Title: Alsin and SOD1G93A Proteins Regulate Endosomal Reactive Oxygen Species Production by Glial Cells and Proinflammatory Pathways Responsible for Neurotoxicity
Abstract: Recent studies have implicated enhanced Nox2-mediated reactive oxygen species (ROS) by microglia in the pathogenesis of motor neuron death observed in familial amyotrophic lateral sclerosis (ALS). In this context, ALS mutant forms of SOD1 enhance Rac1 activation, leading to increased Nox2-dependent microglial ROS production and neuron cell death in mice. It remains unclear if other genetic mutations that cause ALS also function through similar Nox-dependent pathways to enhance ROS-mediate motor neuron death. In the present study, we sought to understand whether alsin, which is mutated in an inherited juvenile form of ALS, functionally converges on Rac1-dependent pathways acted upon by SOD1G93A to regulate Nox-dependent ROS production. Our studies demonstrate that glial cell expression of SOD1G93A or wild type alsin induces ROS production, Rac1 activation, secretion of TNFα, and activation of NFκB, leading to decreased motor neuron survival in co-culture. Interestingly, coexpression of alsin, or shRNA against Nox2, with SOD1G93A in glial cells attenuated these proinflammatory indicators and protected motor neurons in co-culture, although shRNAs against Nox1 and Nox4 had little effect. SOD1G93A expression dramatically enhanced TNFα-mediated endosomal ROS in glial cells in a Rac1-dependent manner and alsin overexpression inhibited SOD1G93A-induced endosomal ROS and Rac1 activation. SOD1G93A expression enhanced recruitment of alsin to the endomembrane compartment in glial cells, suggesting that these two proteins act to modulate Nox2-dependent endosomal ROS and proinflammatory signals that modulate NFκB. These studies suggest that glial proinflammatory signals regulated by endosomal ROS are influenced by two gene products known to cause ALS. Recent studies have implicated enhanced Nox2-mediated reactive oxygen species (ROS) by microglia in the pathogenesis of motor neuron death observed in familial amyotrophic lateral sclerosis (ALS). In this context, ALS mutant forms of SOD1 enhance Rac1 activation, leading to increased Nox2-dependent microglial ROS production and neuron cell death in mice. It remains unclear if other genetic mutations that cause ALS also function through similar Nox-dependent pathways to enhance ROS-mediate motor neuron death. In the present study, we sought to understand whether alsin, which is mutated in an inherited juvenile form of ALS, functionally converges on Rac1-dependent pathways acted upon by SOD1G93A to regulate Nox-dependent ROS production. Our studies demonstrate that glial cell expression of SOD1G93A or wild type alsin induces ROS production, Rac1 activation, secretion of TNFα, and activation of NFκB, leading to decreased motor neuron survival in co-culture. Interestingly, coexpression of alsin, or shRNA against Nox2, with SOD1G93A in glial cells attenuated these proinflammatory indicators and protected motor neurons in co-culture, although shRNAs against Nox1 and Nox4 had little effect. SOD1G93A expression dramatically enhanced TNFα-mediated endosomal ROS in glial cells in a Rac1-dependent manner and alsin overexpression inhibited SOD1G93A-induced endosomal ROS and Rac1 activation. SOD1G93A expression enhanced recruitment of alsin to the endomembrane compartment in glial cells, suggesting that these two proteins act to modulate Nox2-dependent endosomal ROS and proinflammatory signals that modulate NFκB. These studies suggest that glial proinflammatory signals regulated by endosomal ROS are influenced by two gene products known to cause ALS. IntroductionAmyotrophic lateral sclerosis (ALS) is a lethal degenerative neurological disorder characterized by progressive degeneration of motor neurons in the brain and spinal cord (1Ilieva H. Polymenidou M. Cleveland D.W. J. Cell Biol. 2009; 187: 761-772Crossref PubMed Scopus (777) Google Scholar, 2Goodall E.F. Morrison K.E. Expert Rev. Mol. Med. 2006; 8: 1-22Crossref PubMed Google Scholar). The majority of ALS patients have onset of disease between 40 and 50 years of age and about 50% of patients die within 3 years. The majority of ALS cases are categorized as sporadic with no family history of disease. In this context, the causative genes and environmental factors that initiate the disease process remain poorly defined. Only ∼10% of ALS cases have a clearly inherited genetic component and hence are classified as familial ALS (1Ilieva H. Polymenidou M. Cleveland D.W. J. Cell Biol. 2009; 187: 761-772Crossref PubMed Scopus (777) Google Scholar, 2Goodall E.F. Morrison K.E. Expert Rev. Mol. Med. 2006; 8: 1-22Crossref PubMed Google Scholar).The best-characterized forms of familial ALS include those caused by mutations in the gene encoding Cu/Zn-superoxide dismutase (SOD1) 2The abbreviations used are: SODsuperoxide dismutaseROSreactive oxygen speciesH2HFFdihydro-2′,4,5,6,7,7′-hexafluorofluoresceinDHEdihydroethidiumhESChuman embryonic stem cells. (3Rosen D.R. Siddique T. Patterson D. Figlewicz D.A. Sapp P. Hentati A. Donaldson D. Goto J. O'Regan J.P. Deng H.X. et al.Nature. 1993; 362: 59-62Crossref PubMed Scopus (5442) Google Scholar). Approximately 20% of familial ALS cases are caused by a variety of dominant SOD1 mutations (1Ilieva H. Polymenidou M. Cleveland D.W. J. Cell Biol. 2009; 187: 761-772Crossref PubMed Scopus (777) Google Scholar, 3Rosen D.R. Siddique T. Patterson D. Figlewicz D.A. Sapp P. Hentati A. Donaldson D. Goto J. O'Regan J.P. Deng H.X. et al.Nature. 