Credits : Pascal Chhay
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.
My research program focuses on ribosome biogenesis and nucleolar functions. The nucleolus is known as the ribosome production site and has been labeled a “ribosome factory”, but this image has changed over the past 20 years: other nucleolar functions have been discovered, including its participation in cell cycle control, aging or replication of viruses. However, the biogenesis of ribosomes remains its main function. This essential biological process is very complex and finely regulated. More than 200 nucleolar factors are known and as many small non-coding RNAs participate in the manufacture of each ribosome. In a rapidly growing cell, nearly 4000 ribosomes are produced every minute. We understand the importance of properly controlling the entire procedure …
Some genetic diseases result from mutations in factors involved in the production of ribosomes or which are themselves constituents of ribosomes (eg Diamond-Blackfan anemia). These diseases are now grouped under the heading of “ribosomopathies”. What characterizes all ribosomopathies is that they are rare diseases and that 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 yeast molecular genetics tools we have contributed to the advancement of knowledge on such key factors as Dbp4 / DDX10, Kre33 / NAT10 and Shq1 / SHQ1 (yeast / human). Our current work on Kre33 and Shq1 is directly related to rare diseases. Indeed, NAT10 acetyltransferase is linked to Hutchinson-Gilford syndrome (progeria), accelerated aging for which there is no treatment (incidence 1 / 4,000,000). In addition, mutations in SHQ1 have recently been identified in two Canadian women; these children have very serious developmental problems (dystonia, microcephaly, intellectual disability) and we have been contacted by Canadian network “Rare Diseases: Models and Mechanisms” to undertake studies in yeast to elucidate the molecular mechanisms that are altered by the mutations in SHQ1. After starting this work, we learned that an Australian patient had the same phenotype as the two Canadian sisters and had similar mutations in SHQ1. To date, our work in yeast has revealed that the different mutations in SHQ1 cause a significant manufacturing defect in ribosomes. We are convinced that this new rare disease will soon be recognized as a ribosomopathy.
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.
Description of the research program is coming
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.