QUICK SEARCH:   [advanced] Author: Keyword(s):
Home     Help     Feedback     Subscriptions     Archive     Search     Table of Contents

First published online 5 October 2004
doi: 10.1242/jcs.01411

This Article Summary Full Text Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Vanoni, C. Articles by Pietrini, G. Search for Related Content PubMed PubMed Citation Articles by Vanoni, C. Articles by Pietrini, G. Social Bookmarking                         What's this?

Increased internalisation and degradation of GLT-1 glial glutamate transporter in a cell model for familial amyotrophic lateral sclerosis (ALS)

¹ Department of Pharmacology, School of Medicine, C.E.N.D. Center of Excellence on Neurodegenerative Diseases, University of Milan, IN-CNR Cellular and Molecular Pharmacology Section, Via Vanvitelli 32, 20129 Milano, Italy
² Laboratory of Molecular Neurobiology, Department of Neuroscience, Mario Negri Institute for Pharmacological Research, Via Eritrea, 62 20157 Milano, Italy

View larger version (31K): [in a new window]   Fig. 1. The total expression of GLT-1 is downregulated in MDCK cells stably expressing the G93A mutant of SOD1. Forty-eight hours after transfection with cDNAs encoding GLT-1, EAAC1, the chimeric transporters GLT-EAAC or EAAC-GLT, or the neuronal GABA transporter GAT-1 in parental MDCK cells (M), and MDCK cell lines stably expressing wild-type SOD1 (S) or the SOD1 G93A mutant (G), the cells were analysed for total protein expression. (A) 20 µg of total homogenate were separated by 10% SDS-PAGE and immunoblotted with GLT-1 C antibodies to identify GLT-1 and chimera EAAC-GLT, with EAAC C antibodies to identify the EAAC1 isoform and the GLT-EAAC chimera, and with GAT-1 antibodies. The blots were also probed with antibodies to human SOD1 to measure the expression of the wild type and G93A mutant, and with antibodies to LIN-7 as a loading control. The arrowheads indicate the lower molecular weight band stained by SOD1 antibodies corresponding to endogenous SOD1. (B,C) Densitometric analyses of the total expression of glutamate and GABA transporters in G (B) and S cell lines (C). The pixel densities of lanes from seven experiments for GLT-1 and three for EAAC1, the chimeras and GAT-1 (one of which is shown in A) were evaluated using the NIH Image program. All of the values were normalised to LIN-7 and expressed as protein expression compared to that in parental MDCK cells±s.e.m. The G cell lines showed a statistically significant reduction in GLT-1 (P=0.0001) and EAAC-GLT (P=0.043), whereas there was no significant alteration in the transporters or chimeras in the cell lines stably expressing wild-type SOD1 (t-test). (D) Transient co-expression of GLT-1 with wild-type and mutant SOD1 in parental MDCK cells. Forty-eight hours after the co-transfection of GLT-1 with empty vectors, G93A or SOD1 cDNAs, the cells were analysed by SDS-PAGE and immunoprobed with the indicated antibodies. (E) GLT-1 expression in G cells was tested under normal conditions and after exposure to the oxidative reagent menadione (MQ) (20µM; 4 hours), the antioxidant N-acetylcysteine (NAC) (1 mM; 24 hours) or 2-mercaptoethanol (2-ME) (2 mM; 4 hours). Forty-eight hours after the transfection of GLT-1, untreated and treated G93A cells were analysed by SDS-PAGE and immunoprobed with the indicated antibodies.

View larger version (51K): [in a new window]   Fig. 2. Continuous expression of the SOD1 G93A mutant selectively affects the surface distribution of GLT-1 in MDCK cells. (A,B) Forty-eight hours after transfection, the cells were fixed and triple-stained with antibodies against GLT-1, EAAC1 or GLAST (red); SOD1 (green); and ß-catenin, a marker of the lateral junctional domain (blue). Confocal horizontal (A) and vertical (B) sections show the distribution of wild type and chimeric transporters in SOD1- and G93A-expressing cell lines. The confocal merges show surface (apical and basolateral) localisation of the GLT-1 and EAAC-GLT in SOD1-expressing cells, and their intracellular accumulation in G93A-expressing cells. The surface distributions of the neuronal EAAC-1 transporter (apical surface) and the glial GLAST (basolateral surface) are retained in the G93A-expressing MDCK cell lines. The arrows in A indicate perinuclear staining, and the arrows in B the apical cell surface. Bar in A, 5 µm; B, 10 µm.

