Caveolin and GLT-1 gene expression is reciprocally regulated in primary astrocytes: Association of GLT-1 with non-caveolar lipid rafts (original) (raw)
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MLC1 trafficking and membrane expression in astrocytes: Role of caveolin-1 and phosphorylation
Neurobiology of Disease, 2010
Megalencephalic leukoencephalopathy with subcortical cysts (MLC) is a rare congenital leukodystrophy caused by mutations in the MLC1 gene that encodes a membrane protein of unknown function. In the brain MLC1 protein is mainly expressed in astrocyte end-feet, localizes in lipid rafts and associates with the dystrophin glycoprotein complex (DGC). Using pull-down and co-fractionation assays in cultured human and rat astrocytes, we show here that MLC1 intracellular domains pull-down the DGC proteins syntrophin, dystrobrevin, Kir4.1 and caveolin-1, the structural protein of caveolae, thereby supporting a role for DGC and caveolar structures in MLC1 function. By immunostaining and subcellular fractionation of cultured rat or human astrocytes treated with agents modulating caveolin-mediated trafficking, we demonstrate that MLC1 is also expressed in intracellular vesicles and endoplasmic reticulum and undergoes caveolae/raft-mediated endocytosis. Inhibition of endocytosis, cholesterol lowering and protein kinases A-and C-mediated MLC1 phosphorylation favour the expression of membrane-associated MLC1. Because pathological mutations prevent MLC1 membrane expression, the identification of substances regulating MLC1 intracellular trafficking is potentially relevant for the therapy of MLC.
Caveolins in glial cell model systems: from detection to significance
Journal of Neurochemistry, 2007
Glial cells prevail in number and in diversity of cellular phenotypes in the nervous system. They have also gained prominence due to their multiple physiological and pathophysiological roles. Our current knowledge of the asymmetry and heterogeneity of the plasma membrane demands an in depth analysis of the diverse array of membrane microdomains postulated to exist in the context of glial cells. This review focuses and analyzes the studies reported to date on the detection of caveolae membrane rafts and the caveolin family members in glial cell model systems, the conditions leading to changes in their level of expression, and their functional and clinical significance. Outstanding in this work emerge the ubiquitous expression of caveolins, including the typically regarded 'muscle-specific' cav3, in diverse glial cell model systems, their participation in reactive astrogliosis, cancer, and their key relevance to calcium signaling. The knowledge obtained to date demands incorporation of the caveolins and caveolae membrane rafts in our current models on the role of glial cells in heath and neurological disease.
Caveolin-1 Regulates the P2Y2Receptor Signaling in Human 1321N1 Astrocytoma Cells
Journal of Biological Chemistry, 2016
Damage to the CNS can cause a differential spatio-temporal release of multiple factors, such as nucleotides, ATP and UTP. The latter interact with neuronal and glial nucleotide receptors. The P2Y 2 nucleotide receptor (P2Y 2 R) has gained prominence as a modulator of gliotic responses after CNS injury. Still, the molecular mechanisms underlying these responses in glia are not fully understood. Membrane-raft microdomains, such as caveolae, and their constituent caveolins, modulate receptor signaling in astrocytes; yet, their role in P2Y 2 R signaling has not been adequately explored. Hence, this study evaluated the role of caveolin-1 (Cav-1) in modulating P2Y 2 R subcellular distribution and signaling in human 1321N1 astrocytoma cells. Recombinant hP2Y 2 R expressed in 1321N1 cells and Cav-1 were found to co-fractionate in light-density membrane-raft fractions, colocalize via confocal microscopy, and co-immunoprecipitate. Raft localization was dependent on ATP stimulation and Cav-1 expression. This hP2Y 2 R/Cav-1 distribution and interaction was confirmed with various cell model systems differing in the expression of both P2Y 2 R and Cav-1, and shRNA knockdown of Cav-1 expression. Furthermore, shRNA knockdown of Cav-1 expression decreased nucleotide-induced increases in the intracellular Ca 2؉ concentration in 1321N1 and C6 glioma cells without altering TRAP-6 and carbachol Ca 2؉ responses. In addition, Cav-1 shRNA knockdown also decreased AKT phosphorylation and altered the kinetics of ERK1/2 activation in 1321N1 cells. Our findings strongly suggest that P2Y 2 R interaction with Cav-1 in membrane-raft caveolae of 1321N1 cells modulates receptor coupling to its downstream signaling machinery. Thus, P2Y 2 R/Cav-1 interactions represent a novel target for controlling