Dynamic local mRNA localization and translation occurs during the postnatal molecular maturation of perivascular astrocytic processes.

Astrocytes are highly ramified and send out perivascular processes (PvAPs) that entirely sheathe the brain's blood vessels. PvAPs are equipped with an enriched molecular repertoire that sustains astrocytic regulatory functions at the vascular interface. In the mouse, PvAP development starts after birth and is essentially complete by postnatal day (P) 15. Progressive molecular maturation also occurs over this period, with the acquisition of proteins enriched in PvAPs. The mechanisms controlling the development and molecular maturation of PvAPs have not been extensively characterized. We reported previously that mRNAs are distributed unequally in mature PvAPs and are locally translated. Since dynamic mRNA localization and local translation influence the cell's polarity, we hypothesized that they might sustain the postnatal maturation of PvAPs. Here, we used a combination of molecular biology and imaging approaches to demonstrate that the development of PvAPs is accompanied by the transport of mRNA and polysomal mRNA into PvAPs, the development of a rough endoplasmic reticulum (RER) network and Golgi cisternae, and local translation. By focusing on genes and proteins that are selectively or specifically expressed in astrocytes, we characterized the developmental profile of mRNAs, polysomal mRNAs and proteins in PvAPs from P5 to P60. We found that some polysomal mRNAs polarized progressively towards the PvAPs. Lastly, we found that expression and localization of mRNAs in developing PvAPs is perturbed in a mouse model of megalencephalic leukoencephalopathy with subcortical cysts. Our results indicate that dynamic mRNA localization and local translation influence the postnatal maturation of PvAPs.

The ribosome-associated protein RACK1 represses Kir4.1 translation in astrocytes and influences neuronal activity.

The regulation of translation in astrocytes, the main glial cells in the brain, remains poorly characterized. We developed a high-throughput proteomics screen for polysome-associated proteins in astrocytes and focused on ribosomal protein receptor of activated protein C kinase 1 (RACK1), a critical factor in translational regulation. In astrocyte somata and perisynaptic astrocytic processes (PAPs), RACK1 preferentially binds to a number of mRNAs, including Kcnj10, encoding the inward-rectifying potassium (K+) channel Kir4.1. By developing an astrocyte-specific, conditional RACK1 knockout mouse model, we show that RACK1 represses production of Kir4.1 in hippocampal astrocytes and PAPs. Upregulation of Kir4.1 in the absence of RACK1 increases astrocytic Kir4.1-mediated K+ currents and volume. It also modifies neuronal activity attenuating burst frequency and duration. Reporter-based assays reveal that RACK1 controls Kcnj10 translation through the transcript's 5' untranslated region. Hence, translational regulation by RACK1 in astrocytes represses Kir4.1 expression and influences neuronal activity.

Physiopathological changes of ferritin mRNA density and distribution in hippocampal astrocytes in the mouse brain.

Astrocytes are thought to play a crucial role in brain iron homeostasis. How they accomplish this regulation in vivo is unclear. In a recent transcriptomic analysis, we showed that polysomal Ftl1 and Fth1 mRNAs, encoding the ferritin light (Ftl) and heavy (Fth) chains that assemble into ferritin, a critical complex for iron storage and reduction, are enriched in perisynaptic astrocytic processes as compared to astrocytic soma. These data suggested that ferritin translation plays a specific role at the perisynaptic astrocytic interface and is tighly regulated by local translation. Here, we used our recently described AstroDot 3D in situ methodology to study the density and localization of ferritin mRNAs in astrocytes in the hippocampus in three different contexts in which local or systemic iron overload has been documented: aging, the hepcidin knock-out mouse model of hemochromatosis and the APP/PS1dE9 mouse model of Alzheimer's disease (AD). Our results showed that in wild type mice, Fth1 mRNA density was higher than Ftl1 and that both mRNAs were mostly distributed in astrocyte fine processes. Aging and absence of hepcidin caused an increased Fth1/Ftl1 ratio in astrocytes and in the case of aging, led to a redistribution of Fth1 mRNAs in astrocytic fine processes. In contrast, in AD mice, we observed a lower Fth1/Ftl1 ratio. Fth1 mRNAs became more somatic and Ftl1 mRNAs redistributed in large processes of astrocytes proximal to Amyloid beta (Aß) deposits. Hence, we propose that regulation of ferritin mRNA density and distribution in astrocytes contribute to iron homeostasis in physiology and pathophysiology.

