Résumés / Abstracts 2020

Abstract ID
Poster TH1-01

Does the blood-brain barrier contribute to pathological protein spread in Huntington’s disease?

ALPAUGH, MELANIE 1,2, Denis, Hélèna L 1,2, Bousset, Luc 3, Sciacca, Giacomo1,2, Melki, Ronald 3 and Cicchetti, Francesca 1,2

1Centre de Recherche du CHU de Québec - Université Laval, Axe Neurosciences, Québec, QC, Canada, G1V 4G2; 2Département de Psychiatrie & Neurosciences, Université Laval, Québec, QC, Canada, G1K 0A6; 3Laboratory of Neurodegenerative Diseases, Institut François Jacob, MIRCen, CEA, CNRS, Fontenay-aux-Roses, France, 92265


Studies in multiple neurodegenerative conditions have demonstrated that disease-associated pathological proteins can behave in a prion-like manner, contributing to disease manifestation and/or progression. In Huntington’s disease (HD), a dominantly inherited neurodegenerative disease characterized by the expression of a mutated form of the huntingtin protein (mHTT), numerous studies have now demonstrated that similar mechanisms apply to mutant huntingtin (mHTT). Such mechanisms include, evidence of spread from the circulatory system to the brain, suggesting that mHTT can cross the blood-brain barrier (BBB). In addition to crossing the BBB, several reports have indicated that mHTT is present within endothelial cells and that HD patients have vascular abnormalities including decreased integrity of the BBB, changes in the expression of tight-junction proteins and an increase in vessel density.  OBJECTIVE Determine if mHTT can cross the BBB and if the presence of pathological mHTT surrounding the neurovascular unit contributes to a decreased integrity of the BBB and to changes in vascular morphology. METHODS We developed a 3D human BBB model containing human control or HD patient iPSC derived endothelial cells and astrocytes as well as primary human brain pericytes. These BBB were subsequently treated with N-terminal fragments of the HTT protein containing either 19 or 48 polyglutamine repeats. RESULTS Both patient and control-derived microvascular endothelial cells form leak-tight barriers after 96 hours in culture. When these vessels are treated with mHTT and WT HTT for 48 hours, HTT is found to enter endothelial cells and cross the BBB in control and HD patient derived cells. While both forms of HTT show the capacity to cross the BBB, they have different effects on the permeability of the vessel with mHTT causing a greater detrimental change to barrier integrity than WT HTT when added to the lumen of the vessel. In HD vessels the magnitude of this change is increased and more fibrils are observed to cross the barrier. CONCLUSION Using a human cell culture model of the BBB, we observed bi-directional crossing of HTT in a CAG tract size independent manner. Additionally, the presence of fibrils impacts the integrity of the BBB suggesting that mHTT itself may contribute to vascular abnormalities previously reported in HD patients, as well as to the spread/clearance of mHTT.

Poster TH1-02

Optogenetic-mediated spatiotemporal control of α-synuclein aggregation mimics authentic Lewy body formation and triggers neurodegeneration

MORGAN BERARD1,2, Razan Sheta1,2, Sarah Malvaut3,4, Raquel Rodriguez-Aller1,2,3, Maxime Teixeira1,2, Roxanne Turmel1,2, Melanie Alpaugh1,4, Marilyn Dubois1,2, Manel Dahmene1,2, Charleen Sales3,4, Jérôme Lamontagne-Proulx1,5, Marie-Kim St-Pierre1,2, Omid Tavassoly6, Wen Luo6, Raza Qazi7, Jae-Woong Jeong7,8, Thomas M. Durcan6, Luc Vallières1,2, Marie-Eve Tremblay1,2,9, Denis Soulet1,5, Martin Lévesque3,4, Francesca Cicchetti1,4, Edward A. Fon6, Armen Saghatelyan3,4, Abid Oueslati1,2

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. 5Faculty of Pharmacy, Université Laval, Quebec City, Canada. 6McGill Parkinson Program and Neurodegenerative Diseases Group, Montreal Neurological Institute, McGill University, Montreal, Canada. 7Department of Electrical, Computer, and Energy Engineering, University of Colorado, Boulder, CO 80309, USA. 8School of Electrical Engineering Korea Advanced Institute of Science and Technology, Daejeon, 34141, Republic of Korea. 8Division of Medical Sciences, University of Victoria, Victoria, Canada.


α-synuclein (α-syn) aggregation into insoluble deposits, referred to as Lewy bodies (LBs) is the paramount pathological hallmark of Parkinson’s disease (PD) and related α-synucleinopathies. However, how these aggregates affect neuronal homeostasis leading to neurodegeneration remains elusive. This gap in knowledge is mainly due to the lack of proper cellular and animal models to undertake such investigations.
AIMS
We have addressed this limitation by developing a light-inducible protein aggregation system (LIPA). This application is based on the use of a mutant form of the Arabidopsis thaliana photoreceptor cryptochrome 2 (CRY2), which when stimulated with blue light, mutant CRY2 undergoes rapid, reversible and robust protein clustering or aggregation. The use of this application allows for real-time induction of α-syn inclusions formation with remarkable spatial and temporal resolution in both cell culture and in vivo paradigms.
METHODS
We used a gene therapy approach, based on the use of adeno-associated virus (AAV), to overexpress our LIPA system directly into the brains of naive mice. For the delivery of the blue light necessary for the induction of the aggregation and propagation of α-syn, we used implantable micro-devices developed by Amuza Inc.
RESULTS
We report on the development of a light-inducible protein aggregation (LIPA) system that enables real-time induction of α-syn inclusion formation with remarkable spatial and temporal resolution in living cells. We demonstrate that LIPA-α-syn inclusions faithfully mimic key biochemical and ultrastructural features, as well as the seeding capacity of authentic LBs. In vivo, LIPA-α-syn aggregates compromised the nigrostriatal transmission, induced dopaminergic neuronal loss and PD-like behavioral impairment.
CONCLUSION
Our system provides a novel, dependable and invaluable tool to generate, visualize and dissect the role of protein aggregates in PD and possibly other neurodegenerative disorders.

Poster TH1-03

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

BOLDUC CYRIL, Lorente Picón Marina, Dovonou Axelle, Castonguay Anne-Marie, Rioux Véronique, Lévesque Martin

CERVO Brain Research Centre


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.

Poster TH1-04

Testing the efficacy of a human full-length OPG-Fc analog in a model of cardiotoxin-induced skeletal muscle injury and repair

BOUREDJI ZINEB1, Dounia Hamoudi1, Laetitia Marcadet1, Anteneh Argaw1, and Jérôme Frenette1,2

Authors’ affiliations

1 - Centre Hospitalier Universitaire de Québec, Centre de Recherche du Centre Hospitalier de l’Université Laval (CHUQ-CHUL), Axe Neurosciences, Université Laval, Quebec City, QC, Canada G1V 4G2

2 - Département de Réadaptation, Faculté de Médecine, Université Laval, Quebec City, QC, Canada G1V 0A6


INTRODUCTION

Although receptor-activator of nuclear factor (kB) (RANK), its ligand RANKL, and osteoprotegerin (OPG), which are members of the tumor necrosis factor (TNF) superfamily, were first discovered in bone cells, they are also expressed in other cells, including skeletal muscle. We previously showed that the RANK/RANKL/OPG pathway is involved in the physiopathology of Duchenne muscular dystrophy (DMD) and that a mouse full-length OPG-Fc (mFL-OPG-Fc) treatment is superior to muscle-specific RANK deletion in protecting dystrophic muscles. Although mouse mFL-OPG-Fc has a beneficial effect in the context of DMD, the function of human FL-OPG-Fc (hFL-OPG-Fc) during muscle repair is not yet known. In the present study, we investigated the impacts of an hFL-OPG-Fc treatment following the intramuscular injection of cardiotoxin (CTX).

METHODS

The slow twitch soleus (Sol) muscles from 12-week-old-C57BL/10J mice were injected with 15 μL of cardiotoxin (CTX; 20 μM). The mice were then treated daily with either vehicle (PBS) or hFL-OPG-Fc [1 mg/kg/d] for 3 (n=3) or 7 days (n=7-12). The contractile properties were evaluated ex vivo. Sol muscle cross-sections from PBS-treated and hFL-OPG-Fc-treated mice were used  to label inflammation cell infiltration, muscle regeneration and damage. In addition, in vitro experiments were performed on cultured muscle cells.

RESULTS

We show that a 7-d hFL-OPG-Fc treatment improved while muscle-specific RANK deletion had no effect on force production of soleus (Sol) muscle. hFL-OPG-Fc also improved Sol muscle regeneration by increasing satellite cell density and fiber cross-sectional area, attenuating inflammatory cell infiltration, and promoting the switch of macrophages from the M1 phenotype to the M2 phenotype 3- and 7-days post-CTX injury. We also show for the first time that hFL-OPG-Fc improves myotube maturation and fusion in vitro and reduces cytotoxicity and cell apoptosis. These findings demonstrate that hFL-OPG-Fc has therapeutic potential for muscle diseases in which repair and regeneration are impaired.

Poster TH1-05

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

CASTONGUAY, ANNE-MARIE; Gravel, Claude; Lévesque, Martin. (CERVO Research Center)


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.

 

Poster TH1-06

ATLAS DU TRONC CÉRÉBRAL HUMAIN COMBINANT L’IMMUNOHISTOCHIMIE ET L’IMAGERIE PAR RÉSONANCE MAGNÉTIQUE: VERS UNE DESCRIPTION PRÉCISE DU NOYAU PÉDONCULOPONTIN CIBLÉ PAR STIMULATION CÉRÉBRALE PROFONDE

COULOMBE VINCENT, Saikali SP, Goetz L, Takech A, Philippe É, Parent A et Parent M. Centre de recherche CERVO et Département de Psychiatrie et Neurosciences, Faculté de Médicine, Université Laval.


OBJECTIF - OBJECTIVE

Situé dans le tronc cérébral, le noyau pédonculopontin (PPN) suscite un grand intérêt, puisqu’il est impliqué dans diverses fonctions telles que la nociception et le sommeil. Étant également impliqué dans la locomotion, le PPN est souvent ciblé lors de stimulations cérébrales profondes (DBS) permettant de traiter le « freezing de la marche » dont souffrent plusieurs patients parkinsoniens. Les études sur la position exacte qu’occupe le PPN demeurent cependant contradictoires. Ce projet vise à produire un atlas détaillé du tronc cérébral humain afin de localiser précisément le PPN, ainsi que de caractériser la nature neurochimique des neurones qui le composent. 

MÉTHODES - METHODS

Pour atteindre notre objectif, nous avons procédé à une acquisition IRM post-mortem suivie de marquages histologiques d’un tronc cérébral humain. Nous avons également réalisé des immunomarquages chez l’humain et le singe cynomolgus (Macaca fascicularis) pour la choline acétyltransférase (ChAT) ainsi que la nicotiamide adénine dinucléotide-diaphorase (NADPH-δ).

RÉSULTATS - RESULTS

L’IRM, combinée à 70 plans anatomiques constitués de sections histologiques marquées pour le Crésyl Violet et le Luxol Fast Blue, nous a permis de localiser précisément le PPN à la jonction ponto-mésencéphalique. Nos études stéréologiques nous ont conduit à estimer à 58 100 le nombre de neurones ChAT+ du PPN humain contenus à l’intérieur d’un volume de 145 mm3 ainsi qu’à apprécier, suite à une reconstruction 3D, le degré de chevauchement entre le PPN, le noyau latérodorsal tegmentaire, le locus coeruleus et la substance noire. Contrairement à ce que l’on croyait, chez le singe, seulement 71% des neurones ChAT du PPN contiennent la NADPH-δ. 

CONCLUSION

Dans l’ensemble, cette étude pourrait conduire à un meilleur positionnement des électrodes DBS, augmentant ainsi les bienfaits cliniques pour les parkinsoniens, tout en fournissant des données fondamentales, essentielles à une meilleure compréhension du rôle que joue le PPN.

Poster TH1-07

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

DE RUS JACQUET AURELIE1,2,3, Alpaugh Melanie1,2, Tancredi Jenna3, Decaestecker Julien1,4, Beauparlant Charles1,4, Denis Hélèna1,2, Droit Arnaud1,4, O’Shea Erin3, Cicchetti Francesca1,2.

1Centre de Recherche du CHU de Québec - Université Laval, Québec, QC, Canada, G1V 4G2

2Département de Psychiatrie & Neurosciences, Université Laval, Québec, QC, Canada, G1K 0A6

3Janelia Research Campus, Howard Hughes Medical Institute, Ashburn, VA, USA

4Department de Médecine Moléculaire, Faculté de Médecine, Université Laval, Québec, QC, Canada, G1K 0A6


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.

