AstroDot -a new method for studying the spatial distribution of mRNA in astrocytes

Autor: Martine Cohen-Salmon, Noémie Mazaré, Marc Oudart, Romain Tortuyaux, Philippe Mailly, Anne-Cécile Boulay
Přispěvatelé: Centre interdisciplinaire de recherche en biologie (CIRB), Labex MemoLife, École normale supérieure - Paris (ENS Paris), Université Paris sciences et lettres (PSL)-Université Paris sciences et lettres (PSL)-Collège de France (CdF (institution))-Ecole Superieure de Physique et de Chimie Industrielles de la Ville de Paris (ESPCI Paris), Université Paris sciences et lettres (PSL)-École normale supérieure - Paris (ENS Paris), Université Paris sciences et lettres (PSL)-Centre National de la Recherche Scientifique (CNRS)-Institut National de la Santé et de la Recherche Médicale (INSERM), Centre Hospitalier Régional Universitaire [Lille] (CHRU Lille), Oudart, Marc, École normale supérieure - Paris (ENS-PSL), Université Paris sciences et lettres (PSL)-École normale supérieure - Paris (ENS-PSL), Université Paris sciences et lettres (PSL)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS)
Jazyk: angličtina
Rok vydání: 2020
Předmět:
Gene isoform
mRNA
[SDV.NEU.NB]Life Sciences [q-bio]/Neurons and Cognition [q-bio.NC]/Neurobiology
Immunofluorescence
In situ hybridization
APPswe/PS1dE9 mouse
Hippocampus
03 medical and health sciences
Mice
0302 clinical medicine
Alzheimer Disease
Glial Fibrillary Acidic Protein
medicine
[SDV.BBM] Life Sciences [q-bio]/Biochemistry
Molecular Biology
Animals
[SDV.BBM]Life Sciences [q-bio]/Biochemistry
Molecular Biology
RNA
Messenger
Intermediate filament
030304 developmental biology
0303 health sciences
Messenger RNA
Microglia
Glial fibrillary acidic protein
biology
Chemistry
GFAP
[SDV.NEU.NB] Life Sciences [q-bio]/Neurons and Cognition [q-bio.NC]/Neurobiology
Cell Biology
Alzheimer's disease
ImageJ
Cell biology
medicine.anatomical_structure
nervous system
Astrocytes
biology.protein
030217 neurology & neurosurgery
Immunostaining
Astrocyte
Zdroj: Journal of Cell Science
Journal of Cell Science, Company of Biologists, 2020, 133 (7), pp.jcs.239756. ⟨10.1242/jcs.239756⟩
Journal of Cell Science, 2020, 133 (7), pp.jcs.239756. ⟨10.1242/jcs.239756⟩
ISSN: 0021-9533
1477-9137
DOI: 10.1242/jcs.239756⟩
Popis: Cells with a complex shape often use mRNA distribution and local translation to regulate distal functions. These mechanisms have recently been described in astrocytes, the processes of which contact and functionally modulate neighbouring synapses and blood vessels. In order to study the distribution of mRNA in astrocytes, we developed a three-dimensional histological method that combines mRNA detection viain situhybridization with immunostaining of the astrocyte-specific intermediate filament glial fibrillary acidic protein (GFAP). Three-dimensional confocal images were analyzed using AstroDot, a custom Image J plug-in developed in-house for the identification and quantification of mRNAs in GFAP-immunolabelled astrocyte somata, large processes and fine processes. The custom R package AstroStat was used to analyze the AstroDot results. Taking the characterization of mRNAs encoding the astrocyte-specific GFAP α and δ isoforms in the hippocampus as a proof of concept, we showed thatGfapα andGfapδ mRNAs mainly colocalized with GFAP in astrocyte processes.Gfapα mRNA was more abundant thanGfapδ mRNA, and was predominantly found in fine processes. Upon glial activation in the APPswe/PS1dE9 mouse model of Alzheimer’s disease, the same overall patterns were found but we noted strong variations inGfapα andGfapδ mRNA density and distribution as a function of the part of the hippocampus and the astrocyte’s proximity to beta-amyloid (Aβ) plaques. In astrocytes not associated with Aβ, Gfap α mRNA levels were only slightly elevated, and Gfap δ mRNA was distributed within the fine processes; these effects were more prominent in CA3 than in CA1. In contrast, levels of both mRNAs were markedly elevated in the fine processes of Aβ-associated astrocytes in both CA1 and CA3. In order to validate our new method, we confirmed thatRpl4mRNA (a ubiquitously expressed mRNA encoding the large subunit ribosomal protein 4) was present in large and fine processes in both astrocytes and microglia. In summary, we have developed a novel, reliable set of tools for characterizing mRNA densities and distributions in the somata and processes of astrocytes and microglia in physiological or pathological settings. Furthermore, our results suggest that intermediate filaments are crucial for distributing mRNA within astrocytes and for modulating specificGfapmRNA profiles in Alzheimer’s disease.
Databáze: OpenAIRE
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