d’erreur dans la conclusion quantitative selonune étude de John P A Ioannidis en 2005. Citée 7000 fois, elle a été revisitée abondamment par des chercheurs préoccupés par ce constat. Cette faiblesse aggrave le potentiel manque de puissance statistique des échantillons mesurés dans une expérience. De plus, la lenteur de traitement des images par des outils standards interdit de tester plusieurs modèles d’analyse. De nombreuses études incluent maintenant des analyses systématiques et poussées qui surpassent les analyses classiques et viennent ainsi concurrencer les travaux analysés par des méthodes standards.

Solution :  Confier à des experts en analyse numérique le soin d’effectuer l’acquisition d’images et/ou les calculs sur les données massives issues de vos projets. Nous offrons nos compétences en analyses de masse dans les domaines de la biologie, de la physique et des mathématiques (modélisation, apprentissage automatique) pour produire résultats et graphes avec tests d’hypothèses. Nous disposons de nos propres équipements que ce soient des microscopes jusqu’à l’échelle du nanomètre ou des stations de calculs multi-CPU et multi-GPU. Nous disposons de plusieurs algorithmes puissants pour le traitement systématique par lots d’un nombre illimité d’images de très grande taille. Plusieurs modèles d’analyses peuvent être testés simultanément pour cibler le résultat le plus optimal à votre projet.

Conclusion : Au Centre de recherche du CHU de Québec, nous pouvons vous aider à acquérir vos données et à les analyser sans compromis avec des méthodes qui sont sur le point d’être incontournables. Nous traitons avec rigueur et moins de biais vos données et nous certifions les résultats de votre publication à des prix planchers pour vos analyses de routine.

CLÉMENT VINCENT1,3, Roy Vincent1, Touzel-Deschênes1, Dupré Nicolas2, Gros-Louis François3

1CRCHU de Québec, Université Laval; 2 Neurologie CHU de Québec;  3Département de chirurgie, Université Laval

OBJECTIF – OBJECTIVE

La Sclérose Latérale Amyotrophique (SLA) est une maladie neurodégénérative causée par la mort des motoneurones qui mène à la paralysie et la mort des patients 2 à 5 ans après les premiers symptômes. Lorsque Charcot a décrit la SLA pour la première fois, il a observé que les patients alités ne développaient pas de plaies de lit, comme il est commun pour de tels patients de le faire. Depuis, d’autres observations ont été faites reliant le tissus nerveux et la peau de patients SLA, dont la détection d’agrégats cytoplasmiques normalement détectés dans le système nerveux central. Nos objectifs sont: 1) d’investiguer les propriétés de migration, prolifération et de fermeture de plaie de fibroblastes dermiques primaires provenant de patients SLA, et 2) d’étudier les microvésicules sécrétées par ce type cellulaire comme médiateurs potentiels de la communication cellulaire dans la guérison des plaies.

MÉTHODES – METHODS

Nous avons cultivé les fibroblastes dermiques en trois dimensions de patients et de sujets sains, afin de produire des tissus auto-assemblé par génie tissulaire. Après en avoir extrait les exosomes, sécrétés par des fibroblastes dermiques de patients, nous avons analysé leur contenu. Des tests fonctionnels de migration et de prolifération cellulaire in vitro ont également été effectués.

RÉSULTATS – RESULTS

Il y a des évidences montrant que les fibroblastes sécrètent de grandes quantités d’exosomes contenant différentes protéines associées à la matrice extracellulaire (MEC). Nous démontrons que les exosomes dérivés de fibroblastes dermiques ont la capacité de voyager d’une cellule à l’autre, d’induire la migration cellulaire et potentiellement la guérison de plaie. De façon intéressante, ces résultats se trouvent être significativement plus importants lorsque les exosomes dérivés des patients SLA sont utilisés.

CONCLUSION

Ces résultats révèlent un nouveau mécanisme exosome-dépendant de déposition de la MEC et offre une piste d’explication pour les observations jadis fait par Charcot lui-même sur l’absence d’escarres dans la SLA.

ISENBRANDT AMANDINE, Coulombe Katherine, Morissette Marc, Bourque Mélanie, Lamontagne-Proulx Jérôme, Di Paolo Thérèse et Soulet Denis

Centre de Recherche du CHU de Québec-Université Laval – axe neuroscience 

Les cellules microgliales sont des cellules participant à la réponse inflammatoire du cerveau. La morphologie de la microglie est caractéristique de son état d’activation. Une cellule microgliale avec un petit corps cellulaire et de longs embranchements sera caractéristique d’une cellule quiescente alors qu’une cellule microgliale avec un corps cellulaire amiboïde et de courts embranchements sera représentative d’une cellule activée.

OBJECTIF – OBJECTIVE

Dans le cadre de l’analyse de cette activation microgliale dans l’étude des différences sexuelles dans la maladie de Parkinson (MP), nous avons mis en place une technique avancée d’analyse tridimensionnelle de la morphologie de la microglie.

MÉTHODES – METHODS

Une immunofluorescence avec le DAPI (4′,6-diamidino-2-phenylindole) et l’Iba1 (ionized calcium-binding adapter molecule 1) nous ont permis de marquer respectivement les noyaux et les microglies. Les images ont été prises en microscopie confocale avec un grossissement de 60X et un zoom optique de 1.2. Afin d’avoir une analyse tri-dimensionnelle, les images ont été acquises dans le striatum avec 21 plans Z successifs soit un volume final de l’image de 800 000 μm3. Les striosomes ont été exclus de l’analyse. La reconstruction tri-dimensionnelle de chaque cellule microgliale a été réalisée à l’aide du logiciel IMARIS®. Nous avons ainsi pu analyser de nombreux paramètres morphologiques tel que la longueur et le nombre des arborescences, les segments et les branchements ainsi que les intersections de Sholl et le rayon.

RÉSULTATS – RESULTS

Cette analyse à haute résolution nous a notamment permis de mettre en évidence des doublets microgliaux caractérisés par deux cellules microgliales physiquement très proches, mais possédant chacun un noyau distinct et une séparation visible des membranes plasmiques. Ces doublets résultent de la prolifération microgliales en réponse à l’inflammation. À notre connaissance, il s’agit de la première caractérisation de doublets de microglie dans un modèle murin de la MP.

CONCLUSION

En conclusion, cette analyse morphologique très détaillée nous a permis de mettre en évidence une activation microgliale dépendante du sexe biologique, mais indépendante des hormones (obtenue grâce à des souris gonadectomisées) dans notre modèle murin de la MP.

CHUBWA NDIKU ABIGAIL, Seigneur Josée, Bretzner Frédéric

CHU de Québec – Université Laval, Axe Neurosciences

OBJECTIF-OBJECTIVE

Par leur projection dans la moelle épinière, les neurones réticulospinaux de la formation réticulée médullaire ont un accès direct au circuit locomoteur spinal impliqué dans le contrôle des mouvements moteurs et de la marche. Récemment, une sous-population de neurones appelés V2a a été identifiée génétiquement par l’expression de facteurs de transcription Lhx3 et Chx10 durant le développement. Fonctionnellement, ces neurones V2a semblent impliqués dans les virages durant la marche et dans les arrêts locomoteurs. Si cette sous-population de neurones V2a de la formation réticulée médullaire est majoritairement glutamatergique, nous ne savons toujours pas si tous les neurones réticulospinaux glutamatergiques sont V2a.  Nous avons émis l’hypothèse de l’existence d’une autre classe de neurones réticulospinaux glutamatergiques non V2a dans la formation réticulée médullaire.

MÉTHODES -METHODS

Des souris Chx10-CRE de trois semaines ont été injectées dans le segment lombaire de la moelle épinière avec deux virus adéno-associés rétrograde CRE-dépendant. Trois semaines après l’injection, les souris ont été perfusées et disséquées. Les tissus neuronaux ont été congelés et coupés à l’aide d’un cryostat. L’étendue du site d’injection des virus a été confirmée dans la moelle épinière et les sections ont été imagées en microscopie épifluorescente. L’expression de mcherry dans le cytoplasme nous a permis d’identifier les cellules glutamatergiques exprimant CamKIIα, un marqueur de neurones glutamatergiques. L’expression d’une protéine verte (GFP) dans le noyau de cellules CamKIIα exprimant Cre nous a permis d’identifier les neurones glutamatergiques V2a, alors que l’expression de mcherry dans le noyau nous a permis d’identifier les neurones glutamatergiques non V2a. L’organisation topographique et la colocalisation des marqueurs ont été analysées à l’aide de logiciels d’analyses automatisées.

RÉSULTATS-RESULTS

Notre étude a révélé l’existence de deux populations neuronales glutamatergiques à travers la formation réticulée médullaire : une population réticulospinale V2a exprimant CamKIIα et Chx10 et une population réticulospinale glutamatergique non V2a exprimant CamKIIα mais pas Chx10.

CONCLUSION

Nos résultats identifient donc pour la première fois l’existence de neurones réticulospinaux gluamatergiques V2a et non V2a dans la formation réticulée médullaire qui pourraient contribuer de manière différente dans le contrôle moteur et locomoteur.

Canet Geoffrey1, Guisle Isabelle1, Laliberté Francis1, Hébert Sébastien S1,2, Planel Emmanuel1,2

1Université Laval, Faculté de Médecine, Département de Psychiatrie et Neurosciences, Québec, QC, Canada

2Centre de Recherche du CHU de Québec, Axe Neurosciences, Québec, QC, Canada

Aggregates of hyperphosphorylated tau protein are a hallmark of Alzheimer’s disease (AD) and other tauopathies. Sleep disturbances are common in AD patients, and insufficient sleep may be a risk factor for AD. Recent evidence suggests that tau phosphorylation and secretion are dysregulated by sleep disturbances in mice and men. However, the physiological regulation of tau phosphorylation and secretion during the sleep–wake cycle is currently unknown. Previously, we demonstrated that tau phosphorylation level is inversely correlated with body temperature. We thus aimed to determine whether tau phosphorylation and secretion are regulated by circadian rhythms, inherently linked to the sleep-wake cycle and body temperature variations.

We first analyzed tau phosphorylation on relevant epitopes in the brains of awake and sleeping B6 mice by Western blotting, while recording their temperature. We then exposed neuronal cells to different physiological temperatures (from 35°C to 39°C) to assess both intracellular tau and tau-secreted species in the cell medium by ELISA and dot blotting.

