The linkage between mitochondrial dysfunction and neurodegenerative diseases including prion diseases has been frequently reported. As the major deacetylase in mitochondria, SIRT3 plays a crucial part in regulating the function of many mitochondrial proteins. Although SIRT3 was reported to be linked to several neurodegenerative diseases, it is still unknown if SIRT3 is involved in prion diseases. In this study, we have presented a substantially declined status of mitochondrial SIRT3 in both the levels of cultured cells and an experimental rodent model during scrapie prion replication and infection. Such decreased SIRT3 activity led to a decreased deacetylating activity, resulting in increases of the acetylated forms of some substrates of SIRT3 in cells, such as SOD2 and ATP5β. Declined SOD2 and ATP5β activities subsequently caused an increase of intracellular ROS and a reduction of ATP. Furthermore, we have also proposed evidence that the activity of cellular SIRT3 is partially recovered when abnormal prion propagation in the cultured cells is removed by resveratrol. Those data emphasize a close connection between the prion replication and mitochondrial deacetylation due to SIRT3, thereby partially explaining mitochondrial dysfunction in prion diseases.

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BackgroundAcetylation is a reversible post-translational modification in eukaryotic and prokaryotic cells, actively participating in the regulation for biological functions and in the pathogenesis of diseases.MethodsThe acetylated proteins from the cortex regions of scrapie agents 139A- and ME7-infected mice collected at mid-early (80 days post-infection, dpi), mid-late (120 dpi) and terminal stage (180 dpi) were extracted. The global profiles of the brain acetylated proteins were assayed with proteomic mass spectrometry. The acetylated peptides whose levels were >1.5-fold higher or lower than that of age-matched normal controls were considered as differentially expressed acetylated peptides (DEAPs).ResultsA total of 1,485 acetylated peptides were identified. 118, 42 and 51 DEAPs were in the brains of 139A-80 dpi, 120 dpi and 180 dpi, while 390, 227 and 75 DEAPs were in those of ME7-80 dpi, 120 dpi and 180 dpi, respectively. Overwhelming majority of the DEAPs in mid-early stage was down-regulated, while more portions of DEAPs in mid-late and late stage were up-regulated. Approximately 22.1% (328/1485) acetylated peptides were mitochondrial associated, which were mapped to 74 different proteins. Among them, 44 (59.5%) proteins showed differentially expressed at least at one tested time-point. KEEG pathways analysis identified 39, 13, 10 and 55, 25, 18 pathways in the samples of 80, 120 and 180 dpi of 139A- and ME7-infected mice as significantly changed (P<0.05), respectively. Six pathways were commonly involved in all tested samples, including carbon metabolism, metabolic pathways, biosynthesis of amino acids, glycolysis/gluconeogenesis, pyruvate metabolism and citrate cycle (TCA cycle). Moreover, dozens of steps in TAC cycle were affected via down-regulated acetylation for the relevant enzymes in the mid-early stage, while many steps were affected in the mid-late stage via up-regulated acetylation. In the late stage, the affected steps focused on up-regulated acetylation for succinate dehydrogenase, fumarate hydratase and malate dehydrogenase.ConclusionCollectively, Our data here illustrated a picture of global acetylation for brain proteins during prion infection, showing remarkably inhibiting acetylation in the early stage and relatively enhanced acetylation in the late stage.

Aquaporins (AQPs) are widely expressed in various types of tissues, among them AQP1, AQP4 and AQP9 are expressed predominately with relatively special distributing features in various brain regions. The aberrant changes of AQP1 and AQP4 have been observed in the brains of Alzheimer disease (AD). To evaluate the underlying alteration of brain AQPs in prion diseases, scrapie strains of 139A, ME7 and S15 infected mice were tested in this study. Western blots revealed markedly increased levels of AQP1, AQP4 and AQP9 in the brain tissues of all tested scrapie-infected mice collected at terminal stage. Analyses of the AQPs levels in the brain tissues collected at different time-points during incubation period showed time-dependent increased in 139A and ME7-infected mice, especially at the middle-late stage. The AQP1 levels also increased in the cortex regions of some human prion diseases, including the patients with sporadic Creutzfeldt-Jakob disease (CJD), fatal familial insomnia (FFI) and G114V genetic CJD (gCJD). Immunohistochemistry (IHC) assays verified that the AQPs-positive cells were astrocyte-like morphologically; meanwhile, numerous various sizes of AQPs-positive particles and dots were also observable in the brain sections of scrapie-infected mice. Immunofluorescent assays (IFAs) illustrated that the signals of AQPs colocalized with those of the GFAP positive proliferative astrocytes, and more interestingly, appeared to overlap also with the signals of PrP in the brains of scrapie-infected mice. Moreover, IHC assays with a commercial doublestain system revealed that distributing areas of AQPs overlapped not only with that of the activated large astrocytes, but also with that of abundantly deposited PrPSc in the brain tissues of scrapie murine models. Our data here propose the solid evidences that the expressions of brain AQP1, AQP4 and AQP9 are all aberrantly enhanced in various murine models of scrapie infection. The closely anatomical association between the accumulated AQPs and deposited PrPSc in the brain tissues indicates that the abnormally increased water channel proteins participate in the pathogenesis of prion diseases.

Resveratrol shows the ability to block prion replication in a scrapie-infected cell line, SMB-S15, and remove the infectivity of the treated cell lysates in an experimental bioassay. In this study, we compared the effectiveness of three stilbene compounds, resveratrol (Res), pterostilbene (Pte), and piceatannol (Pic), on inhibiting prion propagations in the levels of cell culture, PMCA, and RT-QuIC. All three chemicals showed active suppressions on PrPSc replication in SMB-S15 cells, in which Res seemed to be the most active one, followed by Pic and Pte. Mouse PrP-based PMCA tests using the lysates of SMB-S15 cells and brain homogenates of scrapie agents S15-, 139A-, or ME7-infected mice verified that Res, Pte, and Pic inhibited the amplifications of PK-resistant signals. Res was also the most effective one. Mouse PrP-based RT-QuIC using the above seeds demonstrated that three stilbenes efficiently inhibited the fibril formation. However, Pic was the most effective one, followed by Res and Pte. Furthermore, the inhibition activities of the three stilbenes on the brain-derived prion from a 263K-infected hamster were tested with hamster PrP-based PMCA and RT-QuIC. The results indicated that Pic was the most effective one apparently, followed by Res and Pte. According to the results of Biacore, Res showed binding affinities much stronger than those of Pte, whereas both revealed markedly stronger binding affinities with mouse PrP. Our data here indicate that different stilbenes have the ability to block PrPSc replication in vitro with different prion species. The suppressive effects of stilbene compounds are likely associated with their molecular binding activities with PrPs.