Oxidative stress appears to play an important role in degeneration of dopaminergic neurons of the substantia nigra (SN) associated with Parkinson's disease (PD). The SN of early PD patients have dramatically decreased levels of the thiol tripeptide glutathione (GSH). GSH plays multiple roles in the nervous system both as an antioxidant and a redox modulator. We have generated dopaminergic PC12 cell lines in which levels of GSH can be inducibly down-regulated via doxycycline induction of antisense messages against both the heavy and light subunits of ␥-glutamyl-cysteine synthetase, the rate-limiting enzyme in glutathione synthesis. Down-regulation of glutamyl-cysteine synthetase results in reduction in mitochondrial GSH levels, increased oxidative stress, and decreased mitochondrial function. Interestingly, decreases in mitochondrial activities in GSH-depleted PC12 cells appears to be because of a selective inhibition of complex I activity as a result of thiol oxidation. These results suggest that the early observed GSH losses in the SN may be directly responsible for the noted decreases in complex I activity and the subsequent mitochondrial dysfunction, which ultimately leads to dopaminergic cell death associated with PD.
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...
Exposure of neurons to H 2 O 2 results in both necrosis and apoptosis. Caspases play a pivotal role in apoptosis, but exactly how they are involved in H 2 O 2 -mediated cell death is unknown. We examined H 2 O 2 -induced toxicity in neuronal PC12 cells and the effects of inducible overexpression of the H 2 O 2 -scavenging enzyme catalase on this process. H 2 O 2 caused cell death in a time-and concentration-dependent manner. Cell death induced by H 2 O 2 was found to be mediated in part through an apoptotic pathway as H 2 O 2 -treated cells exhibited cell shrinkage, nuclear condensation and marked DNA fragmentation. H 2 O 2 also triggered activation of caspase 3. Genetic up-regulation of catalase not only signi®cantly reduced cell death but also suppressed caspase 3 activity and DNA fragmentation. While the caspase 3 inhibitor DEVD inhibited both caspase 3 activity and DNA fragmentation induced by H 2 O 2 it did not prevent cell death. Treatment with the general caspase inhibitor ZVAD, however, resulted in complete attenuation of H 2 O 2 -mediated cellular toxicity. These results suggest that DNA fragmentation induced by H 2 O 2 is attributable to caspase 3 activation and that H 2 O 2 may be critical for signaling leading to apoptosis. However, unlike inducibly increased catalase expression and general caspase inhibition both of which protect cells from cytotoxicity, caspase 3 inhibition alone did not improve cell survival suggesting that prevention of DNA fragmentation is insuf®cient to prevent H 2 O 2 -mediated cell death.
The first and rate-limiting reaction in the formation of glutathione is catalyzed by gamma-glutamylcysteine synthetase (GCS), a dimer composed of a catalytic heavy and a regulatory light subunit. We previously found that heavy subunit GCS mRNA appears to be expressed at high levels in the hippocampus, cerebellum, and cortex of murine brain and at lower levels in the neostriatum (Kang et al. [1997] NeuroReport 8:2053). Here we report that variations in expression of light subunit GCS mRNA in murine brain resembles that of the heavy subunit mRNA with a few minor exceptions. Moreover, levels of GCS activity and glutathione levels in various brain regions appear to correspond to levels of expression of both GCS mRNA subunits. Based on these data, differences in the distribution of expression of the GCS subunits in the brain may therefore have major implications for the susceptibility of various brain regions to oxidative stress and/or mitochondrial damage.
The first and rate-limiting reaction in the formation of glutathione is catalyzed by gamma-glutamylcysteine synthetase (GCS), a dimer composed of a catalytic heavy and a regulatory light subunit. We previously found that heavy subunit GCS mRNA appears to be expressed at high levels in the hippocampus, cerebellum, and cortex of murine brain and at lower levels in the neostriatum (Kang et al. [1997] NeuroReport 8:2053). Here we report that variations in expression of light subunit GCS mRNA in murine brain resembles that of the heavy subunit mRNA with a few minor exceptions. Moreover, levels of GCS activity and glutathione levels in various brain regions appear to correspond to levels of expression of both GCS mRNA subunits. Based on these data, differences in the distribution of expression of the GCS subunits in the brain may therefore have major implications for the susceptibility of various brain regions to oxidative stress and/or mitochondrial damage.
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