We found that tau phosphorylation undergoes sleep-driven circadian variations, as it is hyperphosphorylated during sleep, when body temperature is lower. Similar changes in tau phosphorylation were reproduced in neuronal cells exposed to temperatures recorded during the sleep–wake cycle. In addition, we observed that the secretion of tau is temperature-dependant, as higher temperature increased total tau level in the extracellular medium. However, at higher temperatures, tau-secreted species are less phosphorylated on several epitopes. Finally, our results demonstrated that tau cleavage at D421 by caspase-3 regulates its secretion, since the level of extracellular tau was decreased when cells were treated with a selective caspase-3 inhibitor (zDEVD-FMK).

Taken together, these data suggest that tau phosphorylation and secretion follow a circadian rhythm driven mostly by body temperature and sleep. Since AD patients are prone to sleep disturbances and thermoregulation deficits, our study provides new knowledge on how tau pathology could develop and spread.

KARIMI NARGES, Lemieux Maxime, and Bretzner Frédéric

Centre de recherche du CHU de Québec, Université Laval, Québec, Canada 

Although anatomical studies have shown plasticity of the reticulospinal axons of the gigantocellular reticular nucleus (Gi) after spinal cord injury (SCI), little is known about the functional contribution of the Gi to locomotor recovery. Using kinematic and electromyographic measurements in VGluT2-cre mice, we investigated changes in the motor efficacy of glutamatergic neurons of the Gi in relation to locomotor recovery following a lateral thoracic hemisection. Before SCI, short photostimulation delivered in the Gi evoked excitatory motor responses in the flexor muscle of the ipsilesional hindlimb. Although these motor responses decreased in more than half of mice 1 week after SCI, they recovered or increased 7 weeks after SCI. Interestingly, these changes in motor responses correlated with changes in the locomotor performance. Furthermore, long trains of photostimulations delivered in the Gi reduced the variability in the stepping ability and improved the position of the ankle prior to the swing phase. Finally, conditioning photostimulations of the Gi also improved voluntary locomotion 7 weeks after SCI. In summary, our findings show that glutamatergic neurons of the Gi contribute to locomotor recovery after SCI and can improve motor functions in chronic SCI.

Funding: Wings for Life Foundation and Craig H. Neilsen Foundation

Renaud Bernatchez, Theresa Wiesner, Chris Lintott, Renée Hložek, Audrey Durand, Flavie Lavoie-Cardina

Neurodegenerative diseases such as Alzheimer’s disease and ALS are thought to involve structural changes at the synapse level. Super-resolution fluorescence microscopy can be used to image synaptic proteins at the nanoscale, generating images that contain valuable information about the impact of synaptopathies on the organization of these proteins. However, the analysis of this data is difficult due to the large amount of synapses present in the images. We therefore turn to artificial intelligence approaches to automate analysis tasks. Such methods require large amounts of annotated data, which is tedious to obtain for a single expert. Additionally, as there may not be strict ground truth to such images, data labeled by a single expert may not be reliable. Therefore, there is a need for a large and reliable annotated dataset of synaptic protein images to train models to automate analysis approaches.

We propose to generate classification and segmentation datasets of synaptic proteins in super-resolution microscopy images using crowdsourcing. To this end, we use the Zooniverse platform to collect data from two workflows: classification and segmentation. In the former, volunteers classify synaptic protein clusters into different morphology profiles. A secondary task follows, where volunteers mark the center or orientation of clusters. In the second workflow, volunteers choose which of two predetermined segmentations is more appropriate for a protein cluster. We acquire 10 annotations per synapse for each task. This allows both to reduce the variance in the annotations caused by various degrees of skill between volunteers and to capture the actual noise distribution in the labeling.

Over 2000 volunteers have completed over 1.7 million annotations so far. The classification workflow has been fully completed for the current batch of images to be annotated, leading to over 6000 classified and marked synaptic protein clusters. The segmentation dataset is still underway, currently at 80% of annotations acquired.

We aim to use the generated dataset to help train AI models to detect differences in the structure and organization of synaptic proteins in cells from healthy control and disease animal models. This dataset will also serve at  benchmarking new image analysis methods for neuroscience image data.

Marie Roussel, Maxime Lemieux, Frédéric Bretzner.

INTRODUCTION: Electrical stimulation of the midbrain has been shown to improve locomotor recovery after spinal cord injury (SCI). Are part of this functional region: the cuneiform nucleus (CnF) and the pedunculopontine nucleus (PPN). We have recently shown that activation of glutamatergic CnF neurons initiates and accelerates locomotion, whereas glutamatergic and cholinergic PPN neurons decelerate and stop locomotion in the mouse. OBJECTIVE: We hypothesized that these distinct neuronal populations contribute differently to locomotor recovery after SCI. METHODS: Transgenic VGluT2-cre mice were injected with AAV to genetically ablate or photostimulate glutamatergic CnF or PPN neurons. Although mice dragged initially their ipsilesional hindlimb, they recovered locomotor functions by the 3rd week post-SCI. RESULTS: 7 weeks post-SCI, genetic ablation of VGluT2+CnF neurons deteriorated motor functions during walking and swimming, whereas ablation of VGlut2+PPN neurons mildly impaired swimming. Short photostimulations of VGluT2+CnF or PPN neurons evoked phase-dependent electromyographic (EMGs) responses in hindlimb muscles during locomotion. Responses decreased at week 1 post-SCI but recovered by week 4 with locomotor recovery. Furthermore, long trains of photostimulations of VGlut2+CnF neurons improved and accelerated the locomotor pattern and rhythm, whereas VGlut2+PPN neurons failed to improve locomotor functions. CONCLUSION: Although the PPN has been considered as a target in clinical settings, our study argues that glutamatergic neurons of the CnF will be a better neurological target to improve functional locomotor recovery in SCI patients.

Owen Ferguson, Julia Obergasteiger, Kamylle Thériault, William Leclerc, Thomas Durcan, Martin Lévesque, Flavie Lavoie-Cardinal

OBJECTIF – OBJECTIVE

Mitochondria are dynamic and sensitive organelles responsible for providing the majority of the brain’s energy, and their dysfunction has been implicated in Parkinson’s Disease (PD)—one of the most common neurodegenerative diseases. The objective of this project is to characterize mitochondrial dysfunction and morphology in human in vitro models of PD.

MÉTHODES – METHODS

We created human models of PD by differentiating patient-derived induced pluripotent stem cells and their isogenic controls into dopaminergic neurons. The electrical activity of the cultures was measured using a Multi-Electrode array (MEA). To assess mitochondrial health in PD, we used the fluorescent functional reporters Tetramethylrodamine (TMRM) and MitoSox to reveal mitochondrial membrane potential and reactive oxygen species levels. Furthermore, we used super-resolution STimulated Emission Depletion (STED) microscopy to characterize nanoscopic mitochondrial morphology in the context of PD and establish a link between mitochondrial dysfunction and aberrant morphology. Finally, we established an automated semantic segmentation analysis pipeline to identify and characterize the morphology of the mitochondria in dopaminergic neurons.

RÉSULTATS – RESULTS

Preliminary data on the electrical activity of the cultures shows that the isogenic control group tends to fire more often and that the PD-associated group is more synchronized across the culture when there is electrical activity. Preliminary results obtained with fluorescence microscopy in PD-associated neurons show deficits in mitochondrial health through depolarized membrane potentials and increased reactive oxygen species levels alongside increased mitochondrial fragmentation when compared to isogenic controls. Relative to the isogenic controls, the mitochondria in the PD-associated dopaminergic neurons are smaller in terms of both area and volume, are more circular, have a smaller aspect ratio, and have a higher population density.

CONCLUSION

Once we establish the link between mitochondrial dysfunction and aberrant mitochondrial morphology, we aim to apply a similar methodology and use STED microscopy to visualize the mitochondria in PD-associated human neurons that have received neuroprotective treatments. With these methods in place for sample labelling, image acquisition, and image segmentation and analysis, we will be able to quickly quantify the efficacy of novel neuroprotective treatments.

Deschênes, Andréanne; Girard, Antoine;  Gagnon, Samuel; Lebrun, Alexis; Gagnon, Philippe; Paquet, Marie-eve; Boudreau, Denis; Lavoie-Cardinal, Flavie

Increasing scientific evidence suggests that diet and lifestyle can impair the tight and adherens junction (TJ/AJ) structures in both the gut epithelial barrier and in the blood brain barrier (BBB) to the extent that food or bacterial antigens can access the underlying immune system more freely due to alterations in membrane permeability. Certain lifestyle factors, such as chronic stress, may be a cause for mental health disorders including depression and anxiety. To study the alterations to TJ structures and membrane permeability, we will develop a live-cell multi-modal microscopy and spectroscopy platform. This will allow precise measurements of TJ/AJ protein structure and dynamics at the nanoscale combined with label-free imaging of paracellular molecules.

We are developing a multimodal microscope including three complementary microscopy modalities: 1) 2-color parallelized REversible Switchable OpticaL Fluorescence Transitions microscopy (RESOLFT), 2) 3-color Structured Illumination Microscopy, 3) Single Molecule Localisation Microscopy. RESOLFT microscopy uses reversibly switchable fluorescent proteins (rsFPs) and can be parallelized to perform high-speed imaging with optimized resolution and contrast. The proteins of the TJ and AJ will be labeled with rsFPs using CRISPR-Cas9  based gene editing to incorporate the rsFPs directly into the genome, allowing super-resolution imaging of endogenous protein organization. These tools will be used to perform membrane permeability measurements in correlation with imaging of TJ/AJ protein nanoscale organization, with and without stress associated cytokines to simulate pathogenic conditions. These experiments will be conducted in a gut model composed of a monolayer of differentiated Caco-2 cells on a porous membrane between two microfluidic channels reproducing the apical and basal sides of the gut membrane.

We present the design and preliminary integration of different modalities to the multimodal imaging platform including parallelized confocal image acquisition. Using plasmids that express fusion proteins with red and green rsFPs, we performed preliminary RESOLFT experiments on an existing point-scanning microscope. We developed a standardized protocol for the differentiation of Caco-2 cells in order to obtain a monolayer of mature epithelial cells as a model for the intestinal membrane. As a baseline for future live-cell imaging experiments, immunostaining of claudin-3 and f-actin was performed on a mature monolayer of epithelial cells to ensure the morphological integrity of the model.

