SorLA in Glia: Shared Subcellular Distribution Patterns with Caveolin-1 (original) (raw)
Related papers
Glia, 2005
Caveolae represent membrane microdomains acting as integrators of cellular signaling and functional processes. Caveolins are involved in the biogenesis of caveolae and regulate the activity of caveolae-associated proteins. Although caveolin proteins are found in the CNS, the regulation of caveolins in neural cells is poorly described. In the present study, we investigated different modes and mechanisms of caveolin gene regulation in primary rat astrocytes. We demonstrated that activation of cAMP-dependent signaling pathways led to a marked reduction in protein levels of caveolin-1/-2 in cortical astrocytes. Application of transforming growth factor-␣ (TGF-␣) also resulted in a decrease of caveolin-1/-2 expression. Decreased caveolin protein levels were mirrored by diminished caveolin gene transcription. The repressive effect of TGF-␣ on caveolin-1 expression was MAP kinase-independent and partly mediated through the PI3-kinase pathway. Further downstream, inhibition of histone deacetylases abrogated TGF-␣ effects, suggesting that chromatin remodeling processes could contribute to caveolin-1 repression. Intriguingly, alterations of caveolin gene expression in response to cAMP or TGF-␣ coincided with reciprocal and brain-region specific changes in glial glutamate transporter GLT-1 expression. The reciprocal regulation of caveolin-1 and GLT-1 expression might be gated through a common PI3-kinase dependent pathway triggered by TGF-␣. Finally, we showed that GLT-1 is located in non-caveolar lipid rafts of cortical astrocytes. In conclusion, this study highlights the occurrence of the reciprocal regulation of caveolin and GLT-1 expression during processes such as astrocyte differentiation via common signaling pathways. We also provide strong evidence that GLT-1 itself is concentrated in lipid rafts, inferring an important role for glial glutamate transporter function.
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.
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.
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.
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.
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 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
Caveolin-1 upregulation in senescent neurons alters amyloid precursor protein processing
Experimental & Molecular Medicine, 2006
Lipid rafts provide a platform for regulating cellular functions and participate in the pathogenesis of several diseases. However, the role of caveolin-1 in this process has not been elucidated definitely in neuron. Thus, this study was performed to examine whether caveolin-1 can regulate amyloid precursor protein (APP) processing in neuronal cells and to identify the molecular mechanisms involved in this regulation. Caveolin-1 is up-regulated in all parts of old rat brain, namely hippocampus, cerebral cortex and in elderly human cerebral cortex. Moreover, detergent-insoluble glycolipid (DIG) fractions indicated that caveolin-1 was co-localized with APP in caveolae-like structures. In DIG fractions, βAPP secretion was up-regulated by caveolin-1 overexpression, which was modulated via protein kinase C (PKC) in neuroblastoma cells. From these results we conclude that caveolin-1 is selectively expressed in senescent neurons and that it induces the processing of APP by β-secretase via PKC down -regulation.
Identification of caveolae and caveolin in C6 glioma cells
International Journal of Developmental Neuroscience, 1999
AbstractÐCaveolae (CAV) constitute a novel subcellular transport vesicle that has received special attention based on its proven and postulated participation in transcytosis, potocytosis, and in cell signaling events. One of the principal components of CAV are caveolin protein isoforms. Here, we have undertaken the immunochemical identi®cation of CAV and the known caveolin isoforms (1a, 1b, 2 and 3) in cultured rat C6 glioma cells. Immunoblot analysis revealed that particulate fractions from rat C6 glioma cells express caveolin-1 and caveolin-2. The relative detergent-insolubility of these caveolin isoforms was also determined by Western blot analysis. Indirect immuno¯uorescence analysis with caveolin-1 and -2 antibodies revealed staining patterns typical of CAV's known subcellular distribution and localization. For both caveolin isoforms immunocytochemical staining was characterized by intenselȳ uorescent puncta throughout the cytoplasm and diuse micropatches at the level of the plasmalemma. Perinuclear staining was also detected, consistent and suggestive of caveolin's localization in the trans Golgi region. The caveolin-1 and -2 immunoreactivity seen in Western blots and immunocytochemically is related to structurally relevant CAV as supported by the isolation of caveolin-enriched membrane complexes using two dierent methods. Light-density, Triton X-100-insoluble caveolin-1-and caveolin-2-enriched fractions were obtained after fractionation of rat C6 glioma cells and their separation over 5±40% discontinuous sucrose-density gradients. Similar fractions were obtained using a detergent-free, sodium carbonate-based fractionation method. These results further support the localization of CAV and caveolins in glial cells. In addition, they demonstrate that cultured C6 glioma cells can be useful as a model system to study the role of CAV and caveolins in subcellular transport and signal transduction events in glial cells and the brain. #