Noémie Mazaré

Marc Oudart

Martine Cohen-Salmon
email: martine.cohen-salmon@college-de-france.fr

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

Keywords: local translation, mRNA distribution, astrocyte

Together with the compartmentalization of mRNAs in distal regions of the cytoplasm, local translation constitutes a prominent, evolutionarily conserved mechanism for 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, 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.

Introduction

Although it was long thought that translation occurs in the vicinity of the nucleus, it is now known that messenger RNAs (mRNAs) can also be transported to and translated in distal cell compartments as part of a process that helps to regulate protein delivery in space and time [START_REF] Holt | The central dogma decentralized: new perspectives on RNA function and local translation in neurons[END_REF]. The first evidence for compartmentalized mRNA localization was published in 1983; it was found that actin mRNA was present in different regions of the ascidian egg [START_REF] Jeffery | Localization of actin messenger RNA during early ascidian development[END_REF]. Since then, mRNA localization and local translation have been observed in a number of cell types, and particularly in cells with complex morphologies. The bestcharacterized example is the neuron, which can grow an axon of up to one meter in length [START_REF] Biever | Local translation in neuronal processes[END_REF][START_REF] Glock | mRNA transport & local translation in neurons[END_REF][START_REF] Holt | Local translation in neurons: visualization and function[END_REF]. mRNA localization and local translation were observed at active synapses in neurons of the sea slug Aplysia and were shown to contribute to synaptic plasticity [START_REF] Si | A neuronal isoform of CPEB regulates local protein synthesis and stabilizes synapse-specific long-term facilitation in aplysia[END_REF]. Local protein translation has also been observed in dissected squid giant axons [START_REF] Mathur | Demonstration of ion channel synthesis by isolated squid giant axon provides functional evidence for localized axonal membrane protein translation[END_REF]. In Drosophila, mRNA transport in synapses was linked to synaptic plasticity [START_REF] Kuklin | The Long 3'UTR mRNA of CaMKII Is Essential for Translation-Dependent Plasticity of Spontaneous Release in Drosophila melanogaster[END_REF]. In vertebrates, local translation was first described in isolated axons from rabbits and cats (Koenig, 1965a;Koenig, 1965b;Koenig, 1967a;Koenig, 1967b), and has been intensively studied in recent yearsnotably after the development of a number of techniques for tracking local translation events in vitro and in vivo [START_REF] Holt | Local translation in neurons: visualization and function[END_REF]. Overall, the diversity observed in animal models suggests that local translation is an evolutionarily conserved mechanism for the functional polarization of cells.

In glial cells, mRNA localization was first observed in the 1980s; using in situ hybridization, the distal distribution of mRNAs coding for myelin binding protein (which is crucial for building myelin sheaths) was observed in spinal cord sections from mice infected with a demyelinating virus [START_REF] Kristensson | Expression of myelin basic protein gene in the developing rat brain as revealed by in situ hybridization[END_REF]. A later study demonstrated the presence of carbonic anhydrase II mRNA in the processes of primary cultured oligodendrocytes [START_REF] Ghandour | Double-labeling in situ hybridization analysis of mRNAs for carbonic anhydrase II and myelin basic protein: expression in developing cultured glial cells[END_REF]. mRNA isoform transcripts encoding the amyloid precursor protein and Tau protein (both implicated in the pathology of Alzheimer's disease) have also been detected in primary oligodendrocyte processes [START_REF] Garcia-Ladona | Oligodendrocytes express different isoforms of beta-amyloid precursor protein in chemically defined cell culture conditions: in situ hybridization and immunocytochemical detection[END_REF][START_REF] Lopresti | Functional implications for the microtubule-associated protein tau: localization in oligodendrocytes[END_REF]. More recently, several research groups have demonstrated that mRNA localization and local translation also occur in astrocytes -the most abundant population of glial cells in the mammalian brain [START_REF] Boulay | Translation in astrocyte distal processes sets molecular heterogeneity at the gliovascular interface[END_REF][START_REF] Sakers | Astrocytes locally translate transcripts in their peripheral processes[END_REF][START_REF] Mazare | Local Translation in Perisynaptic Astrocytic Processes Is Specific and Changes after Fear Conditioning[END_REF]. Astrocytes are voluminous, morphologically complex cells. They are highly ramified and polarized, and bear processes that form branches, secondary branches and terminations in contact with blood vessels and neurons (Fig. 1). In the CA1 region of the hippocampus, between 60% and 90% of the synapses are contacted by extremely thin (<50 nm) perisynaptic astrocytic processes (PAPs) [START_REF] Reichenbach | Morphology and dynamics of perisynaptic glia[END_REF]. The number of PAPs varies from one region of the brain to another [START_REF] Ventura | Three-dimensional relationships between hippocampal synapses and astrocytes[END_REF]. At the synaptic interface, astrocytes regulate synaptic transmission [START_REF] Dallerac | How do astrocytes shape synaptic transmission? Insights from electrophysiology[END_REF][START_REF] Ghezali | Perisynaptic astroglial processes: dynamic processors of neuronal information[END_REF]. In fact, PAPs can sense changes in the composition of the perisynaptic extracellular space and thus can prevent prolonged neuronal activation and excitotoxicity by clearing ions and neurotransmitters that are released from the synapse [START_REF] Dallerac | Versatile control of synaptic circuits by astrocytes: where, when and how?[END_REF]. PAPs are equipped with transporters (such as glutamate transporters) and channels, which tightly control perisynaptic homeostasis [START_REF] Murphy-Royal | Astroglial glutamate transporters in the brain: Regulating neurotransmitter homeostasis and synaptic transmission[END_REF]. PAPs also release neuroactive factors and influence synaptic functions by dynamically modulating synaptic coverage [START_REF] Bernardinelli | Astrocyte-synapse structural plasticity[END_REF][START_REF] Pannasch | Astroglial networks scale synaptic activity and plasticity[END_REF]. Lastly, PAPs orchestrate synaptogenesis during development and in the mature brain [START_REF] Allen | Cell Biology of Astrocyte-Synapse Interactions[END_REF][START_REF] Chung | Astrocytes Control Synapse Formation, Function, and Elimination[END_REF][START_REF] Stogsdill | The interplay between neurons and glia in synapse development and plasticity[END_REF]. At the vascular interface, perivascular astrocytic processes (PvAPs, often called endfeet) form a continuous layer around the brain vessels [START_REF] Mathiisen | The perivascular astroglial sheath provides a complete covering of the brain microvessels: an electron microscopic 3D reconstruction[END_REF][START_REF] Mccaslin | In vivo 3D morphology of astrocyte-vasculature interactions in the somatosensory cortex: implications for neurovascular coupling[END_REF]. The dimensions of PvAPs vary greatly (from 8 to 198 µm 2 ) -even along the same vessel [START_REF] Wang | Varying perivascular astroglial endfoot dimensions along the vascular tree maintain perivascular-interstitial flux through the cortical mantle[END_REF]. On average, each astrocyte has 3.5 PvAPs (with a range from 1 to 7), which originate from one or more ramifications and that wrap around vessels [START_REF] Bindocci | Three-dimensional Ca(2+) imaging advances understanding of astrocyte biology[END_REF]. Via the PvAPs, astrocytes control several brain vascular functions, including the integrity of the blood-brain barrier, the homeostasis between the brain and the immune system, the transfer of metabolites and the regulation of cerebral blood flow [START_REF] Alvarez | Glial influence on the blood brain barrier[END_REF][START_REF] Cohen-Salmon | Astrocytes in the regulation of cerebrovascular functions[END_REF]. As in PAPs, most of the astrocytes' perivascular functions rely on a specific molecular repertoire that is enriched in PvAPs [START_REF] Cohen-Salmon | Astrocytes in the regulation of cerebrovascular functions[END_REF]. For instance, the water channel aquaporin 4 and the inward-rectifying potassium channel Kir4.1 have critical roles in the regulation of perivascular homeostasis [START_REF] Amiry-Moghaddam | The molecular basis of water transport in the brain[END_REF][START_REF] Cohen-Salmon | Astrocytes in the regulation of cerebrovascular functions[END_REF]. Interestingly, PvAPs are sometimes contiguous with PAPs; this proximity might be critical for coupling the neuronal and vascular activities of astrocytes [START_REF] Boulay | Translation in astrocyte distal processes sets molecular heterogeneity at the gliovascular interface[END_REF] (Fig. 1).

