Rashmi Kothary

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Rashmi Kothary
Full Professor

Room: General Campus
Office: 613-737-8807
Work E-mail: rkothary@ohri.ca

Rashmi Kothary

Biography

Deputy Scientific Director, Ottawa Hospital Research Institute

Senior Scientist, Regenerative Medicine Program, Ottawa Hospital Research Institute

Professor, Department of Cellular and Molecular Medicine, University of Ottawa

Professor, Department of Medicine, University of Ottawa

University Health Research Chair in Neuromuscular Disorders

Research goals and interests

Our laboratory is interested in understanding the mechanisms which maintain/alter muscle and nervous system integrity. In parallel, we are assessing ways to alleviate the pathology of neuromuscular diseases.

Biographical Sketch

Dr. Kothary is an Associate Director and Senior Scientist at the Ottawa Hospital Research Institute. He was originally fascinated by physics, but his interests shifted towards biology, biochemistry, embryology and ultimately neuromuscular disorders. Dr. Kothary received a Ph.D. in Biochemistry from the University of British Columbia and pursued postdoctoral research in the laboratory of Dr. Janet Rossant at the Mount Sinai Hospital Research Institute in Toronto and in the laboratory of Dr. Azim Surani in Cambridge, U.K. It was during these formative years that Dr. Kothary developed his interests in the use of transgenic mice to model disease pathology. Dr. Kothary returned to Canada to begin his independent research career at the Institut du cancer de Montréal. In 1998, Dr. Kothary joined the OHRI as a Senior Scientist. He holds the University Health Research Chair in Neuromuscular Disorders and is a Professor at the University of Ottawa. Dr. Kothary's laboratory is funded by research grants from the Canadian Institutes of Health Research (CIHR), the Muscular Dystrophy Association (USA), Families of Spinal Muscular Atrophy, and the Multiple Sclerosis Society of Canada. His current research focuses on studying the fundamental role of a cytoskeletal linker protein important for intracellular trafficking, investigating signal transduction pathways important for oligodendrocyte mediated myelination and remyelination of the CNS, and understanding Spinal Muscular Atrophy pathogenesis and identifying novel therapeutics for this devastating children's disease.

Research Activities

1. A key cytoskeletal linker protein and the maintenance of cellular integrity:
We are exploring the essential role of cytoskeletal linker proteins in regulation of cellular processes from cytoskeletal dynamics to organelle stability and function. The dystonin class of cytoskeletal linker protein is essential in mediating filament organization, possessing multiple modular domains for cytoskeletal interaction. Loss-of-function of dystonin causes neurodegeneration in dystonia musculorum (dt) mutant mice, pointing to a critical role for these proteins in neuronal function.

We have identified microtubule (MT) associated protein 1B (MAP1B) as an interaction partner for dystonin. Dystonin associates with MAP1B in the area surrounding the centrosome, maintaining acetylation of MTs, stabilizing MTs, maintaining cis-Golgi organization, and promoting anterograde trafficking of motor proteins. In dt, we observe altered MAP1B perikaryal localization, MT deacetylation and instability, Golgi fragmentation, and prevention of anterograde trafficking. Maintenance of MT acetylation mitigated the observed defect. Analysis of dt mice further revealed ultrastructural defects at the endoplasmic reticulum (ER) in sensory neurons corresponding with in vivo induction of ER stress and autophagy. Dystonin deficiency leads to sensory neurodegeneration, and blockade of the ER stress signaling cascade rescued neuronal cell death. Thus, our work provides strong evidence that dystonin is important for mediating MT stability, organelle structure, and flux through the secretory pathway in neurons. The next phase of this study will explore the molecular basis of this activity, and assess isoform-specific functions for dystonin.

Cytoskeletal linkers like dystonin facilitate intracellular transport by regulating organelle organization. Since cytoskeletal defects and MT dysfunction are central features of many neurodegenerative disorders, elucidation of the exact role of dystonin in neurons should not only enhance our understanding of the versatile nature of these large modular proteins, but should help provide fundamental insight into the aetiology of these disorders.

2. Cell extrinsic mechanisms in oligodendrocyte biology:
The oligodendrocyte (OL) is the myelin-forming cell in the central nervous system (CNS) and the myelin sheath is essential for the proper functioning of the nervous system. Aberrant or defective myelination can arise from autoimmunity, infectious agents or genetic causes leading to diseases like multiple sclerosis (MS) and leukodystrophies. The morphological remodeling of the OL is initiated by signals from the extracellular matrix (ECM) and requires cytoskeletal reorganization. However, the mechanisms controlling myelination remain poorly understood. With the long-term goal of identifying ways to repair myelin, we are studying the role of the β1 integrin signaling pathway in OL maturation and CNS myelination and remyelination.

We have generated several transgenic mouse models and have established a primary mouse OL progenitor cell (OPC) culture system, which place us in a unique position to study β1 integrin signaling in CNS myelination/remyelination. Our research will elucidate the molecular mechanisms underlying integrin signaling pathway-mediated cytoskeletal changes in OLs and their importance for OL function.

