Translation in astrocyte distal processes sets molecular heterogeneity at the gliovascular interface

Boulay, Anne; Saubaméa, Bruno; Adam, Nicolas; Chasseigneaux, Stéphanie; Mazaré, Noémie; Gilbert, Alice; Bahin, Mathieu; Bastianelli, Leïla; Blugeon, Corinne; Perrin, Sandrine; Pouch, Juliette; Ducos, Bertrand; Crom, Stéphane Le; Genovesio, Auguste; Chrétien, Fabrice; Declèves, Xavier; Laplanche, Jean‐Louis; Cohen-Salmon, Martine

doi:10.1038/celldisc.2017.5

Cited by 138 publications

(164 citation statements)

References 71 publications

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“…In contrast to previous reports 11,12 , however, our protocol preserved GFAP and enabled us to perform parallel in situ hybridization and GFAP immunodetection. Interestingly, these conditions also allowed us to immunodetect the microglia-specific protein Iba1.…”

Section: Discussionmentioning

confidence: 87%

“…Gfap mRNAs have already been detected in distal perivascular 11 and perisynaptic processes 13 of astrocytes -suggesting that local GFAP translation regulates distal intermediate filament assembly.…”

Section: Gfap a And Gfap D Mrnas Are Distributed In Papsmentioning

confidence: 99%

“…Recent studies of the astrocyte's functional polarity have suggested that mRNA distribution and local translation regulates protein delivery in space and time. In a previous study, we showed that local translation is determined in PvAPs and we characterized the locally translated molecular repertoire 11 . Local translation has also been observed in the radial glia during brain development 12 and in PAPs in the adult cortex 13 .…”

Section: Introductionmentioning

confidence: 99%

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

Oudart

Tortuyaux

Mailly

et al. 2019

Preprint

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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 a and d isoforms in the hippocampus as a proof of concept, we showed that Gfap a and Gfap d mRNAs mainly colocalized with GFAP in astrocyte processes. Gfap a mRNA was more abundant than Gfap d 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 a and Gfap d mRNA density and distribution as a function of the part of the hippocampus and the astrocyte's proximity to beta-amyloid (Ab) plaques. In astrocytes not associated with Ab, Gfap a mRNA levels were only slightly elevated, and Gfap d 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 Ab-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.

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Section: Discussionmentioning

confidence: 87%

Section: Gfap a And Gfap D Mrnas Are Distributed In Papsmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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

Oudart

Tortuyaux

Mailly

et al. 2019

Preprint

Self Cite

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“…Moreover, a very recent study on the transcriptome and translatome of astrocyte foot processes using mice brain microvessels isolation developed in our laboratory, did not evidence any mRNA and protein expression of P‐gp in astrocyte endfeets (Boulay et al . ). Therefore, we cannot exclude that weak amounts of astrocyte foot P‐gp in isolated rat brain microvessels may contaminate whole P‐gp expression in our study and it seems unlikely.…”

Section: Discussionmentioning

confidence: 97%

Targeted unlabeled multiple reaction monitoring analysis of cell markers for the study of sample heterogeneity in isolated rat brain cortical microvessels

Gómez-Zepeda

Chaves

Taghi

et al. 2017

Journal of Neurochemistry

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Liquid chromatography coupled to tandem mass spectrometry-based targeted absolute protein quantification (in fmol of the analyte protein per μg of total protein) is employed for the molecular characterization of the blood-brain barrier using isolated brain microvessels. Nevertheless, the heterogeneity of the sample regarding the levels of different cells co-isolated within the microvessels and bovine serum albumin (BSA) contamination (from buffers) are not always evaluated. We developed an unlabeled targeted liquid chromatography coupled to tandem mass spectrometry method to survey the levels of endothelial cells (ECs), astrocytes, and pericytes, as well as BSA contaminant in rat cortical microvessels. Peptide peak identities were evaluated using a spectral library and chromatographic parameters. Sprague-Dawley rat microvessels obtained on three different days were analyzed with this method complemented by an absolute quantification multiple reaction monitoring method for transporter proteins P-gp, Bcrp, and Na /K ATPase pump using stable isotope labeled peptides as internal standard. Inter-day differences in the cell markers and BSA contamination were observed. Levels of cell markers correlated positively between each other. Then, the correlation between cell marker proteins and transporter proteins was evaluated to choose the best EC marker protein for protein quantification normalization. The membrane protein Pecam-1 showed a very high correlation with the EC-specific transporter P-gp (Pearson product-moment correlation coefficient (r) > 0.89) and moderate to high with Bcrp (r ≥ 0.77), that can be found also in pericytes and astrocytes. Therefore, Pecam-1 was selected as a marker for the normalization of the quantification of the proteins of endothelial cells.

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“…Zhang et al, 2014). This holds true even after enriching for astrocyte endfeet transcripts (Boulay et al, 2017). A study carefully assessing Shh protein and mRNA expression patterns determined that Shh is present in very few astrocytes and the majority of Shh protein in the adult uninjured cortex is made by neurons (Sirko et al, 2013).…”

Section: Introduction (726)mentioning

confidence: 99%

Astrocytes are necessary for blood-brain barrier maintenance in the adult mouse brain

Heithoff

George

Phares

et al. 2020

Preprint

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224/250 words)In the adult brain, multiple cell types are known to produce factors that regulate blood-brain barrier properties, including astrocytes. Yet several recent studies disputed a role for mature astrocytes at the blood-brain barrier. To determine if astrocytes contribute a non-redundant and necessary function in maintaining the adult blood-brain barrier, we used a mouse model of tamoxifen-inducible astrocyte ablation. In adult mice, tamoxifen induction caused sparse apoptotic astrocyte cell death within 2 hours. Indicative of BBB damage, leakage of the small molecule Cadaverine and the large plasma protein fibrinogen into the brain parenchyma indicative of BBB damage was detected as early as astrocyte ablation was present. Vessels within and close to regions of astrocyte loss had lower expression of the tight junction protein zonula occludens-1 while endothelial glucose transporter 1 expression was undisturbed.Cadaverine leakage persisted for several weeks suggesting a lack of barrier repair. This is consistent with the finding that ablated astrocytes were not replaced. Adjacent astrocytes responded with partial non-proliferative astrogliosis, characterized by morphological changes and delayed phosphorylation of STAT3, which restricted dye leakage to the brain and vessel surface areas lacking coverage by astrocytes one month after ablation. In conclusion, astrocytes are necessary to maintain blood-brain barrier integrity in the adult brain. Blood-brain barrier-regulating factors secreted by other cell types, such as pericytes, are not sufficient to compensate for astrocyte loss. Main Points (250 characters)Mature astrocytes are necessary for maintenance of endothelial tight junctions in the adult brain.Ablated astrocytes are not replaced by proliferation or process extension of neighboring astrocytes resulting in long-term blood-brain barrier damage.

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Translation in astrocyte distal processes sets molecular heterogeneity at the gliovascular interface

Cited by 138 publications

References 71 publications

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

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

Targeted unlabeled multiple reaction monitoring analysis of cell markers for the study of sample heterogeneity in isolated rat brain cortical microvessels

Astrocytes are necessary for blood-brain barrier maintenance in the adult mouse brain

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