Development of a drosophila CMT4A model using imago approach

Abstract

Charcot-Marie-Tooth (CMT) disease is the most common inherited peripheral neuropathy. So far, approximately 50 genes and loci have been associated with CMT. Mutations in ganglioside-induced differentiation-associated protein 1 (GDAP1), cause demyelinating, axonal, or intermediate subtypes of CMT with recessive or dominant segregation. GDAP1 is involved in mitochondrial network dynamics and oxidative stress mechanisms in which perturbations lead to neurodegenerative diseases. However, the molecular mechanisms causing CMT pathogenesis are not clearly understood. GDAP1 is a conserved protein in Eukarya and CG4623 is the Drosophila homolog of GDAP1 with 49% similarity and 31% identity at the amino acid level. The aim of this study is to develop a Drosophila model for CMT disease by silencing the CG4623 gene. In the first part of the project, CG4623 was down-regulated spatiotemporally using the RNA interference (RNAi) method. Ubiquitous down-regulation of CG4623 levels resulted in a significant decrease in the mRNA levels by more than 80% compared to control flies. Tissue-specific down-regulation of CG4623 in glia revealed defects in mitochondrial dynamics in peripheral nerves of larvae. However, this observation could not be verified using the available image analysis and quantification software. In the second part of the project, a fly line that harbors a targeting construct for knocking out CG4623 was generated using an integrase-mediated approach for gene knockout (IMAGO). The target vector was injected to Drosophila embryos with the expectation of site-specific integration into the second chromosome. This line was crossed with SceI & Flp lines and the progeny was screened for CG4623 knockout flies. During these crosses it was observed that the construct was integrated into the X chromosome instead of the second. This integration was unexpected; however it does not affect future crosses and analyses. More progeny flies should be screened in order to obtain CG4623 knockout flies.