Poster TH1-08

Identification of a humoral immune response against Huntingtin protein

HÉLÈNA L. DENIS1, Claudia Alvarez2, Melanie Alpaugh1, Martine Saint-Pierre1, Luc Bousset3, Ronald Melki3, Rachel J. Harding2, Francesca Cicchetti1

1 Centre de Recherche du CHU de Québec, Axe Neurosciences and Département de Psychiatrie & Neurosciences, Université Laval, Québec, QC, Canada; 2 Structural Genomics Consortium, University of Toronto, Toronto, Canada; 3 Laboratory of Neurodegenerative Diseases, Institut François Jacob, CNRS, Fontenay-aux-Roses, France


Huntington's disease (HD) is caused by a mutation in the huntingtin (HTT) gene which results in the production/accumulation of an abnormal protein, the mutant huntingtin (mHTT) which is known to confer toxicity to multiple cell types. A well-described aspect of HD is the activation of immune cells throughout the body, which may be particularly relevant to the pathology since they can contribute to the clearance of misfolded pathologic proteins. As demonstrated in Alzheimer disease (AD), antibodies raised against amyloid-ß – the signature misfolded protein of this disease – are significantly increased in the plasma of AD subjects with mild cognitive impairments when compared to controls. To date, the mechanisms of clearance of mHTT protein aggregates in HD have not been fully elucidated. OBJECTIVE. We explored the possibility that the immune system recognizes mHTT as a pathogen and, consequently, develops an antibody-response specific to the mutated protein. METHODS. We developed an ELISA assay to quantify the presence of antibodies against various forms of huntingtin in plasma derived from 1) WT mice injected intravenously with fibrils containing either 25 (HTT) or 48 (mHTT) polyglutamine (Q) repeats, and from 2) human HD samples from premanifest to late stage disease according to the clinical evaluation of the Unified Huntington's Disease Rating Scale (UHDRS). To validate this ELISA assay, we first used a commercial antibody to ensure the specificity of the signal obtained. We then proceeded with the extraction and purification of antibodies from plasma by using an exclusion/concentration Amicon-column with a cut-off at 100kDa. We focused our study on the identification of the immune response against the fibrillar forms of HTT (Q25) or mHTT (Q48) and the full-length HTT protein with either Q23 (HTT) or Q54 (mHTT). RESULTS. Peripheral administration of fibrillar forms to WT mice resulted in a strong antibody response against these fibrillar forms of HTT/mHTT. After purification and concentration of antibodies raised in human plasma, patients with severe symptoms (UHDRS>50) have more antibodies against full-length HTT protein than their associated controls. CONCLUSION. Our findings suggest that the humoral immune response can generate antibodies against different forms of HTT. The presence of antibodies in patients sheds light on a novel aspect of disease.

Poster TH1-09

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

DOVONOU AXELLE (2), De Koninck Yves (2), Metzakopian Emmanouil (1) and Lévesque Martin (2) .

(1) UK Dementia Research Institute, University of Cambridge, Cambridge, United Kingdom.

(2) Centre de recherche CERVO, Université Laval, Québec, QC, Canada.


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.

Poster TH1-10

Using super-resolution microscopy to observe mitochondrial dynamics in Parkinson’s disease

Owen Ferguson, Tiago Cardoso, Martin Lévesque, Flavie Lavoie-Cardinal


OBJECTIVE :Mitochondria are dynamic organelles best known for their role in energy production. These organelles are vital to neuronal health and mitochondrial dysfunction leads to the neuronal abnormalities and cell death present in neurodegenerative diseases like Parkinson’s disease (PD). Mitochondrial dynamics -  or the change in mitochondrial morphology in a changing environment - can provide clues to the underlying mechanisms of mitochondrial dysfunction and possibly even into the pathogenesis of PD. 

 

METHODS : We use super-resolution STED microscopy to visualize and compare mitochondrial morphology in dopaminergic neuronal cultures made from human induced pluripotent stem cells (hiPSC) derived from either a PD patient or a healthy subject. Prior to STED imaging, cells are fixed and we use immunofluorescence to label key mitochondrial proteins such as Tom20 and Cox4 . We visualize the mitochondrial networks in dopaminergic neurons with multi-channel STED and confocal microscopy. 

 

RESULTS : Preliminary results in dopaminergic neurons derived from hiPSCs indicate that we can reveal Tom20 and Cox4 protein expression in combination with the dopaminergic marker Tyrosine Hydroxylase (TH) under multichannel STED and confocal microscopy.

 

CONCLUSION : Based on previous reports, we anticipate that the mitochondrial morphology will be different between the normal neurons that the neurons with PD-related mutations. Developing this approach will help with measuring mitochondrial defects and testing neuroprotective treatments against PD pathology. 

 

Poster TH1-11

REGULATION OF THE AXONAL TRANSLATOME IN DOPAMINERGIC CIRCUITS DURING DEVELOPMENT.

GORA CHARLES (1,2), Metzakopian Emmanouil (3) & Lévesque Martin (1,2).

  1. Centre de recherche CERVO.  
  2. Département de psychiatrie et neurosciences.
  3. UK Dementia Research Institute at the University of Cambridge.

OBJECTIVE

Midbrain dopaminergic (mDA) neurons play important roles in controlling a variety of brain functions, such as voluntary movement, attention, or motivation. An abnormal development of dopaminergic circuits can lead to different brain disorders, namely schizophrenia, attention disorders and depression. Degeneration of mDA neurons is the leading cause of Parkinson’s disease. Dopaminergic neurons in the midbrain form a heterogeneous set of neurons that innervate different regions of the brain. The different functions of these neurons are served by specific axonal connections. However, the mechanisms regulating the precise organisation of these neuronal circuits remain poorly understood. Recent discoveries have revealed that a large proportion of the proteins used for the navigation of developing axons are produced locally in axons. The messenger RNAs (mRNAs) encoding these proteins would be channelled specifically into the different branches of the developing neurons. This project aims to elucidate the cellular and molecular mechanisms regulating the development of dopaminergic circuits.

METHODS

To characterise the axonal translatome, we isolated the mRNAs in the dopaminergic axons at different developmental ages and proceed to the analysis of mRNA content by sequencing. The second part of this project consist of studying the function of these previously identified guidance cues using gain and loss of function analyses in vivo and in vitro.

RESULTS

Our results obtained after mRNA sequencing performed on 1-day, 21-days and adult mouse brain already shown axonal translatome specificity. We were able to identify axonal translatome within the different mDA axon projections, such as Plxna4, Ephb1, Efna4 found in the axons innervating to the dorsal striatum. These genes are known to be involved in the axonal growth and guidance in other cellular context. We are now studying the specific contribution of these genes in the regulation of the mDA axon projections.

CONCLUSION

In addition to uncover for the first time the axonal translatome (axotome) of the dopaminergic system, this project will help to better understand the development of the dopaminergic neurons. This study will also provide a useful resource for understanding changes involved in brain diseases.

Poster TH1-12

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

ESTELLE GOURIOU1,2,3, Thérèse Di Paolo2,4 et Cyril Schneider1,2,3,5

1Laboratoire de Neurostimulation Non-invasive ; 2Centre de recherche du CHU de Québec-Université Laval, Axe Neurosciences ; 3Faculté de Médecine, Université Laval ; 4Faculté de Pharmacie, Université Laval ; 5Département de réadaptation de la faculté de Médecine, université Laval


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. 

 

Poster TH1-13

Identifying sex dependent microglia immune networks after stroke

GURUSWAMY REVATHY, Rahimian Reza, Weng Yang C, Boutej Hejer and Kriz Jasna *

CERVO Brain Research Center, Laval University, Quebec, QC, Canada. 


INTRODUCTION: Microglia is the principal immune cells of the brain inflammatory response to ischemic injury; evidence suggests that any changes in their activation profile may significantly alter the outcome of ischemic stroke. However, at present, the molecular mechanisms involved in the control of microglia polarization profiles remain elusive. To add to the complexity, increasing evidence suggests marked sexual dimorphism in the processes associated with microglial activation after stroke. To address this question, we took advantage of the CD11brGFP mouse model generated and validated in our laboratory. In this transgenic mouse model, the Flag/EGFP is fused to the N-terminus of the larger subunit of ribosomal protein L10a and expressed under the transcriptional control of a myeloid-specific gene promoter. Thus, a simple high-affinity immunoprecipitation assay from brain homogenates allows a pull-down of the transcriptomes and/or proteomes from the targeted cell type. Using this experimental approach, we were able to successfully isolate and analyze microglia mRNA and protein networks following innate immune challenge and brain injuries including stroke.

OBJECTIVE: We hypothesized that after stroke microglial cells develop distinct sex-dependent immune signatures resulting in a sexually dimorphic response to ischemic injury.

METHODS: Ischemic stroke was induced by intraluminal filament occlusion of the middle cerebral artery in CD11brGFP and C57BL/6J male and female mice. The CD11brGFP transgenic mouse model was used to perform translational ribosome affinity purification to isolate ribosomes-attached mRNA and peptides to analyze transcriptome and proteome of activated microglia. Further, to validate proteomics analysis, we purified microglial cells from the ischemic brain by CD11b magnetic microbeads and performed western blot for various microglial markers and identified peptides.

RESULTS: We observed marked differences in microglia proteome between male and female networks after stroke. Further studies are in progress to delineate the underlying molecular mechanism and identify signaling pathways.

CONCLUSION: We anticipate that identifying novel sex-dependent immune targets and / or networks following ischemic brain injury may open new avenues for therapeutic modulation of the innate immune response in microglia / macrophages after stroke.

 

Poster TH1-14

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

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

CHU de Quebec Research Center, Axe Neuroscience and Department of Molecular Medicine, Laval University, Quebec, QC G1V4G2, Canada 


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. 

Poster TH1-15

Induction of autophagy mitigates TDP-43 pathology and translational repression of neurofilament mRNAs in mouse models of ALS/FTD

KUMAR SUNNY, Phaneuf Daniel, Cordeau Pierre, Boutej Hejer, Kriz Jasna, Julien Jean-Pierre

CERVO Brain Research Centre and Department of Psychiatry and Neuroscience, Faculty of Medicine, Université Laval, Quebec City, Canada 


OBJECTIVE:

TDP-43 proteinopathy is a pathological hallmark of many neurodegenerative diseases including amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). So far, there is no therapy available for these neurodegenerative diseases. In addition, the impact of TDP-43 proteinopathy on neuronal translational profile remains unknown. The objective of this study is to understand the impact of TDP-43 proteinopathy on neuronal translational profile and the therapeutic effect of novel NF-ĸB inhibitor (IMS-088) in FTD/ALS.

METHODS:

Biochemical, immunohistology and assay-based studies were done with cell cultures and transgenic mice models. We also used a Ribotag approach combined with microarray and proteomic analyses to investigate the neuronal translational profiles in mouse model of ALS/FTD.

RESULTS:

Here, we report that oral administration of a novel analog (IMS-088) of withaferin-A, an antagonist of nuclear factor kappa-B (NF-ĸB) essential modulator (NEMO), induces autophagy and reduced TDP-43 proteinopathy in the brain and spinal cord of transgenic mice expressing human TDP-43 mutants, models of ALS/FTD. Treatment with IMS-088 ameliorated cognitive impairment, reduced gliosis in the brain of ALS/FTD mouse models. With the Ribotrap method, we investigated the impact of TDP-43 proteinopathy and IMS-088 treatment on the translation profile of neurons of one-year old hTDP-43A315T mice. TDP-43 proteinopathy caused translational dysregulation of specific mRNAs including translational suppression of neurofilament mRNAs resulting in 3 to 4-fold decrease in levels type IV neurofilament proteins. Oral administration of IMS-088 rescued the translational defects associated with TDP-43 proteinopathy and restored the synthesis of neurofilament proteins, which are essential for axon integrity and synaptic function.

CONCLUSION:

Our study revealed that induction of autophagy reduces TDP-43 pathology and ameliorates the translational defect seen in mice models of ALS/FTD. Based on these results, we suggest IMS-088 and perhaps other inducers of autophagy should be considered as potential therapeutics for neurodegenerative disorders with TDP-43 proteinopathies.

 

 

Poster TH1-16

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

LARIVIÈRE-LOISELLE CÉLINE, Bélanger Erik, Marquet Pierre. Centre de recherche CERVO


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.

Poster TH1-17

Signalisation défectueuse du récepteur de l’insuline au niveau de la barrière hématoencéphalique dans la maladie d’Alzheimer.

LECLERC MANON1,2; Caron Vicky1,2; Virgili Jessica1,2; Emond Vincent2; Bourassa Philippe1,2; Bennett David A.3 et Calon Frédéric1,2.

1Faculté de Pharmacie, Université Laval, Québec, Québec, Canada.  2Axe Neurosciences, Centre de Recherche du CHU de Québec-Université Laval, CHUL. 3Rush Alzheimer’s Disease Center, Rush University Medical Center, Chicago, IL, USA


L’insuline est produite en périphérie mais exerce également une action au cerveau. Il est suspecté qu’une signalisation défectueuse de l’insuline au niveau du système nerveux central (SNC) peut jouer un rôle dans la maladie d’Alzheimer (MA). Cependant, l’insuline doit interagir avec la barrière hématoencéphalique (BHE) avant de rejoindre le SNC.

OBJECTIF - OBJECTIVE

Le but de la présente étude était de mieux comprendre les mécanismes sous-jacents au transport et à la signalisation cellulaire de l'insuline au niveau du BHE, et comment ils sont modifiés dans la MA.

MÉTHODES - METHODS

La localisation et l’activation du récepteur de l’insuline (INSR) ont été étudiés par immunobuvardages dans des extraits de microvaisseaux cérébraux murins et humains (Religious Order Study). La perfusion cérébrale in situ a permis de quantifier le transport de l’125I-insuline au travers de la BHE chez la 3xTg-AD (neuropathologie MA) et non-transgénique (NonTg), avec une diète riche en gras (HFD).

