Résumés / Abstracts

Evaluation of the requirement of SLP2 for dopaminergic neurons' survival and susceptibility to neurodegeneration in a mouse model of Parkinson’s disease.

Parkinson’s disease (PD) is characterized by progressive degeneration of dopaminergic (DA) neurons from the substantia nigra pars compacta (SNpc) and the presence of Lewy bodies mainly composed of aggregated α-synuclein (α-syn). There is still no cure for PD, but the development of therapies that aim to protect DA neurons against neurodegeneration is promising. Previous results from our team revealed that SLP2 was efficient to protect DA neurons against degeneration in a mouse model of PD overexpressing the human A53T α-syn.

OBJECTIVE: Our aim was to knock-out (KO) SLP2 in the same mouse model to reveal its requirement in DA neurons' survival and susceptibility to neurodegeneration. 

METHODS: For this purpose, we used a mouse line expressing the Cas9 specifically in DA neurons (DAT-Ires-Cre-LSL-Cas9). To KO SLP2, an AAV encoding a guide RNA against SLP2 has been delivered in the SNpc. Simultaneously, another AAV has been injected in the SNpc to overexpress α-syn. Animals were separated into four groups: 1) Control, 2) SLP2 KO, 3) α-syn, 4) α-syn + SLP2 KO. We performed a motor assessment using cylinder test 10 and 15 weeks following the injection of the AAVs. Animals have been sacrificed four months following viral infection and histologic analysis was performed on coronal brain sections. Neurons in the SNpc have been stained against TH and NeuN and stereological neuron counting was performed using the optical fractionator method. We also quantified the density of DA axon projections in the striatum using the signal intensity for TH immunofluorescence. 

RESULTS: The cylinder test did not reveal any significant difference between the four groups for ipsilateral rotation or preference in the use of a paw. Histologic analysis revealed that α-syn overexpression caused a significant reduction in the number of NeuN+ cell bodies in the SNpc as well as the TH+ axon projections in the striatum. However, when comparing the results from α-syn group to those from α-syn + SLP2 KO group, we did not measure any significant difference. 

CONCLUSION: SLP2 is not required for DA neurons survival and lack of SLP2 does not exacerbate the neurodegenerative process induced by α-syn overexpression.

Single-chain antibody treatment against alpha-synuclein to halt Parkinson’s disease progression

Parkinson’s disease in mainly characterized by the degeneration of dopaminergic neurons from the substantia nigra. Degenerating neurons generally present intracytoplasmic inclusions called Lewy bodies, that are mainly composed of the protein alpha-synuclein. In those insoluble aggregates, alpha-synuclein is found hyperphosphorylated and misfolded. Evidences in various animal and cell models suggest that pathological alpha-synuclein can be transmitted from cell-to-cell in a prion-like manner. Blocking either the aggregation process or the transmission of the pathological protein species across the brain could therefore be an effective therapeutical strategy. OBJECTIVE: We aim to use single-chain variable fragments antibodies (scFvs) against phosphorylated alpha-synuclein to stop the propagation of the pathology in a mouse model of Parkinson’s disease. METHODS: We will synthetize our scFv from a monoclonal antibody specific for the phosphorylated form of alpha-synuclein and will encode it in a viral vector. We will inject pre-formed fibrils of alpha-synuclein in the striatum of transgenic mice overexpressing a mutated form of the protein. We will deliver concomitantly or 2 months later our scFv anti-alpha-synuclein intravenously and will monitor monthly any motor deficits appearing with various behavioral tests. We will sacrifice our mice 4 months after pre-formed fibrils injection and will perform histopathological analysis. We will quantify the dopaminergic neuronal and axonal loss, as well as the extent of the alpha-synuclein pathology in the brain with immunohistochemistry against markers of Lewy bodies. We believe that our scFv will improve the motor deficits and significantly reduce the amount of phosphorylated alpha-synuclein, which will help preserve the dopaminergic neurons. RESULTS: We validated custom monoclonal antibodies specific for phosphorylated alpha-synuclein that work both in immunohistochemistry and Western blotting. We also validated a serotype of viral vector called AAV2/php.EB that offers an impressive penetration of the blood brain barrier and brain cell transduction when injected intravenously. We could achieve a good expression of our control AAV2/php.EB encoding an anti-GFP protein when injected intravenously in a DRD2-GFP mouse. CONCLUSIONS: We are currently developing the tools needed to bring the project forward. We are synthetizing our scFv from the validated monoclonal antibodies and are going to test it in vitro and in vivo shortly. We will be using the serotype php.EB to allow us to eventually move into clinical trial with patients affected by Parkinson’s disease.

The proinflammatory profile of LRRK2 G2019S astrocytes and its impact on the blood-brain barrier

Astrocytes are incredibly versatile cells. Throughout the life of an individual, they harbor a variety of different roles, from metabolic and trophic neuronal support, to the modulation of the brain inflammatory response. They also orchestrate, together with pericytes and brain microvascular endothelial cells (BMECs), the formation of the blood-brain barrier (BBB) to protect the brain from circulating toxins. In Parkinson’s disease (PD), a multifactorial neurodegenerative disorder characterized by neuronal death and astrocyte reactivity, the progressive disruption of the BBB has also been observed. However, while the effects of PD on neurons is under active investigation, little is known about its impact on astrocytes. The OBJECTIVE of this study is to evaluate PD-mediated biochemical and cellular impairments of astrocytes, and the consequences on the integrity of the BBB. METHODS: This study leverages the use of induced pluripotent stem cell (iPSC)-based technologies to model the LRRK2 G2019S familial form of PD. A meta-analysis of published RNA-sequencing studies of iPSC-derived LRRK2 G2019S astrocytes was performed to identify dysregulated biological processes. Astrocytes and BMECs were differentiated from LRRK2 G2019S mutants and their gene-corrected controls, and added to a newly developed human 3D BBB-on-a-chip model that recapitulates the complex cytoarchitecture and cell-cell interactions of the BBB. RESULTS: Our results indicate that LRRK2 G2019S astrocytes are pro-inflammatory, and display a transcriptomic profile suggestive of altered angiogenic properties. This mutation affects the biochemical and functional behavior of astrocytes by reducing the release of trophic factors, activating the MEK/ERK pathway involved in the modulation of the inflammatory response, and thus upregulating the release of cytokines. As a result of this pathological secretome, disease astrocytes prevent the formation of a tight BBB, which may be mediated by a dual effect of oversecretion of cytokines such as IL-8, combined with a lack of pro-angiogenesis factors. CONCLUSION: While studies on PD have largely been focused on cell-autonomous mechanisms of neuron degeneration, a new framework is emerging and suggests to consider the contributions of astrocytes and astrocyte paracrine communication to PD pathology.

Identification et validation de nouvelles cibles thérapeutiques contre la maladie de Parkinson par criblage CRISPR-CAS9

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 (DA) 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, sont associés à la mort des neurones DA.  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.

OBJECTIF: Nous avons pour but d’identifier de nouvelles cibles thérapeutiques contre la maladie de Parkinson.

MÉTHODES: Nous avons procédé à un criblage génétique en appliquant une librairie CRISPR-CAS9. Nous avons identifié 48 gènes candidats où 9 ont montré une forte protection contre la neurotoxine roténone. Le gène le plus prometteur, Lig a été validé in vitro, mais son potentiel rôle neuroprotecteur in vivo nécessite d’être validé. Pour procéder à sa validation in vivo, nous avons supprimé Lig spécifiquement dans les neurones DA de la SNc chez la souris adulte. Nous avons injecté un vecteur AAV2 encodant pour 2 ARN guides dirigés contre Lig dans une souris exprimant Cas9 spécifiquement dans les neurones DA du mésencéphale. À la suite de la suppression de Lig, nous avons induit un phénotype parkinsonien grâce à l’injection intrastriatal de la neurotoxine 6-hydroxydopamine (6-OHDA) ou par la surexpression virale de la forme mutée de l’alpha-synucléine (A53T) dans la SNc.

RÉSULTATS: Une série de tests comportementaux et une analyse stéréologique permettent de confirmer les résultats préalablement observés in vitro. La suppression de Lig protège les neurones DA de la dégénérescence et renverse les phénotypes moteurs induits par 2 modèles de la maladie de Parkinson.

CONCLUSION: Les résultats de notre étude fournissent une validation préclinique de Lig comme nouvelle cible thérapeutique contre la dégénérescence et le parkinsonisme. Nous validons actuellement si la suppression de Lig est aussi efficace à protéger les neurones DA humains différentiés à partir de cellules iPSC dans des gènes causant la maladie de Parkinson. À terme, nos résultats fourniront une évaluation préclinique complète de Lig comme cible thérapeutique contre la maladie de Parkinson.

