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

Oudart, Marc; Tortuyaux, Romain; Mailly, Philippe; Mazaré, Noémie; Boulay, Anne-Cécile; Cohen-Salmon, Martine

doi:10.1101/765784

Cited by 9 publications

(29 citation statements)

References 46 publications

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“…Nevertheless, by comparing the results in PAPs vs. whole astrocytes, we noted that the C-terminal exons of Gfap a and d (84) were differentially distributed: Gfap a was more abundant in PAPs, and GFAP d was more abundant in the cell soma (Table S2). These findings corroborate our recent observation (based on FISH) than GFAP d mRNA is more abundant that GFAP a mRNA in the soma (31), and suggest that alternative splicing may contribute to the PAPs' molecular identity.…”

Section: Discussionsupporting

confidence: 92%

“…We performed a FISH experiment on adult mouse CA1 hippocampal sections in order to detect Slc1a2 mRNA, a transcript coding for the astrocyte-specific glutamate transporter GLT1. The samples were co-immunostained for the astrocyte-specific intermediate filament glial fibrillary acidic protein (GFAP) (31). As shown in Fig.…”

Section: Hippocampal Paps Contain Protein Synthesis and Maturation Ormentioning

confidence: 99%

“…1A, Slc1a2 mRNA FISH dots appeared to be distributed throughout the astrocyte. We used our recently developed AstroDot ImageJ plugin to characterize the mRNA distribution by counting the number of FISH dots localized on GFAPimmunolabeled intermediate filament in the somata and the large and fine processes (31). Since Slc1a2 is specifically expressed by astrocytes in the CA1 region, we considered that mRNA FISH dots localized outside GFAP-immunolabeled processes could be attributed to PAPs, where GFAP is known to be less present (32).…”

Section: Hippocampal Paps Contain Protein Synthesis and Maturation Ormentioning

confidence: 99%

“…5D, E, Table S6). To this end, we used our AstroDot technique to analyze the distribution of fluorescently labeled mRNAs on GFAP-immunolabeled intermediate filaments in control and conditioned mice (31). With the exception of Fth1 and Ccnd2, the amounts of astrocyte mRNA were lower in the fear-conditioned group (Fig.…”

Section: Fear Conditioning Influences Local Translation In Papsmentioning

confidence: 99%

“…Astrocyte-specific FISH dots were recognized on the base of their localization on the GFAP immunolabeling, using the AstroDot ImageJ plug-in. This method has been described in detail elsewhere (31). The Bacillus subtilis dihydrodipicolinate reductase (dapB) gene was used as a negative control.…”

Section: High-resolution Fluorescent In Situ Hybridization and Gfap Cmentioning

confidence: 99%

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Local translation in perisynaptic astrocytic processes is specific and regulated by fear conditioning

Mazaré

Oudart

Moulard

et al. 2020

Preprint

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Local translation is a conserved molecular mechanism conferring cells the ability to quickly respond to local stimuli. It not only permits cells with complex morphology to bypass somatic protein synthesis and transport, but also contributes locally to the establishment of molecular and functional polarity. In the brain, local translation has been extensively studied in neurons and has only been recently reported in astrocytes, whose fine processes contact both blood vessels and synapses. Yet the specificity and regulation of astrocyte local translation remain unknown. Here, we studied hippocampal perisynaptic astrocytic processes (PAPs) and show that they contain all the machinery for translation. Using our recently refined polysome immunoprecipitation technique, we then characterized the pool of polysomal mRNAs in PAPs, referred to as the PAPome, and compared it to the one found in the whole astrocyte. We found that the PAPome encoded an unexpected molecular repertoire, mostly composed of cytoplasmic proteins and of proteins involved in iron homeostasis, translation, cell cycle and cytoskeleton. Among them, ezrin (Ezr), ferritin heavy chain 1 (Fth1) and 60S acidic ribosomal protein1 (Rplp1) were enriched in PAPs compared to perivascular astrocytic processes, indicating that local translation differs at these two interfaces. Remarkably, PAPs were also enriched in transcripts coding for proteins involved in learning and memory, such as ferritin (Ftl1 and Fth1), G1/S-specific cyclin-D2 (Ccnd2), E3 ubiquitin-protein ligase (Mdm2) , Receptor of activated protein C kinase 1 (Gnb2l1) and Elongation factor 1-alpha 1 (Eef1a1).To address their regulation in a physiological context, we assessed their local translation after fear conditioning. We found alterations in their density and/or distribution in astrocytes as well as a drop in their translation specifically in PAPs. In all, our results reveal an unexpected molecular repertoire of hippocampal PAPs, which is regulated by local translation during learning and memory processes.

