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

AVALLONE, MARTINO; PARDO, JOAQUÍN; MERGIYA, TADIWOS F.; RÁJOVÁ, JANA; RÄSÄNEN, ATTE; DAVIDSSON, MARCUS; ÅKERBLOM, MALIN; QUINTINO, LUIS; KUMAR, DARSHAN; BRAMHAM, CLIVE R.; BJÖRKLUND, TOMAS

Frontiers in Molecular Neuroscience

Frontiers Media S.A.

Año: 2023 vol. 16

The activity-regulated cytoskeleton-associated (Arc) protein is essential forsynaptic plasticity and memory formation. The Arc gene, which containsremnants of a structural GAG retrotransposon sequence, produces a proteinthat self-assembles into capsid-like structures harboring Arc mRNA. Arc capsids,released from neurons, have been proposed as a novel intercellular mechanismfor mRNA transmission. Nevertheless, evidence for intercellular transport of Arc inthe mammalian brain is still lacking. To enable the tracking of Arc molecules fromindividual neurons in vivo, we devised an adeno-associated virus (AAV) mediatedapproach to tag the N-terminal of the mouse Arc protein with a fluorescentreporter using CRISPR/Cas9 homologous independent targeted integration (HITI).We show that a sequence coding for mCherry can successfully be knocked in atthe 5′ end of the Arc open reading frame. While nine spCas9 gene editing sitessurround the Arc start codon, the accuracy of the editing was highly sequencedependent,with only a single target resulting in an in-frame reporter integration.When inducing long-term potentiation (LTP) in the hippocampus, we observed anincrease of Arc protein highly correlated with an increase in fluorescent intensityand the number of mCherry-positive cells. By proximity ligation assay (PLA),we demonstrated that the mCherry-Arc fusion protein retains the Arc functionby interacting with the transmembrane protein stargazin in postsynaptic spines.Finally, we recorded mCherry-Arc interaction with presynaptic protein Bassoonin mCherry-negative surrounding neurons at close proximity to mCherry-positivespines of edited neurons. This is the first study to provide support for interneuronalin vivo transfer of Arc in the mammalian brain.