RÉSULTATS - RESULTS

Les concentrations d’INSR étaient nettement plus élevées dans les extraits de microvaisseaux que dans les homogénats totaux, produits à partir de cerveaux humains et de souris. Une diminution d'INSRα-B dans des extraits de microvaisseaux humain a été observée chez les patients atteints de MA (-48%), et associée aux symptômes cognitifs. L’activation de l’INSR après une injection d’insuline était abolie chez les 3xTg-AD sous HFD. Le coefficient de passage de l’125I-insuline est demeuré faible chez les NonTg (0.019 ± 0.005 µl.g-1.s-1) et 3xTg-AD (0.012 ± 0.005 µl.g-1.s-1), mais augmenté par une exposition au HFD (+224 % pour les 3xTg-AD). Pas de changement dans la perméabilité n’a été détecté par co-perfusion d’un marqueur vasculaire.

CONCLUSION

Nos résultats suggèrent que (i) l’INSR est concentré au niveau de la neurovasculature; (ii) la neuropathologie de la MA et la HFD interfèrent avec la signalisation et le transport de l’insuline au niveau de la BHE.

 

Poster TH1-18

ROLE OF INFLAMMATORY BRAIN CELLS IN DEMENTIA FOLLOWING MICROINFARCTS

LECORDIER SARAH, ElAli Ayman


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.

 

Poster TH1-19

Contribution de la protéine tau aux déficits cognitifs associés à la maladie de Huntington

LEPINAY EVA1,2, Alpaugh Melanie1, De Rus Jacquet Aurélie1, Saint-Pierre Martine1, Bousset Luc3, Melki Ronald3, Cicchetti Francesca1,2

1Centre de recherche du CHU de Québec, Axe Neurosciences, Canada

2Département de psychiatrie et de neurosciences, Faculté de Médecine, Université Laval, Québec, Canada

3CEA CNRS, Institut François Jacob (MIRCen), Laboratoire des maladies neurodégénératives, Fontenay-aux-Roses, France


Tau est une protéine qui stabilise et régule la dynamique des microtubules en condition physiologique. De récentes publications ont suggéré que la maladie de Huntington (MH) – une maladie neurodégénérative autosomique dominante caractérisée par des problèmes moteurs, cognitifs et psychiatriques – présente des signes de tauopathie. Les tauopathies sont une sous-classe de maladies neurodégénératives caractérisées par le dépôt de protéines tau phosphorylées dans le tissu cérébral. Effectivement, il a été rapporté que (i) les patients MH présentent des inclusions tau agrégées dans différentes structures du cerveau et que (ii) l'haplotype tau a un impact sur les fonctions cognitives des patients MH. OBJECTIF Le but de ce projet est d'étudier la contribution de la protéine tau aux déficits cognitifs de la MH. METHODES Pour répondre à cette question, nous croisons des souris zQ175 MH et knock-out tau pour évaluer l'évolution des déficits cognitifs en absence de tau. Le comportement des souris sera évalué sur plusieurs mois et une caractérisation neuropathologique sera réalisée. Nous étudions également l’effet des agrégats de mHtt sur la pathologie tau en exposant des fibrilles de mHtt à différents modèles cellulaires; cellules de neuroblastomes (SH-SY5Y), cellules surexprimant des isoformes tau (SH-3R) et cellules souches pluripotentes induites (iPSC). Ces modèles sont différenciés en neurones corticaux en raison d’une grande quantité de tau agrégée retrouvée dans le cortex des patients MH. RESULTATS La différenciation cellulaire a été validée dans chacun de nos modèles. Les premiers tests comportementaux – avant l’apparition de la MH – montrent des résultats similaires entre les génotypes. Nous nous attendons à ce que la suppression génétique de tau prévienne le développement de déficits cognitifs chez la souris MH et, à ce que l’injection de fibrilles mHtt aggrave la pathologie tau dans nos modèles cellulaires. CONCLUSION Ce projet permettra de mieux comprendre le rôle de tau aux déficits cognitifs, un aspect clinique de la MH dont le traitement est sous-optimal.

Poster TH1-20

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

MARTINEZ-DRUDIS LAURA1,2, Pellegrinato Rémi1,2, Sheta Razan1,2, Calon Frédéric1,3, Rivest Serge1,2, Oueslati Abid1,2.
1. CHU de Québec-Université Laval Research Center, Neuroscience Axis.
2. Université Laval, Faculty of Medicine, Department of Molecular Medicine. 
3. Université Laval, Faculty of Pharmacy. 


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.
 

Poster TH1-21

Les anomalies lipidiques postmortem dans le cervelet des patients atteints du tremblement essentiel

Koralie Mélançon B. Sc., Line Berthiaume B. Sc., Cyntia Tremblay M. Sc., Pierre Julien Ph.D, Ali H Rajput M.D., FRCPC, Alex Rajput M.D., FRCPC and Frédéric Calon B.Pharm., Ph.D.

 


OBJECTIF - OBJECTIVE

Malgré que la physiopathologie du tremblement essentiel (TE) reste mal définie, plusieurs études observent un dysfonctionnement du cervelet dans celle-ci. Cette région cérébrale est riche en acides gras polyinsaturés (AGPI) ω3, une sous-classe lipidique jouant un rôle critique dans les fonctions cérébrales de la maladie de Parkinson (MP). Toutefois, le rôle des AG dans la pathologie du TE est inconnu. D'un point de vue thérapeutique, il est capital d’établir la relation entre les AG cérébraux et le TE, car la teneur lipidique dépend de l’alimentation. C’est pourquoi, l'objectif de cette l'étude était de comparer les profils des AG, et plus particulièrement de l’ADH, au sein de différentes fractions de phospholipides (PL) extraites du cervelet de patients décédés atteints de la MP ou du TE. 

MÉTHODES - METHODS

Les lipides ont été extraits du cervelet par la méthode Folch. La chromatographie sur couche mine a permis de séparer les classes de GPL (phosphatidylcholine (PC), phosphatidyléthanolamine (PE), phosphatidylinositol et phosphatidylsérine (PI+PS)), suivi de la chromatographie gazeuse couplée à la détection à ionisation à flamme afin de quantifier les différences entre les groupes témoins (n=17), MP (n=15) et TE (n=15)

RÉSULTATS - RESULTS

Les seuls changements en AG dans la PC sont l’élévation des taux relatifs de 20 :3 ω6 dans le TE comparativement aux témoins. Cependant, des taux inférieurs cérébelleux d’AG totaux, d’AG saturés (AGS) et d’acide docosahexaénoique (ADH) dans le groupe du TE ainsi que d’AGS et d’acide oléique dans le groupe de la MP ont été récupérés par rapport au groupe témoin. La quantité totale d'AG récupérée dans la fraction combinant la PI et la PS était plus élevée chez les patients atteints de la MP par rapport aux autres groupes, comprenant notamment les AGS et les AG monoinsaturés. Finalement, l’âge de la mort a corrélé positivement avec l’ADH et négativement avec l’acide oléique dans le TE, tandis que l’alpha-synucléine phosphorylée a corrélé positivement avec l’ADH dans la MP. 

CONCLUSION

Ces données suggèrent que l'altération des AG peut être impliquée dans la physiopathologie du TE et proposent de nouvelles approches thérapeutiques nutritionnelles basée sur une diète de type méditerranéenne riche en ω3. 

Poster TH1-22

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

NATH BANSHI, Julien Jean-Pierre

CERVO Brain Research Center and  Universite Laval.


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.

Poster TH1-23

Relationships between the number of integrated pressure sensors and step count accuracy using smart insoles

Armelle M. Ngueleu, MSc1 ; Andréanne K. Blanchette, PhD1, 2 ; Stéphane Mandigout, PhD3 ; Charles S. Batcho, PhD1, 2.

1 Centre for Interdisciplinary Research in Rehabilitation and Social Integration (Cirris), Quebec City, Canada.

2 Department of rehabilitation, Faculty of Medicine, Université Laval, Quebec City, Quebec, Canada

3 Laboratory HAVAE EA-6310, University of Limoges, France.


OBJECTIF - OBJECTIVE

Several studies have demonstrated that instrumented insoles enable the quantification of steps taken in people with or without walking limitations. However, the number and location of imbedded pressure sensors in an insole vary considerably in the literature, resulting in variable step count accuracies. The objective of this study was to determine optimum locations to consider with minimum pressure sensors (fewer than 5), without altering the accuracy of step detection.

MÉTHODES - METHODS

Participants were asked to complete a 6-minute Walking Test at self-selected comfortable speed in outdoor setting while wearing an instrumented insole in one of their shoes. This insole, which is equipped with five pressure sensors (FSRs) located mainly under the heel (FSRH), the first (FSRM1), third (FSRM3), and fifth (FSRM5) metatarsal bones, and the great toe (FSRT), was developed and tested in a previous experiment. The step count algorithm used in the present study was also previously developed, based on cumulative sum of pressure signals, and tested with the sensor-equipped insole.

RÉSULTATS - RESULTS

Results showed that there was no statistically significant difference (F=0.895, p=0.423) between the average step count accuracy of combinations from two-to-four pressure signals. The best accuracies per sensor combination were as follows: FSRM1-FSRM5 (99.0±0.9%), FSRM3-FSRM5-FSRT (99.3±0.7%), and FSRH-FSRM3-FSRM5-FSRT (99.5±0.4%).

CONCLUSION

An insole imbedding two pressure sensors located under the first and fifth metatarsal bones enabled to yield a high accuracy in step detection. Therefore, a minimum of two integrated pressure sensors in an insole can yield a high accuracy of step detection. In future research, this combination of two pressure sensors should be validated in people with walking limitations because walking patterns could impact on results.

Poster TH1-24

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

PELAEZ MARI-CARMEN,  Paul Dutchak, Chantelle Sephton

CERVO Research Centre


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.

Poster TH1-25

Role of Polo-like kinase 2 on the processing of the Amyloid precursor protein in Alzheimer’s disease

PELLEGRINATO Rémi, MARTINEZ-DRUDIS Laura, SHETA Razan and OUESLATI Abid


Objective: Alzheimer’s disease (AD) is the major cause of dementia in the aged population, and is characterized by the accumulation of proteinaceous aggregates: amyloid plaques, composed of amyloid beta (Aβ) deposits that result from amyloid precursor protein (APP) cleavage. In vitro and in vivo evidence suggest that abnormal phosphorylation of APP plays a key role in APP processing, and subsequent Aβ formation and aggregation. A recent study demonstrated significant accumulation of Polo-like kinases (Plks), including Plk2, in AD patients’ brains. Collectively, the identification of Polo-like kinases (Plks) accumulation in AD patient brains, particularly Plk2, and recent studies demonstrating a possible direct interaction between Plk2 and APP further support a AD pathogenic role of Plk2. Therefore, our objective is to examine, in vitro, the role of APP phosphorylation by Plk2 on APP processing.

Methods: To this aim, we evaluated the impact of Plk2 on APP protein levels in a cell-based assay by using different molecular, and biochemical approaches.

Results: We observed a Plk2 dose-dependent threonine 743 phosphorylation (pT743) increase of APP correlated with a significant decrease in total APP levels. Interestingly, when using a list of inhibitors against specific degradation pathways, we did not observe variations in APP protein levels. Collectively, our results suggest that Plk2 activity modulates APP protein levels and processing, therefore possibly affecting its accumulation.

Conclusion: Findings from this study will help in shedding more light on the role of Plk2 in AD pathogenesis, thus offering the opportunity to develop new therapeutic strategies for the treatment of AD and related dementia.

Poster TH1-26

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

POIRIER, SARAH-ÈVE, Fossard, Marion et Monetta, Laura

Centre de recherche CERVO


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.

Poster TH1-27

Effets d'anticorps ciblant la protéine TDP-43 chez des souris modèles de la sclérose latérale amyotrophique induite par l'infusion de liquide cérébro-spinal

POULIN-BRIÈRE, AMÉLIE; Pozzi, Silvia; Julien, Jean-Pierre
Centre de recherche CERVO


OBJECTIF - OBJECTIVE

La sclérose latérale amyotrophique (SLA) est une maladie neurodégénérative contre laquelle il n’existe actuellement aucun traitement. De nombreux travaux suggèrent un rôle de la protéine TDP-43. Mutée dans la majorité des cas de SLA, elle est délocalisée vers le cytoplasme et y forme des agrégats impliqués dans la dégénérescence des motoneurones (MN). TDP-43 interagit avec p65 (sous-unité de NFκB) induisant l’activation des cellules gliales. Notre laboratoire a montré que l’anticorps E6 dirigé contre le domaine RRM1 de TDP-43 réduit sa délocalisation dans des souris modèles de la SLA et que le liquide céphalo-rachidien (LCR) de patients SLA induit la délocalisation de TDP-43 dans les neurones. Ce projet vise à étudier l’effet de l’anticorps E6 sur l’agrégation cytoplasmique TDP-43 et l’activité de NFκB sur des cellules en culture exposées au LCR humain, ainsi que sur les performances motrices et cognitives, et sur la pathologie des tissus de souris modèles de la SLA induite par infusion chronique de LCR humain.