Once the microscopy platform is complete, it will be possible to simultaneously  characterize specific TJ/AJ proteins along with cell morphology using RESOLFT and SIM. This platform will allow monitoring of the dynamic structural and functional changes of the TJ/AJ proteins under pathologic conditions off the BBB.

Hoyer R.S., Ginzburg J., Picard M., Labelle C., Albouy P.

Introduction: Working memory refers to the cognitive ability to hold and manipulate information and is supported by fronto-parietal functions (Niendam et al., 2012; Owen et al., 2005; Wager & Smith, 2003). Brain oscillations reflect the rhythmic activity of neural networks. Recent results showed that fronto-parietal activity in the human brain can selectively be entrained by rhythmic sensory stimulations (Albouy et al., 2017): these non-invasive stimulations can causally modulate task performance in another modality (e.g., visual stimulations increase auditory working memory performance; Albouy et al., 2022). More particularly, visual 3D shapes in rotation flickering at 5 Hz before each trial of an auditory working memory task i) enhance theta activity in brain areas supporting working memory and in consequence ii) enhance behavioral performance (Albouy et al., 2022).

Methods: The present study investigates whether the aforementioned supramodal brain entrainment induced by visual rhythmic stimulation can be generalized across sensory modalities. Our hypothesis is that visual rhythmic stimulations can boost working memory performance irrespective of the sensory nature of the manipulated information. To address this hypothesis, we created a new visual working memory task, which was performed by 20 adults (18-30 years old) while their brain activity was recorded using EEG. Participants performed different blocks of the task with and without visual rhythmic stimulation.

Results: Preliminary results show that theta oscillation in the fronto-parietal network (at the sensor and source levels) positively predicts behavioral performance during manipulation periods. Moreover, visual rhythmic stimulation presented at theta rate increases both behavioral performance and brain theta oscillatory activity in the fronto-parietal network.

Discussion: This study enlarges the basic understanding of working memory and supports the assumption that rhythmic sensory stimulation can be used to causally enhance working memory during task performance. The present results also highlight that the beneficial effects induced by this new brain stimulation technic can generalize across sensory modalities.

SALEM SHIREEN, Alpaugh Melanie, Saint-Pierre Martine, Bellande Tracy, Melki Ronald, Cicchetti Francesca

In primary and secondary tauopathies, tau becomes hyperphosphorylated and accumulates into various pathological forms. In Huntington’s disease (HD) -a genetic neurodegenerative disorder- huntingtin (HTT) is the disease associated protein however the presence of hyperphosphorylated tau, accumulation of neurofibrillary tangles and increased expression of 4R tau isoforms in post-mortem brain tissue from HD patients, suggest this disorder may be a secondary tauopathy. Given the presence of both mutant HTT (mHTT) and pathological tau in HD patients, our study aims to evaluate the interactions of these two proteins. We hypothesize that the introduction of tau to cell and animal models of HD exacerbates intracellular HTT aggregation, and modifies disease associated features.

To test this hypothesis, human synthetic recombinant 3R/4R tau were introduced to a cellular model (StHdhQ111/Q111) and a knock-in mouse model (zQ175) of HD, followed by assessment of cellular dysfunction and behavioral changes.

In vitro, control Q7 and HD Q111 cells were treated with various tau doses and tau fibrils significantly altered cell metabolism as measured by a MTT assay. The effect of tau on HTT aggregation was assessed by filter retardation assays, demonstrating that both monomeric and fibrillar 3R tau are sufficient to increase HTT aggregation in HD cells.

In vivo, bilateral intracerebral stereotaxic injections of tau were performed in the prefrontal cortex and hippocampus of wild-type (WT) and zQ175 mice at 3 months of age. A battery of behavioral tests were conducted at baseline and post-surgery. Our behaviour data show that tau fibrils aggravate cognitive and anxiety-like behavior in zQ175 mice. Post-mortem studies revealed that behavioral changes are accompanied by an increase in mHTT aggregation in brains of zQ175 mice treated with fibrils as observed by both filter retardation assays and immunohistochemistry.

Together, our results show that tau treatment alters cellular features associated with HD and worsens behavioral phenotypes in mice with a particularily striking effect of 3R fibrils on HTT aggregation. This supports the idea that tau pathology contributes to HD pathology, and further elucidation of the mechanisms by which this occurs could reveal new therapeutic strategies for HD.

Alana Arrouet1,2, Eduardo Marques-Carneiro1, Pierre Marquet2 and Anne Giersch1

1: INSERM U1114, Cognitive Neuropsychology and Pathophysiology of Schizophrenia, University of

Strasbourg, Strasbourg, France

2: CERVO Brain Research Centre, Québec, QC, Canada

Background: Alterations in time experience and bodily self are found to be related in psychosis and could serve as a predictive marker in high-risk individuals. To objectify these disorders, we explore how the passage of time can be used to anticipate the occurrence of predictable stimuli. To do so, we use a well-known paradigm: the variable foreperiod paradigm, during which a signal is followed at variable delays by a target. In neurotypicals, the more the participant waits for the target, the faster he reacts to it. This is called the ‘hazard function’ (HF) and is altered in patients with disorders of the self. We additionally measure sequential effects (SE) which are used to explore how participants are automatically influenced by what happened on the previous trial to build their temporal prediction for the current trial. Before testing the target populations, we tested neurotypicals to validate our new task. These results are presented in the following.

Method: Participants are asked to perform arm movements inside a box in response to tactile stimuli. A first tactile signal indicates they can start their movement in a straight line. After a short or long delay, a second tactile signal indicates they have to stop their movement as soon as possible. We use two conditions: in one condition participants are instructed to imagine that there is a maze inside the box and to change direction between each trial; in the other condition participants are asked to repeat the same movement at each trial.

Results: In our two conditions, we found an HF on the stopping latency of our participants: they stop faster after a long delay than after a short one. We were also able to collect the exact moment when participants start to slow down their trajectory in anticipation of the stop signal, which is a useful proxy of the temporal prediction abilities. In the condition where participants repeated their movement, we found SE i.e., the current trial performance was influenced by the previous trial.

Conclusion: Our protocol allows us to acquire markers of temporal prediction, and especially indexes of prediction during the action itself (the slowing down of the trajectory). The SE results suggest a role for temporal prediction related to the motor action, which will be useful to explore difficulties related to the bodily self-alterations.

TRABOLSI CHRISTINE1, Alpaugh Melanie2, Saint-Pierre Martine1, Storck Steffen3, Pietrzik Claus4, Cicchetti Francesca1

1 Centre de Recherche du CHU de Québec/ Université Laval, Canada; 2 University of Guelph, Canada; 3 Washington University of St. Louis, United States; 4 University Medical Center of the Johannes Gutenberg-University, Germany

Introduction et objectifs: La maladie de Huntington (MH) est une maladie neurodégénérative héréditaire dominante caractérisée par l’expression d’une forme mutée de la protéine huntingtine (mHtt). La mHtt pourrait être un analogue des prions capable d’endocytose et contribuant à la progression de la maladie de la périphérie vers le système nerveux central (SNC). Diverses études sur les maladies neurodégénératives ont montré que certains transporteurs, retrouvés au niveau de la barrière hématoencéphalique, peuvent favoriser la clairance de protéines pathologiques, notamment le récepteur des lipoprotéines de basse densité (LRP1). Nos données préliminaires suggèrent que les niveaux d’expression de LRP1 sont altérés chez les patients souffrant de la MH et que son inhibition réduit l’endocytose de mHtt in vitro. Nous avons également observé que bloquer LRP1 atténue certains signes de la MH chez un modèle animal. Nous cherchons maintenant à comprendre si l’inhibition du transport de mHtt par un anticorps ciblant LRP1 peut affecter la progression de la MH.

Méthodes: Dans un premier temps, la demi-vie ainsi que la dose optimale de l’anticorps ont été sélectionnées par des études pharmacologiques. Dans un deuxième temps, des injections intrapéritonéales de l’anticorps ou de l’IgG contrôle ont été administrées à un modèle animal de la MH pendant 4 mois (6-10 mois d’âge). Des tests comportementaux ont été effectués à des intervalles de 1 mois. Des analyses post-mortem sont actuellement en cours.

Résultats: À la suite de l’optimisation d’ELISA, nous avons pu mesurer la concentration de l’anticorps dans le plasma du modèle animal, et caractériser ses paramètres pharmacodynamiques; i.e. une injection hebdomadaire de dose 0.8 mg/Kg. Les analyses comportementales et post-mortem sont actuellement en cours.

Conclusion: LRP1 est impliqué dans l’endocytose de mHtt dans le SNC. L’inhibition de LRP1 devrait réduire la quantité de mHtt au sein du SNC et ainsi atténuer les signes pathologiques associés à la MH.

LLINARES JODIE1, Bélanger Erik1, Maziade Michel1, Jourdain Pascal2,Marquet Pierre1,2, 1Centre de recherche CERVO , 2Unité Mixte Internationale, Université Laval et Université de Lausanne, Suisse

OBJECTIVE

Schizophrenia (SZ) is a psychiatric disorder with a neurodevelopmental component affecting about 1% of the population. Alterations of the inhibitory system maturation during the cortical development, inducing perturbations in the excitatory/inhibitory (E/I) balance, are likely to be involved in its pathogenesis. GABA, the main inhibitory neurotransmitter in the adult brain, was shown to be excitatory during early neurodevelopmental stages due to high intracellular chloride (Cl-) concentration resulting from a low expression of the potassium-chloride cotransporter KCC2. It has been proposed that a delay of the GABA transition from excitatory to inhibitory during neurodevelopment could represent a risk biomarker of SZ.

METHODS

We have been developing a methodology based on Digital Holographic Microscopy (DHM), to non-invasively monitor the maturation of the GABAergic system during in vitro neurodevelopment of iPSC-derived cortical neurons obtained from SZ patients. Our first step was to analyze the DHM-quantitative phase signal (QPS), allowing chloride transmembrane movements monitoring, in combination with calcium imaging and gramicidin-perforated patch-clamp in primary cultures of rat cortical neurons.

