Effect of rapamycin on lysosomal accumulation in a CRISPR/Cas9-based cellular model of VPS13A deficiency

VPS13A is a lipid transfer protein localized at different membrane contact sites between organelles, and mutations in the corresponding gene produce a rare neurodegenerative disease called chorea-acanthocytosis (ChAc). Previous studies showed that VPS13A depletion in HeLa cells results in an accumul...

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Detalhes bibliográficos
Autores: Tornero Écija, Alba Rocío, Navas Hernández, María Ángeles, Muñoz Braceras, Sandra, Vincent, Olivier, Escalante Hernández, Ricardo
Formato: artículo
Fecha de publicación:2023
País:España
Recursos:Universidad Complutense de Madrid (UCM)
Repositorio:Docta Complutense
Idioma:inglés
OAI Identifier:oai:docta.ucm.es:20.500.14352/91419
Acesso em linha:https://hdl.handle.net/20.500.14352/91419
Access Level:acceso abierto
Palavra-chave:Autophagy
Chorea-acanthocytosis
CRISPR/Cas9
Rapamycin
VPS13A
Ciencias
Ciencias Biomédicas
24 Ciencias de la Vida
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spelling Effect of rapamycin on lysosomal accumulation in a CRISPR/Cas9-based cellular model of VPS13A deficiencyTornero Écija, Alba RocíoNavas Hernández, María ÁngelesMuñoz Braceras, SandraVincent, OlivierEscalante Hernández, RicardoAutophagyChorea-acanthocytosisCRISPR/Cas9RapamycinVPS13ACienciasCiencias Biomédicas24 Ciencias de la VidaVPS13A is a lipid transfer protein localized at different membrane contact sites between organelles, and mutations in the corresponding gene produce a rare neurodegenerative disease called chorea-acanthocytosis (ChAc). Previous studies showed that VPS13A depletion in HeLa cells results in an accumulation of endosomal and lysosomal markers, suggesting a defect in lysosomal degradation capacity leading to partial autophagic dysfunction. Our goal was to determine whether compounds that modulate the endo-lysosomal pathway could be beneficial in the treatment of ChAc. To test this hypothesis, we first generated a KO model using CRISPR/Cas9 to study the consequences of the absence of VPS13A in HeLa cells. We found that inactivation of VPS13A impairs cell growth, which precludes the use of isolated clones due to the undesirable selection of edited clones with residual protein expression. Therefore, we optimized the use of pool cells obtained shortly after transfection with CRISPR/Cas9 components. These cells are a mixture of wild-type and edited cells that allow a comparative analysis of phenotypes and avoids the selection of clones with residual level of VPS13A expression after long-term growth. Consistent with previous observations by siRNA inactivation, VPS13A inactivation by CRISPR/Cas9 resulted in accumulation of the endo-lysosomal markers RAB7A and LAMP1. Notably, we observed that rapamycin partially suppressed the difference in lysosome accumulation between VPS13A KO and WT cells, suggesting that modulation of the autophagic and lysosomal pathway could be a therapeutic target in the treatment of ChAc.WileyUniversidad Complutense de Madrid20232023-05-1020232023-05-10journal articlehttp://purl.org/coar/resource_type/c_6501info:eu-repo/semantics/articleapplication/pdfhttps://hdl.handle.net/20.500.14352/91419reponame:Docta Complutenseinstname:Universidad Complutense de Madrid (UCM)Inglésengopen accesshttp://purl.org/coar/access_right/c_abf2Attribution 4.0 Internationalhttp://creativecommons.org/licenses/by/4.0/info:eu-repo/semantics/openAccessoai:docta.ucm.es:20.500.14352/914192026-06-02T12:44:21Z
dc.title.none.fl_str_mv Effect of rapamycin on lysosomal accumulation in a CRISPR/Cas9-based cellular model of VPS13A deficiency
title Effect of rapamycin on lysosomal accumulation in a CRISPR/Cas9-based cellular model of VPS13A deficiency
spellingShingle Effect of rapamycin on lysosomal accumulation in a CRISPR/Cas9-based cellular model of VPS13A deficiency
Tornero Écija, Alba Rocío
Autophagy
Chorea-acanthocytosis
CRISPR/Cas9
Rapamycin
VPS13A
Ciencias
Ciencias Biomédicas
24 Ciencias de la Vida
title_short Effect of rapamycin on lysosomal accumulation in a CRISPR/Cas9-based cellular model of VPS13A deficiency
