Kothary Lab

Research Activities

1. A key discovery early in the career of Dr. Kothary was the description of an insertional mouse neurological mutant. The mutation in this mouse caused by transgene insertion was at the dystonia musculorum (dt) locus. This work represented the first reports of insertional mutagenesis in transgenic mice, and spawned the use of "gene-trap" approaches in mice. He subsequently identified the dt gene. Further seminal work included the demonstration that the dt gene encoded a protein, named dystonin, which defined a novel class of cytoskeletal linker proteins since named "plakins". They showed that dystonin is essential for the maintenance of cytoarchitecture integrity in a number of cell types and is important for neuron, muscle and glial cell function. They have continued to work on this protein and have made numerous contributions to show that it interacts with the microtubule associated protein 1B and is essential for neuronal function by regulating cytoskeletal dynamics, organelle stability and function, intracellular trafficking, and autophagy. In ongoing work, they are performing studies to determine the role of dystonin in maintaining neuronal homeostasis by ensuring proper intracellular trafficking and autophagic flux. The goal is to understand the molecular mechanism behind altered tubulin acetylation as well as how dystonin depletion contributes to defects in autophagic flux in neurons. Cytoskeletal linkers like dystonin facilitate intracellular transport flux by regulating microtubule stability and organelle organization. Cytoskeletal defects and microtubule dysfunction are central features of several neurodegenerative disorders. Thus, elucidation of the exact role of dystonin in neurons should not only enhance understanding of these large modular proteins, but should help provide fundamental insight into the aetiology of neurodegenerative disorders, and highlight a link not hitherto appreciated.

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. In multiple sclerosis (MS), immune cells of the body attack the protective coverings of neurons, referred to as myelin. This results in damage to the neurons causing the degenerative symptoms associated with MS. Oligodendrocytes (OLs) are the cell type that produce myelin and are responsible for migrating to the lesion sites and repairing the damaged myelin. Dr. Kothary demonstrated the importance of integrin signaling in myelination and remyelination in the CNS. Further, they developed methods to study OL differentiation in culture that are now widely used by the field. They are now focused on identifying key inhibitory factors at MS lesion sites that prevent the full differentiation of oligodendrocytes and halt remyelination in damaged areas of the CNS. Over the disease time course, the ability of OLs to repair damaged myelin diminishes, partly due to the presence of inhibitory factors like myelin debris, chondroitin sulfate proteoglycans (CSPGs), and netrin at the lesion sites, and thus the severity of the condition increases. To develop therapeutic strategies, one must first understand the mechanisms by which OLs repair damaged myelin. Much work has recently focused on the mechanisms that control OL maturity. The Kothary lab is investigating the molecular mechanism underpinning the inhibitory effect of CSPGs to OL maturation. In addition, they are assessing the role of microRNAs (miRNAs) in the maturation of OLs. They are studying the roles played by miR-145-5p on OL differentiation, myelination and remyelination in both normal biology and in disease. Their model is that the miR-145-5p/myelin regulatory factor (MYRF) axis acts as a critical driver of CNS myelination. The findings will have an impact on development of future therapeutic strategies targeting repair, which is important in the context of MS.

An oligodendrocyte myelinating dorsal root gangion neurite in a co-culture system.

3. Dr. Kothary has taken his expertise in mouse modeling to address the pathogenesis of the devastating human disease spinal muscular atrophy (SMA). SMA is characterized by progressive degeneration of α-motor neurons and consequent muscular atrophy. Accumulating evidence, some of it from the Kothary lab, has indicated that abnormalities in other cell types and organs contribute to the overall disease aetiology in SMA. For example, defects in muscles can exist independent of motor neuron deterioration in SMA. Moreover, profound disruption in the expression level of myogenic genes is associated with Smn reduction in multiple mouse SMA models, suggesting Smn depletion affects regulation of the myogenic program. How exactly Smn deficiency impacts myogenesis, and whether formation and function of resident muscle stem cells, the satellite cells, is disrupted remains largely unknown. Therefore, the Kothary group is interrogating the role of Smn in myogenesis and muscle regeneration. They are conducting a thorough investigation to examine myofibers and muscle satellite cells at multiple stages of disease progression, to assess the ability of Smn-depleted satellite cells to proliferate and differentiate, and to determine the potential of these satellite cells to repopulate and contribute to muscle regeneration. The findings will shed insight into several aspects of muscle biology in Smn-depleted mice, thus expanding our understanding on the requirements of Smn in muscle satellite cell function, and in muscle regeneration. 

The Kothary group has shown that the mouse Smn gene can be induced to undergo alternative splicing of exon 7 by the introduction of mutations within the exonic splice enhancer. This finding opened the way for the generation of mouse SMA models with intermediate phenotypes. His group have shown that mis-regulation of the actin remodeling pathway is a major contributor to the motor neuron loss in SMA. This is a significant finding and will impact on development of therapeutic strategies. Finally, they have shown glucose metabolism and pancreatic developmental defects in mice and humans with SMA. This work points to the need for metabolic assessment and therapeutic intervention when considering clinical care management for SMA patients.

Smn protein expression in single muscle fibers in culture.