1993; 362: 59-62Crossref PubMed Scopus (5442) Google Scholar). There remains great uncertainty as to the primary mechanism(s) by which mutant SOD1 leads to pathology observed in ALS (1Ilieva H. Polymenidou M. Cleveland D.W. J. Cell Biol. 2009; 187: 761-772Crossref PubMed Scopus (777) Google Scholar, 4Glass C.K. Saijo K. Winner B. Marchetto M.C. Gage F.H. Cell. 2010; 140: 918-934Abstract Full Text Full Text PDF PubMed Scopus (2392) Google Scholar). Proposed mechanisms include toxicity associated with misfolding of mutant SOD1, such as ER stress and inhibition of the proteasome, enhanced proinflammatory ROS production, altered axonal transport, excitotoxicity caused by glutamate mishandling, and mitochondrial damage (1Ilieva H. Polymenidou M. Cleveland D.W. J. Cell Biol. 2009; 187: 761-772Crossref PubMed Scopus (777) Google Scholar, 4Glass C.K. Saijo K. Winner B. Marchetto M.C. Gage F.H. Cell. 2010; 140: 918-934Abstract Full Text Full Text PDF PubMed Scopus (2392) Google Scholar). Relevant to the studies in this report are findings demonstrating that SOD1 mutations induce NADPH oxidase-dependent ROS production in microglia of SOD1G93A mice leading to motor neuron death (5Marden J.J. Harraz M.M. Williams A.J. Nelson K. Luo M. Paulson H. Engelhardt J.F. J. Clin. Invest. 2007; 117: 2913-2919Crossref PubMed Scopus (114) Google Scholar, 6Wu D.C. Ré D.B. Nagai M. Ischiropoulos H. Przedborski S. Proc. Natl. Acad. Sci. U.S.A. 2006; 103: 12132-12137Crossref PubMed Scopus (200) Google Scholar). These studies have demonstrated that deletion of the Nox2 isoform of NADPH oxidase, and to a lesser extent also the Nox1 isoform, can prolong survival in SOD1G93A transgenic mice. The importance of SOD1 in regulating cellular ROS production was first revealed by studies demonstrating that SOD1 can directly associate with endosomal Rac1 to regulate its activity (7Harraz M.M. Marden J.J. Zhou W. Zhang Y. Williams A. Sharov V.S. Nelson K. Luo M. Paulson H. Schöneich C. Engelhardt J.F. J. Clin. Invest. 2008; 118: 659-670PubMed Google Scholar). Rac1 is an essential activator of several NADPH oxidases and SOD1 binding to Rac1 slows the hydrolysis of GTP bound to Rac1 in a redox-dependent manner. Thus, SOD1 association with Rac1 is proposed to be a redox-dependent sensor for regulating redox-active NADPH oxidase containing endosomes (called redoxosomes) (7Harraz M.M. Marden J.J. Zhou W. Zhang Y. Williams A. Sharov V.S. Nelson K. Luo M. Paulson H. Schöneich C. Engelhardt J.F. J. Clin. Invest. 2008; 118: 659-670PubMed Google Scholar, 8Oakley F.D. Abbott D. Li Q. Engelhardt J.F. Antioxid. Redox Signal. 2009; 11: 1313-1333Crossref PubMed Scopus (149) Google Scholar). Redoxosomes are important signaling endosomes that regulate proinflammatory receptor signals in a redox-dependent manner through NADPH oxidases (8Oakley F.D. Abbott D. Li Q. Engelhardt J.F. Antioxid. Redox Signal. 2009; 11: 1313-1333Crossref PubMed Scopus (149) Google Scholar, 9Li Q. Zhang Y. Marden J.J. Banfi B. Engelhardt J.F. Biochem. J. 2008; 411: 531-541Crossref PubMed Scopus (47) Google Scholar, 10Li Q. Spencer N.Y. Oakley F.D. Buettner G.R. Engelhardt J.F. Antioxid. Redox Signal. 2009; 11: 1249-1263Crossref PubMed Scopus (92) Google Scholar, 11Li Q. Harraz M.M. Zhou W. Zhang L.N. Ding W. Zhang Y. Eggleston T. Yeaman C. Banfi B. Engelhardt J.F. Mol. Cell. Biol. 2006; 26: 140-154Crossref PubMed Scopus (189) Google Scholar). Redoxosomes are a subpopulation of early endosomes that produce ROS and have been shown to contain early endosomal markers (Rab5 and EEA1), redox effectors (Nox2 or Nox1, Rac1, p47phox, p67phox, and SOD1), and certainly ligand-activated cytokine receptors (TNFR1 or IL-1R1) (8Oakley F.D. Abbott D. Li Q. Engelhardt J.F. Antioxid. Redox Signal. 2009; 11: 1313-1333Crossref PubMed Scopus (149) Google Scholar, 9Li Q. Zhang Y. Marden J.J. Banfi B. Engelhardt J.F. Biochem. J. 2008; 411: 531-541Crossref PubMed Scopus (47) Google Scholar, 10Li Q. Spencer N.Y. Oakley F.D. Buettner G.R. Engelhardt J.F. Antioxid. Redox Signal. 2009; 11: 1249-1263Crossref PubMed Scopus (92) Google Scholar, 11Li Q. Harraz M.M. Zhou W. Zhang L.N. Ding W. Zhang Y. Eggleston T. Yeaman C. Banfi B. Engelhardt J.F. Mol. Cell. Biol. 2006; 26: 140-154Crossref PubMed Scopus (189) Google Scholar). ROS produced by the redoxosome facilitate the redox-dependent recruitment of TRAFs to TNFR1 and IL-1R1 and in this manner facilitate proinflammatory signaling such as NFκB activation (8Oakley F.D. Abbott D. Li Q. Engelhardt J.F. Antioxid. Redox Signal. 2009; 11: 1313-1333Crossref PubMed Scopus (149) Google Scholar, 9Li Q. Zhang Y. Marden J.J. Banfi B. Engelhardt J.F. Biochem. J. 2008; 411: 531-541Crossref PubMed Scopus (47) Google Scholar, 10Li Q. Spencer N.Y. Oakley F.D. Buettner G.R. Engelhardt J.F. Antioxid. Redox Signal. 2009; 11: 1249-1263Crossref PubMed Scopus (92) Google Scholar, 11Li Q. Harraz M.M. Zhou W. Zhang L.N. Ding W. Zhang Y. Eggleston T. Yeaman C. Banfi B. Engelhardt J.F. Mol. Cell. Biol. 2006; 26: 140-154Crossref PubMed Scopus (189) Google Scholar). Interestingly, mutant forms of SOD1 have enhanced redox-independent binding to Rac1 and this has been proposed as a mechanism for enhanced ROS production in microglia of ALS mice (7Harraz M.M. Marden J.J. Zhou W. Zhang Y. Williams A. Sharov V.S. Nelson K. Luo M. Paulson H. Schöneich C. Engelhardt J.F. J. Clin. Invest. 2008; 118: 659-670PubMed Google Scholar, 12Carter B.J. Anklesaria P. Choi S. Engelhardt J.F. Antioxid. Redox Signal. 2009; 11: 1569-1586Crossref PubMed Scopus (30) Google Scholar).Seven known NADPH oxidase catalytic subunits exist (Nox1, Nox2gp91phox, Nox3, Nox4, Nox5, Duox1, and Duox2) (13Lambeth J.D. Nat. Rev. Immunol. 2004; 4: 181-189Crossref PubMed Scopus (2425) Google Scholar, 14Hordijk P.L. Circ. Res. 2006; 98: 453-462Crossref PubMed Scopus (426) Google Scholar). The most widely characterized of these is phagocytic gp91phox (also known as Nox2). Nox2 is also expressed in microglia (6Wu D.C. Ré D.B. Nagai M. Ischiropoulos H. Przedborski S. Proc. Natl. Acad. Sci. U.S.A. 2006; 103: 12132-12137Crossref PubMed Scopus (200) Google Scholar) and a variety of other nonphagocytic cell types. Rac, a small GTPase, is an essential activator of Nox1 and -2, and along with several other subunits (p22phox, p40phox, p47phox, p67phox, NoxO1, and NoxA1) can act to promote Nox complex activation in a cell type-specific fashion (13Lambeth J.D. Nat. Rev. Immunol. 