Protective Effect of PACAP Against Salsolinol and Inflammatory-Mediated Neurotoxicity: Implications for Parkinson's Disease

Parkinson's disease (PD) is a progressive degenerative disorder of the central nervous system (CNS), which results from a progressive decline in dopaminergic cells in the substantia nigra (SN), a region of the midbrain. Several treatments for PD have been developed, but none has been shown to significantly slow or stop the disease progression. Consequently, there is a dire need to find an effective therapy to halt PD disease progression. Recent discoveries suggest that the neuronal degeneration in PD likely involves several cellular and molecular events, including oxidative stress, proapoptotic mechanisms, as well as inflammation through microglial activation. Therefore, effort is being put into finding new therapies for the treatment of PD that targets these various etiologies. One potential drug therapy is pituitary adenylate cyclase-activating polypeptide (PACAP).;PACAP, originally isolated from the sheep hypothalamic extract on the basis of its ability to stimulate cAMP formation in the anterior pituitary cells, activates the pituitary adenylate cyclase (PAC) 1 receptor, which is highly expressed in the SN. PACAP has been shown to be a potent neuroprotective agent in models of ischemic and traumatic brain injuries, and exerts neuroprotection through up-regulation of brain derived neurotrophic factor (BDNF) and its signal transduction pathway: phosphorylated cyclic AMP response element binding protein (p-CREB). Additionally, several studies have shown that PACAP leads to a down-regulation of caspase 3 levels (an apoptotic marker), thus reducing apoptotic mediated cell death.;In this study, we sought to determine whether PACAP could protect SH-SY5Y cells, a human derived dopaminergic cell line used to model PD, against toxicity induced by the direct application of salsolinol (SALS) and lipopolysaccharide (LPS). Furthermore, we tested if PACAP could protect SH-SY5Y cells against the application of inflammatory mediators present in conditioned media (CM) isolated from THP-1 microglia-like cells stimulated with LPS and interferon gamma (IFN-γ). Salsolinol is an endogenous dopamine metabolite with selective toxicity to nigral dopaminergic neurons. LPS (a gram negative derived endotoxin) and IFN-γ (a pro-inflammatory cytokine) are commonly used to activate microglia cells to release inflammatory mediators, which are directly implicated in PD.;The results of this study show that exposure of SH-SY5Y cells to toxicants, direct or indirect lead to significant cell death. Direct exposure to 400 uM SALS for 24 h resulted in approximately 50% cell death. Application of 300 ug/ml LPS directly to the SH-SY5Y cells for 72 h resulted in approximately 35% cell death. Conditioned media isolated from THP-1 cells stimulated with the combination of LPS (1000 ng/ml) + IFN-γ (30 ng/ml) for 72 h resulted in approximately 55% toxicity. The cell death caused by these PD-inducing agents was mediated by apoptosis and increases in caspase-3 levels as determined by cell flow cytometry and western blot analysis. Cellular toxicity was also associated with reductions in BDNF and p-CREB protein expression. Pre-treatment with PACAP dose-dependently attenuated SH-SY5Y cell death and the associated changes in apoptosis and neural protein markers. PACAP receptor antagonist, PACAP 6-38 in turn, dose-dependently blocked the effects of PACAP. Neither PACAP nor PACAP antagonist had any effect of its own on cellular viability.;These results suggest protective effect of PACAP in cellular model of PD. Hence, PACAP or its agonists could be of therapeutic potential in PD.