Transmissible spongiform encephalopathies, or prion diseases, are fatal neurodegenerative diseases with characteristic spongiform lesions, neuronal cell loss, astrocytosis and the accumulation of the pathological form of the prion protein
. The precise mechanisms regulating these processes remain unknown. Genomic approaches are a potential tool to understand the molecular basis of complex mechanisms; in addition, they allow the discovery of new disease biomarkers.
The analysis of gene expression profiling can elucidate the molecular basis of this pathology. Several studies have focused on genomic analyses of brain tissue from animal models of prion diseases, including CJD, scrapie and BSE
[19–24]. However, there are fewer studies involving the mRNA profiles of “natural” human CJD
, bovine BSE
 or ovine scrapie
. We previously reported a genomic analysis performed in tissues obtained from sheep naturally infected with scrapie in terminal stages
. However, the molecular changes can occur before the onset of the disease; for example, PrPSc accumulation occurs early in the disease in both the central nervous system
 and peripheral tissues
. In mouse models, genomic expression profiles revealed the induction of oxidative and endoplasmic reticulum (ER) stress, activated ER and mitochondrial apoptosis pathways, and activated cholesterol biosynthesis in the central nervous system of preclinical mice
. We report here the first transcriptome study of the central nervous system (CNS) in sheep naturally infected with scrapie in preclinal stages that associated the variations in the expression profile with the features of scrapie neuropathology.
Differential gene expression in preclinical scrapie
Our microarray hybridization analysis identified 86 probes with changes in expression greater than 2-fold compared with the controls. Using the same platform, our previous study of sheep clinically affected with scrapie
 revealed 350 differentially regulated probes, indicating that at the early stages of the disease, fewer genes are active or the expression changes are not high enough to be detected by microarray. From the 86 probes, 40 were also differentially regulated in clinical animals
 and 46 were identified only in preclinical sheep (some of these probes are shown in Table
In vitro studies have shown variations in the phospho-proteome of N2a cells after PrPSc infection
 and specific inflammatory profiles in microglia
. In addition, genes involved in defense, the immune response or encoding for secreted extracellular proteins are differentially regulated in murine models during prion infection
. In the clinical phase of classical scrapie, the genes involved in ion-binding and transport, nucleotide binding and the immune system are differentially expressed
. In accordance with the in vitro and murine models and with our previous results in classical scrapie at the late stages of the disease, the differentially regulated genes identified in this study encoded phospho-proteins, extracellular matrix organization proteins and immune response-related proteins. Therefore, these mechanisms seem to be involved in the neuropathology of scrapie from the early phases of the disease.
The microarray analysis was validated using qRT-PCR for 4 genes; their differential regulation was confirmed in all cases. The high adjustment between the fold changes obtained with the microarray and the qRT-PCR of the genes selected for confirmation reflects the high credibility of the microarray and the gene alignment analysis. Three of these genes were downregulated in scrapie tissues. Two of them, CD3G and GNLY, are involved in the immune response. CD3G is part of the T-cell receptor-CD3 complex, which plays an important role in coupling antigen recognition to several intracellular signal-transduction pathways
. The CD3 T cells accumulate near or around blood vessels and in the CNS parenchyma of mice inoculated with scrapie
, suggesting the infiltration of T cells in the brain. In contrast, the downregulation of CD3G observed in our natural model suggests that these cells are decreased in preclinical scrapie. Similarly, we observed a significant decrease in the expression of GNLY, which is a potent antimicrobial protein contained within the granules of CTL and NK cells
. Taking together, our results suggest a decline of immune activity in prion diseases, as described for other neurodegenerative diseases such as Alzheimer’s disease (AD)
Another gene that was downregulated in the preclinical medullae encodes the lysosomal protein transmembrane 4 protein (LAPTM4). The endosomal and lysosomal compartments are implicated in trafficking, recycling and the final degradation of prions
. It has been proposed that autophagy might play a protective role in prion diseases, leading to the degradation of prions. Galectin-3 knockout mice express low levels of lysosomal activation marker (LAMP-2) and autophagy markers, suggesting that endosomal/lysosomal dysfunction in combination with reduced autophagy may contribute to the development of prion diseases
. The downregulation of LAPTM4 is in accordance with these results and might indicate a dysfunction of the lysosomal-endosomal pathway in preclinical scrapie.
One of the genes upregulated in the microarray hybridization analysis was Maguk p55 subfamily member 7 MPP7. The membrane-associated guanylate kinase homologues (MAGUKs) are a family of peripheral membrane proteins that form multiprotein complexes containing distinct sets of transmembrane, cytoskeletal, and cytoplasmic signaling proteins. MPP7 acts as an important adapter that promotes epithelial cell polarity, tight junction formation via its interaction with DLG1 and is involved in the assembly of protein complexes at sites of cell-cell contact
. The cellular prion protein PrPc is also located at cell-cell adhesion sites in polarized/differentiated enterocytes and interacts with desmosomal proteins and with actin and actin-binding proteins at cell-cell junctions
. Moreover, in the CNS, the PrPc is located in the microvascular endothelium and at intercellular junctions of cultured brain endothelial cells of mouse, rat and human origin
. We report here for the first time the upregulation of the gene encoding the MPP7 protein in preclinical scrapie and its positive association with PrPSc deposition, suggesting a possible alteration of cell-cell adhesion the early stages of the disease.
