Cohen-Salmon

Physiology and physiopathology of the gliovascular unit

Principal Investigator: Martine COHEN-SALMON,DR2 Cnrs

Astrocytes are of the most numerous neuroglial cells in the central nervous system (CNS). They display a complex ramified morphology, with processes contacting neurons and the blood vessels. Endfeet-terminated perivascular processes fully cover the brain vascular system. At this interface, astrocytes exert a predominant influence on the cerebrovascular system controlling perivascular homeostasis, blood-brain barrier (BBB) integrity, crosstalk with the peripheral immune system, endothelial transport, and vessel contractility in response to neuronal activity. This morphological and functional polarity is altered in most pathologies associated with vascular dysfunction including epilepsy, ischemic brain damage, Alzheimer’s disease or multiple sclerosis. Thus, the way functional polarization is set in astrocytes and the mechanisms by which astrocytes accomplish their regulatory functions at the brain vascular interface are questions of prime importance on which our group is focusing. Over the last years, we have elaborated an innovative and original research program to investigate how astrocytes regulate the physiology and physiopathology of the cerebrovascular system.

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Figure 1 : Schematic representation of the gliovascular unit. The squared area represents the gliovascular unit. A, astrocyte; AE, astrocyte endfeet; E, endothelial cell; M, mural cell; L, vessel lumen;. An astrocyte is shown in red.

I. Molecular approaches to decipher astrocyte functions at the vascular interface

Most of the molecular and cellular studies concerning the cerebrovascular system have been performed on purified brain vessel cells dissociated by cell-sorting using cell specific reporter mouse strains or immunostaining-based procedures. Although these techniques allow for the isolation of almost pure cerebrovascular cell populations, isolated cells completely lose their in situ morphology and interactions, which in turn, greatly affects their molecular and cellular properties. In our laboratory, we have developed a protocol allowing the isolation of whole cerebrovascular fragments with no need of specific antibodies or transgenic mouse stains, but based on differential centrifugations and filtration of brain homogenates. Through this procedure, we purify intact gliovascular units composed by endothelial cells, mural cells (vascular smooth muscle cells and pericytes) and astrocyte perivascular endfeet attached to the vessel surface. This procedure can be performed at any developmental stage and used to study proteins and mRNAs of the gliovascular unit, as well as gliovascular unit structure and morphology.

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Figure 2: Molecular analysis of the gliovascular unit. A. Purification of the gliovascular unit from the brain. Astrocyte perivascular endfeet remain attached to the vessel surface while astrocyte cell bodies and neural cells are eliminated. B. Purified gliovascular unit immunolabelled for Aqp4 (red) showing that astrocyte perivascular membranes are co-purified with brain vessels. Nuclei are labelled with Hoechst (blue) and blood vessel walls with IB4 (grey).

II. Deciphering the role of perivascular astrocyte endfeet-enriched proteins in the regulation of the cerebrovascular system

To understand the regulatory functions of astrocytes at the vascular interface, we are focusing on the role of astrocyte endfeet enriched proteins such as Connexins (Cx) 30 and Cx43. Cx30 and Cx43 are transmembrane proteins and part of a multigenic family present in almost every cell in vertebrates. Within the cell, Cxs assemble by six in hemichannels (HC) that insert into the plasma membrane, in possible mixed heterotypic or heterodimeric combinations depending on their compatibility. Cx30 and Cx43 can assemble together in common channels. Classically, HCs align head-to-head to form intercellular gap junction (GJ) channels, mediating direct cell-to-cell diffusion of ions and small signaling molecules (<1.2 kDa) and providing electrical and metabolic coupling. HCs can also persist at the cell membrane level in an undocked conformation and mediate the direct exchange of molecules between the intra- and extracellular milieu. Cxs also perform channel-independent functions involving protein interactions and intracellular signaling. In the mouse, Cx30 rises in astrocytes only from postnatal day (P) 10 and concentrates at the perivascular level from P12. In contrast, Cx43 is expressed in the developing brain from embryonic day E10.5 and its accumulation in astrocyte endfeet starts from P2, paralleling the formation of the perivascular astrocyte coverage. At the gliovascular interface, Cx30 and Cx43 gap junction channels have been shown to distribute glucose through the perivascular astroglial network, thereby sustaining neuronal activity. Co-deletion of Cx30 and Cx43, which uncouples astrocytes, leads to the partial loss of two astrocyte perivascular enriched proteins, Aquaporin4 and beta-Dystroglycan, and to the weakening of the blood-brain barrier (BBB). Taken individually, each astroglial Cx exerts distinct functions at the gliovascular interface. The absence of Cx30 does not influence BBB integrity. However, Cx30 channels regulate the transcription level of gamma-Sarcoglycan in the brain vessels. Cx43 inactivation in astrocytes modifies the immune quiescence of the brain. It leads to an abnormal recruitment of lymphocytes in the brain parenchyma and to the development of a specific humoral autoimmune response against an astrocyte extracellular matrix protein. Cx43 is therefore an astroglial factor regulating the crosstalk between the peripheral immune system and the brain.

