Visualizing Arc protein dynamics and localization in the mammalian brain using AAV-mediated in situ gene labeling

Autores
Avallone, Martino; Pardo, Joaquin; Mergiya, Tadiwos F.; Rájová, Jana; Räsänen, Atte; Davidsson, Marcus; Åkerblom, Malin; Quintino, Luis; Kumar, Darshan; Bramham, Clive R.; Björklund, Tomas
Año de publicación
2023
Idioma
inglés
Tipo de recurso
artículo
Estado
versión publicada
Descripción
The activity-regulated cytoskeleton-associated (Arc) protein is essential for synaptic plasticity and memory formation. The Arc gene, which contains remnants of a structural GAG retrotransposon sequence, produces a protein that self-assembles into capsid-like structures harboring Arc mRNA. Arc capsids, released from neurons, have been proposed as a novel intercellular mechanism for mRNA transmission. Nevertheless, evidence for intercellular transport of Arc in the mammalian brain is still lacking. To enable the tracking of Arc molecules from individual neurons in vivo, we devised an adeno-associated virus (AAV) mediated approach to tag the N-terminal of the mouse Arc protein with a fluorescent reporter using CRISPR/Cas9 homologous independent targeted integration (HITI). We show that a sequence coding for mCherry can successfully be knocked in at the 5′ end of the Arc open reading frame. While nine spCas9 gene editing sites surround the Arc start codon, the accuracy of the editing was highly sequence-dependent, with only a single target resulting in an in-frame reporter integration. When inducing long-term potentiation (LTP) in the hippocampus, we observed an increase of Arc protein highly correlated with an increase in fluorescent intensity and the number of mCherry-positive cells. By proximity ligation assay (PLA), we demonstrated that the mCherry-Arc fusion protein retains the Arc function by interacting with the transmembrane protein stargazin in postsynaptic spines. Finally, we recorded mCherry-Arc interaction with presynaptic protein Bassoon in mCherry-negative surrounding neurons at close proximity to mCherry-positive spines of edited neurons. This is the first study to provide support for inter-neuronal in vivo transfer of Arc in the mammalian brain.
Fil: Avallone, Martino. Lund University. Faculty Of Medicine. Department Of Experimental Medical Science;
Fil: Pardo, Joaquin. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto de Investigaciones Bioquímicas de La Plata "Prof. Dr. Rodolfo R. Brenner". Universidad Nacional de la Plata. Facultad de Ciencias Médicas. Instituto de Investigaciones Bioquímicas de La Plata "Prof. Dr. Rodolfo R. Brenner"; Argentina. Lund University. Faculty Of Medicine. Department Of Experimental Medical Science;
Fil: Mergiya, Tadiwos F.. University of Bergen; Noruega
Fil: Rájová, Jana. Lund University. Faculty Of Medicine. Department Of Experimental Medical Science;
Fil: Räsänen, Atte. Lund University. Faculty Of Medicine. Department Of Experimental Medical Science;
Fil: Davidsson, Marcus. Lund University. Faculty Of Medicine. Department Of Experimental Medical Science;
Fil: Åkerblom, Malin. Lund University. Faculty Of Medicine. Department Of Experimental Medical Science;
Fil: Quintino, Luis. Lund University. Faculty Of Medicine. Department Of Experimental Medical Science;
Fil: Kumar, Darshan. Aiforia Technologies Oyj; Finlandia
Fil: Bramham, Clive R.. University of Bergen; Noruega
Fil: Björklund, Tomas. Lund University. Faculty Of Medicine. Department Of Experimental Medical Science;
Materia
AAV
CELL–CELL TRANSFER
HITI
PLA
RETROTRANSPOSON
SYNAPTIC PLASTICITY
Nivel de accesibilidad
acceso abierto
Condiciones de uso
https://creativecommons.org/licenses/by/2.5/ar/
Repositorio
CONICET Digital (CONICET)
Institución
Consejo Nacional de Investigaciones Científicas y Técnicas
OAI Identificador
oai:ri.conicet.gov.ar:11336/226979
Acceder
id CONICETDig_52c867c60a7b6afa1ba9ec8fa535198b
oai_identifier_str oai:ri.conicet.gov.ar:11336/226979
network_acronym_str CONICETDig
repository_id_str 3498
network_name_str CONICET Digital (CONICET)
spelling Visualizing Arc protein dynamics and localization in the mammalian brain using AAV-mediated in situ gene labelingAvallone, MartinoPardo, JoaquinMergiya, Tadiwos F.Rájová, JanaRäsänen, AtteDavidsson, MarcusÅkerblom, MalinQuintino, LuisKumar, DarshanBramham, Clive R.Björklund, TomasAAVCELL–CELL TRANSFERHITIPLARETROTRANSPOSONSYNAPTIC PLASTICITYhttps://purl.org/becyt/ford/1.6https://purl.org/becyt/ford/1The activity-regulated cytoskeleton-associated (Arc) protein is essential for synaptic plasticity and memory formation. The Arc gene, which contains remnants of a structural GAG retrotransposon sequence, produces a protein that self-assembles into capsid-like structures harboring Arc mRNA. Arc capsids, released from neurons, have been proposed as a novel intercellular mechanism for mRNA transmission. Nevertheless, evidence for intercellular transport of Arc in the