TY - JOUR
T1 - In vivo Analysis of CRISPR/Cas9 Induced Atlastin Pathological Mutations in Drosophila
AU - Montagna, Aldo
AU - Vajente, Nicola
AU - Pendin, Diana
AU - Daga, Andrea
N1 - Funding Information:
We thank Jessica Tosetto, Tatiana Trevisan, Giulia Misticoni, Genny Orso, and Alessia Gazziero for generating fly lines and constructs used in this study. Stocks obtained from the Bloomington Drosophila Stock Center (NIH P40OD018537) were used in this study. atl 2 fly line was a kind gift from S. Lee. Funding. This work was supported by the Ministry of Education, University and Research (MIUR; fellowship to NV); Fondazione Cassa di Risparmio di Padova e Rovigo (CARIPARO Foundation, Starting Grant 2015 to DP); Fondazione Telethon (GGP19304 to DP; GGP11189 to AD); and Italian Ministry of Health (5 ? 1000 and RC 2018, 2019, 2020 to AD).
Publisher Copyright:
© Copyright © 2020 Montagna, Vajente, Pendin and Daga.
Copyright:
Copyright 2020 Elsevier B.V., All rights reserved.
PY - 2020/10/15
Y1 - 2020/10/15
N2 - 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.
AB - 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.
KW - atlastin
KW - CRISPR/Cas9
KW - endoplasmic reticulum
KW - Golgi
KW - hereditary spastic paraplegia
KW - mutation
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U2 - 10.3389/fnins.2020.547746
DO - 10.3389/fnins.2020.547746
M3 - Article
AN - SCOPUS:85094838560
VL - 14
JO - Frontiers in Neuroscience
JF - Frontiers in Neuroscience
SN - 1662-4548
M1 - 547746
ER -