2004; 4: 181-189Crossref PubMed Scopus (2425) Google Scholar, 14Hordijk P.L. Circ. Res. 2006; 98: 453-462Crossref PubMed Scopus (426) Google Scholar). In certain cell systems, Rac1 has also been shown to be required for ROS production by Nox4 (15Gorin Y. Ricono J.M. Kim N.H. Bhandari B. Choudhury G.G. Abboud H.E. Am. J. Physiol. Renal Physiol. 2003; 285: F219-229Crossref PubMed Scopus (241) Google Scholar, 16Lee S. Gharavi N.M. Honda H. Chang I. Kim B. Jen N. Li R. Zimman A. Berliner J.A. Free Radic. Biol. Med. 2009; 47: 145-151Crossref PubMed Scopus (58) Google Scholar, 17Meng D. Lv D.D. Fang J. Cardiovasc. Res. 2008; 80: 299-308Crossref PubMed Scopus (118) Google Scholar). Because NADPH oxidases (Nox) generate the O2̇̄ substrate of the SOD1 dismutation reaction (2O2̇̄ + 2H+ → H2O2 + O2), this class of Nox enzymes has recently generated considerable interest in studies of ALS.It is presently unclear if familial and sporadic forms of ALS have common or overlapping molecular mechanisms of disease pathogenesis. A recent genomewide association study in sporadic ALS patients has begun to shed light on this topic (18Dunckley T. Huentelman M.J. Craig D.W. Pearson J.V. Szelinger S. Joshipura K. Halperin R.F. Stamper C. Jensen K.R. Letizia D. Hesterlee S.E. Pestronk A. Levine T. Bertorini T. Graves M.C. Mozaffar T. Jackson C.E. Bosch P. McVey A. Dick A. Barohn R. Lomen-Hoerth C. Rosenfeld J. O'connor D.T. Zhang K. Crook R. Ryberg H. Hutton M. Katz J. Simpson E.P. Mitsumoto H. Bowser R. Miller R.G. Appel S.H. Stephan D.A. N. Engl. J. Med. 2007; 357: 775-788Crossref PubMed Scopus (210) Google Scholar). Several genes that regulate endosomal trafficking, Rac1, and NADPH oxidases were identified in this study, including Nox4, TIAM2, IQGAP2, PTPRT, RAP1GAP, and MAGI2 (12Carter B.J. Anklesaria P. Choi S. Engelhardt J.F. Antioxid. Redox Signal. 2009; 11: 1569-1586Crossref PubMed Scopus (30) Google Scholar). Interestingly, the ALS2 gene product alsin, which when mutated causes juvenile ALS, has also been shown to influence endosomal trafficking and Rac1 activity (19Chandran J. Ding J. Cai H. Mol. Neurobiol. 2007; 36: 224-231Crossref PubMed Scopus (39) Google Scholar, 20Hadano S. Otomo A. Kunita R. Suzuki-Utsunomiya K. Akatsuka A. Koike M. Aoki M. Uchiyama Y. Itoyama Y. Ikeda J.E. PLoS One. 2010; 5: e9805Crossref PubMed Scopus (100) Google Scholar, 21Hadano S. Kunita R. Otomo A. Suzuki-Utsunomiya K. Ikeda J.E. Neurochem. Int. 2007; 51: 74-84Crossref PubMed Scopus (66) Google Scholar, 22Yang Y. Hentati A. Deng H.X. Dabbagh O. Sasaki T. Hirano M. Hung W.Y. Ouahchi K. Yan J. Azim A.C. Cole N. Gascon G. Yagmour A. Ben-Hamida M. Pericak-Vance M. Hentati F. Siddique T. Nat. Genet. 2001; 29: 160-165Crossref PubMed Scopus (658) Google Scholar, 23Hadano S. Hand C.K. Osuga H. Yanagisawa Y. Otomo A. Devon R.S. Miyamoto N. Showguchi-Miyata J. Okada Y. Singaraja R. Figlewicz D.A. Kwiatkowski T. Hosler B.A. Sagie T. Skaug J. Nasir J. Brown Jr., R.H. Scherer S.W. Rouleau G.A. Hayden M.R. Ikeda J.E. Nat. Genet. 2001; 29: 166-173Crossref PubMed Scopus (585) Google Scholar). Alsin appears to serve as a GEF for Rab5 and an effector of Rac1 GTPase activity (24Otomo A. Hadano S. Okada T. Mizumura H. Kunita R. Nishijima H. Showguchi-Miyata J. Yanagisawa Y. Kohiki E. Suga E. Yasuda M. Osuga H. Nishimoto T. Narumiya S. Ikeda J.E. Hum. Mol. Genet. 2003; 12: 1671-1687Crossref PubMed Scopus (212) Google Scholar, 25Topp J.D. Gray N.W. Gerard R.D. Horazdovsky B.F. J. Biol. Chem. 2004; 279: 24612-24623Abstract Full Text Full Text PDF PubMed Scopus (163) Google Scholar, 26Kunita R. Otomo A. Mizumura H. Suzuki-Utsunomiya K. Hadano S. Ikeda J.E. J. Biol. Chem. 2007; 282: 16599-16611Abstract Full Text Full Text PDF PubMed Scopus (64) Google Scholar). These findings are of considerable interest because SOD1 also regulates Rac1 GTPase and NADPH oxidase activity in Rab5-bound early endosomes (7Harraz M.M. Marden J.J. Zhou W. Zhang Y. Williams A. Sharov V.S. Nelson K. Luo M. Paulson H. Schöneich C. Engelhardt J.F. J. Clin. Invest. 2008; 118: 659-670PubMed Google Scholar, 8Oakley F.D. Abbott D. Li Q. Engelhardt J.F. Antioxid. Redox Signal. 2009; 11: 1313-1333Crossref PubMed Scopus (149) Google Scholar, 11Li Q. Harraz M.M. Zhou W. Zhang L.N. Ding W. Zhang Y. Eggleston T. Yeaman C. Banfi B. Engelhardt J.F. Mol. Cell. Biol. 2006; 26: 140-154Crossref PubMed Scopus (189) Google Scholar, 12Carter B.J. Anklesaria P. Choi S. Engelhardt J.F. Antioxid. Redox Signal. 2009; 11: 1569-1586Crossref PubMed Scopus (30) Google Scholar). Both the Rab5-GEF and Rac1-effector functions of alsin appear to influence endocytic mechanisms and endosomal dynamics (20Hadano S. Otomo A. Kunita R. Suzuki-Utsunomiya K. Akatsuka A. Koike M. Aoki M. Uchiyama Y. Itoyama Y. Ikeda J.E. PLoS One. 2010; 5: e9805Crossref PubMed Scopus (100) Google Scholar, 26Kunita R. Otomo A. Mizumura H. Suzuki-Utsunomiya K. Hadano S. Ikeda J.E. J. Biol. Chem. 2007; 282: 16599-16611Abstract Full Text Full Text PDF PubMed Scopus (64) Google Scholar) and alsin appears to protect from motor neuron degeneration in certain SOD1 mutant mice (20Hadano S. Otomo A. Kunita R. Suzuki-Utsunomiya K. Akatsuka A. Koike M. Aoki M. Uchiyama Y. Itoyama Y. Ikeda J.E. PLoS One. 2010; 5: e9805Crossref PubMed Scopus (100) Google Scholar) and motor neurons expressing SOD1 mutants in culture (27Kanekura K. Hashimoto Y. Niikura T. Aiso S. Matsuoka M. Nishimoto I. J. Biol. Chem. 2004; 279: 19247-19256Abstract Full Text Full Text PDF PubMed Scopus (78) Google Scholar, 28Jacquier A. Buhler E. Schäfer M.K. Bohl D. Blanchard S. Beclin C. Haase G. Ann. Neurol. 2006; 60: 105-117Crossref PubMed Scopus (59) Google Scholar). Given the association of Nox1, Nox2, and Nox4 with disease progression in ALS mice (5Marden J.J. Harraz M.M. Williams A.J. Nelson K. Luo M. Paulson H. Engelhardt J.F. J. Clin. Invest. 