MÉTHODES - METHODS

L’anticorps E6 est testé sur des modèles cellulaires et murins exposés au LCR humain. In vitro, l’activité de NFkB est analysée sur des cellules transfectées avec le site de liaison à p65 en amont du gène de la luciférase. La délocalisation de TDP-43 est analysée sur des cellules transfectées avec TDP-43 humaine (TDP-43-WT) par immunofluorescence et par western blot dans les fractions cytoplasmique et nucléaire. In vivo, des souris surexprimant TDP-43-WT traitées avec E6 par injection intrathécale sont soumises à des tests moteurs et cognitifs. L’effet de E6 sur la neuroinflammation et la délocalisation de TDP-43 est analysé par biochimie et immunofluorescence sur les tissus prélevés.

RÉSULTATS - RESULTS

Un traitement de 24h avec l’anticorps E6 tend à réduire la délocalisation de TDP-43 ainsi que l’activité de NFkB de cellules NSC-34 induite par l’exposition au LCR humain. Un pré-traitement et un traitement de 6h avec E6 ne semblent pas avoir d’effet sur l’activité de NFkB de cellules NSC-34. Un pré-traitement avec E6 augmente significativement l’activité de NFkB des cellules BV2, mais ne montre pas d’effet avec un traitement de 24h ou de 6h.

CONCLUSION

Sur base de nos résultats in vitro, nous concluons que E6 réduit l’activation de p65 et la délocalisation de TDP-43 dans les neurones, mais pas l’activation des cellules microgliales. Nous planifions étudier l’effet de E6 in vivo, et nous attendons à observer une réduction de la délocalisation de TDP-43 corrélée à l’amélioration des performances motrices et cognitives.

Poster TH1-28

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

RODRIGUEZ-ALLER RAQUEL1,2,3, malvaut sarah3,4, bérard morgan1,2, sheta razan1,2, saghatelyan armen3,4, oueslati abid1,2

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

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.

Poster TH1-29

Influence de Tau sur la plasticité visuelle adulte

RODRIGUEZ LÉA, Joly Sandrine, Mdzomba Julius Baya, Pernet Vincent

Centre de recherche du CHUQ-UL, Département d'ophtalmologie, Faculté de médecine, Québec, QC, Canada


OBJECTIF - Tau, protéine stabilisatrice des microtubules, est principalement associée à la maladie d’Alzheimer. En conditions physiologiques, Tau pourrait contribuer à la stabilisation des circuits neuronaux dans le système visuel et limiter sa plasticité. Le but de cette étude était d'étudier les effets de la délétion de Tau sur la plasticité visuelle de la souris.

MÉTHODES - Les isoformes 3R et 4R de Tau ont été analysés par immunobuvardage dans des lysats protéiques de rétines et de cortex visuels (CV) de souris âgées de 1 à 360 jours. La plasticité visuelle a été induite par privation monoculaire (PM) en suturant les paupières droites de souris sauvages (WT) et Tau knock-out (Tau KO), à 3 et 20 mois. Après 5 jours de PM, l'expression des isoformes de Tau, d’Erk, marqueur de plasticité visuelle et de tubuline acétylée, marqueur d'instabilité des microtubules, ont été suivies dans le CV. La fonction visuelle a été évaluée avec des enregistrements électrorétinographiques (ERG) et le réflexe optocinétique (OKR). Les projections rétiniennes ont été observées dans le cerveau grâce à des injections intravitréennes de traceur antérograde. La survie des cellules ganglionnaires rétiniennes (CGR) a été quantifiée sur des rétines étalées.

RÉSULTATS - La réduction de Tau 3R et l’augmentation de Tau 4R dans la rétine et le CV coïncidaient avec une baisse de plasticité neuronale à la fin de la période critique. Les ERG et la survie des CGR n'ont montré aucune différence entre WT et Tau KO au cours du vieillissement. Après PM, l’expression de Tau 3R et 4R augmentait dans le CV de l'œil privé, suggérant une implication de Tau dans la plasticité visuelle. L'augmentation de l'OKR induite par PM était statistiquement plus élevée chez les Tau KO, suggérant que Tau limite l'activation de la plasticité neuronale dans le CV. Dans le cortex des Tau KO, l’augmentation de P.Erk et la réduction de tubuline acétylée contribueraient à la réorganisation des connections synaptiques dans les circuits visuels.

CONCLUSION -  Nos résultats dévoilent une nouvelle fonction importante de Tau dans les mécanismes de plasticité dépendante de l’expérience visuelle adultes. Sa modulation représenterait une piste de recherche novatrice pour soigner les maladies visuelles.

Poster TH1-30

Disease associated microglia profiles and deregulation of immunity in ALS.

ROMINA BARRETO-NÚÑEZ1, Louis-Charles Béland1, Hejer Boutej1, Pierre Cordeau1 and Jasna Kriz1.

1CERVO Brain Research Centre and Department of Psychiatry and Neuroscience, Faculty of Medicine, Laval University, Québec, QC G1J2G3, Canada.

 


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.

 

Poster TH1-31

Le modèle organisationnel des ganglions de la base revisité par l’étude neuronale unitaire des efférences du striatum : Considérations méthodologiques

ROULEAU REDURON, LAETITIA, & Parent, Martin. Centre de recherche CERVO & Département de psychiatrie et neurosciences, Faculté de médecine, Université Laval.


OBJECTIF Le modèle actuel des ganglions de la base (GB) ségrègue les efférences striatales (STR) en voies directe et indirecte. Les neurones de projection du STR de la voie directe expriment D1 et co-libèrent avec le GABA les peptides substance P (SP) et dynorphine (DYN). Ceux de la voie indirecte expriment D2 et co-libèrent de l’enképhaline (ENK). L’objectif est de revisiter le modèle organisationnel des GB chez la souris normale et parkinsonienne (MP) par l’étude neuronale unitaire morphologique et neurochimique des efférences du STR. Nous posons l’hypothèse qu’un neurone puisse avoir un axone fortement collatéralisé et co-libérer ses peptides dans différentes structures, représentant un substrat important pour des changements neuroadaptatifs dans la MP.

MÉTHODES Un adénovirus associé (AAV) cre-dépendant est injecté dans le STR de souris D1-cre et D2-cre. Certaines sont lésées à la 6-OHDA pour induire une déafférentation dopaminergique du STR caractéristique de la MP. La fluorescence émise par les neurones D1 ou D2 infectés permet de les visualiser en microscopie confocale. Un marquage immunohistochimique pour la SP, la DYN ou l’ENK avec des reconstructions axonales unitaires complètes permet de caractériser le niveau de collatéralisation et de distribution des peptides dans les axones des neurones infectés.

RÉSULTATS Le travail effectué jusqu’à présent a permis d’identifier deux AAV produisant un marquage puissant des neurones infectés. Toutefois, deux techniques chirurgicales sont en essai afin d’obtenir un marquage tout aussi puissant, mais sur moins de neurones. Une consiste à injecter par pression en insérant un tampon d’huile avant et après l’AAV, réduisant ainsi l’infection indésirable de neurones cre du cortex cérébral lors de la descente de la pipette. L’autre consiste à injecter par microiontophorèse, permettant l’injection de volumes très faibles d’AAV et l’infection d’un nombre restreint de neurones.

CONCLUSION L’optimisation du protocole par un choix judicieux d’AAV combiné à des techniques d’injection intracérébrale améliorées permettra de marquer entièrement et fortement un faible nombre de neurones D1 ou D2 du STR. Ces améliorations chirurgicales sont essentielles à l’étude des projections efférentes du STR. Les prochaines étapes concernent la validation de ces AAV et des techniques de chirurgie et le marquage immunohistochimique de ces neurones afin de poursuivre l’élaboration d’un nouveau modèle des GB en condition normale et pathologique.

Poster TH1-32

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

SAIDI LYDIA, Proulx Christophe, Parent Martin*

CERVO Brain Research Center


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.

Poster TH1-33

Implication de la protéine Tau dans la maladie de Huntington

SALEM SHIREEN1,2, Alpaugh Melanie1,3, Bousset Luc4, de Rus Jacquet Aurélie1,3, Melki Ronald4, Cicchetti Francesca1,2,3
1Centre de Recherche du CHU de Québec, Axe Neurosciences, Québec, QC, Canada; 2Département de Médecine Moléculaire, Université Laval, Québec, QC, Canada; 3Département de Psychiatrie & Neurosciences, Université Laval, Québec, QC, Canada ; 4CEA, Institut François Jacob (MIRcen) and CNRS, Laboratory of Neurodegenerative Diseases, Fontenay-aux-Roses, France


INTRODUCTION. Il a été démontré que la protéine Tau, impliquée dans la stabilisation des microtubules, se présente sous formes toxiques dans plusieurs maladies neurodégénératives dont la maladie de Huntington (MH). La mutation génétique à l'origine de la MH – expansion du nombre de répétitions polyglutamines CAG – engendre l'expression de la protéine huntingtine mutée (mHTT) également toxique. La MH est donc caractérisée par la présence pathologique de Tau et mHTT.

OBJECTIF. Le but du projet est d’élucider le lien entre Tau et mHTT. Une partie in vitro permettra d’étudier l’impact de Tau sur les dysfonctions cellulaires, et une partie in vivo servira à investiguer le rôle de Tau sur la cognition.

MÉTHODES. Deux modèles cellulaires sont étudiés : a) des précurseurs neuronaux de souris, les cellules StHdhQ7/7 (phénotype normal) et StHdhQ111/111 (phénotype MH) contenant respectivement 7 et 111 CAG ; et b) des cellules humaines de neuroblastome SH-SY5Y que l’on transfecte avec des plasmides HTT-Exon1 contenant 16 (phénotype normal) ou 86 (phénotype MH) CAG. Elles ont été triées par FACS selon l’intensité d’expression du plasmide. Les cellules sont exposées à des monomères et fibrilles de Tau. La cytotoxicité de Tau a été étudiée par le test MTT. Des expériences de filter-trap ont permis de mesurer le niveau d’agrégation de mHTT suite au traitement. 17 souris sauvages et 19 zQ175 (modèle MH) ont été injectées à 3 mois avec des formes de Tau. Différents tests comportementaux sont réalisés à différents temps afin d’évaluer la cognition.

RÉSULTATS. A faible dose les fibrilles Tau et non les monomères sont toxiques pour les cellules StHdh. Les formes 3R-Tau, monomères et fibrilles, induisent une agrégation de mHTT dans les StHdhQ111/111. Concernant les formes 4R-Tau, ce sont les fibrilles 1N4R qui entrainent une agrégation de mHTT dans les StHdhQ111/111. Les souris sauvages et zQ175 ne présentent pas de déficits comportementaux – moteurs, cognitifs et anxieux – à 2 mois d’âge (avant injection). Le traitement Tau n’entraine pas d’aggravation des déficits à 5 mois d’âge (2 mois post-injection).

CONCLUSION.Tau semble altérer les aspects cellulaires de la MH, notamment en réduisant les activités métaboliques et augmentant l’agrégation de mHTT. Cette étude rassemblant les aspects moléculaires et cliniques de la pathologie permettra d’apporter des réponses cruciales et innovantes concernant le rôle de Tau dans la MH.

Poster TH1-34

Novel Biomarkers for the Diagnosis of Prodromal Parkinson’s Disease

SOTO LINAN VICTORIA, C. Gora, P. Ruzza, N. Dupré, F. Raymond, M. Hébert, and M. Lévesque

CERVO Brain Research Center

CHU-UL Research Center 

ICB, CNR of Padova


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.

Poster TH1-35

Unveiling the role of the endolysosomal system in the pathogenesis of Parkinson’s disease

TEIXEIRA MAXIME, Bérard Morgan, Sheta Razan, St-Pierre Marie-Kim, Tremblay Marie-Ève, Vallières Luc, Oueslati Abid.

CHU de Quebec Research Center, Axe Neurosciences. Department of Molecular Medicine, Faculty of medicine, Université Laval, Quebec, Canada.


OBJECTIVE

While progress has been made in understanding the neurodegenerative mechanisms that lead to cell death in Parkinson’s disease (PD), early causal pathogenic events are not clear. Converging findings point at endolysosomal system dysfunction as the early mechanism and key pathway affected in PD. However, the exact mechanism by which alpha-synuclein aggregates, also called Lewy Bodies (LBs), disrupt the endolysosomal system remain elusive. To answer this question, our group created a new optogenetic-based model of PD that allows for the real-time induction of α-syn aggregates, under the blue light control, that mimics all cardinal LBs features. This system is referred to as Light-Inducible Protein Aggregation (LIPA) and allows us to explore unsolved questions related to early interactions between the endolysosomal system, LBs and PD pathogenesis.

METHODS

Using the LIPA system inside living cells, we were able to study the direct impact of our aggregates on vesicle homeostasis. To specifically investigate the interactions between our LIPA aggregates and the endolysosomal system, we decided to use the super-resolution microscopy STED in combination with transmission electron microscopy (TEM) to cross-confirm the results observed.

RESULTS

The combination of immunocytochemistry and the super-resolution microscopy STED offered us a better understanding of the interactions between LBs and trafficking vesicles while showing several different. Our LIPA aggregates showed ultrastructural features of authentic LBs, showing interactions with vesicles such as the early/late endosomes (EEA-1/Rab7), exosomes (CD9) but also the degradation vesicles (LAMP1/2A) caracterized by multiple co-localization with those markers. Those results where confirmed using TEM on our LBs, showing that they are composed of multiple vesicles and also shown distorted organelles such as mitochondria, Golgi apparatus or reticulum endoplasmic.

