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. Keywords: time-course

Project description:Transcript profiles of Postia placenta grown on media containing ball-milled aspen or ball-milled pine as the sole carbon source were analyzed. Array design was based on the DoE's Joint Genome Institute's gene models for P. placenta version 1. The research goal is to identtify genes essential for cellulose depolymerization. From a data set of 12,438 unique alleles, each NimbleGen (Madison, WI) array featured 10 unique 60mers per gene, all in triplicate. The dataset was manually annotated to include only the ‘best allelic model’ among CAZY-encoding genes. Total RNA was purified from medium containing ball-milled aspen or ball-milled pine as the sole carbon source. Three biological replicates per medium were used (6 separate arrays). RNA was converted to double-strand cDNA and labeled with the Cy3 fluorophore sample for hybridization to the Postia placenta MAD-698 whole genome expression array by Roche NimbleGen (Iceland). In brief, 10ug of total RNA was incubated with 1X first strand buffer, 10 mM DTT, 0.5mM dNTPs, 100 pM oligo T7 d(T)24 primer, and 200 units of SuperScript II (Invitrogen) for 60 min at 42°C. Second strand cDNA was synthesized by incubation with 1X second strand buffer, 0.2mM dNTPs, 0.07 units per ul DNA ligase (Invitrogen), 0.27 units per ul DNA polymerase I (Invitrogen), 0.013 units per ul RNase H (Invitrogen), at 16°C for 2 hours. Immediately following, 10 units T4 DNA polymerase (Invitrogen) was added for additional 5 minute incubation at 16°C. Double-stranded cDNA was treated with 27ng/ul of RNase A (EpiCenter Technologies) for 10 minutes at 37°C. Treated cDNA was purified using an equal volume of phenol:chloroform:isoamyl alcohol (Ambion), ethanol precipitated, washed with 80% ethanol, and resuspended in 20ul water. One ug of each cDNA sample was amplified and labeled with 1 unit per ul of Klenow Fragment (New England BioLabs) and 1 O.D unit of Cy3 fluorophore (TriLink Biotechnologies, Inc.) for 2 hours at 37°C. Array hybridization was carried out with 6ug of labeled cDNA suspended in NimbleGen hybridization solution for 17 hours at 42°C.

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Balbir Ball Greene Nehaul

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