2007; 117: 2913-2919Crossref PubMed Scopus (114) Google Scholar, 6Wu D.C. Ré D.B. Nagai M. Ischiropoulos H. Przedborski S. Proc. Natl. Acad. Sci. U.S.A. 2006; 103: 12132-12137Crossref PubMed Scopus (200) Google Scholar) and humans (18Dunckley T. Huentelman M.J. Craig D.W. Pearson J.V. Szelinger S. Joshipura K. Halperin R.F. Stamper C. Jensen K.R. Letizia D. Hesterlee S.E. Pestronk A. Levine T. Bertorini T. Graves M.C. Mozaffar T. Jackson C.E. Bosch P. McVey A. Dick A. Barohn R. Lomen-Hoerth C. Rosenfeld J. O'connor D.T. Zhang K. Crook R. Ryberg H. Hutton M. Katz J. Simpson E.P. Mitsumoto H. Bowser R. Miller R.G. Appel S.H. Stephan D.A. N. Engl. J. Med. 2007; 357: 775-788Crossref PubMed Scopus (210) Google Scholar), these findings suggest the intriguing hypothesis that alsin and SOD1 both influence the dynamics of Rac1-dependent, NADPH oxidase-mediated, ROS production by redoxosomes that may impact proinflammatory signaling in ALS. In support of this hypothesis, alsin has been shown to bind three components of the redoxosome (Rac1, Rab5, and SOD1).To test this hypothesis, we investigated whether alsin expression influences SOD1G93A-mediated ROS production by glial cells. Three NADPH oxidases were evaluated as sources of cellular ROS (Nox1, -2, and -4) using shRNA knockdown, based on their association with disease severity in ALS models. Findings from our studies demonstrated that wild type alsin attenuates SOD1G93A-mediated Rac1 activation, ROS production by Nox2, NFκB activation, and TNFα secretion by glial cells and protects neurons from toxicity in co-culture studies. SOD1G93A expression enhanced TNFα-dependent redoxosomal ROS production by Nox2 and this was attenuated by alsin expression. Taken together, our results suggest a potential role for alsin in regulating redox-dependent proinflammatory signals via redoxosomes that are enhanced by SOD1G93A.DISCUSSIONEnhanced ROS production has been proposed to be an important pathophysiologic component for several types of neuronal degenerative diseases including ALS (4Glass C.K. Saijo K. Winner B. Marchetto M.C. Gage F.H. Cell. 2010; 140: 918-934Abstract Full Text Full Text PDF PubMed Scopus (2392) Google Scholar). This is perhaps not surprising because most neuronal degenerative diseases have significant components of inflammation. The challenge in understanding the importance of enhanced ROS production in ALS has been to determine whether primary defects in ROS regulation incite pathophysiologic events responsible for disease progression. The first report to implicate Nox2 as a source of altered ROS production in a FALS (SOD1G93A) mouse model, described by Wu and colleagues (6Wu D.C. Ré D.B. Nagai M. Ischiropoulos H. Przedborski S. Proc. Natl. Acad. Sci. U.S.A. 2006; 103: 12132-12137Crossref PubMed Scopus (200) Google Scholar), was later substantiated by others (5Marden J.J. Harraz M.M. Williams A.J. Nelson K. Luo M. Paulson H. Engelhardt J.F. J. Clin. Invest. 2007; 117: 2913-2919Crossref PubMed Scopus (114) Google Scholar). Additionally, work using hESC differentiated into astrocytes demonstrated that expression of SOD1G37R-induced Nox2 protein production and ROS-mediated killing of hESC-derived motor neurons in co-culture (36Marchetto M.C. Muotri A.R. Mu Y. Smith A.M. Cezar G.G. Gage F.H. Cell Stem Cell. 2008; 3: 649-657Abstract Full Text Full Text PDF PubMed Scopus (350) Google Scholar). Importantly, inhibition of Nox2 with apocynin rescued motor neuron survival in the presence of SOD1G37R-expressing astrocytes (36Marchetto M.C. Muotri A.R. Mu Y. Smith A.M. Cezar G.G. Gage F.H. Cell Stem Cell. 2008; 3: 649-657Abstract Full Text Full Text PDF PubMed Scopus (350) Google Scholar). These in vitro studies by Marchetto and colleagues (36Marchetto M.C. Muotri A.R. Mu Y. Smith A.M. Cezar G.G. Gage F.H. Cell Stem Cell. 2008; 3: 649-657Abstract Full Text Full Text PDF PubMed Scopus (350) Google Scholar) were key to demonstrating that Nox2 plays an important intrinsic role within FALS astrocytes to cause paracrine killing of motor neurons. Together with the finding that SOD1 ALS mutants can also directly regulate Rac1 and Nox2-dependent ROS production, these findings point to dysregulation of NADPH oxidases as an important potential mechanism in the pathology observed in FALS.Studies have also demonstrated that Nox2 is up-regulated in spinal cord microglia of sporadic ALS patients (6Wu D.C. Ré D.B. Nagai M. Ischiropoulos H. Przedborski S. Proc. Natl. Acad. Sci. U.S.A. 2006; 103: 12132-12137Crossref PubMed Scopus (200) Google Scholar), suggesting that the mechanisms of enhanced ROS production through NADPH oxidases may not be limited to SOD1 mutations found in FALS. The potential involvement of NADPH oxidases in FALS disease progression is supported by genomewide association studies demonstrating that Nox4 and several Nox regulators are closely linked to the disease (12Carter B.J. Anklesaria P. Choi S. Engelhardt J.F. Antioxid. Redox Signal. 2009; 11: 1569-1586Crossref PubMed Scopus (30) Google Scholar, 18Dunckley T. Huentelman M.J. Craig D.W. Pearson J.V. Szelinger S. Joshipura K. Halperin R.F. Stamper C. Jensen K.R. Letizia D. Hesterlee S.E. Pestronk A. Levine T. Bertorini T. Graves M.C. Mozaffar T. Jackson C.E. Bosch P. McVey A. Dick A. Barohn R. Lomen-Hoerth C. Rosenfeld J. O'connor D.T. Zhang K. Crook R. Ryberg H. Hutton M. Katz J. Simpson E.P. Mitsumoto H. Bowser R. Miller R.G. Appel S.H. Stephan D.A. N. Engl. J. Med. 2007; 357: 775-788Crossref PubMed Scopus (210) Google Scholar). Furthermore, studies on the juvenile form of ALS caused by mutations in the ALS2 gene encoding for alsin (22Yang Y. Hentati A. Deng H.X. Dabbagh O. Sasaki T. Hirano M. Hung W.Y. Ouahchi K. Yan J. Azim A.C. Cole N. Gascon G. Yagmour A. Ben-Hamida M. Pericak-Vance M. Hentati F. Siddique T. Nat. Genet. 