Evidence for synergism between cell death mechanisms in a cellular model of neurodegeneration in Parkinson's disease.

Delineation of how cell death mechanisms associated with Parkinson's disease (PD) interact and whether they converge would help identify targets for neuroprotective therapies. The purpose of this study was to use a cellular model to address these issues. Catecholaminergic SH-SY5Y neuroblastoma cells were exposed to a range of compounds (dopamine, rotenone, 5,8-dihydroxy-1,4-naphtho-107 quinone [naphthazarin], and Z-Ile-Glu(OBut)-Ala-Leu-al [PSI]) that are neurotoxic when applied to these cells for extended periods of times at specific concentrations. At the concentrations used, these compounds cause cellular stress via mechanisms that mimic those associated with causing neurodegeneration in PD, namely oxidative stress (dopamine), mitochondrial dysfunction (rotenone), lysosomal dysfunction (naphthazarin), and proteasomal dysfunction (PSI). The compounds were applied to the SH-SY5Y cells either alone or in pairs. When applied separately, the compounds produced a significant decrease in cell viability confirming that oxidative stress, mitochondrial, proteosomal, or lysosomal dysfunction can individually result in catecholaminergic cell death. When the compounds were applied in pairs, some of the combinations produced synergistic effects. Analysis of these interactions indicates that proteasomal, lysosomal, and mitochondrial dysfunction is exacerbated by dopamine-induced oxidative stress. Furthermore, inhibition of the proteasome or lysosome or increasing oxidative stress has a synergistic effect on cell viability when combined with mitochondrial dysfunction, suggesting that all cell death mechanisms impair mitochondrial function. Finally, we show that there are reciprocal relationships between oxidative stress, proteasomal dysfunction, and mitochondrial dysfunction, whereas lysosome dysfunction appears to mediate cell death via an independent pathway. Given the highly interactive nature of the various cell death mechanisms linked with PD, we predict that effective neuroprotective strategies should target multiple sites in these pathways, for example oxidative stress and mitochondria.

Mitochondrial dysfunction precedes other sub-cellular abnormalities in an in vitro model linked with cell death in Parkinson's disease.

Dysfunction of mitochondria, the ubiquitin proteasome system (UPS), and lysosomes are believed to contribute to the pathogenesis of Parkinson's disease (PD). If it were possible to rescue functionally compromised, but still viable neurons early in the disease process, this would slow the rate of neurodegeneration. Here, we used a catecholaminergic neuroblastoma cell line (SH-SY5Y) as a model of susceptible neurons in PD. To identify a target early in the cell death process that was common to all neurodegenerative processes linked with PD, cells were exposed to toxins that mimic cell death mechanisms associated with PD. The sub-cellular abnormalities that occur shortly after toxin exposure were determined. 3 h of exposure to either naphthazarin, to inhibit lysosomal function, Z-Ile-Glu(OBu(t))-Ala-Leu-H (PSI), to inhibit the UPS, or rotenone, to inhibit mitochondrial complex I, caused depolarisation of the mitochondrial membrane potential (2.5-fold, twofold, and 4.6-fold change, respectively compared to vehicle), suggesting impaired mitochondrial function. Following 24 h exposure to the same toxins, UPS and lysosomal function were also impaired, and ubiquitin levels were increased. Thus, following exposure to toxins that mimic three important, but disparate cell death mechanisms associated with PD, catecholaminergic cells initially experience mitochondrial dysfunction, which is then followed by abnormalities in UPS and lysosomal function. Thus, mitochondrial dysfunction is an early event in cell stress. We suggest that, in patients with PD, the surviving cells of the substantia nigra pars compacta are most susceptible to mitochondrial impairment. Thus, targeting the mitochondria may be useful for slowing the progression of neurodegeneration in PD.

Development and validation of a screening assay for the evaluation of putative neuroprotective agents in the treatment of Parkinson's disease.

Following initial diagnosis of Parkinson's disease, if it were possible to prescribe a treatment that could halt or prevent further neurodegeneration, disease progression could be prevented. The aim of this study was to generate a quick and reliable assay for assessing putative neuroprotective agents for parkinsonian patients. Abnormalities in mitochondria, proteasome and lysosome function, as well as oxidative stress cause cell death in Parkinson's disease. Thus, we exposed neuroblastoma (SH-SY5Y) cells to EC(50) of toxins that mimic these cell death mechanisms (dopamine to induce oxidative stress; naphthazarin to inhibit lysosome function; proteasome inhibitor N-carbobenzyloxy-Ile-Glu(O-t-butyl)-Ala-leucinal (PSI) to inhibit the UPS (ubiquitin proteasome system) and rotenone to inhibit mitochondria function) in the presence of five compounds previously chosen as neuroprotective agents, and assessed cell viability. Coenzyme Q10 (117 µM) significantly protected against four toxins, dopamine: 16.3 ± 3.3%; naphthazarin: 10.8 ± 1.1%; PSI: 16.2 ± 2.9%; rotenone: 53.2 ± 4.2%; whereas caffeine (140 µM), creatine (25 mM), nicotine (1 µM) and deprenyl (10 µM) provided protection against some, but not all toxins. Interestingly, coenzyme Q10 is the only compound out of the five that showed neuroprotective potential in clinical trials. Thus, there is a direct correlation between the success of disease modifying agents in the clinic and their ability to protect against multiple cell death mechanisms in this assay. We propose that exposure of SH-SY5Y cells to different toxins that recapitulate cell death mechanisms in Parkinson's disease serves as a rapid and reliable method to test neuroprotective agents that may succeed in clinical trials.