RESULTS

During early neurodevelopmental stages, GABA-mediated depolarization via Cl- efflux induces calcium (Ca2+) influx through voltage-gated Ca2+-channels. GABA-induced Ca2+ response was associated with a QPS increase, reflecting Cl- efflux, in 83.1%  and 17.8% of the neurons at 4 and 15 DIV respectively. These QPS and Ca2+ responses were blocked by GABAA receptors antagonists. In 4-6 DIV neurons, the GABA-mediated QPS increase was associated with an inward Cl- current. In contrast, an outward Cl- current was accompanied by a minute QPS decrease in 14-15 DIV neurons.

CONCLUSION

Our results stress that DHM is able to non-invasively monitor the transmembrane Cl- movements mediated by GABA. Excitatory Cl- efflux results in QPS increase in immatures neurons whereas inhibitory Cl- influx leads to minute QPS decrease. This non-invasive DHM based methodology is now ready to explore the GABA maturation in iPSC-derived cortical neurons cultures obtained from patients with SZ, high-risk offspring and controls.

Bader E1, Poulin H1, Lemieux M1, De Koninck P1,2

1 CERVO Brain research Center, Québec City

2 Department of Biochemistry, Microbiology, and Bioinformatics, Université Laval, Québec City

Synaptic plasticity is involved in numerous neurological phenomena, including brain disorders and the mechanisms underlying memory and learning. To learn more on these phenomena and how there are influenced by the exposome, we are exploiting the developing zebrafish. A transgenic zebrafish line is here developed to express fluorescent biomarkers targeting synapses and neuronal membranes in order to monitor their appearance and organisation throughout the first few weeks of development. FingRs (Fibronectin Intrabodies generated with mRNA display) are genetically encoded proteins able to target the post-synaptic proteins gephyrin or PSD95 located at inhibitory and excitatory synapses, respectively. When associated with a fluorescent protein, FingRs act as biomarkers for gephyrin or PSD95, and thus for synapses. To express these biomarkers in zebrafish, plasmid DNA encoding FingRs coupled to a fluorescent protein is injected in fertilised zebrafish eggs. Integration of the plasmid DNA into the genomic DNA is allowed by the presence of a Tol2 transposon system encoded in the plasmid DNA. We use Confocal and 2-Photon microscopy techniques to observe fluorescence in live zebrafish and quantify synaptic organization across the brain. To resolve more refined details in the size and shape of the synapses, we will use STED (stimulated emission depletion) microscopy, which enables resolution beyond the diffraction-limit. We will next perform experiments to manipulate the exposome, including gut microbiota, neurotoxins, and stress, on these zebrafish models to investigate their influence on synaptic plasticity. Finally, we aim to correlate changes in synaptic development with the appearance of social behaviors. These experiments may demonstrate a useful model to study the early phase of synaptic development in vivo, its regulation, and sensitivity to the exposome.

J. Chabbert 1, A. Bilodeau 1,2 , F. Beaupré1,2 , P. De Koninck 1,3, F. Lavoie-Cardinal 1,2,4.

1 Centre de recherche CERVO, Québec, Canada, 2 Institut Intelligence et Données, Québec, Canada, 3 Département de Biochimie, Microbiologie et Bio-informatique, Université Laval, Québec, Canada, 4 Département de Psychiatrie et Neurosciences, Université Laval, Québec, Canada. 

OBJECTIF – OBJECTIVE

Actin, an essential protein for cell architecture, development, and migration, is ubiquitously present in all cell types. Previous studies with super-resolution microscopy techniques revealed a periodical structure of filamentous actin (F-actin) rings, under the membrane of axons and dendrites [1]. Recent observations indicate that the dendritic F-actin ring structure tends to reorganize in a fiber structure following neuronal stimulation in dendrites but not in axons of cultured hippocampal neurons [2]. A weakly-supervised deep-learning image analysis approach had been developed to quantify the prevalence of these two F-actin patterns [2]. We aim to uncover the molecular mechanisms of this structural reorganization and hypothesize that β-Ca2+/Calmodulin-dependent kinase II (βCaMKII), a major Ca2+-sensitive protein activated during synaptic plasticity, is involved.

MÉTHODES – METHODS

Thus, using Stimulated Emission Depletion (STED) nanoscopy in combination with F-actin rings/fibers segmentation algorithms, we are able to investigate dendritic βCaMKII distribution depending on F-actin nanostructures.

RÉSULTATS – RESULTS

As preliminary results we observed that βCaMKII tends to associate with F-actin fibers.

CONCLUSION

Therefore, using super-resolution microscopy techniques combined with single molecule imaging and deep-learning analysis, we will further identify the molecular mechanisms involved in actin remodeling.

Danuzia A. Marques1, Loralie M. Guay1, Aline M A de Souza2, Kathryn Sandberg2, Richard Kinkead1

1 Québec Heart and Lung Institute. Faculty of Medicine. Laval University. Quebec, QC. Canada

2 Division of Nephrology and Hypertension, Department of Medicine, Georgetown University, Washington, District of Columbia. US

BACKGROUND and HYPOTHESIS: Menopause increases the incidence of cardiovascular diseases and hypertension. However, only a subpopulation of menopausal women develops these disorders.  The origins of such distinct aging trajectories are unknown. Since stress has persistent and sex-specific effects on health, we hypothesized that early-life stress exacerbates the cardiovascular consequences of the loss of ovarian function in aging rats.

OBJECTIVE: Understand the neuroendocrine origins of the onset of hypertension associated with the decline of ovarian function in females.

METHODS: Neonatal maternal separation (NMS) is a clinically relevant model of early-life stress. Following birth, rats were either raised under standard conditions (CTRL) or subjected to NMS (3h/day from postnatal days 3 to 12). When the females reached 60 weeks (old age), we measured arterial blood pressure by “tail-cuff” method. We then measured the activity of the angiotensin-converting enzyme (ACE) and its homolog ACE 2 in serum, two key enzymes involved in the homeostasis of the renin-angiotensin system (RAS). Finally, we compared the expression of FosB, a transcription factor indicating neuronal activity, in the periventricular nucleus of the hypothalamus (PVN); this structure is involved in the development and maintenance of hypertension and regulation of stress responses. The PVN contains two major anatomical structures: magnocellular (vasopressin secretion) and parvocellular neuroendocrine-related functional and central autonomic control).

RESULTS: NMS females’ mean arterial blood pressure (MAP) was 12% higher than CTRL but heart rate was 9% lower. Interestingly, this stress effect was not observed in young adults (8 weeks old). The catalyzed activity of ACE was similar between groups, but ACE2 activity was 60% lower in NMS. The number of neurons expressing FosB was 55% higher in the magnocellular region of the PVN in NMS females. Finally, we observed a strong negative correlation between ACE2 activity and MAP (r2= -0.71, p=0.002).

CONCLUSIONS: Unlike CTRL, aged NMS females showed an increased risk of hypertension. The lower activity of ACE2 may lead to an accumulation of Angiotensin II, which stimulates the release of vasopressin. Vasopressin, in turn, inhibits the secretion of renin. Increased renin predisposes individuals to develop hypertension. The high magnocellular activation of PVN indicated that the vasopressin and the RAS might be interacting and contributing to the observed increase in MAP. Therefore, we conclude that loss of ovarian function reveals the effects of early-life stress on cardiovascular homeostasis in females.

ACKNOWLEDGMENTS: DAM is supported by the FRQ-S (268346) and the “Programme de complément de bourse” from RSRQ (2021-22). This research is supported by the Canadian Institutes of Health Research (RK).

1CAPERAA MARGAUX, 1Ladouceur Shanna, 1Poulin Sandrine, 1Lemieux Mado, 1,2Bossé Gabriel.D, 1,3De Koninck Paul.

1CERVO brain research center, Québec, Canada, 2Department of psychiatry and neurosciences, Université Laval, Québec, Canada , 3Department of biochemistry, microbiology and bio-informatics, Université Laval, Québec, Canada

OBJECTIF – OBJECTIVE

Childhood represents a crucial neurodevelopmental phase for the development establishment of complex behaviors such as social interactions, and their underlying neuronal networks. In zebrafish, larvae develop a rich repertoire of behavior over their first two weeks of development, becoming even more sophisticated. To date, little is known about the neuronal mechanisms underlying this complexification. Our goal is to investigate the neuronal circuits supporting the development of social behavior and characterize their neurodevelopmental window.

MÉTHODES – METHODS

Our model is the transparent zebrafish line casper, in which the appearance of skin pigments is suppressed, thus allowing brain optical imaging at larval stages beyond 15 days post-fertilization (dpf). To identify precisely the establishment of early signs of social behavior (between 1 to 28 dpf), we are performing a thorough longitudinal characterization using a series of behavioral assays to measure social interaction with custom-made 3D-printed arenas. Next, our goal is to design an experimental assay that can detect early signs of social interactions, over the same time window, but in larvae head-restrained under the microscope, in order to correlate their onset with changes in neuronal circuits activation. To observe the functional activity of developing neural networks in the larval brain, we use a transgenic line expressing a pan-neuronal nuclear-targeted calcium sensor, GCaMP6. We record calcium oscillations, as a read-out of neuronal activity, with fast, 3D, two-photon microscopy in living larval zebrafish.

RÉSULTATS – RESULTS

However, we anticipate that imaging zebrafish larvae beyond 15 dpf over several days may introduce issues of viability and signal resolution that need to be addressed. We are thus implementing modifications in the imaging conditions. Our preliminary tests indicate that we can monitor circuits activity at the cellular level in live zebrafish larvae up to 28 dpf.

CONCLUSION

Overall, this project will enable us to identify the precise developmental window for sociability development as well as the neuronal networks involved. Considering that several neurological disorders, such as autism, are characterized by early social deficits, such findings may provide insight into the onset of these disturbances

Ruxandra Ungureanu, Shirley Fecteau, Pierre Marquet

Background: With the increasing prevalence rate of severe mental disorders (SMDs), it is becoming critical not only to develop new intervention therapies but also to increase our knowledge of disease onset and progression. While conventional methods to treat SMDs usually involve a combination of psychotherapy and pharmacotherapy, a significant proportion (more than one third of individuals) either do not respond or have adverse effects. One potential alternative is the use of non-invasive brain stimulation techniques, such as repetitive transcranial magnetic stimulation (rTMS), which have been shown to be safe, well-tolerated, and an effective tertiary or adjunctive therapy option for the alleviations of SMD symptoms.