Neurostimulation non invasive dans la maladie de Parkinson : étude de cas

Introduction. La maladie de Parkinson (MP) se manifeste par des symptômes moteurs (bradykinésie, tremblements au repos, pauvreté du mouvement, rigidité musculaire) communément traités par médication. La recherche actuelle teste si la stimulation magnétique (non pharmacologique, non invasive, indolore et sans effet secondaire) du cerveau (rTMS) ou des muscles (rPMS) a un impact sur ces symptômes moteurs. Précisément, la rTMS (stimulation magnétique répétitive transcrânienne) du cortex moteur primaire (M1) influence l’excitabilité corticomotrice ce qui améliore le contrôle moteur. La rPMS (stimulation magnétique répétitive périphérique ou des muscles) permet de nourrir M1 en informations proprioceptives sur le mouvement, ce qui améliore le contrôle posturo-moteur. Mais ces effets des rTMS et rPMS en MP ne persistent pas. 

Objectif. Tester chez une patiente non médicamentée si la combinaison rTMS+rPMS a plus d’effets sur les symptômes parkinsoniens (évalués par UPDRSIII) que la rTMS ou la rPMS administrée seule. 

Méthodes. La participante (52 ans, non médicamentée, MP depuis 10 ans) a été testée dans les trois protocoles de neurostimulation suivants (à une semaine d’intervalle chacun) : rTMSplacebo + rPMSplacebo, rTMSréelles + rPMSréelles, rTMSréelles + rPMSplacebo. Les symptômes moteurs (score UPDRSIII) et l’excitabilité de M1 (testée par TMS) ont été évalués pré/post-stimulation à chaque séance. Les mesures pré-stimulation de la semaine suivante testaient les effets à long terme (à 1 semaine). 

Résultats et perspectives. Seul le protocole de combinaison rTMSréelles + rPMSréelles a permis une amélioration du score UPDRSIII après la stimulation, et ce, en parallèle de changements immédiats d’excitabilité de M1.

Conclusion. Ces résultats soulignent l’efficacité thérapeutique de la combinaison rTMS+rPMS en MP et son influence sur la plasticité cérébrale. Mais ces effets d’une seule session de neurostimulation étaient éteints à 1 semaine. Cette étude de cas encourage à tester un plus grand nombre de personnes avec MP et sur plusieurs sessions. 

Impact of alpha-synuclein aggregation on protein degradation systems and its implication in Parkinson’s Disease pathogenesis

OBJECTIVE

In Parkinson Disease (PD), the aggregation of misfolded proteins called alpha-synuclein (a-syn), represents one of the major cellular hallmarks of the disease. Insoluble property of these aggregates make difficult their degradation, which disrupts the neuronal homeostasis and leads to neurodegeneration. 

Several studies showed correlation between alterations of degradation systems (autophagic or proteasomal), implicated in the protein quality control, and a-syn aggregation. It’s important to know how an alteration of degradation systems is involved in the pathogenesis of PD.

METHODS

Our group recently created a new cellular model in which we control the aggregation of a-syn optogenetically and observe it in real time. This system is called the LIPA (Light-Inducible Protein Aggregation) system, allowing us to induce aggregates in vitro and observe the effects over time. Degradation sytems activity is here mesured by using several specific markers, proteasomal and autophagic, which help us to investigate the relation between a-syn aggregation and the degradations systems.

RESULTS

Using this cellular model, we were able to observe the effect of LIPA-induced aggregates on the proteasomal system in cells for the first time. Moreover, we are interested in the inhibition of proteasomal and autophagic systems and its effect on the aggregation. 

CONCLUSION

Our observations reveal LIPA system's ability to successfuly mimic the potential effects of a-syn aggregation on degradation systems in PD pathogenesis. So, by looking at potential actors implicated in the degradation of alpha-synuclein, we aim to further elucidate ways that can reverse the aberrant accumulation of this protein in PD. 

Microscopie holographique numérique polychromatique : vers l'exploration de la connectivité neuronale

OBJECTIF - OBJECTIVE

La microscopie holographique numérique (DHM) est un système novateur et non-invasif qui revisite le concept d’holographie classique par la reconstruction numérique des hologrammes. Elle est d’un grand intérêt pour l’étude de réseaux neuronaux, car elle offre une méthode d’analyse directe des cellules vivantes et de leurs altérations pathophysiologiques. Bien que la DHM ait démontré ses performances pour l’imagerie de corps cellulaires, il demeure difficile d’étudier l’arborescence neuronale, pourtant d’un grand intérêt, à cause d’un problème intrinsèque à la méthode : le bruit cohérent. Ce type de bruit provenant d’interférences parasites causées par de multiples réflexions dans le microscope n’est pas de nature aléatoire, ce qui rend impossible sa réduction par moyennage de plusieurs images. À l’aide d’une approche expérimentale polychromatique, nous arrivons à réduire ce bruit cohérent et par conséquent à améliorer la qualité des images obtenues en DHM.

MÉTHODES - METHODS

La source laser interne d’un microscope holographique numérique commercial est remplacée par un laser supercontinuum externe dont la longueur d’onde est modulable. Afin de réduire le bruit cohérent, une série de 36 images de phase sont capturées sur deux cibles de phase et sur une culture vivante de neurones de rats pour des longueurs d’onde allant de 500 à 850 nm par incréments de 10 nm.

RÉSULTATS - RESULTS

Les images débruitées par moyennage avec l’approche polychromatique sont comparées aux images obtenues par moyennage de 36 images à une seule longueur d’onde (contrôle). L’approche polychromatique résulte en des images visiblement moins bruitées sur lesquelles l'arborescence dendritique peut être beaucoup mieux appréciée. Des profils de hauteur obtenus à partir des images de phase montrent que le bruit cohérent se déplace avec la longueur d’onde, ce qui explique le succès de la méthode.

CONCLUSION

Nous démontrons que l’approche de débruitage polychromatique réduit drastiquement les perturbations causées par le bruit cohérent sur les images obtenues en DHM et permet de distinguer des ramifications neuronales auparavant dissimulées.

ROLE OF INFLAMMATORY BRAIN CELLS IN DEMENTIA FOLLOWING MICROINFARCTS

BACKGROUND & AIM: Vascular dementia have been recently demonstrated to be a risk factor for dementia. Microinfarcts (Mi) are small lesion caused by the obstruction of small vessels that have a high prevalence in dementia and Alzheimer’s disease which impact mostly women. This type of lesion leads to, a loss of cells involved in different processes such as memory and an activation of cells known as monitoring, cleaning and repairing the brain called microglial cells. However, these cells are less efficient with aging which could accelerate the onset of dementia. Moreover, their function seems to be sex dependent.

OBJECTIVES: So, we propose to study the role of these surveillances’ cells in cerebral Mi and the impact of its malfunction in a sex and age dependent manner. METHODS: First of all, we developed a model of Mi in mice and characterize its neuropathological profile using immunofluorescence to target cell death and degeneration (TUNEL, FJB, caspase3) and behavior to study functional deficits. Then, we will focus on microglial response after Mi and dissect its spatiotemporal activation in a sex and age dependent manner (IBA1, TMEM119, CD68). This will be done on a protein (immunostaining) and genetic levels (RNAscope Fluorescent Multiplex). Inflammation studies by western blot (IL6, IL4) will complement the results. Finally, we will focus on the role of microglia in functional and structural damage repair. Microglia will be manipulated pharmacologically (PLX3397) and genetically (CX3CR1 KO mice) to impair its capacity and study its role in cerebral Mi pathology.

RESULTS: Preliminary results indicate that the model exhibits a high loss of cells associated with an important microglial activation at lesion sites.

CONCLUSION: The findings strongly suggest that cell loss is associated to neurogenesis and inflammation, which are impaired in the aged brain. Further investigations will emphasis on dissecting the role of microglia in Mi. The innovative aspect of this study comes from the model developed specifically in our lab since now a day no experimental models offered the possibility to pursue such investigations. Following these studies, we will be able to evaluate therapeutic approaches aimed at targeting these cells during the pathology.

Role of Polo-like kinase 2 in the pathogenesis and treatment of Alzheimer's Disease

OBJECTIVE

Extracellular amyloid plaques, primarily composed of amyloid beta (Aβ) peptide deposits resulting from amyloid precursor protein (APP) cleavage, and intracellular neurofibrillary tangles of hyperphosphorylated tau deposits represent the major neuropathological hallmarks of Alzheimer’s disease (AD). Increasing evidence suggest that phosphorylation plays an important role in the aggregation and toxicity of Aβ and tau. Our laboratory has reported a dramatic accumulation of Polo-like kinase 2 (PLK2) in the brains of AD patients. This observation, in association with the recent notion of a direct interaction between PLK2 and APP, suggests that the aberrant accumulation and activity of PLK2 may contribute to AD pathogenesis. Our goal is to examine the effect of PLK2 pharmacological inhibition on APP and tau aggregation and toxicity in transgenic mouse models of AD.

