Caspase-3 Cleaves and Inactivates the Glutamate Transporter EAAT2
2006; Elsevier BV; Volume: 281; Issue: 20 Linguagem: Inglês
10.1074/jbc.m600653200
ISSN1083-351X
AutoresWilliam Boston-Howes, Stuart L. Gibb, Eric O. Williams, Piera Pasinelli, Robert H. Brown, Davide Trotti,
Tópico(s)Amino Acid Enzymes and Metabolism
ResumoEAAT2 is a high affinity, Na+-dependent glutamate transporter with predominant astroglial localization. It accounts for the clearance of the bulk of glutamate released at central nervous system synapses and therefore has a crucial role in shaping glutamatergic neurotransmission and limiting excitotoxicity. Caspase-3 activation and impairment in expression and activity of EAAT2 are two distinct molecular mechanisms occurring in human amyotrophic lateral sclerosis (ALS) and in the transgenic rodent model of the disease. Excitotoxicity caused by down-regulation of EAAT2 is thought to be a contributing factor to motor neuron death in ALS. In this study, we report the novel evidence that caspase-3 cleaves EAAT2 at a unique site located in the cytosolic C-terminal domain of the transporter, a finding that links excitotoxicity and activation of caspase-3 as converging mechanisms in the pathogenesis of ALS. Caspase-3 cleavage of EAAT2 leads to a drastic and selective inhibition of this transporter. Heterologous expression of mutant SOD1 proteins linked to the familial form of ALS leads to inhibition of EAAT2 through a mechanism that largely involves activation of caspase-3 and cleavage of the transporter. In addition, we found evidence in spinal cord homogenates of mutant SOD1 ALS mice of a truncated form of EAAT2, likely deriving from caspase-3-mediated proteolytic cleavage, which appeared concurrently to the loss of EAAT2 immunoreactivity and to increased expression of activated caspase-3. Taken together, our findings suggest that caspase-3 cleavage of EAAT2 is one mechanism responsible for the impairment of glutamate uptake in mutant SOD1-linked ALS. EAAT2 is a high affinity, Na+-dependent glutamate transporter with predominant astroglial localization. It accounts for the clearance of the bulk of glutamate released at central nervous system synapses and therefore has a crucial role in shaping glutamatergic neurotransmission and limiting excitotoxicity. Caspase-3 activation and impairment in expression and activity of EAAT2 are two distinct molecular mechanisms occurring in human amyotrophic lateral sclerosis (ALS) and in the transgenic rodent model of the disease. Excitotoxicity caused by down-regulation of EAAT2 is thought to be a contributing factor to motor neuron death in ALS. In this study, we report the novel evidence that caspase-3 cleaves EAAT2 at a unique site located in the cytosolic C-terminal domain of the transporter, a finding that links excitotoxicity and activation of caspase-3 as converging mechanisms in the pathogenesis of ALS. Caspase-3 cleavage of EAAT2 leads to a drastic and selective inhibition of this transporter. Heterologous expression of mutant SOD1 proteins linked to the familial form of ALS leads to inhibition of EAAT2 through a mechanism that largely involves activation of caspase-3 and cleavage of the transporter. In addition, we found evidence in spinal cord homogenates of mutant SOD1 ALS mice of a truncated form of EAAT2, likely deriving from caspase-3-mediated proteolytic cleavage, which appeared concurrently to the loss of EAAT2 immunoreactivity and to increased expression of activated caspase-3. Taken together, our findings suggest that caspase-3 cleavage of EAAT2 is one mechanism responsible for the impairment of glutamate uptake in mutant SOD1-linked ALS. Amyotrophic lateral sclerosis is a fatal, late-onset neurodegenerative disease, resulting from the progressive death of cortical and spinal motor neurons. About 90% of ALS 4The abbreviations used are: ALS, amyotrophic lateral sclerosis; EAAT, excitatory amino acid transporter; (Tr)EAAT2, truncated EAAT2 derived from caspase-3 cleavage at the consensus site; CTE, C-terminal domain of EAAT2; SOD1, Cu2+/Zn2+-superoxide