Credits : Pascal Chhay

RESEARCH PROJECTS

Thanks to the Courtois Foundation, CERMO-FC has enabled 12 recipients to receive a scholarship in 2019. Find below the research summaries of these doctoral or master’s students.

Mariela Gomez Perez, M.Sc  Supervisor: Mircea Alexandru Mateescu (UQAM)

Bio-active agents with copper (II) and their implication in orphan diseases.

Hypertrophic cardioencephalomyopathy (HCEM) is a rare mitochondrial disease caused by mutations in the gene encoding Sco2, a protein implicated in the trafficking of copper to cytochrome c oxidase (COX). This disease is occurring in the first month of life. Recently, we have synthesized solid copper(II) complexes ([Cu(His)2Cl2] and [Cu(Ser)2]) stable in different physiological media with good biocompatibility (viability of neurons and enterocytes over  90%) at concentrations of cupric complexes less than 100 μM. These results opened new perspectives for treatment of HCEM with oral formulations. The project examines a new approach in HCEM therapy considering that copper(II) complexes, properly formulated, can be absorbed at the intestinal level and can improve the COX activity. The understanding of the intestinal absorption system and the transmembrane transport of copper (II) complexes, following their oral administration, will allow the development of new pharmaceutical forms to improve the quality of life of patients.

Baptiste Charrier, M.Sc  Supervisor: Nicolas Pilon (UQAM)

Study of the role of the Nr2f1 nuclear receptor in glial differentiation of neural crest cells

Waardenburg syndrome is a genetic disorder characterized by hair and skin pigmentation defects and hearing and/or balance problems. Type 4 is also combined with aganglionic megacolon, also called Hirschsrpung’s disease (HSCR). Spot mice have a phenotype very similar to that of people affected by WS4, they are depigmented and have spatial orientation defects as well as aganglionic megacolon. These problems have a common origin: a lack of cells derived from neural crest cells (NCC) including neurons and glial cells of the enteric nervous system as well as melanocytes of the skin and the inner ear. The Spot mutation was localized near the Nr2f1 gene and we observed that this gene, as well as several glial genes are significantly overexpressed in the NCCs of Spot embryos. In addition, the NCCs of Spot embryos differentiate prematurely into glial cells at the expense of undifferentiated progenitors. The characterization of the Spot line has identified Nr2f1 as an important gene for the glial differentiation of NCCs. Our goal is now to understand the mechanisms of actions and regulation of Nr2f1 during NCC differentiation.

Confocal images of embryonic intestines at 13.5 days labelled with Sox10 (green) and Nr2f1 (red) by immunofluorescence. Sox10 is a transcription factor expressed by progenitors of neurons and glial cells of the enteric nervous system (ENS). In Spottg/tg embryos, the vast majority of ENS progenitors abnormally expresses Nr2f1 which causes premature glial differentiation at the expense of neurons and undifferentiated progenitors.

Léa Mélin, M.Sc  Supervisor: Alexandre Gagnon (UQAM)

Development of YAP-TEAD inhibitors derived from flufenamic acid

The YAP-TEAD transcriptional complex, the downstream effector of the Hippo signaling pathway, activates the expression of anti-apoptotic genes as well as proliferation ones. In malignant pleural mesothelioma (MPM), a rare and orphan cancer representing less than 1% of known cancers, multiple genetic mutations in the Hippo pathway have been identified. Thus, YAP is overexpressed, in a direct or indirect manner, in more than 70% of MPM cases. Because of its aggressiveness, with a median patient survival of one year after diagnostic, it is now critical to develop effective treatments. As YAP oncogenic function is only activated upon its binding to TEAD, inhibiting YAP-TEAD complex formation should lead to new therapies against MPM. In collaboration with the SGC at Toronto, flufenamic acid derivatives have been designed to bind in a hydrophobic pocket of TEAD, preventing its palmitoylation and therefore YAP-binding. These compounds are synthesized through a Buchwald N-arylation between anilines and corresponding methyl-2-bromobenzoates.

Victoria Cerdeira, B.Sc  Supervisor: Claire Bénard (UQAM)

Neuronal development: roles of messenger RNA stability in the development and plasticity of dendritic spines

Dendritic spines are key structural features required for neurons to communicate and to function. Despite their importance, how dendritic spines develop and are modulated over time remains poorly understood. We propose to study evolutionarily conserved mechanisms driving the development and plasticity of dendritic spines using C. elegans. This powerful model system allows molecular genetic analysis to be done in vivo with single-neuron and single-dendrite resolution at any point of the animal’s life. We will address how the regulation messenger RNAs stability impacts dendritic spines development and plasticity. Indeed, our results indicate that mRNAs stability in neurons is key for the development of dendritic spines in the brain. Very rare neurological conditions are affected by such mechanisms, including the Al-Raqad Syndrome (also called “autosomal recessive rare non-syndromic intellectual disability”) and early-onset schizophrenia. Given the high evolutionary conservation of neuronal development and function, as well as of the mRNA stability machineries between C. elegans and humans, elucidating relationships between mRNA stability and dendritic spines formation and plasticity is expected to provide key insights for the development of strategies to diagnose and treat these rare neurological conditions.