Objective: Although randomised controlled trials have repeatedly claimed excellent outcomes, a number of treatment parameters – including location, frequency, intensity, and duration – remain a significant barrier to properly treating SMDs. Consequently, more research into the mechanisms underlying rTMS is required in order to correctly and efficiently apply this technology to patient groups.

Methods: To this avail, we suggest a novel strategy to investigate the effects of different rMS protocols on cortical neural networks of patients with bipolar disorder and schizophrenia produced from induced pluripotent stem cells (IPSCs) while contrasting them with participants in a control group. Neurodifferentiation will be investigated using a high-resolution multimodal microscopy technique (digital holography microscopy [DHM]), which can monitor fast cellular processes.

Expected results: We expect that different protocols, performed during the neurodifferentiation process, are able to mitigate some of the alterations found in mature neural networks from patients suffering from bipolar disorder and/or schizophrenia.

Discussion: By opening the path for its use as a secure first-line therapy and as a medical tool to prevent the onset of mental health issues, this technique will challenge the present perception of rTMS as a third-line treatment. Moreover, the results may also inform on the ideal dosing for individualised therapy by taking the brain’s integrity and response to rTMS into account.

Antoine Légaré1, Vincent Boily1, Sandrine Poulin1, Mado Lemieux1, Patrick Desrosiers1,2, Paul De Koninck1,3

1Centre de recherche CERVO, Québec, QC, Canada

2Département de physique, de génie physique et d’optique, Université Laval

3Département de biochimie, de microbiologie et de bioinformatique, Université Laval

Les neuromodulateurs comme la dopamine ou la sérotonine exercent un puissant contrôle sur les réseaux neuronaux, leur permettant d’adapter flexiblement leur dynamique selon les états internes et les besoins externes. Pour développer des modèles qui décrivent leur fonctionnement à l’échelle globale du cerveau, nous exploitons l’accessibilité optique et génétique du poisson-zèbre au stade larvaire. À l’aide de microscopie à deux photons, nous effectuons l’imagerie calcique du cerveau entier de larves transgéniques tout en observant leurs mouvements de queue avec une caméra haute-vitesse afin d’investiguer l’influence des neuromodulateurs sur les états cérébraux et comportementaux. Une approche de partitionnement non supervisé nous permet d’extraire un ensemble de patrons d’activation des régions, ou états cérébraux, qui surviennent spontanément dans tous les individus et sur plusieurs séances d’imagerie. La distribution spatiale de l’activité des états est modulaire et contrainte par les connexions structurelles entre les groupes de régions fortement interconnectées du cerveau, tandis que les probabilités de transition entre les états sont similaires d’un individu à l’autre. Pour investiguer le rôle des neuromodulateurs dans l’émergence de ces états, nous utilisons des marquages immunohistochimiques post-hoc suivis d’un recalage numérique pour retrouver les cellules marquées parmi les données fonctionnelles, permettant l’extraction de signaux dopaminergiques et noradrénergiques. L’activité de ces neurones est corrélée aux battements de queue du poisson, ce qui récapitule leur rôle bien connu dans l’initiation du comportement. En mesurant la distribution des récepteurs par hybridation in situ ainsi qu’en perturbant pharmacologiquement le système, nous pourrons relier plus directement les états cérébraux aux systèmes neuromodulateurs qui en seraient la cause. Notre approche expérimentale et computationnelle promet d’approfondir notre compréhension des neuromodulateurs en tant que cibles thérapeutiques de choix dans plusieurs troubles neurologiques.

Gloria N. Edozie (1), Shanna Pigeyre (1), Ariane Gosselin (1), Mari Carmen Pelaez (1), Chantelle F. Sephton (1, 2), Silvia Pozzi (1, 2).

(1) CERVO Brain Research Centre, 2601, Av. de la Canardière, Québec, QC, G1J 2G3, Canada.

(2) Department of Psychiatry and Neuroscience, Université Laval.

INTRODUCTION

Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disease characterized by motor neurons degeneration. Peptidylprolyl cis/trans-isomerase A (PPIA) is a chaperone protein with intracellular isomerase activity and extracellular cytokine-like behaviours. When secreted PPIA binds to its receptor EMMPRIN (extracellular matrix metalloproteinases inducer) activating matrix metalloproteinase 9 (MMP-9) production. In SOD1G93A ALS animal model, PPIA is highly secreted in the CSF and expressed by motor neurons (MN) and glial cells. Moreover, SOD1G93A MN are more sensitive than healthy neurons to PPIA extracellular effect.  Although well characterized in SOD1G93A models, the relevance of this pathway in other familial ALS models, is still unknown.

AIM

Here we aim at characterizing the expression of PPIA and EMMPRIN in various ALS animal models.

METHODS

SOD1G93A, TDP-43A315T, and FUSR5215G mice were sacrificed at different disease stages together with age- and sex-matched non-transgenic mice (NTg). Lumbar spinal cord (SCL) and cerebrospinal fluids (CSF) were isolated. Samples were analyzed by immunoblotting for EMMPRIN and PPIA expression in SC and PPIA secretion in the CSF. Cellular localization was evaluated by immunofluorescence.

RESULTS

We found that SOD1G93A and TDP-43A315T mice present elevated levels of EMMPRIN in the SC at the beginning of their symptoms. The protein is mainly expressed by neurons and astrocytes. As seen in SOD1G93A mice, PPIA is expressed by neuronal and glial cells in TDP-43A315T mice. Differently from the previous models, we did not observe an increased release of PPIA in the CSF of FUSR521G mice.

CONCLUSION

The interaction between extracellular PPIA and EMPPRIN is an interesting therapeutic target for ALS. Our results shows that this pathway can be targeted in mutant SOD1 and TDP-43- forms of ALS, whereas its relevance might be lower in FUS-mediated ALS cases. Moreover, given the specific localization of EMMPRIN in MN and astrocytes, we can hypothesise a higher susceptibility of these cells to the PPIA/EMMPRIN pathway.

Imane Hadj-Aissa1 Chantelle Sephton1 and Paul Dutchak1
1Department of Psychiatry and Neuroscience, Université Laval, CERVO Brain Research Centre, Québec

Epilepsy is a neurological disorder affecting 300,000 people in Canada. GATOR1 is a cytoplasmic protein complex composed of protein subunits called NPRL2, NPRL3 and DEPDC5. A mutation in either subunit can result in loss of function of GATOR1 that will lead to focal epilepsy, autism and SUDEP. GATOR1 functions as an amino acid “sensor” that regulates the balance of protein synthesis and protein degradation by regulating the mTORC1 pathway. mTORC1 is a master protein kinase that regulates several cellular processes including, protein translation, autophagy and metabolism in cells. During amino acid deprivation, GATOR1 is necessary to inhibit mTORC1. Amino acid sufficiency prevents mTORC1 inhibition.

When GATOR1 is mutated, mTORC1 is activated even during amino acids starvation, which leads to increase protein translation and changes in mitochondrial metabolic pathways. Our previous studies have shown that defective GATOR1 activity stimulates a Warburg effect in cells and tissue by increasing oxidative glycolysis. We have started to investigate the effect of GATOR1 activity as a mechanism to control mitochondrial function, particularly its effect on pyruvate dehydrogenase (PDHE1), the gate keeper of pyruvate catabolism through the TCA cycle. When this enzyme is phosphorylated, it’s inhibited, and the pyruvate can’t entry the mitochondria. In our genetic and biochemical studies, we show that GATOR1 is an important regulator of PDHE1 phosphorylation and astrocyte activation.

In the brain, astrocytes have an important functional role to maintain metabolic homeostatic mechanisms that influence neuronal precursors for synaptic transmission. Our hypothesis is that a loss of function of GATOR1 contributes to the epileptic phenotype by affecting the astrocytes.

To test this hypothesis, we have generated a novel genetically engineered mouse model that lacks the expression of the GATOR1 subunit called, NPRL2 in astrocytes and an astrocyte cell-line model using CRISPR mediated deletion of NPRL3.

The data presented here show GFAP-NPRL2 mice have a hyperactivation of mTORC1 and astrogliosis at 5months of age in the hippocampus. We also present evidence that GATOR1 dysfunction in astrocyte cell-lines cause TCA cycle dysregulated through a nitrogen-dependent mechanism.

GOSSELIN Ariane1,2, Godin Antoine1,2 and Pozzi Silvia​1,2

1. CERVO Brain Research Centre

2. Department of Psychiatry & Neuroscience, Laval University

OBJECTIVE

Amyotrophic lateral sclerosis (ALS) is a progressive neurodegenerative disease characterized by the selective death of lower and upper motor neurons, leading to paralysis in voluntary muscles. The extracellular matrix (ECM), a complex network of molecules surrounding cells in tissues, plays a critical role in maintaining the structural integrity of the nervous system and is involved in various physiological processes. Disruption of the ECM has been recently associated with abnormal chloride homeostasis and in dysregulation of inhibition in neurons. Studies have also shown that ECM reorganization occurs in other neurodegenerative diseases, such as Alzheimer’s and Parkinson’s. Nevertheless, the ECM remodeling that happens during ALS progression have never been fully characterized. Since ECM is involved in many cellular processes and provides protection and support for cells, we hypothesize that ECM remodeling may affect the survival of motor neurons during the disease.

Understanding which components are regulated in ALS, at which stage of the pathology, and which are the mechanisms behind this regulation could improve our comprehension of the disease and lead to new therapeutic interventions. The goal of this project is to study the spinal cord ECM organization throughout the progression of the disease.

METHODS

We will use western blots and immunofluorescence on lumbar spinal cord samples from healthy and SOD1G39A mice, an animal model of ALS, to measure and compare ECM changes. The quantification of proteins of interest will be optimized for both methods at the tissue and at single motor neuron levels.

RESULTS

Here, we show preliminary results for the analysis of the main components (aggrecan, brevican, collagen IV, hyaluronan binding protein, neurocan, parvalbumin and versican) composing the ECM. Moreover, we propose a preliminary investigation of aggrecan expression in the ventral horn of the spinal cord of SOD1G93A mice with the progression of the disease.

