Neurodegeneration and gliosis are the main neuropathological features of prion diseases. However, the molecular mechanisms involved in these processes remain unclear. Previous research from our group showed the relationship between altered expression of genes involved in the regulation of apoptosis and scrapie-related lesions [7–9]; however, other factors may participate in scrapie neuropathology.
Formation of protein aggregates in neurons or in the extracellular matrix is an important characteristic in many neurodegenerative processes such as prion diseases, Alzheimer, Parkinson, or Huntington disease. It seems to be evident that the cellular machinery responsible for avoiding anomalous protein accumulation would be implicated in these pathologies. Therefore, chaperones could play an important role in neuronal degeneration . In this context, little is known about natural models of TSE. The aim of this study was to analyse the gene and protein expression of four Hsp chaperones in the CNS of sheep naturally infected with scrapie; the chaperones analysed were chosen according to their known roles in apoptosis modulation or their possible functions in prion diseases.
Over-expression of HSP70 family genes has been reported in brain samples from scrapie-infected mice [25, 27]. Our expression analysis revealed significant over-expression of HSP73 transcripts in scrapie diencephalons. Hsp70 prevents the accumulation and/or promotes the degradation of specific PrP conformers in fly models and protects against PrP neurotoxicity through unknown mechanisms . In our study, HSP73 expression in diencephalons was independent of PrPSc immunolabelling (Table 2) suggesting an alternative role for this chaperone. We reported a lack of apoptosis induction despite the significant increase of BAX and BAK in scrapie diencephalons. Joining these results, we hypothesize that HSP73 could counteract the induction of apoptosis by these mitochondrial factors.
In contrast, in the prefrontal cortex, the expression of HSP90 and HSP27 genes decreased in the scrapie group. This HSP90 pattern is not in agreement with the Hsp90 up-regulation observed in murine models of Bovine Spongiform Encephalopathy (BSE) . It is possible that mRNA levels do not correspond to protein expression. It has been suggested that under inflammation or ischemic processes, Hsp synthesis results from a post-transcriptional regulatory mechanism that involves mRNA stabilisation . This is why we analysed the expression of Hsp proteins by Western blot. Interestingly, no significant differences were detected between controls and scrapie animals. An association analysis established differential distribution of certain polymorphisms in the 5' and 3' regions of the ovine HSP90 AA1 gene in scrapie-susceptible animals . Previously, other authors observed an important role of the 3'UTR region in post-transcriptional regulation of Hsp70 upon heat shock [41, 42]. Thus, the decrease of the HSP90 gene expression observed in the prefrontal cortex could be caused by variation at transcriptional or post-transcriptional levels. Perhaps these mutations do not affect HSP90 translation, or, alternately, Western blot technique is not sensitive enough to detect certain changes in protein expression.
In addition, interindividual variability has been reported for natural scrapie [7–9, 43]. This may mask small differences in gene and protein expression between healthy and scrapie-affected animals. For this reason, we performed an additional statistical analysis to detect whether a relationship exists between the expression pattern and the histopathological changes observed in scrapie animals. This methodology allows the analysis of the molecular mechanisms underlying these processes in the natural form of the disease. Although we are aware that the histopathological valuation does not correspond to a real scale, our procedure can reliably detect the association among different parameters. However, the lack of statistical power due to the limited number of individuals analysed could prevent the detection of additional associations.
Although stepwise regression results were different in the four areas analysed, it is worth mentioning that we identified a negative association between prion protein deposition and the expression of the HSP genes in the cerebellum (HSP27, HSP73 and HSP90) and prefrontal cortex (HSP27, HSP72 and HSP73). It has been demonstrated that Hsp70 members avoid protein aggregation, which leads to neurodegenerative diseases [44–46]. Thus, down-regulation of chaperones could contribute to the accumulation of anomalous proteins and finally to cell stress. A decrease of Hsp27 has been described in early stages of in vitro models of spinocerebellar ataxia type 3  and type 7 , which are neurodegenerative disorders associated with an accumulation of polyglutamine-containing proteins.
