Abstract:
Charcot–Marie–Tooth (CMT) disease is the most common inherited disorder of the peripheral nervous system. It is characterized by progressive distal sensory loss and weakness, muscle atrophy in hands and lower legs, and skeletal deformities. Up to date, approximately 40 genes have been associated with CMT. GDAP1 is one of the few CMTcausative genes in which recessive or dominant mutations lead to demyelinating, axonal or intermediate forms of the disease with ranging onset and/or severity. MTMR2 mutations cause autosomal recessively inherited CMT subtype, CMT4B1 that is a demyelinating form with high severity and early-onset. MTMR2 is known to have a role in endocytosis and membrane trafficking, which are crucial mechanisms for neurons. Therefore, both GDAP1 and MTMR2 are good candidates to study CMT from behavioral and functional aspects. In this study, we aimed to develop Drosophila models for CMT focusing on the fly homologues of these genes, CG4623 and mtm, respectively. In the first part, we verified up- and down-regulation of CG4623 and overexpression of mtm at the mRNA level as we aimed to model the disease by altering the expression levels of these genes. Negative geotaxis assay revealed that altering CG4623 expression levels affected the climbing behavior of the flies in an age-dependent manner recapitulating the CMT phenotype of progressive motor performance decline. In addition, expression level alterations of CG4623 yielded slightly lowered survival with statistical significance. However, RNAi knockdown of mtm resulted in pupal lethality. We have also generated a polyclonal antibody against mtm that allowed us to quantify expression at the protein level and could be further used in functional studies. Lastly, we aimed to verify the fly lines that were generated to knock-out CG4623 and mtm by IMAGO approach. However, our studies showed that IMAGO flies cannot be used in further studies as the targeting vector was integrated incorrectly to their genome. In the scope of this study, we showed that the CMT models that have been developed can be used in further studies to elucidate the CMT pathogenesis and understand the roles of these genes in disease.
