Stimulations of the Culture Medium of Activated Microglia and TNF-Alpha on a Scrapie-Infected Cell Line Decrease the Cell Viability and Induce Marked Necroptosis That Also Occurs in the Brains from the Patients of Human Prion Diseases

Abstract

Activation of microglia and increased expression of TNF-α are frequently observed in the brains of human and animal prion diseases. As an important cytokine, TNF-α participates in not only pro-inflammatory responses but also in cellular communication, cell differentiation, and cell death. However, the role of TNF-α in the pathogenesis of prion disease remains ambiguous. In this study, the activities of a scrapie-infected cell line SMB-S15 and its normal partner SMB-PS exposed to the supernatant of a LPS-activated microglia cell line BV2 were evaluated. After it was exposed to the LPS-stimulated supernatant of BV2 cells, the cell viability of SMB-S15 cells was markedly decreased, whereas that of the SMB-PS cells remained unchanged. The level of TNF-α was significantly increased in the LPS-stimulated supernatant of BV2 cells. Further, we found that the recombinant TNF-α alone induced the decreased cell viability of SMB-S15 and the neutralizing antibody for TNF-α completely antagonized the decreased cell viability caused by the LPS-stimulated supernatant of BV2 cells. Stimulation with TNF-α induced the remarkable increases of apoptosis-associated proteins in SMB-PS cells, such as cleaved caspase-3 and RIP1, whereas an obvious increase of necroptosis-associated protein in SMB-S15 cells, such as p-MLKL. Meanwhile, the upregulation of caspase-8 activity in SMB-PS cells was more significant than that of SMB-S15 cells. The decreased cell viability of SMB-S15 and the increased expression of p-MLKL induced by TNF-α were completely rescued by Necrostatin-1. Moreover, we verified that removal of PrPSc propagation in SMB-S15 cells by resveratrol partially rescues the cell tolerance to the stimulation of TNF-α. These data indicate that the prion-infected cell line SMB-S15 is more vulnerable to the stimulations of activated microglia and TNF-α, which is likely due to the outcome of necroptosis rather than apoptosis. Furthermore, significant upregulation of p-MLKL, MLKL, and RIP3 was detected in the post-mortem cortical brains of the patients of various types of human prion diseases, including sporadic Creutzfeldt-Jakob disease (sCJD), G114 V-genetic CJD (gCJD), and fatal familial insomnia (FFI).

miR32-5p promoted vascular smooth muscle cell calcification by upregulating TNFα in the microenvironment

“…According to the report by Ma et al [19] with some modification, the VSMCs were divided into 6 groups, cultured in 12-well plates with 1 ml DMEM with 10% FBS and 100 U/ml penicillin-streptomycin, and treated as follows: group 1 received 10% BV2 culture supernatant (treated with LPS) and 10 mM β-glycerophosphate (Sigma, Poole, Dorset, UK); group 2 received 10% BV2 culture supernatant (treated with LPS), which was incubated with 7 μl rabbit anti-mouse TNFα antibody (BOSTER Biological Technology Co. Ltd., China) at 4°C for 12 h and 10 mM β-glycerophosphate; group 3 received 10 mM βglycerophosphate and 10 ng/ml mouse TNFα (BOSTER Biological Technology Co. ltd., China); group 4 received 10 ng/ml mouse TNFα; group 5 received 10 mM βglycerophosphate; and group 6 received equal volume of PBS. The VSMCs of all groups were cultured at 37°C and 5% CO 2 for 5 days, and the culture supernatants were replaced at day 3.…”

Different Aberrant Changes of mGluR5 and Its Downstream Signaling Pathways in the Scrapie-Infected Cell Line and the Brains of Scrapie-Infected Experimental Rodents

“…However, we have also identified that lots of abnormally regulated brain proteins in prion-infected animals do not significantly change in SMB-S15 cells or change in opposite direction, such as αΒ-crystalline, brain-derived neurotrophic factor (BDNF) and the relevant factors (TrkB, p-TrkB, GRB2 and p57NTR), metalloproteinase (ADAM10), glucose transporter 3 (GLUT3), and Polo-like kinases 3 (PLK3) ( Wang et al, 2013 ; Chen et al, 2014 ; Yan et al, 2014 ; Ting-Ting Wang et al, 2016 ; Wang et al, 2017 ). Moreover, majority of the abnormally expressed proteins in SMB-S15 cells can be completely or partially converted by removal of prion replication with resveratrol, but some do not, such as RyR2 and caspase 8 ( Shi et al, 2018 ; Ma et al, 2019 ). We assume that although prion-infected cell lines mimic to some extent the prion infection in vivo , it more reflects a situation that the cells adopt the propagation and accumulation of prions; thus, the protein changing profile in prion-infected cell line may differ with that in the brains of prion-infected animals.…”

Activation of IP10/CXCR3 signaling with highly coincidental with PrPScdeposit in the brains of scrapie infected mice

“…Meanwhile, neuroinflammation induce subsequently pathophysiological effects, either neuroprotective or neurotoxic. Active neuroinflammation responses have been well documented in prion diseases, represented by activation of microglia, complement system and numerous inflammatory cytokines [26][27][28][29][30][31]. IP10 is mainly secreted by microglia in CNS after LPS stimulation [32].…”