Local translation in perisynaptic and perivascular astrocytic processes - a means to ensure astrocyte molecular and functional polarity?

Together with the compartmentalization of mRNAs in distal regions of the cytoplasm, local translation constitutes a prominent and evolutionarily conserved mechanism mediating cellular polarization and the regulation of protein delivery in space and time. The translational regulation of gene expression enables a rapid response to stimuli or to a change in the environment, since the use of pre-existing mRNAs can bypass time-consuming nuclear control mechanisms. In the brain, the translation of distally localized mRNAs has been mainly studied in neurons, whose cytoplasmic protrusions may be more than 1000 times longer than the diameter of the cell body. Importantly, alterations in local translation in neurons have been implicated in several neurological diseases. Astrocytes, the most abundant glial cells in the brain, are voluminous, highly ramified cells that project long processes to neurons and brain vessels, and dynamically regulate distal synaptic and vascular functions. Recent research has demonstrated the presence of local translation at these astrocytic interfaces that might regulate the functional compartmentalization of astrocytes. In this Review, we summarize our current knowledge about the localization and local translation of mRNAs in the distal perisynaptic and perivascular processes of astrocytes, and discuss their possible contribution to the molecular and functional polarity of astrocytes.

Astrocytes in the regulation of cerebrovascular functions.

Astrocytes are the most numerous type of neuroglia in the brain and have a predominant influence on the cerebrovascular system; they control perivascular homeostasis, the integrity of the blood-brain barrier, the dialogue with the peripheral immune system, the transfer of metabolites from the blood, and blood vessel contractility in response to neuronal activity. These regulatory processes occur in a specialized interface composed of perivascular astrocyte extensions that almost completely cover the cerebral blood vessels. Scientists have only recently started to study how this interface is formed and how it influences cerebrovascular functions. Here, we review the literature on the astrocytes' role in the regulation of the cerebrovascular system. We cover the anatomy and development of the gliovascular interface, the known gliovascular functions, and molecular factors, the latter's implication in certain pathophysiological situations, and recent cutting-edge experimental tools developed to examine the astrocytes' role at the vascular interface. Finally, we highlight some open questions in this field of research.

Immunoprecipitation of Ribosome-Bound mRNAs from Astrocytic Perisynaptic Processes of the Mouse Hippocampus.

Translation of distally localized mRNAs is an evolutionary mechanism occurring in polarized cells. It has been observed in astrocytes, whose processes contact blood vessels and synapses. Here, we describe a protocol for the purification of the entire pool of ribosome-bound mRNAs in perisynaptic astrocytic processes (PAPs). Our procedure combines the preparation of synaptogliosomes with a refined translating ribosome affinity purification technique. This approach can be used in any brain region to probe the physiological relevance of local translation in PAPs. For complete details on the use and execution of this protocol, please refer to Mazaré et al. (2020).

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

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.

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

Astrocytes are morphologically complex and use local translation to regulate distal functions. To study the distribution of mRNA in astrocytes, we combined mRNA detection via in situ hybridization with immunostaining of the astrocyte-specific intermediate filament glial fibrillary acidic protein (GFAP). mRNAs at the level of GFAP-immunolabelled astrocyte somata, and large and fine processes were analysed using AstroDot, an ImageJ plug-in and the R package AstroStat. Taking the characterization of mRNAs encoding GFAP-α and GFAP-δ isoforms as a proof of concept, we showed that they mainly localized on GFAP processes. In the APPswe/PS1dE9 mouse model of Alzheimer's disease, the density and distribution of both α and δ forms of Gfap mRNA changed as a function of the region of the hippocampus and the astrocyte's proximity to amyloid plaques. To validate our method, we confirmed that the ubiquitous Rpl4 (large subunit ribosomal protein 4) mRNA was present in astrocyte processes as well as in microglia processes immunolabelled for ionized calcium binding adaptor molecule 1 (Iba1; also known as IAF1). In summary, this novel set of tools allows the characterization of mRNA distribution in astrocytes and microglia in physiological or pathological settings.