The genomic association with scrapie-related lesions
Studies of the associations between gene expression and the intensity of scrapie lesions have been shown to be a powerful tool to detect genes potentially involved in the development of these lesions
. In the present study, we found a relatively low number of genes with differential expression in the preclinical tissues; however, the association study allowed the identification of genes that had slight changes (FC < 2) in the microarray hybridization analysis but whose expression was strongly related to the development of the scrapie-related lesions. The genes that displayed a positive regression with prion deposition were involved in several cellular mechanisms, the most frequent of which being protein, metal and ion binding, oxidoreductase activity and transcription factors. The possible role of PrPc to protect cells from oxidative stress is well documented
, as is the capacity of the prion protein to bind Cu2+[63, 64]. In addition, many other roles have been attributed to the prion protein, such as transmembrane signaling or cell adhesion
. Genes involved in these mechanisms have been shown to be associated with prion diseases, astrocytosis or spongiosis, thereby corroborating the reliability of our association study.
The expression of 6 genes and 2 sequences was validated using qRT-PCR. These genes were chosen because of their known role in brain metabolism and/or neurodegeneration, and the sequences displayed the most significant degrees of association with scrapie lesions. We found a slight up-regulation of the amyloid beta (A4) precursor protein (APP). APP and PrP are both cell-surface proteins residing in cholesterol-rich lipid rafts of the cell membrane and play an important role in the development of AD and prion diseases, respectively. These diseases share a number of clinical, pathological and biochemical characteristics
. The brains of CJD patients display the pathology of both prion diseases and AD
. The overexpression of APP in other genetic expression profiling studies in scrapie murine models has been previously reported
. In addition, the overexpression of APP facilitates the rapid development of artificial scrapie
. The overexpression of APP in the preclinical naturally infected animals found in our study are in accordance with these previous studies and highlight the possible early interaction between APP and PrP.
Water metabolism is of major importance in a number of physiological processes in the CNS. Alterations in the distribution of water and cerebrospinal fluid in the brain are a common occurrence in multiple neuropathological conditions, including brain edema, brain tumors, stroke, hyponatremia, head injuries and hydrocephalus. Aquaporin 4 (AQP4) is most likely expressed by activated glial cells, and an increase in its level is indicative of ongoing astrocytosis
. The increase in the expression levels of AQP4 has been reported in Creutzfeldt-Jakob disease, bovine spongiform encephalopathy and scrapie-infected transgenic mice
[22, 43, 44, 70]. Our work confirms the upregulation of AQP4 in the preclinical phases of natural ovine scrapie.
Genes that have never been associated with prion diseases or other neurodegenerative diseases were shown to be significantly regulated. Our microarray data indicated an increase in the expression of a gene similar to calcineurin-like phosphoesterase domain-containing 1 CPPED1 that was confirmed by quantitative RT-PCR. The CPPED1 protein has hydrolase and metal ion-binding activities. To date, no studies have reported the differential regulation of this gene in neurodegenerative diseases. However, PrPc interacts with a range of divalent metal ions and maintains the their homeostasis, and the conformational change that occurs in the formation of PrPSc is induced by the interaction with ions (see
 for review). This gene has a positive association with prion deposition (GEAMM analysis), suggesting a possible role in early scrapie development. However, further analysis will be essential to confirm this conclusion.
Golgi golgin subfamily 4 GOLGA4 may play a role in the delivery of transport vesicles containing GPI-linked proteins from the trans-Golgi network through its interaction with microtubule-actin crosslinking factor 1 (MACF1). The prion protein is attached to the outer leaflet of the plasma membrane by a glycosyl-phosphatidyl-inositol (GPI) anchor (reviewed in
). Our results demonstrate the upregulation of GOLGA4 is positively associated with PrPSc deposition, suggesting that this protein might have a role in PrP trafficking.
We previously reported that anti-apoptotic genes are overexpressed in terminal scrapie, which suggested the activation of neuroprotective mechanisms during the disease
. In accordance with this, we found here that neural tissue-specific epidermal growth factor-like repeat domain-containing protein) NELL2 is overexpressed in preclinical scrapie. NELL2 is a secreted glycoprotein that is predominantly expressed in neural tissues and increases in vitro cell survival under cell death-inducing conditions
. In addition, NELL2 may play an important role in the maintenance of neural functions by regulating the intracellular machinery involving Ca2+ signaling, synaptic transport and/or vesicle release
. In our study, NELL2 displayed a positive association with PrPSc deposition and spongiosis, which suggests a possible role in the pathogenesis of the disease related to the role of PrP in Ca2+ homeostasis
Finally, we observed the downregulation of a serine/arginine-rich splicing factor 3 SRSF3, which seems to be involved in the differential splicing of the low-density lipoprotein receptor (LDLR), a major apolipoprotein E (APOE) receptor that has been associated with cholesterol homeostasis and, possibly, AD development
. This splicing factor is a proto-oncogene
 and is antiapoptotic
. To our knowledge, our work is the first study to describe the differential regulation of this gene in prion diseases. The downregulation of SRSF3 is in accordance with its probable protective activity against neuronal cell death. Further studies will be necessary to investigate the possible role of SRSF3 in the disease.
In addition to the regulation of known genes, several non-annotated sequences were differentially expressed in the preclinical medullae and associated with scrapie lesions. We confirmed the upregulation of two sequences (OSRS1 and OSRS2) that were associated with astrocytosis and spongiosis, respectively. These sequences did not display homology with any known genes (Table
1), but they show a high homology with parts of two published bovine sequences (FQ482089.2 and NW_003104406.1, respectively). These sequences come from an ovine cDNA library generated from the brain and lymphoid tissue of scrapie- and control-infected sheep
. Although further analyses are necessary to confirm their differential regulation in a wider number of animals or in different prion animal models, these custom sequences can represent potential unknown biomarkers useful for the diagnosis of presymptomatic prion disease.