A Caspase-3-cleaved Fragment of the Glial Glutamate Transporter EAAT2 Is Sumoylated and Targeted to Promyelocytic Leukemia Nuclear Bodies in Mutant SOD1-linked Amyotrophic Lateral Sclerosis

Abstract

EAAT2 (excitatory amino acid transporter 2) is a high affinity, Na+-dependent glutamate transporter of glial origin that is essential for the clearance of synaptically released glutamate and prevention of excitotoxicity. During the course of human amyotrophic lateral sclerosis (ALS) and in a transgenic mutant SOD1 mouse model of the disease, expression and activity of EAAT2 is remarkably reduced. We previously showed that some of the mutant SOD1 proteins exposed to oxidative stress inhibit EAAT2 by triggering caspase-3 cleavage of EAAT2 at a single defined locus. This gives rise to two fragments that we termed truncated EAAT2 and COOH terminus of EAAT2 (CTE). In this study, we report that analysis of spinal cord homogenates prepared from mutant G93A-SOD1 mice reveals CTE to be of a higher molecular weight than expected because it is conjugated with SUMO-1. The sumoylated CTE fragment (CTE-SUMO-1) accumulates in the spinal cord of these mice as early as presymptomatic stage (70 days of age) and not in other central nervous system areas unaffected by the disease. The presence and accumulation of CTE-SUMO-1 is specific to ALS mice, since it does not occur in the R6/2 mouse model for Huntington disease. Furthermore, using an astroglial cell line, primary culture of astrocytes, and tissue samples from G93A-SOD1 mice, we show that CTE-SUMO-1 is targeted to promyelocytic leukemia nuclear bodies. Since one of the proposed functions of promyelocytic leukemia nuclear bodies is regulation of gene transcription, we suggest a possible novel mechanism by which the glial glutamate transporter EAAT2 could contribute to the pathology of ALS. EAAT2 (excitatory amino acid transporter 2) is a high affinity, Na+-dependent glutamate transporter of glial origin that is essential for the clearance of synaptically released glutamate and prevention of excitotoxicity. During the course of human amyotrophic lateral sclerosis (ALS) and in a transgenic mutant SOD1 mouse model of the disease, expression and activity of EAAT2 is remarkably reduced. We previously showed that some of the mutant SOD1 proteins exposed to oxidative stress inhibit EAAT2 by triggering caspase-3 cleavage of EAAT2 at a single defined locus. This gives rise to two fragments that we termed truncated EAAT2 and COOH terminus of EAAT2 (CTE). In this study, we report that analysis of spinal cord homogenates prepared from mutant G93A-SOD1 mice reveals CTE to be of a higher molecular weight than expected because it is conjugated with SUMO-1. The sumoylated CTE fragment (CTE-SUMO-1) accumulates in the spinal cord of these mice as early as presymptomatic stage (70 days of age) and not in other central nervous system areas unaffected by the disease. The presence and accumulation of CTE-SUMO-1 is specific to ALS mice, since it does not occur in the R6/2 mouse model for Huntington disease. Furthermore, using an astroglial cell line, primary culture of astrocytes, and tissue samples from G93A-SOD1 mice, we show that CTE-SUMO-1 is targeted to promyelocytic leukemia nuclear bodies. Since one of the proposed functions of promyelocytic leukemia nuclear bodies is regulation of gene transcription, we suggest a possible novel mechanism by which the glial glutamate transporter EAAT2 could contribute to the pathology of ALS. Amyotrophic lateral sclerosis (ALS) 4The abbreviations used are: ALS, amyotrophic lateral sclerosis; SOD1, Cu2+/Zn2+ superoxide dismutase; mutSOD1, mutated SOD1; WT-SOD1, wild type SOD1; SUMO, small ubiquitin modifier protein; PML, promyelocytic leukemia; PML-NB, PML nuclear body; E1, ubiquitin-activating enzyme; E2, ubiquitin carrier protein; E3, ubiquitin-protein isopeptide ligase; PBS, phosphate-buffered saline; CTE, COOH terminus of EAAT2; CHAPS, 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid; mAb, monoclonal antibody; HD, Huntington disease; EGFP, enhanced green fluorescent protein; Tricine, N-[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]glycine; DAPI, 4′,6-diamidino-2-phenylindole.4The abbreviations used are: ALS, amyotrophic lateral sclerosis; SOD1, Cu2+/Zn2+ superoxide dismutase; mutSOD1, mutated SOD1; WT-SOD1, wild type SOD1; SUMO, small ubiquitin modifier protein; PML, promyelocytic leukemia; PML-NB, PML nuclear body; E1, ubiquitin-activating