2001; 29: 160-165Crossref PubMed Scopus (658) Google Scholar) have demonstrated that alsin regulates Rac1 activity (21Hadano S. Kunita R. Otomo A. Suzuki-Utsunomiya K. Ikeda J.E. Neurochem. Int. 2007; 51: 74-84Crossref PubMed Scopus (66) Google Scholar, 25Topp J.D. Gray N.W. Gerard R.D. Horazdovsky B.F. J. Biol. Chem. 2004; 279: 24612-24623Abstract Full Text Full Text PDF PubMed Scopus (163) Google Scholar, 26Kunita R. Otomo A. Mizumura H. Suzuki-Utsunomiya K. Hadano S. Ikeda J.E. J. Biol. Chem. 2007; 282: 16599-16611Abstract Full Text Full Text PDF PubMed Scopus (64) Google Scholar) and thus also has the potential to regulate NADPH oxidases that require Rac1 (i.e. Nox1, -2, and -4). These findings suggest that multiple independent ALS disease-causing and disease-associated genes may converge on regulatory pathways that influence NADPH oxidase-dependent ROS production. In the present study, we sought to determine whether both SOD1G93A and alsin may both regulate the activity of NADPH oxidases in glial cells and the effect of this regulation on neuronal survival.As previously reported for SOD1G37R-expressing astrocytes (36Marchetto M.C. Muotri A.R. Mu Y. Smith A.M. Cezar G.G. Gage F.H. Cell Stem Cell. 2008; 3: 649-657Abstract Full Text Full Text PDF PubMed Scopus (350) Google Scholar), our studies demonstrate that SOD1G93A-expressing glial cells hyperactivate Rac1- and Nox2-dependent ROS production leading to an enhanced proinflammatory state (TNFα and NFκB) and toxicity of a motor neuron-like cell line in co-culture. Nox1 and Nox4 appeared not to play a major role in these processes. The ability of SOD1G93A to activate Rac1 is consistent with previous studies demonstrating that SOD1 binds Rac1-GTP and reduces the rate of GTP hydrolysis and that mutant forms of SOD1 have a higher affinity for Rac1-GTP (7Harraz M.M. Marden J.J. Zhou W. Zhang Y. Williams A. Sharov V.S. Nelson K. Luo M. Paulson H. Schöneich C. Engelhardt J.F. J. Clin. Invest. 2008; 118: 659-670PubMed Google Scholar). Interestingly, expression of full-length wild type alsin together with SOD1G93A in glial cells led to protection of neuronal cells in co-culture by decreasing proinflammatory activation of glial cells (i.e. Rac1 activation, Nox2-dependent ROS production, TNFα production, and NFκB activation). These findings suggest that alsin plays a protective role and is consistent with reports demonstrating alsin knockdown induces motor neuron cell death (20Hadano S. Otomo A. Kunita R. Suzuki-Utsunomiya K. Akatsuka A. Koike M. Aoki M. Uchiyama Y. Itoyama Y. Ikeda J.E. PLoS One. 2010; 5: e9805Crossref PubMed Scopus (100) Google Scholar, 28Jacquier A. Buhler E. Schäfer M.K. Bohl D. Blanchard S. Beclin C. Haase G. Ann. Neurol. 2006; 60: 105-117Crossref PubMed Scopus (59) Google Scholar) and that expression of full-length alsin protects against neurotoxicity caused by SOD1 mutations (27Kanekura K. Hashimoto Y. Niikura T. Aiso S. Matsuoka M. Nishimoto I. J. Biol. Chem. 2004; 279: 19247-19256Abstract Full Text Full Text PDF PubMed Scopus (78) Google Scholar). Interestingly the RhoGEF domain of alsin was required for this protective effect (27Kanekura K. Hashimoto Y. Niikura T. Aiso S. Matsuoka M. Nishimoto I. J. Biol. Chem. 2004; 279: 19247-19256Abstract Full Text Full Text PDF PubMed Scopus (78) Google Scholar), a finding consistent with alsin mediating protection through the Rho-GTPase Rac1.Our studies demonstrate for the first time that alsin can attenuate proinflammatory pathways regulated by Nox2 in SOD1G93A-expressing glial cells and that this effect in turn influences survival of motor neuron-like cells. Previous studies have focused on the function of alsin in motor neurons and have demonstrated similar protective effects by alsin overexpression on SOD1G93A-associated toxicity (27Kanekura K. Hashimoto Y. Niikura T. Aiso S. Matsuoka M. Nishimoto I. J. Biol. Chem. 2004; 279: 19247-19256Abstract Full Text Full Text PDF PubMed Scopus (78) Google Scholar). The mechanism of alsin-mediated protection of motor neurons remains unclear, but previous studies have suggested that protection may be mediated through alsin binding to mutant SOD1 (27Kanekura K. Hashimoto Y. Niikura T. Aiso S. Matsuoka M. Nishimoto I. J. Biol. Chem. 2004; 279: 19247-19256Abstract Full Text Full Text PDF PubMed Scopus (78) Google Scholar) and/or through Rac1 modulation (28Jacquier A. Buhler E. Schäfer M.K. Bohl D. Blanchard S. Beclin C. Haase G. Ann. Neurol. 2006; 60: 105-117Crossref PubMed Scopus (59) Google Scholar). Our studies, demonstrating for the first time protective functions of alsin in glial cells, add to this growing body of literature. Interestingly, however, our studies also demonstrate that overexpression of alsin alone in glial cells (in the absence of SOD1G93A) also drives similar amplification of proinflammatory pathways as seen following expression of SOD1G93A alone (alsin overexpression induced Rac1, glial cell ROS, TNFα production, NFκB activation, and toxicity to motor neuron-like cells in co-culture). Thus, our findings suggest that alsin is not simply a protective modulator of SOD1G93A toxicity in glial cells, but rather that both alsin and SOD1 can act to directly regulate proinflammatory signals by glial cells.Alsin is known to localize to the endosomal compartment where it can serve as a GEF for Rab5, an early endosomal effector GTPase (19Chandran J. Ding J. Cai H. Mol. Neurobiol. 2007; 36: 224-231Crossref PubMed Scopus (39) Google Scholar, 25Topp J.D. Gray N.W. Gerard R.D. Horazdovsky B.F. J. Biol. Chem. 2004; 279: 24612-24623Abstract Full Text Full Text PDF PubMed Scopus (163) Google Scholar, 28Jacquier A. Buhler E. Schäfer M.K. Bohl D. Blanchard S. Beclin C. Haase G. Ann. Neurol. 2006; 60: 105-117Crossref PubMed Scopus (59) Google Scholar). Alsin is also an effector of Rac1 that has been shown to control endocytic mechanisms at the cell membrane (26Kunita R. Otomo A. Mizumura H. Suzuki-Utsunomiya K. Hadano S. Ikeda J.E. J. Biol. Chem. 2007; 282: 16599-16611Abstract Full Text Full Text PDF PubMed Scopus (64) Google Scholar, 52Otomo A. Kunita R. Suzuki-Utsunomiya K. Mizumura H. Onoe K. Osuga H. Hadano S. Ikeda J.E. Biochem. Biophys. Res. Commun. 2008; 370: 87-92Crossref PubMed Scopus (34) Google Scholar) and endolysosomal trafficking (20Hadano S. Otomo A. Kunita R. Suzuki-Utsunomiya K. Akatsuka A. Koike M. Aoki M. Uchiyama Y. Itoyama Y. Ikeda J.E. PLoS One. 2010; 5: e9805Crossref PubMed Scopus (100) Google Scholar). Thus, alsin is a key regulator of endosomal dynamics. We hypothesized that alsin may regulate proinflammatory pathways through redox-active signaling endosomes (i.e. redoxosomes) known to facilitate redox-mediated activation of proinflammatory receptors such as TNFR and IL-1R (8Oakley F.D. Abbott D. Li Q. Engelhardt J.F. Antioxid. Redox Signal. 