The way astrocytes develop and maintain their high level of polarity has not been characterized. The recent discovery of local translation in the distal compartments of astrocytes strongly suggests that (as in neurons) this mechanism might underpin their functional polarity. Local translation requires mRNA transport, mRNA binding to the translation machinery and (for membrane and secreted proteins) proper folding and post-translational modifications. Here, we review the literature on RNA distribution, the detection of local translation in astrocytes, the subcellular organization of astrocytes at the perineuronal and perivascular interfaces, and the machinery for local translation. We conclude with a discussion on open questions in this new field of research.

Detection of mRNA in distal areas of the astrocyte

Several examples of local mRNA distribution in astrocytes were described prior to the discovery of local translation. The analysis of protrusions obtained from primary cultures of astrocytes in a Boyden chamber (a cell culture device allowing cells to extend processes in vitro) provided an initial genome-wide assessment of mRNA localization in these structures (Thomsen et al., 2013a). Glt1a and Glt1b mRNAs were found to be differentially distributed; these mRNAs encode isoforms of the glutamate transporter 1 (GLT1), the most prominent glutamate transporter in astrocytes that is responsible for glutamate uptake from the extracellular space in the brain [START_REF] Murphy-Royal | Astroglial glutamate transporters in the brain: Regulating neurotransmitter homeostasis and synaptic transmission[END_REF]. Elevated amounts of Glt1a mRNA were found in the processes, whereas the Glt1b isoform was more restricted to the cell soma [START_REF] Berger | Cellular and subcellular mRNA localization of glutamate transporter isoforms GLT1a and GLT1b in rat brain by in situ hybridization[END_REF], suggesting that the composition and functions of GLT1 oligomers might differ in these two regions of the cell [START_REF] Berger | Cellular and subcellular mRNA localization of glutamate transporter isoforms GLT1a and GLT1b in rat brain by in situ hybridization[END_REF][START_REF] Chen | The glutamate transporter GLT1a is expressed in excitatory axon terminals of mature hippocampal neurons[END_REF]. A diurnal change in the distribution of fatty acid binding protein 7 (Fabp7) mRNA was detected in mouse hippocampal PAPs, indicating that FABP7 might mediate diurnal changes in neuronal plasticity [START_REF] Gerstner | Time of day regulates subcellular trafficking, tripartite synaptic localization, and polyadenylation of the astrocytic Fabp7 mRNA[END_REF]. mRNA encoding the glial fibrillary acidic protein (GFAP)-α isoform was preferentially detected in primary astrocyte protrusions, whereas mRNA encoding the GFAP-δ isoform was found in the soma (Thomsen et al., 2013b;[START_REF] Moeton | GFAP isoforms control intermediate filament network dynamics, cell morphology, and focal adhesions[END_REF]. We recently confirmed these results in GFAP-immunolabelled hippocampal sections by combining mRNA detection (via in situ hybridization) with an in silico approach to quantify mRNAs in the somata, large processes, and fine processes [START_REF] Oudart | AstroDot -a new method for studying the spatial distribution of mRNA in astrocytes[END_REF]. Changes in the distribution and density of Gfap mRNAs were also detected in a mouse model of Alzheimer's disease, suggesting that astrocyte mRNA transport is dysregulated in this pathology [START_REF] Oudart | AstroDot -a new method for studying the spatial distribution of mRNA in astrocytes[END_REF]. Interestingly, the assembly of GFAP-δ with GFAP-promotes intermediate filament aggregation and dynamic changes [START_REF] Moeton | GFAP isoforms control intermediate filament network dynamics, cell morphology, and focal adhesions[END_REF][START_REF] Perng | Glial fibrillary acidic protein filaments can tolerate the incorporation of assembly-compromised GFAP-delta, but with consequences for filament organization and alphaB-crystallin association[END_REF]. Thus, the differential distribution of Gfap-versus mRNA might regulate intermediate filament dynamics in distal astrocyte processes.

The motility of mRNAs was recently assessed in vivo in radial glial cells [START_REF] Pilaz | Dynamic mRNA Transport and Local Translation in Radial Glial Progenitors of the Developing Brain[END_REF]. The latter are the progenitors of both neurons and astrocytes and possess a basal process that emanates from the cell body, extends up to 450 µm away, and terminates in an endfoot in contact with the meninges [START_REF] Rakic | The radial edifice of cortical architecture: from neuronal silhouettes to genetic engineering[END_REF]. This basal process serves as a scaffold for the migration of excitatory neurons during early development [START_REF] Nadarajah | Two modes of radial migration in early development of the cerebral cortex[END_REF]).

The Ms2 system, which allows to track the movement of mRNAs in vivo [START_REF] Bertrand | Localization of ASH1 mRNA particles in living yeast[END_REF], was used to study Ccnd2 mRNA (encoding cyclin D2) in organotypic slices from embryonic mice [START_REF] Pilaz | Dynamic mRNA Transport and Local Translation in Radial Glial Progenitors of the Developing Brain[END_REF]. This work showed the active localization of the Ccnd2 mRNA in the radial glia endfeet. Lastly, three later studies used high-resolution in situ hybridization and RNA sequencing to demonstrate the presence of mRNAs in purified PvAPs [START_REF] Boulay | Translation in astrocyte distal processes sets molecular heterogeneity at the gliovascular interface[END_REF] and PAPs [START_REF] Sakers | Astrocytes locally translate transcripts in their peripheral processes[END_REF][START_REF] Mazare | Local Translation in Perisynaptic Astrocytic Processes Is Specific and Changes after Fear Conditioning[END_REF] (see below).

