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.