dismutase; mutSOD1, mutant human SOD1; CHAPS, 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid.; DTT, dithiothreitol; Z, benzyloxycarbonyl; fmk, fluoromethyl ketone; PARP, poly(ADP-ribose) polymerase; WT, wild type. cases are sporadic, and the remaining 10% are inherited in a dominant manner (familial ALS). The sporadic and familial forms of ALS are phenotypically indistinguishable suggesting a convergence of common pathogenic mechanisms in this disease. Insights into the pathological mechanisms underlying ALS came with the discovery of causative mutations in the enzyme Cu2+/Zn2+ superoxide dismutase (1Rosen D.R. Siddique T. Patterson D. Figlewicz D.A. Sapp P. Hentati A. Donaldson D. Goto J. O'Regan J.P. 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Watase K. Manabe T. Yamada K. Watanabe M. Takahashi K. Iwama H. Nishikawa T. Ichihara N. Kikuchi T. Okuyama S. Kawashima N. Hori S. Takimoto M. Wada K. Science. 1997; 276: 1699-1702Crossref PubMed Scopus (1481) Google Scholar, 6Williams S.M. Sullivan R.K. Scott H.L. Finkelstein D.I. Colditz P.B. Lingwood B.E. Dodd P.R. Pow D.V. Glia. 2005; 49: 520-541Crossref PubMed Scopus (104) Google Scholar). Several lines of evidence support a prominent role for EAAT2 impairment in the pathogenesis of ALS. Excitotoxicity caused by a consistent reduction in expression and activity of the glutamate transporter EAAT2 is among the various proposed pathogenic mechanisms implicated in playing a role in the propagation of disease (7Cleveland D.W. Rothstein J.D. Nat. Rev. Neurosci. 2001; 2: 806-819Crossref PubMed Scopus (1180) Google Scholar). In the G93A-SOD1 transgenic mouse and rat, the expression levels of EAAT2 are reduced, often focally in the spinal cord ventral gray matter and prior to the onset of the disease (3Howland D.S. Liu J. She Y. Goad B. Maragakis N.J. Kim B. Erickson J. Kulik J. DeVito L. Psaltis G. DeGennaro L.J. Cleveland D.W. Rothstein J.D. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 1604-1609Crossref PubMed Scopus (702) Google Scholar). EAAT2 knockdown achieved by using antisense oligonucleotides in adult rats leads to hind limb paralysis with motor neuron degeneration and denervation similar to ALS (8Rothstein J.D. Dykes-Hoberg M. Pardo C.A. Bristol L.A. Jin L. Kuncl R.W. Kanai Y. Hediger M.A. Wang Y. Schielke J.P. Welty D.F. Neuron. 1996; 16: 675-686Abstract Full Text Full Text PDF PubMed Scopus (2141) Google Scholar). We have reported previously that mutSOD1 proteins, heterologously expressed in Xenopus oocytes exposed to oxidative stress, lead to selective inhibition of EAAT2 by targeting the C-terminal domain (9Trotti D. Rolfs A. Danbolt N.C. Brown Jr., R.H. Hediger M.A. Nat. Neurosci. 1999; 2: 427-433Crossref PubMed Scopus (305) Google Scholar). Moreover, experiments in which EAAT2 was genetically or pharmacologically increased have led to the conclusion that EAAT2 impairment contributes to the progression of motor neuron degeneration in ALS (10Guo H. Lai L. Butchbach M.E. Lin C.L. Mol. Cell. Neurosci. 2002; 21: 546-560Crossref PubMed Scopus (38) Google Scholar, 11Rothstein J.D. Patel S. Regan M.R. Haenggeli C. Huang Y.H. Bergles D.E. Jin L. Hoberg M. Dykes Vidensky S. Chung D.S. Toan S.V. Bruijn L.I. Su Z.Z. Gupta P. Fisher P.B. Nature. 2005; 433: 73-77Crossref PubMed Scopus (1260) Google Scholar, 12Ganel R. Ho T. Maragakis N.J. Jackson M. Steiner J.P. Rothstein J.D. Neurobiol. Dis. 2006; 21: 556-567Crossref PubMed Scopus (81) Google Scholar). Several experimental observations indicate that mutSOD1 toxicity involves sequential activation of caspase-1 and caspase-3 in transgenic mice and in cellular models of ALS (13Pasinelli P. Houseweart M.K. Brown Jr., R.H. Cleveland D.W. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 13901-13906Crossref PubMed Scopus (306) Google Scholar, 14Ando Y. Liang Y. Ishigaki S. Niwa J. Jiang Y. Kobayashi Y. Yamamoto M. Doyu M. Sobue G. Neurochem. Res. 2003; 28: 839-846Crossref PubMed Scopus (21) Google Scholar, 15Guegan C. Przedborski S. J. Clin. Investig. 