Nrf2 Mediated Neuroprotection in In Vitro Models of Parkinson's Disease

Abstract

Oxidative stress and inflammation have long been associated with cell death in Parkinson's disease (PD). As a result, there has been a growing interest in antioxidant pathways and how these pathways might be exploited in order to slow the progressive loss of dopamine (DA) neurons associated with PD. One such pathway that has garnered attention in recent years is the nuclear factor erythroid 2-related factor 2-antioxidant response element (Nrf2-ARE) pathway. A number of groups have shown that activation of the Nrf2-ARE pathway prior to an oxidative insult can protect against DA cell loss both in vitro and in vivo. The next step in establishing its value as a potential therapeutic target is to determine if Nrf2 can be protective when activated either simultaneously with an insult, or when activated after the start of the degenerative process. This question was addressed in two parts: (1) Organotypic nigrostriatal co-cultures were treated acutely with either 6-hydroxydopamine (6-OHDA) or a combination of 6-OHDA and tert-butylhyrdoquinone (tBHQ), a known activator of the Nrf2 pathway. Treatment with 6-OHDA alone led to a significant loss in tyrosine hydroxylase-immunoreactivity (TH-ir). This effect was mitigated when cultures were treated with both 6-OHDA and tBHQ. Protection appeared to be mediated, at least in part, by increased antioxidant activity. Simultaneous treatment with both 6-OHDA and tBHQ increased NQO1 expression 17-fold compared with controls. (2) We developed a novel in vitro model of dopaminergic degeneration using organotypic nigrostriatal co-cultures, enabling us to administer therapeutics after the start of the lesion. Chronic treatment of cultures with low doses of beta-sitosterol glucoside led to a progressive and specific loss of ~50% of TH-ir over the course of two weeks. Furthermore, we observed a loss of DA cell phenotype, as indicated by significant decreases in TH and vesicular monoamine transporter 2 protein levels, before cells underwent apoptosis. Interestingly, activation of Nrf2 with tBHQ during this early stage of the degenerative process was able to restore lost phenotype and mitigate increases in oxidative damage, as evidenced by a decrease in nitrosylated proteins and lipid peroxidation. Combined, these two studies support the hypothesis that activation of the Nrf2 pathway may protect against dopaminergic degeneration.