Altered oligomeric states in pathogenic ALS2 variants associated with juvenile motor neuron diseases cause loss of ALS2‐mediated endosomal function

Abstract

ALS2, the causative gene product for a number of recessive motor neuron diseases, forms a homophilic oligomer and acts as a guanine nucleotide exchange factor (GEF) for the small GTPase Rab5. This oligomerization is crucial for both Rab5 activation and ALS2‐mediated endosome fusion and maturation in cells. Recently, we have shown that pathogenic missense ALS2 variants retaining the Rab5 GEF activity fail to properly localize to endosomes via Rac1‐stimulated macropinocytosis. However, the molecular mechanisms underlying dysregulated distribution of ALS2 variants still unknown. In this study, we clarify the relationship between intracellular localization and oligomeric states of pathogenic ALS2 variants. Upon Rac1 activation, all variants moved from the cytosol to membrane ruffles, but not to macropinosomes and/or endosomes. Further, most wild‐type ALS2 in cultured cells forms tetramers. Importantly, the sizes of an ALS2 complex carrying missense mutations in the N‐terminus of RLD or in‐frame amino acid deletion in the PH domain were shifted toward high molecular weight, whereas the C‐terminal VPS9 domain missense mutant existed as a smaller dimeric or trimeric smaller form. Furthermore, in silico mutagenesis analyses using the RLD protein structure in conjunction with a cycloheximide chase assay in vitro disclosed that these missense mutations led to decrease in the protein stability. These results suggested that disorganized higher structures of ALS2 variants might explain their impaired endosomal localization and the stability, leading to loss of the ALS2 function. Currently, to further clarify the in vivo structure, gel‐filtration analyses of the ALS2 complex in murine tissues, including the brain, spinal cord and liver, are underway.Support or Funding InformationThis work was supported by Japanese Society for Promotion of Science Grants 26290018 and 24650189 and in part by the National Natural Science Foundation of China and JSPS Bilateral Joint Research Project.