P2Y 2 R function after CNS injury. Neurodegenerative conditions are among the leading causes of death and disability in the United States and have dramatically increased in incidence during the last decade (1, 2). The P2 receptors for extracellular nucleotides have emerged as key modulators of the pathophysiology of neurodegeneration (3-6). G protein-coupled P2Y 2 nucleotide receptors (P2Y 2 Rs) 3 have been identified in both neurons and glia as mediators of pro-inflammatory responses, neurotransmission, apoptosis, proliferation, and cell migration (3-5, 7, 8). In addition, the P2Y 2 Rs have also gained prominence, due to their association with some types of neoplasms, spinal cord injury, and the enhancement of neuronal differentiation (7, 9-15). Further insight into the spatio-temporal organization of the P2Y 2 R and its signaling cascades in astrocytic cells is required to expand our knowledge of their role in neurodegenerative diseases. In this context, evidence suggests that receptors and associated signaling molecules are not randomly distributed in plasma membranes but are localized in specialized membrane microdomains, namely membrane rafts (MRs), such as caveolae (Cav) (16-20). MRs are specialized membrane domains enriched in cholesterol and glycosphingolipids (21), which are known to localize signaling molecules, including several types of receptors (e.g. receptor protein kinases and heptahelical receptors), G protein subunits, and an array of signaling molecules (22-25). These scaffolds serve to facilitate protein-protein interactions among signaling molecules, thereby integrating complex signaling pathways. Recently, we have reviewed and established the importance of glial caveolins and the caveolar MR compartment in neurodegenerative conditions, such as Alzheimer disease, aging, among others (26, 27). Caveolin-1 (Cav-1), one of the main raft scaffolding proteins (26, 28-31), has been shown to modulate multiple cellular responses by coupling membrane receptors to downstream signaling molecules (28-32). Because P2Y 2 R expression in 1321N1 astrocytoma cells has been shown to exert glio-protective and neurotrophic actions (33-35), analysis of the subcellular and molecular mechanisms involved in its actions deserve special attention. Although the precise endocytic mechanism of the P2Y 2 R has been partially characterized (36-39), the functional significance of P2Y 2 R trafficking in MRs is only beginning to be recognized (36, 40, 41). Therefore, this study was undertaken to assess the potential role of Cav-1 in modulating P2Y 2 R subcellular distribution and signaling in 1321N1 astrocytoma cells. Results obtained indicate that P2Y 2 R resides in Cav-1 raft microdomains and their interaction
SorLA in Glia: Shared Subcellular Distribution Patterns with Caveolin-1
Cellular and Molecular Neurobiology, 2012
SorLA is an established sorting and trafficking protein in neurons with demonstrated relevance to Alzheimer's disease (AD). It shares these roles with the caveolins, markers of membrane rafts microdomains. To further our knowledge on sorLA's expression and traffic, we studied sorLA expression in various cultured glia and its relation to caveolin-1 (cav-1), a caveolar microdomain marker. RT-PCR and immunoblots demonstrated sorLA expression in rat C6 glioma, primary cultures of rat astrocytes (PCRA), and human astrocytoma 1321N1 cells. PCRA were determined to express the highest levels of…
Neuroscience, 2009
The Golgi complex plays a key role in cholesterol trafficking in cells. Our earlier study demonstrated amyloid β-protein (Aβ) alters cholesterol distribution and abundance in the Golgi complex of astrocytes. We now test the hypothesis that the Aβ-induced increase in Golgi complex cholesterol is due to retrograde movement of the cholesterol carrier protein caveolin-1 from the cell plasma membrane to the Golgi complex in astrocytes. Results with mouse primary astrocytes indicated that Aβ 1-42 -induced increase in cholesterol and caveolin abundance in the Golgi complex was accompanied by a reduction in cholesterol and caveolin levels in the plasma membrane. Transfection of DITNC1 astrocytes with siRNA directed at caveolin-1 mRNA inhibited the Aβ 1-42 -induced redistribution of both cholesterol and caveolin from the plasma membrane to the Golgi complex. In astrocytes not treated with Aβ 1-42 , suppression of caveolin-1 expression also significantly reduced cholesterol abundance in the Golgi complex, further demonstrating the role for caveolin in retrograde transport of cholesterol from the plasma membrane to the Golgi complex. Perturbation of this process by Aβ 1-42 could have consequences on membrane structure and cellular functions requiring optimal levels of cholesterol.