METHODS

Behavioral analysis incorporated evaluation of spatial and reference learning and memory, exploratory behavior, anxiety and sociability. Detailed biochemical and histological analysis of AD neuropathology (APP/Aβ, tau, their phosphorylated forms, and synaptic dysfunction) were additionally performed using immunoblotting, ELISAs and immunohistochemistry.

RESULTS

We observed cognitive decline in symptomatic mice, as well as AD hallmarks in their brains. Moreover, our results showed a decrease in some aspects of APP and tau pathology upon PLK2 inhibition. These observations were reflected by a lessening in the deterioration of behavioral performance, and a reduction in APP and tau accumulation and neurotoxicity.

CONCLUSION

Overall, this project will shed light onto novel mechanisms by which phosphorylation regulates tau and APP aggregation and toxicity, providing a novel therapeutic target for AD and related dementia.
 

Effect of HDAC6 inhibitor on axonal transport in cultured neurons from giant axonal neuropathy mouse model

OBJECTIF - Giant axonal neuropathy (GAN) is a fatal neurodegenerative disease caused by mutations in the gigaxonin gene and characterized by severe deterioration of the central (CNS) and peripheral nervous system (PNS). Gigaxonin is a member of BTB/Kelch proteins family which promotes the proteasomal degradation of substrates including intermediate filaments (IF), MAP1B, MAP8, and TBCB protein in a Cul3-E3-ubiquitin ligase dependent manner. Any loss of function in gigaxonin gene leads to the aggregation of neurofilament (NF) protein in the axonal body of neurons that decreases the axonal transport and promotes neurodegeneration. In this study, we propose to investigate the axonal transport in peripheral neurons of Gan-/- mice and to find possible therapeutic interventions that could improve GAN pathology.

MÉTHODES - Dorsal root ganglia (DRG) explant culture was done using Gan-/- and Wild type mouse embryos at E15. To check the axonal transport live cell imaging was performed at DIV7. HDAC6 inhibitor treatment was also given to check the effect on axonal transport.

RÉSULTATS - On Day 7, live-cell imaging showed a significant reduction of global axonal transport of cell organelles including mitochondria and lysosomes. Our further analysis revealed reduced travel distance and movement of both mitochondria and lysosome in Gan-/- neurons, confirming axonal transport impairments. Histone deacetylase 6 (HDAC6) plays an important role in microtubules organization in neuronal cells by deacetylating α-tubulin. To study the therapeutic benefits in GAN disease, we treated our cultured DRG neurons with HDAC6 inhibitors (Tubastatin A). Treatments with HADC6i (1µM) increased the levels of α-tubulin acetylation as observed by western blot. Moreover, in treated neurons, we observed a significant increase in moving organelles total travel distance and total number compared to vehicle-treated cells.

CONCLUSION - These data suggest that, although the lack of gigaxonin induces impairments in axonal transport, this condition can be improved by inhibition of HDAC6. Further experiments are planned to understand whether the observed rescue corresponds to an increased microtubules re-organization, a reduction of NF and other cytoskeletal protein aggregation, and an improvement in neuronal survival.

INVESTIGATING THE CONTRIBUTION OF NEURONAL CELL POPULATION TO ALS-FUS PATHOLOGY

INTRODUCTION

Mutations in the RNA-binding protein fused in sarcoma (FUS) are causative of amyotrophic lateral sclerosis (ALS) and some cases of frontotemporal dementia (FTD), two neurodegenerative diseases characterised by progressive muscle weakness and cognitive and behavioural abnormalities, respectively. Ubiquitously expressed FUS in mouse models affects both neuronal and glial cells, leading to loss of motor neurons. Interestingly, neuron-specific in vivo studies in other genetic ALS mouse models (SOD1 and TDP43) showed a delay in the disease onset with no effect on the disease progression, suggesting an interplay between autonomous and non-cell autonomous mechanisms in ALS disease. However, the neuronal contribution to ALS in the context of FUS mutations needs to be examined further. In this study, we provide in vivo evidence of the neuron-specific contribution in ALS-FUS pathology.

METHODS 

We generated a neuron-specific transgenic mouse expressing the ALS mutation R521G in FUS using the Cre recombinase approach. Mutant FUS expression and localization were studied by western blot and immunofluorescence. A battery of behavioural analysis was performed to determine the neuronal contribution of mutant FUS to cognitive and motor dysfunction. Moreover, motor neuron’s morphology and survival were analysed.

RESULTS 

FUS-R521G mice presents an initial cognitive impairment that persists during all the later time points analysed. However, no motor impairment was observed before 8 months age; even though an early decrease in motor neuron dendritic branching was detected. In addition, activation of microglia and astrocyte cells was observed.

CONCLUSION

The results provided by this study shows an ALS-FTD phenotype in a neuron-specific mouse model expressing the FUS-R521G mutation. The presence of a persistent cognitive impairment with a delay in the motor deficit, but with motor neuron arborisation alterations, suggests that FUS-R521G mutation in neurons alone is not enough to induce a motor impairment. Other cell contributions, such as astrocytes or microglia could be determinant to the onset of the motor symptoms in ALS disease.

Efficacité de traitements des difficultés de production de phrases en contexte d’aphasie post-AVC

L’aphasie est présente chez le tiers des victimes d’accident vasculaire cérébral (AVC) et en représente l’une conséquences les plus invalidantes, entrainant des difficultés souvent sévères et durables pour l’intégration sociale et la qualité de vie. Les personnes aphasiques peuvent éprouver, entre autres, une difficulté à produire des phrases, ce qui la rend moins compréhensible pour ses interlocuteurs. Ces déficits se manifestant de manière hétérogène d’une personne aphasique à l’autre, il est important de fournir une intervention ciblée. Il existe trois grands types de traitements pour les déficits de production de phrases, qui varient selon ce qu’ils ciblent : les verbes, les structures de phrase ou la morphologie fonctionnelle. Objectifs. L’objectif est de savoir quels types d’interventions ont des données probantes dans la littérature scientifique pour le traitement des déficits de production de phrase en contexte d’aphasie post-AVC et de qualifier l’efficacité de ces interventions en termes d’amélioration de la production de phrases, de généralisation à des items non traités, de maintien des acquis dans le temps et de transfert à d’autres contextes de communication. Méthodes. Une scoping review ainsi qu’une revue systématique de la littérature ont été effectuées. Résultats. Au total, 25 articles ont été retenus pour l’analyse. Parmi ces articles, les trois types de traitements mentionnés plus haut s’y retrouvent avec une surreprésentation des traitements visant la structure de phrase. L’analyse préliminaire de l’efficacité des traitements retenus pour la revue systématique montre que ceux-ci seraient tous efficaces et que la majorité d’entre eux permettent une généralisation des acquis. Toutefois, le transfert et le maintien sont moins mesurés dans les études et les données associées sont plus hétérogènes. Conclusion. En conclusion, les résultats montrent que les trois approches semblent pertinentes pour le traitement de la production des phrases, chez les personnes aphasiques. Or, plusieurs limites rapportées empêchent l’adaptation de certains de ces protocoles à la réalité clinique franco-québécois. Une analyse plus en profondeur des mesures d’efficacité va permettre de préciser les avantages de chaque traitement pour ensuite en adapter un en franco-québécois.

Light-inducible alpha-synuclein aggregation in the midbrain impairs nigrostriatal dopaminergic transmission

OBJECTIF - OBJECTIVE

Parkinson’s disease (PD) is characterized by intracellular inclusions of misfolded alpha-synuclein, known as Lewy bodies, and by the loss of dopaminergic neurons in the substantia nigra pars compacta (SNc) that leads to dopamine (DA) depletion at the striatum. However, it remains unclear how the aggregates of alpha-synuclein can affect the functioning of dopaminergic projections and striatal network, especially due to the absence of proper models that allows to reproduce the spatio-temporal features of PD.

In this context, we have recently developed an in vitro and in vivo model of PD based on the optogenetics technology named LIPA (light-inducible protein aggregation) that controls the aggregation of alpha-synuclein under the control of blue light. We showed that LIPA mimics the histopathological characteristics of PD, and allow thus to study if and how the aggregation of alpha-synuclein in dopaminergic cells of SNc causes a progressive disruption in the nigrostriatal pathway.

MÉTHODES - METHODS

To investigate the physiological impact of LIPA-induced alpha-synuclein aggregation on the dopaminergic projections, we assessed the activity of striatal cells. Briefly, we implanted mini-endoscopes coupled with an optic fiber to induce alpha-synuclein aggregation in the SNc, and analyzed the neuronal activity using the calcium indicator GCaMP6s in the striatum of freely moving mice.

