2009 Southern Regional Meeting Abstracts

Purpose of Study: The earliest signs of autoimmune pathology in NOD mice occur at 5 weeks of age. To gain an understanding of the molecular events that lead to initiation of this autoimmune disease we evaluated gene expression abnormalities at mRNA and protein levels in the autoimmune effector cells, spleen leukocytes.
Methods Used: RNA and protein were extracted from spleen leukocytes of NOD mice and two control strains: NON and C57BL/6 at 2 and 4 weeks of age (n=5 for each age and strain). RNA levels were assayed on expression arrays covering ~39,000 mouse genes. Protein expression was examined by 2-dimensional gel electrophoresis.
Summary of Results: Statistical analysis of the Affymetrix data by 1-way-ANOVA identified 175 and 189 probesets (genes), with highly significant (p<0.005) expression differences at 2 and 4 weeks, respectively, between at least one of the 3 strains. Hierarchical clustering of these gene lists revealed 51 and 76 of these probesets, as differentially expressed in NOD compared to both control strains (45 lower 6 higher in NOD at 2 weeks; 67 lower 9 higher in NOD at 4 weeks). A one-way ANOVA analysis and hierarchical clustering of the proteome data identified 11 and 8 spots as differentially expressed in NOD mice compared to both control strains, at age 2 and 4 weeks, respectively. We have identified 7 and 6 of the respective number of spots. We subjected the four transcriptome/protein gene lists to pathway analysis (www.ingenuity.com). The transcriptome network centered around MYC, TNF, and Ca2+ at age 2 weeks, and around TP53, PRKCA, IL4 and IL15 at age 4 weeks. The networks for the proteome data clustered around MYC, PRKACA and HSPA8* in 2 week old mice, while MYC, PPP5A, CTNNA1, TNF, IL4, hydrogen peroxide as well as HSP90AA1 were central in 4-week old mice.
Conclusions: This data suggests that the basic defects in prediabetic NOD mice involve gene expression abnormalities associated with apoptosis/cellular growth, cellular activation, inflammation, and oxidative stress. Both the transcriptome and proteome data suggests that abnormalities in T-cell differentiation centered on the TH2 cytokine IL4 develop between 2 and 4 weeks of age, before the appearance of autoimmune pathology.