enzyme; E2, ubiquitin carrier protein; E3, ubiquitin-protein isopeptide ligase; PBS, phosphate-buffered saline; CTE, COOH terminus of EAAT2; CHAPS, 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid; mAb, monoclonal antibody; HD, Huntington disease; EGFP, enhanced green fluorescent protein; Tricine, N-[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]glycine; DAPI, 4′,6-diamidino-2-phenylindole. is a fatal neurodegenerative disease, resulting from a progressive death of cortical and spinal motor neurons. About 90% of ALS cases are sporadic, whereas the remaining 10% are inherited in a dominant manner (familial ALS). Transgenic expression of high levels of human mutant SOD1 (mutSOD1) proteins in mice and rats leads to a progressive motor neuron disease that shares most of the clinical features of ALS (1Gurney M.E. Kim H. Chiu A.Y. Dal Canto M.C. Polchow C.Y. Alexander D.D. Caliendo J. Hentati A. Kwon Y.W. Deng H.X. Chen W. Zhai P. Sufit R.L. Siddique T. Science. 1994; 264: 1772-1775Crossref PubMed Scopus (3438) Google Scholar, 2Howland D.S. Kim 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 (696) Google Scholar). Astrocytes are essential partners of motorneurons, providing them with trophic support (3Taylor A.R. Kim D.J. Newbern J.M. Robinson M.B. Strupe J.L. Prevette D. Oppenheim R.W. Milligan C.E. J. Neurosci. 2007; 27: 634-644Crossref PubMed Scopus (23) Google Scholar) and mediating rapid clearance of synaptic glutamate mainly through the action of glial glutamate transporters. The EAAT2 (excitatory amino acid transporter 2) glial isoform of glutamate transporters accounts for ∼95% of total glutamate uptake activity in the brain (4Danbolt N.C. Prog. Neurobiol. 2001; 65: 1-105Crossref PubMed Scopus (3733) Google Scholar). Studies in mutSOD1 mice and in in vitro models of ALS affirmed a role for astroglial involvement in motorneuron death and suggested that motorneuron loss is also the result of toxic contributions of nonneuronal cells of the spinal cord (5Boillee S. Kim K. Lobsiger C.S. Copeland N.G. Jenkins N.A. Kassiotis G. Kollias G. Cleveland D.W. Science. 2006; 312: 1389-1392Crossref PubMed Scopus (1269) Google Scholar, 6Clement A.M. Kim M.D. Roberts E.A. Garcia M.L. Boillee S. Rule M. McMahon A.P. Doucette W. Siwek D. Ferrante R.J. Brown R.H. Jr. Julien J.-P. Goldstein L. S. B. Cleveland D. W. Science. 2003; 302: 113-117Crossref PubMed Scopus (902) Google Scholar, 7Di Giorgio F.P. Kim M.A. Siao M.C. Maniatis T. Eggan K. Nat. Neurosci. 2007; 10: 608-614Crossref PubMed Scopus (634) Google Scholar, 8Nagai M. Kim D.B. Nagata T. Chalazonitis A. Jessell T.M. Wichterle H. Przedborski S. Nat. Neurosci. 2007; 10: 615-622Crossref PubMed Scopus (922) Google Scholar). Reactive astrocytes surrounding motorneurons contain protein inclusions; express inflammatory markers, such as the inducible forms of nitric oxide synthase and cycloxygenase-2; and display increased neural growth factor synthesis (9Pehar M. Kim P. Vargas M.R. Castellanos R. Viera L. Beckman J.S. Estevez A.G. Barbeito L. J. Neurochem. 2004; 89: 464-473Crossref PubMed Scopus (178) Google Scholar), nitrotyrosine immunoreactivity, and down-regulation of EAAT2. Excitotoxicity caused by consistent reduction in expression levels and activity of EAAT2 is among various proposed mechanisms implicated in the propagation of motorneuron death in ALS (10Boillee S. Kim C. Cleveland D.W. Neuron. 2006; 52: 39-59Abstract Full Full PubMed Scopus Google Scholar, P. Kim R.H. Nat. Neurosci. 2006; PubMed Scopus Google Scholar). Studies of mutSOD1 models of ALS and human in EAAT2 expression levels D.S. Kim 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 (696) Google Scholar, G. Kim L. M. T. L. Rothstein J.D. Neuron. Full Full PubMed Scopus Google Scholar, J.D. Kim M. L.J. R.W. PubMed Scopus Google Scholar). in mutSOD1 mice in which the expression levels of EAAT2 that EAAT2 a role in the motor neuron loss and the of the disease H. Kim L. M.E. 2003; PubMed Scopus Google Scholar, Kim M. K. Rothstein J.D. Maragakis N.J. 2006; PubMed Scopus Google Scholar). We that caspase-3 EAAT2 at a in the COOH terminus of the a that could and of caspase-3 as mechanisms in the of ALS. In we that mutSOD1 proteins inhibit EAAT2 a mechanism that of caspase-3 and cleavage of the transporter W. Kim P. Brown R.H. Jr. D. J. 2006; Full Full PubMed Scopus Google Scholar). In this we that the fragment of EAAT2 is by small modifier of to proteins is a