RÉSULTATS - RESULTS

Our results show a progressive decrease in the synchronized activity of striatal cells caused by the aggregation of alpha-synuclein.

CONCLUSION

Altogether, our data showed that the use of this new LIPA-alpha-synuclein system offers a unique tool to elucidate the morphological and physiological changes occurring in the dopaminergic projections in the context of PD.

Disease associated microglia profiles and deregulation of immunity in ALS.

Disease associated microglia profiles and deregulation of immunity in ALS.

Romina Barreto-Núñez, Louis-Charles Béland, Hejer Boutej, Pierre Cordeau and Jasna Kriz.

OBJECTIVE. Recent studies uncovered several disease associated changes in microglia cells at gene/RNA level, but the actual cell specific signature of microglial cells at protein level remains elusive. Our research aims to study and identify the cell specific molecular signature of aberrant microglia at protein level that appears exclusively in the symptomatic phase of disease in SOD1G93A mouse model and determine the role of SRSF3 as a master regulator of innate immune response in microglia in ALS.

METHODS. To assess cell specific changes in microglia at protein level in vitro and in vivo we used the double transgenic mouse model CD11prom-Flag/EGFP-RPL10a;SOD1G93A. We isolated and characterized the aberrant microglial cells from the spinal cord of symptomatic animals. We performed the modified Translating Ribosome Affinity Purification methodology. Microglia proteome/peptides were analyzed by label free high sensitivity mass spectrometry (LC-MS/MS). We determine the role of SRSF3 in the transformation of microglia into aberrant cells in the SOD1G93A mouse model by blocking the expression of SRSF3: *in vitro by treating the cells with antisense oligo morpholinos targeting endogenous SRSF3; *in vivo by treating intraperitoneally animals with antisense oligo morpholinos targeting endogenous SRSF3.

RESULTS. We isolated and characterized the aberrant microglial cells from the spinal cord of symptomatic animals. These cells appeared exclusively in the symptomatic phase of the disease and shown a disease associated profile.  IP injection of morpholinos against SRSF3 achieve a SRSF3 knock down of approximately 50% in the blood. The mice treated with morpholino showed an extended survival of 21 days.

CONCLUSION. We identified a distinct, disease associated protein signature in chronically activated ALS microglia. Functionally, aberrant microglial cells showed a marked deregulation of innate immune response and phagocytic activity. Our results provide the first comprehensive analysis of disease associated microglia proteome. We provide evidence that SRSF3 is involved in the transformation of microglia into aberrant/disease associated microglia in ALS and the therapeutic modulation of microglia with morpholinos against SRSF3 may help slow the disease progression and the symptoms of ALS in the SOD1 mouse model.

The role of glutamate co-transmission by serotonin neurons of the dorsal raphe nucleus in the expression of L-Dopa-induced dyskinesia

Parkinson’s disease is 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 adverse effects such as abnormal involuntary movements (AIMs), also termed L-Dopa-induced dyskinesia (LID). Recent studies have shown the crucial role of serotonin (5-HT) neurons in LID expression. OBJECTIVE 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. METHODS We used CRIPSR-Cas9 technology and viral injections to knock-out or overexpress the atypical vesicular glutamate transporter 3 (VGluT3), specifically in 5-HT neurons of the DRN in adult mice. Two weeks later, these mice were injected with 6-OHDA in the medial forebrain bundle to selectively lesion dopaminergic axons, and then treated with L-Dopa to induce AIMs. RESULTS Post-mortem analysis confirmed the depletion or overexpression of VGluT3 in AAV-infected 5-HT neurons of the DRN and the striatal dopamine denervation. After dopamine lesion and L-Dopa administration, VGluT3-depleted mice tend to show exacerbated AIMs caused by low dose L-Dopa (1 mg/kg) administration compared to controls and transgenic mice overexpressing VGluT3. CONCLUSION Glutamate that is co-released by 5-HT neurons of the DRN appears to be involved in the expression of LID.

Novel Biomarkers for the Diagnosis of Prodromal Parkinson’s Disease

OBJECTIVE

Parkinson's is correlated with Lewy body inclusions and the deterioration of nigral dopaminergic neurons. By the diagnostic timepoint reliant on motor symptoms, there is severe neurodegeneration. Thus, efficient biomarkers for an early diagnosis are needed. As non-motor symptomatology is present decades prior, the project uses non-invasive techniques to detect emerging peripheral effects reflecting central dysfunction during the prodromal phase.  

Whereas the ERG is ideal for measuring the repercussions on functional output in retinal cellular populations before morphological compromise, abnormal compositional changes in oral bacterial abundance can also reflect peripheral α-synucleinopathy. The neurodegeneration caused by a central lesion is hypothesized to sustain neuroinflammation, dysbiosis, and compensation mechanisms in the periphery. 

METHODS

We will start with a model of M83 transgenic mice expressing human A53T variant α-synuclein, complemented by a striatal stereotaxic injection with preformed fibrils of α-synuclein. Mice will undergo a battery of tests monthly for six months. Motor symptoms will be tracked with the cylinder, open-field, and rotor rod test, followed by the swab collection of an oral microbiota sample. Finally, the retinal electrical function will be analyzed with the dark/light-adapted ERG. Analysis of neural dysfunction, retinal morphology, and salivary gland compromise will be performed on harvested tissue.

Next, we will recruit an inclusive cohort of afflicted patients and healthy individuals. Their two-day visit layout will include the ERG, oral bacteria Salivette swabs, and unstimulated saliva collection for complementary proteomic/metabolomic analysis.

RESULTS

Based on preliminary data from a mouse PD model of AAV-α-syn overexpression, we expect a sustained retinal sensitivity in the ERG despite aging, i.e., peripheral compensation for developing α-synucleinopathies. Moreover, increases in the Streptococcaceae family might be tied with the salivary amylase increase seen in patients.

CONCLUSION

The current project can reveal non-invasive tools to track central neurological changes, helping us detect Parkinson’s early and providing a wider window for neuroprotective therapeutic intervention.

Investigating perineuronal nets in patient-derived induced pluripotent stem cells and brain with Huntington’s disease

Perineuronal net (PNN) is a specialized extracellular matrix structure that acts as a physical barrier between the neuron and its external environment, protecting it from oxidative stress and toxic protein species. Alterations in PNN structure and function have been identified in neurological diseases such as Alzheimer’s disease, multiple sclerosis and stroke. However, the role of PNNs in Huntington’s disease is unknown. OBJECTIVE: The objective of this study is to investigate the distribution and role of PNNs in HD human and mouse brain as well as in patient-derived induced pluripotent cells (iPSC). METHODS: Free-floating tissue sections were immunolabelled with WFA (PNN marker), EM48 (mutant huntingtin protein marker, mHTT) and MAP2 (neuronal marker). Sections from R6/2 HD mice were immunolabelled with WFA followed by tissue enlargement using Expansion microscopy technique. For the in vitro study, iPSCs derived from control and HD (180 CAG repeats) patients (Corriell Institute) were maintained at 37°C/5%CO2 and differentiated into neurons on Matrigel®-coated dishes. To ensure specific labelling of PNNs, neurons were treated with 0.1U/ml of ChondroitinaseABC enzyme. RESULTS: WFA-labelled PNNs were detected around a subset of neurons in layer II-IV of the human cortex. However, striatal neurons did not express the usual lattice-like PNN structure. mHTT aggregates colocalized and adhered to the PNNs in both cortical and striatal neurons. PNN structure in R6/2 HD cortex remained intact despite 4.5X expansion. PNNs were detected in both Neuronal progenitor cells (NPCs) and differentiated neurons in control and HD cell lines. The NPCs exhibited structurally less mature PNNs whereas the differentiated neurons presented PNNs with discrete lattice-like structure. ChondroitinaseABC treatment eliminated PNN labelling in the differentiated neurons indicating a specific detection of PNNs using WFA lectin. CONCLUSION: PNNs are expressed in striatal and cortical neurons of human and mouse HD brain as well as in patient-derived iPSCs, and HD-related structural differences in PNNs can be studied using expansion microscopy. Ongoing in vivo and in vitro studies will shed more light onto the role of PNNs in HD.  

Post-mortem analysis of Parkinson’s disease brains after 11 and 12 years of deep brain stimulation of the subthalamic nucleus

OBJECTIF - OBJECTIVE

Deep brain stimulation (DBS) of the subthalamic nucleus (STN) is an effective surgical treatment for Parkinsons disease (PD), often completely ameliorating the symptoms and restoring quality of life to the patient. However, research into the long term effects of DBS on the human brain is lacking. The aim of the current study is to investigate how long term DBS could alter the morphology and physiological functioning of the affected areas, and how this could translate to clinical effectiveness.

