Loss-of-function mutation in Hippo suppressed enlargement of lysosomes and neurodegeneration caused by dFIG4 knockdown

Abstract

Charcot–Marie–Tooth disease (CMT) is the most common hereditary neuropathy, and more than 80 CMT-causing genes have been identified to date. CMT4J is caused by a loss-of-function mutation in the Factor-Induced-Gene 4 (FIG4) gene, the product of which plays important roles in endosome–lysosome homeostasis. We hypothesized that Mammalian sterile 20-like kinase (MST) 1 and 2, tumor-suppressor genes, are candidate modifiers of CMT4J. We therefore examined the interaction between dFIG4 and Hippo (hpo), Drosophila counterparts of FIG4 and MSTs, respectively, using the Drosophila CMT4J model with the knockdown of dFIG4. The loss-of-function allele of hpo improved the rough eye morphology, locomotive dysfunction accompanied by structural defects in the presynaptic terminals of motoneurons, and the enlargement of lysosomes caused by the knockdown of dFIG4. Therefore, we identified hpo as a modifier of phenotypes induced by the knockdown of dFIG4. These results in Drosophila may provide an insight into the pathogenesis of CMT4J and contribute toward the development of disease-modifying therapy for CMT. We also identified the regulation of endosome–lysosome homeostasis as a novel probable function of Hippo/MST.

Genetic modifiers and non-Mendelian aspects of CMT

“…Knockdown of dFIG4 in this model resulted in aberrant motor neuron morphology, a phenotype that was improved through the downregulation of hpo. 63…”

Identification of Rpd3 as a novel epigenetic regulator of Drosophila FIG 4, a Charcot-Marie-Tooth disease-causing gene

“…Synapses were visualized as described previously [7,9]. In brief, third instar larvae were dissected and fixed in 4% paraformaldehyde in PBS at 25°C for 30 min.…”

“…Samples were incubated in PBS containing 0.1% Triton X-100 and FITC-conjugated goat anti-HRP IgG at 1:1000 (Jackson ImmunoResearch, Pennsylvania, USA). Samples were examined under a confocal laser scanning microscope (Olympus FLUOVIEW FV10i, Tokyo, Japan) [7,9]. The length of the longest synaptic branch at the NMJ of muscle 4 was measured and analyzed using MetaMorph Imaging System 7.7 (Molecular Devices, California, USA) [7,9].…”

“…Drosophila contains a single ortholog of human FIG 4, designated as dFIG 4 [7]. Knockdown of dFIG 4 in pan-neurons reduced the locomotive ability accompanying a reduced length of synapses at the neuromuscular junctions (NMJs) [7][8][9]. The loss-of-function dFIG 4 mutants also exhibited enlarged vacuoles in muscles and neurons, resulting in deficient flying ability [10].…”

“…Defects in epigenetic regulators, such as HDAC1, may therefore explain the diverse symptoms of human disorders associated with FIG 4 mutations. w; UAS-dFIG 4-IR 605-714 ; + (CG17840) and w; UAS-dFIG 4-IR 516-523 ; + (CG17840) obtained from the Vienna Drosophila RNAi Center (VDRC) and the Bloomington Drosophila Stock Center (BDSC), respectively, were fully characterized previously [7,9,11]. Flies carrying w; +; P[GAL4-elav.L] 3 , w; +; P[nSyb-GAL4.S] 3 , w; +; P[Lsp2-GAL4], w; +; Rpd3 def24 /TM6B, w; +; Rpd3 303 or w; +; Rpd3 04556 were obtained from BDSC.…”