Alternative complement pathway is activated in the brains of scrapie-infected rodents.

Activation of complement system in central nervous system (CNS) of the patients suffering from prion diseases or animal models infected with prion agents experimentally is reported repeatedly, but which pathways are involved in the complement system during prion infection is not well documented. Here, we evaluated the level of complement factor B (CFB), which is the key factor that triggers alterative pathway (AP) of complement in the brain tissues of scrapie-infected mice with various methodologies. We found that the levels of mRNA and protein of CFB significantly increased in the brain tissues of scrapie-infected mice. Morphologically, the increased CFB-specific signal overlapped with the elevated C3 signal in brain sections of scrapie-infected mice, meanwhile overlapped with damaged neurons and activated microglia, but not with the proliferative astrocytes. Additionally, the level of complement factor P (CFP), the key positive regulator of AP, also increased remarkably in the brain tissues of infected mice. The transcriptional levels of CD55 and CD46, two negative regulators of AP, decreased without significance in brain tissues of scrapie-infected mice at the terminal stage. However, the mRNA and protein levels of CFH, another negative regulator of AP, increased. Through the dynamic analyses of the expressions of CFB, CFP, and CFH in brain sections of 139A-infected mice, which were collected at different time-points during incubation period, illustrated time-dependent increase levels of each factor during the incubation period of scrapie infection. Taken together, our data here demonstrate that the AP of complement cascade is activated in the CNS microenvironment during prion infection.

Decrease of RyR2 in the prion infected cell line and in the brains of the scrapie infected mice models and the patients of human prion diseases.

The levels of ryanodine receptors (RyRs) are usually increased in the brains of human Alzheimer disease (AD) and AD animal models. To evaluate the underlying alteration of brain RyRs in prion disease, scrapie infected cell line SMB-S15 and its infected mice were tested. RyR2 specific Western blots revealed markedly decreased RyR2 levels both in the cells and in the brains of infected mice. Assays of the brain samples of other scrapie (agents 139A and ME7) infected mice collected at different time-points during incubation period showed time-dependent decreases of RyR2. Immunofluorescent assays (IFA) verified that the expression of RyR2 locates predominantly in cytoplasm of SMB cells and overlapped with the neurons in the brain slices of mice. Furthermore, significant down-regulation of RyR2 was also detected in the postmortem cortical brains of the patients of various types of human prion diseases, including sporadic Creutzfeldt-Jakob disease (sCJD), fatal familial insomnia (FFI) and G114V-genetic CJD. Our data here propose the evidences of remarkably decreased brain RyR2 at terminal stages of both human prion diseases and prion infected rodent models. It also highlights that the therapeutic strategy with antagonist of RyRs in AD may not be suitable for prion disease.

Remarkable increases of α1-antichymotrypsin in brain tissues of rodents during prion infection.

α1-Antichymotrypsin (α1-ACT) belongs to a kind of acute-phase inflammatory protein. Recently, such protein has been proved exist in the amyloid deposits which is the hallmark of Alzheimer's disease, but limitedly reported in prion disease. To estimate the change of α1-ACT during prion infection, the levels of α1-ACT in the brain tissues of scrapie agents 263K-, 139A- and ME7-infected rodents were analyzed, respectively. Results shown that α1-ACT levels were significantly increased in the brain tissues of the three kinds of scrapie-infected rodents, displaying a time-dependent manner during prion infection. Immunohistochemistry assays revealed the increased α1-ACT mainly accumulated in some cerebral regions of rodents infected with prion, such as cortex, thalamus and cerebellum. Immunofluorescent assays illustrated ubiquitously localization of α1-ACT with GFAP positive astrocytes, Iba1-positive microglia and NeuN-positive neurons. Moreover, double-stained immunofluorescent assays and immunohistochemistry assays using series of brain slices demonstrated close morphological colocalization of α1-ACT signals with that of PrP and PrPSc in the brain slices of 263K-infected hamster. However, co-immunoprecipitation does not identify any detectable molecular interaction between the endogenous α1-ACT and PrP either in the brain homogenates of 263K-infected hamsters or in the lysates of prion-infected cultured cells. Our data here imply that brain α1-ACT is increased abnormally in various scrapie-infected rodent models. Direct molecular interaction between α1-ACT and PrP seems not to be essential for the morphological colocalization of those two proteins in the brain tissues of prion infection.

Increases of Galectin-1 and its S-nitrosylated form in the Brain Tissues of Scrapie-Infected Rodent Models and Human Prion Diseases.

Galectin-1 (Gal-1) shows neuroprotective activity in brain ischemia, spinal cord injury, and autoimmune neuroinflammation. To evaluate the Gal-1 situation in the brains of prion disease, the brain levels of Gal-1 in several scrapie-infected experimental rodent models were tested by Western blot, including agents 263K-infected hamsters, 139A-, ME7-, and S15-infected mice. Remarkable increases of brain Gal-1 were observed in all tested scrapie-infected rodents at the terminal stage. The brain levels of Gal-1 showed time-dependent increases along with the prolonging of incubation times. Immunohistochemical assays illustrated much stronger stainings in the brain sections of scrapie-infected rodents. Quantitative RT-PCR of Gal-1 gene demonstrated increased transcription in the brains of scrapie-infected mice. Gal-1 was colocalized with GFAP- and NeuN-positive cells, but not with Iba-1-positive cells in immunofluorescent test. Increases of Gal-1 were also detected in the several postmortem cortex regions of human prion diseases. Moreover, the S-nitrosylated forms of Gal-1 in the brains of scrapie-infected rodents were significantly higher than those of normal ones. Our finding here demonstrates markedly increased brain Gal-1 and S-nitrosylated Gal-1 both in scrapie-infected rodents and human prion diseases.