Spine-shaped structures along the C. elegans motor neuron DDD dendrite. Dendritic spines are indicated by white arrows.

Layal El Cheikh Hussien, B.Sc  Supervisor: Marc Lussier (UQAM)

Characterization of synaptic regulation of AMPA receptors by phosphorylation and ubiquitination

Synaptic functional changes can lead to loss of plasticity and consequently alter the storage of information in the brain. Examples include Alzheimer’s disease, which affects the synaptic expression of AMPA-type glutamate receptors (AMPAR), and schizophrenia, which has abnormal regulation of glutamate receptor activity including AMPARs. AMPARs are ionotropic receptors that mediate the majority of fast excitatory neurotransmission in the central nervous system. The synaptic expression of AMPARs is controlled by several factors such as endocytosis, recycling and lysosomal degradation. Several recent studies have shown the importance of post-translational modifications such as phosphorylation and ubiquitination in the synaptic regulation of these receptors. Ubiquitination is an enzymatic process resulting in the covalent addition of a small molecule ubiquitin to a substrate such as AMPARs. In addition, AMPARs can be phosphorylated by different kinases such as PKC, PKA and CaMKII. The goal of this project is to dissect the crosstalk between phosphorylation and ubiquitination in order to better understand their roles in the regulation of AMPARs. We hope that our work will help find the right treatments for Alzheimer’s disease or schizophrenia.

Study design regulating postsynaptic expression of AMPA receptors. AMPA receptors (AMPAR) are stabilized at the PSD by GRIP1 proteins. Binding of glutamate neurotransmitters allows lateral diffusion of AMPAR. The serine S880 of the GluA2 subunit is phosphoryled by PKC. This change increases its affinity to PICK1 protein. In the endocytic vesicles (EV), AMPAR subunits are ubiquitinated by UBE3 ligase RNF167. Following endocytosis, AMPARs are found in early endosomes (EE). A loss of ubiquitination allows membrane recycling of receptors via recycling endosomes (RE). Ubiquitinated AMPAR are trafficked to lysosomal degradation via late endosomes (LE). 

Neija Lassoued, M.Sc  Supervisor: Nicolas Pilon (UQAM)

Characterization of the therapeutic effects of neurotrophic factor GDNF on the digestive system of mouse models for Hirschsprung disease

Hirschsprung’s disease is characterized by the absence of ganglia of enteric nervous system in the colon. To avoid death by constipation and enterocolitis, the only strategy applicable to treat this disease is the surgical removal of the affected area.

The Pilon laboratory has developed an alternative regenerative strategy of enteric ganglia at the aganglionnal zone in different mouse models and in human patient tissues. This approach is based on the administration of GDNF, a powerful neurotrophic factor. We found that about 35% of the neoformed ganglia are derived from Schwann cells. We therefore seek to identify the other cell types that generate 65 % of ganglia by a cell tracking using the Cre/Loxp genetic system. Enteric glial cells are suspected to be a target of GDNF. To confirm this, three triple-transgenic mouse lines with tamoxifen-inducible Cre-ERT2 recombinase expression were generated. Studies are underway to determine optimal tamoxifen doses. Ganglia formation is evaluated by immunofluorescence.

We are also studying the effect of GDNF on the immune system of the colon by flow cytometry. The choice of the optimal method of cell dissociation of mouse colons as well as markers of myeloid and lymphoid cells was made. At the same time, we aim to continue studying the other curative aspects of GDNF, including the composition of the intestinal flora in our mice models. This allows us to consider that our treatment will be effective for patients with Hirschsprung’s disease.

Immunofluorescent staining of colon of Holstein; Nestin-CreERT2; R26R-YFP mice at P20.
The mice were treated with tamoxifen (2mg/ml; 50 μl) by oral gavage from P3 to P8 (P: Post-natal Day). The efficacy of tamoxifen treatment to induce Cre recombinase was tested at P20. We see some Nestin+ cells that are GFP+ (YFP+). Thus, at the molecular level, the dose of tamoxifen introduced by oral gavage appears to be capable of activating Cre recombinase in some Nestin+ cells.  

Comparison of three cell dissociation techniques from the colon of FVB mice (WT) at P20 by flow cytometry. (A) Mechanical method Versus (B) Enzymatic method: collagease I + dispase Versus (C) Enzymatic method: collagease V + liberase.
According to the results, it seems that the mechanical technique will be our method of choice. Enzymatic methods have altered the surface markers of lymphocytic cells, including CD4 and CD8.

Margaryta Babych, M.Sc  Supervisor: Steve Bourgault (UQAM)

Role of conformational transitions in self-assembly and cytotoxicity of amyloidogenic peptides

Amyloidoses are a set of diseases associated to depositions of insoluble protein aggregates, named amyloid fibrils. The majority of these diseases remain orphans due to challenges in their rarity or challenges in their diagnostic and treatment. The common trait of these diseases is the peptide/protein misfolding that aggregate in insoluble amyloid deposits. These deposites can be localized or systemic, at the level of several tissues or organs. Considering that prefibrillar species are highly toxic for cells and that they seem to lie at the origin of the tissue degeneration, a better understanding of molecular events preceding amyloid fibrils formation will allow the development of pre-emptive diagnostic assays and/or new therapeutic approaches to inhibit the formation of transitionary cytotoxic species. Our hypothesis is that conformational transitions of oligomeric aggregates to a stable nucleus play a key role in the mechanistic basis of amyloid fibrils formation and in the existence of toxic species that form a large conformational ensemble. For this reason we propose to study the effect of perturbations of non-structured oligomer formation by favoring a secondary structure at the monomer level to control the kinetics of amyloid auto-assembly and the presence of cytotoxic species.

