Stimulations of the Culture Medium of Activated Microglia and TNF

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Stimulations of the culture medium of activated microglia and TNF-alpha on 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 Yue Ma, Qi Shi, Kang Xiao, Jing Wang, Cao Chen, Li-Ping Gao, Chen Gao, and Xiaoping Dong ACS Chem. Neurosci., Just Accepted Manuscript • DOI: 10.1021/acschemneuro.8b00354 • Publication Date (Web): 06 Nov 2018 Downloaded from http://pubs.acs.org on November 7, 2018

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ACS Chemical Neuroscience

Stimulations of the culture medium of activated microglia and TNF-alpha on 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 Yue Ma1,#, Qi Shi1#,*, Kang Xiao1, #, Jing Wang1, Cao Chen1, Li-Ping Gao1, Chen Gao1, Xiao-Ping Dong1,2,*

1State

Key Laboratory for Infectious Disease Prevention and Control, Collaborative

Innovation Center for Diagnosis and Treatment of Infectious Diseases (Zhejiang University), National Institute for Viral Disease Control and Prevention, Chinese Center for Disease Control and Prevention, Chang-Bai Rd 155, Beijing 102206, China 2

Center of Global Public Health, Chinese Center for Disease Control and Prevention,

Chang-Bai Rd 155, Beijing 102206, China

# The *

first three authors contribute equally to this study

Corresponding authors Fax: +86-10-58900815. Email address: [email protected]

and [email protected]

ACS Paragon Plus Environment

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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 LPS-activated microglia cell line BV2 were evaluated. After exposed to the LPS-stimulated supernatant of BV2 cells, the cell viability of SMB-S15 cells was markedly decreased, while that of 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 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


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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 up-regulation of p-MLKL, MLKL and RIP3 was detected in the postmortem cortical brains of the patients of various types of human prion diseases, including sporadic Creutzfeldt-Jakob disease (sCJD), G114V-genetic CJD (gCJD) and fatal familial insomnia (FFI). Keywords: Prion, scrapie, microglia, TNF-alpha, apoptosis, necroptosis



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Introduction Prion

diseases

are

a

kind

of

fatal,

transmissible,

and

progressive

neurodegenerative disease, which may affect human and various species of animals 1, 2.

The neuropathology of prion diseases is characterized by progressive neuronal

degeneration, neuronal vacuolation, gliosis as well as progressive accumulation of a pathological form of scrapie prion protein (PrPSc), which is believed to derive from a host-encoded normal cellular prion protein (PrPC), in the central nerve system (CNS) 3.

The activation of innate immune system is emerging as a crucial component of many neurodegenerative diseases, such as Alzheimer’s disease4-6, amyotrophic lateral sclerosis7, Huntington’s disease8, prion disease and so on9, following with the excessive production and release of pro-inflammatory cytokines, such as interleukin-1β (IL-1β) and tumour necrosis factor-alpha (TNF-α)9. TNF-α is one of the most important players within the complicated network of cytokines. It participates in not only pro-inflammatory responses, but also in cellular communication, cell differentiation and cell death10-12. Via binding to its receptors (TNFR1 or TNFR2), TNF-α may exert different functions. Ligand-binding to TNFR1 results in the recruitment of the adaptor molecule TNFR1-associated death domain protein (TRADD) and the assembly of distinct signaling complexes, termed complexes I, IIa and IIb, which lead to distinct functional outcomes13, 14. Complex I induces the expression of NF-κB and AP-1 target genes that are important in inflammation, host defense and cell proliferation and survival14. Complex IIa leads to


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the activation of caspase-8 and a caspase cascade that results in cell death via apoptosis15. Complex IIb causes the activation of the necroptosis effector mixed lineage kinase domain-like protein (MLKL) by receptor interacting protein 3 (RIP3)-dependent mechanism13, 16. MLKL is the key protein involved in the execution of necroptosis16. The activation of MLKL leads to mitochondrial uncoupling, lipid peroxidation, and eventually cell death16, 17. Necroptosis is also a programmed form of cell death, whose result may contribute to inflammation13. The activation of necroptosis has been reported in multiple sclerosis18, amyotrophic lateral sclerosis19 and Alzheimer’s disease20, but is rarely described in prion disease. However, activation of microglia has been repeatedly reported in the brain tissues of prion diseases and their rodent models21, 22. During this process, the brain level of cytokine TNF-α is usually increased22. What is the contribution of TNF-α to the cell death in prion disease remains ambiguous. In the current study, we demonstrated that the scrapie infectious cell line SMB-S15 was more vulnerable to the supernatant of a cultured microglia BV2 cells activated by LPS than its normal partner cell line SMB-PS. Meanwhile, cytokine TNF-α, which was one of the main components in the supernatant of BV2 cells activated by LPS, induced the similar effect on the cell viability of SMB-S15 cells. Compared with that of SMB-PS cells, relatively lower levels and activities of caspase-8 and caspase-3 were detected in SMB-S15 cells, in the situations treated with or without TNF-α. Activation of MLKL, a marker of necroptosis, was observed in the TNF-α treated SMB-S15 cells and in the postmortem cortical brains of the



patients of various types of human prion diseases. Removal of PrPSc propagation in SMB-S15 cells by resveratrol (SMB-S15-Res) partially rescued the cell tolerance to the stimulation of TNF-α, showing unchanged cell viability in the reaction exposed to low concentration of TNF-α. These data indicate that the cultured supernatant of the activated microglia, as well as TNF-α, can induce more cell death on the cells with the propagation of PrPSc, which is possibly associated with the activation of necroptosis.


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Results SMB-S15 cells are more vulnerable to the conditioned medium of BV2 activated by LPS One of the neuropathological features of prion disease is activation of microglia, usually accompanying with the releases of a serial of cytokines3,

23.

To test the

potential effect of the activated microglia on the prion infected cells, a cultured microglial cell line BV2 was exposed to LPS (1 μg/mL) for 24 h and the supernatants of the BV2 cells treated with or without LPS were collected separately. The prion infected cell line SMB-S15 and its normal partner cell line SMB-PS were exposed to the same volume of the supernatants of BV2 cells stimulated with or without LPS for 18 h, meanwhile, both SMB-S15 and -PS cells were also exposed to the medium containing 1 μg/mL of LPS as the control. Evaluations of the cell viabilities by CCK-8 assays revealed that the treatment of LPS (1 μg/mL) alone did not induce detectable effect on both two SMB cells (Fig 1A). The supernatant of BV2 cells with the stimulation of LPS also appeared no effect on the SMB-PS cells, whereas induced a rightly 20% decrease of the cell viability on SMB-S15 cells (P=0.0005) (Fig 1B). Furthermore, the SMB cells were stained with Hoechst and Annexin V-FITC after exposed to the conditioned medium of BV2 cells for 18 h. The images captured by Operetta illustrated more Annexin V-FITC stained (brilliant green) signals in both SMB cells when cultured with the supernatant of BV2 cells with the stimulation of LPS than that without the stimulation of LPS (Fig 1C). After cultured with the LPS-stimulated supernatant of BV2 cells, significantly more green signals were



observed in both cell lines, revealing statistical differences in SMB-S15 cells (P

Stimulations of the Culture Medium of Activated Microglia and TNF

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