P4‐164: Dimebon (Latrepirdine) protects from cell death‐induced by mitochondrial stressors and alpha‐synuclein over‐expression, and decreases alpha‐synuclein protein levels in a Parkinson's disease cell model. (2011)

Aggregation of alpha-synuclein (a-syn) and mitochondrial dysfunction are important for the development of several neurodegenerative diseases including Parkinson's disease (PD) and Alzheimer's disease (AD). Increased levels of a-syn are detected in brains of patients with early-onset familial and sporadic PD and a-syn protein aggregates are associated with cases of AD. Evidences suggest that aggregation of a-syn is an important intermediate in the development of mitochondrial dysfunction and neurodegeneration in PD. Dimebon was originally developed as an antihistamine drug. This drug was later found to have cognition-enhancing effects in rodents and neuroprotective and pro-neurogenic functions, possibly through a mitochondrial-related mechanism, and is currently in Phase 3 clinical trials for the treatment of AD and Huntington's disease (HD). SH-SY5Y cells were treated with hydrogen peroxide (300 uM), the calcium ionophore 4-Br-A23187 (2 uM) or the mitochondrial toxin rotenone (1- 50 uM) in the presence of vehicle or Dimebon for 12 and 24 hours and cell viability measured by lactate dehydrogenase (LDH) release. In addition, we used a Tet-off system to induce the expression of wild type a-syn in SH-SY5Y cells (Vekrellis K et al. J. of Neurochemistry 2009; 109: 1348) to study the effect of Dimebon on a-syn mRNA and protein expression and cytotoxicity. Cells were treated with retinoic acid (10 uM) (RA) and vehicle or Dimebon in the absence of doxycycline. Cultures were maintained with RA and drugs treatments for 14 days. Cell viability was by LDH release and MTS tetrazolium reduction assays. Expression of a-syn protein was by western blotting and by immunocytochemistry and levels of a-syn mRNA was by quantitative PCR. Levels of a-syn mRNA were normalized to beta-tubulin mRNA. Dimebon at concentrations of between 1 and 100 nM were associated with a statistically significant reduction in LDH release in SH-SY5Y cells treated with hydrogen peroxide (15-20% reduction), 4-Br-A23187 (15-20%) and rotenone (55-60%). Elimination of doxycycline from the culture media induces a-syn expression by the Tet/off SH-SY5Y cells and increases LDH release and reduces MTS activity. Dimebon treatment (0.1 to 100 nM) was associated with a statistically significant improvement in cell viability as measured by the LDH and MTS assays. Dimebon (10 nM) treatment for 14 days decreases total levels of a-syn protein, measured by western blot analysis and immunocytochemistry. By western blotting it was found that levels of Triton X-100 insoluble a-syn aggregates as wells soluble monomers were reduced. No significant differences were found in the levels of a-syn mRNA measured by quantitative PCR. These results indicate that in cell cultures Dimebon enhances the viability of neuroblastoma cells under different cytotoxic conditions including oxidative stress (hydrogen peroxide), high intracellular calcium (4-Br-A23187), mitochondrial poison (rotenone) and high a-syn protein levels. Dimebon reduces a-syn protein but not mRNA suggesting an effect on protein turnover rather than synthesis. These studies suggest that Dimebon has mitochondrial actions that could play a role in modulating cell processes involving protein turnover which in the setting of neurodegenerative proteinopathies may be beneficial.