Project description:A key event in the pathogenic process of prion diseases is the conversion of the cellular prion protein (PrPC) to an abnormal and protease-resistant isoform (PrPSc). Mice lacking PrP are resistant to prion infection, and down-regulation of PrPC during prion infection prevents neuronal loss and the progression to clinical disease. These results are suggestive of the potential beneficial effect of silencing PrPC during prion diseases. However, the silencing of a protein that is widely expressed throughout the CNS could be detrimental to brain homeostasis. The physiological role of PrPC remains still unclear, but several putative functions have been proposed. Among these, several lines of evidence support PrPC function in neuronal development and maintenance. To assess the influence of PrPC on gene expression profile during development in the mouse brain, we undertook a microarray analysis by using RNA isolated from the hippocampus, at two different developmental stages: newborn (4-day-old) and adult (3-month-old) mice, both from Prnp+/+ and Prnp0/0 animals. The comparison of the different datasets allowed us to identify “commonly” co-regulated genes and “uniquely” de-regulated genes during postnatal development in these animal models. The lack of PrPC during neuronal development affected several biological pathways, among which the most representative were cell signaling, cell-cell communication and transduction process. In addition, the absence of PrPC influenced genes involved in calcium homeostasis, nervous system development, synaptic transmission and cell adhesion. There was only a moderate alteration of the gene expression profile during neuronal development in the animal models we studied. PrPC deficiency does not lead to a dramatic alteration of gene expression profile, and produces moderate altered gene expression levels from young to adult animals. Thus, our results may provide additional support to silencing endogenous PrPC levels as a therapeutic approach to prion diseases.

Project description:A key event in the pathogenic process of prion diseases is the conversion of the cellular prion protein (PrPC) to an abnormal and protease-resistant isoform (PrPSc). Mice lacking PrP are resistant to prion infection, and down-regulation of PrPC during prion infection prevents neuronal loss and the progression to clinical disease. These results are suggestive of the potential beneficial effect of silencing PrPC during prion diseases. However, the silencing of a protein that is widely expressed throughout the CNS could be detrimental to brain homeostasis. The physiological role of PrPC remains still unclear, but several putative functions have been proposed. Among these, several lines of evidence support PrPC function in neuronal development and maintenance. To assess the influence of PrPC on gene expression profile during development in the mouse brain, we undertook a microarray analysis by using RNA isolated from the hippocampus, at two different developmental stages: newborn (4-day-old) and adult (3-month-old) mice, both from Prnp+/+ and Prnp0/0 animals. The comparison of the different datasets allowed us to identify “commonly” co-regulated genes and “uniquely” de-regulated genes during postnatal development in these animal models. The lack of PrPC during neuronal development affected several biological pathways, among which the most representative were cell signaling, cell-cell communication and transduction process. In addition, the absence of PrPC influenced genes involved in calcium homeostasis, nervous system development, synaptic transmission and cell adhesion. There was only a moderate alteration of the gene expression profile during neuronal development in the animal models we studied. PrPC deficiency does not lead to a dramatic alteration of gene expression profile, and produces moderate altered gene expression levels from young to adult animals. Thus, our results may provide additional support to silencing endogenous PrPC levels as a therapeutic approach to prion diseases. To analyze the influence of PrPC expression on CNS gene expression profile during development, we investigated WT PrPC (Prnp+/+) and Prnp0/0 mice at two different developmental stages: in neonatal animals (postnatal day 4, P4) and in adult animals (3 months old). For each developmental stage, hippocampi of 3 (pups) or 4 (adult) animals were dissected immediately after animal sacrifice and promptly processed for RNA extraction and purification, for a total of 14 samples.

Project description:Sheep scrapie (Sc) is the classical transmissible spongiform encephalopathy (prion disease). The conversion of normal cellular prion protein (PrPC) to disease-associated prion protein (PrPSc) is a fundamental component of prion disease pathogenesis. The molecular mechanisms contributing to prion diseases and the impact of PrPSc accumulation on cellular biology are not fully understood. To define the molecular changes associated with PrPSc accumulation, primary sheep microglia were inoculated with PrPSc and then the transcriptional profile of these PrPSc-accumulating microglial cells was compared to the profile of PrPSc-lacking microglial cells using the Affymetrix Bovine Genome Array. The experimental design included three biological replicates, each with three technical replicates, and samples that were collected at the point of maximal PrPSc accumulation levels as measured by ELISA. The array analysis revealed 19 upregulated genes and 30 downregulated genes in PrPSc-accumulating microglia. Three transcripts (CCL2, SGK1, and AASDHPPT) were differentially regulated in a direction similar to previous reports from mouse or human models, whereas the response of three other transcripts (MT1E, NR4A1, PKP2) conflicted with previous reports. Overall, the results demonstrated a limited transcriptional response to PrPSc accumulation, when compared to microglia and macrophage cultures infected with other agents such as viruses and bacteria. This is the first microarray-based analysis of prion accumulation in primary cells derived from a natural TSE-host. Experiment Overall Design: Primary sheep microglial cells were either inoculated with PrPSc (Inoc) or sham-inoculated (Mock) Experiment Overall Design: Three biological replicates per treatment. Experiment Overall Design: Three technical replicates per biological replicate. Experiment Overall Design: biological replicate: Inoc12A, Inoc12B.2, Inoc12C Experiment Overall Design: biological replicate: Mock12A, Mock12B.2, Mock12C Experiment Overall Design: technical replicate - extract: Inoc12A.1, Inoc12A.2, Inoc12A.3 Experiment Overall Design: technical replicate - extract: Inoc12B.2.1, Inoc12B.2.2, Inoc12B.2.3 Experiment Overall Design: technical replicate - extract: Inoc12C.1, Inoc12C.2, Inoc12C.3 Experiment Overall Design: technical replicate - extract: Mock12A.1, Mock12A.2, Mock12A.3 Experiment Overall Design: technical replicate - extract: Mock12B.2.1, Mock12B.2.2, Mock12B.2.3 Experiment Overall Design: technical replicate - extract: Mock12C.1, Mock12C.2, Mock12C.3

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Chemo-omic pipeline enables discovery of prion synaptotoxic pathways and inhibitory drugs

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