Detection of local translation events in astrocytes

The localization of mRNAs in astrocytic distal processes raises the question of their translational status. To address this issue, Pilaz et al. linked a Dendra2 photoconvertible reporter to the Ccnd2 3'UTR and tracked the translation of this mRNA in radial glia endfoot preparations; green Dendra2 was irreversibly photoconverted to red, and time-lapse imaging over the following 45 minutes revealed a steady increase in green fluorescence recovery in the endfeet, suggesting de novo synthesis [START_REF] Pilaz | Dynamic mRNA Transport and Local Translation in Radial Glial Progenitors of the Developing Brain[END_REF]. Local translation of the Gja1 mRNA encoding connexin 43 (an astrocyte gap-junction protein strongly expressed in PvAPs) was measured in an ex vivo assay [START_REF] Boulay | Translation in astrocyte distal processes sets molecular heterogeneity at the gliovascular interface[END_REF]. Cx43 is known to have a very dynamic life cycle, with a turnover time of 1.5 to 5 h [START_REF] Fallon | Five-hour half-life of mouse liver gap-junction protein[END_REF][START_REF] Laird | Gap junction turnover, intracellular trafficking, and phosphorylation of connexin43 in brefeldin A-treated rat mammary tumor cells[END_REF][START_REF] Laird | Turnover and phosphorylation dynamics of connexin43 gap junction protein in cultured cardiac myocytes[END_REF]. Freshly isolated PvAPs attached to the surface of mechanically purified brain vessels [START_REF] Boulay | Purification of Mouse Brain Vessels[END_REF] were treated with cycloheximide (an inhibitor of protein synthesis) for 6 hours. The level of Cx43 (assessed via Western blots) was lower upon cycloheximide treatment than in untreated samples, indicating that Cx43 turnover in PvAPs relies on local translation [START_REF] Boulay | Translation in astrocyte distal processes sets molecular heterogeneity at the gliovascular interface[END_REF]. Other recently developed techniques for visualizing local translation in astrocytes include the use of modified amino-acid analogs, such as homopropargylglycine (HPG) or tRNA analogs (e.g. puromycin). The methionine analog HPG inserts into the nascent protein chain and can be subsequently detected by a chemoselective ligation ("click") reaction with a fluorescent protein reporter [START_REF] Horisawa | Specific and quantitative labeling of biomolecules using click chemistry[END_REF]. The aminoglycoside antibiotic puromycin, which can be detected by immunofluorescence, mimics tRNA-Tyr; it incorporates into the ribosome A binding site and induces the premature termination of translation by ribosome-catalyzed covalent incorporation into the C-terminal of the nascent peptide [START_REF] Schmidt | SUnSET, a nonradioactive method to monitor protein synthesis[END_REF][START_REF] Pestka | Inhibitors of ribosome functions[END_REF][START_REF] Pestka | Studies on the formation of transfer ribonucleic acid-ribosome complexes. IV. Effect of antibiotics on steps of bacterial protein synthesis: some new ribosomal inhibitors of translocation[END_REF]). An HPG protein-synthesis assay of freshly purified brain vessels gave a strong signal in the copurified PvAPs [START_REF] Boulay | Translation in astrocyte distal processes sets molecular heterogeneity at the gliovascular interface[END_REF]. Another study reported that, after incubating acute brain slices with puromycin, about 73% of the puromycin punctae were located more than 9 µm away from the center of the cortical astrocyte nucleus, suggesting that translation occurs more in distal processes than in the soma [START_REF] Sakers | Astrocytes locally translate transcripts in their peripheral processes[END_REF]. More recently, quantification of the puromycin signal in hippocampal PAPs on acute brain sections showed that immunolabelled synapses that are found within 1 µm of puromycin-and GFP-labelled astrocytic ribosome signals accounted for about 3% of all synapses contacted by eGFP-labelled astrocytic ribosomes. Although this proportion is not large and might be due to technical issues related to the low level of puromycin incorporated into astrocytes, these results indicate the presence of local translation in PAPs [START_REF] Mazare | Local Translation in Perisynaptic Astrocytic Processes Is Specific and Changes after Fear Conditioning[END_REF].

Identification of ribosome-bound mRNAs in astrocyte processes

To further analyze translation in PvAPs and PAPs, several recent studies focused on ribosome-bound mRNAs in astrocyte processes (Fig. 2). All the studies were based on the use of a transgenic mouse expressing the chimeric ribosomal protein L10A-eGFP under the control of the Aldh1l1 astrocyte-specific promoter (Aldh1l1 L10A-eGFP) and the purification of eGFP-tagged polysomes by so-called translating ribosome affinity purification (TRAP) [START_REF] Doyle | Application of a translational profiling approach for the comparative analysis of CNS cell types[END_REF][START_REF] Heiman | Cell type-specific mRNA purification by translating ribosome affinity purification (TRAP)[END_REF][START_REF] Heiman | A translational profiling approach for the molecular characterization of CNS cell types[END_REF]. Importantly, this transgenic model was also instrumental in the visualization of ribosomes in PAPs and PvAPs [START_REF] Boulay | Translation in astrocyte distal processes sets molecular heterogeneity at the gliovascular interface[END_REF][START_REF] Mazare | Local Translation in Perisynaptic Astrocytic Processes Is Specific and Changes after Fear Conditioning[END_REF][START_REF] Sakers | Astrocytes locally translate transcripts in their peripheral processes[END_REF]. Our initial study enabled the identification of the most abundant ribosome-bound mRNAs in PvAPs from whole brain [START_REF] Boulay | Translation in astrocyte distal processes sets molecular heterogeneity at the gliovascular interface[END_REF] (Fig. 2A). Firstly, we extracted total mRNAs from purified brain vessels and brain vessels that had been partially depleted of PvAPs by basal lamina digestion. The comparison of these two samples allowed us to identify mRNAs that were relatively abundant in PvAPs, compared to the vascular compartment. Secondly, we investigated the ribosome-bound status of these mRNAs by comparing ribosome-bound mRNAs from whole astrocytes or PvAPs that were extracted by TRAP from either whole brains or purified brain vessels from Aldh1l1 L10A-eGFP mice. Only mRNAs detected in both preparations were considered, since all mRNAs present in PvAPs should also be detected in whole astrocytes. The intersection between the total mRNA preparation and the TRAP preparation allowed us to identify 28 mRNAs that constituted the "endfeetome", i.e. the pool of most abundant ribosome-bound mRNAs in PvAPs [START_REF] Boulay | Translation in astrocyte distal processes sets molecular heterogeneity at the gliovascular interface[END_REF]. Some of these mRNAs encoded proteins involved in vascular functions, such as Aqp4, Kir4.1, and Cx43 all of which are transmembrane proteins known to have crucial roles in blood-brain barrier homeostasis. More details on the functions of these proteins can be found in a recent review [START_REF] Cohen-Salmon | Astrocytes in the regulation of cerebrovascular functions[END_REF].

Interestingly, a comparison of total mRNA versus ribosome-bound mRNAs in PvAPs indicated that some PvAP mRNAs were not bound to ribosomes and so might remain silent after their transport [START_REF] Boulay | Translation in astrocyte distal processes sets molecular heterogeneity at the gliovascular interface[END_REF].

