Limited transcriptional response of ovine microglia to prion accumulation.
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ABSTRACT:
SUBMITTER: Stanton JB
PROVIDER: S-EPMC2746661 | biostudies-literature | 2009 Aug
REPOSITORIES: biostudies-literature
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Limited transcriptional response of ovine microglia to prion accumulation.
Stanton James B JB Knowles Donald P DP Call Douglas R DR Mathison Bruce A BA Baszler Timothy V TV
Biochemical and biophysical research communications 20090610 2
The conversion of normal cellular prion protein to disease-associated prion protein (PrP(Sc)) is a fundamental component of prion disease pathogenesis. The molecular mechanisms contributing to prion conversion and the impact of PrP(Sc) accumulation on cellular biology are not fully understood. To further define the molecular changes associated with PrP(Sc) accumulation in cultured cells, the transcriptional profile of PrP(Sc)-accumulating primary ovine microglia was compared to the profile of Pr ...[more]
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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. Keywords: disease state analysis
2008-09-06 | GSE12688 | GEO
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
2008-11-04 | E-GEOD-12688 | biostudies-arrayexpress
Project description:Susceptibility to infection by prions is highly dependent on the amino acid sequence and host expression of the cellular prion protein (PrPC); however, cellular expression of a genetically susceptible PrPC is insufficient. As an example, it has been shown in cultured cells that permissive and resistant sublines derived from the same parental population often have similar expression levels of PrPC. Thus, additional cellular factors must influence susceptibility to prion infection. The aim of this study was to elucidate the factors associated with relative permissiveness and resistance to scrapie prions in cultured cells derived from a naturally affected species. Two closely related ovine microglia clones with different prion susceptibility, but no detectable differences in PrPC expression levels, were inoculated with either scrapie-positive or scrapie-negative sheep brainstem homogenates. Five passages post-inoculation, the transcriptional profiles of mock and infected clones were sequenced using Illumina technology. Comparative transcriptional analyses identified twenty-two differentially transcribed genes, most of which were upregulated in poorly permissive microglia. This included genes encoding for selenoprotein P, endolysosomal proteases, and proteins involved in extracellular matrix remodeling. Furthermore, in highly permissive microglia, transforming growth factor ?-induced, retinoic acid receptor response 1, and phosphoserine aminotranspherase 1 gene transcripts were upregulated. Gene Set Enrichment Analysis identified proteolysis, translation, and mitosis as the most affected pathways and supported the upregulation trend of several genes encoding for intracellular proteases and ribosomal proteins in poorly permissive microglia. This study identifies new genes potentially involved in scrapie prion propagation, corroborates results from other studies, and extends those results into another cell culture model.
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Project description:Prion diseases are fatal neurodegenerative disorders by which the native cellular prion protein (PrPC) is misfolded into an accumulating, disease-associated isoform (PrPD). To improve the understanding of prion pathogenesis and develop effective treatments, it is essential to elucidate factors contributing to cellular permissiveness. We previously isolated five clones from an immortalized subline of ovine microglia, two of which had demonstrated differential permissiveness to a natural isolate of sheep scrapie and distinct transcriptomic profiles. To more robustly identify factors contributing to this activity, relative permissiveness, cell proliferation, selected gene transcript level, and matrix metalloproteinase 2 (MMP2) activity were compared amongst all five clones. Differences in cell proliferation were not detected between clones; however, significant correlations were identified between relative permissiveness and genes associated with cell growth (i.e., RARRES1 and PTN), protein degradation (i.e., CTSB and SQSTM1), and heparin binding (i.e., SEPP1). MMP2 activity varied amongst clones, but did not correlate with permissiveness. These associations support the contribution of cell division and protein degradation on the permissiveness of cultured ovine microglia to PrPD.
