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

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 via in situ hybridization 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 and isoforms in the hippocampus as a proof of concept, we showed that Gfap alpha and Gfap delta mRNAs mainly colocalized with GFAP in astrocyte processes. Gfap alpha mRNA was more abundant than Gfap delta 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 in Gfap alpha and Gfap delta mRNA density and distribution as a function of the part of the hippocampus and the astrocyte s proximity to beta-amyloid (Abeta plaques). In astrocytes not associated with Abeta, Gfap alpha mRNA levels were only slightly elevated, and Gfap delta 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 Abeta-associated astrocytes in both CA1 and CA3. In order to validate our new method, we confirmed that Rpl4 mRNA (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 specific Gfap mRNA profiles in Alzheimer s disease.