is to since mechanisms The of an an and a from the of Full Full PubMed Scopus Google Scholar). of a that is a The is since a and be Kim M. J. 2001; Full Full PubMed Scopus Google Scholar, Kim Chen S. J. 2004; Full Full PubMed Scopus Google Scholar). is by Kim M. A. Sci. 2003; Full Full PubMed Scopus Google Scholar). with are for functions for to ubiquitin J.M. Kim Full Full PubMed Scopus Google Scholar), of of K. Kim K. U. 2007; PubMed Scopus Google Scholar), regulation of factor activity S. Kim M. J.S. Y. A. J. Full Full PubMed Scopus Google Scholar), of S. Kim M.L. Goldstein Full Full PubMed Scopus Google Scholar, M.D. Kim K. D. M.R. Proc. Natl. Acad. Sci. U. S. A. 2007; PubMed Scopus Google Scholar), and of nuclear J. Kim M. J. 2002; PubMed Scopus Google Scholar). We that the sumoylated CTE is targeted to a as promyelocytic leukemia nuclear bodies. PML nuclear bodies are of PML protein by Kim T.M. 2006; Full Full PubMed Scopus Google Scholar). sumoylated proteins are targeted to these Full Full PubMed Scopus Google Scholar). The of functions to to be that a fragment of EAAT2 is accumulates the course of ALS, and to that it an role in the of the disease. In support of is a of protein as a mechanism implicated in a of neurodegenerative ALS Kim 2007; PubMed Scopus Google Scholar). type human SOD1 and G93A-SOD1 transgenic mice, mice, transgenic and the R6/2 mouse model of Huntington disease used for this The mouse and and and in The of the M. Kim M. M. P. Brown R.H. Jr. Y. J. Neurosci. 2001; PubMed Google Scholar). R6/2 mice from spinal from mice and in in the tissue for in in and by the tissue through a in from and from The through a cell and in 10% Astrocytes and in culture for to for and CTE of and in primary astrocytes as using CTE and CTE-SUMO-1 by and the in and an at the of the CTE and CTE-SUMO-1 cells in and at of and nuclear from cells and with the to the used for the amino of EAAT2 COOH terminus from which we termed The and EAAT2 from C. and in S. Kim Y. J.L. D. N.C. PubMed Scopus Google Scholar, S. Kim N.C. 2006; PubMed Scopus Google Scholar, N.C. Kim Y. PubMed Scopus Google Scholar). The by This is a in the mouse EAAT2 amino acid from the caspase-3 at of the mouse EAAT2 transporter The to be of the that are for this with and EAAT2 used in this and and with and with prepared for at and with and 10% in with in with and for at in also for in with and in expression by of spinal cord homogenates in and at with protein for at with the and the by the of of protein for at The with of and for in and with from using and the and The two contain a for and and the of the of EAAT2 from amino acid to for for for The and the by in with the for The cell used to analysis using an to protein of the expected in the to that the in not the by that which is an for the We used the the and the The with from a human brain The as previously J. Kim H. R. Full PubMed Scopus Google Scholar). proteins from the in in for and which the of proteins with and expression by and growth In by of and in of and with for at to and at The homogenates prepared with the termed the cleavage of of total for at in of cleavage 10% the with in the presence of human The not inhibit caspase-3 W. Kim P. Brown R.H. Jr. D. J. 2006; Full Full PubMed Scopus Google Scholar). and cleavage at for in of and by and for and by In of EAAT2 and CTE cells to and with in the presence of and of for In the EAAT2 of of to the total of to the in inhibit and by of a 10% to the of expression of EAAT2 CTE of protein from the a and the proteins by the with of the of the by with as from of the of EAAT2 in the of ALS at a than we previously that caspase-3 the glutamate transporter EAAT2 at a in W. Kim P. Brown R.H. Jr. D. J. 2006; Full Full PubMed Scopus Google Scholar). cleavage of in vitro EAAT2 two and CTE, with molecular of and W. Kim P. Brown R.H. Jr. D. J. 2006; Full Full PubMed Scopus Google Scholar) We for the presence of the in spinal cord homogenates of G93A-SOD1 mice at of disease and previously a progressive loss of EAAT2 with the which also a accumulation at of disease of a to the fragment W. Kim P. Brown R.H. Jr. D. J. 2006; Full Full PubMed Scopus Google Scholar). the homogenates with the of which is the amino of the EAAT2 COOH for the CTE we a at and in as the disease and This also by the is the CTE fragment of the caspase-3 cleavage also a in spinal cord homogenates of rats not of the by a in EAAT2 that the of the and the of EAAT2 disease We