title_full Effect of rapamycin on lysosomal accumulation in a CRISPR/Cas9-based cellular model of VPS13A deficiency
title_fullStr Effect of rapamycin on lysosomal accumulation in a CRISPR/Cas9-based cellular model of VPS13A deficiency
title_full_unstemmed Effect of rapamycin on lysosomal accumulation in a CRISPR/Cas9-based cellular model of VPS13A deficiency
title_sort Effect of rapamycin on lysosomal accumulation in a CRISPR/Cas9-based cellular model of VPS13A deficiency
dc.creator.none.fl_str_mv Tornero Écija, Alba Rocío
Navas Hernández, María Ángeles
Muñoz Braceras, Sandra
Vincent, Olivier
Escalante Hernández, Ricardo
author Tornero Écija, Alba Rocío
author_facet Tornero Écija, Alba Rocío
Navas Hernández, María Ángeles
Muñoz Braceras, Sandra
Vincent, Olivier
Escalante Hernández, Ricardo
author_role author
author2 Navas Hernández, María Ángeles
Muñoz Braceras, Sandra
Vincent, Olivier
Escalante Hernández, Ricardo
author2_role author
author
author
author
dc.contributor.none.fl_str_mv Universidad Complutense de Madrid
dc.subject.none.fl_str_mv Autophagy
Chorea-acanthocytosis
CRISPR/Cas9
Rapamycin
VPS13A
Ciencias
Ciencias Biomédicas
24 Ciencias de la Vida
topic Autophagy
Chorea-acanthocytosis
CRISPR/Cas9
Rapamycin
VPS13A
Ciencias
Ciencias Biomédicas
24 Ciencias de la Vida
description VPS13A is a lipid transfer protein localized at different membrane contact sites between organelles, and mutations in the corresponding gene produce a rare neurodegenerative disease called chorea-acanthocytosis (ChAc). Previous studies showed that VPS13A depletion in HeLa cells results in an accumulation of endosomal and lysosomal markers, suggesting a defect in lysosomal degradation capacity leading to partial autophagic dysfunction. Our goal was to determine whether compounds that modulate the endo-lysosomal pathway could be beneficial in the treatment of ChAc. To test this hypothesis, we first generated a KO model using CRISPR/Cas9 to study the consequences of the absence of VPS13A in HeLa cells. We found that inactivation of VPS13A impairs cell growth, which precludes the use of isolated clones due to the undesirable selection of edited clones with residual protein expression. Therefore, we optimized the use of pool cells obtained shortly after transfection with CRISPR/Cas9 components. These cells are a mixture of wild-type and edited cells that allow a comparative analysis of phenotypes and avoids the selection of clones with residual level of VPS13A expression after long-term growth. Consistent with previous observations by siRNA inactivation, VPS13A inactivation by CRISPR/Cas9 resulted in accumulation of the endo-lysosomal markers RAB7A and LAMP1. Notably, we observed that rapamycin partially suppressed the difference in lysosome accumulation between VPS13A KO and WT cells, suggesting that modulation of the autophagic and lysosomal pathway could be a therapeutic target in the treatment of ChAc.
publishDate 2023
dc.date.none.fl_str_mv 2023
2023-05-10
2023
2023-05-10
dc.type.none.fl_str_mv journal article
http://purl.org/coar/resource_type/c_6501
dc.type.openaire.fl_str_mv info:eu-repo/semantics/article
format article
dc.identifier.none.fl_str_mv https://hdl.handle.net/20.500.14352/91419
url https://hdl.handle.net/20.500.14352/91419
dc.language.none.fl_str_mv Inglés
eng
language_invalid_str_mv Inglés
language eng
dc.rights.none.fl_str_mv open access
http://purl.org/coar/access_right/c_abf2
Attribution 4.0 International
http://creativecommons.org/licenses/by/4.0/
dc.rights.openaire.fl_str_mv info:eu-repo/semantics/openAccess
rights_invalid_str_mv open access
http://purl.org/coar/access_right/c_abf2
Attribution 4.0 International
http://creativecommons.org/licenses/by/4.0/
eu_rights_str_mv openAccess
dc.format.none.fl_str_mv application/pdf
dc.publisher.none.fl_str_mv Wiley
publisher.none.fl_str_mv Wiley
dc.source.none.fl_str_mv reponame:Docta Complutense
instname:Universidad Complutense de Madrid (UCM)
instname_str Universidad Complutense de Madrid (UCM)
reponame_str Docta Complutense
collection Docta Complutense
repository.name.fl_str_mv
repository.mail.fl_str_mv
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score 15,300719

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