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Project description:Microglia (MG) are critical to host defense during prion infection, but the mechanism(s) of this neuroprotection are poorly understood. To better examine the influence of MG during prion infection, we reduced MG in the brains of C57BL/10 mice using PLX5622 and assessed prion clearance and replication using multiple approaches that included bioassay, immunohistochemistry, and Real-Time Quaking Inducted Conversion (RT-QuIC). We also utilized a strategy of intermittent PLX5622 treatments to reduce MG and allow MG repopulation to test whether new MG could alter prion disease progress. Lastly, we investigated the influence of MG using tga20 mice, a rapid prion model that accumulates fewer pathological features and less PrPres in the infected brain. In C57BL/10 mice we found that MG were excluded from the inoculation site early after infection, but Iba1 positive infiltrating monocytes/macrophage were present. Reducing MG in the brain prior to prion inoculation did not increase susceptibility to prion infection. Short intermittent treatments with PLX5622 in prion infected C57BL/10 mice after 80 dpi were unsuccessful at altering the MG population, gliosis, or survival. Additionally, MG depletion using PLX5622 in tga20 mice had only a minor impact on prion pathogenesis, indicating that the presence of MG might be less important in this fast model with less prion accumulation. In contrast to the benefits of MG against prion disease in late stages of disease, our current experiments suggest MG do not play a role in early prion pathogenesis, clearance, or replication.
| S-EPMC9612458 | biostudies-literature
Project description:Prion diseases are transmissible, neurodegenerative disorders associated with misfolding of the prion protein. Previous studies show that reduction of microglia accelerates central nervous system (CNS) prion disease and increases the accumulation of prions in the brain, suggesting that microglia provide neuroprotection by phagocytosing and destroying prions. In Csf1rΔFIRE mice, the deletion of an enhancer within Csf1r specifically blocks microglia development, however, their brains develop normally and show none of the deficits reported in other microglia-deficient models. Csf1rΔFIRE mice were used as a refined model in which to study the impact of microglia-deficiency on CNS prion disease. Although Csf1rΔFIRE mice succumbed to CNS prion disease much earlier than wild-type mice, the accumulation of prions in their brains was reduced. Instead, astrocytes displayed earlier, non-polarized reactive activation with enhanced phagocytosis of neuronal contents and unfolded protein responses. Our data suggest that rather than simply phagocytosing and destroying prions, the microglia instead provide host-protection during CNS prion disease and restrict the harmful activities of reactive astrocytes.
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Project description:Jaagsiekte sheep retrovirus (JSRV) is the etiologic agent of ovine pulmonary adenocarcinoma (OPA), a neoplastic lung disease of sheep. OPA is an important economic and welfare issue for sheep farmers and a valuable naturally occurring animal model for human lung adenocarcinoma. Here, we used RNA sequencing to study the transcriptional response of ovine lung tissue to infection by JSRV. We identified 1,971 ovine genes differentially expressed in JSRV-infected lung compared to noninfected lung, including many genes with roles in carcinogenesis and immunomodulation. The differential expression of selected genes was confirmed using immunohistochemistry and reverse transcription-quantitative PCR. A key finding was the activation of anterior gradient 2, yes-associated protein 1, and amphiregulin in OPA tumor cells, indicating a role for this oncogenic pathway in OPA. In addition, there was differential expression of genes related to innate immunity, including genes encoding cytokines, chemokines, and complement system proteins. In contrast, there was little evidence for the upregulation of genes involved in T-cell immunity. Many genes related to macrophage function were also differentially expressed, reflecting the increased abundance of these cells in OPA-affected lung tissue. Comparison of the genes differentially regulated in OPA with the transcriptional changes occurring in human lung cancer revealed important similarities and differences between OPA and human lung adenocarcinoma. This study provides valuable new information on the pathogenesis of OPA and strengthens the use of this naturally occurring animal model for human lung adenocarcinoma.IMPORTANCE Ovine pulmonary adenocarcinoma is a chronic respiratory disease of sheep caused by jaagsiekte sheep retrovirus (JSRV). OPA is a significant economic problem for sheep farmers in many countries and is a valuable animal model for some forms of human lung cancer. Here, we examined the changes in host gene expression that occur in the lung in response to JSRV infection. We identified a large number of genes with altered expression in infected lung, including factors with roles in cancer and immune system function. We also compared the data from OPA to previously published data from human lung adenocarcinoma and found a large degree of overlap in the genes that were dysregulated. The results of this study provide exciting new avenues for future studies of OPA and may have comparative relevance for understanding human lung cancer.