MÉTHODES - METHODS

The primary subjects were two brains of PD patients who had received 11 and 12 years of DBS in the STN respectively. For each brain, 3D graphical representations of the basal ganglia (BG) and DBS electrode were produced to determine the electrical current propagation. Fluorescent immunohistochemistry along with confocal microscopy was used on sections of the BG to determine the expression of various proteins and the quantification and morphology of cells and blood vessels.

RÉSULTATS - RESULTS

Along the length of the electrode path, there was GFAP positive fibrillary gliosis along with elevated expression of GDNF, a marker for neuroprotective activity. Near the stimulated part of the STN, astrocytes showed higher varicose processes against blood vessels, microglial count was decreased with no change in CD68 expression, a marker of phagocytic activity, and there was increased expression of VEGF and Claudin-5, markers of angiogenesis and blood brain barrier integrity respectively. Additionally, microglia presented with a morphology suggesting a more active state near the electrode in the STN, with morphology returning to more inactive states at furthering distances from the electrode. Finally, subventricular zone thickness was restored to amounts comparible to healthy brains.

CONCLUSION

It is the hope that these results will further our understanding of the effects that DBS has on the brain, thereby assisting in its development as an effective treatment.

Adolescent idiopathic scoliosis: asymmetric amplitude of the vestibular-evoked postural response relates to the severity of the spine deformation

OBJECTIF - OBJECTIVE: Adolescent idiopathic scoliosis (AIS) is the most common spine deformation in pediatrics orthopedics. Despite its unknown origins, evidence suggests that it may involve vestibular alteration. In this study, we assessed the vestibulomotor transformation in adolescents with moderate and severe spine deformation and healthy adolescents by measuring the direction and amplitude of the vestibular-evoked postural responses during and following left and right electrical vestibular stimulation (EVS). We hypothesized that the orientation of the vestibular-evoked postural responses in AIS should deviate from the interaural direction compared to healthy adolescents. The amplitude of the vestibular-evoked response should also be asymmetrical. These results would confirm alteration in the vestibulomotor transformation suspected in AIS. MÉTHODES - METHODS: To evaluate whether spine deformation altered balance control without sensory manipulation, we calculated the variation (root mean square value, RMS) of the net ground reaction force before EVS. To characterize the vestibulomotor transformation, we calculated the amplitude and the orientation of the vestibular-evoked postural response from the mean net ground reaction force, either during or immediately after left and right EVS. RÉSULTATS - RESULTS: The results revealed that balance control performance (i.e., RMS value) was alike across groups without sudden vestibular information changes. However, during and after EVS, the net force's amplitude was more asymmetrical in adolescents with severe spine deformation. The orientation of the postural responses did not differ either during or after EVS. CONCLUSION: These results suggest asymmetrical changes in the gain but not in the direction of the vestibulomotor transformation in AIS related to the severity of spine deformation.

Expression of Acid-sensing ion channels in primary sensory neurons

OBJECTIF - OBJECTIVE: It is well established that Acid- Sensing Ion Channels (ASICs) have been implicated in normal functions and in several pathological conditions. Four genes (ASIC1-4), encoding 6 different subunits (ASIC1a, ASIC1b, ASIC2a, ASIC2b, ASIC3, and ASIC4) through alternative splicing, have been identified in rodents. ASICs and more specifically ASIC1a and ASIC3 have been shown to be implicated in pain since both subunits are expressed in nociceptors in the Dorsal Root Ganglia (DRGs). ASIC1, ASIC2 and ASIC3 expression was identified in adult mouse DRGs, however their detailed expression pattern has not been investigated yet.

MÉTHODES - METHODS: The aim of the present study is to examine thoroughly the expression pattern of different ASIC subunits in different neuronal populations. To overcome the obstacle of poor specificity of anti-ASICs antibodies, we used a very sensitive in situ hybridization technique (RNAscope) to identify the expression of the genes of interest. We combined this methodology with immunohistochemistry to reveal specific neuronal populations in DRGs. More specifically we targeted Isolectin IB4 (IB4), Calcitonin gene-related peptide (CGRP) and Neurofilament 200 (NF200) positive neurons. All experiments were performed on frozen sections of fixed DRGs obtained from 3-6 adult male mice.

RÉSULTATS - RESULTS: ASIC1a and ASIC1b are not expressed in IB4+ neurons, but in about 28% of CGRP+ and the majority of NF200+ neurons. ASIC2a is expressed in about 70% of IB4+, 11% of CGRP+ and half of NF200+ neurons. ASIC2b is expressed in most DRG neurons, including almost all IB4+ and CGRP+ neurons, as well as 80% of NF200+ neurons. ASIC3 is expressed in 10%, 65% and 82% of IB4+, CGRP+ and NF200+ neurons, respectively. Overall, different ASIC subunits show a distinct expression pattern in DRG neurons. The only two subunits with similar expression patterns are ASIC1a and ASIC1b; they are present in most of NF200+ neurons but are not in IB4+ neurons. In mice subjected to peripheral nerve injury, the expression level of ASIC1a and ASIC1b in L4 DRG and ASIC3 in L5 DRG were altered in CGRP+ neurons, but not in IB4+ neurons.

CONCLUSION: The distinct expression patterns of different ASICs in DRG neurons, indicates that they may be involved in different types of somatosensation.

STIMULATING GLUTAMATERGIC NEURONS OF THE MESENCEPHALIC LOCOMOTOR REGION TO IMPROVE LOCOMOTION AFTER SPINAL CORD INJURY

OBJECTIF - OBJECTIVE

Recently, electrical stimulation of the mesencephalic locomotor region has been shown to improve locomotor recovery after spinal cord injury (SCI). The cuneiform nucleus (CnF), a cluster of glutamatergic neurons, and the pedunculopontine nucleus (PPN), a cluster of glutamatergic and cholinergic neurons, are part of this functional region. It has been shown, that these three neuronal populations provide distinct contributions to locomotor control: glutamatergic neurons of the CNF can initiate and accelerate locomotion, whereas cholinergic and glutamatergic neurons of the PPN decelerate and stop locomotion. Here, we propose to identify and characterize the functional contribution of these distinct neuronal populations to locomotor recovery after SCI.

MÉTHODES - METHODS

Using transgenic mice expressing ChR2 in glutamatergic (VGluT2) neurons, we photostimulated VGluT2+CnF or PPN neurons before and after a thoracic lateral hemisection.

RÉSULTATS - RESULTS

7 weeks after SCI, the ipsilesional limb still showed functional deficits including a slower locomotor rhythm and a decrease in the amplitude of the intralimb coordination and step height. During treadmill locomotion, long photostimulations (10 ms pulses at 20 Hz for 1 s) of VGluT2+CnF neurons accelerated the locomotor rhythm, increased postural tone, and improved step height, whereas photostimulations of VGluT2+PPN neurons evoked decelerations and locomotor arrests. Likewise, during swimming, long photostimulations of VGluT2+CnF neurons improved the fluidity and amplitude of intralimb coordination.

CONCLUSION

In summary, our results argue that VGluT2+CnF neurons could be a neurological target to improve functional locomotor recovery following SCI.

MICRA-Net: MICRoscopy Analysis Neural Network to solve detection, classification, and segmentation from a single simple auxiliary task

OBJECTIF - OBJECTIVE

High throughput quantitative analysis of microscopy images presents a challenge due to the complexity of the image content and the difficulty to retrieve precisely annotated datasets. In recent years, fully-supervised deep learning has provided researchers with a novel tool capable of handling such demanding analysis. However, to achieve this unprecedented performance on several challenging tasks (e.g. segmentation, enumeration, and classification) such algorithms require a high number of precisely annotated datasets. While weakly-supervised deep learning was introduced to train on less spatially informative annotations, therefore reducing the required annotation time, they often lack the precision of full-supervision.

MÉTHODES - METHODS

We introduce a weakly-supervised MICRoscopy Analysis neural network (MICRA-Net) that can be trained on a simple main classification task using image-level annotations to solve multiple more complex auxiliary tasks, such as segmentation, detection, and enumeration. MICRA-Net relies on the latent information embedded within a trained model to achieve performances similar to state-of-the-art fully-supervised learning. This learned information is extracted from the network using gradient class activation maps, which are combined to generate precise feature maps of the biological structures of interest.

RÉSULTATS - RESULTS

We demonstrate how MICRA-Net significantly alleviates the expert annotation process on various microscopy datasets by using solely image-level labels for training. We show the performance of MICRA-Net on 4 different imaging modalities (fluorescence, phase contrast, differential interference, and electron microscopy) at solving a segmentation, detection or classification auxiliary tasks. MICRA-Net reduces the number of human decisions required to annotate a training set by more than 3 folds and can reduce the annotation period by more than 7 folds on average. 