CONCLUSION

Those results allowed us to observe and to better understand how alpha-synuclein aggregates impact the trafficking of the vesicles inside the cell and how those aggregates where managed in the cell overtime.

 

Poster TH1-36

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

ANWER MEHWISH1, Alpaugh Melanie1, Cicchetti Francesca1

1Centre de Recherche du CHU de Québec, Axe Neurosciences, Universite Laval, Québec, Canada


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.  

Poster TH1-37

Enhancing the potassium chloride co-transporter KCC2 reverses functional deficits associated with Alzheimer’s disease-related mutations in mice

KERAMIDIS IASON1, McAllister Brendan2, Mehla Jorgender3, Godin G Antoine1, Mohajerani Majid2, De Koninck Yves1

1. CERVO Brain Research Center, Quebec Mental Health Institute, Quebec City, Qc, Canada 

2. Canadian Centre for Behavioural Neuroscience, University of Lethbridge, Lethbridge, AB, Canada

3. Department of Neurosurgery, Washington University School of Medicine, St. Louis, Missouri, USA


OBJECTIF - OBJECTIVE

Growing evidence indicates that during early stages of Alzheimer’s disease (AD) an abnormal brain activity appears due to disruption of GABAA-mediated transmission. While disrupted GABAA signaling may result from several mechanisms, recent evidence points to deficits in the potassium-chloride cotransporter KCC2, responsible for maintaining low intracellular chloride in neurons to maintain robust inhibition. In this study, we validate whether KCC2 is downregulated in two transgenic lines that accumulate AD-like symptoms and whether by restoring KCC2 function we can alleviate deficits associated with AD

MÉTHODES - METHODS

The 5xFAD transgenic and APPNL-G-F knock-in lines are used in this study. Immunofluorescence analysis is conducted in layer II/III of the medial prefrontal cortex (mPFC) to assess the total and membrane KCC2 protein levels. The network activity of the CA1 pyramidal layer is recorded with a chronic electrode bundle. The memory and cognitive deficits are measured with the Morris Water Maze, Fear Conditioning and Elevated Plus Maze tests

RÉSULTATS - RESULTS

We found a decrease in total and membrane protein levels of KCC2 in 4-month-old 5xFAD mPFC. In addition, the hippocampal gamma oscillation power was decreased in 9-month-old APPNL-G-F mice, consistent with predictions from deficits in KCC2. Treatment with CLP290, a KCC2 activity enhancer, restored the gamma oscillation power. Acute administration of CLP290 in APPNL-G-F mice also improved memory performance in the Morris Water Maze test the Contextual Fear Conditioning test

CONCLUSION

These results indicate that KCC2 may be a viable target for reversing deficits in GABAA-mediated inhibition in AD and attenuating several symptoms associated with AD pathology

Poster TH1-38

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

DESMEULES FRANCIS, Munro Jonathan, 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 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.

Poster TH2-01

Effet de la présence d’un conflit sensorimoteur sur la performance motrice et l’activité cérébrale chez des personnes atteintes de fibromyalgie

AUGIERE TANIA, Brun, C., Pinard, A. M., Blouin, J., Mouchnino, L., Simoneau, M., Mercier, C., Centre interdisciplinaire de recherche en réadaptation et insertion sociale (CIRRIS)


La fibromyalgie est une pathologie musculosquelettique caractérisée par une douleur constante et diffuse. Plusieurs études rapportent des altérations de l’intégration sensorimotrice chez les personnes souffrant de cette pathologie mais, à notre connaissance, peu d’études se sont penchées sur l’influence de ces altérations sur le contrôle et la planification du mouvement, et aucune au niveau cérébral. Les tâches de conflit sensorimoteur (i.e. dans lesquelles les informations sensorielles et motrices sont non congruentes) permettent d’étudier l’intégration sensorimotrice lors de la réalisation d’un mouvement. OBJECTIF Dans une tâche visuomotrice nouvelle, nos objectifs sont d’établir l’effet de la présence de conflit, par rapport à l’absence de conflit, 1) sur la performance motrice et 2) sur l’activité cérébrale, chez des personnes atteintes de fibromyalgie comparées à des individus sains. MÉTHODES Les participants traceront le contour de figures géométriques en ne voyant pas leur main directement. Les informations visuelles fournies seront soit congruentes avec les informations proprioceptives et motrices (condition Sans conflit), soit non congruentes (condition Conflit). L’activité cérébrale sera mesurée avec l’électroencéphalographie et exprimée en changement de synchronisation et de désynchronisation. Cette étude prédictive quasi-expérimentale inclura 20 participants atteints de fibromyalgie et 20 participants sains appariés selon l’âge, le sexe et la latéralité manuelle. Des analyses de variance mixte permettront de comparer l’effet des facteurs sur la performance motrice et des tests de permutation permettront de conclure sur leurs effets sur l’activité cérébrale.

 

Poster TH2-02

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

CYR JEAN-PHILIPPE1,2,

Mercier Pierre3,

Pialasse Jean-Philippe1,

Simoneau Martin 1,2

 

1 Faculté de médecine, Département de kinésiologie, Université Laval, Québec, Canada

2 Centre interdisciplinaire de recherche en réadaptation et intégration sociale (Cirris) du CIUSSS de la Capitale Nationale, Québec, Canada

3 Clinique d’orthopédie pédiatrique de Québec, Québec, Canada


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.

 

Poster TH2-03

Un vêtement instrumenté pour évaluer les altérations du contrôle moteur et de la fatigue musculaire précoce chez le lombalgique chronique : protocole.

Frasie Antoine1,2, Pagé Isabelle2,3, Roy Jean-Sébastien1,2, Blanchette Andréanne K1,2, Massé-Alarie Hugo1,2, Bouyer Laurent1,2.

1Département de rééducation, Faculté de médecine, Université Laval, Québec ville, Québec, Canada;

2Centre interdisciplinaire de recherche en réadaptation et intégration sociale (Cirris), Québec ville, Québec, Canada;

3Département de chiropratique, Université du Québec à Trois-Rivières, Trois-Rivières, Québec, Canada.


CONTEXTE : La lombalgie chronique est la première cause des troubles musculosquelettiques liées au travail. Ce trouble est associé à des changements biomécaniques du rachis et des altérations du contrôle moteur. Ces perturbations et la présence de douleur peuvent engendrer secondairement de la fatigue précoce des muscles érecteurs du rachis. La fatigue musculaire correspond à la diminution de capacité à générer une force ou une puissance. Elle peut être mesurée de façon indirecte à l’aide d’électromyogrammes de surface (sEMG) et la chute de fréquence médiane du spectre de puissance (CFM). Plusieurs études sur ce sujet ont été effectué en laboratoire. Les nouveaux outils miniatures, sans fils tels que les sEMG pourraient permettre des mesures continues en condition réelle.

OBJECTIF : Évaluer la validité d’un nouveau type d’électrodes, intégrables dans un vêtement, lors de la mesure de l’activité des muscles érecteurs du rachis lors de tâches de flexion-relaxation, de force et d’endurance.

METHODE : Trente sujets sains vont être recrutés. Deux paires de fibres vont être placées bilatéralement sur les muscles érecteurs du rachis (T12-L1 et L4-L5), en parallèle avec des capteurs commerciaux (Delsys Trigno). Le test de flexion-relaxation consiste, debout, à se pencher vers l’avant puis se redresser. En flexion totale, les muscles spinaux sont relâchés en condition normale. L’endurance sera évaluée avec le test de Biering – Sorensen. Pour ce test, le participant est positionné sur une table de traitement en décubitus ventral, le tronc sans appui sur celle-ci. Le participant doit maintenir le tronc parallèle au sol contre gravité jusqu’à épuisement. La mesure de force (avant, pendant la tâche d’endurance, après) sera faite à l’aide d’une jauge de force fixée au sol et reliée au sujet grâce à un harnais. La fatigue sera mesurée directement par la jauge de force et indirectement par les sEMG.

RÉSULTATS ATTENDUS :  le nouveau type d’électrodes permettra une mesure de l’activité EMG (amplitude et CFM) avec une bonne corrélation par rapport aux systèmes de mesures de référence (ICC>0.75).

CONCLUSION : des électrodes nouvelle génération, de bas coût, intégrable à un vêtement ont le potentiel de mesurer l’activité EMG (contraction/relaxation; fatigue) des muscles érecteurs du rachis. Cela ouvre de nouvelles possibilités de mesures en condition réelle afin de mieux comprendre les conséquences sensorimotrices de la douleur rachidienne chronique.

Poster TH2-04

Expression of Acid-sensing ion channels in primary sensory neurons

PAPALAMPROPOULOU-TSIRIDOU MELINA, Wang Feng, De Koninck Yves


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.

 

Poster TH2-05

L’EFFET DE LA DOULEUR EXPÉRIMENTALE SUR L’EXCITABILITÉ CORTICOSPINALE CHEZ L’HUMAIN : UNE REVUE SYSTÉMATIQUE AVEC MÉTA-ANALYSE.

ROHEL ANTOINEb, Bouffard Jasonb, Patricio Philippeb, Mavromatis Nicolasb, Billot Maximeb, Roy Jean-Sébastiena,b, Bouyer Laurenta,b, Mercier Catherinea,b, Masse-Alarie Hugoa,b,*

a Département de réadaptation, Faculté de médecine, Université Laval, Quebec, Quebec, Canada

b Centre interdisciplinaire de recherche en réadaptation et intégration sociale, Quebec, Quebec, Canada


OBJECTIF - OBJECTIVE

La douleur influence le contrôle moteur, en partie, par son effet inhibiteur sur l’excitabilité des projections corticospinales qui est testée par la stimulation magnétique transcrânienne. Cependant l’influence des différents facteurs tels que le type de douleur, sa localisation ou le tissu visé restent peu explorée. L’objectif de cette revue était de déterminer l’influence de la douleur expérimentale et de ces facteurs sur l’excitabilité corticospinale. 

MÉTHODES - METHODS

Trois bases de données ont été utilisées (Embase, Pubmed, Web of Science). Des méta analyses ont été réalisées selon trois niveaux pour réduire la variabilité: (1) toutes les études; (2) même localisation de la douleur; (3) même tissu visé, localisation et état musculaire (repos vs. activité). Le niveau de preuve a été évalué pour chaque analyse. 

RÉSULTATS - RESULTS

40 études ont été incluses dans la revue et 26 dans la méta-analyse. En général, une diminution de l’excitabilité corticospinale a été observée pendant et après la douleur tonique (preuves conflictuelles/modérées). En considérant la même localisation de la douleur, seules les douleurs à la main et au visage induisaient une diminution de l’excitabilité corticospinale (preuves limitées). Les douleurs cutanées et musculaires induisaient également une réduction d’excitabilité (preuves limitées/conflictuelles). Des résultats similaires ont été observés pour la douleur phasique (preuves limitées). 

CONCLUSION

Nos résultats confirment l’effet inhibiteur de la douleur sur l’excitabilité corticospinale pour la douleur phasique et tonique. Cette réduction était spécifique à la main et au visage. Un effet similaire était retrouvé pour les douleurs musculaires et cutanées. Le niveau de preuves reste tout de même limité/conflictuel à cause de l’hétérogénéité des méthodes/résultats,  les risques de biais et le faible nombre de participants. Plus d’études de bonne qualité sont nécessaires pour confirmer nos résultats.

Poster TH2-06

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

MARIE ROUSSEL, Frédéric Bretzner.


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.

 

Poster TH3-01

EXPLORATION DU POTENTIEL PRO-ANGIOGENIQUE DES PERICYTES DANS LA REPARATION DE L’UNITE NEUROVASCULAIRE SUITE AUX ACCIDENTS VASCULAIRES CEREBRAUX

BERNARD MAXIME, ElAli Ayman, Axe neurosciences, CHU centre de recherche de Québec (CHUL)


CONTEXTE: Les accidents vasculaires cérébraux (AVC) constituent une cause majeure de décès et de handicap au Canada. Au cours des dernières années, des recherches ont été effectuées afin de trouver de nouvelles approches thérapeutiques dont l’angiogenèse thérapeutique qui pourrait être promu par les péricytes, et contribuent à la stabilité de l’unité neurovasculaire (UNV). De plus, les péricytes possèdent une grande plasticité et seraient capables de se différencier en cellules de différentes lignées en conditions pathologiques. Du fait de ces propriétés, les péricytes seraient impliqués dans des mécanismes de remodelage de l’UNV après un AVC et constituent une cible intéressante afin de développer de nouvelles interventions thérapeutiques.

OBJECTIFS: Mes objectifs sont d’étudier le rôle de PDGF-D qui est impliqué après un AVC dans les mécanismes de survie et d’angiogenèse, de comprendre les mécanismes amenant à la reprogrammation des péricytes dans le but de promouvoir l’angiogenèse, et transplanter des péricytes afin de restaurer l’ensemble des fonctions de l’UNV.

