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In vivo Analysis of CRISPR/Cas9 Induced Atlastin Pathological Mutations in Drosophila

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 mainte...

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Autores principales: Montagna, Aldo, Vajente, Nicola, Pendin, Diana, Daga, Andrea
Formato: Online Artículo Texto
Lenguaje:English
Publicado: Frontiers Media S.A. 2020
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7593789/
https://www.ncbi.nlm.nih.gov/pubmed/33177972
http://dx.doi.org/10.3389/fnins.2020.547746
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author Montagna, Aldo
Vajente, Nicola
Pendin, Diana
Daga, Andrea
author_facet Montagna, Aldo
Vajente, Nicola
Pendin, Diana
Daga, Andrea
author_sort Montagna, Aldo
collection PubMed
description 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.
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spelling pubmed-75937892020-11-10 In vivo Analysis of CRISPR/Cas9 Induced Atlastin Pathological Mutations in Drosophila Montagna, Aldo Vajente, Nicola Pendin, Diana Daga, Andrea Front Neurosci Neuroscience 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. Frontiers Media S.A. 2020-10-15 /pmc/articles/PMC7593789/ /pubmed/33177972 http://dx.doi.org/10.3389/fnins.2020.547746 Text en Copyright © 2020 Montagna, Vajente, Pendin and Daga. http://creativecommons.org/licenses/by/4.0/ This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.
spellingShingle Neuroscience
Montagna, Aldo
Vajente, Nicola
Pendin, Diana
Daga, Andrea
In vivo Analysis of CRISPR/Cas9 Induced Atlastin Pathological Mutations in Drosophila
title In vivo Analysis of CRISPR/Cas9 Induced Atlastin Pathological Mutations in Drosophila
title_full In vivo Analysis of CRISPR/Cas9 Induced Atlastin Pathological Mutations in Drosophila
title_fullStr In vivo Analysis of CRISPR/Cas9 Induced Atlastin Pathological Mutations in Drosophila
title_full_unstemmed In vivo Analysis of CRISPR/Cas9 Induced Atlastin Pathological Mutations in Drosophila
title_short In vivo Analysis of CRISPR/Cas9 Induced Atlastin Pathological Mutations in Drosophila
title_sort in vivo analysis of crispr/cas9 induced atlastin pathological mutations in drosophila
topic Neuroscience
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7593789/
https://www.ncbi.nlm.nih.gov/pubmed/33177972
http://dx.doi.org/10.3389/fnins.2020.547746
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