Molecular Pharmacology, 2009
Lipid rafts and caveolae are specialized membrane microdomains implicated in regulating G protein-coupled receptor signaling cascades. Previous studies have suggested that rafts/ caveolae may regulate -adrenergic receptor/G␣ s signaling, but underlying molecular mechanisms are largely undefined. Using a simplified model system in C6 glioma cells, this study disrupts rafts/caveolae using both pharmacological and genetic approaches to test whether caveolin-1 and lipid microdomains regulate G s trafficking and signaling. Lipid rafts/caveolae were disrupted in C6 cells by either short-term cholesterol chelation using methyl--cyclodextrin or by stable knockdown of caveolin-1 and -2 by RNA interference. In imaging studies examining G␣ s -GFP during signaling, stimulation with the AR agonist isoproterenol resulted in internalization of G␣ s -GFP; however, this trafficking was blocked by methyl--cyclodextrin or by caveolin knockdown. Caveolin knockdown significantly MM (2006) Agonist induced internalization of G alpha s regulates adenylyl cyclase (Abstract). FASEB J 20: A694; and Rasenick MM (2007) Regulation of G protein signaling by cytoskeletal components and membrane microdomains. Experimental Biology; April 28 -May 2, 2007; Washington DC.
Glutamate regulates caveolin expression in rat hippocampal neurons
Journal of Neuroscience Research, 2003
Caveolae are cholesterol-rich, membrane microdomains that appear critical to signaling between extracellular and intracellular macromolecules as well as cholesterol homeostasis. Caveolae formation is modulated by caveolin, a protein family that is the proteinaceous hallmark of caveolae. Very little is known regarding the events that modulate caveolin expression and regulation in neurons. To detect caveolin expression in neurons, primary rat hippocampal neurons were harvested at embryonic day 18, maintained for 7 days in vitro, and then analyzed for caveolin immunofluorescence. Caveolin-1 immunoreactivity was detected in cells that were identified as neurons by morphology and concurrent microtubule-associated protein (MAP2) staining. Changes in caveolin-1 expression were evaluated by reverse transcriptase-polymerase chain reaction (RT-PCR) analyses of RNA isolated from hippocampal neurons treated with glutamate receptor agonists. Glutamate induced a concentration-dependent increase in caveolin-1 mRNA. The largest increases in caveolin-1 mRNA were detected after 6 hours of treatment. Kainate and AMPA both mimicked glutamate effects on caveolin-1 mRNA expression. Western blot analyses revealed that caveolin was induced at the protein level as well. Taken together, these data suggest that glutamate can regulate caveolin expression through kainate and AMPA ionotropic glutamate receptors.
Expression of caveolin-1 in human brain microvessels
Neuroscience, 2002
öCaveolae are microinvaginations of the cell plasma membrane involved in cell transport and metabolism as well as in signal transduction; these functions depend on the presence of integral proteins named caveolins in the caveolar frame. In the brain, various caveolin subtypes have been detected in vivo by immunocytochemistry: caveolin-1 and-2 were found in rat brain microvessels, caveolin-3 was revealed in astrocytes. The aim of this study was to identify the site(s) of cellular expression of caveolin-1 in the microvessels of the human cerebral cortex by immuno£uorescence confocal microscopy and immunogold electron microscopy. Since in the barrier-provided brain microvessels tight relations occur between the endothelium^pericyte layer and the surrounding vascular astrocytes, double immunostaining with caveolin-1 and the astroglia marker, glial ¢brillary acidic protein, was also carried out. Immunocytochemistry by confocal microscopy revealed that caveolin-1 is expressed by endothelial cells and pericytes in all the cortex microvessels; caveolin-1 is also expressed by cells located in the neuropil around the microvessels and identi¢ed as astrocytes. Study of the cortex microvessels carried out by immunoelectron microscopy con¢rmed that in the vascular wall caveolin-1 is expressed by endothelial cells, pericytes, and vascular astrocytes, and revealed the association of caveolin-1 with the cell caveolar compartment. The demonstration of caveolin-1 in the cells of the brain microvessels suggests that caveolin-1 may be involved in blood^brain barrier functioning, and also supports coordinated activities between these cells.
