Studies on postmortem brains from Parkinson's patients reveal elevated iron in the substantia nigra (SN). Selective cell death in this brain region is associated with oxidative stress, which may be exacerbated by the presence of excess iron. Whether iron plays a causative role in cell death, however, is controversial. Here, we explore the effects of iron chelation via either transgenic expression of the iron binding protein ferritin or oral administration of the bioavailable metal chelator clioquinol (CQ) on susceptibility to the Parkinson's-inducing agent 1-methyl-4-phenyl-1,2,3,6-tetrapyridine (MPTP). Reduction in reactive iron by either genetic or pharmacological means was found to be well tolerated in animals in our studies and to result in protection against the toxin, suggesting that iron chelation may be an effective therapy for prevention and treatment of the disease.

Parkinson's disease (PD) and 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) toxicity are both associated with dopaminergic neuron death in the substantia nigra (SN). Apoptosis has been implicated in this cell loss; however, whether or not it is a major component of disease pathology remains controversial. Caspases are a major class of proteases involved in the apoptotic process. To evaluate the role of caspases in PD, we analyzed caspase activation in MPTP-treated mice, in cultured dopaminergic cells, and in postmortem PD brain tissue. MPTP was found to elicit not only the activation of the effector caspase-3 but also the initiators caspase-8 and caspase-9, mitochondrial cytochrome c release, and Bid cleavage in the SN of wild-type mice. These changes were attenuated in transgenic mice neuronally expressing the general caspase inhibitor protein baculoviral p35. These mice also displayed increased resistance to the cytotoxic effects of the drug. MPTP-associated toxicity in culture was found temporally to involve cytochrome c release, activation of caspase-9, caspase-3, and caspase-8, and Bid cleavage. Caspase-9 inhibition prevented the activation of both caspase-3 and caspase-8 and also inhibited Bid cleavage, but not cytochrome c release. Activated caspase-8 and caspase-9 were immunologically detectable within MPP(+)-treated mesencephalic dopaminergic neurons, dopaminergic nigral neurons from MPTP-treated mice, and autopsied Parkinsonian tissue from late-onset sporadic cases of the disease. These data demonstrate that MPTP-mediated activation of caspase-9 via cytochrome c release results in the activation of caspase-8 and Bid cleavage, which we speculate may be involved in the amplification of caspase-mediated dopaminergic cell death. These data suggest that caspase inhibitors constitute a plausible therapeutic for 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...

Apoptosis is a cell-suicide process that appears to play a central role not only during normal neuronal development but also in several neuropathological disease states. An important component of this process is a proteolytic cascade involving a family of cysteine proteases called caspases. Caspase inhibitors have been demonstrated to be effective in inhibiting neuronal cell death in various apoptotic paradigms. We have created transgenic mice that neuronally express the baculoviral caspase inhibitor p35. Neuronal expression of the p35 protein was found to confer functional caspase inhibitory activity and prevent apoptosis in isolated cerebellar granular cultures induced to undergo apoptosis either via staurosporine treatment or through withdrawal of extracellular potassium. Neuronal expression of p35 was also found to attenuate neurodegeneration associated with the excitotoxic glutamate analogue kainic acid (KA) in vitro and in vivo. Organotypic hippocampal cultures isolated from p35 transgenics demonstrated lowered caspase activity and decreased apoptosis compared with wild type when exposed to KA. In vivo injection of KA also produced decreased caspase activity and cell death in p35 transgenics vs. wild type. These results suggest that the presence of p35 in neurons in vivo is protective against various types of apoptosis, including seizure-related neurodegeneration, and that caspases may be attractive potential targets for preventing neuronal injury associated with diseases such as epilepsy. These mice also provide a valuable tool for exploring the role of caspases in other neuropathological conditions in which apoptosis has been implicated. A poptosis is a highly ordered, morphologically distinct process of cell death involving the activation of a family of cysteine proteases called caspases (1). Caspases were first implicated in apoptosis by the discovery of the Caenorhabditis elegans ced-3 gene (2). Since then, a large family of these caspases has been described in a wide variety of organisms. Caspases are expressed as proenzymes and are activated during apoptosis either by autocatalytic cleavage or via other caspases (3).Much interest in the process of neuronal apoptosis has been generated recently because of a growing body of evidence suggesting that inappropriate apoptosis may contribute to the pathology associated with several neurological disorders (4-6). In several instances, inhibition of caspases has been shown to functionally rescue neurons from death. After permanent focal ischemia, for example, transgenic mice expressing a dominantnegative form of caspase-1 display significantly reduced brain injury and behavioral deficits (7). The presence of this transgene also delays the appearance of symptoms and increases survival rates in mouse models of both amyotrophic lateral sclerosis and Huntington's disease (8, 9).To further explore the role of caspases in various neuropathological processes, we have created transgenic mice that neuronally express the baculoviral caspase inhibitor protein, p35.Expre...