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Figure 3: Deciphering the functions of astrocyte perivascular endfeet enriched molecules: The example of Cx43. A. Confocal microscopy projection of a 20 µm-large blood vessel in the adult mouse cortex showing the enrichement of Cx43 in astrocyte perivascular endfeet. Endothelial cells are immunolabeled with Pecam1 (gray), and astrocyte endfeet enwrapping the vessel are immunolabeled with GFAP (green) and Connexin 43 (red). B. Tranmission electron microscopy image showing a leukocyte (arrow) migrating across blood-brain barrier in absence of astroglial Connexin43 (L, lumen; AE, astrocyte endfeet; BL, basal lamina; E, endothelial cell)

III. Local translation in astrocyte perivascular endfeet: a window to understand astrocyte functions at the vascular interface

We recently demonstrated that distal compartmentalization of mRNAs and local translation take place in astrocyte perivascular endfeet. Combining our gliovascular unit protocol with the translating-ribosome affinity purification approach, we have purified and characterized the endfeetome, the astroglial polysomal repertoire of astrocyte endfeet. These observations went with our description of the astrocyte perivascular endfeet ultrastructure showing that astrocytes are equipped at the perivascular interface with endoplasmic reticulum and Golgi apparatus, and suggesting the existence of local routes for protein maturation at the gliovascular interface. Altogether, these findings conceptually change the way we think about astrocyte-vascular signaling. Until now, proteins required for astrocyte functions were thought to be produced and matured in the cell body (with the exception of mitochondrial proteins). We now propose that distribution of mRNAs and local translation in astrocyte perivascular endfeet might set astrocyte functional polarization at the vascular interface. Moreover, we propose that the endfeetome might be a molecular repertoire dedicated to gliovascular-polarized functions.

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Figure 4: Astrocytes set protein synthesis in distal perivascular processes mRNAs are present in astrocyte perivascular processes and endfeet.
A. Detection of GFAP mRNAs (red) (white arrows)in astrocyte perivascular endfeet by FISH. Astrocytes are immunostained for GFAP (green). Aqp4 or GFAP mRNAs are detected in PVAPs and endfeet. The astrocyte somata are indicated by an asterisk. The boxed area indicates the gliovascular unit.
B. Graphical abstract illustrating local translation in astrocyte perivascular endfeet. Astrocytes, the most numerous neuroglial cells in the central nervous system, are multipolar cells. They extend long processes terminated by endfeet (in green) at the surface of brain vessels (composed by mural cells in blue and endothelial cells in grey) and regulate vascular functions. In the present study, we demonstrate that some mRNAs (gray lines) are transported in astrocyte perivascular endfeet and bound to ribosomes (black dots) suggesting that they are translated on site. We also show that protein synthesis (red dots) occurs in endfeet. Finally, we show that endfeet are equipped with smooth and rough endoplasmic reticulum (gray) and the Golgi apparatus (purple), suggesting that the maturation of membrane and secreted proteins may occur locally. These results suggest that alternative routes for the translation, maturation and secretion of a specific pool of proteins are organized in the astrocyte perivascular endfeet. Proteins synthesized there might be either translated in the cytosol or in the RER. They can be further maturated in the local Golgi apparatus, inserted in the membrane or secreted in the perivascular space.

Selected publications

- Gilbert, A., Vidal, X.E., Estevez, R., Cohen-Salmon, M., and Boulay, A.-C. (2019). Postnatal development of the astrocyte perivascular MLC1/GlialCAM complex defines a temporal window for the gliovascular unit maturation. Brain Struct Funct.