Study of the kinetics of amyloid fiber formation by thioflavin-T and transmission electron microscopy

Fabio Luiz Bandeira Ferreira, B.Sc  Supervisor: Krista Heinonen (INRS)

Evaluation of hematopoietic stem cell (HSC) activation and exhaustion in mice from a Leishmania major infection model

Secondary haemophagocytic lymphohistiocytosis (HLH) (ORPHA: 158041) can occur at any age, often as a result of various infections, including leishmaniasis. It is characterized by an exacerbated immune response, especially by macrophages (also called histiocytes), leading to uncontrolled tissue damage. However, the exact causes and mechanisms are still unknown. Our work is based on the hypothesis that infection-induced emergency haematopoiesis contributes directly to the pathogenesis of HLH by promoting accumulation of macrophages responsible for bone marrow destruction and failure. We will use the intradermal mouse ear infection model by Leishmania major to better understand the involvement of hematopoiesis in HLH pathogenesis. We will quantify cell populations as well as cytokines/chemokines present in bone marrow and other hematopoietic organs in the context of either self-healing or chronic infection by different strains of the parasite. These data will help us identify those populations that may contribute to parasite persistence and the development of HLH. Subsequently, these populations can be eliminated to determine their actual importance. Our work will allow us to better understand the immune processes involved in the development and pathogenesis of secondary HLH.

Amira Aoussim, B.Sc  Supervisor: Élise Duchesne (UQAC)

Identification of proteomic and transcriptomic markers associated to the positive response induced by physical training observed in myotonic dystrophy type 1 patients

Myotonic dystrophy type 1 (DM1) is a multisystem inherited disease. The highest global prevalence is found in the Saguenay–Lac-St-Jean region (1/500). Skeletal muscle is severely impaired; individuals suffering from DM1 present, among others, muscular atrophy and a progressive loss of the maximum muscle strength. Strength training represents an interesting therapeutic avenue to slow the progression of muscular deficiencies. Recent data from our research group suggest a significant improvement in muscle strength and functional capacity after a strength training program (Figure 1). Skeletal muscle adaptation is possible in DM1 despite the genetic defect, but the underlying biological processes remain unknown. Since several biological processes and molecular functions are definitely involved in these strength and muscle mass gains, a study of muscle proteome was carried out by our group to explore the biological pathways explaining them. The objectives of this project are to measure the expression levels of the proteins of interest modulated by the training and of the genes encoding them by immunoblotting and qRTPCR, respectively. These analyses will increase knowledge about an orphan disease, DM1, and in particular about the related muscle deficiencies.

Figure 1: Average percentage of progression of different strength and functional tests for 11 men with DM1 who have completed a 12-week strength training program. Measurements were taken before (0-week), during (6-week), immediately after (12-week) the program, and maintained benefits were measured at month 6 and 9 post-program. A) 1 repetition maximum (1RM), B) quantified muscle testing (QMT), 30 second sit to stand (30ssts) and 10 meter walk test (10mwt) are presented as an average of progression with week 0 being the baseline. * p<0.05

Séphora Sallis, M.Sc  Supervisor: Nicolas Pilon (UQAM)

Study of the regulation mechanism of FAM172A, and its key role in co-transcriptional alternative splicing.

CHARGE Syndrome is a rare developmental disorder which affects 1/ 10 000 newborns and has a very complex clinical presentation. Heterozygous mutation of CHD7 (Chromodomain helicase DNA-binding domain) is currently the main genetic cause of CHARGE Syndrome. This condition belongs to the neurocristopathies family, which are diseases of neural crest cell development, but underlying molecular mechanisms are not well understood.

The Pilon lab recently identified FAM172A as a new candidate gene for CHARGE Syndrome. Preliminary studies revealed that FAM172A could regulate the co-transcriptional alternative splicing machinery, through interaction with Argonaute 2 (AGO2), DNA and RNA. We hypothesize that dysregulation of alternative splicing, along with transcriptional landscape perturbation specific to neural crest cells are the cause of CHARGE Syndrome. Bioinformatics-based analyses further predicted several functional domains in FAM172A, such as a consensus motif for Casein Kinase II (CK2) phosphorylation, and an esterase-like serine hydrolase activity. In order to better understand the molecular mechanism of FAM172A and its role in co-transcriptional alternative splicing events, as well as its implication in CHARGE Syndrome, these two motifs will be carefully studied during my PhD project.

