Interdependence of Laminin-mediated Clustering of Lipid Rafts and the Dystrophin Complex in Astrocytes

Abstract

Astrocyte endfeet surrounding blood vessels are active domains involved in water and potassium ion transport crucial to the maintenance of water and potassium ion homeostasis in brain. A growing body of evidence points to a role for dystroglycan and its interaction with perivascular laminin in the targeting of the dystrophin complex and the water-permeable channel, aquaporin 4 (AQP4), at astrocyte endfeet. However, the mechanisms underlying such compartmentalization remain poorly understood. In the present study we found that AQP4 resided in Triton X-100-insoluble fraction, whereas dystroglycan was recovered in the soluble fraction in astrocytes. Cholesterol depletion resulted in the translocation of a pool of AQP4 to the soluble fraction indicating that its distribution is indeed associated with cholesterol-rich membrane domains. Upon laminin treatment AQP4 and the dystrophin complex, including dystroglycan, reorganized into laminin-associated clusters enriched for the lipid raft markers GM1 and flotillin-1 but not caveolin-1. Reduced diffusion rates of GM1 in the laminin-induced clusters were indicative of the reorganization of raft components in these domains. In addition, both cholesterol depletion and dystroglycan silencing reduced the number and area of laminininduced clusters of GM1, AQP4, and dystroglycan. These findings demonstrate the interdependence between laminin binding to dystroglycan and GM1-containing lipid raft reorganization and provide novel insight into the dystrophin complex regulation of AQP4 polarization in astrocytes.The basement membrane is a specialized extracellular matrix (ECM) 2 composed of collagen, fibronectin, perlecan, agrin, and laminin. Several studies have focused on the involvement of these ECM molecules in the formation and maturation of neuromuscular junctions (1-4) and interneuronal synapses (5). More recently, much effort has been made by our group and others to understand the role of these molecules at the interface of astroglia and blood vessels (6 -8). Laminin is highly expressed at the perivascular ECM, and the laminin receptor, dystroglycan (␣-DG), together with many other components of the dystrophin-associated protein (DAP) complex, is particularly enriched at astrocyte endfeet abutting the blood vessels (9 -11). The binding of laminin to ␣-DG at these specialized astrocyte domains in brain plays a key role in the polarized distribution of components of the DAP complex (6, 12).Multiple lines of evidence indicate that the DAP complex is crucial for the functional distribution both of the water-permeable channel, AQP4, and the inwardly rectifying potassium channel, Kir4

Physiological Roles of Aquaporin-4 in Brain

“…Evidence in support of this was provided by biochemical analyses and coimmunoprecipitation experiments (116). It is now well documented that the entire DAPC is held in place through its attachment to laminin, a principal component of the pericapillary basal lamina (5,49,116,131,200). This mode of anchoring explains why the density of AQP4 drops as soon as the endfoot plasma membrane is reflected away from the capillary (114,129).…”

Glial dystrophin-associated proteins, laminin and agrin, are downregulated in the brain of mdx mouse

“…56 Moreover, the correct AQP4 and Kir 4.1 clustering on glial membranes required a-DG and laminin. 15,30,57 In glioma cells, agrin deficiency is coupled with redistribution of AQP4 and syntrophins, loss in a-DG and absence of OAPs. 12,58 It is important to note that agrin is also able to induce AQP4 clustering into OAP membranes, 15,59 suggesting that AQP4 and DG are components of OAPs.…”

“…15,30,57 In glioma cells, agrin deficiency is coupled with redistribution of AQP4 and syntrophins, loss in a-DG and absence of OAPs. 12,58 It is important to note that agrin is also able to induce AQP4 clustering into OAP membranes, 15,59 suggesting that AQP4 and DG are components of OAPs. 60 The findings that AQP4 and OAPs are concentrated on the glial endfeet as opposed to the basement membrane, 4,7 and are reduced in the neurological disorders affecting the BBB including brain tumors, in which edema and AQP4 redistribution on glioma cell membrane occurred, 12,16,17,61 suggest a role of basement membrane components in the polarity and functions of the astroglial endfeet.…”

“…7 Moreover, OAPs contain AQP4 associated with Kir 4.1 12 and their polarized distribution on the glial endfeet seems to be dependent by the presence of the perivascular basement membrane. 15 AQP4 controls BBB functioning, modifying its expression and polarization when BBB is damaged with a consequent increase in vascular permeability and edema. 7,16,17 AQP4 is also a component of the dystrophin-associated proteins (DAPs) complex, including dystroglycan (DG), syntrophin, dystrobrevin and sarcoglycans associated with dystrophin or its homolog utrophin.…”

Dystrophin–Glycoprotein Complex in Blood Cells

“…During embryogenesis, the follicle-cell epithelium (FCE) maintains the cell polarity promoted by the association between perlecan and Dg [68], while in astrocytes, end-feet in brain laminin induced a dramatic, polarized redistribution of cell-surface clusters or macrodomains, which colocalized extensively with β-Dg and AQP4 [69].…”