Expression, purification of recombinant apolipoprotein E4 under non-denaturing conditions, and mitochondrial morphology analysis in a neuronal clonal cell line

Autores
Serrano, Ezequiel Antonio; Valdivieso, Ángel Gabriel; Barrantes, Francisco Jose
Año de publicación
2021
Idioma
inglés
Tipo de recurso
documento de conferencia
Estado
versión publicada
Descripción
Apolipoprotein E (ApoE) is the key risk factor for Alzheimer disease. The APOE4 allele increases the probability of developing the disease up to 15 times in homozygote carriers. Among other effects, ApoE4 has been associated with alterations in mitochondrial dynamics. In order to learn about the effects of ApoE4 on neuronal cells, we aimed at purifying the recombinant protein expressed in E. coli and assaying it on mitochondria of a neuronal clonal cell line, CNh. E. coli BL21 strain expressing the ApoE4 (pET32-E43C, containing His and Trx-tags) and 3C-protease were grown in LB medium and induced with 1 mM of IPTG for 1.5 and 4 h at 37 and 30Cº, respectively. Crude soluble ApoE was purified by affinity chromatography using a Ni-NTA resin. The 3C-protease was purified by FPLC (Superose-12 size exclusion). ApoE4 and 3C-protease were incubated at 4ºC in a 25:1 ratio to release the His-Trx tag. ApoE4 was next purified by size exclusion chromatography, with a yield of ~5 mg/L without denaturing/renaturing steps and no apparent contaminant bands in SDS polyacrylamide gels. We next studied mitochondrial morphology in neuronal CNh cells using confocal fluorescence microscopy. Cells were incubated with17mg/mL ApoE4 and mitochondria labelled with MitoTracker Orange and imaged in vivo. Mitochondrial morphology was analyzed with the Mitochondrial Network Analysis plugin for Fiji. Individual counts (<1 branch), network counts (>1 branch), network length and mitochondrial footprint (area covered) were quantified. ApoE4 induced an increase in mitochondrial footprint (p<0.001) and mitochondrial network (p<0.001) indicating an ApoE4-induced increase in mitochondrial proliferation.
Fil: Serrano, Ezequiel Antonio. Pontificia Universidad Católica Argentina "Santa María de los Buenos Aires". Facultad de Ciencias Médicas. Instituto de Investigaciones Biomédicas; Argentina
Fil: Valdivieso, Ángel Gabriel. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Investigaciones Biomédicas. Universidad de Buenos Aires. Facultad de Medicina. Instituto de Investigaciones Biomédicas; Argentina. Pontificia Universidad Católica Argentina "Santa María de los Buenos Aires". Facultad de Ciencias Médicas. Instituto de Investigaciones Biomédicas; Argentina
Fil: Barrantes, Francisco Jose. Pontificia Universidad Católica Argentina "Santa María de los Buenos Aires". Facultad de Ciencias Médicas. Instituto de Investigaciones Biomédicas; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Investigaciones Biomédicas. Universidad de Buenos Aires. Facultad de Medicina. Instituto de Investigaciones Biomédicas; Argentina
LXVI Reunión anual de la Sociedad Argentina de Investigación Clínica; LXIX Reunión anual de la Sociedad Argentina de Immunología; LIII Reunión anual de la Asociación Argentina de Farmacología Experimental y XI Reunión anual de la Asociación Argentina de Nanomedicinas
Buenos Aires
Argentina
Sociedad Argentina de Inmunología
Asociación Argentina de Farmacología Experimental
Sociedad Argentina de Investigación Clínica
Asociación Argentina de Nanomedicinas
Materia
Alzheimer
Apolipoprotein E
APOE4
Cholesterol
Nivel de accesibilidad
acceso abierto
Condiciones de uso
https://creativecommons.org/licenses/by-nc-sa/2.5/ar/
Repositorio
CONICET Digital (CONICET)
Institución