Although an mRNA's ribosome-bound status does not necessarily reflect its translation as ribosome-bound mRNAs might also be silent if they are compacted in granules, our results were the first to highlight potential translation events in a distal compartment of mature astrocytes. Given that transcripts in the endfeetome are known to have critical roles in the regulation of the brain vascular systems, we hypothesized that local translation may be crucially involved in maintaining the vascular functions of astrocytes [START_REF] Boulay | Translation in astrocyte distal processes sets molecular heterogeneity at the gliovascular interface[END_REF]. In a second study, a similar subtractive approach was used to identify ribosome-bound mRNAs that were abundant in PAPs from the cortex [START_REF] Sakers | Astrocytes locally translate transcripts in their peripheral processes[END_REF] (Fig. 2B). Here, the pool of ribosome-bound mRNAs in PAPs was extracted by performing TRAP on Aldh1l1 L10A-eGFP purified cortical synaptoneurosomes, which in addition to PAPs comprise the pre-and post-synaptic compartments of neurons [START_REF] Sakers | Astrocytes locally translate transcripts in their peripheral processes[END_REF]. The authors first compared the transcriptomes of total brain cortex and synaptoneurosomes, in order to identify mRNAs that were abundant in the latter structures. In parallel, they performed TRAP extraction on Aldh1l1 L10A-eGFP synaptoneurosomes and compared purified mRNAs with total mRNAs extracted from synaptoneurosomes, in order to identify ribosome-bound mRNAs that were highly abundant in PAPs [START_REF] Sakers | Astrocytes locally translate transcripts in their peripheral processes[END_REF]. Comparison of the two lists allowed the identification of 224 abundant ribosome-bound mRNAs in astrocyte PAPs [START_REF] Sakers | Astrocytes locally translate transcripts in their peripheral processes[END_REF]. These mRNAs coded for (i) proteins involved in glutamate metabolism, GABA metabolism, and the biosynthesis of unsaturated fatty acids, (ii) cytoskeletal proteins, such as ezrin, which might have a role in PAP remodeling [START_REF] Lavialle | Structural plasticity of perisynaptic astrocyte processes involves ezrin and metabotropic glutamate receptors[END_REF], and (iii) synaptogenic factors, such as the secreted protein acidic and rich in cysteine (Sparc), which regulates the synapse number [START_REF] Lopez-Murcia | SPARC triggers a cell-autonomous program of synapse elimination[END_REF]. Taken as a whole, these two first studies identified a set of ribosome-bound mRNAs that were more abundant in PvAPs and PAPs than in whole astrocytes. The results thus suggested that mRNA distribution and local translation could sustain the polarity of astrocytes at the perisynaptic and perivascular interfaces [START_REF] Boulay | Translation in astrocyte distal processes sets molecular heterogeneity at the gliovascular interface[END_REF][START_REF] Sakers | Astrocytes locally translate transcripts in their peripheral processes[END_REF]. Despite these promising results, these initial studies focused solely on mRNAs that were either highly expressed in or specific to astrocytes. In fact, the technical limitations of the TRAP protocol prevented the detection of ubiquitous transcripts. Further work was thus required to characterize the entire pool of local ribosome-bound mRNAs at the perisynaptic and perivascular interfaces. To overcome these limitations, we recently refined the TRAP protocol by adding additional precleaning and blocking steps that reduced the background noise caused by unspecific mRNA binding (Mazaré et al., 2020) (Fig. 2C). The use of this protocol eliminated contamination by neuronal mRNA and enabled us to extract a pool of 844 astroglial ribosome-bound mRNAs in PAPs from dorsal hippocampus synaptogliosomes. It should be noted that our extraction protocol differed slightly from that used in the synaptoneurosome study by Sakers et al. because we did not include ultracentrifugation on discontinuous Percoll-sucrose density gradients [START_REF] Westmark | Preparation of synaptoneurosomes from mouse cortex using a discontinuous percoll-sucrose density gradient[END_REF]. By analogy with the above-mentioned "endfeetome" in PvAPs, we referred to this repertoire as the "PAPome" [START_REF] Mazare | Local Translation in Perisynaptic Astrocytic Processes Is Specific and Changes after Fear Conditioning[END_REF]. Interestingly, our study revealed a wholly new, unexpected set of enriched ribosome-bound transcripts in PAPs compared to those found previously [START_REF] Sakers | Astrocytes locally translate transcripts in their peripheral processes[END_REF]; the most abundant mRNAs encoded ubiquitous proteins involved in iron homeostasis, translation, the cell cycle, and the cytoskeleton [notably ezrin, as also identified by [START_REF] Sakers | Astrocytes locally translate transcripts in their peripheral processes[END_REF]] (Mazaré et al., 2020). Remarkably, a large proportion of ribosome-bound transcripts in PAPs encoded ribosomal proteins and elongation factors (Mazaré et al., 2020), which is reminiscent of observations in neuronal processes [START_REF] Deglincerti | Insights into the roles of local translation from the axonal transcriptome[END_REF][START_REF] Giustetto | Axonal transport of eukaryotic translation elongation factor 1alpha mRNA couples transcription in the nucleus to long-term facilitation at the synapse[END_REF][START_REF] Moccia | An unbiased cDNA library prepared from isolated Aplysia sensory neuron processes is enriched for cytoskeletal and translational mRNAs[END_REF][START_REF] Shigeoka | On-Site Ribosome Remodeling by Locally Synthesized Ribosomal Proteins in Axons[END_REF].

Overall, our results strongly suggest that local translation in PAPs might be sustained by local synthesis of the translation machinery, with either the de novo assembly of translational complexes or the replacement of damaged proteins, as recently suggested for ribosomal proteins in axons [START_REF] Shigeoka | On-Site Ribosome Remodeling by Locally Synthesized Ribosomal Proteins in Axons[END_REF].

The subcellular organization of local protein synthesis and maturation in astrocytes

To become functional, most secreted and membrane proteins must undergo post-translational modifications. In PvAPs, most of the endfeetome mRNAs encode membrane proteins that require folding and/or glycosylation as they pass through the endoplasmic reticulum (ER) and the Golgi apparatus. Accordingly, we observed smooth, rough and mixed ERs in all PvAPs (Fig. 3). A full Golgi apparatus was also detected in 7% of PvAPs, and these specific PvAPs might therefore contain the same canonical translation machinery as the soma [START_REF] Boulay | Translation in astrocyte distal processes sets molecular heterogeneity at the gliovascular interface[END_REF]. PAPs are extremely thin structures (<50 nm in diameter) [START_REF] Reichenbach | Morphology and dynamics of perisynaptic glia[END_REF], and their subcellular organization has not yet been fully explored. However, by using an adeno-associated virus strategy, we were able to show that 32% of the hippocampal glutamatergic synapses in an astrocyte territory are contacted by PAPs containing ERGIC-53, an integral membrane protein located in the intermediate region between the ER and the Golgi apparatus [START_REF] Mazare | Local Translation in Perisynaptic Astrocytic Processes Is Specific and Changes after Fear Conditioning[END_REF]. Furthermore, expression of the Golgi tracker pGolt [START_REF] Mikhaylova | A Dendritic Golgi Satellite between ERGIC and Retromer[END_REF] was detected in PAPs surrounding 38% of the glutamatergic synapses, suggesting the existence of detached Golgi particles (possibly outposts) in PAPs [START_REF] Mazare | Local Translation in Perisynaptic Astrocytic Processes Is Specific and Changes after Fear Conditioning[END_REF] (Fig. 3). A recent electron microscopy study provided evidence for ER in PvAPs and PAPs, and showed that the contact between the ER and mitochondria in PvAPs changed upon brain injury [START_REF] Göbel | Mitochondria-Endoplasmic Reticulum Contacts in Reactive Astrocytes Promote Vascular Remodeling[END_REF]. The authors suggested that the subcellular organization of astrocyte processes might be dynamically regulated [START_REF] Göbel | Mitochondria-Endoplasmic Reticulum Contacts in Reactive Astrocytes Promote Vascular Remodeling[END_REF]. Finally, to go further, it would be useful to determine whether polysomes and/or monosomes are present in PvAPs as well as PAPs. Indeed, monosomes display specific translational properties [START_REF] Heyer | Redefining the Translational Status of 80S Monosomes[END_REF] and have been shown to translate key synaptic transcripts in dendrites and axons [START_REF] Biever | Monosomes actively translate synaptic mRNAs in neuronal processes[END_REF].

