Dataset Information

Greene-5P01NS017771-220003

ABSTRACT: Parkinson's disease is a prevalent neurodegenerative disorder for which there is no cure. The cause of PD symptoms is loss of dopamine neurons in the midbrain, but it is not known why these neurons die. Pesticide exposure is epidemiologically associated with PD, and administration of the organic pesticide rotenone to rats recapitulates most of the behavioral, neurochemical, and neuropathological findings in PD, including specific death of dopamine neurons. We have developed an in vitro model of rotenone toxicity using a dopaminergic cell line (SK-N-MC neuroblastoma cells) that mimics many of the cellular changes seen with in vivo rotenone toxicity and with PD, such as alpha-synuclein aggregation and oxidative damage. We are currently using this simple model to explore mechanisms of dopaminergic neurodegeneration, with our ultimate goal being the discovery of novel mechanisms for dopaminergic neuroprotection in PD. We will examine gene expression profiles of cultured SK-N-MC cells at several time points during rotenone exposure to determine pathways involved in rotenone toxicity and dopaminergic degeneration. We will compare these profiles to baseline profiles of rodent dopaminergic neurons that we have already obtained, as well as to profiles of dopamine neurons from rotenone-treated rats that we will obtain in the near future. We will also compare these data to published results from SN neurons from human PD patients. This technique will not only help us to detect gene expression changes relevant to dopaminergic neurodegeneration in PD, but it will allow us to determine if the SK-N-MC system can be reliably used to screen for neuroprotective therapies for PD. We anticipate that SK-N-MC cells will show a relevant subset of the gene changes seen in dopamine neurons in vivo and that this will guide us in the sorts of mechanisms and drugs that can be screened in this system. Chronic exposure to low levels of rotenone causes changes in gene expression in SK-N-MC cells that sensitize the cells to toxic insults. We also hypothesize that there are several compensatory protective pathways that are stimulated by chronic rotenone, although these pathways are ultimately ineffective at preventing damage. We anticipate that gene expression profiling of rotenone-treated cells over time will suggest several novel strategies for neuroprotective intervention. SK-N-MC cells will be grown in three different media: media only, vehicle (EtOH), and rotenone (5 nM). All current experimental evidence in our lab indicates that vehicle-treated cells are indistinguishable from media-only cells. Rotenone-treated cellls have a stereotypical response in culture. At one week, the only noticed change is an increase in alph-synuclein aggregation. At two weeks, evidence of increased oxidative stress appears (increased protein carbonyls and lipid peroxidation). At four weeks, the cells are markedly sensitized to oxidative challenge with H2O2. Therefore, we will examine gene expression at baseline, and during 1, 2, and 4 weeks of rotenone treatment. Three experiments will be performed, each lasting 4 weeks. For each experiment, three separate dishes of vehicle-treated, and rotenone-treated cells will be harvested at 1, 2, and 4 weeks (18 independent samples). Untreated, media-only cells will be harvested after 1 week in vitro to serve as baseline cells. Total RNA will be isolated. An equal amount of RNA from one dish per experiment per group will be used to compose the final samples. Therefore, each independent sample will consist of RNA from 3 separate experiments. This will allow us to take advantage of a pooling strategy, yet not sacrifice technical and biological replication. 21 samples will be sent to the Consortium. Three will be from untreated cells. Nine will be vehicle-treated at 1, 2, and 4 weeks (3 each). Nine will be rotenone-treated at 1, 2 , and 4 weeks (3 each). Each sample will be labeled and hybridized to one Affymetrix Human Genome U133 Plus 2.0 Gene Chip. With assistance of the consortium, we will analyze the data using the Signifiance Analysis of Microarrays (SAM) program and self-organizing map algorithms. Keywords: time-course