Glial reaction is the other classical sign of natural scrapie. We previously reported a positive association between astrogliosis and the extrinsic pathway of apoptosis in the medulla oblongata and prefrontal cortex. In addition to apoptosis regulation, factors involved in this pathway can participate in inflammation , reactive gliosis and neuroinflammation [50, 51]. Hsp are also secreted as inflammatory mediators that bind surface receptors and induce the production of nitric oxide, cytokines, and other immunoregulatory molecules. Levels of the small heat shock protein Hsp25 increased during reactive astrogliosis in a murine model of BSE , and Hsp70 expression has been reported in reactive astrocytes following ischemia [53, 54]. Microglial activation by Hsp proteins, such as Hsp70 and Hsp90, results in clearance of amyloid peptide aggregates [55, 56]. In agreement with this, in the present work GFAP immunolabelling was positively associated with the expression of several HSP genes and proteins in the areas where these genes were negatively associated with prion protein immunolabelling (cerebellum and prefrontal cortex). Then, high levels of HSP gene or protein expression would be associated to the prevention or degradation of prion protein aggregates and the presence of reactive astrocytosis in natural scrapie. Although a strong positive association was found between HSP73 gene expression and microglia staining in the cerebellum, our association results are not conclusive with respect to the role of HSP in microglia activation.
The Hsp analysed in this work were selected due to their known role in apoptosis regulation. In a previous study, searching for possible evidence of cell death mechanisms in natural scrapie by immunodetection of activated caspase-3 , we observed weak staining in all CNS sections without finding significant differences between control and scrapie animals. However, a positive association was found between caspase-3 and the mitochondrial pathway of apoptosis . Since Hsp work as anti-apoptotic factors [15, 17, 19] we analysed the relationship between caspase-3 expression and Hsp gene and protein expression. Only Hsp70 protein expression in the cerebellum showed a positive correlation with caspase-3. The absence of an association in the remaining areas may reflect that, in general, Hsp are not involved in the regulation of neuronal death during natural disease, as was observed in the acute phase of epilepsy .
Finally, when cases of natural disease are studied, changes detected in certain cell populations are frequently not correlated with variations in gene or protein expression in the final tissue mixture, as we observed for Bax protein determination in the CNS of scrapie sheep . For this reason, in order to confirm the previous results and elucidate the cell type involved in possible expression variability, an immunohistochemical assay of Hsp was performed in each area of the CNS. In general, diffuse and highly variable staining for Hsp70 and Hsp90 was observed in the four CNS areas of both groups of animals. Nevertheless, the over-expression of Hsp in Purkinje cells of scrapie cerebella was remarkable. Intense staining for Hsp72 in this cell type was also observed in Creutzfeldt-Jakob disease (CJD) patients ; accumulation of this protein in Purkinje cells supports the proposed neuroprotective role of Hsp in the natural form of the disease. Similarly, although caspase-3 immunostaining was present in cerebellar granular cells, we did not observe this staining in Purkinje cells, in either scrapie-affected or control sheep [8, 9]. To our knowledge, there is no clear evidence of prion protein toxicity in Purkinje cells. Only over-expression of the Doppel gene in Prnp-deficient mice has been correlated with Purkinje cell loss [59, 60]. Our results support the possible protective function of Hsp70 in Purkinje cells and suggest a role of Hsp90 in this mechanism. Further analysis would be necessary to clarify the role of Hsp over-expression in these cells in prion diseases.
In CJD , the regions with severe spongiosis showed low immunoreactivity for Hsp72. In agreement with this report, we detected low expression of Hsp70 protein in one infected animal showing the greatest degree of spongiform alteration. In fact, the stepwise regression analysis revealed a negative association of Hsp70 protein with spongiosis in the cerebellum and diencephalon, suggesting that the reduction in the cytoprotective effect of Hsp70 could be involved in neuronal loss, at least in these two areas. However, the results of correlation and stepwise regression must be taken with caution due to the reduced amount of available data. Nevertheless, they illustrate the joint effect of several lesions of the gene or protein expression on the tissues analysed and pointed out the complexity of causes of modifications of gene and protein expression.
In conclusion, Hsp seem to be involved in gliosis and inflammatory reactions rather than in anti-apoptotic processes in natural scrapie. However, the intense immunoreactivity of Purkinje cells for Hsp70 and Hsp90, as well as the negative association of Hsp70 with prion protein immunolabelling and spongiosis, suggests a neuroprotective effect in this cell type against the stress related to ovine encephalopathy.