mammalian brain is still lacking. To enable the tracking of Arc molecules from individual neurons in vivo, we devised an adeno-associated virus (AAV) mediated approach to tag the N-terminal of the mouse Arc protein with a fluorescent reporter using CRISPR/Cas9 homologous independent targeted integration (HITI). We show that a sequence coding for mCherry can successfully be knocked in at the 5′ end of the Arc open reading frame. While nine spCas9 gene editing sites surround the Arc start codon, the accuracy of the editing was highly sequence-dependent, with only a single target resulting in an in-frame reporter integration. When inducing long-term potentiation (LTP) in the hippocampus, we observed an increase of Arc protein highly correlated with an increase in fluorescent intensity and the number of mCherry-positive cells. By proximity ligation assay (PLA), we demonstrated that the mCherry-Arc fusion protein retains the Arc function by interacting with the transmembrane protein stargazin in postsynaptic spines. Finally, we recorded mCherry-Arc interaction with presynaptic protein Bassoon in mCherry-negative surrounding neurons at close proximity to mCherry-positive spines of edited neurons. This is the first study to provide support for inter-neuronal in vivo transfer of Arc in the mammalian brain.Fil: Avallone, Martino. Lund University. Faculty Of Medicine. Department Of Experimental Medical Science;Fil: Pardo, Joaquin. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto de Investigaciones Bioquímicas de La Plata "Prof. Dr. Rodolfo R. Brenner". Universidad Nacional de la Plata. Facultad de Ciencias Médicas. Instituto de Investigaciones Bioquímicas de La Plata "Prof. Dr. Rodolfo R. Brenner"; Argentina. Lund University. Faculty Of Medicine. Department Of Experimental Medical Science;Fil: Mergiya, Tadiwos F.. University of Bergen; NoruegaFil: Rájová, Jana. Lund University. Faculty Of Medicine. Department Of Experimental Medical Science;Fil: Räsänen, Atte. Lund University. Faculty Of Medicine. Department Of Experimental Medical Science;Fil: Davidsson, Marcus. Lund University. Faculty Of Medicine. Department Of Experimental Medical Science;Fil: Åkerblom, Malin. Lund University. Faculty Of Medicine. Department Of Experimental Medical Science;Fil: Quintino, Luis. Lund University. Faculty Of Medicine. Department Of Experimental Medical Science;Fil: Kumar, Darshan. Aiforia Technologies Oyj; FinlandiaFil: Bramham, Clive R.. University of Bergen; NoruegaFil: Björklund, Tomas. Lund University. Faculty Of Medicine. Department Of Experimental Medical Science;Frontiers Media2023-06info:eu-repo/semantics/articleinfo:eu-repo/semantics/publishedVersionhttp://purl.org/coar/resource_type/c_6501info:ar-repo/semantics/articuloapplication/pdfapplication/pdfhttp://hdl.handle.net/11336/226979Avallone, Martino; Pardo, Joaquin; Mergiya, Tadiwos F.; Rájová, Jana; Räsänen, Atte; et al.; Visualizing Arc protein dynamics and localization in the mammalian brain using AAV-mediated in situ gene labeling; Frontiers Media; Frontiers in Molecular Neuroscience; 16; 6-2023; 1-181662-5099CONICET DigitalCONICETenginfo:eu-repo/semantics/altIdentifier/url/https://www.frontiersin.org/articles/10.3389/fnmol.2023.1140785/fullinfo:eu-repo/semantics/altIdentifier/doi/10.3389/fnmol.2023.1140785info:eu-repo/semantics/openAccesshttps://creativecommons.org/licenses/by/2.5/ar/reponame:CONICET Digital (CONICET)instname:Consejo Nacional de Investigaciones Científicas y Técnicas2025-09-29T10:34:28Zoai:ri.conicet.gov.ar:11336/226979instacron: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:34982025-09-29 10:34:28.537CONICET Digital (CONICET) - Consejo Nacional de Investigaciones Científicas y Técnicasfalse
dc.title.none.fl_str_mv Visualizing Arc protein dynamics and localization in the mammalian brain using AAV-mediated in situ gene labeling
title Visualizing Arc protein dynamics and localization in the mammalian brain using AAV-mediated in situ gene labeling
spellingShingle Visualizing Arc protein dynamics and localization in the mammalian brain using AAV-mediated in situ gene labeling
Avallone, Martino
AAV
CELL–CELL TRANSFER
HITI
PLA
RETROTRANSPOSON
SYNAPTIC PLASTICITY
title_short Visualizing Arc protein dynamics and localization in the mammalian brain using AAV-mediated in situ gene labeling
title_full Visualizing Arc protein dynamics and localization in the mammalian brain using AAV-mediated in situ gene labeling
title_fullStr Visualizing Arc protein dynamics and localization in the mammalian brain using AAV-mediated in situ gene labeling
title_full_unstemmed Visualizing Arc protein dynamics and localization in the mammalian brain using AAV-mediated in situ gene labeling
title_sort Visualizing Arc protein dynamics and localization in the mammalian brain using AAV-mediated in situ gene labeling
dc.creator.none.fl_str_mv Avallone, Martino
Pardo, Joaquin
Mergiya, Tadiwos F.