2003; 111: 153-161Crossref PubMed Scopus (159) Google Scholar). Of particular interest is the role that caspase-3, a key proteolytic enzyme executioner of apoptosis (16Fischer U. Janicke R.U. Schulze-Osthoff K. Cell Death Differ. 2003; 10: 76-100Crossref PubMed Scopus (889) Google Scholar), plays in ALS. Oxidative stress delivered to the N2a cell model of mutSOD1-linked ALS triggered caspase-3 activation (17Pasinelli P. Borchelt D.R. Houseweart M.K. Cleveland D.W. Brown Jr., R.H. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 15763-15768Crossref PubMed Scopus (190) Google Scholar). Evidence for activation of caspase-3 was also reported in vivo in mutSOD1 transgenic mice, both in motor neurons and astrocytes of the spinal cord at the time of onset of ALS-like symptoms (13Pasinelli P. Houseweart M.K. Brown Jr., R.H. Cleveland D.W. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 13901-13906Crossref PubMed Scopus (306) Google Scholar). In addition, modulation of caspase-3 activity by overexpression of Bcl-2 or by infusion of the caspase inhibitor Z-VAD-fmk provided protective benefits in a transgenic mouse model of ALS (18Vukosavic S. Stefanis L. Jackson-Lewis V. Guegan C. Romero N. Chen C. Dubois-Dauphin M. Przedborski S. J. Neurosci. 2000; 20: 9119-9125Crossref PubMed Google Scholar, 19Kostic V. Jackson-Lewis V. de Bilbao F. Dubois-Dauphin M. Przedborski S. Science. 1997; 277: 559-562Crossref PubMed Scopus (437) Google Scholar, 20Li M. Ona V.O. Guegan C. Chen M. Jackson-Lewis V. Andrews L.J. Olszewski A.J. Stieg P.E. Lee J.P. Przedborski S. Friedlander R.M. Science. 2000; 288: 335-339Crossref PubMed Scopus (639) Google Scholar). Although it is not clear whether activation of caspase-3 initiates ALS, the observations accumulated thus far strongly indicate that this mechanism contributes substantially to the progression of the disease. EAAT2 has a putative consensus site for caspase-3 cleavage located in the cytoplasmic C-terminal domain. In this study, we provide evidence that the C-terminal domain of EAAT2 is cleaved by caspase-3 and that this proteolytic process functionally impairs the transporter activity. Cleavage and inactivation of EAAT2 are triggered by mutSOD1 when challenged by oxidative stress in vitro. Moreover, we found evidence of a truncated EAAT2 form likely derived from caspase-3 cleavage in the spinal cord of G93A-SOD1 transgenic ALS mice, suggesting that this event also occurs in vivo. Animals—Wild type, G93A-SOD1 transgenic mice, and nontransgenic control mice were used for this study. The mouse colonies were B6SJL-TgN (G93A-SOD1)1Gur (stock number 002726; ∼30 copies of the transgene; The Jackson Laboratory, Bar Harbor, ME), and B6SJL-TgN(SOD1)2Gur (stock number 002297; ∼8 copies of the transgene) and were maintained in-house. These lines expressed approximately the same amount of human SOD1 protein as assessed on immunoblot. Genotyping and determination of transgene copies number were performed by PCR analysis. Transfections and Protein Extractions—HEK 293 cells were maintained in Dulbecco's modified Eagle's medium containing 10% fetal bovine serum and 1% penicillin/streptomycin. EAAT2 and D505N-EAAT2 cloned in pcDNA3.1 were transiently transfected in HEK 293 cells at ∼70% confluence using Lipofectamine 2000 (Invitrogen). Transfected cells were harvested 48 h later in buffer containing CHAPS (1%), 150 mm NaCl, 10 mm NaPi, pH 7.4, and Complete™ protease inhibitor mixture (Roche Applied Bioscience), briefly sonicated, and stored at –80 °C for Western blot analysis or immediately processed for caspase cleavage reactions. In Vitro Cleavage Reactions—Mice were euthanized by intraperitoneal injection of xylazine/ketamine according to institutional guidelines. Spinal cords were collected and immediately homogenized on ice (glass-Teflon homogenizer; 1,000 rpm) in 30 volumes of hypotonic solution containing 2 mm EDTA, 10 mm NaPi, pH 7.4, and protease inhibitors (Complete Mini™ with EDTA). The homogenates were centrifuged (39,000 × g, 15 min), supernatants removed, and the "crude" membrane pellet