CONCLUSION

The proposed technique alleviates the annotation task and allows to precisely identify and locate different biological structures for high-throughput analysis of optical microscopy images. The training from MICRA-Net binary labels opens new possibilities to minimize the inter- and intra-expert variability which is inherent to precise contouring of structures.

APEX2 as a new genetic tool to study the ultrastructural features of serotonin neurons and their axonal projections in the subthalamic nucleus

OBJECTIF - OBJECTIVE

Electron microscopy (EM) remains the best methodological approach to precisely describe the fine morphological features of neurons. In order to characterize subgroups of neuronal elements, most researchers rely on immunohistochemistry with the inherent constraint imposed by a limited penetration of antibodies, thus precluding 3D reconstructions through large volumes of brain tissue. The use of specific neuronal labeling with the cre-lox system is constantly increasing in research. It has been largely used with fluorescent proteins for confocal microscopy but very little has been done for EM. In this study, we used an innovative genetic approach to label entirely and specifically serotonin (5-HT) neurons for EM analysis. We took advantage of the cre-lox system and an engineered peroxidase called APEX2 that catalyzes the oxidation of a wide range of substrates, including 3,3’-diaminobenzidine (DAB), that can readily be detected by EM.

MÉTHODES - METHODS

Design of the viral construct– A plasmid containing the second generation of engineered ascorbate peroxidase (APEX2) was purchased from Addgene (product # 49386) and packaged into an adeno associated virus of serotype 9 (AAV9). Using the cre-lox system, this viral construct allowed a specific expression of the APEX2 gene (Figure 1).

Genetic labelling - In ePet-cre transgenic mice, 4 stereotaxic viral injections were performed in the dorsal raphe nucleus. Animals were perfused 21 days after viral injections with 50mL of phosphate buffer pH7.4 followed by 150mL of 4% paraformaldehyde and 1% glutaraldehyde solution. Mouse brains were cut into 50 µm-thick transverse sections with a vibratome. The efficient activity of the small APEX2 peroxidase introduced in cre positive infected 5-HT neurons was then revealed using DAB as the chromogen (at room temperature 1 hour DAB pre-incubation without H₂O₂ then 1 hour DAB incubation with H₂O₂.

3D electron microscopy – Mouse brain sections were processed according to a modified version of the NCMIR protocol and prepared for ultramicrotomy. Ultrathin serial sections of 80 nm were collected on silicon wafer. Arrays were then automatically and consecutively imaged with a scanning electron microscope (SEM) at a resolution of 5 nm / pixel (Figure 2). Images obtained from ultrathin serial sections were then aligned with FIJI’s plugins and further alignment, segmentation, 3D measurements and 3D rendering were done using the Reconstruct® software.

RÉSULTATS - RESULTS

Serotonin neurons of the dorsal raphe nucleus – Following the peroxidase reaction, infected 5-HT neurons expressing the cre peptide presented a dense DAB precipitate (Figure 3.a-c). Localization and identification of DAB-labeled neuronal elements was possible at both the light and electron microscope levels. Using light microscopy, we were able to show that the entire infected neurons were labeled, including their whole dendritic and axonal processes. Using EM, we showed that the ultrastructural features of DAB-labeled neuronal elements were not affected by viral infections and both dendritic and axonal profiles could be identified. Importantly, DAB - labeled elements were found to be present throughout the whole thickness of the 50 µm-thick section. Volume measurements and 3D rendering of connectivity could be provided for a 13.5µm serotonin dendritic segment (Figure 3.f-l).

Serotonin axons in the subthalamic nucleus – Following the DAB-peroxidase reaction, axons arising from 5-HT infected neurons of the dorsal raphe nucleus (DRN) and projecting to or passing through the subthalamic nucleus (STN) could be identified at both light and electron microscope levels. Again, genetically-labeled 5-HT axons observed in the STN showed a well- preserved ultrastructure. No synaptic contacts were observed in the reconstructed axonal segment found in the STN.

CONCLUSION

APEX2 restricted expression under the cre enzyme represents a powerful genetic labeling approach for electron microscopy. Our method provides strong DAB labeling of the entire axonal arborization of 5-HT neurons, evenly through large volumes of brain tissue. In addition, the lack of reagents in the preparation helps to preserve tissue integrity. We exploited this new technique to specifically label 5-HT neurons, their dendrites as well as their axonal projections in their target sites such as the STN. By using a large volume EM analysis approach, we are able to selectively label and analyze the morphological and relational features of axons arising from the DRN 5-HT neurons and provide detailed morphometric data. With GFP being co-expressed with APEX2, we are now conducting correlative light and electron microscopy experiments.

Influence de la direction du courant de la stimulation magnétique transcrânienne sur l’excitabilité intra cortical et corticospinal de la représentation des muscles multifides

OBJECTIF

Les muscles multifides superficiel (sMF) sont impliqués dans le contrôle moteur de la colonne vertébrale lors de tâches posturales et volontaires. En fonction de la tâche, les sMF seraient contrôlés par différents réseaux de neurones. Les régions prémotrices dont l’aire motrice supplémentaire (SMA) semblent impliquées dans le contrôle postural alors que le cortex moteur primaire (M1) est le centre d’exécution du mouvement volontaire. La stimulation magnétique transcrânienne (TMS) permettrait de tester ces circuits en manipulant le sens du courant (postéro-antérieur (PA) : M1 / antéro-postérieur (AP) : prémoteur/SMA).

L’objectif est de déterminer si la direction du courant influence différemment les circuits du M1 et l’excitabilité des projections corticospinales impliquées dans le contrôle des sMF

MÉTHODES

Vingt-quatre participants en santés ont pris part à deux expérimentations et ont été testés avec deux directions de courant de la TMS (PA vs AP).

1ère expérimentation (n=12) : la force de connexion corticospinale a été mesurée par une courbe de recrutement (90 à 160% du seuil moteur actif [AMT]). Les mécanismes de facilitation et d’inhibition intra corticales ont été testés par des stimulations pairées (80% puis 120% AMT, intervalles inter stimulus : 2-3-10 et 15ms).

2ème expérimentation (n=12) : la représentation corticale du sMF a été évaluée par mapping (36 sites de stimulations) sur des régions corticales motrices. Quatre stimulations par sites ont été réalisées à 110% AMT.

RÉSULTATS

La direction AP produit une plus grande inhibition que la direction PA aux intervalles 2ms (p=0.0447) et 3ms (p=0.0342). Aucune différence n’a été observé entre les directions de courant aux intervalles 10ms et 15ms. La courbe de recrutement ne diffère pas entre les directions de courant. Aucune différence de représentation corticale (centre de gravité et volume) n’a été observée entre les directions de courants.

CONCLUSION

La direction du courant influencerait différemment les réseaux de neurones impliqués dans l’inhibition intra corticale de la représentation des sMF.

Electrophysiological properties of glutamatergic reticulospinal and brainstem neurons

OBJECTIF - OBJECTIVE

Although the spinal cord contains the neural networks responsible for movement, these networks remain under the control of higher-order brain areas that initiate locomotor activity. The motor command reaches the spinal cord through the medullary reticular formation including the gigantocellular reticular nucleus (Gi) and the lateral paragigantocellular nucleus (LPGi). Here, we propose to characterize the intrinsic and extrinsic properties of reticular neurons and their connectivity to downstream circuits.

MÉTHODES - METHODS

AAV2-retro-DiO-mcherry was injected in the spinal cord to express mcherry in glutamatergic neurons of the Gi or LPGi. Brainstem slices were prepared to perform whole-cell patch-clamp recordings to characterize intrinsic and extrinsic properties of these glutamatergic Gi or LPGi neurons. we were able to record from 10 juvenile (3-4 week-old) or 27 young adult (2 month-old) mice but not from advanced adult (3-4 month-old).

RÉSULTATS - RESULTS

We identified and characterized the intrinsic properties of a total of 42 neurons in the medulla, 30 of those neurons were in the gigantocellular reticular nucleus (Gi), 24 out of these 30 neurons displayed a regular spiking pattern (RS) with some spike frequency adaption and 6 out of 30 cells displayed a fast spiking firing pattern (FS) without spike frequency adaptation. The morphology of the RS neurons was round while that of FS neurons was ovoid. RS neurons displayed a mean initial firing frequency of 24 +/- 2.2 Hz, while FS neurons displayed a mean initial firing frequency of 40.82+/- 8.3 Hz. 30% of the neurons display some sag to hyperpolarizing current. Mean membrane resistance was 388 +/- 47 MΩ for RS neurons and 285 +/- 22 MΩ for FS neurons. Cre immunostaining confirmed that glutamatergic Gi neurons displayed a RS pattern in 6 neurons.