View larger version (12K): [in a new window]   Fig. 3. Decreased GLT-1 level on the cell surface of G93A-expressing MDCK cell lines. (A) Biotinylation of surface GLT-1 in parental MDCK cells (M) and MDCK cells expressing wild-type SOD1 (S) or the G93A mutant (G). Forty-eight hours after GLT-1 transfection, the cells were pre-incubated with 5 mM EDTA to open the tight junctions and allow biotinylation of lateral surface proteins with sulfo-NSH-biotin; the surface biotinylated proteins were recovered on streptavidin beads. Equal volumes of total cell lysates (Tot), biotinylated surface proteins (Biot) and intracellular proteins (Ic) were separated by 10% SDS-PAGE and immunostained for GLT-1. The biotinylated proteins were also probed for E-cadherin (E-CAD) as surface markers. (B-C) Densitometric analysis of the expression of GLT-1 in M, S and G cell lines. The pixel densities of lanes from three separate experiments (one of which is shown in A) were evaluated using the NIH Image program. All of the values were expressed as the percentage of surface transporter compared to total expression of GLT-1 in each cell line±s.e.m. Statistical an alysis (t-test) revealed a highly significant reduction in surface GLT-1 in the G93A-expressing cell line (P=0.0063). (C) The pixel densities of lanes from the experiments shown in A (Tot, Biot) were evaluated using the NIH Image program and expressed as OD units.

View larger version (71K): [in a new window]   Fig. 4. GLT-1 accumulates in the endocytotic compartments of G93A-expressing MDCK cell lines. (A) Morphological endocytosis assay. Forty-eight hours after GLT-1 transfection, the cells were labelled with FITC-WGA for 30 minutes at 0°C, washed to remove unbound WGA, and cultured for 60 minutes in regular medium to allow the internalisation of surface WGA-labelled glycoproteins prior to fixation and staining with GLT-1 antibody. The merged confocal horizontal section shows the colocalisation (yellow staining) of GLT-1 (red) with the internalised labelled glycoproteins (green). (B) Surface redistribution of GLT-1 in G93A-expressing MDCK cell lines after the inhibition of endocytosis by hypertonic media. Forty-eight hours after GLT-1 transfection, the cells were treated with 0.45 M sucrose for 30 minutes, fixed and double-stained with GLT-1 (red) and ß-catenin (green). The horizontal confocal sections taken at the plane of the lateral surface show the colocalisation of GLT-1 with ß-catenin at the cell surface (yellow), and the disappearance of intracellular perinuclear GLT-1 staining. Bar, 5 µm.

View larger version (99K): [in a new window]   Fig. 5. GLT-1 is targeted to degradative acidic compartments in G93A-expressing MDCK cell lines. Forty-eight hours after GLT-1 transfection, the cells were fixed and stained for EEA1 (green), a marker of early endosomal compartments, and GLT-1 (red). To verify whether GLT-1 accumulates in the sub apical slow recycling compartment labelled by the mutant 5BGT epithelial GABA transporter (Perego et al., 1999), the G93A-expressing MDCK cells were co-transfected with GLT-1 and myc-tagged 5BGT, and fixed and double-stained for GLT-1 (red) and myc (green) after 48 hours. Confocal analysis of the double indirect immunofluorescence staining showed no colocalisation of GLT-1 with EEA1 or 5BGT, whereas colocalisation of GLT-1 (yellow) with acidic organelles was revealed by labelling with the acidotropic FITC-lysotracker (0.5 mM) for 1 hour at 37°C before fixation and staining for GLT-1. Bar in upper panels, 5 µm; middle panels, 2 µm; lower panels, 3 µm.

View larger version (16K): [in a new window]   Fig. 6. Increased rate of degradation of GLT-1 in G93A-expressing MDCK cell lines. (A) Forty-eight hours after GLT-1 transfection, the indicated cell lines were treated for 6 hours with 60 µg/ml cycloheximide (CHX) to inhibit protein synthesis or overnight with 100 µM chloroquine (CHQ) to inhibit lysosomal degradation. Equal amounts of proteins were separated by 10% SDS-PAGE and immunoblotted for GLT-1. (B) The pixel densities of the lanes from the experiment shown in A were evaluated using the NIH Image program. The values were expressed as fold increases in protein expression in treated compared to untreated cells. When protein synthesis was inhibited by cycloheximide, GLT-1 levels were reduced to 18% in G cells, whereas a negligible reduction in GLT-1 was observed in M or S cells. Chloroquine treatment protected GLT-1 from lysosomal degradation mainly in G cells.

CiteULike

Complore

Connotea

Del.icio.us

Digg

Reddit

Technorati

Twitter