Overall, the subcellular organization of PvAPs and PAPs is heterogenous; it is likely that the full functional diversity of translational and post-translational mechanisms in these structures has yet to be discovered [START_REF] Rangaraju | Local translation in neuronal compartments: how local is local?[END_REF].

The molecular bases of translation in astrocytes

Local translation is mediated by cis-acting elements that include RNA motifs and secondary structures influencing the binding of trans-acting proteins, also known as RNA-binding proteins (RBPs) [START_REF] Harvey | Trans-acting translational regulatory RNA binding proteins[END_REF]. RNA-binding motifs are mostly present in UTRs in general and the 3'UTR in particular. Interestingly, a gene can give rise to different 3'UTR isoforms through alternative polyadenylation, which therefore modulates the ability of an mRNA to bind to an RBP. mRNAs bind to RBPs in the nucleus and soma to form ribonucleoparticles (RNPs, also referred to as granules). Depending on the nature of the cis-and trans-acting elements, the RNPs are transported along the cytoskeleton as cargo by kinesin and dynein molecular motor complexes [START_REF] Pushpalatha | Local Translation in Axons: When Membraneless RNP Granules Meet Membrane-Bound Organelles[END_REF]. Although transcriptomic data indicate that RBPs are expressed in astrocytes (sometimes more strongly than in neurons), their roles in astrocytes have been poorly explored [START_REF] Zhang | An RNA-sequencing transcriptome and splicing database of glia, neurons, and vascular cells of the cerebral cortex[END_REF] (Table 1). Fmr1 encodes the fragile X mental retardation protein (FMRP) and is mutated in fragile X syndrome (FXS, a neurodevelopmental disorder resulting in intellectual disability and autism) [START_REF] Penagarikano | The pathophysiology of fragile x syndrome[END_REF]. One of the several possible functions of FMRP in neurons is its ability to act as an RBP and silence the translation of mRNAs encoding synaptic proteins [START_REF] Darnell | FMRP stalls ribosomal translocation on mRNAs linked to synaptic function and autism[END_REF]. Interestingly, FMRP might be more strongly expressed in astrocytes than in neurons (Table 1) [START_REF] Zhang | An RNA-sequencing transcriptome and splicing database of glia, neurons, and vascular cells of the cerebral cortex[END_REF]. It has been shown that FMRP controls mRNA transport in radial glia PvAPs in vivo and binds to mRNAs that encode autism-related signaling proteins and cytoskeletal regulators [START_REF] Pilaz | Dynamic mRNA Transport and Local Translation in Radial Glial Progenitors of the Developing Brain[END_REF]. In astrocytes, the selective loss of FMRP was shown to dysregulate protein synthesis in general and expression of the glutamate transporter GLT1 (impairing neurotransmission and astrocytic glutamate uptake) in particular [START_REF] Higashimori | Selective Deletion of Astroglial FMRP Dysregulates Glutamate Transporter GLT1 and Contributes to Fragile X Syndrome Phenotypes In Vivo[END_REF]. In astrocytes, the expression of a pathological form of FMRP linked to late-onset FXS/ataxia syndrome was found to impair motor performance in the mouse [START_REF] Wenzel | Astroglial-targeted expression of the fragile X CGG repeat premutation in mice yields RAN translation, motor deficits and possible evidence for cell-to-cell propagation of FXTAS pathology[END_REF]. These results strongly suggest that translational control by astroglial FMRP is involved in the pathogenesis of FXS.

The RBP quaking (QKI) is also strongly expressed in glial cells [START_REF] Zhang | An RNA-sequencing transcriptome and splicing database of glia, neurons, and vascular cells of the cerebral cortex[END_REF]. QKI was initially implicated in the regulation of Mbp mRNA transport in oligodendrocyte processes [START_REF] Li | Destabilization and mislocalization of myelin basic protein mRNAs in quaking dysmyelination lacking the QKI RNA-binding proteins[END_REF][START_REF] Doukhanine | The QKI-6 RNA binding protein regulates actin-interacting protein-1 mRNA stability during oligodendrocyte differentiation[END_REF][START_REF] Larocque | The QKI-6 and QKI-7 RNA binding proteins block proliferation and promote Schwann cell myelination[END_REF][START_REF] Wang | The QKI-6 RNA binding protein localizes with the MBP mRNAs in stress granules of glial cells[END_REF]. Furthermore, the QKI-7 cytosolic isoform was shown to regulate Gfap mRNA translation in human primary astrocytes [START_REF] Radomska | RNA-binding protein QKI regulates Glial fibrillary acidic protein expression in human astrocytes[END_REF]. More recently, it has been demonstrated that the ribosome-bound mRNAs that are preferentially found in PAPs contain a larger number of QKI-binding motifs [START_REF] Sakers | Astrocytes locally translate transcripts in their peripheral processes[END_REF]. This study also suggested that the inactivation of QKI-6 (another cytosolic isoform of QKI) in astrocytes altered the binding of certain mRNAs (e.g. Sparc) to ribosomes and thus influenced translation (Sakers et al., 2020, preprint). The role of QKI in brain development was recently studied in a conditional knock-out (KO) model of Qki in neural stem cells (NSCs), which showed that QKI regulates the differentiation of NSCs into glial precursor cells by upregulating several genes involved in gliogenesis [START_REF] Takeuchi | Identification of Qk as a Glial Precursor Cell Marker that Governs the Fate Specification of Neural Stem Cells to a Glial Cell Lineage[END_REF]. Lastly, QKI has been reported as a candidate gene in schizophrenia [START_REF] Aberg | Human QKI, a new candidate gene for schizophrenia involved in myelination[END_REF]. Thus, as with FXS, the regulation of QKI-mediated translation in astrocytes might be involved in schizophrenia.

Human antigen R (HuR, also referred to as ELAV-like protein 1) is one of the best-known RBPs in astrocytes. It binds to AU-rich elements (AREs) and stabilizes mRNAs [START_REF] Brennan | HuR and mRNA stability[END_REF]. Although HuR is predominantly located in the nucleus, it translocates to the cytoplasm, transports the bound mRNAs to polysomes, and promotes their translation and stabilization [START_REF] Fan | Overexpression of HuR, a nuclear-cytoplasmic shuttling protein, increases the in vivo stability of ARE-containing mRNAs[END_REF]. This mode of translational regulation has been observed in vivo following spinal cord injury (Kwan et al., 2017a) and in vitro in a stretch injury model of primary astrocytes, in which HuR activated the expression of inflammatory mediators such as interleukin-1β, tumor necrosis factor-α, and matrix metalloprotease-12 (Kwan et al., 2017b) . Furthermore, translocation of HuR from the nucleus to the cytoplasm has been found to upregulate the translation of the cysteine-glutamate antiporter (also known as the xCT system), following the treatment of mouse cortical primary astrocytes with interleukin-1 [START_REF] Shi | Interleukin 1beta Regulation of the System xc-Substratespecific Subunit, xCT, in Primary Mouse Astrocytes Involves the RNA-binding Protein HuR[END_REF]. HuR might therefore be a key factor for astrocyte translation in inflammatory contexts. Interestingly, HuR in primary astrocytes was also found to bind to the 3'UTR of Tardbp, which encodes TDP-43, an RBP linked to amyotrophic lateral sclerosis (ALS) [START_REF] Lu | Hu antigen R (HuR) is a positive regulator of the RNA-binding proteins TDP-43 and FUS/TLS: implications for amyotrophic lateral sclerosis[END_REF]. Lastly, experiments in a glioma cell line showed that HuR bound to and stabilized the mRNA encoding B-cell lymphoma 2 (Bcl2, an important regulator of cell death) by activating its translation [START_REF] Filippova | The RNA-binding protein HuR promotes glioma growth and treatment resistance[END_REF].