CONCLUSION

These results represent the foundation for our further study of the ECM remodeling during the progression of ALS.

MAELINE MULLER1 ; Jasmine Bélanger1 ; Chantelle Sephton1 ; Paul Dutchak1 

1Department of Psychiatry and Neuroscience, Université Laval, CERVO Brain Research Center, Québec 

The mammalian target of rapamycin (mTOR) signaling pathway is involved in multiple processes including cell growth, protein synthesis or autophagy. mTOR signaling pathway is widely involved in the regulation of physiological processes in the brain and disruption of this pathway have been associated with multiple neurological diseases such as epilepsy.

The mTORC1 complex functions to integrate intracellular amino acid content and growth factor signaling in the cell. GATOR1 is a cytoplasmic protein complex formed by DEPDC5, NPRL2 and NPRL3 and respond to a low level in amino acid by hydrolyzing the RAG-GTP and inhibit mTORC1 activity. When GATOR1 is not functional and unable to play its inhibiting role, mTORC1 is permanently activated by GTP-bound RAGs even in presence of a low level of amino acid in the cell. Mutations that impair the functionality of GATOR1 complex have been associate to a wide spectrum of focal epilepsy suggesting that too high activation of mTORC1 is detrimental for the brain.

Both the localization of mTORC1 on lysosome via amino acid pathway and activation of the complex by the small GTP bound protein Rheb, via the PI3K/AKT pathway in response to growth factor, is required to have a full activation of mTORC1. It is still unclear how and why the balance between these two pathways is regulated depending on nutriment availability, and cell type specific growth factor signaling pathways. Thus, although a dysfunction in GATOR1 function directly impacts the amino acid sensing role of mTORC1, few studies have focused on the impact of these mutations on cross-talk with major growth factor signaling pathways. This include the PI3K/AKT pathway, an important signaling cascade that responds to growth factors, such as Brain-derived neurotrophic factor (BDNF), which plays an important role in neuronal survival and growth.

Here we investigate the coordinate regulation of mTORC1 by the GATOR1 amino acid-dependent signaling pathway and PI3K/AKT pathways to develop a mechanistic understanding of this pathway in epilepsy linked GATOR1 dysfunction.

Shanna Pigeyre1
, Morgan Bérard2
, Maxime Teixeira2
, Abid Oueslati2,3 and Silvia Pozzi1,4
1- Centre de recherche CERVO, 2601, Av. de la Canardière, Québec, QC, G1J 2G3, Canada
2- Centre de rechercher CHUL, 2705 Bd Laurier, Québec, QC G1V 4G2
3- Département de médecine moléculaire, Université Laval
4-Département de Psychiatrie et Neuroscience, Université Laval

Amyotrophic Lateral Sclerosis (ALS) is a neurodegenerative disease that causes the
degeneration of motor neurons and results in muscle atrophy. The “dying-back”
hypothesis, proposed for ALS, suggests that pathological events occur at the peripheral
sites, such as nerves, muscles, and the neuromuscular junction (NMJ), contributing to
motor neurons death.
TDP-43 (TAR-DNA binding protein 43) is a pathological protein in ALS. In the disease,
it mislocalisesfrom the nucleus to the cytoplasm forming phosphorylated and ubiquitinated
aggregates that induce cell death. This event is called TDP-43 proteinopathy and it is well
studied in motor neurons. Recently, several studies have demonstrated TDP-43
proteinopathy also in muscle of ALS patients and animal models, but its consequences in
these cells are still unknown.
A Light Inducible Protein Aggregation (LIPA) system has been recently developed to
obtain spatiotemporal aggregation of alpha-synuclein, a hallmark of Parkinson’s disease.
The general aim of this project is to generate a LIPA-TDP-43 system to study the TDP-43
proteinopathy at the level of the NMJ. Here we show the preliminary results obtained to
optimize and characterize the LIPA-TDP43 system in Hek-293 cells.
We subcloned TDP-43-WT fragment into a plasmid containing the Cry2 fragment (mutant
form of the Arabidopsis thaliana photoreceptor cryptochrome 2) and the mcherry tag (to
monitor the expressed construct) and used different concentration of plasmid to transiently
transfect Hek-293 cells. Cells were then stimulated with blue light for different time points.
TDP-43 aggregation was evaluated by immunofluorescence.
In transfected Hek-293 cells stimulated with blue light we were able to detect cytoplasmic
mcherry aggregates that were positive for TDP-43.
Thus, we showed that the produced LIPA-TDP-43-WT plasmid works in Hek-293 cells,
confirming that this system can be a good tool to study the consequences of TDP-43
proteinopathy. We are now implementing the LIPA-TDP-43 system in murine skeletal
muscles immortalized cells (C2C12 cell line). This model will allow us to study the
consequences of TDP-43 proteinopathy in muscle cells and at the level of the NMJ.

Odessa Tanvé1, Gabriel Byatt1, Mado Lemieux1, Marie-Eve Paquet1, Sylvain Moineau2, 3, Paul De Koninck1, 2

1CERVO Brain Research Center, Québec, QC, Canada, 2Université Laval, Département de Biochimie, microbiologie et bio-informatique, Québec, QC, Canada, 3Centre de référence Félix d’Hérelle pour les virus bactériens, Québec, QC, Canada

The gut microbiota is composed of thousands of different bacteria interacting with each other by the means of molecules released in the lumen of the intestine. The nature and amounts of these metabolites depend on the bacterial populations colonizing the intestine and the different conditions of the gut (available nutriments, oxygen, etc.). Several studies indicate that metabolites coming from the gut can reach the brain and impact its development and function.  We aim to learn more on the cellular and molecular mechanisms that regulate this gut-brain axis. To this end, we are studying the impact of the gut microbiota on the activity of microglia, cells involved in brain development, function and homeostasis. Through their phagocytic activity, during brain development, microglia eliminate debris and dying cells as well as prune synapses thereby controlling the formation of neuronal networks. We established a zebrafish model to follow the phagocytic activity of microglia with two-photon microscopy: a cross between two transgenic lines reporting either glutamatergic neurons in red (DsRed) or microglia in green (GFP). Phagocytic function is detected by the inclusion of DsRed from the neurons inside the microglia, as phagosome-like structures. Larvae from 3-16dpf (day post-fertilization), conventionally raised or axenic, exposed  to different concentrations of complex sugars, are imaged live to monitor the dynamic phagocytic activity.  We find that the phagocytic activity of microglia changes significantly over the period 3-10dpf. Our preliminary results suggest that while the phagocytosis by microglia drops at 10dpf in control larvae, it persists in axenic larvae. We find that this change in microglial activity is modulated by the metabolism of complex sugars by the gut microbiota.  We aim to identify the metabolites controlling this microglial activity and to determine how they affect the establishment of functional neural circuits.

Marion Boyer (1), Frédérique Crépeau (1), Silvia Pozzi (1,2)
(1) CERVO Brain Research Centre, 2601, Av. de la Canardière, Québec, QC, G1J 2G3, Canada
(2) Department of Psychiatry and Neuroscience, Université Laval.

Peptidyl-prolyl isomerase A (PPIA) has been described as a translational biomarker for amyotrophic
lateral sclerosis (ALS). PPIA is a chaperone protein with cis/trans isomerase activity involved in protein
folding and assembly. In the central nervous system of ALS models (SOD1G93A mice) PPIA is highly
expressed by motoneurons and glial cells. Once released, extracellular PPIA (ePPIA) binds to its
receptor EMMPRIN (extracellular matrix metalloproteinase receptor), inducing proinflammatory events
and the release of MMPs (matrix metalloproteinases). It is known that MMP-9 is implicated in motor
neuron death and MMP-7 has a role in Schwann cells migration and myelination of axons. The role of
the PPIA/EMMPRIN pathway in the peripheral nervous system (PNS) of ALS remains still unknown.
We hypothesized that the PPIA/EMMPRIN pathway is implicated in the communication between
Schwann cells, motoneurons and muscle cells, and that PPIA might contribute to motor neurons
degeneration and glial cells activation in the PNS of ALS conditions.
This study assesses PPIA and EMMPRIN expression in the PNS of SOD1G93A mice model of ALS at
different stage of the disease.
Sciatic nerves (SN) and tibialis anterior (TA) muscles were collected from non-transgenic mice (Ntg)
and SOD1G93A mice at different disease stages. Immunofluorescence staining was performed to unravel
PPIA and EMMPRIN localization and western blot experiments to investigate their levels in PNS
tissues.
Both western blot and immunofluorescence analyses showed that PPIA and EMMPRIN expression are
altered in SOD1G93A PNS tissues with the progression of the disease. Immunofluorescence experiments
revealed the presence of PPIA and EMMPRIN in motor neurons and glial cells of the PNS.
Although preliminary, this study shows alterations of PPIA levels in the PNS of SOD1G93A mice. Given
the relevance of PPIA in the central nervous system of SOD1G93A mice, both as an intracellular and
extracellular protein, our results highlight a possible implication of PPIA and of the PPIA/EMMPRIN
pathway also in the peripheral nervous system.

OYE MINTSA MI-MBA MÉRYL-FARELLE 1,2, Landry Olivier 1,2, François Arnaud 2, Traversy Marie-Thérèse 1,2, Tremblay Cyntia 2, Émond Vincent 2, Bennett David A. 3, Buxbaum Joseph 4 and Calon Frédéric 1,2

1Faculty of Pharmacy, Université Laval, Quebec City, Canada 2CHU de Québec – Université Laval Research Center, Quebec City, Canada, 3 Rush Alzheimer’s Disease Center, Rush University Medical Center, Chicago, IL, USA and 4Seaver Autism Center for Research and Treatment, Mount Sinai School of Medicine, NY.

OBJECTIF – OBJECTIVE

Alzheimer’s disease (AD) is an age-related pathology characterized not only by an accumulation of Aß plaques and hyperphosphorylation of tau protein, but also by synaptic loss, indexed by a reduction of pre and post-synaptic proteins. However, the consequences of lowering the expression of synaptic protein remain elusive. The postsynaptic protein Shank3 (SH3 and multiple ankyrin repeat domains) may be an exception because the loss of a single allele of the Shank3 gene is sufficient to cause profound cognitive symptoms in children. We therefore hypothesized that Shank3 deficiency contribute to the development or worsening of cognitive symptoms and neuropathology in AD.