2009; 11: 1313-1333Crossref PubMed Scopus (149) Google Scholar, 10Li Q. Spencer N.Y. Oakley F.D. Buettner G.R. Engelhardt J.F. Antioxid. Redox Signal. 2009; 11: 1249-1263Crossref PubMed Scopus (92) Google Scholar, 11Li Q. Harraz M.M. Zhou W. Zhang L.N. Ding W. Zhang Y. Eggleston T. Yeaman C. Banfi B. Engelhardt J.F. Mol. Cell. Biol. 2006; 26: 140-154Crossref PubMed Scopus (189) Google Scholar). Our findings demonstrating that alsin recruitment to endomembranes is enhanced by SOD1G93A expression supports its potential function on redoxosomes. The recruitment of SOD1 to redoxosomes, following TNFα or IL-1β stimulation, is important for controlling Rac1-dependent NADPH oxidase activation, redox-dependent TRAF recruitment to receptor complexes, and ultimately activation of NFκB (8Oakley F.D. Abbott D. Li Q. Engelhardt J.F. Antioxid. Redox Signal. 2009; 11: 1313-1333Crossref PubMed Scopus (149) Google Scholar). Our novel findings that SOD1G93A expression induces redoxosomal ROS production by TNFα, and that this ROS is attenuated by alsin overexpression, provide a mechanistic link for anti-inflammatory effects of alsin in the presence of SOD1G93A. Thus, our findings are consistent with alsin being a modulator of proinflammatory Nox2-dependent redoxosomal activation.In summary, our studies demonstrate that SOD1G93A expression in glial cells modulates proinflammatory signaling through redoxosomes in a Nox2-dependent fashion. This elevated proinflammatory state in turn leads to secreted factors that are toxic to neurons. Alsin appears to directly attenuate glial cell-dependent neurotoxicity by reducing Nox2-mediated signaling by redoxosomes. This protective effect appears to be mediated by the ability of alsin to decrease Rac1 activation in the presence of SOD1G93A. Such findings provide insights into potential common regulatory pathways controlled by NADPH oxidases that influence proinflammatory signaling and neurotoxicity in two independent genetic forms of ALS. IntroductionAmyotrophic lateral sclerosis (ALS) is a lethal degenerative neurological disorder characterized by progressive degeneration of motor neurons in the brain and spinal cord (1Ilieva H. Polymenidou M. Cleveland D.W. J. Cell Biol. 2009; 187: 761-772Crossref PubMed Scopus (777) Google Scholar, 2Goodall E.F. Morrison K.E. Expert Rev. Mol. Med. 2006; 8: 1-22Crossref PubMed Google Scholar). The majority of ALS patients have onset of disease between 40 and 50 years of age and about 50% of patients die within 3 years. The majority of ALS cases are categorized as sporadic with no family history of disease. In this context, the causative genes and environmental factors that initiate the disease process remain poorly defined. Only ∼10% of ALS cases have a clearly inherited genetic component and hence are classified as familial ALS (1Ilieva H. Polymenidou M. Cleveland D.W. J. Cell Biol. 2009; 187: 761-772Crossref PubMed Scopus (777) Google Scholar, 2Goodall E.F. Morrison K.E. Expert Rev. Mol. Med. 2006; 8: 1-22Crossref PubMed Google Scholar).The best-characterized forms of familial ALS include those caused by mutations in the gene encoding Cu/Zn-superoxide dismutase (SOD1) 2The abbreviations used are: SODsuperoxide dismutaseROSreactive oxygen speciesH2HFFdihydro-2′,4,5,6,7,7′-hexafluorofluoresceinDHEdihydroethidiumhESChuman embryonic stem cells. (3Rosen D.R. Siddique T. Patterson D. Figlewicz D.A. Sapp P. Hentati A. Donaldson D. Goto J. O'Regan J.P. Deng H.X. et al.Nature. 1993; 362: 59-62Crossref PubMed Scopus (5442) Google Scholar). Approximately 20% of familial ALS cases are caused by a variety of dominant SOD1 mutations (1Ilieva H. Polymenidou M. Cleveland D.W. J. Cell Biol. 2009; 187: 761-772Crossref PubMed Scopus (777) Google Scholar, 3Rosen D.R. Siddique T. Patterson D. Figlewicz D.A. Sapp P. Hentati A. Donaldson D. Goto J. O'Regan J.P. Deng H.X. et al.Nature. 1993; 362: 59-62Crossref PubMed Scopus (5442) Google Scholar). There remains great uncertainty as to the primary mechanism(s) by which mutant SOD1 leads to pathology observed in ALS (1Ilieva H. Polymenidou M. Cleveland D.W. J. Cell Biol. 2009; 187: 761-772Crossref PubMed Scopus (777) Google Scholar, 4Glass C.K. Saijo K. Winner B. Marchetto M.C. Gage F.H. Cell. 2010; 140: 918-934Abstract Full Text Full Text PDF PubMed Scopus (2392) Google Scholar). Proposed mechanisms include toxicity associated with misfolding of mutant SOD1, such as ER stress and inhibition of the proteasome, enhanced proinflammatory ROS production, altered axonal transport, excitotoxicity caused by glutamate mishandling, and mitochondrial damage (1Ilieva H. Polymenidou M. Cleveland D.W. J. Cell Biol. 2009; 187: 761-772Crossref PubMed Scopus (777) Google Scholar, 4Glass C.K. Saijo K. Winner B. Marchetto M.C. Gage F.H. Cell. 2010; 140: 918-934Abstract Full Text Full Text PDF PubMed Scopus (2392) Google Scholar). Relevant to the studies in this report are findings demonstrating that SOD1 mutations induce NADPH oxidase-dependent ROS production in microglia of SOD1G93A mice leading to motor neuron death (5Marden J.J. Harraz M.M. Williams A.J. Nelson K. Luo M. Paulson H. Engelhardt J.F. J. Clin. Invest. 2007; 117: 2913-2919Crossref PubMed Scopus (114) Google Scholar, 6Wu D.C. Ré D.B. Nagai M. Ischiropoulos H. Przedborski S. Proc. Natl. Acad. Sci. U.S.A. 2006; 103: 12132-12137Crossref PubMed Scopus (200) Google Scholar). These studies have demonstrated that deletion of the Nox2 isoform of NADPH oxidase, and to a lesser extent also the Nox1 isoform, can prolong survival in SOD1G93A transgenic mice. The importance of SOD1 in regulating cellular ROS production was first revealed by studies demonstrating that SOD1 can directly associate with endosomal Rac1 to regulate its activity (7Harraz M.M. Marden J.J. Zhou W. Zhang Y. Williams A. Sharov V.S. Nelson K. Luo M. Paulson H. Schöneich C. Engelhardt J.F. J. Clin. Invest. 