the of the fragment using this not because of the of fragment an we an We spinal cord homogenates of mice with The a at which by and not by of which the caspase-3 cleavage the terminus In to the the also that caspase-3 at in the EAAT2 COOH whereas the a in EAAT2 not the fragment at as one caspase-3 to at the the The of the fragment in vitro in spinal cord the in that the EAAT2 fragment in the spinal cord of ALS mice from caspase-3 in vitro at the and a fragment of W. Kim P. Brown R.H. Jr. D. J. 2006; Full Full PubMed Scopus Google Scholar). for the one the that in a this fragment forms an EAAT2 is at a by other that are by the caspase-3 to the cleavage the EAAT2 fragment is EAAT2 a to and D. Kim Y. 2004; PubMed Scopus Google Scholar). the at of the CTE we cells with the for the CTE fragment and the with the The analysis showed that CTE as a single at the of the of the EAAT2 the that the in an of also that the fragment from other since the caspase-3 cleavage in the presence of a of of could cleavage of EAAT2 at an cleavage by other R.W. Kim H. R. J. 2003; Full Full PubMed Scopus Google Scholar). this not be the since EAAT2 is not to cleavage by other such as W. Kim P. Brown R.H. Jr. D. J. 2006; Full Full PubMed Scopus Google Scholar) as as and that the fragment from the activity of these of the EAAT2 the in and the most for the is that CTE is by this we to the CTE fragment to ubiquitin two occur at The CTE fragment a for Kim M. A. Sci. 2003; Full Full PubMed Scopus Google Scholar), by the at the of the EAAT2 COOH terminus the CTE fragment spinal cord homogenates from stage G93A-SOD1 and mice with monoclonal and ubiquitin and and The a at in the G93A-SOD1 stage that by the that this fragment with the a by with an molecular of Furthermore, also at high molecular of the EAAT2 In in to in mice, a and G93A-SOD1 stage The proteins also to a at that the CTE fragment at not other at and high molecular and of EAAT2 at disease stage EAAT2 fragments not at presymptomatic and early not We also CTE could be by and of such not of to the CTE fragment the molecular by because at not K. Kim D. C. G. PubMed Scopus Google Scholar), the resulting molecular be this we an of of with J.S. Kim J. K. T. J.L. Science. 2004; PubMed Scopus Google Scholar). The of a CTE-SUMO-1 protein since the in EAAT2 is at the of the The of CTE and in analysis of of cells with CTE-SUMO-1 an molecular of which to the fragment to the amino the by the these suggest that CTE is rise to that at EAAT2 COOH with of the to that the of be to EAAT2. this the we a of proteins in the with other proteins in neurodegenerative for with the COOH terminus of EAAT2. The of EAAT2 to and used as in a The with and other with and whereas with and and showed with of synthesis and The and as a for a K. Kim B. P. S. Google Scholar, B. Kim S. J. PubMed Scopus Google Scholar). in the presence of analysis of a of of the EAAT2 COOH terminus with the EAAT2 in a In of EAAT2 and the CTE we for EAAT2 and CTE in cells with the CTE fragment the EAAT2 transporter with and the proteins from the cell by with analysis of the from cells a at by the and the We that EAAT2 also of sumoylated in with higher than the CTE of cells with and to of EAAT2 the of at the that the glutamate transporter EAAT2 and fragment are sumoylated the course of ALS and that this be in a model of the EAAT2 in of the in sumoylated fragment of EAAT2 in the spinal cord of G93A-SOD1 mice at higher levels with mice accumulation in the fragment accumulation to ALS, since we to the EAAT2 fragment in homogenates of areas of the R6/2 mouse model of Huntington disease and we an of the to EAAT2 also This in molecular weight of EAAT2 and spinal the that and ALS mice a in EAAT2 expression levels Kim B. A. D. L. 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Neurosci. 2006; PubMed Scopus Google Scholar). is that EAAT2 sumoylated as a result of increased stress to the presence of mutant SOD1 is by and the sumoylated CTE is to the it is to Since are the the novel not at as the disease and levels of the CTE-SUMO-1 the is such that it nuclear The role of in the is the we CTE-SUMO-1 in the the is we express the in cells to the that the CTE-SUMO-1 is an and is a with The result is accumulation of sumoylated CTE at it a of In of the of a of astrocytes the of motor in ALS and that the is to it is to that could an is that the is to for of CTE-SUMO-1 targeted to We are to C. of for providing the glutamate transporter and