Rájová, Jana
Räsänen, Atte
Davidsson, Marcus
Åkerblom, Malin
Quintino, Luis
Kumar, Darshan
Bramham, Clive R.
Björklund, Tomas
author Avallone, Martino
author_facet Avallone, Martino
Pardo, Joaquin
Mergiya, Tadiwos F.
Rájová, Jana
Räsänen, Atte
Davidsson, Marcus
Åkerblom, Malin
Quintino, Luis
Kumar, Darshan
Bramham, Clive R.
Björklund, Tomas
author_role author
author2 Pardo, Joaquin
Mergiya, Tadiwos F.
Rájová, Jana
Räsänen, Atte
Davidsson, Marcus
Åkerblom, Malin
Quintino, Luis
Kumar, Darshan
Bramham, Clive R.
Björklund, Tomas
author2_role author
author
author
author
author
author
author
author
author
author
dc.subject.none.fl_str_mv AAV
CELL–CELL TRANSFER
HITI
PLA
RETROTRANSPOSON
SYNAPTIC PLASTICITY
topic AAV
CELL–CELL TRANSFER
HITI
PLA
RETROTRANSPOSON
SYNAPTIC PLASTICITY
purl_subject.fl_str_mv https://purl.org/becyt/ford/1.6
https://purl.org/becyt/ford/1
dc.description.none.fl_txt_mv The activity-regulated cytoskeleton-associated (Arc) protein is essential for synaptic plasticity and memory formation. The Arc gene, which contains remnants of a structural GAG retrotransposon sequence, produces a protein that self-assembles into capsid-like structures harboring Arc mRNA. Arc capsids, released from neurons, have been proposed as a novel intercellular mechanism for mRNA transmission. Nevertheless, evidence for intercellular transport of Arc in the mammalian brain is still lacking. To enable the tracking of Arc molecules from individual neurons in vivo, we devised an adeno-associated virus (AAV) mediated approach to tag the N-terminal of the mouse Arc protein with a fluorescent reporter using CRISPR/Cas9 homologous independent targeted integration (HITI). We show that a sequence coding for mCherry can successfully be knocked in at the 5′ end of the Arc open reading frame. While nine spCas9 gene editing sites surround the Arc start codon, the accuracy of the editing was highly sequence-dependent, with only a single target resulting in an in-frame reporter integration. When inducing long-term potentiation (LTP) in the hippocampus, we observed an increase of Arc protein highly correlated with an increase in fluorescent intensity and the number of mCherry-positive cells. By proximity ligation assay (PLA), we demonstrated that the mCherry-Arc fusion protein retains the Arc function by interacting with the transmembrane protein stargazin in postsynaptic spines. Finally, we recorded mCherry-Arc interaction with presynaptic protein Bassoon in mCherry-negative surrounding neurons at close proximity to mCherry-positive spines of edited neurons. This is the first study to provide support for inter-neuronal in vivo transfer of Arc in the mammalian brain.
