Rotenone induces cell death in primary dopaminergic culture by increasing ROS production and inhibiting mitochondrial respiration

Abstract

Although the definite etiology of Parkinson's disease is still unclear, increasing evidence has suggested an important role for environmental factors such as exposure to pesticides in increasing the risk of developing Parkinson's disease. In the present study, primary cultures prepared from embryonic mouse mesencephala were applied to investigate the toxic effects and underlying mechanisms of rotenone-induced neuronal cell death relevant to Parkinson's disease. Results revealed that rotenone destroyed dopaminergic neurons in a dose- and time-dependent manner. Consistent with the cytotoxic effect of rotenone as evidenced by dopaminergic cell loss, it significantly increased the release of lactate dehydrogenase into the culture medium, the number of necrotic cells in the culture and the number of nuclei showing apoptotic features. Rotenone exerted toxicity by decreasing the mitochondrial membrane potential, increasing reactive oxygen species production and shifting respiration to a more anaerobic state.

Introduction

Parkinson's disease (PD) is one of the most common neurodegenerative disorders and is characterized by the selective loss of dopaminergic neurons in the substantia nigra pars compacta (SNpc) and subsequent decrease of dopamine levels in the striatum, the main target innervated by these neurons (Shimizu et al., 2003). Although, the disease symptoms and neuropathology have been well characterized, the exact etiology and the mechanisms underlying the degeneration of nigral dopaminergic neurons are still unknown. On the other hand, several epidemiological and case control studies revealed that certain pesticides and other environmental factors such as farming, rural living and well water consumption have a strong correlation with an increased incidence of non-familial PD (Kitazawa et al., 2001, Gao et al., 2002, Wang et al., 2002, Sherer et al., 2003, Di Monte, 2003, Shimizu et al., 2003). Non-familial PD represents more than 90% of all PD cases and its onset occurs typically after 50 years of age (Moghal et al., 1994, Langston, 1998). An additional environmental risk for PD is occupational exposure to certain metals, most notably manganese (Gorell et al., 1999, Racette et al., 2001). Moreover, many pesticides and manganese are able to cause mitochondrial dysfunction by inhibiting complex I of the respiratory chain (Hollingworth et al., 1994, Gavin et al., 1999).

Rotenone, a naturally occurring compound derived from the roots of Derris and Lonchorcarpus plants species, is the active ingredient in hundreds of pesticide products widely used as a household insecticide and as a tool for eradicating nuisance fish populations in lakes and reservoirs (Betarbet et al., 2000). Because rotenone is extremely hydrophobic, it crosses biological membranes easily and does not depend on the dopamine transporter for access to the cytoplasm of dopaminergic neurons (Betarbet et al., 2000, Greenamyre et al., 2001). Rotenone is also a classical, well-characterized and high-affinity specific inhibitor of mitochondrial NADH dehydrogenase (complex I), one of the five enzymes complexes of the inner mitochondrial membrane involved in oxidative phosphorylation (Betarbet et al., 2000, Thiffault et al., 2000).

Treatment with rotenone or paraquat as new model substances for selective nigrostriatal damage is a matter of debate. On the one hand, rotenone has been shown to lead to selective dopaminergic cell death in vivo. In this context, Betarbet et al. (2000) and Sherer et al. (2003) reported selective nigrostriatal dopaminergic degeneration in rats infused intravenously or subcutaneously with rotenone, although rotenone also induced degeneration of non-dopaminergic neurons in both the basal ganglia and the brainstem (Hirsch et al., 2003). On the other hand, in in vitro models, it was reported that dopaminergic neurons were more sensitive to rotenone-induced toxicity than other neuronal cells and glial cells (Ahmadi et al., 2003, Kweon et al., 2004, Moon et al., 2005), but non-dopaminergic neurons were reduced by rotenone in primary mesencephalic culture as well (Nakamura et al., 2000, Sakka et al., 2003).

Here, we used primary dopaminergic cultures from embryonic mouse mesencephala to investigate in detail potential molecular mechanisms underlying the degeneration of dopaminergic neurons induced by rotenone. The direct toxicity of rotenone to dopaminergic neurons was identified by immunocytochemical staining. Fluorescence staining techniques were applied to measure necrosis/apoptosis, mitochondrial membrane potential (Δψ_m) and reactive oxygen species (ROS) formation in the overall culture. Determination of lactate dehydrogenase (LDH) and lactate in the culture medium reflected the condition of the overall culture as well. We demonstrate that rotenone destroyed dopaminergic neurons in a dose- and time-dependent manner. Furthermore, blocking of mitochondrial respiration and increasing ROS formation played an important role in rotenone toxicity.