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Localisation of novel forms of glutamate transporters and the cystine-glutamate antiporter in the choroid plexus: Implications for CSF glutamate homeostasis.

ABSTRACT: The choroid plexus is a structure within each ventricle of the brain that is composed of fenestrated vessels surrounded by secretory epithelial cells. The epithelial cells are linked by tight junctions to create a permeability barrier. The epithelial cells are derived from neuroectoderm, and are thus defined by some authors as a subtype of macroglia. Glutamate is a tightly regulated substance in the CSF, as it is in the rest of the brain. In the brain macroglia express multiple sodium dependent and independent glutamate transporters and are the main regulators of extracellular glutamate. However, the identities of the transporters in the choroid plexus and their localisations have remained poorly defined. In this study we examined the expression and distribution of multiple splice variants of classical sodium-dependent glutamate transporters, as well as the cystine-glutamate antiporter, and the PDZ protein NHERF1, (which acts as a molecular anchor for proteins such as the glutamate transporter GLAST). We identified three forms of sodium-dependent transporters (GLAST1a, GLAST1c and GLT1b) that are expressed at the apical surface of the epithelial cells, a location that matches the distribution of NHERF1 and the cystine-glutamate antiporter. We propose that this coincident localisation of GLAST1a/GLAST1c/GLT1b and the cystine-glutamate antiporter would permit the cyclical trafficking of glutamate and thus optimise the accumulation of cystine for the formation of glutathione in the choroid plexus.

SUBMITTER: Lee A

PROVIDER: S-EPMC3299572 | biostudies-literature | 2012 Jan

REPOSITORIES: biostudies-literature

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Localisation of novel forms of glutamate transporters and the cystine-glutamate antiporter in the choroid plexus: Implications for CSF glutamate homeostasis.

Lee Aven A Anderson Ashley R AR Rayfield Andrew J AJ Stevens Melissa G MG Poronnik Philip P Meabon James S JS Cook David G DG Pow David V DV

Journal of chemical neuroanatomy 20110929 1

The choroid plexus is a structure within each ventricle of the brain that is composed of fenestrated vessels surrounded by secretory epithelial cells. The epithelial cells are linked by tight junctions to create a permeability barrier. The epithelial cells are derived from neuroectoderm, and are thus defined by some authors as a subtype of macroglia. Glutamate is a tightly regulated substance in the CSF, as it is in the rest of the brain. In the brain macroglia express multiple sodium dependent ...[more]

PMID: 21982839

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Project description:BACKGROUND:Cerebrospinal fluid (CSF) is mainly produced by the choroid plexus (CP) located in brain ventricles. Although derived from blood plasma, it is nearly protein-free (~ 250-fold less) and contains about 2-20-fold less free amino acids, with the exception of glutamine (Gln) which is nearly equal. The aim of this study was to determine which amino acid transporters are expressed in mouse CP epithelium in order to gain understanding about how this barrier maintains the observed amino acid concentration gradient. METHODS:Expression of amino acid transporters was assessed in isolated choroid plexuses (CPs) by qRT-PCR followed by localization studies using immunofluorescence with specific antibodies. The impact of LAT2 (Slc7a8) antiporter deletion on CSF amino acids was determined. RESULTS:The purity of isolated choroid plexuses was tested on the mRNA level using specific markers, in particular transthyretin (Ttr) that was enriched 330-fold in CP compared to cerebral tissue. In a first experimental round, 14 out of 32 Slc amino acid transporters tested on the mRNA level by qPCR were selected for further investigation. Out of these, five were considered highly expressed, SNAT1 (Slc38a1), SNAT3 (Slc38a3), LAT2 (Slc7a8), ASC1 (Slc7a10) and SIT1 (Slc6a20b). Three of them were visualized by immunofluorescence: SNAT1 (Slc38a1), a neutral amino acid-Na+ symporter, found at the blood side basolateral membrane of CP epithelium, while SNAT3 (Slc38a3), an amino acid-Na+ symporter and H+ antiporter, as well as LAT2 (Slc7a8), a neutral amino acid antiporter, were localized at the CSF-facing luminal membrane. In a LAT2 knock-out mouse model, CSF Gln was unchanged, whereas other amino acids normally 2-20-fold lower than in plasma, were increased, in particular the LAT2 uptake substrates leucine (Leu), valine (Val) and tryptophan (Trp) and some other amino acids such as glutamate (Glu), glycine (Gly) and proline (Pro). CONCLUSION:These results suggest that Gln is actively transported by SNAT1 from the blood into CP epithelial cells and then released luminally into CSF via SNAT3 and LAT2. Its efflux via LAT2 may drive the reuptake from the CSF of essential amino acid substrates of this antiporter and thereby participates to maintaining the amino acid gradient between plasma and CSF.

