Abstract
The endoplasmic reticulum (ER) is a highly dynamic network whose shape is thought to be actively regulated by membrane resident proteins. Mutation of several such morphology regulators cause the neurological disorder Hereditary Sp astic Paraplegia (HSP), suggesting a critical role of ER shape maintenance in neuronal activity and function. Human Atlastin-1 mutations are responsible for SPG3A, the earliest onset and one of the more severe forms of dominant HSP. Atlastin has been initially identified in Drosophila as the GTPase responsible for the homotypic fusion of ER membrane. The majority of SPG3A-linked Atlastin-1 mutations map to the GTPase domain, potentially interfering with atlastin GTPase activity, and to the three-helix-bundle (3HB) domain, a region critical for homo-oligomerization. Here we have examined the in vivo effects of four pathogenetic missense mutations (two mapping to the GTPase domain and two to the 3HB domain) using two complementary approaches: CRISPR/Cas9 editing to introduce such variants in the endogenous atlastin gene and transgenesis to generate lines overexpressing atlastin carrying the same pathogenic variants. We found that all pathological mutations examined reduce atlastin activity in vivo although to different degrees of severity. Moreover, overexpression of the pathogenic variants in a wild type atlastin background does not give rise to the loss of function phenotypes expected for dominant negative mutations. These results indicate that the four pathological mutations investigated act through a loss of function mechanism.
Highlights
Atlastin-1 is one of a three-member family of dynamin-like GTPases present in vertebrate genomes, single homologs of atlastin are present in invertebrates, yeast and plants (Hu and Rapoport, 2016)
R192Q/R217Q is completely defective in dimerization, GTPase and fusion activities (Bian et al, 2011; Byrnes and Sondermann, 2011; Ulengin et al, 2015); R214C/R239C is essentially indistinguishable from wild type under all in vitro experimental paradigms (Byrnes and Sondermann, 2011; Ulengin et al, 2015), despite being the most common pathological mutation; the C350R/C375R variant has not been studied in vitro because it is insoluble due to protein folding or stability issues (Byrnes and Sondermann, 2011; Ulengin et al, 2015); M383T/M408T dimerization and GTPase activities have been shown to be slightly lower but comparable to wild type while its fusion activity has not been tested (Bian et al, 2011; Byrnes and Sondermann, 2011)
Our study exploits Drosophila as an in vivo system to comprehend the role of organelle morphology in the onset of SPG3A-hereditary spastic paraplegia (HSP) disease and to model the mechanism whereby highly conserved pathological mutations cause this disease
Summary
Atlastin-1 is one of a three-member family of dynamin-like GTPases present in vertebrate genomes, single homologs of atlastin are present in invertebrates, yeast and plants (Hu and Rapoport, 2016). The atlastins are membrane proteins, embedded in the endoplasmic reticulum (ER) bilayer, whose main function is to mediate ER homotypic membrane fusion, a process crucial for proper ER morphogenesis and maintenance (Hu and Rapoport, 2016). To date, this ability to promote fusion has been demonstrated exclusively for invertebrate atlastins (Orso et al, 2009; Anwar et al, 2012; Zhang et al, 2013; Wu et al, 2014). The genotype-phenotype correlation remains utterly unclear (Zhao and Liu, 2017)
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