MÉTHODES: Une culture primaire de péricytes humains à été utilisée. Une hybridation in situ a été réalisé afin de caractériser l’expression de PDGF-D. Un AVC a été induit chez la souris via l'occlusion transitoire de l'artère cérébrale moyenne, et des péricytes ont été transplantées dans le cerveau via la voie intranasale. RESULTATS: Un AVC induit l’expression de Kruppel-like factor-4 (KLF4), impliqué dans la reprogrammation des cellules pluripotentes. Les péricytes reprogrammés exposés à un milieu endothéliale conditionné développent des traits endothéliaux. Les cellules transplantées migrent vers le site de la lésion en utilisant la vasculature comme échafaudage. Leur recrutement est accompagné par une régression de la lésion et une diminution des déficits neurologiques. CONCLUSION: La transplantation des péricytes semble renforcer le système vasculaire au niveau du site la lésion grâce à une angiogenèse contrôlée. Les péricytes contribuent activement à la réparation de l’intégrité du tissu cérébral après un AVC. L’ensemble des résultats suggèrent ainsi que les péricytes constitue un nouvel outil pour le développement de nouvelles thérapies dans le cadre des AVC.

Poster TH3-02

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

BILODEAU ANTHONY, V.L. Delmas Constantin, Parent Martin, De Koninck Paul, Durand Audrey, Lavoie-Cardinal Flavie - Centre de recherche CERVO


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.

Poster TH3-03

Task Guided Conditional GAN for Resolution Enhancement in Fluorescence Microscopy

BOUCHARD, Catherine, Wiesner, T., Deschênes, A., Gagné, C., Lavoie-Cardinal, F


OBJECTIVE

Understanding the dynamics of molecular processes in living neurons is highly dependent on the possibility to visualize them. In super-resoltuion microscopy, acquiring multiple images of the same region of interest in living cells is challenging due to phototoxicity and photobleaching effects. The project aims at developing a method where super-resolved information is extracted from diffraction-limited confocal acquisitions thereby limiting the invasiveness of the approach while maximizing the spatiotemporal resolution.

 

METHODS

A generative adversarial network (GAN) is used to transform a confocal image into its stimulated emission depletion (STED) counterpart. To compel the generator to not only translate the confocal features into realistic STED features but also to generate accurately the sub-diffraction details of interest, an additional network is trained parallely to the generator. This network uses the real STED images to learn a task that is impossible to perform on confocal images, like the segmentation of F-actin rings, and applies corrections on the generator to make this task solvable on the synthetic generations. Once trained, the network is included directly in the imaging workflow to generate a synthetic STED from a confocal acquisition. In addition, stochasticity included in the generator informs about which regions are worth imaging in super-resolution to gain the most information at a minimal imaging cost; all in real-time and without external user intervention.

 

RESULTS

The automated approach reduces photobleaching enough to enable the visualisation of the dynamic remodelling of F-actin rings into elongated fibers in living cultured hippocampal neurons. With this technique, the nanoscale organization of F-actin can be monitored for more than 30 minutes, which was impossible using a standard STED imaging protocol. It enables for the first time to observe the dynamic remodelling of F-actin in living neurons that had been documented recently in fixed cells only. 

 

CONCLUSION

The method developed shows promising results for F-actin in live cells, and is not limited to this specific application; it can be applied to an extensive number of tasks and modalities. The method improves super-resolution systems by limiting the undesirable effects, like photo-bleaching and photo-toxicity, without compromising on the quality of the information acquired.

Poster TH3-04

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

DELMAS CONSTANTIN 1, Savage Julie 2, Landry Marc 3, Tremblay Marie-Ève 2 and Parent Martin 1 *

1 CERVO Brain Research Centre, Quebec City, Canada

2 CHU de Québec, Quebec City, Canada

3 IINS, Université de Bordeaux, UMR CNRS 5297, Bordeaux, France


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.

Poster TH3-05

Classification of super-resolution images of synaptic proteins according to their spatial orientation

DESCHÊNES, ANDRÉANNE1, Bouchard,Catherine1, Michaud-Gagnon, Albert1, Wiesner, Theresa1, Lavoie-Cardinal, Flavie1,2

1CERVO Brain research centre

2Département de psychiatrie et neurosciences, Faculté de médecine, Université Laval


OBJECTIF - OBJECTIVE

Super-resolution (SR) optical microscopy allows us to observe the molecular dynamics and interactions of synaptic proteins at their scale : the nanoscale. The evaluation of synaptic organization, such as the measurement of the distance between pre- and post-synaptic proteins, depends on the orientation of the synapses in reference to the imaging plane. In particular, it would be useful to automatically identify the orientation of associated synaptic protein clusters in the SR microscopy images. Typically, this classification must be done manually. The goal of this project is to develop an automatic synapse detection procedure that can classify the synapse orientation in SR microscopy images of cultured neurons.

MÉTHODES - METHODS

STED (Stimulated Emission Depletion) microscopy of pre- and post-synaptic proteins was performed in hippocampal neuronal cultures for different stimulation paradigms. The images were pre-processed using Statistical Object Distance Analysis (pySODA) to characterize the morphology and coupling (spatial association) of the protein  clusters in the STED images. The synaptic protein pairs were manually annotated into three categories: 1) parallel, 2) perpendicular, and 3) diagonal orientation to the imaging plane.  Machine learning (ML) clustering algorithms and a convolutional neural network (CNN) were used to solve the classification task. The performance of each method was evaluated on the testing dataset and compared to the inter-expert accuracy measured on the manual labelling of two experts. The classification accuracy of the automatic methods was also compared to a naive but prevalently used analysis where a single user-selected morphological feature is used to set a cutoff between the classes.

RÉSULTATS - RESULTS

The ML clustering and CNN outperformed all the methods against which they were compared with classification accuracies of 78.8% for the CNN, 73.9% for the best ML clustering approach, and 72.8% for the user-study.

CONCLUSION

The presented tool will enable automatic selection of synapses that are in a precise orientation for super-resolution image analysis of synaptic proteins. In the future, this tool will be useful to study the nanometric reorganization of proteins during synaptic potentiation.

Poster TH3-06

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

DESMONS MIKAËL1,2, Rohel Antoine1,2, Desgagnés Amélie1,2, Mercier Catherine1,2, Massé-Alarie Hugo1,2.

1Centre interdisciplinaire de recherche en réadaptation et intégration (CIRRIS); 2Département de réadaptation, Université Laval.


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.

Poster TH3-07

Clustering dendritic spines structural properties with machine learning after odour stimulation and learning.

FERREIRA AYMERIC1,2, Constantinescu Vlad1,3, Malvaut Sarah1,3, Saghatelyan Armen1,3, Hardy Simon1,2,4

1CERVO Brain Research Center, Quebec City, QC G1J 2G3, Canada

2Department of biochemistry, microbiology and bio-informatics, Faculté des sciences et de génie, pavillon Alexandre-Vachon 1045, av. de la Médecine, local 3261, Québec (Québec), G1V 0A6, Canada

3Department of Psychiatry and Neuroscience, Université Laval, Quebec City, QC G1V 0A6, Canada

4Department of Computer Science and Software Engineering, Université Laval, Pavillon Adrien-Pouliot, 1065, av. de la Médecine, Québec, Canada, G1V 0A6


OBJECTIF - OBJECTIVE

The ability to store new information and adapt to new environments is a crucial neurobiological process underlying our everyday life. This property has been linked to modifications in the efficacy of synapses partly due to activity-dependent structural modification of dendritic spines. Moreover, neurodevelopmental disorders such as schizophrenia, autism, Fragile X and others are linked to alterations in the dendritic structure of neurons in the brain. We propose a pipeline to reconstruct dendritic spines from confocal microscopy into a 3D model, while also performing the reconstruction of spine neck, which is difficult without super-resolution microscopy. After the reconstruction, we provide a tool for further processing and analysis of morphological features.

MÉTHODES - METHODS

First, we acquired confocal, high resolution images of distal dendritic segments from adult born neurons in the olfactory bulb (OB) after odour learning using a simple and complex go/no-go odour discrimination paradigm and after sensory deprivation. The reconstruction of dendritic spines from images is done in 5 steps i) correction of image stacks via deconvolution and diffraction correction, ii) 3D segmentation using the Chan-Vese and morphologic-snake algorithm, iii) reconstruction via marching-cubes algorithm into a 3D mesh, iv) extraction of spines and measure of features and v) analysis and clustering.

Recent papers suggest that since spine morphologies are continuous, using spine clusterization using morphological measures instead of qualitative spine classification provides a more precise description. We assessed several spine features such as the surface, volume, gaussian and mean curvature, open angle, etc. These features are analyzed using dimensional reduction method such as Principal Component Analysis (PCA), t-distributed Stochastic Neighbor Embedding (t-SNE), or Uniform Manifold Approximation and Projection (UMAP). The optimal number of clusters is found based on the silhouette score and the KMeans algorithm. Once the spines are grouped into clusters, a physiological role is investigated for each cluster. To perform these tasks, we developed a tool written in Python and validated with a standard dataset (iris) but also with a real biological dataset based on cell migration.

Poster TH3-08

Electrophysiological properties of glutamatergic reticulospinal and brainstem neurons

HAILI YACINE , Josée Seigneur , Frédéric Bretzner

Centre de Recherche du CHU de Québec-Université Laval, CHUL-Neurosciences

Université Laval, Department of Psychiatry and Neurosciences


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.

Poster TH3-09

Chronic social defeat stress differently alters synaptic transmission of LHb neural outputs

HERNANDEZ SILVA JOSE CESAR, Pausic Nikola and Proulx Christophe 

Universite Laval CERVO research center

Quebec QC Canada


OBJECTIVE - OBJECTIVE

The lateral habenula, the main disappointment center of the brain, has been shown to be hyperactive in depressive disorders. However, how synaptic transmission at its neural outputs is affected in depression is not known. Here, we use optogenetics and electrophysiology to examine synaptic transmission from the LHb to three of its main output targets : the serotoninergic dorsal raphe nucleus (DRN), the rostromedial tegmental nucleus (RMTg), and the ventral tegmental area (VTA), in mice subjected to chronic social defeat stress (CSDS).

METHODS - METHODS

To selectively activate LHb efferent, an AAV-ChR2-mCherry is first injected in the LHb. Ten days later, mice are subjected to 10 days of CSDS, and tested in the social interaction test to determine their resilience or susceptibility to chronic defeat stress. Acute brain slices are obtained from control, susceptible and resilient mice and synaptic transmission is examined using whole-cell patch clamp recordings.

RESULTS - RESULTS

 At the LHb-DRN synapses, chronic stress did not change paired-pulse ratio (PPR) but increased the evoked AMPAr/NMDAr ratio in susceptible mice. At the LHb-RMTg synapses, CSDS decreased paired-pulse ratio both in susceptible and resilient mice while decreasing evoked AMPAr/NMDAr ratio in resilient mice. Finally, at the LHb-VTA synapses, CSDS decreased AMPAr/NMDAr ratio in susceptible and resilient mice while no change was observed for PPR.

CONCLUSION

Taken together, these results suggest that LHb neural outputs are differently altered following CSDS, and these synaptic changes may contribute to distinct symptoms found in depressive disorders.

 

Poster TH3-10

EFFECT OF DCC HETEROZYGEOUS MUTATION ON THE LOCOMOTOR CONTROL

LEMAIRE CHLOɹ, Lemieux Maxime¹, Rasqtar Ali¹, Ferent Julien², Bretzner Frédéric¹

¹ Centre de Recherche du CHU de Québec-Université Laval

² Montreal Clinical Research Institute (IRCM), Université de Montréal, McGill University


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.

Poster TH3-11

Lateral hypothalamic neural outputs control motivated behaviors

MARTIANOVA EKATERINA, Pageau Alicia, Doucet Gentiletti Tommy, Leblanc Danahé, Proulx Christophe D.


OBJECTIF - OBJECTIVE

The lateral hypothalamus (LH) sends neural outputs to brain regions known to control reward and motivated behaviors. Here we studied how these distinct LH neural outputs process information to control behavior.

MÉTHODES - METHODS

Using fiber photometry calcium imaging in freely moving mice, we examined the role of three major LH neural projections targets, the lateral habenula (LHb), the ventral tegmental area (VTA), and the dorsal raphe nucleus (DRN), in motivated behavioral responses.

RÉSULTATS - RESULTS

We found a significant and coherent increase in activity at LH axon terminals in all three outputs in mice engaging motivated behaviors, which decrease during immobility. These pathways were also activated by cues predicting aversive foot shock in an active avoidance task.Optogenetic activation of individual LH neural outputs significantly increased mobility in open field and tail suspension tests, but had different effects on passive avoidance and on sucrose consumption, supporting complementary mechanisms used by LH outputs to control behavioral responses.

CONCLUSION

Together, our results support the complementary role of three LH neural outputs in controlling spontaneous motivated behaviors.

Poster TH3-12

Dickkopf-1 induction impairs short- and long-term structural and functional recovery after ischemic stroke.

Menet Romain, Bernard Maxime, Lecordier Sarah, Aldib Natija, Khajjad-Mallat Rayan and ElAli Ayman


OBJECTIF - OBJECTIVE

Les accidents vasculaires cérébraux (AVC) constituent la cause majeure de décès et d'invalidité chez les adultes dans le monde. L'AVC ischémique (AVCi), qui survient à la suite d'une obstruction soudaine de l’artère cérébrale due à une embolie ou à un thrombus, représente 87% des cas. Nous avons récemment publié un article qui montre qu'après un AVCi, la voie canonique Wnt est dérégulée. De plus, il a récemment été démontré que les taux de Dicckopf-1 (DKK1), une protéine extracellulaire sécrétée, augmente chez des patients et des modèles animaux d’AVCi. DKK1, un inhibiteur endogène de la voie canonique Wnt, empêche les ligands Wnt de se lier au complexe formé par la low-density lipoprotein receptor-related protein-5/6 (LRP5/6) et les récepteurs frizzled (Fzd). La liaison des ligands Wnt/ Fzd/ LRP5/6 stabilise la β-caténine dans le cytosol et stimule sa translocation vers le noyau pour réguler la transcription des gènes cibles impliqués dans le maintien des fonctions neurovasculaires. Le but de notre étude est d’élucider le rôle de DKK1 après un AVCi.

