Wild-type and mutant alpha-synuclein induce a multi-component gene expression profile consistent with shared pathophysiology in different transgenic mouse models of PD.

The pathophysiological processes that cause Parkinson's disease (PD) affect dopamine neurons residing in the substantia nigra with devastating consequences for normal movement. One important gene involved in both familial and sporadic PD is alpha-synuclein. We have generated three strains of alpha-synuclein transgenic mice to study the pathologic consequences of the targeted expression of mutant or wild-type human alpha-synuclein in a model system. We have analyzed gene expression patterns in these mice using high throughput microarrays in anatomical regions implicated in disease (substantia nigra and brainstem). Our study reveals gene dosage-dependent dysregulation of several genes important for the dopaminergic phenotype in mice over-expressing wild-type human alpha-synuclein in the substantia nigra at time points preceding neuronal cell death. Analysis of mutant alpha-synuclein mice at a time point when pathology is advanced reveals several new candidate genes that may play a role in neuronal demise and/or protein accumulation.

Robust dysregulation of gene expression in substantia nigra and striatum in Parkinson's disease.

Large-scale genomics approaches are now widely utilized to study a myriad of human diseases. These powerful techniques, when combined with data analysis tools, detect changes in transcript abundance in diseased tissue relative to control. We hypothesize that specific differential gene expression underlies important pathogenic processes in Parkinson's disease, which is characterized by the gradual loss of dopaminergic neurons in the substantia nigra and consequent loss of dopamine in the striatum. We have therefore examined gene expression levels in the human parkinsonian nigrostriatal pathway, and compared them with those of neurologically normal controls. Using unsupervised clustering methods, we demonstrate that relatively few genes' expression levels can effectively distinguish between disease and control brains. Further, we identify several interesting patterns of gene expression that illuminate pathogenic cascades in Parkinson's disease. In particular is the robust loss of synaptic gene expression in diseased substantia nigra and striatum.

Use of a mixed tissue RNA design for performance assessments on multiple microarray formats.

The comparability and reliability of data generated using microarray technology would be enhanced by use of a common set of standards that allow accuracy, reproducibility and dynamic range assessments on multiple formats. We designed and tested a complex biological reagent for performance measurements on three commercial oligonucleotide array formats that differ in probe design and signal measurement methodology. The reagent is a set of two mixtures with different proportions of RNA for each of four rat tissues (brain, liver, kidney and testes). The design provides four known ratio measurements of >200 reference probes, which were chosen for their tissue-selectivity, dynamic range coverage and alignment to the same exemplar transcript sequence across all three platforms. The data generated from testing three biological replicates of the reagent at eight laboratories on three array formats provides a benchmark set for both laboratory and data processing performance assessments. Close agreement with target ratios adjusted for sample complexity was achieved on all platforms and low variance was observed among platforms, replicates and sites. The mixed tissue design produces a reagent with known gene expression changes within a complex sample and can serve as a paradigm for performance standards for microarrays that target other species.

The External RNA Controls Consortium: a progress report.

Standard controls and best practice guidelines advance acceptance of data from research, preclinical and clinical laboratories by providing a means for evaluating data quality. The External RNA Controls Consortium (ERCC) is developing commonly agreed-upon and tested controls for use in expression assays, a true industry-wide standard control.

Characterization and gene expression profiling of a stable cell line expressing a cell cycle GFP sensor.

The use of stable cell lines expressing fusions with green fluorescent protein (GFP) has increased significantly in recent years. In this study we have used a range of complimentary analytical techniques to examine the characteristics of a cell line stably expressing a EGFP cell cycle sensor relative to parental U2OS cells. Analysis of cell cycle duration and cell cycle phase distribution by cell growth assays and flow cytometry revealed that the two cell lines had identical doubling times and cell cycle distributions. Measurement of EGFP fusion protein mRNA by quantitative RT-PCR indicated a EGFP sensor expression level equivalent to endogenous Cyclin B1 (7000 copies/cell in G2). Microarray analysis showed a 0.9% (>2 fold at p<0.001 across 20,000 genes) difference in global gene expression levels between parental and EGFP expressing U2OS cells, with no significant differences in expression of A, B, C, D, E, F, G, H, I, K, L, M or T type Cyclins between the two cell types. These results confirm that engineering a stable cell line for low expression of a EGFP cell cycle sensor is minimally perturbing to the cell cycle and cellular gene expression.

Dysregulation of gene expression in the 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine-lesioned mouse substantia nigra.

Parkinson's disease pathogenesis proceeds through several phases, culminating in the loss of dopaminergic neurons of the substantia nigra (SN). Although the 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) model of oxidative SN injury is frequently used to study degeneration of dopaminergic neurons in mice and non-human primates, an understanding of the temporal sequence of molecular events from inhibition of mitochondrial complex 1 to neuronal cell death is limited. Here, microarray analysis and integrative data mining were used to uncover pathways implicated in the progression of changes in dopaminergic neurons after MPTP administration. This approach enabled the identification of small, yet consistently significant, changes in gene expression within the SN of MPTP-treated animals. Such an analysis disclosed dysregulation of genes in three main areas related to neuronal function: cytoskeletal stability and maintenance, synaptic integrity, and cell cycle and apoptosis. The discovery and validation of these alterations provide molecular evidence for an evolving cascade of injury, dysfunction, and cell death.