resuspended in extraction buffer containing SDS or CHAPS (1%), 150 mm NaCl, 10 mm NaPi, pH 7.4, and Complete™ protease inhibitor mixture (Roche Applied Bioscience). Extracts were briefly sonicated, centrifuged (1,000 × g, 4 min) to remove unsolubilized material, and immediately analyzed or stored at –80 °C. The homogenates prepared with the above protocol were termed SDS or CHAPS extracts according to the extraction detergent used. For the caspase cleavage experiments, 5 μl of CHAPS extract, containing 10–25 μgof total proteins, were incubated for 1–7 h at 37 °C in 50 μl of caspase cleavage buffer containing 10% sucrose, 20 mm HEPES/Na, pH 7.4, 100 mm NaCl, 1 mm EDTA, 10 mm DTT, the protease inhibitors mixture Complete™, in the presence or absence of purified human recombinant active caspases (BD Biosciences). We determined that the protease inhibitors of the Complete™ mixture did not prevent caspase-3-mediated cleavage in vitro of its well characterized substrate PARP (16Fischer U. Janicke R.U. Schulze-Osthoff K. Cell Death Differ. 2003; 10: 76-100Crossref PubMed Scopus (889) Google Scholar). Reactions were terminated by adding SDS-containing sample buffer, boiling for 5 min, and then analyzing by immunoblot on polyvinylidene difluoride membranes. Proteins were visualized by chemiluminescence (ECL Plus). Glutamate transporter cDNAs were translated in vitro using the TnT®-coupled reticulocyte lysate kit and [35S]methionine labeling (Promega, Madison, WI). Five μl of the in vitro translated reactions were used for caspase cleavage experiments. In vitro translations and autoradiography protocols were performed according to the manufacturer's instructions. Site-directed Mutagenesis and PCRs—D505N-EAAT2 was generated using the QuikChange™ site-directed mutagenesis kit (Stratagene, La Jolla, CA), confirmed by sequencing and cloned in pcDNA3.1 for mammalian expression or pOX for oocyte expression. Asparagine was chosen rather than the more commonly used alanine because the structural similarity of asparagine to aspartate did not alter the activity and trafficking of D505N-EAAT2 in oocytes, which was a crucial requisite to assess the impact of the caspase-3 treatment on the transporter function. The coding region of the truncated EAAT2 ((Tr)EAAT2) was generated by PCR and the cDNAs subcloned in pcDNA3.1. Antibodies—Affinity-purified polyclonal antibodies against peptides of the glutamate transporters EAAT1–3 were referred to by capital letters A, B, and C, respectively, followed by numbers indicating the corresponding peptide of the rat transporter sequence. For this study we used A522-541 (0.2 μg/ml, rabbit 8D0161, EAAT1 cytoplasmic C terminus), B12-26 (0.2 μg/ml, rabbit 26970, EAAT2 cytoplasmic N terminus), B493-508 (0.1 μg/ml, rabbit 84946, EAAT2 cytoplasmic C terminus), and C161-177 (0.5 mg/ml, EAAT3 extracellular loop, Zymed Laboratories Inc.). Moreover, an antibody raised against the last 17 amino acids of EAAT2 C terminus was purchased from Affinity BioReagent (which we termed ABR556-573, catalogue number PA3-040; 1:1,000–10,000). These anti-glutamate transporter antibodies, although raised against the rat sequence, cross-react with mouse and human isoforms except for B518-536, which does not cross-react with the mouse isoform. Other antibodies used are anti-human SOD1 (Calbiochem) and anti-p17 fragment of caspase-3 (Cell Signaling). Protein Expression in Oocytes—Linearized plasmids were transcribed in vitro using the mMessage mMachine kit (Ambion, Austin, TX). Stage V Xenopus oocytes were enzymatically defolliculated, injected with cRNAs (20–50 ng/oocyte), incubated at 18 °C in L-15 solution (Specialty Media) supplemented with gentamycin sulfate (100 μg/ml) and used for experiments 1–3 days post-injection. Glutamate Uptake Measurements—Glutamate uptake current and uptake of l-[3H]glutamate were measured in oocytes as described previously (9Trotti D. Rolfs A. Danbolt N.C. Brown Jr., R.H. Hediger M.A. Nat. Neurosci. 