MÉTHODES - METHODS

Tout d’abord, nous avons réalisé la chirurgie de l’occlusion de l’artère cérébrale moyenne (MCAo) chez des souris DKK1 inductibles pour disséquer le rôle de DKK1 dans la phase subaiguë suite à un AVCi. Ensuite, nous avons neutralisé le DKK1 24 heures après AVCi chez des souris de type sauvage en utilisant le WAY262611, un inhibiteur spécifique de DKK1, pour explorer le potentiel thérapeutique de son inhibition sur la réparation neurovasculaire.

RÉSULTATS - RESULTS

L’induction systémique de DKK1 aggrave la taille de l’infarctus sans moduler l’œdème (Cresyl violet), augmente la perméabilité vasculaire (IgG) et empire les facultés sensori-motrice suite aux AVCi (Barre horizontale et Rotarod) dans la phase subaiguë. Fait très intéressant, la neutralisation pharmacologique de DKK1 améliore les fonctions sensori-motrices (Corner et Pole test) dès la première injection et réduit la taille de l’infarctus 7 jours après induction de l’AVCi. De plus, la neutralisation de DKK1 atténue la dégénérescence neuronale au niveau du cortex mais aussi du striatum et de l’hippocampe (FJB+ cells) et diminue la perméabilité vasculaire.

CONCLUSION

Prises ensemble, nos données suggèrent que DKK1 constitue une cible thérapeutique élégante pour favoriser la neurorestoration suite à un AVCi.

Poster TH3-13

AUTOMATED ONLINE MULTI-OBJECTIVE OPTIMIZATION OF SPLIT-STED SUPER-RESOLUTION MICROSCOPY UNDER A BANDIT FRAMEWORK

MICHAUD GAGNON ALBERT1, Deschênes Andréanne1 , Lavoie-Cardinal Flavie1,2, Durand Audrey3,4. 1CERVO Brain Research Center; 2Département de psychiatrie et de neurosciences, faculté de médecine, Université Laval; 3Département de génie électrique et de génie informatique, Faculté des sciences et de génie, Université Laval; 4Département d!informatique et de génie logiciel, Faculté des sciences et de génie, Université Laval.


OBJECTIVE: Super-resolution (SR) optical microscopy techniques provide the necessary spatial resolution to observe and characterize biological structures whose size is significantly lower than the resolution of conventional, diffraction-limited, optical microscopes (around 250 nm). However, a common drawback of these SR methods is that several imaging parameters need to be properly tuned in order to achieve good results with regard to image quality and sample photodamage/photobleaching. Adjusting these parameters is tedious and time-consuming, especially when multiple parameters need to be considered. The objective of this project is to implement an efficient automated method for tuning parameters in SPLIT-STED (STimulated Emission Depletion) microscopy, a super-resolution modality which improves the spatial resolution by using stacks of super-resolved images that were acquired at various excitation and depletion powers. Once implemented, the machine learning-assisted SPLIT-STED approach will be used for SR imaging of synaptic proteins in living neurons at low light levels. METHODS: Previous work introduced an online multi-objective optimization method based on Thompson sampling with Gaussian kernel (non-parametric) regression under the multi-armed bandit framework for tuning STED parameters. The current project extends this technique to the SPLIT-STED modality. Experiments were conducted on simulated data, where objective functions and noise were estimated from real image data. The considered objectives were photobleaching, spatial resolution, signal-to-noise ratio, and quality (measured automatically using nanoJ-SQUIRREL). Finally, a variant of the optimization algorithm using parametric (polynomial) regression was also investigated. RESULTS: As previously observed in STED experiments, online multi-objective optimization allowed quick convergence of the parameters and good sample efficiency, especially compared to a random grid search. In some scenarios, optimization based on a parametric model was able to improve convergence and sample efficiency. CONCLUSION: Online automated optimization under the multi-armed bandit framework with parametric regression seems a promising method for efficiently tuning the parameters in SPLIT-STED. In the future, this approach will be applied to SR microscopy of synaptic proteins in living cultured neurons and brain slices.

 

Poster TH3-14

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

SCOVRONEC, ANAÏS (1)(2), Iceta, Sylvain (2), Dadar, Mahsa (4), Pelletier, Mélissa (2), Nadeau, Mélanie (2), Richard, Denis (2), Biertho, Laurent (3), Tchernof, André (1)(2) et Michaud, Andréanne (1)(2).

(1) École de nutrition, Université Laval, Québec

(2) Centre de recherche de l’Institut universitaire de cardiologie et pneumologie de Québec, Université Laval, Québec

(3) Département de chirurgie, Institut universitaire de cardiologie et pneumologie de Québec, Université Laval, Québec

(4) Institut neurologique de Montréal, Département de Neurologie et de Neurochirurgie, Université McGill


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.

Poster TH3-15

Focus-tunable microscope for imaging small neuronal processes in freely moving animals

TABOURIN LOIC1,2, Bagramyan Arutyun1,2, Rastqar Ali2, Karimi Narges2, Bretzner Frédéric2 and Galstian Tigran1.

1 Center for Optics, Photonics and Lasers (COPL), Faculty of Science and Engineering, Department of Physics, Engineering Physics and Optics, Université Laval.

2 Centre de Recherche du CHU de Québec-Université Laval, CHUL-Neurosciences, Université Laval, Faculty of Medicine, Department of Psychiatry and Neurosciences.


INTRODUCTION

Miniature 1-photon microscopes are widely used for the imaging of the brain of freely behaving animals. However, these systems are usually limited to a cellular resolution and a fixed focal plane imaging. We developed a compact and a lightweight 1-photon device with an integrated electrical tunable liquid crystal lens to enable a motionless depth scan of subcellular structures. Our device has high acquisition rate (50 fps), high magnification (8.7 x), low aberrations, and 1.4 µm resolution.

OBJECTIF - OBJECTIVE

We propose to test this new endoscope for imaging calcium activity in fine cortical neuronal processes.

MÉTHODES - METHODS

GCaMP6s was conditionally expressed in the motor cortex of adult VGluT2-IRES-Cre mice. A gradient index (GRIN) lens was then implanted chronically above this region. After recovery, movements of the animal and calcium activity of the cortex were recorded during different motor tasks in freely behaving mice.  

RÉSULTATS - RESULTS

Using our device, calcium dynamics of various neuronal structures were correlated according to the behavior (walking and grooming). The tunable liquid crystal lens allowed us to reveal fine neuronal features, such as dendritic processes and spines.

CONCLUSION

This new device allowed us to image calcium activity cortical neurons and fine processes in freely moving animals.

Poster TH3-16

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

TING WINDSOR KWAN-CHUN, Éthier Christian

Centre de Recherche CERVO


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.

Poster TH3-17

Élaboration d’une tâche pour étudier le rôle du faisceau oblique dans la production de la parole

VILLENEUVE Marjorie, Mailloux Sophia, Joyal Marilyne, Tremblay Pascale

Département de réadaptation, Université Laval 

Centre de recherche CERVO


PROBLÉMATIQUE. Le faisceau oblique, reliant l’aire motrice pré-supplémentaire (pré-SMA) et le gyrus frontal inférieur (IFG), influencerait l'initiation et la fluidité de la parole, car il serait impliqué dans la planification/programmation des sons volontaires de la parole. La connexion entre la pré-SMA et l’IFG a toutefois été très peu étudiée et peu de données existent quant à l’amélioration de la parole lorsque ces régions sont stimulées. La stimulation magnétique transcrânienne répétitive (rTMS) permet d’augmenter ou de réduire temporairement l’excitabilité de régions spécifiques du cortex cérébral, entraînant une modulation des fonctions soutenues par ces régions. OBJECTIF. L’objectif du projet est de créer un protocole novateur de rTMS pour stimuler le faisceau oblique via la pré-SMA et l’IFG gauches et d’étudier sa contribution à la production de la parole. Ce protocole sera testé chez des personnes âgées en santé afin d’étudier comment ces régions communiquent entre elles et de déterminer s’il est possible d’améliorer l’initiation et la fluidité de la parole. MÉTHODES. Une tâche de parole assez complexe pour qu’une amélioration de la parole des participants puisse être observée et assez courte pour être effectuée lorsque les effets de la rTMS sont maximaux est en cours d’élaboration. Il s’agit d’une tâche de répétition et de manipulation syllabique de non-mots contrôlés pour plusieurs facteurs psycholinguistiques. 25 participants âgés de 50 ans et plus complèteront cette tâche à la suite d’une stimulation de la pré-SMA et/ou de l’IFG dans cinq conditions de stimulation, dont une placebo, afin d’observer les effets de la stimulation de l’une des régions lorsque l'activité de l’autre est inhibée, excitée, ou non stimulée. La stimulation se fera au moyen d'un Magstim Super Rapid2 et d’un système de neuronavigation (Brainsight TMS). Des images anatomiques IRM du cerveau de chaque participant seront acquises avant la séance de stimulation. RÉSULTATS. Dans un premier temps, les résultats à la tâche seront pilotés chez 10 personnes pour valider son utilisation dans l’étude de rTMS. CONCLUSION. Ce projet novateur fera avancer les connaissances sur le faisceau oblique et sur son potentiel de plasticité chez les personnes âgées. Ces données pourraient par la suite être utiles pour le traitement de l’apraxie de la parole, pouvant être liée à une lésion du faisceau oblique, que l’on retrouve dans certaines maladies neurodégénératives ou à la suite d’un AVC. 

 

Poster TH3-18

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

WIESNER, THERESA1, Bilodeau, Anthony1, Bernatchez, Renaud1, Deschênes, Andréanne1, Raulier, Bastian1, De Koninck, Paul1,2, Lavoie-Cardinal, Flavie1,3; 1CERVO Brain Research Center, 2Department of Biochemistry, Molecular Biology and Bioinformatics, Université Laval, 3Department of Psychiatry and Neuroscience, Université Laval, Quebec, Canada


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.

 

Poster TH3-19

Blocking CD36 in neonatal mice alters immune profile of activated microglia

Shruti Gururaj Gadagkar, Melanie Lalancette-Hébert, Sai Sampath Thammisetty, Jasna Kriz

 


Objective:

Neonatal immune response to brain injury is associated with a robust microglial activation and induction of Toll-like Receptors (TLRs). To date, the role of scavenger receptor CD36 has been studied in adults to be consorted with the TLRs, particularly TLR2, consequentially affecting microglial activities such as migration, phagocytosis and production of inflammatory mediators.

In this study, we examined the extent to which CD36 influences microglial activation and inflammatory profile in neonates.

 

Methods:

Using an anti-CD36 blocking antibody at post-natal day 8 (P8), we evaluated the response of neonates to systemic lipopolysaccharide (LPS) challenge. To assess the effects in vivo we took advantage of the transgenic mouse model bearing the dual reporter system luciferase/GFP under transcriptional control of a murine TLR2 promoter (TLR2-luc-GFP). We then proceeded to check the effect on the inflammatory pattern by western blot.

 

Results:

Our results revealed that treatment with anti-CD36-blocking antibody reduces the pro-inflammatory profile of LPS-induced activated microglia. Further, we noted that CD36 is involved in TLR2 and TLR3 mediated inflammation, as TLR4-IRF3 pathway remains unaffected upon CD36 blocking. In order to validate our results in human microglia, the effects of anti-CD36 blocking antibody were investigated using human embryonic microglial cell line. Our analysis revealed that that the TLRs induction patterns as well as effects of anti-CD36 blocking antibody were rather similar in both human and mouse microglia.

 

Conclusion:

Our results indicate that blocking CD36 alters the inflammatory profile of mouse and human microglia, suggesting its therapeutic aspect in fine-tuning of neuroinflammation.

 

 

 

Poster TH3-20

pySTED: STED Microscopy Simulator for Reinforcement Learning Applications

TURCOTTE BENOIT, Bilodeau Anthony, Durand Audrey, Lavoie-Cardinal Flavie - Centre de recherche CERVO


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.