Consejo Nacional de Investigaciones Científicas y Técnicas
OAI Identificador
oai:ri.conicet.gov.ar:11336/280751
Acceder
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oai_identifier_str oai:ri.conicet.gov.ar:11336/280751
network_acronym_str CONICETDig
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network_name_str CONICET Digital (CONICET)
spelling Expression, purification of recombinant apolipoprotein E4 under non-denaturing conditions, and mitochondrial morphology analysis in a neuronal clonal cell lineSerrano, Ezequiel AntonioValdivieso, Ángel GabrielBarrantes, Francisco JoseAlzheimerApolipoprotein EAPOE4Cholesterolhttps://purl.org/becyt/ford/3.1https://purl.org/becyt/ford/3Apolipoprotein E (ApoE) is the key risk factor for Alzheimer disease. The APOE4 allele increases the probability of developing the disease up to 15 times in homozygote carriers. Among other effects, ApoE4 has been associated with alterations in mitochondrial dynamics. In order to learn about the effects of ApoE4 on neuronal cells, we aimed at purifying the recombinant protein expressed in E. coli and assaying it on mitochondria of a neuronal clonal cell line, CNh. E. coli BL21 strain expressing the ApoE4 (pET32-E43C, containing His and Trx-tags) and 3C-protease were grown in LB medium and induced with 1 mM of IPTG for 1.5 and 4 h at 37 and 30Cº, respectively. Crude soluble ApoE was purified by affinity chromatography using a Ni-NTA resin. The 3C-protease was purified by FPLC (Superose-12 size exclusion). ApoE4 and 3C-protease were incubated at 4ºC in a 25:1 ratio to release the His-Trx tag. ApoE4 was next purified by size exclusion chromatography, with a yield of ~5 mg/L without denaturing/renaturing steps and no apparent contaminant bands in SDS polyacrylamide gels. We next studied mitochondrial morphology in neuronal CNh cells using confocal fluorescence microscopy. Cells were incubated with17mg/mL ApoE4 and mitochondria labelled with MitoTracker Orange and imaged in vivo. Mitochondrial morphology was analyzed with the Mitochondrial Network Analysis plugin for Fiji. Individual counts (<1 branch), network counts (>1 branch), network length and mitochondrial footprint (area covered) were quantified. ApoE4 induced an increase in mitochondrial footprint (p<0.001) and mitochondrial network (p<0.001) indicating an ApoE4-induced increase in mitochondrial proliferation.Fil: Serrano, Ezequiel Antonio. Pontificia Universidad Católica Argentina "Santa María de los Buenos Aires". Facultad de Ciencias Médicas. Instituto de Investigaciones Biomédicas; ArgentinaFil: Valdivieso, Ángel Gabriel. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Investigaciones Biomédicas. Universidad de Buenos Aires. Facultad de Medicina. Instituto de Investigaciones Biomédicas; Argentina. Pontificia Universidad Católica Argentina "Santa María de los Buenos Aires". Facultad de Ciencias Médicas. Instituto de Investigaciones Biomédicas; ArgentinaFil: Barrantes, Francisco Jose. Pontificia Universidad Católica Argentina "Santa María de los Buenos Aires". Facultad de Ciencias Médicas. Instituto de Investigaciones Biomédicas; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Investigaciones Biomédicas. Universidad de Buenos Aires. Facultad de Medicina. Instituto de Investigaciones Biomédicas; ArgentinaLXVI Reunión anual de la Sociedad Argentina de Investigación Clínica; LXIX Reunión anual de la Sociedad Argentina de Immunología; LIII Reunión anual de la Asociación Argentina de Farmacología Experimental y XI Reunión anual de la Asociación Argentina de NanomedicinasBuenos AiresArgentinaSociedad Argentina de InmunologíaAsociación Argentina de Farmacología