A zebrafish model of Letm 1 and Letm 2 deficiency verfasst

Page 81 6. Abstract Wolf Hirschhorn Syndrome (WHS) is severe gene deletion syndrome associated with a wide range of related symptoms. Different studies showed that WHS patients with seizures are LETM1 haploinsufficient. The leucine zipper-EF-hand containing transmembrane protein 1 (LETM1) is an evolutionary conserved and nucleus encoded mitochondrial protein located in the inner membrane. Recent studies showed that LETM1 regulates the mitochondrial potassium homeostasis and is therefore a factor of the Potassium/Proton-Exchanger (KHE) complex. Disruption of LETM1 in various model systems revealed a temporary growth retardation and mitochondrial swelling, associated with mitophagy and cell death. Furthermore, while DmLetm1 deficient flies are less mobile than the controls, mice do not show any characteristic phenotype. However, injection of a convulsant substance induced prolonged and higher frequent seizures in the mutant rodents, compared to healthy specimens. Besides LETM1, most eukaryotes also encode a LETM1-like protein, termed LETM2. Sequence alignments showed that LETM2 shares various domains with LETM1. However, the role and function of the novel protein is still elusive. The current study was addressed to investigate several aspects of LETM1 and LETM2 in the zebrafish and human cells. Sequence alignments and a phylogenetic analysis of the studied proteins revealed their highly conservation throughout the animal kingdom. Furthermore, it was also found that especially the transmembrane and calcium binding domains are very well preserved. Visualization (In situ hybridization) and quantification (qPCR) of the letm1 and letm2 gene expression in larval and adult tissue samples was further conducted to determine their expression pattern in the zebrafish. The first method revealed a ubiquitous expression of both investigated proteins in larvae and adult fish brains. Quantification of the letm1 expression levels showed a higher abundance in the head than in the residual body during development. However, the levels of letm2 stayed almost even. Interestingly, while the letm2 expression is modestly higher during some developmental stages, adult fish clearly display higher levels of letm1 in all observed organs. A novel letm1 and letm2 haploinsufficient zebrafish strain was generated by means of the TALEN and CRISPR/Cas9 technology. The locomotor behavior of the generated letm1 mutants was further assessed, but did not show an abnormal movement behavior. Furthermore, the established fish lines will serve as important models to investigate additional aspects of Letm1 and Letm2. Moreover, to conduct localization studies two different sets of expression vectors, carrying the fluorescent tagged fish