 

Poster TH4-01

Mécanismes moléculaires impliqués dans la régulation transcriptionnelle de la prolifération microgliale in vivo

SARAH BELHOCINE, André Machado Xavier, Félix Distéfano-Gagné, Stéphanie Fiola, David Gosselin

Département de médecine, Université Laval, CRCHU de Québec - CHUL, Axe Neurosciences


Les microglies sont les macrophages résidents du système nerveux central (SNC), dont les fonctions sont nécessaires au développement du SNC et au maintien de son homéostasie au cours de la vie adulte. La perturbation des fonctions microgliales est associée au développement de nombreuses maladies neurodégénératives et neurodéveloppementales. Tôt dans le développement du SNC, les microglies prolifèrent massivement pour permettre à l’organe neural de se développer. La prolifération des microglies dépend de l’expression coordonnée de dizaines des gènes associés au cycle de prolifération, ce qui implique des changements dans l’épigénome de ces cellules. Or, les mécanismes épigénomiques sous-jacents au cycle cellulaire des microglies n’ont jamais été caractérisés auparavant. Objectif : Notre projet a pour but d’identifier les éléments génomique régulateurs qui coordonnent la prolifération microgliale dans les contextes neurodéveloppemental et de lésion neuro-inflammatoire. Méthodologie : Pour ce faire nous avons isolé par cytométrie des microglies en état de prolifération ou non, puis effectué des analyses de ChIP-seq (Chromatin ImmunoPrecipitation-sequencing) afin de mesurer l’état d’activation des promoteurs et des éléments amplificateurs, en se basant sur l’abondance de la marque d’activité épigénétique H3K27ac. Résultats : Les analyses bio-informatiques confirment que les microglies prolifératives ont des programmes épigénomique et transcriptionnel différents des microglies non-prolifératives. De plus, les facteurs de transcriptions des familles KLF et MEF2 semblent être impliqués dans la coordination de la prolifération des microglies. Enfin, nous montrons aussi que les programmes épigénomique et transcriptionnel des microglies prolifératives dans un contexte de neurodéveloppement sont différents de ceux des microglies qui prolifèrent dans un contexte de lésion. Ainsi, le programme épigénomique associé à la prolifération microgliales est « contexte-dépendent ».

Poster TH4-02

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

CADORET ALICE1, Dion-Albert Laurence1, Doney Ellen1, Lebel Manon1, Ménard Caroline1

1Centre de recherche CERVO, Université Laval, Québec, Canada


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.  

Poster TH4-03

Altérations de l’Intégrité de la Matière Blanche avec l’Obésité: Méta-Analyse des Études d’Imagerie par Tenseur de Diffusion

DAOUST JUSTINE, Schaffer Joelle, Dagher Alain, Garcia-Garcia Isabel et Michaud Andréanne. 

Centre de recherche de l’Institut universitaire de cardiologie et de pneumologie de Québec (IUCPQ), Université Laval.


OBJECTIF: Des études d’imagerie par tenseur de diffusion (DTI), une technique permettant d’évaluer l'intégrité des faisceaux de la matière blanche par la mesure d’anisotropie fractionnelle (FA), ont montré qu’une perte d'intégrité de la matière blanche est associée à une altération des fonctions cognitives chez les individus souffrant d’obésité. Toutefois, les études dans le domaine ne sont pas unanimes et rapportent parfois des résultats contradictoires. Nous avons donc mené une méta-analyse afin d’identifier les faisceaux de la matière blanche qui présentent des changements significatifs avec l’obésité. MÉTHODES : Une revue systématique des études de DTI évaluant la relation entre les mesures d’obésité et l’intégrité de la matière blanche, rapportée en FA, a été réalisée dans la base de données PubMed. Nous avons effectué une méta-analyse basée sur les coordonnées significatives générées par les études en utilisant le logiciel Anisotropic Effect Size-Signed Differential Mapping (AES-SDM) version 5.15. RÉSULTATS : Dix-huit bases de données ont été incluses dans la méta-analyse représentant 4453 participants (55% de femmes) âgés entre 18 et 92 ans. Les résultats obtenus montrent que les individus obèses sont caractérisés par une réduction des valeurs de FA dans la partie antérieure droite du corps calleux (SDM-Z = -3.282; MNI = 26, 32, 22; p < 0.001). Nos analyses indiquent également que nos résultats ont un niveau élevé de reproductibilité (Jackknife, 15/18) et aucun biais de publication (test d’Egger, p=0.218). CONCLUSION : Bien que les mécanismes expliquant ces résultats ne soient pas complètement élucidés, les résultats de cette méta-analyse suggèrent que l’obésité est associée à une réduction de l’intégrité de la matière blanche dans une zone du cerveau qui relie les cortex préfrontal et orbitofrontal, des régions impliquées dans les processus cognitifs. Des études futures sont nécessaires pour valider les mécanismes et vérifier si les changements cérébraux associés à l’obésité sont permanents ou s'ils peuvent être renversés à la suite d’une perte de poids.

Poster TH4-04

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

DION-ALBERT, LAURENCE*1, Cadoret, Alice1, Doney E1, Neutzling Kaufmann, Fernanda1, Dudek, Katarzyna A1, Samba, Nalia2, Lebel, Manon1, Mechawar, Naguib3, Ménard, Caroline1

1 Département de psychiatrie et neurosciences, centre de recherche CERVO, Université Laval, Québec (Canada)
2 Département de biologie intégrative et psychologie, Université Sorbonne, Paris (France)
3 Département de psychiatrie, Douglas – Bell Canada Brain Bank, Université McGill, Montréal (Canada)


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.

 

Poster TH4-05

Sex-specific effects of chronic stress on the intestinal barrier integrity

DONEY, ELLEN, Coulombe-Rozon, Francois, Dion-Albert, Laurence, Dudek, Katarzyna, Neutzling-Kaufmann, Fernanda, Lebel, Manon, Ménard, Caroline


OBJECTIF - OBJECTIVE : To investigate how the effects of chronic stress can influence manifestations of intestinal permeability in both male and female mouse models of depression. We hypothesize that stress may induce changes to gut barrier integrity in a sex-specific manner, playing a role in vulnerability or resilience. Subsequently, we will assess the function of novel peptide polymer, designed with the specific purpose of being able to structurally stabilize and reduce gut permeability by coating the intestinal tract. We will contribute to its investigation by testing the therapeutic potential in chronic stress animal models of depression.

 

MÉTHODES - METHODS: Mice are subjected to various stress paradigms: 6 day or 28 day chronic variable stress, or 10 day chronic social defeat stress (CSDS). CSDS is followed by a social interaction test to determine the phenotype of susceptibility or resilience to stress.Gene and protein expression of tight junctions are analyzed from intestinal tissues. Results are correlated with markers of BBB permeability and behavioral outcomes to provide a whole-body profile of biological alterations associated with social stress vulnerability vs resilience. The second portion of the study, mice will undergo 10 days CSDS while receiving dietary supplementation of a peptide polymer. Tissues will be analyzed to determine the effects of the supplement on the overall integrity of macro structures such as villi, crypts, and mucous layer as well as changes to tight junction expression. As well, to investigate potential biomarkers of gut permeability associated with stress, blood is collected before and after the paradigm for serum analysis of markers related to gut dysregulation and microbial translocation.

 

RÉSULTATS - RESULTS: Chronic stress induces changes to intestinal tight junction expression in the jejunum. Specifically, alteration in the expression of different tight junctions are related type of stress and duration. As well, sex-specific changes are seen across the various stress paradigms. In CSDS, changes in tight junction expression are associated with resilience or susceptibility to chronic social stress and correlated with phenotype severity. In the pilot trial of the peptide polymer, mice tolerated the supplement, maintaining healthy weight and showing no evidence of behavioral alterations.

 

CONCLUSION: By investigating individual and sex-differences, our results will be contributing to the knowledge of molecular mechanisms underlying vulnerability or resilience to chronic stress. In the next steps we will contribute to the investigation of the peptide polymer as a potential therapeutic intervention. By targeting the intestinal barrier and potentially promoting barrier integrity, treatments could have positive downstream effects on peripheral and central inflammatory pathways implicated in depression.

Poster TH4-06

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

Katarzyna Anna Dudek , Olivier Lavoine, Fernanda Neutzling Kaufmann , Laurence Dion-Albert , Dr. Manon LeBel , Dr. Claudia Manca , Prof. Cristoforo Silvestri , Prof. Vincenzo Di Marzo , Prof. Caroline Menard


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.

Poster TH4-07

L’adiposité viscérale est associé à une diminution du volume du cortex cingulaire antérieur et à la sévérité des accès hyperphagiques chez les femmes souffrant d'obésité

ICETA Sylvain(1), Dadar Masha (2), Scovronec Anais (1), Daoust Justine (1), Legault Marianne (1), Pelletier Melissa (1), Biertho Laurent (3), Richard Denis (1), Tchernof André (1), Michaud Andréanne (1)(4)

1) Centre de recherche de l’Institut universitaire de cardiologie et pneumologie de Québec, Université Laval, (2) Département de radiologie, Faculté de médecine, Université Laval, (3) Département de chirurgie, Université Laval, (4) Institut sur la nutrition et les aliments fonctionnels, Université Laval.


OBJECTIF 

Des études d’imagerie par résonance magnétique (IRM) montrent que les désordres métaboliques associés à l’obésité pourraient entrainer des altérations de la structure du cerveau. L’accumulation de tissu adipeux viscéral est un marqueur du risque métabolique et peut être estimé par l'indice d'adiposité viscérale (VAI), calculé avec le tour de taille, l’IMC, les taux de triglycérides et de cholestérol-HDL. Comparer la densité des régions cérébrales impliquées dans la régulation alimentaire chez des individus obèses avec un score VAI faible versus élevé et examiner le lien avec les accès hyperphagiques, dépendamment du sexe.

MÉTHODES 

Des IRM anatomiques du cerveau (pondération T1) ont été obtenues pour 20 hommes et 59 femmes sévèrement obèses. La sévérité des accès hyperphagiques (Binge Eating Scale, BES) a été mesurée. Les participants ont été divisés en 2 groupes selon la médiane du score VAI (faible ou élevé). La densité de la matière grise (MG) a été mesurée par morphométrie cérébrale (VBM). Les valeurs de VBM ont été extraites pour des régions d’intérêt (ROIs) : 1) cortex préfrontal dorsolatéral et ventromédian (prise de décision et inhibition), 2) cortex orbitofrontal et insula (réactivité aux stimuli alimentaires) et 3) cortex cingulaire antérieur, CCA (régulation cognitive et émotionnelle). L’influence des ROIs dans la relation entre les scores VAI et le BES a été testée par une analyse de médiation.

RÉSULTATS

Les femmes avec un VAI élevé ont un score plus élevé à la BES (13.8 ±6.7 vs 9.1±5.8; p<0.05) et une densité diminuée de la partie caudale du CCA (0.56±0.08 vs 0.62±0.08; p=0.007). Les différences entre les groupes n’étaient pas significatives pour les autres ROIs ou chez les hommes. La densité de la région caudale du CCA influence la relation entre les scores VAI et BES.

CONCLUSION

Chez la femme, une adiposité viscérale élevée est liée à une plus grande sévérité des accès hyperphagiques. La baisse de la densité de la partie caudale du CCA, une région impliquée dans la régulation cognitive, semble influencer cette relation.

 

Poster TH4-08

Characterization and optimization of human iPSCs derived mixed neuronal cultures to identify with high-resolution multimodal microscopy techniques risk biomarkers of Schizophrenia.

Niraj Patel, Catherine Giroux, Isabelle St Amour, Erik Belanger, Jean-Michel Mugnès, Hugo Poulin, Thomas Paccalet, Michel Maziade and Pierre Marquet


Schizophrenia (SZ) is a neuropsychiatric disorder affecting over 1% of the world’s population, characterized by distortions in thinking, perception, emotion, language, sense of self, and behavior. In many neuropsychiatric disorders such as Schizophrenia, a complex interaction between genetic susceptibility and environmental insults leading to alteration of the brain development has been reported. Specifically, profound modifications in anatomy, physiology, and functional properties of cortical networks during adolescence has been described. Various abnormal gene expression's synergistic effects lead to various neurotransmitter abnormalities, making disease phenotypes and characteristics extremely heterogeneous. Alterations in neuronal connectivity may alter the homeostasis plasticity (HP) of neuronal circuits during the brain development, making them particularly vulnerable to environmental stressors. This is sometimes called childhood risk syndrome, which may lead to Schizophrenia and other neuropsychiatric disorders. It has been shown, In vitro neural differentiation of induced pluripotent stem cells (iPSCs) recapitulates at some extent some important steps of the in vivo neurodevelopment. Therefore, my study's first step is to develop a methodology to generate from iPSCs cells, coming from patients and their high-risk offspring, cultures of mixed cortical neurons. An important part of this work is to optimize and characterize this neuro-differentiation process to obtain highly reproducible neuronal cultures, recapitulating some critical step of the forebrain development and representing an in vitro model of cortical diseases, including psychiatric disorders. 

The advanced cell reprogramming tools helped us successfully develop the human-iPSCs from urothelial cells obtained from schizophrenic patients and healthy controls using integration-free methods. Then iPSCs are differentiated into neurons following commercially available protocol and culture media. The characterization was performed using flow cytometry and immunostaining tools. These cultures exhibit different kinds of neurons, including glutamatergic, dopaminergic, GABAergic neurons, and glial cells. The functional activity was measured by the whole-cell patch-clamp technique and calcium imaging. In next step, we plan to analyze with multimodal digital holographic microscopy (QP-DHM) the activity and maturation of these human iPSC-derived neuronal cultures. Specifically, the excitatory/inhibitory (E/I) balance, which has been shown to be altered in neurodevelopmental psychiatric disorders, will be explored. QP-DHM is a high-resolution non-invasive microscopy technique that assesses cell structure and dynamics with a nanoscale axial sensitivity. Such an approach, combining cutting edge imaging techniques with in vitro model of cortical diseases, is highly promising to identify cellular risk biomarkers of major neurodevelopmental psychiatric disorders, including Schizophrenia. Our approach could easily be applied to identify some vulnerability versus resilient states within the northern populations.