CONCLUSION

These results will allow us to get a better understanding of the physiological properties and network connectivity of brainstem neurons important for locomotion.

EFFECT OF DCC HETEROZYGEOUS MUTATION ON THE LOCOMOTOR CONTROL

OBJECTIF - OBJECTIVE

Axon guidance receptors such as Dcc contribute to the normal formation of neural circuits, and their mutations are associated with neural defects. In humans, heterozygous mutations in DCC have been linked to congenital mirror movements, which are involuntary movements on one side of the body that mirror voluntary movements of the opposite side. In mice, mirror-movement-like hopping phenotypes have only been reported for bi-allelic Dcc mutations, while heterozygous mutant have not been closely examined. Since Dcc is important for the normal development of spinal commissural interneurons, we hypothesized that Dcc heterozygous mice may reveal impaired spinal motor functions.

MÉTHODES - METHODS

Adult DCC+/- and wild-type littermates were assessed in the cylinder test and during beam and ladder locomotion. Spinal cords were isolated from neonatal mice to assess the spinal locomotor circuit upon bath-application of low and high concentrations of neurotransmitters mimicking the descending control of the brain.

RÉSULTATS - RESULTS

In comparison to controls, adult DCC+/- mice showed no change in the cylinder test or during beam and or ladder locomotion. Neonatal DCC+/- spinal cords showed a normal left-right and flexor-extensor coordination regardless of the locomotor frequency. However, the locomotor rhythm and pattern were increased in mutant spinal cords at a high locomotor frequency in contrast to their wild-type controls.

CONCLUSION

Heterozygous Dcc mutation seems to impair transiently the developing spinal locomotor circuit, but this does not persist through adulthood.

Modulation of the biological activity of DKK1 decisively influences structural and functional neurovascular recovery after ischemic stroke.

BACKGROUND

Stroke constitutes a major cause of death and disability in Canada. Stroke, which occurs as a result of a sudden obstruction within a cerebral artery due to an embolus or thrombus. We have previously shown that upon stroke, the canonical Wnt pathway, which plays key roles in regulating neurovascular functions, is deregulated. Interestingly, Dickkopf-1 (DKK1), a secreted inhibitor of the pathway, is induced after stroke in patients and animal models. Although DKK1 levels have been shown to increase, its implication in stroke pathobiology and therapy remains unknown.

OBJECTIVE

Our study aims to elucidate the role of DKK1 after stroke. We developed the following objectives: 1) investigating the impact of conditional DKK1 induction in TOPGAL-iDKK1 mice on brain functions, 2) evaluating the consequences of temporal DKK1 induction (early and prolonged induction) after stroke, and 3) assessing the potential of DKK1 inhibition, using the DKK1 inhibitor WAY262611, on brain recovery.

METHODS AND RESULTS

For the first objective, we found that DKK1 genetic induction for 2 weeks by doxycycline was sufficient to initiate Dkk1 mRNA upregulation and β-catenin mRNA reduction in the brain, thus outlining the downregulation of the canonical Wnt pathway in the TOPGAL-iDKK1 mice. Interesting, blockage of the pathway results in a poor motor coordination. For the second objective, we found that DKK1 induction 2 weeks before MCAo impaired motor functions, structural damages and decreased survival rate 1 week after stroke.  In addition, DKK1 induction induced neuronal degeneration and impaired neurogenesis notably by a decreased level of DCX+ in the SVZ and TUJ1+ in the perilesional and infarcted areas. Finally, DKK1 induction impaired angiogenesis and blood flow reperfusion. For the third objective, we treated MCAo mice with WAY262611 (5 or 10mg/kg) at 24 hours and every 2 days until sacrifice at 1 week. DKK1 inhibition strongly restored motor dysfunctions and decreased structural damages.  In addition, DKK1 inhibition decreased neuronal degeneration, allowed neurogenesis and angiogenesis 1 week following stroke.

CONCLUSION

Our findings suggest that genetical induction of DKK1 is directly implicated in the poor diagnosis after stroke whereas DKK1 inhibition as the opposite effect that could constitute a very interesting and safe therapeutic approach to attenuate stroke pathology. DKK1 inhibition are now being assessed in various clinical trials for other diseases, thus increasing the translational potential of our study.

Associations entre la circonférence du cou, les altérations métaboliques associées à l’obésité et la densité de matière grise cérébrale chez les individus souffrant d’obésité sévère

OBJECTIF : L’obésité abdominale mesurée par la circonférence de la taille est associée à des altérations métaboliques et une atrophie de la matière grise (MG) dans des régions du cerveau impliquées dans le contrôle cognitif, chez les individus souffrant d’obésité. Toutefois, la circonférence du cou semble être un meilleur marqueur des altérations métaboliques chez les individus souffrant d’obésité sévère. Nous avons donc vérifié les hypothèses que la circonférence du cou est le meilleur indicateur des altérations métaboliques chez cette population et qu’elle est associée à des changements de densité de MG dans des régions impliquées dans le contrôle cognitif. MÉTHODE : 79 individus (âge : 44,8 ± 8,6 ans; IMC : 43,6 ± 4,1 kg/m2) devant subir une chirurgie bariatrique à l’IUCPQ ont été recrutés. Avant chaque chirurgie, nous avons mesuré la taille, les circonférences du cou et de la taille, ainsi que la composition corporelle et le poids par bioimpédance. Les paramètres sanguins de l’homéostasie du glucose et du profil lipidique ont été mesurés à jeun. La densité de MG a été mesurée par morphométrie cérébrale (VBM) à partir des scans T1 obtenus par IRM. Les valeurs de VBM ont été extraites pour des régions d’intérêt (ROIs) précédemment associées au contrôle cognitif et à l’obésité: 1) gyrus frontal inférieur, 2) cortex préfrontal dorsolatéral et 3) cervelet. RÉSULTATS : La circonférence du cou était positivement associée aux niveaux plasmatiques d’insuline (r=0.39, p<0.001), à l’indice de résistance à l’insuline HOMA-IR (r=0.30, p<0.01) et négativement au taux de cholestérol-HDL (r=-0.31, p<0.01). Des analyses de régressions multivariées ont montré que la circonférence du cou était le meilleur indicateur des mesures de résistance à l’insuline et des niveaux plasmatiques de cholestérol-HDL, expliquant 32-62% (p<0.05) de la variance (modèles incluant la circonférence de la taille, l’indice de masse corporelle et la masse grasse totale). La circonférence du cou était négativement associée à la densité de MG au niveau du cervelet (r=-0.24, p=0.03). Aucune association significative n’a été observée avec les autres ROIs. CONCLUSION : La circonférence du cou semble être un meilleur marqueur des altérations métaboliques chez les individus souffrant d’obésité sévère et nos résultats préliminaires montrent qu’elle est aussi associée à une atrophie de la MG au niveau du cervelet, une région impliquée dans le mouvement ainsi que la régulation émotionnelle et cognitive.

Paired Stimulation for Induction of Plasticity at the Cortical and Spinal Levels in Rodents

OBJECTIF - OBJECTIVE

We sought to determine whether neuronal plasticity can be induced at the cortical and/or spinal levels in rodents through paired electrical stimulation. Identification of effective means to potentiate the circuits along the corticomotor pathway has potent implications for developing effective interventions for those with stroke and spinal cord injury.

MÉTHODES - METHODS

In experiments under anaesthesia and awake rats, we conducted paired electrical associative stimulation between the contralateral motor cortex / ipsilateral cervical spinal cord and a target forelimb muscle using a variety of stimulation approaches (intracortical, intraspinal, epidural) and protocols (trains and single pulses, with modulation of interleaved parameters). The paired stimulation approach was aimed at activating inputs to motoneurons and motoneurons themselves (antidromically) in a timely manner to induce synaptic potentiation through spike-timing-dependent plasticity.

RÉSULTATS - RESULTS

Potentiation of putatively spinal motor circuits controlling the extensor carpi radialis (ECR) muscle could be induced under urethane/ketamine-xylazine anaesthesia, using a single pulse protocol and intraspinal approach. Depending on the protocol used, this led to a persistent hour-long effect and close to two-fold elevation (relative to baseline) in spinal evoked potentials induced via electrical stimulation. Paired motor cortex and ECR muscle stimulation was unsuccessful, both in exploratory experiments under anaesthesia and in a published study using single pulse protocols in a chronic implementation.

CONCLUSION

Pending an ongoing sufficiently powered validation study of this protocol with extensive controls, and further work to delineate the mechanisms involved, spinal PAS (sPAS) may be a new method of inducing persistent beneficial plasticity for eventual therapeutic applications after neurological insult.

