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 Noxdependent ROS production. Our studies demonstrate that glial cell expression of SOD1 G93A or wild type alsin induces ROS production, Rac1 activation, secretion of TNF␣, and activation of NFB, leading to decreased motor neuron survival in co-culture. Interestingly, coexpression of alsin, or shRNA against Nox2, with SOD1 G93A 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 TNF␣-mediated endosomal ROS in glial cells in a Rac1-dependent manner and alsin overexpression inhibited SOD1 G93A -induced endosomal ROS and Rac1 activation. SOD1 G93A 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 NFB. These studies suggest that glial proinflammatory signals regulated by endosomal ROS are influenced by two gene products known to cause ALS.Amyotrophic lateral sclerosis (ALS) is a lethal degenerative neurological disorder characterized by progressive degeneration of motor neurons in the brain and spinal cord (1, 2). 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 ϳ10% of ALS cases have a clearly inherited genetic component and hence are classified as familial ALS (1, 2).The best-characterized forms of familial ALS include those caused by mutations in the gene encoding Cu/Zn-superoxide dismutase (SOD1) 2 (3). Approximately 20% of familial ALS cases are caused by a variety of dominant SOD1 mutations (1, 3). There remains great uncertainty as to the primary mechanism(s) by which mutant SOD1 leads to pathology observed in ALS (1, 4). 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 (1, 4). Relevant to the studi...

The NADPH Oxidase Pathway Is Dysregulated by the P2X7 Receptor in the SOD1-G93A Microglia Model of Amyotrophic Lateral Sclerosis

“…In addition, several reports describe NOX2 as a source of damaging ROS in ALS (7,9,10,12). Consistent with this, in the present study we found a basal upregulation of the Rac1-NOX2 pathway in the model of SOD1-G93A primary microglia, in line with previous results obtained in cell lines transiently infected with ALS-related genes (7,12), and we also demonstrated that activation of the P2X 7 receptor further enhances this pathway, thus producing additional oxidative stress. Furthermore, by overexpression of a dominant-negative or constitutively active form of Rac1 in ALS microglia, we established that p67 phox subunit translocation on membranes is totally dependent on Rac1 recruitment and not on alternative pathways; thus, Rac1 activation is a prerequisite for NOX2 activation by the P2X 7 receptor.…”

“…Indeed, in a previous work we demonstrated that the extracellular ATP pool is better preserved and the P2X 7 receptor is upregulated in SOD1-G93A microglia, which translates into an increase in released proinflammatory factors and neurotoxicity induced by microglia upon P2X 7 receptor stimulation (21). In addition, several reports describe NOX2 as a source of damaging ROS in ALS (7,9,10,12). Consistent with this, in the present study we found a basal upregulation of the Rac1-NOX2 pathway in the model of SOD1-G93A primary microglia, in line with previous results obtained in cell lines transiently infected with ALS-related genes (7,12), and we also demonstrated that activation of the P2X 7 receptor further enhances this pathway, thus producing additional oxidative stress.…”

“…Consistently, NOX2 is upregulated in activated ALS microglia, and the deletion of NOX2 can improve disease progression and survival of SOD1-G93A mice (9)(10)(11). Furthermore, uncontrolled ROS, together with inflammatory cytokines produced by microglia, mediate disease progression, causing direct neurotoxicity and increasing motor neuron susceptibility to ALS-triggering factors (2,12). Among these, extracellular ATP was recently suggested as a possible candidate.…”

Atorvastatin Protects NSC-34 Motor Neurons Against Oxidative Stress by Activating PI3K, ERK and Free Radical Scavenging

“…As H 2 O 2 levels increase, thiols are reduced and thiolate-dependent enzymes are inhibited, leading to oxidative damage to lipids, proteins, and DNA. Furthermore, superoxide and NO released from activated microglia may act as exogenous toxins to motor neurons [41]. Oxidative stress probably accelerates disease progression.…”

New Insights onNOXEnzymes in the Central Nervous System

“…(171,182) and novel studies on the role of NOX isoforms in these new models are nowadays needed (107). Taken together, evidence from NOX2-(and, to a lesser extent, NOX1-) deficient mice suggest that NOX activation is causally involved in the progression of ALS.…”