1999; 2: 427-433Crossref PubMed Scopus (305) Google Scholar). To assess the effect of caspases on glutamate-evoked EAAT2 uptake current, 0.2–4 ng of active recombinant purified caspases (BD Biosciences) in 50 nl of buffer were injected into the oocytes, and the uptake current was recorded before and after the injection. Injection buffer contained (final concentration in the oocyte in mm) the following: 1 Tris-HCl, 2 NaCl, 1 imidazole, 10 KPi, pH 7.4, 0.01% CHAPS, 0.1 EDTA, 1 DTT, and 0.2% glycerol. Glutamate uptake was measured for 5 min by incubating the oocytes with 10 μm glutamate isotopically diluted with l-[3H]glutamate (specific activity 51.9 Ci/mmol; PerkinElmer Life Sciences) in 1 ml of frog Ringer solution (115 mm NaCl, 2 mm KCl, 1.8 mm CaCl2·H2O, 10 mm HEPES, pH 7.1–7.3). The uptake reaction was stopped by rinsing the oocytes with cold sodium-free frog Ringer solution (0 Na+, 115 mm choline. The oocytes were then dissolved in 10% SDS and counted to quantify the incorporation of glutamate. Caspase Activity Assay—Ten oocytes/group were incubated for 1 h with H2O2 (150 μm). Each group was lysed in 400 μl of buffer containing 50 mm HEPES, pH 7.4, 0.1% CHAPS, 1 mm DTT and incubated for 5 min in an ice bath. The lysate was centrifuged at 16,000 × g (15 min at 4 °C), and 5 μl of supernatant was used to determine caspase-3 cleavage activity and kinetics (>12 h) using a fluorometric assay (BD Biosciences). Fluorescence emission was determined by a Fluo-star BMG fluorometer (excitation 380 nm and emission 420 nm). l-[3H]Glutamate uptake was measured for 5 min in oocytes treated for 1 h with 150 μm H2O2 or control as described in the legend to Fig. 6. Biotinylation of Cell Surface Transporters—Biotinylation reactions were performed using the biotinylation kit from Pierce and following the manufacturer's protocol with minor adaptations for Xenopus oocytes. Briefly, 20–25 oocytes/group were used for these experiments. The high number of oocytes per group was intended to minimize intra-group variability because of unpredictable variations in the expression levels of EAAT2 molecules targeted at the cell surface of the oocyte. Caspase-3 Cleaves the Cytoplasmic C-terminal Domain of EAAT2 at Aspartate 505 —The cytoplasmic C-terminal domain of the glutamate transporter EAAT2 has a unique putative caspase-3 cleavage motif located at aspartate 505 (human EAAT2 sequence numbering). This aspartate (Asp-505) is conserved among isoforms from different species, including rat and mouse (Fig. 1A). To test whether EAAT2 could be cleaved by caspase-3, we treated mouse spinal cord homogenates with increasing amounts of purified caspase-3 (active form of the recombinant human caspase-3, concentration range 10–100 nm), and the reaction was then probed on Western blot with three polyclonal anti-EAAT2 antibodies directed against different domains of the transporter (Fig. 1A). Caspase-3 concentrations used in this experiment are comparable with those of other studies reporting caspase-3 cleavage of different substrates, such as PARP or amyloid precursor protein (21Gervais F.G. Xu D. Robertson G.S. Vaillancourt J.P. Zhu Y. Huang J. LeBlanc A. Smith D. Rigby M. Shearman M.S. Clarke E.E. Zheng H. Van Der Ploeg L.H. Ruffolo S.C. Thornberry N.A. Xanthoudakis S. Zamboni R.J. Roy S. Nicholson D.W. Cell. 1999; 97: 395-406Abstract Full Text Full Text PDF PubMed Scopus (716) Google Scholar). The treatment led to a dose-dependent loss of EAAT2 immunoreactivity and a simultaneous dose-dependent appearance of a lower band that corresponded to the shortened form of EAAT2 (Fig. 1B, (Tr)EAAT2) when the blot was probed with the anti-EAAT2 N-terminal antibody B12-26. The same reaction probed with the antibodies B493-508 (Fig. 1B) and ABR556-573 (Fig. 1B) showed dose-dependent loss of EAAT2 immunoreactivity and no evidence for (Tr)EAAT2 accumulation, as expected if the cleavage occurred at the unique site at aspartate 505. Failure to recognize (Tr)EAAT2 by the B493-508 antibody, whose epitope sequence encompasses the putative caspase-3 consensus site in EAAT2, argues that the immunoreactivity of this antibody is mainly directed against