ExperimentalSociedad Argentina de Investigación ClínicaAsociación Argentina de NanomedicinasFundación Revista Medicina2021info:eu-repo/semantics/publishedVersioninfo:eu-repo/semantics/conferenceObjectReuniónJournalhttp://purl.org/coar/resource_type/c_5794info:ar-repo/semantics/documentoDeConferenciaapplication/pdfapplication/pdfapplication/pdfapplication/pdfhttp://hdl.handle.net/11336/280751Expression, purification of recombinant apolipoprotein E4 under non-denaturing conditions, and mitochondrial morphology analysis in a neuronal clonal cell line; LXVI Reunión anual de la Sociedad Argentina de Investigación Clínica; LXIX Reunión anual de la Sociedad Argentina de Immunología; LIII Reunión anual de la Asociación Argentina de Farmacología Experimental y XI Reunión anual de la Asociación Argentina de Nanomedicinas; Buenos Aires; Argentina; 2021; 1-1CONICET DigitalCONICETenginfo:eu-repo/semantics/altIdentifier/url/https://www.reunionbiociencias.com.ar/wp-content/uploads/2021/11/Revista-Medicina-2021.pdfNacionalinfo:eu-repo/semantics/openAccesshttps://creativecommons.org/licenses/by-nc-sa/2.5/ar/reponame:CONICET Digital (CONICET)instname:Consejo Nacional de Investigaciones Científicas y Técnicas2026-02-26T10:12:16Zoai:ri.conicet.gov.ar:11336/280751instacron:CONICETInstitucionalhttp://ri.conicet.gov.ar/Organismo científico-tecnológicoNo correspondehttp://ri.conicet.gov.ar/oai/requestdasensio@conicet.gov.ar; lcarlino@conicet.gov.arArgentinaNo correspondeNo correspondeNo correspondeopendoar:34982026-02-26 10:12:17.273CONICET Digital (CONICET) - Consejo Nacional de Investigaciones Científicas y Técnicasfalse
dc.title.none.fl_str_mv Expression, purification of recombinant apolipoprotein E4 under non-denaturing conditions, and mitochondrial morphology analysis in a neuronal clonal cell line
title Expression, purification of recombinant apolipoprotein E4 under non-denaturing conditions, and mitochondrial morphology analysis in a neuronal clonal cell line
spellingShingle Expression, purification of recombinant apolipoprotein E4 under non-denaturing conditions, and mitochondrial morphology analysis in a neuronal clonal cell line
Serrano, Ezequiel Antonio
Alzheimer
Apolipoprotein E
APOE4
Cholesterol
title_short Expression, purification of recombinant apolipoprotein E4 under non-denaturing conditions, and mitochondrial morphology analysis in a neuronal clonal cell line
title_full Expression, purification of recombinant apolipoprotein E4 under non-denaturing conditions, and mitochondrial morphology analysis in a neuronal clonal cell line
title_fullStr Expression, purification of recombinant apolipoprotein E4 under non-denaturing conditions, and mitochondrial morphology analysis in a neuronal clonal cell line
title_full_unstemmed Expression, purification of recombinant apolipoprotein E4 under non-denaturing conditions, and mitochondrial morphology analysis in a neuronal clonal cell line
title_sort Expression, purification of recombinant apolipoprotein E4 under non-denaturing conditions, and mitochondrial morphology analysis in a neuronal clonal cell line
dc.creator.none.fl_str_mv Serrano, Ezequiel Antonio
Valdivieso, Ángel Gabriel
Barrantes, Francisco Jose
author Serrano, Ezequiel Antonio
author_facet Serrano, Ezequiel Antonio
Valdivieso, Ángel Gabriel
Barrantes, Francisco Jose
author_role author
author2 Valdivieso, Ángel Gabriel
Barrantes, Francisco Jose
author2_role author
author
dc.subject.none.fl_str_mv Alzheimer
Apolipoprotein E
APOE4
Cholesterol
topic Alzheimer
Apolipoprotein E
APOE4
Cholesterol
purl_subject.fl_str_mv https://purl.org/becyt/ford/3.1
https://purl.org/becyt/ford/3
