| Prion diseases, also known as transmissible spongiform encephalopathy (TSEs), are a group of rare, fatal neurodegenerative diseases, including Creutzfeldt-Jakob disease (CJD), Kuru, fatal familial insomnia (FFI) and Gerstmann- Straussler-Scheinker syndrome (GSS) in human, bovine spongiform encephalopathy (BSE) in cow, scrapie in sheep and so on. The clinical manifestations of TSEs include progressive dementia, ataxia, tremor, cognitive impairment, and eventually lead to death. Pathological features show characteristic spongy change, loss of neurons and glial cell proliferation in central nervous system, etc. A possible pathogenesis of TSEs is a transformation of normal prion protein (PrPc) into an abnormal form (PrPSc) via misfolding of the protein and the accumulation of the abnormal prion protein in central nervous system, eventually leading to neuron death.Mitochondria are the organelles of aerobic respiration and production of ATP in eukaryotic cells, as well as are the main source of energy needed for cell metabolism. The mitochondria are highly dynamic organelles that in a dynamic process of persistently fission, fusion, transfer and autophagy. Mitochondria keep a balance of their shape and distribution through continuous fission and fusion. When the balance was damaged, the functions of mitochondria will be affected. The fission and fusion of mitochondria are regulated by a group of mitochondrial dynamics related proteins, such as dynamin related proteinl (Drpl), and its effect factors including Fisl, Mff, MID49 and MID51; mitochondria fusion protein Mitofusin 1 (Mfnl), Mitofusin 2 (Mfn2), optic atrophy proteinl (Opal), etc. These proteins regulate mitochondrial morphology and distribution by adjusting the mitochondrial fission and fusion, and play an important role in the maintenance of mitochondria function.Under normal circumstances, Drpl is mainly distributed in the cytoplasm, just before the mitochondria fission, Drpl is recruited to a division site around a circular spiral, hydrolysis GTP to release energy, then micropipe ring shrink and cut off the mitochondria. Opal locates on inner mitochondrial membrane and has the function of promoting mitochondria fusion, playing an important role in maintaining crest shape. Studies have shown that disorder of mitochondria fission and fusion occurs in a variety of neurodegenerative diseases. In addition to the changes of the expression level, protein modification after translation also shows significance on the biological activities. Abnormal changes of protein S-nitrosylation have been demonstrated in a variety of neurodegenerative diseases. Drpl protein may process a series of post-translation modifications, such as phosphorylation, ubiquitin, and S-nitrosylation. In Alzheimer's disease (AD), the total level of Drpl in the brain tissues is decreased, while the level of S-nitrosylated Drpl (SNO-Drpl rose significantly. Currently, the relationship between Drpl S-nitrosylation and nerve impair of prion disease is still unclear.To investigate the alteration of mitochondrial dynamics in the brain tissues of scrapie-infected animal models, the situations of Drpl and Opal in the brains during prion infection were evaluated with various scrapie experimental mouse models, including scrapie agents 139A- and ME7-infected mice, agents 263K-infected hamsters, as well as S15-infected mice that were infected with the cellular extracts from a prion infectious cell line SMB-S15.Westerm blots revealed that in the terminal stages of the diseases, the brain levels of Drpl in 139A-infected mice,263K-infected hamsters and S15-infected mice (controlled with the mice inoculated with the extracts from its normal partner cells SMB-PS), were significantly decreased, while Drpl level in ME7-infected mice increased but without statistical significance. Dynamic assays of brain Drpl in the samples collected at different time-points during incubation period of 139A-and ME7-mice (0,90,120,150, 180 day post-infection (dpi)) showed a similar alterative trend, which declined to the bottom at the middle stage and rose again at terminal stage. Similar changing pattern was also observed in the brain samples of the hamsters infected with agent 263K collected at different time points (0,20,40,60,80 dpi). IHC assays confirmed that compared with the observations of numerous large brown-colored cells in the normal controls, there were markedly less brown signals in the cortex and cerebellum regions of the 139A-and ME7-infected mice.Compared with the individual normal controls, the brain Opal levels of the four tested scrapie rodents by Western blots also revealed decrease tendency at the terminal stage, but only that of 263K-infected hamsters showed statistical significance. Further dynamic analyses of Opal levels in the brains collected at different time-points during infection identified time-dependent declines. IHC assays illustrated that Opal-specific signals in the brains of 139A- and ME7-infected mice were relatively small and weak, compared with amounts of large and deep-stained cells in the normal mice.Furthermore, the distribations of Drpl and Opal in neurons and glial cells were accessed with immunofluorescent assay (IFA). It showed that both Drpl and Opal are mainly distributed mainl within neurons in the central nervous system.To figure out the possible changes of S-nitrosylation of Drpl and Opal during scrapie infection, the total S-nitrosylated (SNO) proteins were extracted from the brain tissues of 139A-and ME7-infected mice at terminal stage and age-matched normal ones. The extracts were subsequently subjected into Drpl and Opal specific Western blots. It demonstrated that the levels of S-nitrosylated forms of Drpl in the brains of three 139A-and three ME7-infected mice were obviously increased compared with the data of three normal controls, showing statistical difference in quantitative assay. Surprisingly, no S-nitrosylated form of Opal was detectable both in the brains of infected or normal mice. Dynamic assays of SNO-Drpl in the incubation period of prion infection illustrated the level of SNO-Drpl in the incubation period of prion infection. It seems that accompanying with the decrease of total Drpl in the brains, the SNO form of Drpl increases during prion infection with time-dependent alteration.In summary, our study here has proposed the molecular evidences that Drpl and Opal, which were two essential factors for fission and fusion of mitochondria, decreased obviously in the scrapie infected mouse and hamster animal models including 139A, ME7, SMB-S15 and 263K. But the quantities of S-nitrosylated forms of Drpl in the brains of 139A- and ME7-infected mice were obviously increased compared with the data of normal controls. All these changes of mitochondria maybe the important pathogenesis of prion diseases and in the end the changes of mitochondria will lead to irreversible damage to nerve cells in the central nervous system. |
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