Stduy On The Impairment Of Wnt/β-catenin Signaling And Relavant Molecular Mechanisms During Prion Infections In Vivo And In Vitro

Prion diseases are a kind of fatal neurodegenerative diseases affecting central nervous system of humans and animals. The pathogen is a type of infectious, insoluble, limited protease digestion resistant scrapie prion protein (PrPSc). which is derived from the normal cellular prion protein (PrPC) on the cell membrane. Prion diseases include Creutzfeldt-Jakob disease (CJD), fatal familial insomnia (FFI), Gerstmann-Straussler-Scheinker syndrome (GSS). and Kuru in humans. CJD subdivides into sporadic CJD, genetic CJD, iatrogenic CJD, and variant CJD. Animal prion diseases include chronic wasting disease (CWD) in deer, scrapie in sheep and goat and bovine spongiform encephalopathy (BSE) in cattle. The neuropathologic changes are characterized by neuronal loss, spongiform degeneration, astrogliosis and aggregation of PrPSc in the central nervous system.The Wnt signaling pathway is a highly evolutionarily conserved pathway in eukaryotes that regulates cell proliferation, differentiation, renew, metabolism and migrate. The Wnt signaling pathway can be activated by 19 Wnt ligands, according to whether the β-catenin is involved in the signal transduction, and this pathway is divides into the canonical Wnt (Wnt/β-catenin) and the non-canonical Wnt pathway, and the latter is further subdivided into planar cell polarity pathway and the non-canonical Wnt/calcium pathway. Among these pathways, canonical Wnt/p-catenin signaling is well documented. Wnt signaling pathways contribute to neurogenesis and synaptogenesis physiological process.A number of studies have reported that the deregulation of Wnt/β-catenin signaling was related to the pathogenesis of neurodegenerative diseases, such as Alzheimer’s disease (AD) and Parkinson’s disease (PD), but few report is addressed on prion disease. In order to investigate the functional state of Wnt/β-catenin signaling in the CNS tissues during the progression of prion disease, the components of Wnt/p-catenin signaling in the brains of the scrapie agents 139A-and ME7-infected mice were evaluated.Compared with the normal controls, the brain levels of phosphor-β-catenin (Ser33’37, Thr41), which are normal degraded by ubiquitin-proteasome pathway, in 139A-and ME7-infected mice were significantly increased, while those of cyclin D1, which is one of the target genes of canonical Wnt signaling, were decreased. These data indicate an impairment of Wnt/β-catenin pathway at the end stage of prion infection.As glycogen synthase kinase-3β (GSK-3β) directly controls the level of β-catenin phosphorylation and the phosphorylation on the position of Ser9 is believed to be able to repress the activity of GSK-3β, we further investigated the changes of the ratio of phosphor-GSK-3β Ser9 to total GSK-3β.Compared with the normal controls, the ratio of phosphor-GSK-3β Ser9 to total GSK-3β in 139A-and ME7-infected mice were significantly decreased, implying an enhanced GSK-3P activity in scrapie infected mice.Furthermore, two other factors, Wnt-3 and Dishevelled-3 (Dvl-3), which are the positive agents for the activity of Wnt signaling, were tested with individual specific Western blots. We observed slightly decreased Wnt-3 and unchanged Dvl-3 in the brains of the infected mice. Dickkopf-1 (DKK-1) has the ability to inhibit canonical Wnt signaling. Both Western blot and immunohistochemical assays revealed a remarkable increase of DKK-1 in the brain of 139A-and ME7-infected mice, More numbers of DKK-1 positively stained cells and signals were detected in the regions of cortex and cerebellum of the 139A-and ME7-infected mice. Meanwhile, colocalization of the overexpressed DKK-1 signals were obeserved in neurons using immunofluorescent tests.In order to investigate the dynamic changes of some Wnt signaling components in the brains of scrapie-infected mice during incubation periods, brain samples of the scrapie agents 139A-and ME7-infected mice were collected on the different time points during incubation period, and the expression levels of phosphor-β-catenin Ser33,37, Thr and DKK-1 were comparatively evaluated by Western blot. Our data illustrate an impairment of Wnt/β-catenin pathway in the brains of prion infection, which shows a time-dependent progression along with the extension of incubation period.SMB-S15 is a mouse brain cell line infected with scrapie agent Chandler strain, which substantly supported the republication of PrPSc in vitro, while SMB-PS is the partern cell line cured by pentosan sulfate to remove PrPSc. Compared with the control cell line SMB-PS, the transcriptions of Wnt/β-catenin signaling target genes cyclin D1, c-myc, survivin were down-regulated in scrapie infected cell line SMB-S15 in the assays of quatitative real time-PCR.Western blots revealed that the levels of phosphor-β-catenin Ser33,37 and Thr41 in SMB-S15 cell line were much higher than that of SMB-PS cell, while that of cyclin D1, one of the target genes of Wnt signaling, was decreased. The level of phosphor-GSK-3β Ser9 was markedly reduced, representing an enhanced GSK-3β activity in SMB-S15 cells. Meanwhile, the level of cyclin D1 was significant decreased. Moreover, two positive agents in Wnt signalling pathway, Wnt-3 and Dvl-3 were tested with individual specific Western blots. We observed slightly decreased Wnt-3 and Dvl-3 in SMB-S15 cell line, but the antagonist DKK-1 in SMB-PS cell line was much higher than that of SMB-S15 cell.In summary, we uncovered the impairment of canonical Wnt signaling pathway in prion infections in vivo and in vitro.