- Boulay, A.-C., Gilbert, A., Oliveira Moreira, V., Blugeon, C., Perrin, S., Pouch, J., Le Crom, S., Ducos, B., and Cohen-Salmon, M. (2018). Connexin 43 Controls the Astrocyte Immunoregulatory Phenotype. Brain Sci 8.

- Mazaré, N., Gilbert, A., Boulay, A.-C., Rouach, N., and Cohen-Salmon, M. (2018). Connexin 30 is expressed in a subtype of mouse brain pericytes. Brain Struct Funct 223, 1017–1024.

- Boulay, A.-C., Saubaméa, B., Adam, N., Chasseigneaux, S., Mazaré, N., Gilbert, A., Bahin, M., Bastianelli, L., Blugeon, C., Perrin, S., et al. (2017). Translation in astrocyte distal processes sets molecular heterogeneity at the gliovascular interface. Cell Discov 3, 17005.

- Boulay, A.-C., Cisternino, S., and Cohen-Salmon, M. (2016). Immunoregulation at the gliovascular unit in the healthy brain: A focus on Connexin 43. Brain Behav. Immun. 56, 1–9.

- Boulay, A.-C., Saubaméa, B., Declèves, X., and Cohen-Salmon, M. (2015). Purification of Mouse Brain Vessels. J Vis Exp e53208.

- Boulay, A.-C., Mazeraud, A., Cisternino, S., Saubaméa, B., Mailly, P., Jourdren, L., Blugeon, C., Mignon, V., Smirnova, M., Cavallo, A., et al. (2015). Immune quiescence of the brain is set by astroglial connexin 43. J. Neurosci. 35, 4427–4439.

- Boulay, A.-C., Saubaméa, B., Cisternino, S., Mignon, V., Mazeraud, A., Jourdren, L., Blugeon, C., and Cohen-Salmon, M. (2015). The Sarcoglycan complex is expressed in the cerebrovascular system and is specifically regulated by astroglial Cx30 channels. Front Cell Neurosci 9, 9.

- Pannasch, U., Freche, D., Dallérac, G., Ghézali, G., Escartin, C., Ezan, P., Cohen-Salmon, M., Benchenane, K., Abudara, V., Dufour, A., et al. (2014). Connexin 30 sets synaptic strength by controlling astroglial synapse invasion. Nat. Neurosci. 17, 549–558.

- Boulay, A.-C., del Castillo, F.J., Giraudet, F., Hamard, G., Giaume, C., Petit, C., Avan, P., and Cohen-Salmon, M. (2013). Hearing is normal without connexin30. J. Neurosci. 33, 430–434.

- Boulay, A.-C., Burbassi, S., Lorenzo, H.-K., Loew, D., Ezan, P., Giaume, C., and Cohen-Salmon, M. (2013). Bmcc1s interacts with the phosphate-activated glutaminase in the brain. Biochimie 95, 799–807.

- Ezan, P., André, P., Cisternino, S., Saubaméa, B., Boulay, A.-C., Doutremer, S., Thomas, M.-A., Quenech’du, N., Giaume, C., and Cohen-Salmon, M. (2012). Deletion of astroglial connexins weakens the blood-brain barrier. J. Cereb. Blood Flow Metab. 32, 1457–1467.

- Arama, J., Boulay, A.-C., Bosc, C., Delphin, C., Loew, D., Rostaing, P., Amigou, E., Ezan, P., Wingertsmann, L., Guillaud, L., et al. (2012). Bmcc1s, a novel brain-isoform of Bmcc1, affects cell morphology by regulating MAP6/STOP functions. PLoS ONE 7, e35488.

- Ezan, P., André, P., Cisternino, S., Saubaméa, B., Boulay, A.-C., Doutremer, S., Thomas, M.-A., Quenech’du, N., Giaume, C., and Cohen-Salmon, M. (2012). Deletion of astroglial connexins weakens the blood-brain barrier. J. Cereb. Blood Flow Metab. 32, 1457–1467.

People

Group leader:
Cohen-Salmon Martine, DR2 CNRS

Postdoctoral fellows, PhD Students & Master student:
Boulay Anne-Cécile, Postdoctoral fellow
Mazare Noemie, PhD student
Gilbert Alice, PhD student
Oudart Marc, PhD student
Augustin Emma, M2 (fev-juin)
Tortuyaux Romain, M2 (jan-juil)