2008; 118: 659-670PubMed Google Scholar). Rac1 is an essential activator of several NADPH oxidases and SOD1 binding to Rac1 slows the hydrolysis of GTP bound to Rac1 in a redox-dependent manner. Thus, SOD1 association with Rac1 is proposed to be a redox-dependent sensor for regulating redox-active NADPH oxidase containing endosomes (called redoxosomes) (7Harraz M.M. Marden J.J. Zhou W. Zhang Y. Williams A. Sharov V.S. Nelson K. Luo M. Paulson H. Schöneich C. Engelhardt J.F. J. Clin. Invest. 2008; 118: 659-670PubMed Google Scholar, 8Oakley F.D. Abbott D. Li Q. Engelhardt J.F. Antioxid. Redox Signal. 2009; 11: 1313-1333Crossref PubMed Scopus (149) Google Scholar). Redoxosomes are important signaling endosomes that regulate proinflammatory receptor signals in a redox-dependent manner through NADPH oxidases (8Oakley F.D. Abbott D. Li Q. Engelhardt J.F. Antioxid. Redox Signal. 2009; 11: 1313-1333Crossref PubMed Scopus (149) Google Scholar, 9Li Q. Zhang Y. Marden J.J. Banfi B. Engelhardt J.F. Biochem. J. 2008; 411: 531-541Crossref PubMed Scopus (47) Google Scholar, 10Li Q. Spencer N.Y. Oakley F.D. Buettner G.R. Engelhardt J.F. Antioxid. Redox Signal. 2009; 11: 1249-1263Crossref PubMed Scopus (92) Google Scholar, 11Li Q. Harraz M.M. Zhou W. Zhang L.N. Ding W. Zhang Y. Eggleston T. Yeaman C. Banfi B. Engelhardt J.F. Mol. Cell. Biol. 2006; 26: 140-154Crossref PubMed Scopus (189) Google Scholar). Redoxosomes are a subpopulation of early endosomes that produce ROS and have been shown to contain early endosomal markers (Rab5 and EEA1), redox effectors (Nox2 or Nox1, Rac1, p47phox, p67phox, and SOD1), and certainly ligand-activated cytokine receptors (TNFR1 or IL-1R1) (8Oakley F.D. Abbott D. Li Q. Engelhardt J.F. Antioxid. Redox Signal. 2009; 11: 1313-1333Crossref PubMed Scopus (149) Google Scholar, 9Li Q. Zhang Y. Marden J.J. Banfi B. Engelhardt J.F. Biochem. J. 2008; 411: 531-541Crossref PubMed Scopus (47) Google Scholar, 10Li Q. Spencer N.Y. Oakley F.D. Buettner G.R. Engelhardt J.F. Antioxid. Redox Signal. 2009; 11: 1249-1263Crossref PubMed Scopus (92) Google Scholar, 11Li Q. Harraz M.M. Zhou W. Zhang L.N. Ding W. Zhang Y. Eggleston T. Yeaman C. Banfi B. Engelhardt J.F. Mol. Cell. Biol. 2006; 26: 140-154Crossref PubMed Scopus (189) Google Scholar). ROS produced by the redoxosome facilitate the redox-dependent recruitment of TRAFs to TNFR1 and IL-1R1 and in this manner facilitate proinflammatory signaling such as NFκB activation (8Oakley F.D. Abbott D. Li Q. Engelhardt J.F. Antioxid. Redox Signal. 2009; 11: 1313-1333Crossref PubMed Scopus (149) Google Scholar, 9Li Q. Zhang Y. Marden J.J. Banfi B. Engelhardt J.F. Biochem. J. 2008; 411: 531-541Crossref PubMed Scopus (47) Google Scholar, 10Li Q. Spencer N.Y. Oakley F.D. Buettner G.R. Engelhardt J.F. Antioxid. Redox Signal. 2009; 11: 1249-1263Crossref PubMed Scopus (92) Google Scholar, 11Li Q. Harraz M.M. Zhou W. Zhang L.N. Ding W. Zhang Y. Eggleston T. Yeaman C. Banfi B. Engelhardt J.F. Mol. Cell. Biol. 2006; 26: 140-154Crossref PubMed Scopus (189) Google Scholar). Interestingly, mutant forms of SOD1 have enhanced redox-independent binding to Rac1 and this has been proposed as a mechanism for enhanced ROS production in microglia of ALS mice (7Harraz M.M. Marden J.J. Zhou W. Zhang Y. Williams A. Sharov V.S. Nelson K. Luo M. Paulson H. Schöneich C. Engelhardt J.F. J. Clin. Invest. 2008; 118: 659-670PubMed Google Scholar, 12Carter B.J. Anklesaria P. Choi S. Engelhardt J.F. Antioxid. Redox Signal. 2009; 11: 1569-1586Crossref PubMed Scopus (30) Google Scholar).Seven known NADPH oxidase catalytic subunits exist (Nox1, Nox2gp91phox, Nox3, Nox4, Nox5, Duox1, and Duox2) (13Lambeth J.D. Nat. Rev. Immunol. 2004; 4: 181-189Crossref PubMed Scopus (2425) Google Scholar, 14Hordijk P.L. Circ. Res. 2006; 98: 453-462Crossref PubMed Scopus (426) Google Scholar). The most widely characterized of these is phagocytic gp91phox (also known as Nox2). Nox2 is also expressed in microglia (6Wu D.C. Ré D.B. Nagai M. Ischiropoulos H. Przedborski S. Proc. Natl. Acad. Sci. U.S.A. 2006; 103: 12132-12137Crossref PubMed Scopus (200) Google Scholar) and a variety of other nonphagocytic cell types. Rac, a small GTPase, is an essential activator of Nox1 and -2, and along with several other subunits (p22phox, p40phox, p47phox, p67phox, NoxO1, and NoxA1) can act to promote Nox complex activation in a cell type-specific fashion (13Lambeth J.D. Nat. Rev. Immunol. 2004; 4: 181-189Crossref PubMed Scopus (2425) Google Scholar, 14Hordijk P.L. Circ. Res. 2006; 98: 453-462Crossref PubMed Scopus (426) Google Scholar). In certain cell systems, Rac1 has also been shown to be required for ROS production by Nox4 (15Gorin Y. Ricono J.M. Kim N.H. Bhandari B. Choudhury G.G. Abboud H.E. Am. J. Physiol. Renal Physiol. 2003; 285: F219-229Crossref PubMed Scopus (241) Google Scholar, 16Lee S. Gharavi N.M. Honda H. Chang I. Kim B. Jen N. Li R. Zimman A. Berliner J.A. Free Radic. Biol. Med. 2009; 47: 145-151Crossref PubMed Scopus (58) Google Scholar, 17Meng D. Lv D.D. Fang J. Cardiovasc. Res. 2008; 80: 299-308Crossref PubMed Scopus (118) Google Scholar). Because NADPH oxidases (Nox) generate the O2̇̄ substrate of the SOD1 dismutation reaction (2O2̇̄ + 2H+ → H2O2 + O2), this class of Nox enzymes has recently generated considerable interest in studies of ALS.It is presently unclear if familial and sporadic forms of ALS have common or overlapping molecular mechanisms of disease pathogenesis. A recent genomewide association study in sporadic ALS patients has begun to shed light on this topic (18Dunckley T. Huentelman M.J. Craig D.W. Pearson J.V. Szelinger S. Joshipura K. Halperin R.F. Stamper C. Jensen K.R. Letizia D. Hesterlee S.E. Pestronk A. Levine T. Bertorini T. Graves M.C. Mozaffar T. Jackson C.E. Bosch P. McVey A. Dick A. Barohn R. Lomen-Hoerth C. Rosenfeld J. O'connor D.T. Zhang K. Crook R. Ryberg H. Hutton M. Katz J. Simpson E.P. Mitsumoto H. Bowser R. Miller R.G. Appel S.H. Stephan D.A. N. Engl. J. Med. 2007; 357: 775-788Crossref PubMed Scopus (210) Google Scholar). Several genes that regulate endosomal trafficking, Rac1, and NADPH oxidases were identified in this study, including Nox4, TIAM2, IQGAP2, PTPRT, RAP1GAP, and MAGI2 (12Carter B.J. Anklesaria P. Choi S. Engelhardt J.F. Antioxid. Redox Signal. 