ORGANISM(S): Homo sapiens

PROVIDER: GSE4773 | GEO | 2006/06/06

SECONDARY ACCESSION(S): PRJNA95655

REPOSITORIES: GEO

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Project description:Parkinson's disease is a prevalent neurodegenerative disorder for which there is no cure. The cause of PD symptoms is loss of dopamine neurons in the midbrain, but it is not known why these neurons die. Pesticide exposure is epidemiologically associated with PD, and administration of the organic pesticide rotenone to rats recapitulates most of the behavioral, neurochemical, and neuropathological findings in PD, including specific death of dopamine neurons. We have developed an in vitro model of rotenone toxicity using a dopaminergic cell line (SK-N-MC neuroblastoma cells) that mimics many of the cellular changes seen with in vivo rotenone toxicity and with PD, such as alpha-synuclein aggregation and oxidative damage. We are currently using this simple model to explore mechanisms of dopaminergic neurodegeneration, with our ultimate goal being the discovery of novel mechanisms for dopaminergic neuroprotection in PD. We will examine gene expression profiles of cultured SK-N-MC cells at several time points during rotenone exposure to determine pathways involved in rotenone toxicity and dopaminergic degeneration. We will compare these profiles to baseline profiles of rodent dopaminergic neurons that we have already obtained, as well as to profiles of dopamine neurons from rotenone-treated rats that we will obtain in the near future. We will also compare these data to published results from SN neurons from human PD patients. This technique will not only help us to detect gene expression changes relevant to dopaminergic neurodegeneration in PD, but it will allow us to determine if the SK-N-MC system can be reliably used to screen for neuroprotective therapies for PD. We anticipate that SK-N-MC cells will show a relevant subset of the gene changes seen in dopamine neurons in vivo and that this will guide us in the sorts of mechanisms and drugs that can be screened in this system. Chronic exposure to low levels of rotenone causes changes in gene expression in SK-N-MC cells that sensitize the cells to toxic insults. We also hypothesize that there are several compensatory protective pathways that are stimulated by chronic rotenone, although these pathways are ultimately ineffective at preventing damage. We anticipate that gene expression profiling of rotenone-treated cells over time will suggest several novel strategies for neuroprotective intervention. SK-N-MC cells will be grown in three different media: media only, vehicle (EtOH), and rotenone (5 nM). All current experimental evidence in our lab indicates that vehicle-treated cells are indistinguishable from media-only cells. Rotenone-treated cellls have a stereotypical response in culture. At one week, the only noticed change is an increase in alph-synuclein aggregation. At two weeks, evidence of increased oxidative stress appears (increased protein carbonyls and lipid peroxidation). At four weeks, the cells are markedly sensitized to oxidative challenge with H2O2. Therefore, we will examine gene expression at baseline, and during 1, 2, and 4 weeks of rotenone treatment. Three experiments will be performed, each lasting 4 weeks. For each experiment, three separate dishes of vehicle-treated, and rotenone-treated cells will be harvested at 1, 2, and 4 weeks (18 independent samples). Untreated, media-only cells will be harvested after 1 week in vitro to serve as baseline cells. Total RNA will be isolated. An equal amount of RNA from one dish per experiment per group will be used to compose the final samples. Therefore, each independent sample will consist of RNA from 3 separate experiments. This will allow us to take advantage of a pooling strategy, yet not sacrifice technical and biological replication. 21 samples will be sent to the Consortium. Three will be from untreated cells. Nine will be vehicle-treated at 1, 2, and 4 weeks (3 each). Nine will be rotenone-treated at 1, 2 , and 4 weeks (3 each). Each sample will be labeled and hybridized to one Affymetrix Human Genome U133 Plus 2.0 Gene Chip. With assistance of the consortium, we will analyze the data using the Signifiance Analysis of Microarrays (SAM) program and self-organizing map algorithms.

