Mitochondrial oxidative stress is a major factor in several neurodegenerative disorders including Parkinson's disease (PD) and amyotrophic lateral sclerosis (ALS). One of the main consequences of mitochondrial oxidative stress is activation of an intrinsic apoptosis cascade that can lead to neuronal death. Bcl-2 family proteins are principal regulators of mitochondrial oxidative stress and intrinsic apoptosis. In many instances of neurodegeneration, pro-survival Bcl-2 is downregulated while pro-apoptotic Bax and Bcl-2 homology-3 domain (BH3)-only proteins (e.g., Bim) are upregulated. Consequently, either overexpression of Bcl-2 or deletion of Bax is neuroprotective in animal models of PD and ALS. The divergent effects of Bcl-2 and Bax on neuronal survival are most often attributed to their opposing actions on the intrinsic apoptosis pathway where Bcl-2 suppresses cytochrome c release while Bax promotes this effect. In addition, these proteins demonstrate opposing effects on mitochondrial oxidative stress; Bcl-2 acts in an antioxidant-like manner while Bax (and some BH3-only proteins) induces a pro-oxidant state at this organelle. Presently, it is unknown whether the antioxidant actions of Bcl-2 and the pro-oxidant effects of Bax at mitochondria are related at the molecular level. We have utilized small molecule BH3 mimetics, which bind in a hydrophobic surface groove of Bcl-2 (the BH3 groove), as tools to study this relationship in primary cultures of cerebellar granule neurons. BH3 mimetics induce glutathione (GSH)-sensitive mitochondrial oxidative stress and neuronal apoptosis, suggesting that these compounds inhibit the antioxidant function of Bcl-2. In this context, we have identified a novel GSH-binding property of Bcl-2 that is inhibited by BH3 mimetics in vitro, in isolated rat brain mitochondria, and in intact neurons. Moreover, BH3 mimetics inhibit GSH uptake into isolated rat brain mitochondria and displace GSH from mitochondria of intact neurons. Based on these data, we hypothesize that GSH-binding and regulation of GSH transport are essential components of Bcl-2's antioxidant function at mitochondria. Furthermore, we predict that pro-apoptotic Bax induces a pro-oxidant state at mitochondria by disrupting these novel functions of Bcl-2 in a BH3-dependent manner. We will test our hypothesis with the following specific aims: 1) to create a GSH-binding-deficient mutant of Bcl-2 and determine if its expression sensitizes neurons to mitochondrial oxidative stress; 2) to determine if Bcl-2 is a critical regulator of mitochondrial GSH transport; 3) to determine if pro-apoptotic Bcl-2 family members elicit their pro-oxidant effects at mitochondria by antagonizing the GSH-binding and GSH transporter functions of Bcl-2; and 4) to evaluate the GSH-binding potential of Bcl-2 and the GSH uptake capacity of mitochondria in a mouse model of ALS. These studies may reveal a molecular mechanism to explain the opposing effects of Bcl-2 and Bax on the redox state of mitochondria. The data obtained may be particularly relevant in elucidating mechanisms of neuronal death in degenerative disorders (such as ALS) where Bcl-2 loss-of-function and mitochondrial oxidative stress play critical roles.
Neurodegenerative diseases like Parkinson's disease and amyotrophic lateral sclerosis (Lou Gehrig's disease) significantly reduce quality of life. Like all neurodegenerative diseases, nerve cell dysfunction and death underlie the pathology of these disorders. Although Bcl-2 protein signaling and oxidative stress at mitochondria (organelles that supply the cell with energy) markedly influence nerve cell survival and death, little is known about how these pathways interact. The proposed project is focused on the role of Bcl-2 family proteins in the regulation of mitochondrial antioxidant pools and oxidative stress. These studies should provide novel information about the molecular mechanisms underlying mitochondrial dysfunction and nerve cell death that contribute to neurodegenerative diseases. PHS 398/2590 (Rev. 09/04, Reissued 4/2006) Page Continuation Format Page ? ?