Fil: Avallone, Martino. Lund University. Faculty Of Medicine. Department Of Experimental Medical Science;
Fil: Pardo, Joaquin. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto de Investigaciones Bioquímicas de La Plata "Prof. Dr. Rodolfo R. Brenner". Universidad Nacional de la Plata. Facultad de Ciencias Médicas. Instituto de Investigaciones Bioquímicas de La Plata "Prof. Dr. Rodolfo R. Brenner"; Argentina. Lund University. Faculty Of Medicine. Department Of Experimental Medical Science;
Fil: Mergiya, Tadiwos F.. University of Bergen; Noruega
Fil: Rájová, Jana. Lund University. Faculty Of Medicine. Department Of Experimental Medical Science;
Fil: Räsänen, Atte. Lund University. Faculty Of Medicine. Department Of Experimental Medical Science;
Fil: Davidsson, Marcus. Lund University. Faculty Of Medicine. Department Of Experimental Medical Science;
Fil: Åkerblom, Malin. Lund University. Faculty Of Medicine. Department Of Experimental Medical Science;
Fil: Quintino, Luis. Lund University. Faculty Of Medicine. Department Of Experimental Medical Science;
Fil: Kumar, Darshan. Aiforia Technologies Oyj; Finlandia
Fil: Bramham, Clive R.. University of Bergen; Noruega
Fil: Björklund, Tomas. Lund University. Faculty Of Medicine. Department Of Experimental Medical Science;
description The activity-regulated cytoskeleton-associated (Arc) protein is essential for synaptic plasticity and memory formation. The Arc gene, which contains remnants of a structural GAG retrotransposon sequence, produces a protein that self-assembles into capsid-like structures harboring Arc mRNA. Arc capsids, released from neurons, have been proposed as a novel intercellular mechanism for mRNA transmission. Nevertheless, evidence for intercellular transport of Arc in the mammalian brain is still lacking. To enable the tracking of Arc molecules from individual neurons in vivo, we devised an adeno-associated virus (AAV) mediated approach to tag the N-terminal of the mouse Arc protein with a fluorescent reporter using CRISPR/Cas9 homologous independent targeted integration (HITI). We show that a sequence coding for mCherry can successfully be knocked in at the 5′ end of the Arc open reading frame. While nine spCas9 gene editing sites surround the Arc start codon, the accuracy of the editing was highly sequence-dependent, with only a single target resulting in an in-frame reporter integration. When inducing long-term potentiation (LTP) in the hippocampus, we observed an increase of Arc protein highly correlated with an increase in fluorescent intensity and the number of mCherry-positive cells. By proximity ligation assay (PLA), we demonstrated that the mCherry-Arc fusion protein retains the Arc function by interacting with the transmembrane protein stargazin in postsynaptic spines. Finally, we recorded mCherry-Arc interaction with presynaptic protein Bassoon in mCherry-negative surrounding neurons at close proximity to mCherry-positive spines of edited neurons. This is the first study to provide support for inter-neuronal in vivo transfer of Arc in the mammalian brain.
publishDate 2023
dc.date.none.fl_str_mv 2023-06
dc.type.none.fl_str_mv info:eu-repo/semantics/article
info:eu-repo/semantics/publishedVersion
http://purl.org/coar/resource_type/c_6501
info:ar-repo/semantics/articulo
format article
status_str publishedVersion
dc.identifier.none.fl_str_mv http://hdl.handle.net/11336/226979
Avallone, Martino; Pardo, Joaquin; Mergiya, Tadiwos F.; Rájová, Jana; Räsänen, Atte; et al.; Visualizing Arc protein dynamics and localization in the mammalian brain using AAV-mediated in situ gene labeling; Frontiers Media; Frontiers in Molecular Neuroscience; 16; 6-2023; 1-18
1662-5099
CONICET Digital
CONICET
url http://hdl.handle.net/11336/226979
identifier_str_mv Avallone, Martino; Pardo, Joaquin; Mergiya, Tadiwos F.; Rájová, Jana; Räsänen, Atte; et al.; Visualizing Arc protein dynamics and localization in the mammalian brain using AAV-mediated in situ gene labeling; Frontiers Media; Frontiers in Molecular Neuroscience; 16; 6-2023; 1-18
1662-5099
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.frontiersin.org/articles/10.3389/fnmol.2023.1140785/full
info:eu-repo/semantics/altIdentifier/doi/10.3389/fnmol.2023.1140785
dc.rights.none.fl_str_mv info:eu-repo/semantics/openAccess
https://creativecommons.org/licenses/by/2.5/ar/
eu_rights_str_mv openAccess
rights_invalid_str_mv https://creativecommons.org/licenses/by/2.5/ar/
dc.format.none.fl_str_mv application/pdf
application/pdf
dc.publisher.none.fl_str_mv Frontiers Media
publisher.none.fl_str_mv Frontiers Media
dc.source.none.fl_str_mv reponame:CONICET Digital (CONICET)
instname:Consejo Nacional de Investigaciones Científicas y Técnicas
reponame_str CONICET Digital (CONICET)
collection CONICET Digital (CONICET)
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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score 13.070432

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