Priyanka Jamadagni, M.Sc  Supervisor: Kessen Patten (INRS)

Understanding the role of CHD7 in brain development: CHARGE syndrome and ASD

Mutations in the gene coding chromodomain-helicase-DNA-binding protein 7 (CHD7), an ATP-dependent chromatin remodeller, are the primary cause of CHARGE syndrome and have been associated to CHARGE syndrome. However, the neuropathological mechanisms underlying brain abnormalities in these disorders, upon CHD7 mutations, remain unknown. It is well-known that CHD7 regulates gene transcription. I therefore hypothesize that CHD7 regulates genes that are crucial for proper neural network development, function and maintenance in the brain. Using the zebrafish – a powerful tool for studying such a neurodevelopmental disorder, we have created a zebrafish chd7 mutant using CRISPR / Case 9. Using this model and transgenic fish that express fluorescent protein in specific neuron types, I plan to first characterize the defects in the mutant brain. Further, an RNA-seq experiment will identify the genes that are dysregulated upon chd7 mutation, and guide us to identifying pathways that could be involved. This will help in understanding the mechanisms of the CHARGE syndrome with a particular emphasis on the brain defects. Lastly, we also propose to perform a drug screen of FDA-approved compounds in their abilities to restore / ameliorate symptoms of CHARGE syndrome in a simple genetic model. Our findings have the potential to be translated into the clinic for human trials.

Olivier Reynaud, M.Sc  Supervisor: Gilles Gouspillou (UQAM)

Impact of Parkin overexpression in a mouse model of Duchenne’s disease

Duchenne muscular dystrophy (DMD) is an inherited disease resulting from mutations in the gene encoding dystrophin, which leads to absence of this subsarcolemmal cytoskeletal protein from striated muscle cells. Although much progress has been made in our understanding of DMD, the precise mechanisms involved in the progression of the disease still remain unclear. The absence of dystrophin is obviously central in the pathogenesis of DMD since it renders the sarcolemma fragile and instable. Several studies in DMD patients and mdx mice (the most frequently employed animal model of the disease) reported data indicating that mitochondrial dysfunction are involved in the progression of DMD and that improving mitochondrial fitness (i.e. mitochondrial content and function) is a promising therapeutic strategy to improve muscle function in DMD. However, to date, no study has investigated whether stimulating mitophagy, the process in charge of the removal of damaged/dysfunctional mitochondria, can attenuate mitochondrial dysfunction in dystrophic muscle and slow-down the progression of DMD. The aim of the present project is therefore to define whether overexpressing Parkin, an E3 ligase regulating the recognition and removal of damaged mitochondria, can improve skeletal muscle and mitochondrial function of mdx mice.

Thanks to the Université du Québec à Montreal (UQAM), one of the CERMO-FC competitions allows UQAM faculty members of CERMO-FC to devote more time to their research activities, thus increasing their scientific productivity and their competitiveness in biomedical and biopharmaceutical research. Find below the research programs of the winners of the 2019 edition.

Learn more about Pr Dragon

My research program focuses on ribosome biogenesis and nucleolar function. The nucleolus is a specialized compartment of the cell nucleus: it is the site of ribosome production, and it is often seen as a “ribosome factory”. However, this view has changed over the last 20 years because other functions have been discovered for the nucleolus, including roles in cell-cycle control, aging and viral replication. Nevertheless, the principal function of the nucleolus remains ribosome biogenesis. This biological process is very complex and finely tuned: more than 200 nucleolar factors (proteins) and as many small non-coding RNAs participate in the making of each ribosome. In a rapidly growing cell, nearly 4000 ribosomes are produced every minute. It is therefore crucial to properly control the entire process…
A number of genetic diseases result from mutations in factors involved in the production of ribosomes, or even ribosomes constituents themselves (e.g. Diamond-Blackfan anemia caused by mutations in ribosomal proteins). Diseases affecting ribosome structure and/or function are now grouped under the heading of “ribosomopathies”. These are rare diseases, and the molecular mechanisms underlying the “manufacturing defects” of ribosomes are poorly understood. In my laboratory we use the yeast Saccharomyces cerevisiae as a model to study the early stages of ribosome formation. Using the powerful tools of yeast molecular genetics, we have contributed to the advancement of knowledge on key nucleolar factors such as Dbp4/DDX10, Kre33/NAT10 and Shq1/SHQ1 (yeast/human). Our current work on Kre33 and Shq1 is directly related to rare diseases. Indeed, the RNA acetyltransferase NAT10 is linked to Hutchinson-Gilford progeria syndrome, an accelerated aging syndrome for which there is no treatment (incidence 1/4,000,000). Mutations in the gene SHQ1 have recently been identified in two Canadian siblings; these children have severe developmental problems (dystonia, microcephaly, intellectual disability), and we have been contacted by the Canadian “Rare Diseases: Models and Mechanisms” Network to undertake studies in yeast in order to elucidate the molecular mechanisms that are altered by mutations in SHQ1. Later we learned that an Australian patient presented the same phenotype as the two Canadian sisters and had similar mutations in SHQ1. Our work in yeast revealed that the different mutations in SHQ1 cause a significant ribosome manufacturing defect. We are convinced that this novel rare disease will soon be recognized as a ribosomopathy.