Consistently, the cytoplasmic level of HuR is positively correlated with the tumor grade in human glioma tissues [START_REF] Bolognani | mRNA stability alterations mediated by HuR are necessary to sustain the fast growth of glioma cells[END_REF].

Other known RBPs have been less extensively studied with regard to their possible effects on translation in astrocytes. This is the case for the cytoplasmic polyadenylation element binding protein 1 (CPEB1), which regulates polyA tail length and binds cytoplasmic polyadenylation elements (CPEs) in mRNA [START_REF] Hake | CPEB is a specificity factor that mediates cytoplasmic polyadenylation during Xenopus oocyte maturation[END_REF]. CPEB1 also regulates synaptic plasticity [START_REF] Alarcon | Chromatin acetylation, memory, and LTP are impaired in CBP+/-mice: a model for the cognitive deficit in Rubinstein-Taybi syndrome and its amelioration[END_REF] and binds to RNA granules in dendrites [START_REF] Ohashi | Cataloguing and Selection of mRNAs Localized to Dendrites in Neurons and Regulated by RNA-Binding Proteins in RNA Granules[END_REF]. In primary astrocytes, Fabp7 mRNA (containing CPEs in its 3'UTR) co-immunoprecipitates with CPEB1 [START_REF] Gerstner | Time of day regulates subcellular trafficking, tripartite synaptic localization, and polyadenylation of the astrocytic Fabp7 mRNA[END_REF]. Interestingly, the length of the polyA tail in Fabp7 mRNA in mouse brain samples varies with the time of day, and CPEB1 might thus have a role in the astrocytemediated neuronal plasticity linked to circadian-rhythm, as previously suggested [START_REF] Gerstner | Time of day regulates subcellular trafficking, tripartite synaptic localization, and polyadenylation of the astrocytic Fabp7 mRNA[END_REF]. Furthermore, CPEB1 controls the division of rat primary astrocytes [START_REF] Kim | Cyclin B1 expression regulated by cytoplasmic polyadenylation element binding protein in astrocytes[END_REF]. Upon the stimulation of cell division, CPEB1 is phosphorylated, binds to cyclinB1 mRNA and lengthens the mRNA polyA tail; in turn, this increases the rate of cyclinB1 translation and activates cell proliferation [START_REF] Kim | Cyclin B1 expression regulated by cytoplasmic polyadenylation element binding protein in astrocytes[END_REF]. The far upstream element-binding protein 2 (FUSE-binding protein 2 (FUBP2), also known as KH type-splicing regulatory protein (KSRP)) is an RBP that destabilizes ARE-containing mRNAs [START_REF] Bird | KSRP modulation of GAP-43 mRNA stability restricts axonal outgrowth in embryonic hippocampal neurons[END_REF]. In an in vitro luciferase reporter assay, FUSE-binding protein 2 was shown to downregulate the translation of cytokines. The knockdown of FUSE-binding protein 2 in rat primary astrocytes induced cortical neuron toxicity and astrocyte migration [START_REF] Li | KSRP: a checkpoint for inflammatory cytokine production in astrocytes[END_REF].

In conclusion, only a few in vivo studies have been conducted regarding astrocytic RBPs and data on their mode of action and their function in astrocytes are scarce. In particular, their possible role in the regulation of local translation remains to be addressed.

Conclusions and perspectives

Local translation in astrocytes is an emerging field of research, and many questions have yet to be addressed.

Firstly, the role of local translation in the establishment and maintenance of the functional polarity of astrocytes at their perivascular and perisynaptic interfaces is still an open question. Astrocyte heterogeneity constitutes a technical challenge in this respect. Indeed, astrocytes are diverse in terms of both morphology, for instance with voluminous, bushy, protoplasmic astroglia in the gray matter compared to elongated, fibrous astroglia in the white matter, and functionality, particularly with regard to their neuronal and vascular microenvironment [START_REF] Farmer | Resolving Astrocyte Heterogeneity in the CNS[END_REF][START_REF] Miller | Astrocyte Heterogeneity in the Adult Central Nervous System[END_REF]. For instance, the gliovascular interface greatly differs from one region of the brain to another and from one type of vessel to another, i.e. capillaries, arteries or veins (Cohen-Salmon et al., 2020). The same reasoning applies to PAPs, which display functional and morphological differences throughout the brain. Another inherent difficulty in studies of astrocyte (compared to neurons) lies in the absence of in vitro systems, in which astrocytes can reliably develop polarized interfaces with vessels and/or neurons.

The emergence of new in vitro microfluidic or organoid methods might offer greater opportunities in the future.

If local translation does sustain astrocyte polarity, it might occur differently in the soma and in the various processes. As mentioned above, the three TRAP studies found clear differences in the repertoire of ribosome-bound mRNAs between astrocyte soma and the processes, suggesting that local translation might sustain molecular polarity in PAPs and PvAPs [START_REF] Boulay | Translation in astrocyte distal processes sets molecular heterogeneity at the gliovascular interface[END_REF][START_REF] Mazare | Local Translation in Perisynaptic Astrocytic Processes Is Specific and Changes after Fear Conditioning[END_REF][START_REF] Sakers | Astrocytes locally translate transcripts in their peripheral processes[END_REF]. One of the most striking results obtained in our latest TRAP study of PAPs from the dorsal hippocampus was the enrichment in several ribosome-bound mRNAs encoding proteins of the large and small ribosomal subunits in PAPs compared to whole astrocytes [START_REF] Mazare | Local Translation in Perisynaptic Astrocytic Processes Is Specific and Changes after Fear Conditioning[END_REF]. These results suggest that the ribosomal compositions of the astrocyte soma and PAPs could differ. Ribosomal proteins exhibit different functions in ribosomes [START_REF] Castello | Insights into RNA biology from an atlas of mammalian mRNA-binding proteins[END_REF], and mutations in individual ribosomal proteins give rise to distinct qualitative effects rather than an overall loss of protein synthesis [START_REF] Shi | Translating the genome in time and space: specialized ribosomes, RNA regulons, and RNA-binding proteins[END_REF]. Hence, differences in ribosomal protein composition between astrocytic soma and PAPs might have important functional consequences. This question has yet to be resolved and will require a detailed proteomics study. The comparison of astrocytic interfaces might be an important way to determine the role of local translation in astrocyte polarity. PvAPs and PAPs differ in their subcellular organization; they both contain ER, but a full Golgi apparatus is observed only in approximately 7% of PvAPs, suggesting that protein maturation is regulated differently (even among PvAPs) [START_REF] Boulay | Translation in astrocyte distal processes sets molecular heterogeneity at the gliovascular interface[END_REF]. Interestingly, studies of dendrites have shown that membrane proteins bypassing the Golgi apparatus display atypical N-glycosylation profiles and thus probably have specific properties [START_REF] Hanus | Unconventional secretory processing diversifies neuronal ion channel properties[END_REF]. A link between Golgi outposts, microtubule branching, cell polarity, and myelination was recently observed in oligodendrocytes [START_REF] Fu | The Golgi Outpost Protein TPPP Nucleates Microtubules and Is Critical for Myelination[END_REF]. These findings suggest that the differences in ultrastructural organization between PvAPs and PAPs might underpin a diversity of translational, post-translational mechanisms and perhaps other functions yet to be discovered. Lastly, and if local translation indeed sustains astrocyte polarity, PvAPs and PAPs might differ in the repertoire of ribosome-bound mRNAs. This question is still unresolved because the endfeetome has been characterized in whole brain and only covered transcripts that were abundant in astrocytes [START_REF] Boulay | Translation in astrocyte distal processes sets molecular heterogeneity at the gliovascular interface[END_REF], and the full ribosome-bound repertoire in PAPs was assessed specifically in the dorsal hippocampus (Mazaré et al., 2020). Our recent refinement of the TRAP protocol now makes it possible to compare ribosome-bound mRNAs in PAPs and PvAPs from a given region of the brain (Mazaré et al., 2020). Incidentally, this technical refinement might also be useful for reinvestigating ribosomebound mRNAs in axons and dendrites because the earlier TRAP preparations of synapses or neurites contained astrocyte-specific mRNAs [START_REF] Ouwenga | Transcriptomic Analysis of Ribosome-Bound mRNA in Cortical Neurites In Vivo[END_REF][START_REF] Shigeoka | Dynamic Axonal Translation in Developing and Mature Visual Circuits[END_REF]. Interestingly, our study of some of the most abundant mRNAs in the PAPome revealed differences between hippocampal PAPs and PvAPs, suggesting that local translation might indeed govern functional polarity (Mazaré et al., 2020). One of the ribosome-bound mRNAs found to be more abundant in PAPs than in PvAPs was Rplp1, which encodes a ribosomal protein, suggesting that PvAPs and PAPs might also differ in the composition of their ribosomes, which might give rise to specific translational properties [START_REF] Castello | Insights into RNA biology from an atlas of mammalian mRNA-binding proteins[END_REF][START_REF] Shi | Translating the genome in time and space: specialized ribosomes, RNA regulons, and RNA-binding proteins[END_REF].