the last amino acids of the C-terminal portion of the epitope. This is also supported by evidence that B493-508 immunoreactivity toward EAAT2 is abolished by incubating the antibody with the peptide 506–516 (100 μm, human EAAT2 sequence numbering), whereas the peptide 493–505, although it slightly decreased the affinity of the antibody for EAAT2, it did not block the antibody immunoreactivity (not shown). Treatment of rat spinal cord homogenates with increasing concentrations of active caspase-3 and probed with the B518-536 antibody led to similar dose-dependent loss of EAAT2 immunoreactivity as seen with the ABR556-573 antibody in mouse homogenates (not shown). The cleavage of EAAT2 is specific for caspase-3 as other caspases, such as caspase-1, -6, -7, and -8, were ineffective (Fig. 1C). Quantitation of the proteolytic reaction indicates that EAAT2 cleavage occurs with relevant kinetics properties (kcat/Km = 3 × 105 m–1 s–1) similar to other validated caspase-3 substrates, such as PARP or amyloid precursor protein (21Gervais F.G. Xu D. Robertson G.S. Vaillancourt J.P. Zhu Y. Huang J. LeBlanc A. Smith D. Rigby M. Shearman M.S. Clarke E.E. Zheng H. Van Der Ploeg L.H. Ruffolo S.C. Thornberry N.A. Xanthoudakis S. Zamboni R.J. Roy S. Nicholson D.W. Cell. 1999; 97: 395-406Abstract Full Text Full Text PDF PubMed Scopus (716) Google Scholar) (Fig. 1, B and D). To ascertain whether the cleavage occurred at the predicted site in the EAAT2 sequence, we disrupted the putative consensus site in EAAT2 by mutating aspartate (D) at position 505 to asparagine (N). CHAPS extracts of HEK 293 cells transfected with wild type and D505N-EAAT2 mutant were then treated with caspase-3 and analyzed by immunoblot. As expected, the D505N mutation prevented the cleavage from occurring (Fig. 2A). We also tested whether other nonconventional sites could possibly be used in addition to the canonical one. In vitro translated EAAT2 labeled with [35S]methionine was incubated with caspase-3 and the reaction analyzed by autoradiography. EAAT2 has 24 methionine residues evenly scattered in the primary structure, including 2 methionines located downstream of the caspase-3 consensus motif, ensuring even labeling of all domains of the transporter. Caspase-3 cleaved EAAT2 but not D505N-EAAT2, forming two fragments at ∼55 kDa ((Tr)EAAT2) and 8 kDa (termed CTE as for the C-terminal domain of EAAT2 excised by caspase-3) (Fig. 2B). It follows that the canonical motif for caspase-3 cleavage is the only site used to cleave EAAT2. EAAT1, the other major astroglial glutamate transporter, lacks conventional cleavage sites for caspase-3 and was indeed insensitive to caspase-3 treatment (Fig. 2C and see also Fig. 4E). On the contrary, the neuronal glutamate transporter EAAT3 has two caspase-3 cleavage sites defined by the sequence -DXXD-, also located at the cytoplasmic C terminus. Cleavage at these sites would generate small fragments of ≤3 kDa, which is hard to detect on the gel. Moreover, these fragments do not contain methionines and therefore could not be visualized by autoradiography. Indeed, treatment of the in vitro translated EAAT3 with caspase-3 did not produce proteolytic fragments. Nevertheless, we detected a small but reproducible downward shift of the EAAT3 band, suggesting that the cleavage may have occurred (Fig. 2D and see also Fig. 4C). EAAT2 Cleavage by Caspase-3 Leads to Uptake Inhibition—Despite EAAT2 being an astroglial protein, previous studies have shown that primary astrocytes in culture do not express physiological levels of EAAT2. In vitro manipulation of astrocyte cultures with cell differentiation-inducing compounds, such as cAMP analogues or epidermal growth factor, promoted expression of EAAT2 (22Robinson M.B. J. Neurochem. 2002; 80: 1-11Crossref PubMed Scopus (175) Google Scholar). However, these manipulations resulted in expression of non-functional transporters, raising the concern that EAAT2 was not properly targeted to the plasma membrane or was incorrectly processed in this system, therefore questioning the validity of the astrocytes as an experimental model system to study the function of EAAT2 (23Swanson R.A. Liu J. Miller J.W. Rothstein J.D. Farrell K. Stein B.A. Longuemare M.C. J. Neurosci. 