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Section: Discussionsupporting

confidence: 92%

Section: Hippocampal Paps Contain Protein Synthesis and Maturation Ormentioning

confidence: 99%

Section: Hippocampal Paps Contain Protein Synthesis and Maturation Ormentioning

confidence: 99%

Section: Fear Conditioning Influences Local Translation In Papsmentioning

confidence: 99%

Section: High-resolution Fluorescent In Situ Hybridization and Gfap Cmentioning

confidence: 99%

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Local translation in perisynaptic astrocytic processes is specific and regulated by fear conditioning

Mazaré

Oudart

Moulard

et al. 2020

Preprint

Self Cite

View full text Add to dashboard Cite

show abstract

Local Translation in Perisynaptic Astrocytic Processes Is Specific and Changes after Fear Conditioning

et al. 2020

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Summary Local translation is a conserved mechanism conferring cells the ability to quickly respond to local stimuli. In the brain, it has been recently reported in astrocytes, whose fine processes contact blood vessels and synapses. Yet the specificity and regulation of astrocyte local translation remain unknown. We study hippocampal perisynaptic astrocytic processes (PAPs) and show that they contain the machinery for translation. Using a refined immunoprecipitation technique, we characterize the entire pool of ribosome-bound mRNAs in PAPs and compare it with the one expressed in the whole astrocyte. We find that a specific pool of mRNAs is highly polarized at the synaptic interface. These transcripts encode an unexpected molecular repertoire, composed of proteins involved in iron homeostasis, translation, cell cycle, and cytoskeleton. Remarkably, we observe alterations in global RNA distribution and ribosome-bound status of some PAP-enriched transcripts after fear conditioning, indicating the role of astrocytic local translation in memory and learning.

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In mice and humans, the brain’s blood vessels mature postnatally to acquire barrier and contractile properties

Slaoui

Gilbert

Federici

et al. 2021

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The brain dense vascular network is essential for distributing oxygen and nutrients to neural cells. The network develops during embryogenesis and leads to the formation of the endothelial blood-brain barrier (BBB). This barrier is surrounded by mural cells (pericytes and vascular smooth muscle cells (VSMCs)) and fibroblasts. Here, we compared the molecular and functional properties of brain vascular cells on postnatal day (P)5 vs. P15, via a transcriptomic analysis of purified mouse cortical microvessels (MVs) and the identification of vascular-cell-type-specific or -preferentially expressed transcripts. We found that endothelial cells (ECs), VSMCs and fibroblasts follow specific molecular maturation programs over this time period. In particular, ECs acquire P-glycoprotein (P-gP)-mediated efflux capacities. The arterial VSMC network expands, acquires contractile proteins (such as smooth muscle actin (SMA) and myosin heavy chain 11 (Myh11)) and becomes contractile. We also analyzed samples of human brain cortex from the early prenatal stage through to adulthood: the expression of endothelial P-gP increased at birth and Myh11 in VSMCs acts as a developmental switch (as in the mouse) at birth and up to the age of 2 of 5 years. Thus, in both mice and humans, the early postnatal phase is a critical period during which the essential properties of cerebral blood vessels (i.e. the endothelial efflux of xenobiotics and other molecules, and the VSMC contractility required for vessel tone and brain perfusion) are acquired and mature.

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

Cited by 9 publications

References 46 publications

Local translation in perisynaptic astrocytic processes is specific and regulated by fear conditioning

Local translation in perisynaptic astrocytic processes is specific and regulated by fear conditioning

Local Translation in Perisynaptic Astrocytic Processes Is Specific and Changes after Fear Conditioning

In mice and humans, the brain’s blood vessels mature postnatally to acquire barrier and contractile properties

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