MÉTHODES – METHODS

We first confirmed a significant postmortem reduction of Shank3 in the parietal cortex of AD patients (Braak stage 4-5) as well as a correlation between Shank3 levels and antemortem cognitive scores in these patients. To investigate the cause-effect relationship in AD, we developed an animal model by crossing Shank3-deficient (Shank3Δex4-9) and non-Shank3-deficient mice with a 3xTg-AD mouse model showing AD-associated neuropathology. The model was validated by in situ hybridization on sections and in Western blot on PSD extracts from the parietotemporal cortex. Results showed that Shank3 protein and mRNA expression were significantly reduced by 30-50% in Shank3-deficient mice compared to non-deficient animals, consistent with the results found in AD patients.

RÉSULTATS – RESULTS

We observed synergistic deleterious effects of Shank3 deficiency and AD neuropathology on object recognition memory at 9, 12 and 18 months of age and on anxious behavior at 9 and 12 months of age in hemizygous Shank3Δex4-9-3xTg-AD mice. Western blot analyses of PSD extracts from the parietotemporal cortex showed that levels of synaptic proteins such as PSD-95, drebrin, homer1 remained unchanged in hemizygous Shank3 mice. ELISA and Western blot analyses showed that Shank3 deficiency increased the levels of soluble Aß42 and human tau at 18 months of age compared to 3xTg-AD mice with normal Shank3 expression.

CONCLUSION

The results of this study in human brain samples and in transgenic mice are consistent with the hypothesis that SHANK3 deficiency plays a key role in the apparition of cognitive impairment in AD. Further investigations using dietary docosahexaenoic acid (DHA) treatment to increase Shank3 levels in the brain and lead to an improvement of cognitive symptoms are underway.

DOMINIC BÉLANGER, Benoit Mailhot, Steve Lacroix

Axe Neurosciences, Centre de recherche du CHU de Québec–Université Laval

Chez les individus souffrant de maladies auto-immunes inflammatoires telles que la sclérose en plaques (SEP), l’arthrite rhumatoïde (AR) et l’ostéo-arthrite (OA), la douleur chronique est présente chez plus de 50% des patients. La douleur est transmise par les nocicepteurs vers la moelle épinière et le cerveau par l’intermédiaire des ganglions de la racine dorsale (DRGs), où sont situés les corps cellulaires des neurones sensoriels. Une même cytokine pro-inflammatoire, l’interleukine-1 β (IL-1β), serait capable de déclencher la réponse inflammatoire de même que la douleur.

OBJECTIF – OBJECTIVE

Identifier les récepteurs et voies de signalisation médiant la réponse inflammatoire et la douleur induites par l’IL-1β.

MÉTHODES – METHODS

Nous avons caractérisé par immunofluorescence l’identité des neurones exprimant l’IL-1R1. Puis, nous avons injecté la forme recombinante de l’IL-1β à des souris ayant une délétion ou une restauration d’Il1r1. Nous avons par la suite réalisé différents tests de comportement. Finalement, une amplification rapide de l’extrémité 5’ de l’ADNc à partir d’ARN total provenant de DRGs a été effectuée.

RÉSULTATS – RESULTS

L’IL-1R1 est exprimé dans un sous-type de nocicepteurs des DRGs coexprimant le récepteur TRPV1 reconnu pour son rôle majeur dans la douleur chronique. L’invalidation d’Il1r1 spécifiquement dans ces nocicepteurs empêche l’apparition de douleur mécanique chez des souris atteintes d’encéphalomyélite auto-immune expérimentale (EAE) – un modèle murin de la SEP – d’AR ou d’OA sans pour autant affecter les autres signes cliniques de ces maladies. De plus, la restauration d’Il1r1 spécifiquement dans ces nocicepteurs restaure entièrement la douleur chez ces animaux. Enfin, nous avons détecté à partir d’ARN total extrait de DRGs la présence de sites d’initiation de la transcription d’une forme d’IL-1R1 qui pourrait être distincte et correspondrait à une forme tronquée, τIL-1R1.

CONCLUSION

IL-1β déclenche à la fois une réponse inflammatoire et douloureuse qui pourraient être indépendantes l’une de l’autre. La réponse douloureuse est médiée par des nocicepteurs TRPV1+ IL-1R1+. Nous croyons que ces nocicepteurs exprimeraient τIL-1R1 régissant la réponse douloureuse.

REVERET LOUISE1,3, Leclerc Manon1,3, Emond Vincent3, Loiselle Andréanne3, Bourassa Philippe1,3, Tremblay Cyntia3, Bennett David4, Hébert Sébastien2,3, Calon Frédéric,3;

1Faculté de pharmacie, Université Laval, Québec, Québec, Canada;  2Université Laval, Faculté de Médecine, Département de Psychiatrie et Neurosciences, Québec, QC, Canada; 3Axe Neurosciences, Centre de recherche du CHU-Q (Pavillon CHUL), Québec, Québec, Canada; 4Rush Alzheimer’s Disease Center, Rush University Medical Center, Chicago, IL, USA

OBJECTIF – OBJECTIVE

The emergence of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is a major cause of death, particularly in the elderly. The geriatric population in which cognitive decline due to Alzheimer’s disease (AD) is frequent was disproportionately affected by the pandemic.  In addition, central nervous system (CNS) manifestations have been reported in a significant subset of SARS-CoV-2 infected patients.

MÉTHODES – METHODS

Since the principal entry receptor utilized by SARS-COV-2 is Angiotensin-Converting Enzyme 2 (ACE2), we examined whether post-mortem ACE2 protein and mRNA levels were altered in parietal cortex samples from two different AD cohorts, totalling 142 cases.

RÉSULTATS – RESULTS

Both immunoblot and RT-qPCR analysis revealed higher concentrations of ACE2 protein and mRNA in persons with a neuropathological diagnosis of AD, compared to age-matched controls. Brain levels of ACE2 were inversely correlated with antemortem cognitive scores. We found that ACE2 protein was highly enriched in microvessels of mice compared to brain parenchyma, but not in humans. These observations were confirmed with immunostaining in both species.  Detachment of ACE2 from brain cell membranes was strongly associated with pericytes loss.

CONCLUSION

Our data suggest that cognitive impairment is associated with higher levels of ACE2 in the brain, which might contribute the higher risk of CNS SARS-CoV-2 infection in cognitively impaired individuals and AD patients.

Ana Boisvert1, Adrian Castellanos-Molina1, Nicolas Vallieres1, Martine Lessard1, Éric Boilard2, and Steve Lacroix1

1 Neurosciences Axis, CHU de Québec-Université Laval Research Center, Quebec City, QC, Canada;

2 Infectious and Immune Diseases Axis, CHU de Québec-Université Laval Research Center, Quebec City, QC, Canada.

Spinal cord injury (SCI) results in loss of sensation and voluntary movements below the site of injury, for which there are still no cure and few drugs under development. SCI divides into primary damage, occasioned by the physical impact, and secondary damage which begins with the release of alarmins by necrotic cells into the spinal cord parenchyma, thus initiating neuroinflammation. During secondary damage, the blood-spinal cord barrier (BSCB) experiences changes that increase permeability. In addition, endothelial cells (ECs) are activated, enhancing immune cell recruitment. We recently discovered that the alarmin IL-1α is released by necrotic microglia immediately after SCI. Thus, we injected recombinant mouse IL-1α intra-cisterna magna in adult mice to study its effects on the spinal cord. We found that IL-1α induces infiltration of neutrophils, disruption of the BSCB, and the death of mature oligodendrocytes within 24 hours after injection. Notably, the permeabilization of the BSCB was correlated with the infiltration of immunoglobulin type G (IgG) and their internalization inside neurons of the spinal cord, midbrain, and brainstem. Taking advantage of IL-1R1-restored mice, which exhibit an IL-1R1 knockout phenotype that can be reversed in a cell-specific manner by Cre-mediated recombination, we restored IL-1R1 expression in ECs. We found that IL-1R1 signaling in ECs directly drives the disruption of the BSCB, as well as the infiltration of the IgG. Using anti-Gr1 antibody, we performed a depletion of myeloid cells, showing that the absence of these cells prevented BSCB disruption, while infiltration of IgG still occurred. This suggests that crosstalk between myeloid cells and ECs is at the origin of BSCB permeability, but IgG infiltration must occur by mechanisms independent of the loss of the integrity of the BSCB.

Josue Valentin 1,2, T-M. Vu, C1,2, Tremblay 1,2, B. Andersen3, F. Calon 1,2

1Faculty of pharmacy, Université Laval, Québec, Qc, Canada; 2 Neuroscience Axis, CHU de Québec-Université Laval; 3Department of Diabetes and Obesity Biology, Novo Nordisk A/S, Denmark

Background

Alzheimer’s disease is an incurable form of dementia that is recognized to have a strong metabolic component.  This opens the possibility to target metabolic deficits as therapeutic strategies for AD. FGF21 is a hormone with important metabolic effects that is being tested in clinical trials for diabetes and obesity. There is evidence showing that FGF21 can also act on the CNS and modulate brain functions, but such mechanisms are less understood.

Methods

We used a transgenic mouse model of AD (3xTgAD) that develops tau and amyloid pathologies over time. Mice were fed either a CD (5% w/w) or a HFD (35% w/w) for 9 months and received rhFGF21 (1 mg/kg/day, osmotic minipump) or vehicle from 15 to 16 months of age. Metabolic and cognitive tests were performed during the last week of treatment and protein analysis in the hippocampus and liver were performed post-mortem.

Results

FGF21 administration profoundly improved metabolic determinants in NonTg and 3xTgAD, including decreased levels of fasting glucose and insulin, and improved glucose tolerance.  The novel object recognition test (NOR) and Dark-light box test (DLB) revealed that FGF21 improved anxiety and memory in 3xTgAD mice. Moreover, tau phosphorylation was not affected in the hippocampus while the Aβ42/ Aβ40 ratio was reduced female 3xTgAD mice. FGF21 increased FGFR1 phosphorylation in the liver, but not in the brain.