Caveolin isoform expression during differentiation of C6 glioma cells
International Journal of Developmental Neuroscience, 2005
Caveolae, a specialized form of lipid rafts, are cholesterol-and sphingolipid-rich membrane microdomains implicated in potocytosis, endocytosis, transcytosis, and as platforms for signal transduction. One of the major constituents of caveolae are three highly homologous caveolin isoforms (caveolin-1, caveolin-2, and caveolin-3). The present study expands the analysis of caveolin isoform expression in C6 glioma cells. Three complementary approaches were used to assess their differential expression during the dibutyryl-cyclic AMP-induced differentiation of C6 cells into an astrocyte-like phenotype. Immunoblotting, conventional RT-PCR, and real-time RT-PCR analysis established the expression of the caveolin-3 isoform in C6 cells, in addition to caveolin-1 and caveolin-2. Similar to the other isoforms, caveolin-3 was associated with light-density, detergent-insoluble caveolae membrane fractions obtained using sucrose-density gradient centrifugation. The three caveolin isoforms display different temporal patterns of mRNA/protein expression during the differentiation of C6 cells. Western blot and real-time RT-PCR analysis demonstrate that caveolin-1 and caveolin-2 are up-regulated during the late stages of the differentiation of C6 cells. Meanwhile, caveolin-3 is gradually down-regulated during the differentiation process. Indirect immunofluorescence analysis via laser-scanning confocal microscopy reveals that the three caveolin isoforms display similar subcellular distribution patterns. In addition, co-localization of caveolin-1/caveolin-2 and caveolin-1/caveolin-3 was detected in both C6 glioma phenotypes. The findings reveal a differential temporal pattern of caveolin gene expression during phenotypic differentiation of C6 glioma cells, with potential implications to developmental and degenerative events in the brain.
Caveolin-1 Regulates P2Y2 Receptor Signaling in Human 1321N1 Astrocytoma Cells
Damage to the CNS can cause a differential spatio-temporal release of multiple factors, such as nucleotides like ATP and UTP. The latter interact with neuronal and glial nucleotide receptors. The P2Y2 nucleotide receptor (P2Y2R) has gained prominence as a modulator of gliotic responses after CNS injury. Still, the molecular mechanisms underlying these responses in glia are not fully understood. Membrane-raft microdomains, such as caveolae, and their constituent caveolins, modulate receptor signaling in astrocytes; yet, their role in P2Y2R signaling has not been adequately explored. Hence, this study evaluated the role of caveolin-1 (Cav-1) in modulating P2Y2R subcellular distribution and signaling in human 1321N1 astrocytoma cells. Recombinant hP2Y2R expressed in 1321N1 cells and Cav-1 were found to co-fractionate in light-density membrane-raft fractions, co-localize via confocal microscopy, and co-immunoprecipitate. Raft localization was dependent on ATP stimulation and Cav-1 expression. This hP2Y2R/Cav-1 distribution and interaction was confirmed with various cell model systems differing in the expression of both P2Y2R and Cav-1, and shRNA knockdown of Cav-1 expression. Furthermore, shRNA knockdown of Cav-1 expression decreased nucleotide-induced increases in the intracellular Ca2+ concentration in 1321N1 and C6 glioma cells without altering TRAP-6 and carbachol Ca2+ responses. In addition, Cav-1 shRNA knockdown also decreased AKT phosphorylation and altered the kinetics of ERK1/2 activation in 1321N1 cells. Our findings strongly suggest that P2Y2R interaction with Cav-1 in membrane-raft caveolae of 1321N1 cells modulates receptor coupling to its downstream signaling machinery. Thus, P2Y2R/Cav-1 interactions represent a novel target for controlling P2Y2R function after CNS injury.