Project description:The pesticide rotenone, a neurotoxin that inhibits the mitochondrial complex I, and destabilizes microtubules (MT) has been linked to Parkinson disease (PD) etiology and is often used to model this neurodegenerative disease (ND). Many of the mechanisms of action of rotenone are posited mechanisms of neurodegeneration; however, they are not fully understood. Therefore, the study of rotenone-affected functional pathways is pertinent to the understanding of NDs pathogenesis. This report describes the transcriptome analysis of a neuroblastoma (NB) cell line chronically exposed to marginally toxic and moderately toxic doses of rotenone. The results revealed a complex pleiotropic response to rotenone that impacts a variety of cellular events, including cell cycle, DNA damage response, proliferation, differentiation, senescence and cell death, which could lead to survival or neurodegeneration depending on the dose and time of exposure and cell phenotype. The response encompasses an array of physiological pathways, modulated by transcriptional and epigenetic regulatory networks, likely activated by homeostatic alterations. Pathways that incorporate the contribution of MT destabilization to rotenone toxicity are suggested to explain complex I-independent rotenone-induced alterations of metabolism and redox homeostasis. The postulated mechanisms involve the blockage of mitochondrial voltage-dependent anions channels (VDACs) by tubulin, which coupled with other rotenone-induced organelle dysfunctions may underlie many presumed neurodegeneration mechanisms associated with pathophysiological aspects of various NDs including PD, AD and their variant forms. Thus, further investigation of such pathways may help identify novel therapeutic paths for these NDs. SK-N-MC cells were grown in three different conditions: media + vehicle (EtOH), media + a sublethal dose (5 nM) of rotenone, and media + a slightly toxic dose (50 nM) of rotenone. Gene expression was examined at 1 and 4 weeks of rotenone treatment. Three experiments were performed, each lasting 1 and 4 weeks. For each experiment, 5 separate dishes of vehicle-treated, and rotenone-treated cells were harvested at 1 and 4 weeks (30 independent samples at each time point) and pooled into 3 samples (vehicle treated,and rotenone treated with 5 nM and 50 nM) at each time point for a total of 18 individual samples from all thre experiments. Total RNA isolated from each of the 18 individual samples was used to prepared labeled cRNA and hybridized to one Genechip (Affymetrix) Human Genome array HG-U133A at the UCLA microarray core facility (http://microarray.genetics.ucla.edu/). After QC check, the data was normalized and used to assess expression indexes and fold changes (FC > 2.0, compared to vehicle-treated controls) using the model-based expression indexes (MBEI) method implemented in dCHIP, ( http://biosun1.harvard.edu/complab/dchip/), and the Signifiance Analysis of Microarrays (SAM) software for multiple test corrections. Enrichment analysis was performed by the functional annotation tools implemented in DAVID ( http://david.abcc.ncifcrf.gov), to ascertain sets of rotenone DRGs that are enriched in certain biological annotations.

Project description:The cardinal clinical features of Parkinson's disease (PD) (rigidity, rest tremor, bradykinesia, and postural instability) result from selective loss of midbrain dopaminergic neurons. More specifically, dopaminergic neurons in the substantia nigra pars compacta (SNc) are much more susceptible to damage than the adjacent dopaminergic neurons in the ventral tegmental area (VTA). This dichotomy is not only seen in human Parkinson's disease, but also in many animal models of PD, including administration of the mitochondrial toxin rotenone to rats, which replicates many of the behavioral and neuropathological features of PD. The factors underlying this selective vulnerability are unknown, but could be related to differences in neuronal circuitry, differences in glial support, or intrinsic differences between the neuronal populations of the two regions. Elucidation of these factors may lead to a greater understanding of the pathogenesis and treatment of Parkinson's disease. We will determine gene expression profiles of untreated rat SNc and VTA dopaminergic neurons using laser capture microscopy to obtain region-specific neuronal mRNA. There are intrinsic differences in gene expression between dopaminergic neurons in the rat SNc and VTA that result in greater susceptibility of SNc neurons to degeneration in experimental parkinsonism. These differences may be related to dopamine metabolism, oxidative metabolism and stress, protein aggregation, or other unforseen pathways. We will compare gene expression profiles between SNc and VTA dopaminergic neurons in normal rats. No treatment or time points will be studied in this experiment. Animals will be anesthetized, sacrificed by decapitation, and brains frozen on dry ice. Frozen sections will be collected onto glass microscope slides and rapidly immunostained for tyrosine hydroxylase to identify dopaminergic neurons. SNc and VTA neurons (approx. 200 per sample) will be isolated using laser capture microscopy. Total RNA will be extracted and poly-A RNA will be amplified using a modified Eberwine protocol. aRNA will be sent to the centers for labeling and hybridization to Affymetrix rat U34A arrays. We have confirmed with the center that our aRNA protocol is compatible with the centers amplification protocols; in fact, it is essentially identical. We will be providing a two-round amplification product to the center for labeling and hybridization. We recognize that using RNA after three rounds of amplification may decrease sensitivity for low copy number transcripts, but favor this approach versus pooling our samples (which are inherently paired) at this point. We have discussed this point in detail with the center. SNc and VTA samples from eight animals (16 samples total) will be provided to mitigate differences specific to individual animals. With the assisatnce of the center, paired t-tests will be used to determine differential expression between the two regions. Permutational t-test analysis and/or Benjamini and Hochberg analysis of expression ratios will be used to protect against multiple comparisons. Selected differentially expressed genes will be validated on separate tissue samples using quantitative RT-PCR or in situ hybridization.

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Greene-5P01NS017771-220003

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