Learn more about Pre Bénard

Our research program has two interrelated axes aimed at understanding the cellular and molecular mechanisms of (1) nervous system development, and (2) long-term nervous system protection. For our studies, we use the C. elegans microscopic nematode as a powerful genetic and molecular model. C. elegans has already largely contributed to the decoding the processes of brain development and function, as well as aging processes, and the fundamental mechanisms of these processes are remarkably preserved between C. elegans and humans. For the axis (1) we study the mechanisms ensuring the migration of neurons and their axons through complex environments in order to establish connections of functional neural circuits. In particular, we aim to elucidate the regulation and coordination of guidance signals (e.g. netrin) by proteoglycans with heparan sulfate chains. In addition, once formed, the nervous system must be preserved throughout life, despite growth, maturation, and mechanical stress related to movement. For this axis (2), we work to identify molecules regulating the interactions of neurons with their environment and to elucidate their modes of action to ensure the maintenance of the architecture and neuronal connectivity. The results of our research help to elucidate the cellular and molecular mechanisms involved in neurodevelopment and neuroprotection, which are processes that are affected by many orphan diseases of a neurodevelopmental nature (eg, microvillous inclusions disease / congenital mirror movements, Al-Raqad syndrome) and / or neurodegenerative (eg, some cases of rare schizophrenia). Thus, we provide important information that has the potential to contribute to the development of strategies to diagnose, prevent or reduce the consequences of such orphan diseases. In addition, we have active collaborations to validate our results in murine models, and assist in a transition to the clinic.

Learn more about Pre Oubaha

Description of the research program is coming

 

 

 

Learn more about Pr Cappadocia

My research program concerns the structure-function study of post-translational modifications of proteins and, more specifically, the study of SUMOylation of proteins. This type of modification, ubiquitous in eukaryotes, regulates localization, function, protein stability and protein-protein interactions. As a result, SUMOylation is involved in multiple biochemical processes and plays a role in several human pathologies such as certain cancers or certain neurodegenerative diseases. Many proteins are thus SUMOylated in humans due to the sequential action of an activation enzyme E1, a conjugation E2 enzyme and an E3 ligase that brings substrate and E2 activated within a single protein complex to facilitate the transfer of SUMO from the active site of E2 to the substrate. Although most SUMO E3 ligases belong to the RING domain family of proteins, my post-doctoral studies have contributed to the structural and mechanistic characterization of smaller E3 ligases collectively referred to as “atypical SUMO E3 ligases”. A SUMO E3 ligase activity has also been proposed for several other proteins, but the molecular bases of this activity remain very often elutrive, which limits the possibilities of manipulation, particularly for therapeutic purposes. I am now planning to develop a new line of research aimed at understanding the SUMO E3 ligase activity of these proteins at the molecular level.

Thanks to the Courtois Foundation, CERMO-FC has launched a first edition of a scholarship contest in 2018. Find below the research summaries of the 12 doctoral or master’s students, laureates of this first edition.

Noé Quittot, M.Sc. Supervisor: Steve Bourgault (UQAM)

Roles of glycosaminoglycans in membrane perturbation and cytotoxicity induced by amyloidogenic proteins

Amyloidosis includes numerous orphan diseases characterized by the tissue accumulation of insoluble proteins in the form of amyloid fibrils. For instance, in patients afflicted by AL amyloidosis, the protein immunoglobulin light chain accumulates in numerous tissues. Recently, it has been shown that cell and tissue degeneration is mainly mediated by intermediates of the amyloidogenic cascade and not by the deposition of amyloid fibrils. Besides, many biological macromolecules, such as glycosaminoglycans, can modulate the self-assembly and toxicity of amyloidogenic polypeptides. Thus, a better understanding of the mechanisms of toxicity as well as the involvement of biological macromolecules in the pathological process is critical to treat amyloidosis. In this study, two amyloidogenic polypeptides will be used, an isoform of the immunoglobulin light chain and the peptide islet amyloid polypeptide, which is associated with type II diabetes. The self-assembly of these amyloidogenic polypeptides in a complex biological environment will be studied using state-of-the-art biophysical approaches. In parallel, membrane disruption induced by the self-assembly process will be assessed by confocal microscopy and cell-based assays. Overall, this project will provide a better understanding of the molecular mechanisms of amyloidosis and could lead to the identification of novel therapeutic targets.


Image by transmission electron microscopy of amyloid fibers of the polypeptide amyloid islet. Scale : 100 nm.

Priyanka Jamadagni, M.Sc. Supervisor: Kessen Patten (INRS-IAF)