ACTIVITY-DEPENDENT REMODELING OF SYNAPTIC PROTEIN ORGANIZATION REVEALED BY HIGH THROUGHPUT ANALYSIS OF STED IMAGES

OBJECTIVE: The nanoscale organization of synaptic proteins is thought to play a pivotal role in the establishment of synaptic strength and plasticity. In this study we assessed whether neuronal activity induces remodelling of pre- and postsynaptic scaffold proteins at the nanoscale. METHODS: We investigated the impact of synaptic activity on the organization, morphology and alignment of presynaptic (Bassoon, RIM1/2) and postsynaptic (PSD95, Homer1c) scaffold proteins in dissociated cultured hippocampal circuits. Their distribution inside single synapses was characterized using multicolor STED nanoscopy following treatments to either reduce or enhance excitatory synaptic activity. We adapted a method based on statistical object distance analysis (pySODA) to allow high throughput quantitative assessment of the spatial organization of synaptic proteins.  The degree of association of the detected synaptic scaffold protein clusters was quantified by measuring their coupling probability. We also used machine learning-based feature projection for multidimensional analysis of morphology and organization of diverse synapse types present in cultured neuronal circuits. RESULTS: This high throughput analysis framework allowed us to characterize the activity dependent remodelling of scaffold protein pairs in functional synapses at the population level. Furthermore we show that different activity paradigms change the morphological features of synaptic protein clusters, allowing us to identify morphological subtypes of functional synapses.  CONCLUSION: Our approach revealed an enlarged spectrum of synaptic features, providing a basis to further explore the different molecular mechanisms of synaptic protein remodeling.

pySTED: STED Microscopy Simulator for Reinforcement Learning Applications

OBJECTIVE Optical microscopy methods can benefit greatly from machine learning (ML) methods to solve different tasks such as automated control, parameter optimization and online image analysis. However, it is difficult to obtain large amounts of data to train ML algorithms, especially when imaging biological samples or using high-end techniques such as STimulated Emission Depletion (STED) microscopy. The objective of this project is to develop a simulation platform for STED microscopy applied to nanoscale imaging of neuronal structures. METHODS We introduce a simulator for STED microscopy that was developed in Python (pySTED). This tool simulates the acquisition of STED images, allowing for the generation of large quantities of STED data with diverse acquisition parameters. This simulated data will then be used to train reinforcement learning (RL) algorithms that would otherwise require unrealistically large training datasets to be applicable to biological specimens. The simulation platform includes imaging schemes aimed at reducing photobleaching while acquiring the maximum amount of important information for super-resolution imaging of living neurons. RESULTS We demonstrate that the pySTED allows to generate realistic STED image stacks. It simulates image acquisition schemes with various parameters that influence for example the image quality, spatial resolution, and acquisition speed. Using datamaps that simulate a dynamic neuronal arborisation, we can evaluate the impact of the imaging protocols on our ability to resolve subcellular neuronal remodelling with STED microscopy. CONCLUSION The pySTED simulation tool will be essential to investigate how RL can be applied to STED microscopy and improve its performance in living neurons, while minimizing invasiveness.

Les effets d’une expérience de mémoire sur le système vasculaire du cerveau selon les sexes

OBJECTIF - OBJECTIVE

Des expériences montrent que les émotions, positives comme négatives, altèrent la mémoire et ont un impact sur plusieurs régions du cerveau reliées à cette fonction cognitive. La barrière hémato-encéphalique (blood brain barrier, ou BBB) subit des changements morphologiques et fonctionnels suite à différentes émotions, tel que le stress. Le lien entre la formation de la mémoire avec ou sans valence émotionnelle et les adaptations neurovasculaires de la BBB n’est pas encore connu puisque la plupart des études se penchent sur les fonctionnements neuronaux de l’acquisition de la mémoire. Mon projet s’intéresse aux impacts d’une expérience de mémoire avec impact émotionnel neutre sur le système vasculaire de différentes régions du cerveau.

MÉTHODES - METHODS

Le test de reconnaissance d’un nouvel objet (novel object recognition, ou NOR) a été utilisé à cet effet avec des souris mâles et femelles. Le comportement des animaux a été évalué ainsi que le changement d’expression de gènes endothéliaux de la BBB par qPCR.

RÉSULTATS - RESULTS

Les gènes d’angiogenèse Vegfa, Fgf2, Bdnf, d’astrocyte Gfap, et endothéliaux Cldn5, Cd31 sont modulés de différentes façons dans l’hippocampe dorsal, ventral et l’amygdale, de façon spécifique au sexe. L’expression des gènes varie également selon l’environnement où se déroule le test, dépendamment de la taille de l’arène durant le NOR.

CONCLUSION

Ces résultats suggèrent que les gènes endothéliaux et d’angiogenèse reliés à la BBB sont modulés lors d’un test de mémoire à impact neutre chez la souris. Les changements sont spécifiques à certaines régions impliquées dans la formation de la mémoire et sont spécifique au sexe. Pour la suite du projet, j’utiliserai des modèles animaux pour réaliser des expériences de mémoire avec valence émotionnelle positive et négative afin de comparer les effets sur la BBB.  

Chronic stress induces sex-specific blood brain barrier dysfunction promoting depression

Major depressive disorder (MDD) is the leading cause of disabilities worldwide and women have a roughly 2x higher risk. Only 30% of patients completely remit from MDD, suggesting that neuron-centric traditional treatments do not address important causal biological factors. Studies report higher prevalence of MDD in patients suffering from cardiovascular diseases or stroke, indicating that vascular dysfunction may contribute to depression pathogenesis. Most studies on MDD have been conducted exclusively in males, leading to causal biological factors being omitted. Recent evidence shows that chronic social stress in male mice decreases expression of tight-junction protein cldn5 and induces blood-brain barrier (BBB) leakiness in the nucleus accumbens (NAc), a critical structure for stress response. This promotes infiltration of harmful peripheral immune signals into the brain and establishment of depressive behaviors. These effects have not yet been studied in female mice.

OBJECTIF - OBJECTIVE
I aim to study neurovascular function under chronic stress conditions, in a sex-specific manner. I hypothesize that MDD leads to sex-specific neurovascular adaptations, which may explain heightened vulnerability in women and sex-symptomatology observed in MDD.

MÉTHODES - METHODS
I investigate BBB function under a 10-day chronic social defeat stress (CSDS) paradigm, a mouse model of depression mimicking human bullying. Transcriptional patterns of key BBB genes will be investigated in qPCR and validated at protein level using immunofluorescence. Human postmortem brain samples of depressed male and female subjects will be processed to add translational value to my animal findings.  

RÉSULTATS - RESULTS
After CSDS, cldn5 gene expression is unchanged in the NAc of stress-susceptible females but decreased in the prefrontal cortex (PFC), a brain region regulating social behaviors. Immunostaining confirmed cldn5 protein reduction in the female PFC. Importantly, this sexual dimorphism of stress-induced neurovascular adaptations was confirmed in postmortem human brain samples from depressed individuals. Viral-mediated functional manipulation confirmed the causality of cldn5 loss in the female PFC in the establishment of depressive behaviors and possibly sex-specific MDD symptomatology.

CONCLUSION
My results suggest that stress-induced neurovascular changes are not occurring in the same brain regions in stressed females. It is imperative to study mental health as whole-body maladaptive responses and consider sex differences to develop innovative therapeutic strategies.

Role of the endocannabinoid system in stress resilience and depression: a master regulator of neurovascular health

Only 30 to 50% of major depressive disorder (MDD) patients completely remit, making it a leading cause of disability worldwide. This lack of efficacy suggests that current neuron-centric treatments do not address important biological factors. Chronic stress, the main environmental risk for MDD development, has been known to trigger a whole-body response including neuroimmune and neurovascular adaptations. We recently reported that chronic social stress causes a detrimental increase in blood-brain barrier (BBB) permeability, promoting infiltration of circulating inflammatory mediators and development of depressive-like behaviours in mice. Those pathological changes have been confirmed in brain samples of MDD patients. However, biological mechanisms underlying these molecular changes in response to stress remain elusive. Interestingly, the endocannabinoid system (ECS) is a crucial regulator of stress responses. Moreover, ECS was shown to regulate BBB permeability under homeostatic and pathological conditions. Here we combine molecular, cellular and morphological analyzes to behavioral studies and show that the ECS is actively involved in stress resilience to chronic social defeat stress, a mouse model of depression, in a sex- and brain region-specific manner. Based on those results, we propose that stress-induced increased in BBB permeability could be due to pathological changes in the ECS system, enabling release of inflammatory signals into the circulation, vascular dysfunction and establishment of depressive behaviours.