Combining ROS Reduction and Allotopic ATP6 Expression in LS and NARP Patient Neurons to Increase ATP Synthesis

The mitochondria are the power plants of the cell; when they are unable to meet the brain’s substantial energy demands, neurodegeneration occurs. Leigh syndrome (LS) and Neuropathy, Ataxia, and Retinitis Pigmentosa (NARP) are two such neurodegenerative diseases, and are caused by dysfunctional ATP synthase. This enzyme is composed of fifteen subunits that work together to couple the flow of protons into the mitochondrial matrix to a rotating complex that catalyzes the ADP + Pi ATP reaction (Baracca et al., 2000; Elston et al., 1998). Specifically, the ATP6 gene that is encoded in the mitochondrial genome forms the proton channel subunit of ATP synthase (Baracca et al., 2000; Elston et al., 1998). In LS and NARP patients, the ATP6 gene contains either a T8993G (Holt et al., 1990) or much less common T8993C (deVries et al., 1993) nucleotide mutation. Consequently, proton conductance is reduced and ATP synthesis as well as holoenzyme assembly is decreased (Trounce et al., 1994; Garcia et al., 2000; Baracca et al., 2000). Because ATP6 is a mitochondrial gene, LS and NARP syndromes are maternally inherited and result when the cellular heteroplasmy (mutant dosage) is 90-95% and 70-90%, respectively (Tautch et al., 1992). My research interests focus on the development of a gene therapy solution for LS and NARP syndromes that increases ATP synthesis. In addition to ATP synthase dysfunction, the T8993G mutation results in increased levels of reactive oxygen species (ROS), which induce an increase in the expression of the free-radical scavenging superoxide dismutases (SOD) and trigger apoptosis (Geromel et al., 2001). Furthermore, oxidative stress damages mitochondrial lipids and impairs function of the election transport chain, consequently decreasing ATP synthesis (Mattiazzi et al., 2004). ATP synthesis increases after antioxidant treatment of 100% (homoplasmic) T8993G cybrids synthesized from enucleated plate cells and osteosarcoma cells (Mattiazzi et al, 2004). The most promising possibility for increasing ATP synthesis and ATP synthase function in LS and NARP cells arises from the success of allotropic expression of wild-type (WT) ATP6 within homoplasmic T8993G Human Embryonic Kidney (HEK) cells (Manfredi et al., 2002). This is achieved through the use of a recombinant Adeno Associated Virus (AAV) to deliver WT-ATP6 into the nuclear genome (i.e. Allotopic Expression; Manfredi et al., 2002). ATP synthesis is significantly increased in HEK cells allotopically expressing WT-ATP6 compared to non-treated mutants; however, it remains to be discovered whether the increase in WT-ATP6 expression will increase ATP ____________________________________________