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.

In mice and humans, brain microvascular contractility matures postnatally.

Although great efforts to characterize the embryonic phase of brain microvascular system development have been made, its postnatal maturation has barely been described. 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 (EC), vascular smooth muscle cells (VSMC) and fibroblasts (FB) follow specific molecular maturation programs over this time period. Focusing on VSMCs, we showed that the arteriolar VSMC network expands and becomes contractile resulting in a greater cerebral blood flow (CBF), with heterogenous developmental trajectories within cortical regions. Samples of the human brain cortex showed the same postnatal maturation process. Thus, the postnatal phase is a critical period during which arteriolar VSMC contractility required for vessel tone and brain perfusion is acquired and mature.

PAI-1 production by reactive astrocytes drives tissue dysfibrinolysis in multiple sclerosis models.

BACKGROUND: In multiple sclerosis (MS), disturbance of the plasminogen activation system (PAS) and blood brain barrier (BBB) disruption are physiopathological processes that might lead to an abnormal fibrin(ogen) extravasation into the parenchyma. Fibrin(ogen) deposits, usually degraded by the PAS, promote an autoimmune response and subsequent demyelination. However, the PAS disruption is not well understood and not fully characterized in this disorder. METHODS: Here, we characterized the expression of PAS actors during different stages of two mouse models of MS (experimental autoimmune encephalomyelitis-EAE), in the central nervous system (CNS) by quantitative RT-PCR, immunohistofluorescence and fluorescent in situ hybridization (FISH). Thanks to constitutive PAI-1 knockout mice (PAI-1 KO) and an immunotherapy using a blocking PAI-1 antibody, we evaluated the role of PAI-1 in EAE models and its impact on physiopathological processes such as fibrin(ogen) deposits, lymphocyte infiltration and demyelination. RESULTS: We report a striking overexpression of PAI-1 in reactive astrocytes during symptomatic phases, in two EAE mouse models of MS. This increase is concomitant with lymphocyte infiltration and fibrin(ogen) deposits in CNS parenchyma. By genetic invalidation of PAI-1 in mice and immunotherapy using a blocking PAI-1 antibody, we demonstrate that abolition of PAI-1 reduces the severity of EAE and occurrence of relapses in two EAE models. These benefits are correlated with a decrease in fibrin(ogen) deposits, infiltration of T4 lymphocytes, reactive astrogliosis, demyelination and axonal damage. CONCLUSION: These results demonstrate that a deleterious overexpression of PAI-1 by reactive astrocytes leads to intra-parenchymal dysfibrinolysis in MS models and anti-PAI-1 strategies could be a new therapeutic perspective for MS.

Megalencephalic leukoencephalopathy with subcortical cysts is a developmental disorder of the gliovascular unit.

Absence of the astrocyte-specific membrane protein MLC1 is responsible for megalencephalic leukoencephalopathy with subcortical cysts (MLC), a rare type of leukodystrophy characterized by early-onset macrocephaly and progressive white matter vacuolation that lead to ataxia, spasticity, and cognitive decline. During postnatal development (from P5 to P15 in the mouse), MLC1 forms a membrane complex with GlialCAM (another astrocytic transmembrane protein) at the junctions between perivascular astrocytic processes. Perivascular astrocytic processes along with blood vessels form the gliovascular unit. It was not previously known how MLC1 influences the physiology of the gliovascular unit. Here, using the Mlc1 knock-out mouse model of MLC, we demonstrated that MLC1 controls the postnatal development and organization of perivascular astrocytic processes, vascular smooth muscle cell contractility, neurovascular coupling, and intraparenchymal interstitial fluid clearance. Our data suggest that MLC is a developmental disorder of the gliovascular unit, and perivascular astrocytic processes and vascular smooth muscle cell maturation defects are primary events in the pathogenesis of MLC and therapeutic targets for this disease.

CC17 group B Streptococcus exploits integrins for neonatal meningitis development.