Besides polarity, there is a need for further investigations into the physiological and pathophysiological relevance of local translation in astrocytes. Interestingly, we observed that fear-conditioning in mice altered the levels of several enriched ribosome-bound mRNAs in dorsal hippocampal PAPs; this hints at a physiological role of astrocyte local translation in memory and learning and, more generally, in the cellular response to environmental cues (Mazaré et al., 2020). During development, local translation in axons has a preponderant role in growth cone guidance, axon elongation and membrane remodeling [START_REF] Wu | Local translation of RhoA regulates growth cone collapse[END_REF][START_REF] Yao | An essential role for beta-actin mRNA localization and translation in Ca2+-dependent growth cone guidance[END_REF][START_REF] Campbell | Chemotropic responses of retinal growth cones mediated by rapid local protein synthesis and degradation[END_REF][START_REF] Leung | Asymmetrical beta-actin mRNA translation in growth cones mediates attractive turning to netrin-1[END_REF][START_REF] Ming | Adaptation in the chemotactic guidance of nerve growth cones[END_REF][START_REF] Piper | Signaling mechanisms underlying Slit2-induced collapse of Xenopus retinal growth cones[END_REF][START_REF] Cagnetta | Rapid Cue-Specific Remodeling of the Nascent Axonal Proteome[END_REF][START_REF] Hengst | Axonal elongation triggered by stimulus-induced local translation of a polarity complex protein[END_REF][START_REF] Gracias | Local translation of TC10 is required for membrane expansion during axon outgrowth[END_REF]. Results on axonal growth cone raised the intriguing possibility that local translation conditions the growth and formation of PvAPs and PAPs. Dysregulation of local translation in neurons has been linked to diseases such as FXS [START_REF] Kao | Altered mRNA transport, docking, and protein translation in neurons lacking fragile X mental retardation protein[END_REF], spinal muscular atrophy [START_REF] Jablonka | Co-regulation of survival of motor neuron (SMN) protein and its interactor SIP1 during development and in spinal muscular atrophy[END_REF][START_REF] Zhang | Active transport of the survival motor neuron protein and the role of exon-7 in cytoplasmic localization[END_REF], ALS [START_REF] Alami | Axonal transport of TDP-43 mRNA granules is impaired by ALS-causing mutations[END_REF][START_REF] Fallini | The ALS disease protein TDP-43 is actively transported in motor neuron axons and regulates axon outgrowth[END_REF], and Alzheimer´s disease [START_REF] Baleriola | Axonally synthesized ATF4 transmits a neurodegenerative signal across brain regions[END_REF][START_REF] Kobayashi | Local Somatodendritic Translation and Hyperphosphorylation of Tau Protein Triggered by AMPA and NMDA Receptor Stimulation[END_REF][START_REF] Li | Somatodendritic accumulation of Tau in Alzheimer's disease is promoted by Fynmediated local protein translation[END_REF][START_REF] Walker | Abeta1-42 triggers the generation of a retrograde signaling complex from sentinel mRNAs in axons[END_REF]. Furthermore, local translation might also be essential for the restoration of axon outgrowth after axon injury [START_REF] Koley | Translating regeneration: Local protein synthesis in the neuronal injury response[END_REF]. With regard to the glia, translation of myelin binding protein in oligodendrocyte distal cell processes in vitro is impaired by exposure to amyloid -peptide [START_REF] Quintela-Lopez | Abeta oligomers promote oligodendrocyte differentiation and maturation via integrin beta1 and Fyn kinase signaling[END_REF]. We recently observed that the distribution of mRNAs encoding the GFAP isoforms  and  differed in an Alzheimer's disease mouse model, particularly in astrocytes close to amyloid deposits [START_REF] Oudart | AstroDot -a new method for studying the spatial distribution of mRNA in astrocytes[END_REF]. As discussed above, most of the RBPs linked to neuronal dysfunction are also expressed in astrocytes. Alteration of astrocyte polarity is a hallmark in neuropathology [START_REF] Cohen-Salmon | Astrocytes in the regulation of cerebrovascular functions[END_REF][START_REF] Dossi | Human astrocytes in the diseased brain[END_REF]. Hence, changes in local translation in astrocytes are likely to be linked to diseases by altering the perivascular and perisynaptic functions of astrocytes.

In conclusion, the discovery of local translation in astrocytes raises a new repertoire of questions, in particular regarding the way astrocytes regulate their high level of polarity in normal and pathological contexts. Given the critical functions of astrocytes in the regulation of synaptic and vascular functions, characterization of local translation in these cells might also reveal important and novel aspects of the brain physiology.

