Analysis of transcriptional profiles and functional clustering of global cerebellar gene expression in PCD3J mice

Abstract

The Purkinje cell degeneration (PCD) mutant mouse is characterized by a degeneration of cerebellar Purkinje cells and progressive ataxia. To identify the molecular mechanisms that lead to the death of Purkinje neurons in PCD mice, we used Affymetrix microarray technology to compare cerebellar gene expression profiles in pcd3J mutant mice 14 days of age (prior to Purkinje cell loss) to unaffected littermates. Microarray analysis, Ingenuity Pathway Analysis (IPA) and expression analysis systematic explorer (EASE) software were used to identify biological and molecular pathways implicated in the progression of Purkinje cell degeneration. IPA analysis indicated that mutant pcd3J mice showed dysregulation of specific processes that may lead to Purkinje cell death, including several molecules known to control neuronal apoptosis such as Bad, CDK5 and PTEN. These findings demonstrate the usefulness of these powerful microarray analysis tools and have important implications for understanding the mechanisms of selective neuronal death and for developing therapeutic strategies to treat neurodegenerative disorders.

Figure 1

Global functional category comparison of genes sets up-regulated or down-regulated in the homozygous mutant groups compared to wild type litter mates. Genes linked to cell death, developmental and reproductive dysfunctions were differentially expressed in the homozygous mutant group. Increasing value of −log (significance) indicated increased confidence for each category.

Figure 2

(A) Network analysis. This network from is IPA analysis of gene sets up- or down- regulated in the homozygous mutant compared to wild type litter mates The network is displayed as nodes (genes or gene products) and edges (lines; biological relationships between nodes). Solid lines denote direct interactions, while dotted lines represent indirect interactions between the genes. Genes colored green displayed two-fold or larger decreases, while genes colored red displayed two-fold or larger increases. Table 2 is a description on the genes represented in this network. (B) Real-time RT-PCR analysis of transcripts from pcd3J mice. The RNA expression of selected genes was assessed by real-time RT-PCR in pcd/+ and pcd/pcd mice and compared to wild-type mice.

Figure 3

Bad protein expression in the Purkinje cell layer of the cerebellum of the P14 Nna1+/+ and Nna1-/- mutant mice. Cells in the Purkinje cell layer were counterstained with 4′,6-diamidino-2-phenylindole (DAPI), a nuclear label (A, D). Bad staining was absent in the Purkinje cell layer of Nna1 +/+ mice (B) but expression was detected in Purkinje cells of Nna1-/- mutants (E). Figures C and F are merged images indicating co-localization of DAPI and Bad in NNa1+/+ (C) and Nna1-/- mice (F). The scale bar is 20 μm.