Group B Streptococcus (GBS) is the major cause of human neonatal infections. A single clone, designated CC17-GBS, accounts for more than 80% of meningitis cases, the most severe form of the infection. However, the events allowing blood-borne GBS to penetrate the brain remain largely elusive. In this study, we identified the host transmembrane receptors α5ß1 and αvß3 integrins as the ligands of Srr2, a major CC17-GBS-specific adhesin. Two motifs located in the binding region of Srr2 were responsible for the interaction between CC17-GBS and these integrins. We demonstrated in a blood-brain-barrier cellular model that both integrins contributed to the adhesion and internalization of CC17-GBS. Strikingly, both integrins were overexpressed during the postnatal period in the brain vessels of the blood-brain barrier and blood-cerebrospinal fluid barrier and contributed to juvenile susceptibility to CC17 meningitis. Finally, blocking these integrins decreased the ability of CC17-GBS to cross into the CNS of juvenile mice in an in vivo model of meningitis. Our study demonstrated that CC17-GBS exploits integrins in order to cross the brain vessels, leading to meningitis. Importantly, it provides host molecular insights into neonate's susceptibility to CC17-GBS meningitis, thereby opening new perspectives for therapeutic and prevention strategies of GBS-elicited meningitis.

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.

Dp71 contribution to the molecular scaffold anchoring aquaporine-4 channels in brain macroglial cells.

Intellectual disability in Duchenne muscular dystrophy has been associated with the loss of dystrophin-protein 71, Dp71, the main dystrophin-gene product in the adult brain. Dp71 shows major expression in perivascular macroglial endfeet, suggesting that dysfunctional glial mechanisms contribute to cognitive impairments. In the present study, we investigated the molecular alterations induced by a selective loss of Dp71 in mice, using semi-quantitative immunogold analyses in electron microscopy and immunofluorescence confocal analyses in brain sections and purified gliovascular units. In macroglial pericapillary endfeet of the cerebellum and hippocampus, we found a drastic reduction (70%) of the polarized distribution of aquaporin-4 (AQP4) channels, a 50% reduction of ß-dystroglycan, and a complete loss of α1-syntrophin. Interestingly, in the hippocampus and cortex, these effects were not homogeneous: AQP4 and AQP4ex isoforms were mostly lost around capillaries but preserved in large vessels corresponding to pial arteries, penetrating cortical arterioles, and arterioles of the hippocampal fissure, indicating the presence of Dp71-independent pools of AQP4 in these vascular structures. In conclusion, the depletion of Dp71 strongly alters the distribution of AQP4 selectively in macroglial perivascular endfeet surrounding capillaries. This effect likely affects water homeostasis and blood-brain barrier functions and may thus contribute to the synaptic and cognitive defects associated with Dp71 deficiency.

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.

Astroglial Connexin 43 Deficiency Protects against LPS-Induced Neuroinflammation: A TSPO Brain µPET Study with [18F]FEPPA.

Astroglial connexin 43 (Cx43) has been recognized as a crucial immunoregulating factor in the brain. Its inactivation leads to a continuous immune recruitment, cytokine expression modification and a specific humoral autoimmune response against the astrocytic extracellular matrix but without brain lesions or cell lysis. To assess the impact of Cx43 deletion on the brain's inflammatory response, TSPO expression was studied by positron emission tomography (PET) imaging with a specific radioligand, [18F]FEPPA, in basal conditions or upon Lipopolysaccharides (LPS)-induced inflammatory challenge. Astroglial Cx43-deleted mice underwent [18F]FEPPA PET/CT dynamic imaging with or without LPS injection (5 mg/kg) 24 h before imaging. Quantification and pharmacokinetic data modelling with a 2TCM-1K compartment model were performed. After collecting the mice brains, TSPO expression was quantified and localized by Western blot and FISH analysis. We found that astroglial Cx43 deficiency does not significantly alter TSPO expression in the basal state as observed with [18F]FEPPA PET imaging, FISH and Western blot analysis. However, deletion of astrocyte Cx43 abolishes the LPS-induced TSPO increase. Autoimmune encephalopathy observed in astroglial Cx43-deleted mice does not involve TSPO overexpression. Consistent with previous studies showing a unique inflammatory status in the absence of astrocyte Cx43, we show that a deficient expression of astrocytic Cx43 protects the animals from LPS-induced neuroinflammation as addressed by TSPO expression.

Postnatal development of the astrocyte perivascular MLC1/GlialCAM complex defines a temporal window for the gliovascular unit maturation.

