Nrf2 (NF-E2-related factor 2) has been demonstrated to play a central role in the gene expression of detoxification enzymes and antioxidant genes through binding the antioxidant responsive element (ARE). Importantly, in in vitro and in vivo models, this system has been shown to be effective at blocking neurotoxicity resulting from glutathione depletion, lipid peroxidation, intracellular calcium overload, excitotoxins, and disruption of the mitochondrial electron transport chain. In particular, increased levels of glutathione (GSH) seem to be a major component of the protection observed by Nrf2 activation. Glutamate-cysteine ligase catalytic (GCLC) and modifier (GCLM) subunits make up the rate-limiting enzyme complex for GSH synthesis and Nrf2 transcriptionally regulated both enzymes. Interestingly, the Nrf2-ARE pathway activation is isolated to astrocytes that confer resistance to neurons. Several studies have demonstrated that secreted GSH can protect neurons acting as an antioxidant in the extracellular compartment and boosting GSH levels in neurons by increasing the availability of precursors for GSH synthesis. We are particularly intrigued by astrocyte-motor neuron interaction and how the Nrf2-ARE pathway could be involved in this intimate relationship, as well as the level of importance of GSH to motor neuron survival, in amyotrophic lateral sclerosis (ALS). Some of the inherited forms of ALS are associated with mutations in human Cu/Zn superoxide dismutase 1 (hSOD1). Mouse models have been generated using mutant forms of hSOD1 that recapitulate much of the cellular and phenotypic changes observed in human ALS patients. We hypothesize that Nrf2-ARE activation and GSH production in the astrocyte will protect motor neurons in models of ALS in vitro and in vivo. We have generated glial fibrillary acidic protein (GFAP)-Nrf2 mice that selectively overexpress Nrf2 in astrocytes. Preliminary data in co-cultures of astrocytes and motor neurons indicate that hSOD1G93A astrocytes are sick and that motor neurons die when plated on these sick astrocytes. Strikingly, if Nrf2 is overexpressed in the same sick astrocytes, the motor neurons do not die. Initial in vivo studies show an increase in lifespan of the hSOD1G93A mice when crossed with GFAP-Nrf2 mice. Finally, ongoing experiments using the GCLM-/- mice are underway. In this case, hSOD1G93A mice on GCLM-/- background show dramatic muscle weakness (0 sec on wire hang) and initial signs of paralysis by 45 days of age compared to 120 days of age on a GCLM+/+ background. The specific aims of this proposal are: Aim 1. To determine the significance of Nrf2 and GSH in astrocyte-mediated motor neuron survival in vitro; Aim 2. To evaluate the role of GSH in the Nrf2- mediated delay in onset and progression of disease in hSOD1G93A mice; and Aim 3. Test the efficacy of clinically relevant approaches for the treatment o ALS using viral-mediated delivery of Nrf2 or non- viral delivery of Keap1 siRNA. PUBLIC HEALTH RELEVANCE Pound 5,600 people in the United States are diagnosed with ALS every year and there are about 30,000 Americans with the disease at any given time. Approximately 80% of ALS suffers will die within 5 years of diagnosis. Thus, the lack of any significant therapeutic intervention for ALS is an important medical issue. The majority of ALS cases are sporadic with unknown cause. Environmental exposure is thought to play a major role in sporadic disease and is believed to be a principal component behind the increased incidence in ALS among Gulf War veterans and people serving in the military. Irrespective of the underlying cause of ALS, the approaches outlined in proposal are focused on a pathologic processes seen in both sporadic and familial disease, and could lead to new ways to treat ALS. In addition to looking at the onset of the disease in these animal models, specific experiments will examine disease progression that more accurately represent the human clinical situation. The hope is to gain a greater understanding of the disease process and discover ways to treat ALS that will stop or at least slow disease progression.