Conclusions

FGF21 is an important metabolic regulator. The effects on cognitive endpoints observed here suggest that FGF21 can also present a therapeutic potential for CNS diseases, such as AD. Whether its impact on the brain is direct or indirect through the periphery remains to be determined.

BOUCHARD SARAH-JULIE, Gauthier Léa-Maude, Obergasteiger Julia, Zhuo Yizhou, Proulx Christophe, Volta Mattia, Li Yulong, Lévesque Martin, Breton-Provencher Vincent

(U Laval et al) CERVO Brain Research Center, Quebec City, Quebec, Canada

Psychiatry and Neurosciences Department, Université Laval, Quebec City, Quebec, Canada

(YL et al) State Key Laboratory of Membrane Biology, Peking University School of Life Sciences, Beijing, China 

(Volta) Institute for Biomedicine, Eurac research, Bolzano, Italy

The dopamine system carries critical learning signals through the mesocorticolimbic pathway, in which dopaminergic neurons from the ventral tegmental area send dopamine to the ventral striatum and the cortex. How homogeneous dopamine release along the mesocorticolimbic pathway is during reinforcement learning remains unknown. OBJECTIVE Here, we measured dopamine signals in two critical areas of this pathway: the nucleus accumbens (NAc), known for its role in reward processing, and the medial prefrontal cortex (mPFC), known for its role in associative learning. METHODS To compare fast kinetics of dopamine release, fiber optics were simultaneously implanted above the NAc and mPFC to measure fluorescence of a novel and improved dopamine sensor (GRAB-DA3) with multi-site fiber photometry. Head-fixed mice were conditioned over several sessions to obtain a reward following an auditory tone of a fixed frequency. Following these initial sessions, we varied the task contingencies to measure the dopamine reinforcement signal to reward prediction error, reward magnitude and reinforcement stimuli of variable valence.  RESULTS Our preliminary data show that dopamine linked to reward prediction error and reward magnitude is signaled homogeneously throughout the mesocorticolimbic pathway. However, negative reinforcement produces relatively larger dopamine release in mPFC compared to NAc. To understand the origin of these heterogeneous signals, we are carrying anatomical tracing studies to measure how many neurons from the ventral tegmental area have mPFC-only projections. Since recent evidence shows a discrepancy between somatic activity of dopaminergic neurons and the release of dopamine in target regions, our ongoing experiments are also testing cellular mechanisms that could locally regulate dopamine release. One important candidate could be the protein Rin, coded by the Rit2 gene, which we found to interact with the dopamine transporter DAT and could play a role in dopamine release. CONCLUSION Together, our results indicate that dopamine release signals aversive stimuli primarily in mPFC, yet dopamine release linked to reward is a prevalent feature of the mesocorticolimbic pathway.

GODBOUT KELLY, Leblanc Maxeen, Rousseau Joël, Tremblay, Jacques P.

Centre de recherche du CHU de Québec-Université Laval

Université Laval, Département de médecine moléculaire.

OBJECTIF – OBJECTIVE

Notre objectif est de développer un traitement de thérapie génique pour les myopathies causées par une mutation du gène RYR1. Ce gène code pour un canal à calcium dans les fibres musculaires. Sa défaillance cause chez les patients des crampes et des faiblesses musculaires pouvant dégénérer en incapacité motrice. Pour le moment, il n’existe pas de traitements efficaces pour ces maladies. Nous tentons donc de développer une thérapie génique par Prime editing pour corriger la mutation et restaurer la séquence saine du gène. Ces maladies sont principalement causées par la mutation d’un seul nucléotide. Pour notre traitement, le Prime editing, qui permet justement de modifier un nucléotide précis dans l’ADN, sera utilisé. Cela permettra de remettre la séquence du gène intacte.

MÉTHODES – METHODS

Dérivé de la technologie CRISPR/Cas9, le Prime editing est composé d’une Cas9, d’une transcriptase inverse (RT) et d’un prime editing guide (peg). Côté expérimental, nous avons tout d’abord construit les composantes nécessaires au Prime editing par clonage. Plusieurs versions du peg ont été construites pour optimiser l’efficacité du prime editing. Le traitement a été expérimenté dans plusieurs types cellulaires (HEK293T, fibroblastes de patient, myoblastes, C2C12 et cellules primaires de souris). Par clonage manuel, nous avons également généré par prime editing une lignée de myoblastes portant la mutation T4709M dans le gène RYR1 à l’état homozygote, et ce à partir de myoblastes normaux.

RÉSULTATS – RESULTS

L’édition atteint présentement 63 % en une seule transfection dans des cellules HEK293T. Dans des myoblastes humains, ce taux s’élève à 24 % et 33 % pour la mutation silencieuse adjacente à la mutation, après respectivement un et trois traitements par électroporation.

CONCLUSION

Ce projet aura un impact significatif dans le traitement d’autres maladies héréditaires. En effet, cette méthode peut s’appliquer à d’autres mutations génétiques, et dans chaque cas, seul le peg serait à modifier !

TANGUAY EMMERAUDE1,2,3, Breton-Provencher  Vincent1,2, De Koninck Paul1,3

1. CERVO Brain Research Center, Quebec City, Quebec, Canada, 
2. Psychiatry and Neurosciences Department, Université Laval, Quebec City, Quebec, Canada
3. Biochemistry, Microbiology and Bioinformatics Department, Université Laval, Quebec City, QC, Canada

The noradrenergic system modulates sensory processing, attention, learning and memory formation. To support this role in cognition, noradrenergic axons densely innervate the cortex suggesting that noradrenaline affects cortical circuits. However, the cellular mechanisms underlying this effect remains poorly characterized. Here, we aim at identifying how noradrenergic projections are distributed, what neuronal subtypes noradrenaline mainly targets, and how the system varies in mouse cortex. We labeled the mRNA of noradrenergic receptors and interneuron molecular subtypes to analyze the distribution of noradrenergic receptors expression in inhibitory neurons across the cortex. In parallel, we quantified the density and entropy of noradrenergic projections with immunohistochemistry targeting of the noradrenaline vesicular transporter. To analyze these data across the cortex, we developed a pipeline to automatically detect nuclei and axons and register these data to a reference atlas. Our preliminary data show that noradrenergic axons density peaked in the first cortical layer. We also observed a different layer-dependent distribution of noradrenergic receptors between VIP and NDNF interneurons. We are currently extending this analysis to include other interneuron subtypes and pyramidal neurons. Our plan is to determine how this heterogeneous distribution of projection and receptors affects cortical activity of selected neuronal types using in vivo multiphoton microscopy. Our experiments should help dissecting the roles of the noradrenergic modulation on fundamental brain function and may serve to model disfunctions of these pathways.

Yaoyao Lu1,2，Cedric Happi Mbakam1,2，Nathalie Majeau1,2，Jacques-P, Tremblay1,2

1. CHU de Québec Research Center, Laval University, Quebec City, QC, Canada.
2. Department of Molecular Medicine, Laval University, Quebec City, QC, Canada.

Introduction: The CRISPR-Cas9 system is a promising technology that revolutionized genome editing applications for the potential treatment of a variety of genetic diseases. However, efficiently delivering the CRISPR-Cas9 components to the target organ or cell remains a significant challenge. Our study aimed to encapsulate CRISPR/Cas9 protein into extracellular vesicles (EVs) by protein S-palmitoylation, which is the reversible addition of fatty acids to the cysteine residues of proteins. It is an important post-translational modification that regulates multiple aspects of protein function, including the localization to membranes, intracellular trafficking, protein interactions, protein stability, and protein conformation. In this study, we demonstrate that S-palmitoylation proteins were preferentially encapsulated into EVs.

Methods: We obtained the peptide sequence from the N-terminus of a few membrane-related proteins, which is the ideal substrate for S-acyltransferase, the enzyme that catalyzes S-palmitoylation. We fused those peptides onto the N-terminus of both eGFP and SpCas9, which promoted the palmitoylation and encapsulation of those proteins into EVs. The encapsulation efficiency of palmitoylated eGFP was verified by flow cytometry and western blotting. Meanwhile, the packaging efficiency of SpCas9 was verified in reporter cells.

Results: The palmitoylation modification permitted 87.2% of eGFP entry into EVs. The N-terminus palmitoylation of SpCas9 did not affect its activity and was successfully encapsulated into EVs. EVs coated with VSV-G encapsulating palmitoylated SpCas9/sgRNA complex restored 12.3% mCherry expression from the Ai9 reporter cells.

Conclusion: Our study provides a novel approach to encapsulating CRISPR/Cas9-sgRNA complex into EVs. This may open an effective avenue for using EVs as vehicles to deliver CRISPR/Cas9 for genome editing.

Iason Keramidis, Brendan B McAllister, Julien Bourbonnais, Feng Wang, Dominique Isabel, Romain Sansonetti, Phil Degagne, Justin P Hamel, Mojtaba Nazari, Samsoon Inayat, Annie Barbeau, Lionel Froux, Antoine G Godin, Majid H Mohajerani, Yves De Koninck

Disinhibition during early stages of Alzheimer’s disease is postulated to cause network dysfunction and hyperexcitability leading to cognitive deficits. However, the underlying molecular mechanism remains unknown. In this study we show that in mouse lines carrying Alzheimer’s disease-related mutations, a loss of neuronal membrane potassium-chloride co-transporter KCC2, responsible for maintaining the robustness of GABAA-mediated inhibition, occurs pre-symptomatically in the hippocampus and prefrontal cortex. KCC2 downregulation was inversely correlated with the aged-dependent increase in amyloid-β 42 (Aβ42). Loss of KCC2 resulted in impaired chloride homeostasis. Acute administration of Aβ42 caused a downregulation of membrane KCC2. Preventing the decrease in KCC2 using long term treatment with CLP290 protected against deterioration of learning and cortical hyperactivity. In turn, restoring KCC2, using short term CP290 treatment, following the transporter reduction effectively reversed spatial memory deficits and social dysfunction, linking chloride dysregulation with Alzheimer’s disease-related cognitive decline. This reveals KCC2 hypofunction as a viable target for treatment of Alzheimer’s disease-related cognitive decline, it confirms target engagement, where the therapeutic intervention takes place, and its effectiveness.