Xiangmei Zhou

The NALP3 inflammasome is involved in neurotoxic prion peptide-induced microglial activation

BackgroundPrion diseases are neurodegenerative disorders characterized by the accumulation of an abnormal disease-associated prion protein, PrPSc. In prion-infected brains, activated microglia are often present in the vicinity of PrPSc aggregates, and microglial activation is thought to play a key role in the pathogenesis of prion diseases. Although interleukin (IL)-1β release by prion-induced microglia has been widely reported, the mechanism by which primed microglia become activated and secrete IL-1β in prion diseases has not yet been elucidated. In this study, we investigated the role of the NACHT, LRR and PYD domains-containing protein (NALP)3 inflammasome in IL-1β release from lipopolysaccharide (LPS)-primed microglia after exposure to a synthetic neurotoxic prion fragment (PrP106-126).MethodsThe inflammasome components NALP3 and apoptosis-associated speck-like protein (ASC) were knocked down by gene silencing. IL-1β production was assessed using ELISA. The mRNA expression of NALP3, ASC, and pro-inflammatory factors was measured by quantitative PCR. Western blot analysis was used to detect the protein level of NALP3, ASC, caspase-1 and nuclear factor-κB.ResultsWe found that that PrP106-126-induced IL-1β release depends on NALP3 inflammasome activation, that inflammasome activation is required for the synthesis of pro-inflammatory and chemotactic factors by PrP106-126-activated microglia, that inhibition of NF-κB activation abrogated PrP106-126-induced NALP3 upregulation, and that potassium efflux and production of reactive oxygen species were implicated in PrP106-126-induced NALP3 inflammasome activation in microglia.ConclusionsWe conclude that the NALP3 inflammasome is involved in neurotoxic prion peptide-induced microglial activation. To our knowledge, this is the first time that strong evidence for the involvement of NALP3 inflammasome in prion-associated inflammation has been found.

p75NTR activation of NF-κB is involved in PrP106-126-induced apoptosis in mouse neuroblastoma cells

Neuronal death is a pathological hallmark of prion diseases. Synthetic prion peptide PrP106-126 can convert PrP(C) into protease-resistant aggregates, which can cause neurotoxicity in vivo and in vitro. Various cell surface proteins can participate in the infection process of prions. p75(NTR) can interact with PrP106-126 and has a neurotoxic effect on neurons. However, for p75(NTR) lacking intrinsic catalytic activity domain in cytoplasm, p75(NTR) -associated signaling molecular and the signaling events in cytoplasm in p75(NTR)-mediated apoptosis responding to PrP106-126 remain still unknown. Thus p75(NTR) -associated NF-kappaB signaling pathway was investigated in this study. Herein PrP106-126-induced apoptosis in mouse neuroblastoma cell line N2a, PrP106-126 significantly up-regulated p75(NTR) expression on mRNA and protein levels. For the first time we found that PrP106-126 induced activation of NF-kappaB by Western blot assay, and blocking the interaction of p75(NTR) with PrP106-126 by p75(NTR) polyclonal antibody sc-6189 or pretreatment with inhibitor NF-kappaB SN50 reduced the activation of NF-kappaB and attenuated the apoptotic effect by PrP106-126. This study offers a possible interpretation that NF-kappaB signaling pathway was activated by the interaction of PrP106-126 with p75(NTR), and NF-kappaB activity showed the pro-apoptotic effect in PrP106-126-induced apoptosis in N2a cells. Involvement of NF-kappaB signaling pathway in p75(NTR)-mediated apoptosis may partially account for the PrP106-126-induced neurotoxicity in N2a cells.

The AIM2 Inflammasome Is Involved in Macrophage Activation During Infection With Virulent Mycobacterium bovis Strain

BACKGROUND Mycobacterium bovis, the causative agent of bovine tuberculosis, infects host macrophages and triggers production of the proinflammatory cytokine interleukin 1β (IL-1β). The mechanism by which macrophages become activated and secrete IL-1β in tuberculosis has not yet been elucidated. METHODS In this study, we investigated the role of the absence in melanoma 2 (AIM2) inflammasome in IL-1β release from macrophages infected with pathogenic M. bovis strain. RESULTS We found that the AIM2 inflammasome activation is involved in the production of IL-1β in primary and immortalized mouse macrophage upon M. bovis infection; that the activation process requires cytoplasmic potassium efflux, mycobacterial internalization, but not reactive oxygen species (ROS) or IFN-β release; that the AIM2 inflammasome contributes to the synthesis of proinflammatory and chemotatic factors in M. bovis-infected macrophages; and that the activation of the AIM2 inflammasome is due, at least in part, to mycobacterial translocation into the cytosol. CONCLUSIONS We conclude that the AIM2 inflammasome is involved in macrophage activation during infection with virulent M. bovis strain. To our knowledge, this is the first evidences for the involvement of the AIM2 inflammasome in M. bovis infection.