Understanding the role of CHD7 in brain development: CHARGE syndrome and Autism

Mutations in the chromodomain-helicase-DNA-binding protein 7 (CHD7) gene, which is involved in chromatin remodeling, are the main cause of CHARGE syndrome and have been associated with the spectrum of autism.However, the neuropathological mechanisms by which these mutations induce brain abnormalities in these syndromes remain unknown. It is known that CHD7 regulates gene transcription. I therefore hypothesize that CHD7 regulates the genes essential for the proper development and maintenance of neural networks in the brain. Using zebrafish – a powerful tool for the study of neurodevelopmental disorder and humans, we created a mutant chd7 using the CRISPR / Cas9 technique. Using this model and transgenic fish expressing fluorescent proteins in certain types of neurons, I would characterize the defects induced in the mutant brain. Then, a transcriptome study, the mutated genes, would be identified in these chd7 mutants, and would guide us to identify pathways that could be involved in the development of neural networks in the brain. This will help to understand the mechanisms that particularly induce brain defects in the CHARGE syndrome. Finally, we propose to screen FDA-approved compounds for their ability to restore / improve CHARGE syndrome symptoms in our genetic model. Our results could be rapidly transposed to clinical trials for humans. I would characterize the defects induced in the mutant brain. Then, a transcriptome study, the mutated genes, would be identified in these chd7 mutants, and would guide us to identify pathways that could be involved in the development of neural networks in the brain. This will help to understand the mechanisms that particularly induce brain defects in the CHARGE syndrome. Finally, we propose to screen FDA-approved compounds for their ability to restore / improve CHARGE syndrome symptoms in our genetic model. Our results could be rapidly transposed to clinical trials for humans. I would characterize the defects induced in the mutant brain. Then, a transcriptome study, the mutated genes, would be identified in these chd7 mutants, and would guide us to identify pathways that could be involved in the development of neural networks in the brain. This will help to understand the mechanisms that particularly induce brain defects in the CHARGE syndrome. Finally, we propose to screen FDA-approved compounds for their ability to restore / improve CHARGE syndrome symptoms in our genetic model. Our results could be rapidly transposed to clinical trials for humans. and would guide us in identifying pathways that could be involved in the development of neural networks in the brain. This will help to understand the mechanisms that particularly induce brain defects in the CHARGE syndrome. Finally, we propose to screen FDA-approved compounds for their ability to restore / improve CHARGE syndrome symptoms in our genetic model. Our results could be rapidly transposed to clinical trials for humans. and would guide us in identifying pathways that could be involved in the development of neural networks in the brain. This will help to understand the mechanisms that particularly induce brain defects in the CHARGE syndrome. Finally, we propose to screen FDA-approved compounds for their ability to restore / improve CHARGE syndrome symptoms in our genetic model. Our results could be rapidly transposed to clinical trials for humans. we propose screening for FDA-approved compounds for their ability to restore / improve CHARGE syndrome symptoms in our genetic model. Our results could be rapidly transposed to clinical trials for humans. we propose screening for FDA-approved compounds for their ability to restore / improve CHARGE syndrome symptoms in our genetic model. Our results could be rapidly transposed to clinical trials for humans.

Grégoire Bonnamour,M.Sc. Supervisor : Nicolas Pilon (UQAM)

Characterization of melanocyte development defects in Waardenburg syndrome

Waardenburg syndrome is characterized by skin and hair pigmentation defects, as well as by inner ear dysfunction affecting hearing and/or balance. These anomalies are believed to be due to defective development of neural crest-derived melanocytes (pigment cells). In the inner ear, we previously reported using the Spot mouse model (which overexpresses Nr2f1 in neural crest cells) that balance problems are due to a specific lack of melanocytes in the vestibule. In the skin, preliminary results show that melanocytes are not properly located along the epidermis in Spot embryos. As melanocytes can be derived from two pathways, either directly from neural crest cells or indirectly from a Schwann cell intermediate, the question is which of these pathways is affected in Spot animals. This is currently addressed using a cell lineage tracing approach based on the Schwann cell specific transgene Plp1-CreERT2 and the R26R-YFP Cre activity reporter. In combination with specific labeling of melanoblasts/melanocytes, it will enable to determine if the observed defects in the skin and inner ear are related to the failure of one or both of the two sources of melanocytes. Then, via time-lapse imaging and marker analyses, we will determine whether these defects are due to a problem of migration, differentiation, proliferation or survival of melanocyte precursors. In the end, this study will significantly increase our understanding of Waardenburg syndrome.

Immunostaining of melanocytes from a mouse hair follicle.

The nuclei of the cells are stained blue. All melanocytes express c-Kit growth factor (in red) but only mature melanocytes produce melanin, which causes hair and skin staining, thanks to the enzyme dopachrome tautomerase (in green). The loss of this mature melanocyte population may be responsible for the partial depigmentation of skin and hair in the type 4 Waardenburg syndrome.

Raphaël Dima, M.Sc. Supervisor: Claire Bénard (UQAM)

Study of the molecular mechanisms that regulate the establishment of polarized projections

The mechanisms involved in brain development, including the guidance of migrating neurons, have been extensively studied over the past two decades. However, how neuronal morphology is regulated, in particular the establishment of the exact number of neuronal projections, remains poorly understood. We have discovered new molecular actors that play a key role in these steps: heparan sulfate proteoglycans (HSPG). To study the role of HSPGs and their partners, we use molecular genetics and fluorescence microscopy approaches in vivo using the C. elegans model. This nematode allows cellular and molecular analysis with single-neuron resolution. Our studies will elucidate mechanisms controlling the development of an appropriate polarized morphology of neurons and other cells. They will help to better understand the molecular bases of some orphan diseases that affect the balance between the structure and function of some cell types, such as the microvillus inclusion disease. Our work will also help to characterize the impact of guidance signals and their receptors involved in rare human genetic diseases, such as Rett-atypic syndrome and congenital mirror movement disorder.

