Low dose rotenone treatment causes selective transcriptional activation of cell death related pathways in dopaminergic neurons in vivo
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.
References (65)
- et al.
Rotenone destroys dopaminergic neurons and induces parkinsonian symptoms in rats
Behav. Brain Res.
(2002) - et al.
Nuclei and subnuclei gene expression profiling in mammalian brain
Brain Res.
(2002) - et al.
Effects of gender on nigral gene expression and Parkinson disease
Neurobiol. Dis.
(2007) - et al.
Behavioral and immunohistochemical effects of chronic intravenous and subcutaneous infusions of varying doses of rotenone
Exp. Neurol.
(2004) - et al.
Peroxisome proliferator-activated receptor {gamma} Up-regulates the Bcl-2 anti-apoptotic protein in neurons and induces mitochondrial stabilization and protection against oxidative stress and apoptosis
J. Biol. Chem.
(2007) - et al.
Profiling genes related to mitochondrial function in mice treated with N-methyl-4-phenyl-1,2,3,6-tetrahydropyridine
Biochem. Biophys. Res. Commun.
(2003) - et al.
Overexpression of VDUP1 mRNA sensitizes HeLa cells to paraquat
Biochem. Biophys. Res. Commun.
(2002) - et al.
Robust dysregulation of gene expression in substantia nigra and striatum in Parkinson's disease
Neurobiol. Dis.
(2006) - et al.
Identification of thioredoxin-binding protein-2/vitamin D(3) up-regulated protein 1 as a negative regulator of thioredoxin function and expression
J. Biol. Chem.
(1999) - et al.
Cloning and characterization of a novel GRP78-binding protein in the rat brain
J. Biol. Chem.
(2003)
Cirp protects against tumor necrosis factor-alpha-induced apoptosis via activation of extracellular signal-regulated kinase
Biochim. Biophys. Acta.
Mitochondrial complex I deficiency in Parkinson's disease
Lancet
Expression of PEX11beta mediates peroxisome proliferation in the absence of extracellular stimuli
J. Biol. Chem.
Peroxisomes and oxidative stress
Biochim. Biophys. Acta
Hyperglycemia promotes oxidative stress through inhibition of thioredoxin function by thioredoxin-interacting protein
J. Biol. Chem.
N-myristoyltransferase 2 expression in human colon cancer: cross-talk between the calpain and caspase system
FEBS Lett.
Subcutaneous rotenone exposure causes highly selective dopaminergic degeneration and alpha-synuclein aggregation
Exp. Neurol.
High levels of mitochondrial DNA deletions in substantia nigra neurons in aging and Parkinson disease
Nat. Genet.
Chronic systemic pesticide exposure reproduces features of Parkinson's disease
Nat. Neurosci.
Idiopathic Parkinson's disease: possible routes by which vulnerable neuronal types may be subject to neuroinvasion by an unknown pathogen
J. Neural. Transm.
Protective action of the peroxisome proliferator-activated receptor-gamma agonist pioglitazone in a mouse model of Parkinson's disease
J. Neurochem.
‘Rejuvenation’ protects neurons in mouse models of Parkinson's disease
Nature
Vitamin D3 upregulated protein 1 (VDUP1) is a regulator for redox signaling and stress-mediated diseases
J. Dermatol.
Deficiency of the zinc finger protein ZPR1 causes neurodegeneration
Proc. Natl. Acad. Sci. U. S. A.
Two N-myristoyltransferase isozymes play unique roles in protein myristoylation, proliferation, and apoptosis
Mol.Cancer Res.
RNA interference targeting the M2 subunit of ribonucleotide reductase enhances pancreatic adenocarcinoma chemosensitivity to gemcitabine
Oncogene
Empirical bayes methods and false discovery rates for microarrays
Genet. Epidemiol.
Small heat-shock protein Hsp20 phosphorylation inhibits beta-agonist-induced cardiac apoptosis
Circ. Res.
Ageing and Parkinson's disease: substantia nigra regional selectivity
Brain
Identification of a neural-specific cDNA, NPDC-1, able to down-regulate cell proliferation and to suppress transformation
Proc. Natl. Acad. Sci. U. S. A.
Gene expression analysis in N-methyl-4-phenyl-1,2,3,6-tetrahydropyridine mice model of Parkinson's disease using cDNA microarray: effect of R-apomorphine
J. Neurochem.
Gene expression profiling of parkinsonian substantia nigra pars compacta; alterations in ubiquitin–proteasome, heat shock protein, iron and oxidative stress regulated proteins, cell adhesion/cellular matrix and vesicle trafficking genes
J. Neural. Transm.
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Present address: CNRS UMR 5227, Universite Victor Segalen Bordeaux 2, 33076 Bordeaux, France.