CD36 Participates in PrP106–126-Induced Activation of Microglia

Microglial activation is a characteristic feature of the pathogenesis of prion diseases. The molecular mechanisms that underlie prion-induced microglial activation are not very well understood. In the present study, we investigated the role of the class B scavenger receptor CD36 in microglial activation induced by neurotoxic prion protein (PrP) fragment 106–126 (PrP106–126). We first examined the time course of CD36 mRNA expression upon exposure to PrP106–126 in BV2 microglia. We then analyzed different parameters of microglial activation in PrP106–126-treated cells in the presence or not of anti-CD36 monoclonal antibody (mAb). The cells were first incubated for 1 h with CD36 monoclonal antibody to block the CD36 receptor, and were then treated with neurotoxic prion peptides PrP106–126. The results showed that PrP106–126 treatment led to a rapid yet transitory increase in the mRNA expression of CD36, upregulated mRNA and protein levels of proinflammatory cytokines (IL-1β, IL-6 and TNF-α), increased iNOS expression and nitric oxide (NO) production, stimulated the activation of NF-κB and caspase-1, and elevated Fyn activity. The blockade of CD36 had no effect on PrP106–126-stimulated NF-κB activation and TNF-α protein release, abrogated the PrP106–126-induced iNOS stimulation, downregulated IL-1β and IL-6 expression at both mRNA and protein levels as well as TNF-α mRNA expression, decreased NO production and Fyn phosphorylation, reduced caspase-1 cleavage induced by moderate PrP106–126 –treatment, but had no effect on caspase-1 activation after treatment with a high concentration of PrP106–126. Together, these results suggest that CD36 is involved in PrP106–126-induced microglial activation and that the participation of CD36 in the interaction between PrP106–126 and microglia may be mediated by Src tyrosine kinases. Our findings provide new insights into the mechanisms underlying the activation of microglia by neurotoxic prion peptides and open perspectives for new therapeutic strategies for prion diseases by modulation of CD36 signaling.

c-Abl Tyrosine Kinase Mediates Neurotoxic Prion Peptide-Induced Neuronal Apoptosis via Regulating Mitochondrial Homeostasis

Prion diseases are neurodegenerative disorders characterized by the accumulation of a disease-associated prion protein and apoptotic neuronal death. Previous studies indicated that the ubiquitous expression of c-Abl tyrosine kinase transduces a variety of extrinsic and intrinsic cellular signals. In this study, we demonstrated that a synthetic neurotoxic prion fragment (PrP106-126) activated c-Abl tyrosine kinase, which in turn triggered the upregulation of MST1 and BIM, suggesting the activation of the c-Abl-BIM signaling pathway. The peptide fragment was found to result in cell death via mitochondrial dysfunction in neuron cultures. Knockdown of c-Abl using small interfering RNA protected neuronal cells from PrP106-126-induced mitochondrial dysfunction, production of reactive oxygen species, and apoptotic events inducing translocation of Bax to the mitochondria, cytochrome c release into the cytosol, and activation of caspase-9 and caspase-3. Blocking the c-Abl tyrosine kinase also prevented neuronal cells from PrP106-126-induced apoptotic morphological changes. This is the first study reporting that c-Abl tyrosine kinase as a novel upstream activator of MST1 and BIM plays an important role in prion-induced neuron apoptosis via mitochondrial dysfunction. Our findings suggest that c-Abl tyrosine kinase is a potential therapeutic target for prion disease.

Aspirin inhibits cytotoxicity of prion peptide PrP106-126 to neuronal cells associated with microglia activation in vitro.

The synthetic peptide consisting of amino acid residues 106-126 of the human prion protein PrP106-126 has been demonstrated to generate fibrils, which damage neurons either directly by interacting with components of the cell surface to trigger cell apoptosis signaling or indirectly by activating microglia to produce inflammatory mediators. In our study, rat microglia cells were treated with PrP106-126 or scramble PrP106-126 (Scr PrP). Activated nuclear factor kappaB (NF-kappaB) was determined using immunofluorescence staining and the expression of TNF-alpha and IL-1beta mRNA was measured by quantitative RT-PCR. Inhibitory activity of aspirin on neurotoxicity of PrP106-126 associated with microglia activation was determined using an apoptosis detection kit. Treatment of microglia with 25 microM PrP106-126, but not Scr PrP, resulted in activation and translocation of NF-kappaB, which peaked after 20 min of treatment. The activation of NF-kappaB was followed by increased mRNA expression of TNF-alpha and IL-1beta peaking at about 20 h. In the presence of microglia, aspirin significantly inhibited neuro-2a cell death induced by PrP106-126. The number of neuro-2a cells in apoptosis and necrosis with 5 mM aspirin was about 3-fold lower than the cell culture without aspirin (P<0.05). These data suggest that increased production of cytokines by microglia cells in prion disease is probably regulated by NF-kappaB translocation and may contribute to neurotoxicity of prions, and neurotoxicity of PrP106-126 may be inhibited by aspirin.