1997; 17: 932-940Crossref PubMed Google Scholar, 24Schlag B.D. Vondrasek J.R. Munir M. Kalandadze A. Zelenaia O.A. Rothstein J.D. Robinson M.B. Mol. Pharmacol. 1998; 53: 355-369Crossref PubMed Scopus (281) Google Scholar). To assess whether caspase-3 proteolytic cleavage had a functional impact on EAAT2, we therefore took advantage of the oocyte expression system and the two-electrode voltage clamp technique to measure EAAT2 uptake current. Xenopus oocytes injected with cRNA for EAAT2 abundantly express a functional transporter (25Trotti D. Aoki M. Pasinelli P. Berger U.V. Danbolt N.C. Brown Jr., R.H. Hediger M.A. J. Biol. Chem. 2000; 276: 576-582Abstract Full Text Full Text PDF Scopus (156) Google Scholar). EAAT2-expressing oocytes were then injected with the active caspase-3, and EAAT2 uptake current was measured before and after the injection. At –50 mV, EAAT2 uptake current was rapidly and progressively inhibited by the injection of caspase-3 (∼45% in 20 min), whereas the uptake current of the D505N-EAAT2 mutant was unaltered, suggesting that the proteolytic cleavage at the caspase-3 consensus site in EAAT2 was responsible for the inhibition (Fig. 3, A and B). Affinity for substrate (i.e. Km for glutamate) calculated from the glutamate-evoked current of the D505N-EAAT2 mutant transporter expressed in oocytes was statistically not different from wild type EAAT2 (20.2 ± 4.5 μm for WT-EAAT2 versus 17.7 ± 5 μm for D505N-EAAT2, n = 3 oocytes; data not shown); in addition, no change in glutamate uptake velocity was also observed (23 ± 4 for WT-EAAT2 versus 25 ± 3 nmol/mg protein/15 min for D505N-EAAT2, n = 10 oocytes/group, measured at 10 μm glutamate; see "Experimental Procedures"). A similar uptake activity between wild type and D505N-EAAT2 is consistent with a lack of significant structural alterations in the mutant D505N-EAAT2. The transport of glutamate is coupled to the movements of cations across the plasma membrane (Na+, K+, and H+) that are necessary for transporter cycling and glutamate uptake, as well as chloride anions that are uncoupled to the flux of glutamate. The relative proportion of the uptake current generated by ion-coupled glutamate transport (stoichiometric current) and the glutamate-gated chloride conductance varies in intensity among EAAT isoforms (26Seal R.P. Amara S.G. Annu. Rev. Pharmacol. Toxicol. 1999; 39: 431-456Crossref PubMed Scopus (282) Google Scholar). In EAAT2, the stoichiometric current is predominant compared with the chloride conductance (26Seal R.P. Amara S.G. Annu. Rev. Pharmacol. Toxicol. 1999; 39: 431-456Crossref PubMed Scopus (282) Google Scholar). However, to rule out that the caspase-3-mediated inhibition of the uptake current was because of alterations in the glutamate-gated chloride current component rather than the transport of glutamate, we measured the effect of caspase-3 on EAAT2 uptake current at the chloride equilibrium potential (–20 mV), whereby the contribution of this conductance to the total uptake current is nullified. The extent of inhibition (∼45% at 20 min at –20 mV; data not shown) was the same as at –50 mV, suggesting that the glutamate transport current was the EAAT2 uptake current component affected by the caspase-3 cleavage. EAAT2 inhibition increased at increasing amounts of injected caspase-3 (Fig. 3C). Other caspases like caspase-7 (Fig. 3A) or caspase-1 and -6 (not shown) did not affect EAAT2, an observation in accord with the lack of effect of these caspases on EAAT2 in the in vitro cleavage reaction. Despite the fact that caspase-8 did not directly cleave EAAT2 in vitro (Fig. 1C), when injected into oocytes expressing EAAT2, it caused a progressive inhibition of the transporter-mediated uptake current, although with much slower kinetics because 50% inhibition was achieved in 60 min post-injectio
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