Elsevier

Neurobiology of Disease

Volume 33, Issue 2, February 2009, Pages 182-192
Neurobiology of Disease

Low dose rotenone treatment causes selective transcriptional activation of cell death related pathways in dopaminergic neurons in vivo

https://doi.org/10.1016/j.nbd.2008.10.001Get rights and content

Abstract

Mitochondrial complex I inhibition has been implicated in the degeneration of midbrain dopaminergic (DA) neurons in Parkinson's disease. However, the mechanisms and pathways that determine the cellular fate of DA neurons downstream of the mitochondrial dysfunction have not been fully identified. We conducted cell-type specific gene array experiments with nigral DA neurons from rats treated with the complex I inhibitor, rotenone, at a dose that does not induce cell death. The genome wide screen identified transcriptional changes in multiple cell death related pathways that are indicative of a simultaneous activation of both degenerative and protective mechanisms. Quantitative PCR analyses of a subset of these genes in different neuronal populations of the basal ganglia revealed that some of the changes are specific for DA neurons, suggesting that these neurons are highly sensitive to rotenone. Our data provide insight into potentially defensive strategies of DA neurons against disease relevant insults.

Introduction

Although many neuronal populations are affected in Parkinson disease (PD), dopaminergic (DA) neurons in the substantia nigra pars compacta (SNC) are among the most severely affected and their death leads to major neurological disability (Hirsch et al., 1988, Fearnley and Lees, 1991). With the exception of rare hereditary forms of the disease, the causes of PD are not known. Deletions of mitochondrial DNA (Bender et al., 2006) and a decreased activity of complex I of the respiratory chain (Schapira et al., 1989, Schapira et al., 1990, Mann et al., 1992, Swerdlow et al., 1996) have implicated mitochondrial dysfunction in the degenerative process occurring in sporadic PD. This hypothesis is further supported by evidence that two ion channels that are expressed by SNC-DA neurons render these neurons more susceptible to oxidative stress (Liss et al., 2005, Chan et al., 2007). Despite increasing knowledge about the mechanisms responsible for the vulnerability of DA neurons to oxidative stress, the downstream molecular responses to the insult are not well understood.

To identify cellular pathways and mechanisms that are activated at an early stage of mitochondrial dysfunction in SNC-DA neurons we performed transcriptional analyses of these neurons in rats treated with low doses of the mitochondrial complex I inhibitor rotenone (Sherer et al., 2003a). Rotenone has been shown to reproduce key features of PD, including motor deficits and a variable loss of DA neurons and terminals (Betarbet et al., 2000, Alam and Schmidt, 2002, Sherer et al., 2003b, Fleming et al., 2004). However, the vulnerability of SNC-DA neurons to rotenone, and the selectivity of the insult highly depend on experimental conditions and vary in individual animals (Höglinger et al., 2003, Zhu et al., 2004).

For the present study, our goal was to produce a low level insult that possibly affected DA neurons without inducing rapid cell loss. Our previous studies have shown that after administration of 2.0 mg/kg/day s.c. for 3 weeks, a high percentage of surviving animals have normal cell counts of DA neurons in the SNC and normal TH protein expression levels in the striatum despite their clearly detectable motor impairment (Fleming et al., 2004, Zhu et al., 2004). To further reduce the risk of inducing a structural deficit, we reduced the exposure time to one week. We then selected a subset of animals with both weight loss and behavioral impairment but without a loss of tyrosine hydroxylase- (TH) positive fibers in the striatum, and performed transcriptome analyses of laser-capture microdissected DA neurons (Bonaventure et al., 2002, Kamme et al., 2004). The gene array experiments identified large scale transcriptional alterations in SNC-DA neurons with genes involved in the regulation of cell death comprising one of the most prominent functional categories. Quantitative PCR (qPCR) analyses of a subset of these genes in GABAergic neurons of the substantia nigra (SN) and striatum revealed that the transcriptional activation is specific for SNC-DA neurons, indicating selectivity in the molecular response elicited by low cumulative doses of rotenone in this neuronal population.

Section snippets

In vivo rotenone administration

Adult male Lewis rats (Charles River Labs, Michigan), which have previously been shown to develop consistent rotenone induced lesions (Betarbet et al., 2000), were used in the current study. All experiments were performed in accordance with the National Research Council Guide for the Care and Use of Laboratory Animals and were approved by the University of California, Los Angeles and the US Air Force Research Laboratory (AFRL)-Brooks Institutional Animal Care and Use Committee. 34 animals

Animals selected for the transcriptome analyses showed a mild weight loss and decreased exploratory motor behavior

In the present study 34 male Lewis rats received continuous subcutaneous rotenone infusions of 2 mg/kg/day for one week. This dose had only mild toxic effects as indicated by the high survival rate (33 out of 34 animals), and previously demonstrated absence of DA neuron loss in the SNC (Fleming et al., 2004, Zhu et al., 2004). Except for the animal that died on day four of the treatment period, there were no signs of general distress, such as irritability or reduced responsiveness to external

Discussion

The main goal of our experiments was to study the transcriptional response of DA neurons in the SNC to mild rotenone-induced cellular stress. Therefore, dose and duration of treatment were designed to minimize histologically detectable structural defects of the nigrostriatal system. Our goal was to avoid the relatively acute cell death induced by higher doses of the toxin (Betarbet et al., 2000, Sherer et al., 2003a, Sherer et al., 2003b, Höglinger et al., 2003), which would likely bias the

Acknowledgments

We thank Lee Goodglick for providing access to the Arcturus PixCell II laser-capture microscope and Desmond Smith for usage of his ABI 7900HT sequence detection system.

Funding: The work was supported by US Army MRMC contract DAMD17-94-C-4069 and Public Health Service awards U54 ES12078 and P50 NS38367.

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    Present address: CNRS UMR 5227, Universite Victor Segalen Bordeaux 2, 33076 Bordeaux, France.

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