Agrin plays a major role in the coalescence of the aquaporin‐4 clusters induced by gamma‐1‐containing laminin

Abstract

The basement membrane that seperates the endothelial cells and astrocytic endfeet that comprise the blood-brain barrier is rich in collagen, laminin, agrin, and perlecan. Previous studies have demonstrated that the proper recruitment of the water-permeable channel aquaporin-4 (AQP4) to astrocytic endfeet is dependent on interactions between laminin and the receptor dystroglycan. In this study, we conducted a deeper investigation into how the basement membrane might further regulate the expression, localization, and function of AQP4, using primary astrocytes as a model system. We found that treating these cells with laminin causes endogenous agrin to localize to the cell surface, where it co-clusters with β-dystroglycan (β-DG). Conversely, agrin sliencing profoundly disrupts β-DG clustering. As in the case of laminin111, Matrigel™, a complete basement membrane analog, also causes the clustering of AQP4 and β-DG. This clustering, whether induced by laminin111 or Matrigel™ is inhibited when the astrocytes are first incubated with an antibody against the γ1 subunit of laminin, suggesting that the latter is crucial to the process. Finally, we showed that laminin111 appears to negatively regulate AQP4-mediated water transport in astrocytes, suppressing the cell swelling that occurs following a hypoosmotic challenge. This suppression is abolished if DG expression is silenced, again demonstrating the central role of this receptor in relaying the effects of laminin.

Insights into Cell Surface Expression, Supramolecular Organization, and Functions of Aquaporin 4 Isoforms in Astrocytes

“…In vivo studies supported the data obtained in cultured cells by showing that interaction between perivascular laminin and α-dystroglycan enables AQP4 positioning in perivascular astrocyte endfeet, which is also supported by a similar distribution of AQP4, α-syntrophin, and dystrophin-dystroglycan complex [46,47]. The effect of laminin on the positioning of agrin in hippocampal rat astrocytes, where laminin induced trafficking of endogenous agrin to the plasma membrane, also triggered clustering of AQP4 and β-dystroglycan [43]. Neither laminin nor agrin affect the overall expression of AQP4a (M1) or AQP4c (M23) isoforms, nor their ratio of expression [43], suggesting that clustering into OAPs and their reorganization depend on their recruitment from the cell cytoplasm to the cell surface and vice versa.…”

“…Neither laminin nor agrin affect the overall expression of AQP4a (M1) or AQP4c (M23) isoforms, nor their ratio of expression [43], suggesting that clustering into OAPs and their reorganization depend on their recruitment from the cell cytoplasm to the cell surface and vice versa. The induced clustering of AQP4 triggered by addition of extracellular laminin into growth medium significantly attenuated astrocyte swelling in hypoosmotic conditions [43]. Again, this finding corroborates the hypothesis that OAPs regulate the propensity of astrocytes to the extent of cell volume changes.…”

“…The effect of laminin on the positioning of agrin in hippocampal rat astrocytes, where laminin induced trafficking of endogenous agrin to the plasma membrane, also triggered clustering of AQP4 and β-dystroglycan [43]. Neither laminin nor agrin affect the overall expression of AQP4a (M1) or AQP4c (M23) isoforms, nor their ratio of expression [43], suggesting that clustering into OAPs and their reorganization depend on their recruitment from the cell cytoplasm to the cell surface and vice versa. The induced clustering of AQP4 triggered by addition of extracellular laminin into growth medium significantly attenuated astrocyte swelling in hypoosmotic conditions [43].…”

“…Laminin, another of the main structural proteins of the perivascular basal lamina, has also been demonstrated to induce plasma membrane clustering of not only AQP4 but also β-dystroglycan and Kir4.1 channels in hippocampal and cortical astrocytes [42,43]. Coating of growth substrate with laminin affects the abundance of AQP4 expression at the plasmalemma, without affecting the size of OAPs [21,42].…”

Organisation of extracellular matrix proteins laminin and agrin in pericapillary basal laminae in mouse brain

“…The extracellular matrix proteins, laminin and agrin, have long been recognised as important structural components of basal laminae in brain (Thomsen et al 2017). Only more recently has it become clear that these molecules serve additional roles and that they are essential for upholding the functional specialisation of the gliovascular interface (Fallier-Becker et al 2011;Noel et al 2019). Notably, it is now realised that the functional polarisation of astrocytes depends on these matrix proteins (Camassa et al 2015;Lunde et al 2015;Neely et al 2001).…”