Astrocytes, the most abundant glial cells of the central nervous system are morphologically complex. They display numerous processes interacting with synapses and blood vessels. At the vascular interface, astrocyte endfeet-terminated processes almost entirely cover the blood vessel surface and participate to the gliovascular unit where important vascular properties of the brain are set such as the blood-brain barrier (BBB) integrity. How specific morphological and functional interactions between astrocytes and the vascular compartment develop has not been fully investigated. Here, we elaborated an original experimental strategy to study the postnatal development of astrocyte perivascular endfeet. Using purified gliovascular units, we focused on the postnatal expression of MLC1 and GlialCAM, two transmembrane proteins forming a complex enriched at the junction between mature astrocyte perivascular endfeet. We showed that MLC1 and GlialCAM were enriched and assembled into mature complexes in astrocyte perivascular endfeet between postnatal days 10 and 15, after the formation of astrocyte perivascular Aquaporin 4 water channels. These events correlated with the increased expression of Claudin-5 and P-gP, two endothelial-specific BBB components. These results illustrate for the first time that astrocyte perivascular endfeet differentiation is a complex and progressive process which correlates with BBB maturation. Moreover, our results suggest that maturation of the astrocyte endfeet MLC1/GlialCAM complex between postnatal days 10 and 15 might be a key event in the gliovascular unit maturation.

Preparing the Astrocyte Perivascular Endfeet Transcriptome to Investigate Astrocyte Molecular Regulations at the Brain-Vascular Interface.

Astrocytes send out long processes that are terminated by endfeet at the vascular surface and regulate vascular functions in particular through the expression of a specific molecular repertoire in perivascular endfeet. We recently proposed that local translation might sustain this structural and functional polarization. More specifically we showed that a subset of mRNAs is distributed in astrocyte endfeet and characterized this transcriptome. We also identified among these endfeet RNAs, the ones bound to ribosomes, the polysomal astrocyte endfeet mRNAs, which we called the endfeetome. Here, we describe experimental strategies to identify mRNAs and polysomes in astrocyte perivascular endfeet, which are based on the combination of gliovascular unit purification and astrocyte-specific translating ribosome affinity purification.

Isolation and differential transcriptome of vascular smooth muscle cells and mid-capillary pericytes from the rat brain.

Brain mural cells form a heterogeneous family which significantly contributes to the maintenance of the blood-brain barrier and regulation of the cerebral blood flow. Current procedures to isolate them cannot specifically separate their distinct subtypes, in particular vascular smooth muscle cells (VSMCs) and mid-capillary pericytes (mcPCs), which differ among others by their expression of smooth muscle actin (SMA). We herein describe an innovative method allowing SMA+ VSMCs and SMA- mcPCs to be freshly isolated from the rat cerebral cortex. Using differential RNA-Seq analysis, we then reveal the specific gene expression profile of each subtype. Our results refine the current description of the role of VSMCs in parenchymal cortical arterioles at the molecular level and provide a unique platform to identify the molecular mechanisms underlying the specific functions of mcPCs in the brain vasculature.

Connexin 43 Controls the Astrocyte Immunoregulatory Phenotype.

Astrocytes are the most abundant glial cells of the central nervous system and have recently been recognized as crucial in the regulation of brain immunity. In most neuropathological conditions, astrocytes are prone to a radical phenotypical change called reactivity, which plays a key role in astrocyte contribution to neuroinflammation. However, how astrocytes regulate brain immunity in healthy conditions is an understudied question. One of the astroglial molecule involved in these regulations might be Connexin 43 (Cx43), a gap junction protein highly enriched in astrocyte perivascular endfeet-terminated processes forming the glia limitans. Indeed, Cx43 deletion in astrocytes (Cx43KO) promotes a continuous immune recruitment and an autoimmune response against an astrocyte protein, without inducing any brain lesion. To investigate the molecular basis of this unique immune response, we characterized the polysomal transcriptome of hippocampal astrocytes deleted for Cx43. Our results demonstrate that, in the absence of Cx43, astrocytes adopt an atypical reactive status with no change in most canonical astrogliosis markers, but with an upregulation of molecules promoting immune recruitment, complement activation as well as anti-inflammatory processes. Intriguingly, while several of these upregulated transcriptional events suggested an activation of the γ-interferon pathway, no increase in this cytokine or activation of related signaling pathways were found in Cx43KO. Finally, deletion of astroglial Cx43 was associated with the upregulation of several angiogenic factors, consistent with an increase in microvascular density in Cx43KO brains. Collectively, these results strongly suggest that Cx43 controls immunoregulatory and angiogenic properties of astrocytes.

Connexin 30 is expressed in a subtype of mouse brain pericytes.

