Medulla oblongata transcriptome changes during presymptomatic natural scrapie and their association with prion-related lesions

Abstract

Background: The pathogenesis of natural scrapie and other prion diseases is still poorly understood. Determining the variations in the transcriptome in the early phases of the disease might clarify some of the molecular mechanisms of the prion-induced pathology and allow for the development of new biomarkers for diagnosis and therapy. This study is the first to focus on the identification of genes regulated during the preclinical phases of natural scrapie in the ovine medulla oblongata (MO) and the association of these genes with prion deposition, astrocytosis and spongiosis.

Results: A custom microarray platform revealed that 86 significant probes had expression changes greater than 2-fold. From these probes, we identified 32 genes with known function; the highest number of regulated genes was included in the phosphoprotein-encoding group. Genes encoding extracellular marker proteins and those involved in the immune response and apoptosis were also differentially expressed. In addition, we investigated the relationship between the gene expression profiles and the appearance of the main scrapie-associated brain lesions. Quantitative Real-time PCR was used to validate the expression of some of the regulated genes, thus showing the reliability of the microarray hybridization technology.

Conclusions: Genes involved in protein and metal binding and oxidoreductase activity were associated with prion deposition. The expression of glial fibrillary acidic protein (GFAP) was associated with changes in the expression of genes encoding proteins with oxidoreductase and phosphatase activity, and the expression of spongiosis was related to genes encoding extracellular matrix components or transmembrane transporters. This is the first genome-wide expression study performed in naturally infected sheep with preclinical scrapie. As in previous studies, our findings confirm the close relationship between scrapie and other neurodegenerative diseases.

Figure 1

Quantification of PrP^Scdeposition, glial fibrillary acidic protein expression and spongiform degeneration. The values represent the means (± standard deviation) of intensity of the DAB color (PrP^Sc and astrocytosis) and Haematoxiline & Eosine (spongiosis) obtained from ImageJ software (A). Grey bar correspond to scrapie-affected sheep and black bar to control sheep. Significant differences were detected using Student´s t test (**P < 0.01, *P < 0.05). A generalized increase in the expression of the astroglial marker glial fibrillary acidic protein (GFAP) was observed in the brains of the scrapie-affected sheep (P < 0.01). Hyperplasia and hypertrophy of the stellate GFAP-positive cells were observed in the medulla oblongata of the affected sheep, which is consistent with reactive astrocytosis. PrP^Sc staining in control (B) and scrapie medulla oblongata sample (C). GFAP staining in control (D) and scrapie medulla oblongata sample (E). Haematoxylin/Eosin staining in control (F) and scrapie medulla oblongata sample (G).

Figure 2

Condition trees of the clustering analysis. The hierarchical cluster analysis (Euclidean distance clustering algorithm) was performed using PermutMatrix [32], and 86 clones/genes differed significantly. Each colored bar represents a gene. The color represents the level of expression, and the sample information is listed across the top. The names of the known genes are indicated. Note the distinct patterns of altered gene expression between the positive and control animals.

Figure 3

Relationship between gene expression profiles and scrapie histopathological lesions. Proteins encoded by genes whose expression is associated with PrP^Sc deposition, glial fibrillary acidic protein expression and spongiosis. Only the highly significant related genes are shown (P < 3.5x10^-3). The slope of regression between histopathological lesions and gene expression was obtained under a Mixed Model approach.

Figure 4

Real-time RT-PCR confirmation of the microarray results. Differential expression of selected sequences/genes analyzed by microarray and quantitative RT-PCR: amyloid beta (A4) precursor (APP), aquaporin 4 (AQP4), CD3 gamma chain (CD3G), calcineurin-like phosphoesterase domain-containing 1 (CPPED1), granulysin (GNLY), golgi golgin subfamily 4 (GOLGA4), lysosomal protein transmembrane 4 beta (LAPTM4B), maguk p55 subfamily member 7 (MPP7), nell2 (NELL2), ovine scrapie related sequence 1 (OSRS1), ovine scrapie related sequence 2 (OSRS2) and serine/arginine-rich splicing factor 3 (SRSF3).