Parkinson's disease is a neurodegenerative disorder characterized by the preferential loss of midbrain dopaminergic neurons in the substantia nigra (SN). One of the earliest detectable biochemical alterations that occurs in the Parkinsonian brain is a marked reduction in SN levels of total glutathione (glutathione plus glutathione disulfide), occurring before losses in mitochondrial complex I (CI) activity, striatal dopamine levels, or midbrain dopaminergic neurodegeneration associated with the disease. Previous in vitro data from our laboratory has suggested that prolonged depletion of dopaminergic glutathione results in selective impairment of mitochondrial complex I activity through a reversible thiol oxidation event. To address the effects of depletion in dopaminergic glutathione levels in vivo on the nigrostriatal system, we created genetically engineered transgenic mouse lines in which expression of ␥-glutamyl cysteine ligase, the rate-limiting enzyme in de novo glutathione synthesis, can be inducibly downregulated in catecholaminergic neurons, including those of the SN. A novel method for isolation of purified dopaminergic striatal synaptosomes was used to study the impact of dopaminergic glutathione depletion on mitochondrial events demonstrated previously to occur in vitro as a consequence of this alteration. Dopaminergic glutathione depletion was found to result in a selective reversible thiol-oxidation-dependent mitochondrial complex I inhibition, followed by an age-related nigrostriatal neurodegeneration. This suggests that depletion in glutathione within dopaminergic SN neurons has a direct impact on mitochondrial complex I activity via increased nitric oxide-related thiol oxidation and age-related dopaminergic SN cell loss.

Age-related increases in brain monoamine oxidase B (MAO-B) and its ability to produce reactive oxygen species as a by-product of catalysis could contribute to neurodegeneration associated with Parkinson's disease. This may be via increased oxidative stress and/or mitochondrial dysfunction either on its own or through its interaction with endogenous or exogenous neurotoxic species. We have created genetically engineered dopaminergic PC12 cell lines with subtly increased levels of MAO-B mimicking those observed during normal aging. In our cells, increased MAO-B activity was found to result in increased H 2 O 2 production. This was found to correlate with a decrease in mitochondrial complex I activity which may involve both direct oxidative damage to the complex itself as well as oxidative effects on the tricarboxylic acid cycle enzyme ␣-ketoglutarate dehydrogenase (KGDH) which provides substrate for the complex. Both complex I and KGDH activities have been reported to be decreased in the Parkinsonian brain. These in vitro events are reversible by catalase addition. Importantly, MAO-B elevation was found to abolish the spare KGDH threshold capacity, which can normally be significantly inhibited before it affects maximal mitochondrial oxygen consumption rates. Our data suggest that H 2 O 2 production via subtle elevations in MAO-B levels can result in oxidative effects on KGDH that can compromise the ability of dopaminergic neurons to cope with increased energetic stress.

Parkinson's disease (PD) is characterized by the presence of proteinaceous neuronal inclusions called Lewy bodies in susceptible dopaminergic midbrain neurons. Inhibition of the ubiquitin-proteasome protein degradation pathway may contribute to protein build-up and subsequent cell death. Ubiquitin is normally activated for transfer to substrate proteins by interaction with the E1 ubiquitin ligase enzyme via a thiol ester bond. Parkinson's disease is also characterized by decreases in midbrain levels of total glutathione which could impact on E1 enzyme activity via oxidation of the active site sulfhydryl. We have demonstrated that increasing reductions in total glutathione in dopaminergic PC12 cells results in corresponding decreases in ubiquitin-protein conjugate levels suggesting that ubiquitination of proteins is inhibited in a glutathione-dependent fashion. Decreased ubiquitinated protein levels appears to be due to inhibition of E1 activity as demonstrated by reductions in endogenous E1-ubiquitin conjugate levels as well as decreases in the production of de novo E1-ubiquitin conjugates when glutathione is depleted. This is a reversible process as E1 activity increases upon glutathione restoration. Our data suggests that decreases in cellular glutathione in dopaminergic cells results in decreased E1 activity and subsequent disruption of the ubiquitin pathway. This may have implications for neuronal degeneration in PD. Ubiquitin is a highly conserved, 8.5 kDa, 76 residue protein found in all eukaryotes. It is present either in a free state or covalently bound to a variety of cytoplasmic, nuclear and integral membrane proteins, e.g. cell cycle regulators, transcription factors, tumor suppressors and oncoproteins. It is primarily known for its involvement in a pathway that regulates the bulk of intracellular protein turnover (Ciechanover and Schwartz 1994; Harshko and Ciechanover 1998). Ubiquitin acts as a covalent tag to mark damaged or shortlived proteins for degradation by the ATP-dependent multiprotease 26S proteasome complex (Wilkinson 1997(Wilkinson , 1999. Ubiquitin is first activated by conjugation of its carboxy terminal glycine residue to the thiol group of a cysteine residue on the E1 activating enzyme via an ester bond . It is then transferred to a cysteine thiol group on one of the several substrate-specific E2 conjugating enzymes and finally either conjugated directly to the side chain group of a lysine residue on an acceptor protein substrate or indirectly via an E3 ligating enzyme (Hershko et al. 1983;Pickart and Rose 1985;Haas and Bright 1988). Ubiquitin forms self-linked polyubiquitin chains on the protein substrate via isopeptide linkages between its lysine 48 residues. These polyubiquitin adducts act as the preferred substrates for rapid proteolysis by the 26S proteasome complex, which degrades the tagged protein to small peptides (Ciechanover and Schwartz 1994). If the ubiquitin pathway is not functioning properly, this can result in the build-up of proteins in the cytoplasm ultimate...

The catecholamine-oxidizing enzyme monoamine oxidase-B (MAO-B) has been hypothesized to be an important determining factor in the etiology of both normal aging and age-related neurological disorders such as Parkinson's disease (PD). Catalysis of substrate by the enzyme produces H2O2 which is a primary originator of oxidative stress which in turn can lead to cellular damage. MAO-B increases with age as does predisposition towards PD which has also been linked to increased oxidative stress. Inhibition of MAO-B, along with supplementation of lost dopamine via L-DOPA, is one of the major antiparkinsonian therapies currently in use. In this review, we address several factors contributing to a possible role for MAO-B in normal brain aging and neurological disease and also discuss the use of MAO-B inhibitors as drug therapy for these conditions.

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