Comparative prion disease gene expression profiling using the prion disease mimetic, cuprizone

Abstract

Identification of genes expressed in response to prion infection may elucidate biomarkers for disease, identify factors involved in agent replication, mechanisms of neuropathology and therapeutic targets. Although several groups have sought to identify gene expression changes specific to prion disease, expression profiles rife with cell population changes have consistently been identified. Cuprizone, a neurotoxicant, qualitatively mimics the cell population changes observed in prion disease, resulting in both spongiform change and astrocytosis. The use of cuprizone-treated animals as an experimental control during comparative expression profiling allows for the identification of transcripts whose expression increases during prion disease and remains unchanged during cuprizone-triggered neuropathology. In this study, expression profiles from the brains of mice preclinically and clinically infected with Rocky Mountain Laboratory (RML) mouse-adapted scrapie agent and age-matched controls were profiled using Affymetrix gene arrays. In total, 164 genes were differentially regulated during prion infection. Eighty-three of these transcripts have been previously undescribed as differentially regulated during prion disease. A 0.4% cuprizone diet was utilized as a control for comparative expression profiling. Cuprizone treatment induced spongiosis and astrocyte proliferation as indicated by glial fibrillary acidic protein (Gfap) transcriptional activation and immunohistochemistry. Gene expression profiles from brain tissue obtained from cuprizone-treated mice identified 307 differentially regulated transcript changes. After comparative analysis, 17 transcripts unaffected by cuprizone treatment but increasing in expression from preclinical to clinical prion infection were identified. Here we describe the novel use of the prion disease mimetic, cuprizone, to control for cell population changes in the brain during prion infection.

Figure 1

Expression of Gfap during cuprizone treatment and prion disease. Relative Gfap mRNA levels from 3, 4, 6, 7, 8 and 10 weeks of cuprizone treatment and 108, 158 and 198 dpi RML infection were generated by the Pfaffl method after qPCR, as indicated by fold change versus age-matched controls. After 8 and 10 weeks of cuprizone intoxication, Gfap levels most closely resemble those observed at 158 dpi of prion disease. Tbp was used as an endogenous control for normalization.

Figure 2

Spongiosis and astrocytosis in mice treated with cuprizone, infected with RML or controls. (A, D, G, J and M) Hematoxylin and eosin staining shows spongiosis in the white matter of the cerebellum after eight weeks of cuprizone treatment and after 158 and 198 days of RML infection. (B, E, H, K and N) Anti-GFAP immunohistochemistry shows star-shaped astrocytosis in the frontal cortex after cuprizone treatment and after 158 and 198 days of RML infection. (C, F, I, L and O) PrP^TSE staining in the medulla. Red, positive staining can be seen at 158 and 198 dpi and is not observed after cuprizone treatment. Scale bars: (A, D, G, J, M, F, L and O) = 50 µm; (B, E, H, K, N, C and I) = 100 µm.

Figure 3

Functional analysis of transcripts differentially regulated in the brains of mice treated with cuprizone. The DAVID annotational database was utilized to determine which gene ontology biological processes were affected by cuprizone treatment (p ≤ 0.05). Upregulated transcripts are in green; downregulated transcripts are in blue.

Figure 4

Comparative analysis between transcripts increasing in expression during prion disease and unchanging during cuprizone treatment. (A) After SOM clustering analysis, one cluster emerged containing transcripts that increased in expression over the time course of prion infection (shown here, 333 total transcripts). (B) The cuprizone data set was mined for one transcript whose expression was unchanged as compared to control (Tbp). Using the Euclidean similarity metric, transcripts with 100-85% similar expression patterns to Tbp were identified (22,729 total transcripts). (C) Venn diagram illustrating the comparative gene expression analysis of both data sets described above. In total, 17 probe sets belonged to both data sets and represent transcripts whose expression increased during prion disease but did not change with cuprizone treatment. This overlap signifies transcripts whose expression is related to prion disease and not associated to the changing cell population that occurs during neuroinflammation.

Figure 5

Annotated transcripts upregulated during prion disease and unrelated to cuprizone-induced neuroinflammation. Of the seventeen transcripts that increased in expression over the course of prion disease and remained unchanged during cuprizone treatment, fourteen were found to be annotated. The log₂ transformed expression ratios versus control are shown here for ease in visualization, allowing gene expression to be centered on zero. The shaded region represents non-significant, unchanging transcript levels. Unchanging was classified as |0.0-0.3| log₂ transformed expression ratio versus control.

Figure 6

Validation of selected microarray results by qPCR. The expression of Rsad2, ApoD and Socs3 was analyzed from RML-infected brain homogenate obtained at two preclinical time points (108 and 158 dpi) and at clinical disease (198 dpi) as well as after eight weeks of cuprizone treatment. Pooled RNA samples from eight infected/treated and eight controls were studied at each time point. The chart above shows relative mRNA levels, as indicated by fold change versus age-matched controls, generated by the Pfaffl method after qPCR, with a threshold line at 1.5-fold. Tbp was used as an endogenous control for normalization. Samples used for qPCR were from the same samples used for microarray analysis. Microarray data was taken from Table 1 and Supplemental Table 2.