dc.description.none.fl_txt_mv Apolipoprotein E (ApoE) is the key risk factor for Alzheimer disease. The APOE4 allele increases the probability of developing the disease up to 15 times in homozygote carriers. Among other effects, ApoE4 has been associated with alterations in mitochondrial dynamics. In order to learn about the effects of ApoE4 on neuronal cells, we aimed at purifying the recombinant protein expressed in E. coli and assaying it on mitochondria of a neuronal clonal cell line, CNh. E. coli BL21 strain expressing the ApoE4 (pET32-E43C, containing His and Trx-tags) and 3C-protease were grown in LB medium and induced with 1 mM of IPTG for 1.5 and 4 h at 37 and 30Cº, respectively. Crude soluble ApoE was purified by affinity chromatography using a Ni-NTA resin. The 3C-protease was purified by FPLC (Superose-12 size exclusion). ApoE4 and 3C-protease were incubated at 4ºC in a 25:1 ratio to release the His-Trx tag. ApoE4 was next purified by size exclusion chromatography, with a yield of ~5 mg/L without denaturing/renaturing steps and no apparent contaminant bands in SDS polyacrylamide gels. We next studied mitochondrial morphology in neuronal CNh cells using confocal fluorescence microscopy. Cells were incubated with17mg/mL ApoE4 and mitochondria labelled with MitoTracker Orange and imaged in vivo. Mitochondrial morphology was analyzed with the Mitochondrial Network Analysis plugin for Fiji. Individual counts (<1 branch), network counts (>1 branch), network length and mitochondrial footprint (area covered) were quantified. ApoE4 induced an increase in mitochondrial footprint (p<0.001) and mitochondrial network (p<0.001) indicating an ApoE4-induced increase in mitochondrial proliferation.
Fil: Serrano, Ezequiel Antonio. Pontificia Universidad Católica Argentina "Santa María de los Buenos Aires". Facultad de Ciencias Médicas. Instituto de Investigaciones Biomédicas; Argentina
Fil: Valdivieso, Ángel Gabriel. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Investigaciones Biomédicas. Universidad de Buenos Aires. Facultad de Medicina. Instituto de Investigaciones Biomédicas; Argentina. Pontificia Universidad Católica Argentina "Santa María de los Buenos Aires". Facultad de Ciencias Médicas. Instituto de Investigaciones Biomédicas; Argentina
Fil: Barrantes, Francisco Jose. Pontificia Universidad Católica Argentina "Santa María de los Buenos Aires". Facultad de Ciencias Médicas. Instituto de Investigaciones Biomédicas; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Investigaciones Biomédicas. Universidad de Buenos Aires. Facultad de Medicina. Instituto de Investigaciones Biomédicas; Argentina
LXVI Reunión anual de la Sociedad Argentina de Investigación Clínica; LXIX Reunión anual de la Sociedad Argentina de Immunología; LIII Reunión anual de la Asociación Argentina de Farmacología Experimental y XI Reunión anual de la Asociación Argentina de Nanomedicinas
Buenos Aires
Argentina
Sociedad Argentina de Inmunología
Asociación Argentina de Farmacología Experimental
Sociedad Argentina de Investigación Clínica
Asociación Argentina de Nanomedicinas
description Apolipoprotein E (ApoE) is the key risk factor for Alzheimer disease. The APOE4 allele increases the probability of developing the disease up to 15 times in homozygote carriers. Among other effects, ApoE4 has been associated with alterations in mitochondrial dynamics. In order to learn about the effects of ApoE4 on neuronal cells, we aimed at purifying the recombinant protein expressed in E. coli and assaying it on mitochondria of a neuronal clonal cell line, CNh. E. coli BL21 strain expressing the ApoE4 (pET32-E43C, containing His and Trx-tags) and 3C-protease were