Frédérik Desmarais, M.Sc  Supervisor: Karl-Frédéric Bergeron (UQAM)

Cholesterol transport by apoD neural lipocalin: compensatory mechanism during Niemann-Pick-C disease

The Niemann-Pick-C disease (NPC) is a rare disease affecting 1 child in 100,000. The NPC normally manifest itself early in the patients by causing a degeneration of the neurones. The disease is caused by a mutation in either the gene Npc1 or Npc2 which induces a toxic cholesterol accumulation in the neurones.

Apolipoprotein D (apoD) is overexpressed in the cerebral zones that are the most touched by the disease. ApoD is a soluble neural protein which role is to transport small hydrophobic ligands, one of which is cholesterol. ApoD is implicated in the transport of lipids and enables the transfer of lipids from neurons to glial cells. Recent data from our laboratory also shows that apoD can escape the confines of the central nervous system (CNS) and accumulates in the liver and urine. This indicates that apoD could play a potential role in the recycling and excretion of its ligands.

Our objectives are to evaluates whether an overproduction of apoD in the CNS can attenuates the neuronal cholesterol accumulation by transporting the excess cholesterol outside of the neurones and the CNS and therefore increasing the longevity of mice afflicted by the NPC.

Ons Ousji, M.Sc Supervisor: Lekha Sleno (UQAM)

Metabolomic Analysis of Epidermolysis bullosa simplex

Epidermolysis bullosa simplex (EBS) is a rare genetic disease, involving mutation in the keratin genes. Therefore, there is a failure of keratinization, which affects the integrity and ability of the skin to withstand stress conditions by causing localized skin blistering and hyperpigmentation, among other clinical manifestations. Some gene expression studies have been performed to better understand this disease, however, there still remains many questions about the different physiopathological effects associated to this disease, including metabolomic signature and stress responses.

Through collaboration with Catherine Laprise (UQAC) in genomics and rare diseases, we will have privileged access to clinically relevant cell lines. The first analysis would be an untargeted metabolomics assay by LC-HRMS/MS to access hundreds, if not thousands, of metabolites. In addition, we will use derivatization chemistry to study polar metabolites that are difficult to analyze with traditional chromatographic techniques. This will also allow us to introduce the use of isotopic labeling for quantitative purposes

Mariela Gomez Perez, M.Sc  Supervisor: Mircea Alexandru Mateescu (UQAM)

Bio-active agents with copper (II) and their implication in rare diseases

Menkes’ disease (MD) and occipital horn syndrome (OHS) are severe multisystemic orphan diseases caused by a mutation in the gene encoding the ATP7A protein responsible for copper transport. Both diseases are occurring in the first month of life. The [Cu(His)2] complex was used to treat Menkes’ disease by subcutaneous administration. However, the instability of [Cu(His)2] limits its application as a therapeutic agent. Recently, we have synthesized solid complexes of copper(II) ([Cu(His)2Cl2] and [Cu(Ser)2]) stable in different physiological media and with good neurocompatibility (cell viability of about 90%) at concentrations of copper(II) complexes less than 200 μM. These results opened new perspectives for treatment of MD and OHS with oral formulations. The project examines a new approach in MD and OHS therapy by considering that properly formulated copper(II) complexes can be absorbed at the intestinal level by a mechanism different from that of copper. The understanding of the system of absorption and transmembrane transport of copper(II) complexes following its oral administration will allow to develop new pharmaceutical forms and routes of administration to improve the quality of life of patients.

Sophie Sleiman, M.Sc  Superviseur: Francois Dragon (UQAM)

Effets de la mutation SHQ1

My project aims at understanding the molecular defects caused by mutations in a gene implicated in a novel rare disease. There is presently no name for this disease, but mutations cause a very severe phenotype (e.g. dystonia and microcephaly). This gene is required for the biogenesis of small nucleolar ribonucleoproteins that participate in the making of ribosomes, the molecular machines that synthesize proteins in all living cells. Because the mutated gene is highly conserved amongst eukaryotes, we sought to study the effects of human mutations in our favorite model system, the yeast Saccharomyces cerevisiae. To this end we generated a conditional yeast strain to specifically inactivate the endogenous gene of the yeast and replace it by the human gene (either wild-type or mutated versions). Although yeast cells expressing the human gene are viable, those that carry the various mutated forms cannot grow, indicating that all human mutations are lethal when expressed in yeast. Further analyses revealed that the biogenesis of ribosomes is altered in mutant cells: the mutations lead to an important reduction in ribosome production. We foresee that this rare disease will join the list of “ribosomopathies”, which comprise diseases caused by mutations in genes required for the biogenesis or function of ribosomes.

Crystallographic modeling of a ribosome

The ribosome is the association of two subunits, one large (above) and one small (below). It consists mainly of RNA (in red) and some small proteins represented in dark blue in the small subunit and in green / turquoise in the large subunit. Incorrect assembly or ribosome dysfunction could be the cause of a rare disease.