Pericytes are mural cells of blood microvessels which play a crucial role at the neurovascular interface of the central nervous system. They are involved in the regulation of blood-brain barrier integrity, angiogenesis, clearance of toxic metabolites, capillary hemodynamic responses, and neuroinflammation, and they demonstrate stem cell activity. Morphological and molecular studies to characterize brain pericytes recently pointed out some heterogeneity in pericyte population. Nevertheless, a clear definition of pericyte subtypes is still lacking. Here, we demonstrate that a fraction of brain pericytes express Connexin 30 (Cx30), a gap junction protein, which, in the brain parenchyma, was thought to be exclusively found in astrocytes. Cx30 could thus be a candidate protein in the composition of the gap junction channels already described between endothelial cells and pericytes. It could also form hemichannels or acts in a channel-independent manner to regulate pericyte morphology, as already observed in astrocytes. Altogether, our results suggest that Cx30 defines a novel brain pericyte subtype.

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

Astrocytes send out long processes that are terminated by endfeet at the vascular surface and regulate vascular functions as well as homeostasis at the vascular interface. To date, the astroglial mechanisms underlying these functions have been poorly addressed. Here we demonstrate that a subset of messenger RNAs is distributed in astrocyte endfeet. We identified, among this transcriptome, a pool of messenger RNAs bound to ribosomes, the endfeetome, that primarily encodes for secreted and membrane proteins. We detected nascent protein synthesis in astrocyte endfeet. Finally, we determined the presence of smooth and rough endoplasmic reticulum and the Golgi apparatus in astrocyte perivascular processes and endfeet, suggesting for local maturation of membrane and secreted proteins. These results demonstrate for the first time that protein synthesis occurs in astrocyte perivascular distal processes that may sustain their structural and functional polarization at the vascular interface.

Immunoregulation at the gliovascular unit in the healthy brain: A focus on Connexin 43.

In the brain, immune cell infiltration is normally kept at a very low level and a unique microenvironment strictly restricts immune reactions and inflammation. Even in such quiescent environment, a constant immune surveillance is at work allowing the brain to rapidly react to threats. To date, knowledge about the factors regulating the brain-immune system interrelationship in healthy conditions remains elusive. Interestingly, astrocytes, the most abundant glial cells in the brain, may participate in many aspects of this unique homeostasis, in particular due to their close interaction with the brain vascular system and expression of a specific molecular repertoire. Indeed, astrocytes maintain the blood-brain barrier (BBB) integrity, interact with immune cells, and participate in the regulation of intracerebral liquid movements. We recently showed that Connexin 43 (Cx43), a gap junction protein highly expressed by astrocytes at the BBB interface, is an immunoregulating factor. The absence of astroglial Cx43 leads to a transient endothelial activation, a continuous immune recruitment as well as the development of a specific humoral autoimmune response against the von Willebrand factor A domain-containing protein 5a, an extracellular matrix protein expressed by astrocytes. In this review, we propose to gather current knowledge on how astrocytes may influence the immune system in the healthy brain, focusing on their roles at the gliovascular interface. We will also consider pathological situations involving astrocyte-specific autoimmunities. Finally, we will discuss the specific role of astroglial Cx43 and the physiological consequences of immune regulations taking place on inflammation, cognition and behavior in the absence of Cx43.

Purification of Mouse Brain Vessels.

In the brain, most of the vascular system consists of a selective barrier, the blood-brain barrier (BBB) that regulates the exchange of molecules and immune cells between the brain and the blood. Moreover, the huge neuronal metabolic demand requires a moment-to-moment regulation of blood flow. Notably, abnormalities of these regulations are etiological hallmarks of most brain pathologies; including glioblastoma, stroke, edema, epilepsy, degenerative diseases (ex: Parkinson's disease, Alzheimer's disease), brain tumors, as well as inflammatory conditions such as multiple sclerosis, meningitis and sepsis-induced brain dysfunctions. Thus, understanding the signaling events modulating the cerebrovascular physiology is a major challenge. Much insight into the cellular and molecular properties of the various cell types that compose the cerebrovascular system can be gained from primary culture or cell sorting from freshly dissociated brain tissue. However, properties such as cell polarity, morphology and intercellular relationships are not maintained in such preparations. The protocol that we describe here is designed to purify brain vessel fragments, whilst maintaining structural integrity. We show that isolated vessels consist of endothelial cells sealed by tight junctions that are surrounded by a continuous basal lamina. Pericytes, smooth muscle cells as well as the perivascular astrocyte endfeet membranes remain attached to the endothelial layer. Finally, we describe how to perform immunostaining experiments on purified brain vessels.