grown in LB medium and induced with 1 mM of IPTG for 1.5 and 4 h at 37 and 30Cº, respectively. Crude soluble ApoE was purified by affinity chromatography using a Ni-NTA resin. The 3C-protease was purified by FPLC (Superose-12 size exclusion). ApoE4 and 3C-protease were incubated at 4ºC in a 25:1 ratio to release the His-Trx tag. ApoE4 was next purified by size exclusion chromatography, with a yield of ~5 mg/L without denaturing/renaturing steps and no apparent contaminant bands in SDS polyacrylamide gels. We next studied mitochondrial morphology in neuronal CNh cells using confocal fluorescence microscopy. Cells were incubated with17mg/mL ApoE4 and mitochondria labelled with MitoTracker Orange and imaged in vivo. Mitochondrial morphology was analyzed with the Mitochondrial Network Analysis plugin for Fiji. Individual counts (<1 branch), network counts (>1 branch), network length and mitochondrial footprint (area covered) were quantified. ApoE4 induced an increase in mitochondrial footprint (p<0.001) and mitochondrial network (p<0.001) indicating an ApoE4-induced increase in mitochondrial proliferation.
publishDate 2021
dc.date.none.fl_str_mv 2021
dc.type.none.fl_str_mv info:eu-repo/semantics/publishedVersion
info:eu-repo/semantics/conferenceObject
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http://purl.org/coar/resource_type/c_5794
info:ar-repo/semantics/documentoDeConferencia
status_str publishedVersion
format conferenceObject
dc.identifier.none.fl_str_mv http://hdl.handle.net/11336/280751
Expression, purification of recombinant apolipoprotein E4 under non-denaturing conditions, and mitochondrial morphology analysis in a neuronal clonal cell line; LXVI Reunión anual de la Sociedad Argentina de Investigación Clínica; LXIX Reunión anual de la Sociedad Argentina de Immunología; LIII Reunión anual de la Asociación Argentina de Farmacología Experimental y XI Reunión anual de la Asociación Argentina de Nanomedicinas; Buenos Aires; Argentina; 2021; 1-1
CONICET Digital
CONICET
url http://hdl.handle.net/11336/280751
identifier_str_mv Expression, purification of recombinant apolipoprotein E4 under non-denaturing conditions, and mitochondrial morphology analysis in a neuronal clonal cell line; LXVI Reunión anual de la Sociedad Argentina de Investigación Clínica; LXIX Reunión anual de la Sociedad Argentina de Immunología; LIII Reunión anual de la Asociación Argentina de Farmacología Experimental y XI Reunión anual de la Asociación Argentina de Nanomedicinas; Buenos Aires; Argentina; 2021; 1-1
CONICET Digital
CONICET
dc.language.none.fl_str_mv eng
language eng
dc.relation.none.fl_str_mv info:eu-repo/semantics/altIdentifier/url/https://www.reunionbiociencias.com.ar/wp-content/uploads/2021/11/Revista-Medicina-2021.pdf
dc.rights.none.fl_str_mv info:eu-repo/semantics/openAccess
https://creativecommons.org/licenses/by-nc-sa/2.5/ar/
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application/pdf
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dc.coverage.none.fl_str_mv Nacional
dc.publisher.none.fl_str_mv Fundación Revista Medicina
publisher.none.fl_str_mv Fundación Revista Medicina
dc.source.none.fl_str_mv reponame:CONICET Digital (CONICET)
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reponame_str CONICET Digital (CONICET)
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instname_str Consejo Nacional de Investigaciones Científicas y Técnicas
repository.name.fl_str_mv CONICET Digital (CONICET) - Consejo Nacional de Investigaciones Científicas y Técnicas
repository.mail.fl_str_mv dasensio@conicet.gov.ar; lcarlino@conicet.gov.ar
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