2009; 11: 1569-1586Crossref PubMed Scopus (30) Google Scholar). Interestingly, the ALS2 gene product alsin, which when mutated causes juvenile ALS, has also been shown to influence endosomal trafficking and Rac1 activity (19Chandran J. Ding J. Cai H. Mol. Neurobiol. 2007; 36: 224-231Crossref PubMed Scopus (39) Google Scholar, 20Hadano S. Otomo A. Kunita R. Suzuki-Utsunomiya K. Akatsuka A. Koike M. Aoki M. Uchiyama Y. Itoyama Y. Ikeda J.E. PLoS One. 2010; 5: e9805Crossref PubMed Scopus (100) Google Scholar, 21Hadano S. Kunita R. Otomo A. Suzuki-Utsunomiya K. Ikeda J.E. Neurochem. Int. 2007; 51: 74-84Crossref PubMed Scopus (66) Google Scholar, 22Yang Y. Hentati A. Deng H.X. Dabbagh O. Sasaki T. Hirano M. Hung W.Y. Ouahchi K. Yan J. Azim A.C. Cole N. Gascon G. Yagmour A. Ben-Hamida M. Pericak-Vance M. Hentati F. Siddique T. Nat. Genet. 2001; 29: 160-165Crossref PubMed Scopus (658) Google Scholar, 23Hadano S. Hand C.K. Osuga H. Yanagisawa Y. Otomo A. Devon R.S. Miyamoto N. Showguchi-Miyata J. Okada Y. Singaraja R. Figlewicz D.A. Kwiatkowski T. Hosler B.A. Sagie T. Skaug J. Nasir J. Brown Jr., R.H. Scherer S.W. Rouleau G.A. Hayden M.R. Ikeda J.E. Nat. Genet. 2001; 29: 166-173Crossref PubMed Scopus (585) Google Scholar). Alsin appears to serve as a GEF for Rab5 and an effector of Rac1 GTPase activity (24Otomo A. Hadano S. Okada T. Mizumura H. Kunita R. Nishijima H. Showguchi-Miyata J. Yanagisawa Y. Kohiki E. Suga E. Yasuda M. Osuga H. Nishimoto T. Narumiya S. Ikeda J.E. Hum. Mol. Genet. 2003; 12: 1671-1687Crossref PubMed Scopus (212) Google Scholar, 25Topp J.D. Gray N.W. Gerard R.D. Horazdovsky B.F. J. Biol. Chem. 2004; 279: 24612-24623Abstract Full Text Full Text PDF PubMed Scopus (163) Google Scholar, 26Kunita R. Otomo A. Mizumura H. Suzuki-Utsunomiya K. Hadano S. Ikeda J.E. J. Biol. Chem. 2007; 282: 16599-16611Abstract Full Text Full Text PDF PubMed Scopus (64) Google Scholar). These findings are of considerable interest because SOD1 also regulates Rac1 GTPase and NADPH oxidase activity in Rab5-bound early endosomes (7Harraz M.M. Marden J.J. Zhou W. Zhang Y. Williams A. Sharov V.S. Nelson K. Luo M. Paulson H. Schöneich C. Engelhardt J.F. J. Clin. Invest. 2008; 118: 659-670PubMed Google Scholar, 8Oakley F.D. Abbott D. Li Q. Engelhardt J.F. Antioxid. Redox Signal. 2009; 11: 1313-1333Crossref PubMed Scopus (149) Google Scholar, 11Li Q. Harraz M.M. Zhou W. Zhang L.N. Ding W. Zhang Y. Eggleston T. Yeaman C. Banfi B. Engelhardt J.F. Mol. Cell. Biol. 2006; 26: 140-154Crossref PubMed Scopus (189) Google Scholar, 12Carter B.J. Anklesaria P. Choi S. Engelhardt J.F. Antioxid. Redox Signal. 2009; 11: 1569-1586Crossref PubMed Scopus (30) Google Scholar). Both the Rab5-GEF and Rac1-effector functions of alsin appear to influence endocytic mechanisms and endosomal dynamics (20Hadano S. Otomo A. Kunita R. Suzuki-Utsunomiya K. Akatsuka A. Koike M. Aoki M. Uchiyama Y. Itoyama Y. Ikeda J.E. PLoS One. 2010; 5: e9805Crossref PubMed Scopus (100) Google Scholar, 26Kunita R. Otomo A. Mizumura H. Suzuki-Utsunomiya K. Hadano S. Ikeda J.E. J. Biol. Chem. 2007; 282: 16599-16611Abstract Full Text Full Text PDF PubMed Scopus (64) Google Scholar) and alsin appears to protect from motor neuron degeneration in certain SOD1 mutant mice (20Hadano S. Otomo A. Kunita R. Suzuki-Utsunomiya K. Akatsuka A. Koike M. Aoki M. Uchiyama Y. Itoyama Y. Ikeda J.E. PLoS One. 2010; 5: e9805Crossref PubMed Scopus (100) Google Scholar) and motor neurons expressing SOD1 mutants in culture (27Kanekura K. Hashimoto Y. Niikura T. Aiso S. Matsuoka M. Nishimoto I. J. Biol. Chem. 2004; 279: 19247-19256Abstract Full Text Full Text PDF PubMed Scopus (78) Google Scholar, 28Jacquier A. Buhler E. Schäfer M.K. Bohl D. Blanchard S. Beclin C. Haase G. Ann. Neurol. 2006; 60: 105-117Crossref PubMed Scopus (59) Google Scholar). Given the association of Nox1, Nox2, and Nox4 with disease progression in ALS mice (5Marden J.J. Harraz M.M. Williams A.J. Nelson K. Luo M. Paulson H. Engelhardt J.F. J. Clin. Invest. 2007; 117: 2913-2919Crossref PubMed Scopus (114) Google Scholar, 6Wu D.C. Ré D.B. Nagai M. Ischiropoulos H. Przedborski S. Proc. Natl. Acad. Sci. U.S.A. 2006; 103: 12132-12137Crossref PubMed Scopus (200) Google Scholar) and humans (18Dunckley T. Huentelman M.J. Craig D.W. Pearson J.V. Szelinger S. Joshipura K. Halperin R.F. Stamper C. Jensen K.R. Letizia D. Hesterlee S.E. Pestronk A. Levine T. Bertorini T. Graves M.C. Mozaffar T. Jackson C.E. Bosch P. McVey A. Dick A. Barohn R. Lomen-Hoerth C. Rosenfeld J. O'connor D.T. Zhang K. Crook R. Ryberg H. Hutton M. Katz J. Simpson E.P. Mitsumoto H. Bowser R. Miller R.G. Appel S.H. Stephan D.A. N. Engl. J. Med. 2007; 357: 775-788Crossref PubMed Scopus (210) Google Scholar), these findings suggest the intriguing hypothesis that alsin and SOD1 both influence the dynamics of Rac1-dependent, NADPH oxidase-mediated, ROS production by redoxosomes that may impact proinflammatory signaling in ALS. In support of this hypothesis, alsin has been shown to bind three components of the redoxosome (Rac1, Rab5, and SOD1).To test this hypothesis, we investigated whether alsin expression influences SOD1G93A-mediated ROS production by glial cells. Three NADPH oxidases were evaluated as sources of cellular ROS (Nox1, -2, and -4) using shRNA knockdown, based on their association with disease severity in ALS models. Findings from our studies demonstrated that wild type alsin attenuates SOD1G93A-mediated Rac1 activation, ROS production by Nox2, NFκB activation, and TNFα secretion by glial cells and protects neurons from toxicity in co-culture studies. SOD1G93A expression enhanced TNFα-dependent redoxosomal ROS production by Nox2 and this was attenuated by alsin expression. Taken together, our results suggest a potential role for alsin in regulating redox-dependent proinflammatory signals via redoxosomes that are enhanced by SOD1G93A.