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Project description:BACKGROUND: Genetic predisposition to scrapie in sheep is associated with several variations in the peptide sequence of the prion protein gene (PRNP). DNA-based tests for scoring PRNP codons are essential tools for eradicating scrapie and for evaluating rare alleles for increased resistance to disease. In addition to those associated with scrapie, there are dozens more PRNP polymorphisms that may occur in various flocks. If not accounted for, these sites may cause base-pair mismatching with oligonucleotides used in DNA testing. Thus, the fidelity of scrapie genetic testing is enhanced by knowing the position and frequency of PRNP polymorphisms in targeted flocks. RESULTS: An adaptive DNA sequencing strategy was developed to determine the 771 bp PRNP coding sequence for any sheep and thereby produce a consensus sequence for targeted flocks. The strategy initially accounted for 43 known polymorphisms and facilitates the detection of unknown polymorphisms through an overlapping amplicon design. The strategy was applied to 953 sheep DNAs from multiple breeds in U.S. populations. The samples included two sets of reference sheep: one set for standardizing PRNP genetic testing and another set for discovering polymorphisms, estimating allele frequencies, and determining haplotype phase. DNA sequencing revealed 16 previously unreported polymorphisms, including a L237P variant on the F141 haplotype. Two mass spectrometry multiplex assays were developed to score five codons of interest in U.S. sheep: 112, 136, 141, 154, and 171. Reference tissues, DNA, trace files, and genotypes from this project are publicly available for use without restriction. CONCLUSION: Identifying ovine PRNP polymorphisms in targeted flocks is critical for designing efficient scrapie genetic testing systems. Together with reference DNA panels, this information facilitates training, certification, and development of new tests and knowledge that may expedite the eradication of sheep scrapie.
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Project description:BackgroundAlzheimer's disease (AD) is a neurodegenerative disorder and is the most common cause of late-onset dementia. Microglia, the primary innate immune cells of the central nervous system (CNS), have a complex role in AD neuropathology. In the initial stages of AD, microglia play a role in limiting pathology by removing amyloid-β (Aβ) by phagocytosis. In contrast, microglia also release pro-inflammatory cytokines and chemokines to promote neuroinflammation and exacerbate AD neuropathology. Therefore, investigating microglial gene networks could identify new targets for therapeutic strategies for AD.ResultsWe identified 465 differentially expressed genes (DEG) in 5XFAD versus wild-type mice by microarray, 354 DEG in lipopolysaccharide (LPS)-stimulated N9 microglia versus unstimulated control cells using RNA-sequencing (RNA-seq), with 32 DEG common between both datasets. Analyses of the 32 common DEG uncovered numerous molecular functions and pathways involved in Aβ phagocytosis and neuroinflammation associated with AD. Furthermore, multiplex ELISA confirmed the induction of several cytokines and chemokines in LPS-stimulated microglia.ConclusionsIn summary, AD triggered multiple signaling pathways that regulate numerous genes in microglia, contributing to Aβ phagocytosis and neuroinflammation. Overall, these data identified several regulatory factors and biomarkers in microglia that could be useful in further understanding AD neuropathology.
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Project description:Microglial activation is a hallmark of most neurodegenerative disorders, and is particularly conspicuous in prion diseases. However, the role of microglia, which function as both primary immune effector cells and professional phagocytes in the central nervous system, remains contentious in the context of neurodegeneration. Here, we evaluated the effect of microglial depletion/deficiency on prion pathogenesis. We found that ganciclovir-mediated microglial ablation on tga20/CD11b-thymidine kinase of Herpes simplex virus (HSVTK) cerebellar organotypic cultured slices markedly aggravated prion-induced neurotoxicity. A similar deterioration of disease was recapitulated in in vivo microglial depletion in prion-infected tga20/CD11b-HSVTK mice. Additionally, deficiency of microglia in interleukin 34 knockout (IL34(-/-)) mice again resulted in significantly augmented proteinase K-resistant prion protein deposition and accelerated prion disease progression. These results provide unambiguous evidence for a general protective role of microglia in prion pathogenesis.
| S-EPMC4886355 | biostudies-literature