Meagan Collins, B.Sc  Supervisor : Zoha Kibar (McGill)

Genetics and Mechanisms of Congenital Mirror Movements

Congenital Mirror Movements (CMM) is a rare neurodevelopmental disorder, characterized by voluntary movements from one side of the body that are mirrored by involuntary movements on the opposite side. It is considered a disorder of axonal guidance, and affected individuals have abnormalities in the corticospinal tract. We have performed whole exome sequencing in a large autosomal dominant family with CMM and identified a frameshift variant in ARHGEF7 segregating with the CMM phenotype, c.1751_1752del that leads to p.Asn584Thrfs*90. The purpose of this study is to validate the role of ARHGEF7 in the pathogenesis of CMM. We are using the zebrafish model to investigate whether this variant is pathogenic and to further understand its mode of action. We have created a CRISPR-induced knockout mutant at Arhgef7, and we will conduct overexpression assays in zebrafish. We will perform behavioral analyses and examine axonal defects using immunostaining. This project aligns with the missions of CERMO-FC because CMM is considered an orphan disease, with no known effective treatments. With over 35 individuals diagnosed with CMM in Quebec alone, our research findings will be presented at scientific conferences to further the public’s knowledge of this disease and will directly impact the community.

Fatiha Azouz, B.Sc.  Supervisor: Nicolas Pilon (UQAM)

Analysis of the regulatory mechanisms of the Subcellular Localization of FAM172A Protein

CHARGE syndrome is a rare genetic disorder that is characterized by a complex set of abnormalities that gave it its name: Coloboma of the eye, Heart problems, Atresia of the choanae, Retarded growth, Genital abnormalities and Ear abnormalities. In Dr. Pilon’s laboratory we identified different mutations of the Fam172a gene and identified them as being responsible for the CHARGE syndrome. The Pilon’s lab has generated a mouse model with a mutation of Fam172a that captured all the anomalies of this syndrome. Our previous work was based on the existence of a single isoform of Fam172a though; we recently discovered the presence of 3 different isoforms attributed to alternative splicing. The aim of this work is to characterize the regulatory mechanisms of the subcellular localization of the different isoforms by using an immunofluorescent labelling in transfected cells. To determine the pattern of spatiotemporal expression of Fam172a isoforms during the embryonic development of the mouse by Western Blot. Validate some Fam172a interaction partners, such as Argonaute2, using the BiFC technique. Our results showed that the short and the intermediate forms of Fam172a are predominantly localized in the nucleus, whereas, the long form is predominantly localized in the cytoplasm. The most expressed form during embryonic development is the intermediate form.

Percentage of transfected Co7 and N2A cells relative to the subcellular localization of the different isoforms of Fam172a

Virginie Desse, B.Sc. Supervisor: Claire Bénard (UQAM)

The role of the sax-7/L1CAM gene in the maintenance of neuronal architecture

Whereas remarkable advances have been made in understanding brain development, the mechanisms that protect the nervous system architecture throughout life are unknown. Indeed, how the structural and functional integrity of a nervous system established during embryogenesis is maintained lifelong, despite the growth and movements of the body, brain maturation and aging, remains unanswered. To study the maintenance of neuronal architecture, we use the powerful genetic and molecular model Caenorhabditis elegans. Our research has identified genes involved in neural maintenance, one of them being sax-7, an evolutionary conserved gene that encodes the homologue of cell adhesion molecule L1CAM in mammals. It ensures the structural maintenance of the nervous system in worms, it is required for proper brain function in adult mice, and in humans, L1CAM mutations lead to neurodevelopmental conditions, referred to as the C.R.A.S.H syndrome. This syndrome affects less than 0.05% of the population and treatments are still lacking. Thus, elucidating the molecular and cellular mechanisms by which the molecule SAX-7/L1CAM and its interactors participate in the neuronal maintenance is expected to help in the development of new detection and treatment strategies of this orphan syndrome and of other neurodevelopmental and neurodegenerative conditions in humans.

Oscar Gamboa, Supervisor: Charles Gauthier (INRS-IAF)

Development of inhibitors of exopolysaccharide biosynthesis in Burkholderia pseudomallei as an anti-virulence strategy against melioidosis

Melioidosis is a highly infectious disease caused by the Gram-negative bacterium Burkholderia pseudomallei, which is characteristic from tropical regions such as Southeast Asia and Northern Australia. The bacterium can be transmitted through direct contact with contaminated water or soil or by inhalation of dust and contaminated water droplets suspended in the air. Representing a potential biological weapon, melioidosis is a largely neglected disease and has not been taken seriously by international public health organizations including the World Health Organization. New antibiotic alternatives are actually needed to treat B. pseudomallei infections due to its high lethality once transmitted.

My research project is focused on the synthesis of 3-deoxy-D-manno-oct-2-ulosonic acid (Kdo) derivatives as potential inhibitors of exopolysaccharide (EPS) biosynthesis process. EPS is an important virulence factor for B. pseudomallei. By blocking its incorporation into the bacteria biofilm, we hope to destabilize its growth or even facilitate the incorporation of actual antibiotic treatments. In a multidisciplinary work involving the participation of experts in carbohydrate chemistry and microbiology, new Kdo-like molecules will be synthetized and tested in in vitro as well as in vivo in order to evaluate their antibiotic potential. This work could ultimately lead to a new generation of antibiotic molecules able to treat effectively melioidosis.