Functional and morphological evaluation of drosophila CMT models

Abstract

Charcot-Marie-Tooth (CMT) disease is the most frequent inherited peripheral neuropathy in humans with a prevalence of 1 in 2500. It comprises a large group of genetically heterogeneous hereditary motor and sensory neuropathies with mutations identified in 40 causative genes. It is clinically characterized with progressive distal muscle weakness and atrophy, foot deformities, distal sensory loss and depressed tendon reflexes. Mutations in the GDAP1 gene are associated with demyelinating, axonal or mixed forms of the disease with recessive or dominant modes of inheritance. Additionally, GDAP1 is a mitochondrial fission factor with glutathione S-transferase activity and involved in mitochondrial dynamics. MTMR2, on the other hand, is a phosphatase that is suggested to regulate vesicular trafficking in nervous system. Loss of function mutations in MTMR2 gene are linked with autosomal recessive demyelinating CMT type 4B1 neuropathy with myelin outfoldings. Both GDAP1 and MTMR2 genes are good candidates to model CMT disease in terms of their involvement in CMT pathogenesis. In this study, our aim was to generate Drosophila models for CMT disease, specifically for its subtype CMT4, by altering the expression levels of GDAP1 and MTMR2 genes. We have up-or-down regulated Drosophila homologs of GDAP1 and MTMR2, which are CG4623 and mtm in order. In the first part of the study, a CG4623 antibody was generated allowing us to investigate expression pattern of the protein using Western blot and immunohistochemistry assays. With these experiments, ubiquitous expression of CG4623 was shown to be more prominent in the brain, neurons and imaginal discs. Later, we performed synaptic electrophysiology to elucidate functional roles of CG4623 and mtm at the synapses. Our results implicated that both proteins had an effect on synaptic transmission and neurotransmitter release. Lastly, we have investigated changes in neuromuscular junction (NMJ) morphology, bouton structure and mitochondrial dynamics. Since we did not observe any drastic change in the morphology of the mentioned structures in the third instar larvae, we attributed this phenomenon to progressive characteristics of CMT. With this study, we showed that the generated CMT models and the newly developed CG4623 antibody can be further used to illuminate the CMT pathogenesis.