Project description:BackgroundThe roles of the choroid plexus (CP) and cerebrospinal fluid (CSF) production have drawn increasing attention in Alzheimer's disease (AD) research. Specifically, studies document markedly decreased CSF production and turnover in moderate-to-severe AD. Moreover, reduced CP function and CSF turnover lead to impaired clearance of toxic metabolites, likely promote neuroinflammation, and may facilitate neuronal death during AD progression. We analyzed CP gene expression in AD compared with control subjects, specifically considering those genes involved with CSF production and CP structural integrity.MethodsThe Brown-Merck Gene Expression Omnibus (GEO) database (CP transcripts) was mined to examine changes in gene expression in AD compared to controls with a focus on assorted genes thought to play a role in CSF production. Specifically, genes coding for ion transporters in CP epithelium (CPE) and associated enzymes like Na-K-ATPase and carbonic anhydrase, aquaporins, mitochondrial transporters/enzymes, blood-cerebrospinal fluid barrier (BCSFB) stability proteins, and pro-inflammatory mediators were selected for investigation. Data were analyzed using t test p-value and fold-change analysis conducted by the GEO2R feature of the GEO database.ResultsSignificant expression changes for several genes were observed in AD CP. These included disruptions to ion transporters (e.g., the solute carrier gene SLC4A5, p = 0.004) and associated enzyme expressions (e.g., carbonic anhydrase CA4, p = 0.0001), along with decreased expression of genes involved in BCSFB integrity (e.g., claudin CLDN5, p = 0.039) and mitochondrial ATP synthesis (e.g., adenosine triphosphate ATP5L, p = 0.0004). Together all changes point to disrupted solute transport at the blood-CSF interface in AD. Increased expression of pro-inflammatory (e.g., interleukin IL1RL1, p = 0.00001) and potential neurodegenerative genes (e.g., amyloid precursor APBA3, p = 0.002) also implicate disturbed CP function.ConclusionsBecause the altered expression of numerous transcripts in AD-CP help explain decreased CSF production in AD, these findings represent a first step towards identifying novel therapeutic targets in AD.

Project description:BACKGROUND:In Alzheimer's disease, there are striking changes in CSF composition that relate to altered choroid plexus (CP) function. Studying CP tissue gene expression at the blood-cerebrospinal fluid barrier could provide further insight into the epithelial and stromal responses to neurodegenerative disease states. METHODS:Transcriptome-wide Affymetrix microarrays were used to determine disease-related changes in gene expression in human CP. RNA from post-mortem samples of the entire lateral ventricular choroid plexus was extracted from 6 healthy controls (Ctrl), 7 patients with advanced (Braak and Braak stage III-VI) Alzheimer's disease (AD), 4 with frontotemporal dementia (FTD) and 3 with Huntington's disease (HuD). Statistics and agglomerative clustering were accomplished with MathWorks, MatLab; and gene set annotations by comparing input sets to GeneGo ( http://www.genego.com ) and Ingenuity ( http://www.ingenuity.com ) pathway sets. Bonferroni-corrected hypergeometric p-values of < 0.1 were considered a significant overlap between sets. RESULTS:Pronounced differences in gene expression occurred in CP of advanced AD patients vs. Ctrls. Metabolic and immune-related pathways including acute phase response, cytokine, cell adhesion, interferons, and JAK-STAT as well as mTOR were significantly enriched among the genes upregulated. Methionine degradation, claudin-5 and protein translation genes were downregulated. Many gene expression changes in AD patients were observed in FTD and HuD (e.g., claudin-5, tight junction downregulation), but there were significant differences between the disease groups. In AD and HuD (but not FTD), several neuroimmune-modulating interferons were significantly enriched (e.g., in AD: IFI-TM1, IFN-AR1, IFN-AR2, and IFN-GR2). AD-associated expression changes, but not those in HuD and FTD, were enriched for upregulation of VEGF signaling and immune response proteins, e.g., interleukins. HuD and FTD patients distinctively displayed upregulated cadherin-mediated adhesion. CONCLUSIONS:Our transcript data for human CP tissue provides genomic and mechanistic insight for differential expression in AD vs. FTD vs. HuD for stromal as well as epithelial components. These choroidal transcriptome characterizations elucidate immune activation, tissue functional resiliency, and CSF metabolic homeostasis. The BCSFB undergoes harmful, but also important functional and adaptive changes in neurodegenerative diseases; accordingly, the enriched JAK-STAT and mTOR pathways, respectively, likely help the CP in adaptive transcription and epithelial repair and/or replacement when harmed by neurodegeneration pathophysiology. We anticipate that these precise CP translational data will facilitate pharmacologic/transgenic therapies to alleviate dementia.

Project description:The cystine-glutamate antiporter (system xc-) is a Na+-independent amino acid transporter that exchanges extracellular cystine for intracellular glutamate. It is thought to play a critical role in cellular redox processes through regulation of intracellular glutathione synthesis via cystine uptake. In gliomas, system xc- expression is universally up-regulated while that of glutamate transporters down-regulated, leading to a progressive accumulation of extracellular glutamate and excitotoxic cell death of the surrounding non-tumorous tissue. Additionally, up-regulation of system xc- in activated microglia has been implicated in the pathogenesis of several neurodegenerative disorders mediated by excess glutamate. Consequently, system xc- is a new drug target for brain cancer and neuroinflammatory diseases associated with excess extracellular glutamate. Unfortunately no potent and selective small molecule system xc- inhibitors exist and to our knowledge, no high throughput screening (HTS) assay has been developed to identify new scaffolds for inhibitor design. To develop such an assay, various neuronal and non-neuronal human cells were evaluated as sources of system xc-. Human glioma cells were chosen based on their high system xc- activity. Using these cells, [14C]-cystine uptake and cystine-induced glutamate release assays were characterized and optimized with respect to cystine and protein concentrations and time of incubation. A pilot screen of the LOPAC/NINDS libraries using glutamate release demonstrated that the logistics of the assay were in place but unfortunately, did not yield meaningful pharmacophores. A larger, HTS campaign using the 384-well cystine-induced glutamate release as primary assay and the 96-well 14C-cystine uptake as confirmatory assay is currently underway. Unexpectedly, we observed that the rate of cystine uptake was significantly faster than the rate of glutamate release in human glioma cells. This was in contrast to the same rates of cystine uptake and glutamate release previously reported in normal human fibroblast cells.

Dataset Information

Localisation of novel forms of glutamate transporters and the cystine-glutamate antiporter in the choroid plexus: Implications for CSF glutamate homeostasis.

Publications

Localisation of novel forms of glutamate transporters and the cystine-glutamate antiporter in the choroid plexus: Implications for CSF glutamate homeostasis.

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