| Prion diseases or transmissible spongiform encephalopathies (TSEs) are a class of fatal neurodegenerative diseases affecting both humans and animals, with a long incubation period and a 100% rate of mortality. There is no effective method of treatment. The infectious particle of prion disease is diseased prion protein. There are two isforms of prion protein, the cellular prion protein and the diseased prion protein. Although they are identical in primary structure of protein, they are different in secondary structure. PrPc is mainly composed ofα-helix, while PrPsc mainly consists ofβ-sheet. PrPc is normal, sensitive to proteinase and non-infectious, while PrPsc is diseased, resistant to proteinase and infectious. A conformational transformation from an a-helix rich normal, soluble, sensitive to proteinase and non-infectious prion protein (PrPc) to aβ-sheet rich, insoluble, partially protease resistant and infectious disease-associated form of the protein (PrPsc) results in the accumulation of the abnormally folded protein in the central nervous system and pathogenesis of prion disease. So, Prion disease is also named protein conformational disease. The typical pathological features of prion disease consist of spongiform degeneration, gliosis, PrPsc accumulation and neuronal death. It is reported that prion disease is associated with disorder of ion metabolism, oxidative stress and apoptosis. However, it is poorly understood at the following issues. 1) In conformation change of PrPc to PrPsc, molecular chaperones such as Hsp60 and Grb2 involved in the conformation. It is not clear whether other chaperones take part in this process. 2) It is controversial that as a pathogen whether PrPsc could lead to inflammation. 3) The biomarkers of prion disease on diagnosis is few, more biomarkers are needed to be explored. 4) The relationship of prion disease and miRNAs are obscure. It is the hot spot for researchers. In this study, a model of scrapie of 22L strain was established on BALB/c mice. Gene expression profiling, microarry of miRNA and proteomic analysis were applied to reveal the concrete mechanism of prion disease and new biomarkers for diagnosis of prion disease. The works in this dissertation are as follow:1 Establishment of mouse model of scrapie of 22L strain on BALB/c mouse 22L strain is from scrapie of prion disease. The mice were intracerebrally inoculated with 22L strain at the right side of the brain with a concentration of 10-2. The general state of health, clinical symptoms and survival times were observed. About 120 days post inoculation (dpi), rough coat, weight loss, hunch back and hyper-reactivity appeared in 22L-infected mice. After lasting for 2 weeks, continuously weight loss, depression, ataxia and finally the mice were on the brink of death. The average survival period was 142.6±5.0 d. The pathological changes of brain tissue at the end point were observed by HE staining and immunohistochemistry. Different extent of vacuolation was seen in cortex, hippocampus and cerebellum. The cerebellum showed more vacuoles. There were not vacuoles in control mice. In immunohistochemistry, more GFAP positive cells were observed in 22L-infected mice than control mice in cortex, hippocampus and cerebellum. PrPsc expression was only detected in 22L-infected mice in cortex, hippocampus and cerebellum. In Western blotting analysis, GFAP expression in brain of 22L-infected mice were much higher than that in control mice (P<0.05). Specific bands were detected in 22L-infected mice brain samples digested by proteinase K. Not bands were detected in control mice brain samples digested by proteinase K. In order to analyze the feature of 22L strain in BALB/c mice, we evaluated the vacuoles, GFAP expression and PrPsc accumulation in different region of the brain. It showed that the cerebellum was the most diseased region in prion disease of scrapie of 22L strain on BALB/c mice. The above results indicated that we successfully established a BALB/c mice model of prion disease of 22L strain of scrapie. The typical pathological changes were observed in 22L-infected mice. The cerebellum showed the most diseased region. The following experiments were mainly on cerebellum.2 Genes expression profiling and miRNAs profiling in the cerebellum of 22L-infected mice and control mice.1) Genes profiling in the cerebellum of 22L-infected mice and control mice.To investigate the transcriptional changes of all the genes in the progression of prion disease of 22L strain. The cerebellum was analyzed by microarry at 60, 90, 120 and 145 dpi. The differentially expressed genes with a fold change cut-off of±2.0 were gained. At 60 dpi, 369 genes were down-regulated while 322 genes were up-regulated. At 90 dpi, 127 genes were down-regulated while 337 genes were up-regulated. At 120 dpi, 633 genes were down-regulated while 557 genes were up-regulated. At the end point (145 dpi), 384 genes were down-regulated while 346 genes were up-regulated. The differentially expressed genes were analyzed by IPA software in IPKB. The main function of these genes was analyzed. Inflammatory responses were the main functions of the differentially expressed genes at 120 dpi and 145 dpi. It is suggested that inflammatory responses might play a role in prion disease. To validate this deduction, the core molecules of the function analysis were chose for real time PCR analysis. The molecules were IL-1β, TNFа, TGFβ1, CCL4, CCL5 and TLR1-13. At different time point, mRNA expression of IL-1β,TNFα, TGFβ1, CCL4, CCL5 and TLR1-13 except TLR4 in 22L-infected mice were