Inhibition of phagocytosis and lysosomal acidification suppresses neurotoxic prion peptide-induced NALP3 inflammasome activation in BV2 microglia

Prion diseases are neurodegenerative disorders characterized by the accumulation of misfolded prion protein. In a previous study, we showed that neurotoxic prion peptide (PrP106-126) induced NALP3 inflammasome activation in mouse primary and immortalized microglia. In the present work, we examined the relevance of phagocytosis and lysosomal acidification to the activation of the NALP3 inflammasome in PrP106-126-stimulated microglia. Our results showed that the inhibition of phagocytosis or lysosomal acidification significantly reduced IL-1β and IL-18 production, downregulated NALP3 and ASC expression, and decreased the expression of proinflammatory factors. We concluded that phagocytosis and lysosomal acidification are necessary for PrP106-126-induced NALP3 activation in BV2 cells.

Cellular prion protein participates in the regulation of inflammatory response and apoptosis in BV2 microglia during infection with Mycobacterium bovis.

The cellular prion protein (PrPC) is a glycoprotein anchored by glycosylphosphatidylinositol to the cell surface and is abundantly expressed in the central nervous system. A previous study has shown that PrPC contributes to the establishment of infections with intracellular bacteria in macrophages. In the present work, we investigated the role of PrPC in the response of BV2 microglia to Mycobacterium bovis infection. For this purpose, we examined the mRNA expression of prion protein gene (PRNP) upon M. bovis infection and analyzed the effect of siRNA-mediated disruption of PRNP on different parameters of microglial activation and apoptosis in M. bovis-infected microglia. We found that M. bovis infection induced a gradual increase in PRNP mRNA level and that siRNA-mediated silencing of PRNP in M. bovis-infected microglia reduced M. bovis-induced upregulation of pro-inflammatory factors, increased the rate of apoptosis in infected microglia, promoted the intrinsic apoptotic pathway, and downregulated the extrinsic apoptotic pathway. We conclude that PrPC participates in the regulation of the response of microglia to M. bovis infection through the upregulation of pro-inflammatory cytokines and the modulation of apoptosis by interference with the intrinsic apoptotic pathway.

Phagolysosome maturation of macrophages was reduced by PE_PGRS 62 protein expressing in Mycobacterium smegmatis and induced in IFN-γ priming.

Mycobacterium bovis parasitizes host macrophages and has developed strategies to survive within macrophages. Research on mycobacteria-specific PE_PGRS genes indicates that they code for cell surface proteins that may influence virulence. To further elucidate the molecular pathogenesis of tuberculosis and host response to M. bovis, we explored the mechanisms by which PE_PGRS62 protein increase persistence of mycobacterium within host macrophages. We found that the M. smegmatis strain expressing M. bovis PE_PGRS 62 protein reduced phagolysosome maturation in human macrophages, and significantly decreased the mRNA expression of IL-1β in a dose- and time-dependent. We identified that IFN-γ priming of macrophages immediately prior to infection with PE_PGRS62 expressing M. smegmantis, enhanced the maturation of phagolysosomes and induced IL-1β production both that the protein and mRNA levels and further activated the NF-κB pathway. Overall, we demonstrated that PE_PGRS62 protein altered the immune environment of the host cells, which suggested that the pathogenic PE_PGRS62 protein altering the immune mechanism might be involved in the pathogenesis of mycobacterial disease and hence influenced host cell responses to M. bovis infection.

Polymorphism of prion protein gene in sheep of Inner Mongolian, China

Polymorphisms of the prion protein gene (Prnp), especially the amino acid residue alterations at codons 136, 154, and 174, in sheep have been found to be associated with susceptibility to scrapie disease. We investigated Prnp polymorphisms in three local sheep breeds in Inner Mongolia, China. Blood samples were collected from 46 Ujumqin, 34 Sunite, and 22 Mongolian sheep. The genetic DNA of blood samples was extracted, amplified and sequenced, and amino acid alignment was determined. Polymorphisms were detected at 8 codons, among which M157I, Q220H, and R223K have not been previously reported. The frequency of the amino acid residues ARQ/ARQ at codons 136, 154, and 171, respectively, which is associated with medium-high susceptibility to scrapie, was 74.5%, and the frequency of scrapie-resistant genotype ARR/ARR was 7.9%. The highly susceptible genotype VRQ/VRQ at these codons as not detected from the tested sheep. Of the three sheep breeds, Ujumqin sheep had the highest frequency (15.2%) of scrapie-resistant amino acid sequence, ARR/ARR at codons 136, 154, and 171, respectively, accounting for 87.5% sheep that carry these polymorphisms. Our findings are of special importance for both live sheep export and sheep breeding.