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From: TSS ()
BMC Genomics 2006, 7:114 doi:10.1186/1471-2164-7-114 Published 16 May 2006 Background Transmissible spongiform encephalopathies (TSEs) or prion diseases are fatal neurodegenerative disorders which occur in humans and various animal species. Examples include Creutzfeldt-Jakob disease (CJD) in humans, bovine spongiform encephalopathy (BSE) in cattle, chronic wasting disease (CWD) in deer and elk, and scrapie in sheep, and experimental mice. To gain insights into TSE pathogenesis, we made and used cDNA microarrays to identify disease-associated alterations in gene expression. Brain gene expression in scrapie-infected mice was compared to mock-infected mice at pre-symptomatic and symptomatic time points. Three strains of mouse scrapie that show striking differences in neuropathology were studied: ME7, 22L, and Chandler/RML. Results In symptomatic mice, over 400 significant gene expression alterations were identified. In contrast, only 22 genes showed significant alteration in the pre-symptomatic animals. We also identified genes that showed significant differences in alterations in gene expression between strains. Genes identified in this study encode proteins that are involved in many cellular processes including protein folding, endosome/lysosome function, immunity, synapse function, metal ion binding, calcium regulation and cytoskeletal function. Conclusions These studies shed light on the complex molecular events that occur during prion disease, and identify genes whose further study may yield new insights into strain specific neuropathogenesis and ante-mortem tests for TSEs. http://www.biomedcentral.com/1471-2164/7/114 full text concusions; Conclusions We identified alterations in gene expression that occur in the brains of mice infected with scrapie strains ME7, 22L, and RML-Chandler at two times post infection. While it is difficult to compare our results with other previously performed gene expression studies in related TSE systems, because of the multiple differences in the methodologies used, the considerable overlap in the genes identified in our study and those identified in previous studies validate the importance of these alterations in gene expression in scrapie and suggest that these genes may generally play important roles in TSEs. We identified for the first time novel scrapie-associated alterations in expression in numerous genes with known and as of yet unknown function. Many of the identified alterations in brain gene expression are likely important contributors to disease. In support of this notion, Klein et al., showed that complement gene knock out mice are resistant to intraperitoneal but not intracerebral inoculation of scrapie indicating that a single gene can be important to early events in peripheral transmission, propagation, and dissemination of scrapie in mice [33]. There are several genes identified in our studies that stand out as likely cofactors in TSE pathogenesis. For example, a large percentage of the genes we identified function in the endosome/lysosome system. These genes are of particular interest because the conversion of PrPc to PrPsc is thought to occur on the cell membrane or in the endosome/lysosome system and PrPsc accumulates in endosomes and lysosomes of brain cells [34-37]. Over twenty percent of the scrapie-associated alterations identified in our study encode proteins that function in protein folding, protein degradation or localize to the endosomes/lysosomes system. We hypothesize that many of these genes are cofactors in prion protein misfolding and accumulation. Other important scrapie-associated alterations identified in this study include genes that function in immunity, neuronal synapses, metal ion binding, calcium regulation, mitochondria and the cytoskeleton. Alterations in immunity genes are likely largely associated with microglial and astrocyte activation. Alterations in synapse genes may be important factors in the degeneration of neuronal dendrites. Many of the genes identified in this study bind metal ions, consistent with a role for disruption of metal ion homeostasis in scrapie neuropathogenesis. Alterations in genes that function in calcium homoeostasis and the mitochondria may be important factors in the degeneration of neurons, as maintenance of calcium homeostasis and energy production is critical for neuronal survival. Cytoskeleton gene alterations are likely reflective of migration of microglia, but may also be factors in other changes in cellular activity important to prion diseases such as vacuole formation in neurons. C57Bl mice infected intracerebrally with scrapie strains ME7, 22L and RML-Chandler have similar time courses of disease and develop clinical symptoms within a week of each other at approximately 140 dpi. However, mice infected with these three strains of scrapie differ in the extent and localization of vacuolization and plaque formation. The intensity of vacuolation is highest in the anterior of the brain in mice infected with ME7, similar in various brain regions with RML-Chandler, and highest in the posterior of the brain with strain 22L. Strain 22L also shows a unique brain pathology in that it induces vacuolation in the cerebellar cortex and the vacuoles are larger in size compared to those induced by other strains of scrapie [29, 31, 32]. Strain specific alterations in gene expression detected in this study may indicate genes that contribute to the molecular mechanisms underlying strain specific neuropathology. However, from this study we can’t distinguish alterations in gene expression between strains that are due to differences in neuropathology and those due to mice being at slightly differently stages of infection. In future experiments we hope to better understand the process of vacuolization by the analysis of regional expression of genes identified as strain-specific in our studies at multiple time-points post infection using in situ hybridization and immunohistochemical approaches. Analysis of the cellular localization of the known genes showed that over 40 genes encode extracellular proteins (indicated with an asterisk in Table 1, Table 2 and additional file 1). If in future studies these proteins show TSE-specific differential accumulation in the cerebrospinal fluid or other accessible tissues, they could provide novel ante-mortem diagnostic markers for TSEs using simple antibody based or enzyme detection assays. The studies reported here add to the handful of previously reported gene profiling studies of TSE pathogenesis using DNA microarrays. Our study included analysis of scrapie strain 22L which has not previously been reported in gene expression profiling studies, and implemented refined microarray analysis methodologies that allowed for identification of significant diseaseassociated alterations in gene expression using minimal numbers of mice. These studies are important because they shed light on the molecular mechanisms underlying TSE pathogenesis, identify potential surrogate markers for TSE diagnosis, and identify potential targets for drug treatment for TSEs. However, information gleaned from these studies represents just the tip of the iceberg of all of the genomic and other cellular and molecular changes that occur during TSEs. Additional studies are needed in order to unravel the rest of the mystery. The strain specific as well as strain non-specific alterations in gene expression identified in this study need to be evaluated using in situ hybridization and immunohistochemical approaches at multiple times post infection in order to determine the cell types in which alterations occur, the precise time post infection that they occur, and to determine whether specific TSE pathology including PrPsc accumulation is associated with cellular populations showing alterations in gene expression. In addition, studies are needed to investigate gene expression changes in animals and humans infected with TSEs that have different genetic backgrounds including different alleles of PrP. Indeed Booth et al., found differences in alterations in gene expression in C57Bl/6 mice compared to VM mice infected with scrapie strain 22A [10]. Genomic studies in TSEs have thus far been confined to brain and need to be expanded to other tissues such as spleen and blood. Gene knock-out studies are needed to determine the extent to which each of the TSE-associated alterations in gene expression contributes to PrPsc formation, and disease pathogenesis and progression. Cross-species studies are needed to determine alterations that are species specific and species non-specific. A comparison to other infectious and neurodegenerative diseases is needed to establish TSE-specific alterations from non-specific alterations. Studies are also needed to evaluate the use of genomic changes as surrogate markers for disease diagnosis, as well as the targeting genomic changes for potential drug therapies to treat TSEs. Thus, the scrapie-associated alterations in gene expression identified in the studies presented here and in those previously described have taken an important step in furthering our understanding the molecular mechanisms underlying TSE disease pathogenesis and disease progression. These discoveries provide a foundation for future studies that will take the next steps towards fully understanding TSEs, developing novel genomic based diagnostic assays for TSEs, and identifying targets for drug therapies to treat TSEs. snip...full text ; http://www.biomedcentral.com/content/pdf/1471-2164-7-114.pdf TSS
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