higher than that of control mice. The increase was more obvious at the end point. The mRNA levels of TGFβ1, TNFα, TLR1, TLR2, TLR5, TLR7, TLR 8 and TLR9 were increased in a time-dependent manner in 22L-infected mice and were significantly increased at 120 dpi. Protein level of TLR1, TLR2, TLR7, TLR8 and TLR9 were analyzed by Western blotting at 145 dpi, TLR7 and TLR9 expression were increased significantly. The results suggested again that inflammatory responses might play roles in prion disease. In CNS, the inflammatory responses were mediated by glial cells. To explore the relationship between inflammatory responses in prion disease and glial cells, GFAP and PrPsc expression at 60, 90, 120 and 145 dpi were analyzed by Western blotting. The data showed that PrPsc and GFAP expression was in a time dependent manner, too. It indicated that PrPsc accumulation resulted in inflammatory responses of gliosis which induced pro-inflammatory cytokines and chemokines release. Moreover, TLRs are mainly expressed on neurons and glial cells. In prion disease, neurons were loss while glial cells were increased. The up-regulation of TLRs in 22L-infected mice attributed to gliosis.2) MiRNAs profiling in the cerebellum of 22L-infected mice and control miceTo investigate whether miRNAs expression was changed in 22L-infected mice, the cerebellum was chosen to analyze miRNAs profiling at the end point. There were 25 differentially expressed miRNAs,8 up-regulated and 17 down-regulated. Nine of them were chosen for real time PCR. The results showed that miR-448 and miR-142-3P were up-regulated, indicating their regulatory effects in prion disease.3 The proteomic analysis of brain in 22L-infected mice and control mice1) Differentially expressed protein selecting in the brain of 22L-infected and control mice.To search the key molecules involving in prion disease and new biomarkers, LC-MS/MS was used to analyze differentially expressed protein in the brain of 22L-infected mice and control mice. The data showed that with a P-value cut-off of 0.05, 137 differentially expressed protein were gained, 108 up-regulated and 29 down-regulated. After analyzed by IPA, it is shown that the differentially expressed protein correlated with neurological disease. Two of down-regutated protein (Hsp60 and Calreticulin) and 4 of up-regulated protein (Alpha-internexin, Cofilin 1, Neurofascin and Contactin 1) were chosen for validation by Western blotting and immunofluorescence. The results of Western blotting confirmed the differential expression of the protein. The up-regulated expression of Alpha-internexin was in Purkinje cells. Previous study reported that over-expression of alpha-internexin results in intermediate filaments accumulation in Purkinje cells which leads to numerous swellings of the proximal portions of the axons of Purkinje cells and neurodegeneration. It indicated that up-regulation of alpha-internexin in the brain of 22L-infected mice might result in neurodegeneration. Alpha-internexin probably is a new biomarker for prion disease. It is meaningful for diagnosis of prion disease. In addition, Hsp60 and Careticulin were down-regulated in 22L-infected mice. In previous study, Hsp60 and Careticulin are chaperones which help with correct protein folding and clearing misfolded proteins. It indicates that the down-regulation of Hsp60 and Careticulin leads to not clear misfolded protein promptly. The misfolded protein accumulated in CNS. Hsp60 and Careticulin might be new therapeutic target for prion disease. Moreover, Cofilin 1 was up-regulated and it can make G-actin to F-actin. Neurofascin and contactin-1 expression was also up-regulated. It indicates that Cofilin 1, neurofascin and contactin 1 might be important in prion disease, but it is not clear.2) Differentially expressed polypeptides selecting in the brain of 22L-infected and control mice.To find new biomarkers for prion disease, MALDI-TOF IMS was used to analyze differentially expressed polypeptides in brain tissue sections. The results showed that 21 polypeptides were down-regulated in 22L-infected mice. m/z 758.7725 and m/z 5432.2069 were significantly decreased (P<0.05). The peptides probably are biomarkers, which might play important roles in pathogenesis of prion disease. This study provides new thinking for diagnosis of prion disease.Taken together, we successfully established a BALB/c mice model of prion disease of 22L strain of scrapie. The typical pathological changes were observed in 22L-infected mice. The cerebellum showed the most diseased region. PrPsc accumulation resulted in inflammatory responses of gliosis which induced pro-inflammatory cytokines, chemokines release and TLRs increase. MiR-448 and miR-142-3P were up-regulated in prion disease, indicating their regulatory effects in prion disease. Up-regulation of Alpha-internexin in the brain of 22L-infected mice might result in neurodegeneration. Alpha-internexin probably is a new biomarker for prion disease. It is meaningful for diagnosis of prion disease. Down-regulation of Hsp60 and Careticulin leads to not clear misfolded protein promptly. The misfolded protein accumulated in CNS. Hsp60 and Careticulin might be new therapeutic target for prion disease. Cofilin 1, neurofascin and contactin 1 might be important in prion disease, but they are not clear. The peptides of m/z 758.7725 and m/z 5432.2069 probably are biomarkers, which might play important roles in pathogenesis of prion disease. This study provides new thinking for diagnosis of prion disease. |
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