RBP Gene

Expression HuR increases the level of cytokine mRNAs (Kwan et al., 2017a;Kwan et al., 2017b) In vitro primary cortical astrocytes IL-1 promotes HuR translocation to the cytoplasm. HuR's binding to the 3'UTR of xCT mRNA increases the latter's half-life, protein level, and functions linked to the xCT system. [START_REF] Shi | Interleukin 1beta Regulation of the System xc-Substratespecific Subunit, xCT, in Primary Mouse Astrocytes Involves the RNA-binding Protein HuR[END_REF] In vitro primary cortical astrocytes from the G93A SOD1 mouse [START_REF] Zhang | An RNA-sequencing transcriptome and splicing database of glia, neurons, and vascular cells of the cerebral cortex[END_REF] HuR's binding to the 3'UTR of Tardbp and Fus mRNAs controls their translational efficiency. HuR KO in astrocytes leads to neuronal toxicity. [START_REF] Lu | Hu antigen R (HuR) is a positive regulator of the RNA-binding proteins TDP-43 and FUS/TLS: implications for amyotrophic lateral sclerosis[END_REF] In vitro U251 cells and glioblastoma xenograft HuR upregulates bcl-2 mRNA translation and promotes cell survival. [START_REF] Filippova | The RNA-binding protein HuR promotes glioma growth and treatment resistance[END_REF] Human glioma tissue and cell lines

The HuR protein level is increased in gliomas and correlates with the tumor grade. (Bolognani et al., [START_REF] Briata | Diverse roles of the nucleic acid-binding protein KHSRP in cell differentiation and disease: Diverse roles of the nucleic acid-binding protein KHSRP[END_REF] In vitro primary cortical astrocytes from KSRP-KO and WT mice KSRP downregulates some cytokine mRNAs and mediates an inflammatory response in astrocytes. [START_REF] Li | KSRP: a checkpoint for inflammatory cytokine production in astrocytes[END_REF]) The experimental set up used in [START_REF] Boulay | Translation in astrocyte distal processes sets molecular heterogeneity at the gliovascular interface[END_REF] to analyze ribosome-bound mRNAs in perivascular astrocytic processes (PvAPs) from the whole brain. mRNAs from blood vessels (including the basal lamina, endothelial cells, and mural cells) were mechanically purified from whole brain and thereby retained the PvAPs. They were then compared with mRNAs extracted from blood vessels after the basal lamina was enzymatically digested to remove PvAPs. mRNAs found to be more abundant in the preparation that retained PvAPs were identified. In parallel, translating ribosome affinity purification (TRAP) was applied to mechanically isolated blood vessels from whole brains of Aldh1l1:L10a-eGFP mice, expressing an eGFP fusion to the ribosomal protein L10A specifically in astrocytes, in order to extract ribosome-bound mRNAs from PvAPs (second row). This preparation was then compared with the set of ribosome-bound mRNAs from whole astrocytes (extracted from whole brains using TRAP). Ribosome-bound mRNAs that were more abundant in PvAPs than in whole astrocytes were selected and then compared with ribosome-bound mRNAs in PvAPs. The overlapping mRNAs constituted the endfeetome, a set of highly expressed, ribosome-bound mRNAs in PvAPs.

B. Identification of ribosome-bound mRNAs present in PAPs and highly enriched in the

synaptoneurosomes of the cortex. The experimental design used by [START_REF] Sakers | Astrocytes locally translate transcripts in their peripheral processes[END_REF] to analyze ribosomebound mRNAs in perisynaptic processes (PAPs) from the cortex. mRNAs from cortical synaptoneurosomes (including the PAP, pre-synapse and postsynapse) were purified and compared with mRNAs from whole cortices and the mRNAs that were more abundant in synaptoneurosomes were determined. In parallel, TRAP was used to extract the set of ribosome-bound mRNAs from cortical PAPs (the PAP-TRAP fraction, second row), and the latter were compared with mRNAs from whole cortical synaptoneurosomes to determine the mRNAs that are towards blood vessels. The astrocytic endoplasmic reticulum (ER) forms a continuous network that is in contact with both PAPs and PvAPs. PvAPs also contain rough ER to which ribosomes are bound, as well as mitochondria, ribosomes, mRNAs and mRNA-ribosome complexes that translate new proteins. A Golgi apparatus is observed in ~7% of the PvAPs in the cortex and the hippocampus. In contrast, PAPs only contain small Golgi particles (pGolt particles), and small, round mitochondria can also be observed.

Fig. 1 .

 1 Fig. 1. Comparison of perivascular and perisynaptic astrocytic processes. Schematic representation of an astrocyte extending perisynaptic astrocytic processes (PAPs) towards a synapse, comprising the pre-and the post-synapse. Astrocytes also send perivascular astrocytic processes (PvAPs) towards blood vessels; together with mural cells, endothelial cells and the basal lamina, the PvAPs form the gliovascular unit. Synapses can be located adjacent to PvAPs (centre). A confocal microscopy image of an astrocyte filled with eGFP is shown on the left. The astrocyte comes from the pyramidal layer of the hippocampus in a transgenic mouse line expressing GFP under the control of the astrocyte-specific Gfap promoter. The synapses are labelled with the pre-and postsynaptic markers VGluT1 and Homer1. Scale bar: 10 µm. The inset shows PAPs in contact with synapses. On the right, a confocal microscopy image of a hippocampal astrocyte from a transgenic mouse expressing eGFP under the control of the Gfap promoter is shown. The astrocyte extends a PvAP (boxed area) that wraps around the blood vessel (labelled with isolectin B4; IB4). Scale bar: 10 µm. The inset highlights an astrocyte PvAP surrounding the blood vessel. The electron micrograph below the scheme shows a PvAP contiguous with PAPs: Two synapses are abutting a PvAP around a blood vessel.

Fig. 2 .

 2 Fig. 2. Identification of ribosome-bound mRNAs in the perivascular and perisynaptic processes of astrocytes. A. Identification of ribosome-bound mRNAs present in PvAPs and highly expressed in astrocytes in the whole brain. The experimental set up used in[START_REF] Boulay | Translation in astrocyte distal processes sets molecular heterogeneity at the gliovascular interface[END_REF] to analyze ribosome-bound

  more abundant in the PAP-TRAP fraction. Next, mRNAs that were more abundant in synaptoneurosomes were compared with ribosome-bound mRNAs from PAPs; and overlap between the two sets yielded a set of ribosome-bound mRNAs present in PAPs and highly enriched in astrocytes. C. Identification of the entire pool of ribosome-bound mRNAs enriched in PAPs of the dorsal hippocampus. The experimental design used by(Mazaré et al., 2020) to analyze ribosome-bound mRNAs that are more abundant in PAPs compared with whole astrocytes from the dorsal hippocampus. Starting with synaptogliosomes from dorsal hippocampi and whole dorsal hippocampi, a refined TRAP protocol was used to collect ribosome-bound mRNAs present in PAPs or whole astrocytes. The overlap between the two sets yielded the entire pool of ribosome-bound mRNAs that were enriched in PAPs from the dorsal hippocampus.

Fig. 3 .

 3 Fig. 3. Subcellular organization of perisynaptic and perivascular astrocytic processes. Astrocytes extend perisynaptic astrocytic processes (PAPs) towards the synapse and perivascular astrocytic processes (PvAPs)

Acknowledgments

This work was funded by the Fondation pour la Recherche Médicale (FRM) (grant: AJE20171039094), the Fondation pour l'aide à la recherche sur la sclérose en plaques (ARSEP), the Association Européenne contre les Leucodystrophies (ELA), and the Fondation France Alzheimer to M. Cohen-Salmon. N. Mazaré's work was funded by the ED3C doctoral student program and the FRM (grant: FDT201904008077). M. Oudart's work was funded by the ED3C doctoral student program. Creation of the Center for Interdisciplinary Research in Biology (CIRB) was funded by the Fondation Bettencourt Schueller.