Results from the proposed work will allow us to better understand the role of β1 integrin signaling in CNS myelination/remyelination. This is an essential step towards the development of therapeutic strategies to enhance myelin repair in patients suffering from demyelinating disorders such as MS.

3. Pathogenesis and therapeutics of Spinal Muscular Atrophy (SMA):
Spinal muscular atrophy (SMA) is an inherited and often fatal disease of young children that destroys the nerves controlling voluntary muscle movement, which affects crawling, walking, head and neck control, and swallowing. This neuropathy affects 1/10,000 children and at present there is no cure, even though the gene responsible, Survival Motor Neuron 1 (SMN1), has been known since 1995. SMN protein dosage is inversely correlated with the severity of SMA. Although low levels of SMN are sufficient to allow embryonic development, alpha motor neurons in the spinal cord eventually succumb to the reduced SMN dosage. We have generated a hypomorphic allelic series of mice by the introduction of subtle mutations within the exonic splice enhancer in exon 7 of the murine Smn gene that mimic human SMN2 splicing and reduce Smn protein dosage. Our mice represent the first models of SMA with type II phenotypes.

SMN is a ubiquitously expressed protein. Thus, why some cell types are specifically affected in SMA is not clear but it has been proposed that this may be due to distinct cell-specific roles for SMN. The known ubiquitous role of SMN is in snRNP biogenesis and RNA splicing. However, whether it is the disruption of this housekeeping role or the perturbation of another cell-specific function of SMN that is directly responsible for SMA pathogenesis remains to be addressed.

Although SMA is primarily a motor neuron disease, the involvement of muscle in the pathophysiology has not been entirely ruled out. Based on recent findings showing Smn localization at Z-discs, it would appear that Smn might have a specific function other than snRNP biogenesis in muscle. Furthermore, skeletal muscle is severely affected by Smn-depletion as demonstrated in a conditional knock-out mouse model. These mice display a severe dystrophic phenotype and myocytes that have compromised sarcolemma. In a study from our group, C2C12 myoblasts with reduced Smn expression levels display defects such as abnormal proliferation, aberrant myoblast fusion and malformed myotubes. These studies demonstrate that intrinsic muscle defects are associated with a decrease in Smn levels. That said, the above-mentioned mouse and cell culture model are not necessarily representative of the true SMA pathology given the lack of motor neuron signal in the C2C12 cell model and the absence of residual full length SMN in skeletal muscle of the conditional knock-out mice. Therefore, we are performing a series of histological, ultrastructural and physiological studies on muscle from SMA model mice. Collectively, these studies will allow for a better understanding of the muscle contribution to the SMA phenotype and will lead the field to re-evaluate whether SMA is uniquely a motor neuron disease.

We are now in a position to understand the contribution of various cell types to SMA disease pathogenesis. Determining the cell-specific functions for SMN will elucidate the molecular mechanism of the disease process. Our work is crucial for understanding the pathophysiology of SMA and for the identification of novel targets for therapy. In the long term, the mouse model that we have generated and described here will be critical for evaluating new therapeutics.

4. Defining a role for SMN protein in actin dynamics and SMA pathogenesis:
Recently, SMN has been attributed a role in neuromuscular junction (NMJ) development. Indeed, pre-synaptic defects such as poor terminal arborization, intermediate filament aggregates, and denervation have been observed in SMA mouse models. All of these observations therefore suggest a role for SMN in neurodevelopment and/or neuromaintenance. Our research focuses on the effects of SMN depletion on differentiation, development and maintenance of neuronal cells. Many studies have found correlations between SMN and axon growth. Work using PC12 cells revealed that upon differentiation, the nuclear and cytoplasmic levels of SMN were upregulated, suggesting a role for SMN during this cellular process. Further, SMN's interaction with β-actin and profilin, an actin-binding protein, points towards a possible function for SMN in the regulation of actin dynamics. Since the tight regulation of actin dynamics through specific signaling pathways is crucial for correct neuronal differentiation, we propose that SMN is an essential component for neuritogenesis and neuromaintenance. RhoA and Cdc42 are small GTPases that play an important role in the regulation of actin cytoskeletal dynamics. We have recently shown that active RhoA is increased in spinal cord of the SMA mice and further that Rho-kinase (ROCK) inhibition had a positive impact on the survival and phenotype of these mice. We are presently assessing the impact of a U.S. FDA-approved ROCK inhibitor, fasudil, on the survival and phenotype of the SMA mice. We will continue to assess the potential of using ROCK inhibitors as a therapeutic for SMA.

Key Words:

neurodegeneration, cytoskeleton pre-clinical models, pathogenesis, translational research

Fields of Interest

  • Understanding the mechanisms which maintain/alter muscle and neurvous system integrity in an effort to develop new strategies for the treatment of disorders such as spinal muscular atrophy and Multiple Sclerosis
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