The low levels of nerve growth factor and its upstream regulatory kinases in prion infection is reversed by resveratrol.

Resveratrol shows ability to eliminate prion replication, but the exact mechanism for prion eradication was not clear yet. Our previous studies demonstrate a downregulation of brain-derived nerve growth factor (BDNF) during prion infection, meanwhile recovery of cerebral nerve growth factor (NGF) level by resveratrol treatment has been reported in other neurodegenerative models. To obtain the possible changes of brain NGF and its upstream regulatory cascade during prion infection and after removal of prion propagation, the levels of NGF and its upstream regulatory factors in various prion-infected and prion-eradicated SMB cell lines and mice brains inoculated with various SMB cellular lysates were assessed with various methodologies. The levels of NGF were significantly decreased during prion replication, while recovered after removal of PrPSc by resveratrol in vitro. Morphological assays revealed that the NGF signals mainly colocalized within neurons, but not in the proliferative astrocytes and microglia. The upstream positive regulatory kinases, such as p-CREB, p-CaMKIV, CaMKK2 were decreased in the prion infected cells and mice brains, whereas the negative regulatory one, p-CaMKK2, was increased. The aberrant situations of those kinases in prion infected cell lines or mice brains could be also partially reversed by removal of prion agent. Moreover, we demonstrated that the signals of CaMKK2 and p-CaMKK2 were also distributed predominately in neurons in the brain tissues. The data illustrate a direct linkage of abnormally repressive NGF and its upstream regulatory kinases with prion infection. Resveratrol has not only the ability to inhibit prion replication, but also to improve the expression of NGF via CaMKK2/CaMKIV cascade, which might benefit the microenvironment in brains.

Stilbene Compounds Inhibit the Replications of Various Strains of Prions in the Levels of Cell Culture, PMCA, and RT-QuIC Possibly via Molecular Binding.

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.

Significant enhanced expressions of aquaporin-1, -4 and -9 in the brains of various prion diseases.

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.

Aberrant Decrease of the Endogenous SIRT3 and Increases of Acetylated Proteins in Scrapie-Infected Cell Line SMB-S15 and in the Brains of Experimental Mice.

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 ATP5ß. Declined SOD2 and ATP5ß 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.