1. Molecular mechanisms of CMT associated with mutations in tRNA synthetases
Heterozygous mutations in five distinct genes encoding cytoplasmic aminoacyl tRNA synthetases (aaRSs) have been identified as a genetic cause of axonal and intermediate forms of CMT: glycyl- (GlyRS), tyrosyl- (TyrRS), alanyl- (AlaRS), histidyl- (HisRS) and methionyl-tRNA synthetase (MetRS). aaRSs are enzymes that covalently attach amino acids to their cognate tRNAs (tRNA aminoacylation), thus catalyzing the first step of mRNA translation. Surprisingly, at least for some CMT-aaRS mutations, loss of aminoacylation activity is not required to cause disease. Rather, a mutation-induced acquired toxicity, a so-called gain-of-toxic-function mechanism, likely underlies peripheral neuropathy.
We have previously generated and characterized Drosophila models for CMT-TyrRS (Storkebaum et al. 2009, Proc. Natl. Acad. Sci. USA) and CMT-GlyRS (Niehues, Bussmann et al. 2015, Nature Communications), which recapitulate several hallmarks of the human disease, including progressive motor deficits, electrophysiological evidence of neuronal dysfunction, axonal degeneration, sensory neuron and neuromuscular junction (NMJ) morphology defects, and progressive muscle denervation with distal muscles being more severely affected. The use of a novel method based on non-canonical amino acid tagging (NCAT), which, for the first time, allows to cell-type-specifically monitor translation in Drosophila in vivo (Erdmann et al. 2015, Nature Communications) revealed that selective expression of each of six distinct GlyRS or TyrRS mutants in either motor or sensory neurons substantially reduced global protein translation, indicating that impaired translation may constitute a common pathogenic mechanism underlying CMT-aaRS (Niehues, Bussmann et al. 2015, Nature Communications).
We are currently evaluating whether or not all CMT mutations in different aaRSs cause disease through similar or disparate molecular mechanisms, and whether this mechanism involves inhibition of mRNA translation. Furthermore, we aim at unraveling the detailed molecular mechanism underlying the translation defect in CMT-aaRS, using Drosophila and mouse models.
2. Deciphering molecular mechanisms of FUS-associated ALS and FTD
FUS is a DNA- and RNA-binding protein involved in regulation of transcription, mRNA splicing, mRNA subcellular localization, and microRNA biogenesis. Mutations in FUS cause familial ALS, and FUS-positive cytoplasmic aggregates are not only found in ALS-FUS, but also in ~10% of frontotemporal dementia (FTD) patients. FUS is one of the protagonists of a group of DNA/RNA-binding proteins (including TDP-43, TAF15, EWSR1, hnRNPA2B1, hnRNPA1 and matrin-3) that have all been implicated in ALS and FTD, suggesting that defects in RNA biogenesis may causally contribute to ALS and possibly FTD pathogenesis.
It has been unclear whether ALS-FUS is caused by a gain-of-toxic-function mechanism, or by (partial) loss of nuclear FUS function. To study both possible scenarios, we have generated (conditional) loss of function and knock-in Drosophila and mouse models. In Drosophila, loss of function of the Drosophila FUS ortholog cabeza (caz) selectively in neurons is both necessary and sufficient to cause motor deficits, indicating that caz has a key role in neurons (Frickenhaus et al. 2015, Sci Rep). In mice, we compared knock-out to knock-in mice, in which a truncated FUS protein that lacks the C-terminal nuclear localization signal is expressed, which mislocalizes to the cytoplasm. Both homozygous (Scekic-Zahirovic, EMBO J, 2016) and heterozygous (Scekic-Zahirovic, Acta Neuropathol, 2017) Fus knock-in, but not knock-out mice, displayed progressive motor neuron loss and motor deficits, indicating that gain-of-toxic-function of cytoplasmically mislocalized FUS underlies these phenotypes. Furthermore, we could show that the truncated mislocalized FUS protein is intrinsically toxic to motor neurons.
We currently aim at elucidating the molecular mechanisms underlying caz mutant phenotypes in Drosophila, and we try to identify the molecular mechanism by which the truncated mislocalized FUS protein induces motor neuron degeneration in mice.