Atl (atlastin) regulates mTor signaling and autophagy in Drosophila muscle through alteration of the lysosomal network

Abstract

ABSTRACT The hereditary spastic paraplegias (HSPs) represent a family of genetic disorders comprising at least 72 different genes with the common pathology of progressive locomotor deficits and spasticity. ATL1/SPG3A (atlastin GTPase 1) encodes an ER fusion protein that controls ER morphology, which implicates ER structure as a causal factor in HSP. Here we use Drosophila to study effects of decreased atl (atlastin) on properties of the larval body wall muscle. We found that muscle atl loss causes accumulation of aggregates containing polyubiquitin (polyUB), mostly bound to the autophagy receptor ref(2)P/SQSTM1/p62. Muscle atl loss also decreased volume and complexity of the endolysosomal network and decreased lysosome number. To determine effects of these lysosomal deficits on progression through the basal autophagy pathway, we expressed Atg8a tagged with both GFP and mCherry in a wild-type and atl mutant background. We found numerous structures containing mCherry but not GFP fluorescence in wild type, indicating that Atg8a was found mostly in mature autolysosomes. In contrast, muscles lacking atl exhibited significant amounts of GFP signal, indicating failure of autophagosome maturation with acidic lysosomes. Many of these GFP-positive puncta contained the late-endosome marker Rab7 but not Lamp1, indicating that some autophagy cargo was accumulating within amphisomes. We also found that this autophagy block was accompanied by an inability to activate the mTor kinase. Our results provide mechanistic insights into the role of atl in maintaining proper function of the autophagy pathway and suggests that certain pathologies in patients with mutations in ATL1/SPG3A might result from altered MTOR signaling. Abbreviations atl atlastin; ALR autophagic lysosome reformation; ER endoplasmic reticulum; GFP green fluorescent protein; HSP hereditary spastic paraplegia; Lamp1 lysosomal associated membrane protein 1 PolyUB polyubiquitin; RFP red fluorescent protein; spin spinster; mTor mechanistic Target of rapamycin; VCP valosin containing protein