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

Primers used for real-time PCR and cloning.

<p>Primers used for real-time PCR and cloning.</p

Role of CD36 in PrP_106–126-induced production of nitric oxide and iNOS expression in BV2 and primary microglia.

<p>Cells were first pre-incubated or not with anti-CD36mAb (1 µg/ml) or irrelevant rabbit IgG (Ab) and then treated for 12 hours with PBS, 50 µM PrP_106–126 (PrP), or scrambled PrP_106–126 (Scr). A. The level of NO was measured in supernatant from primary microglia culture with nitrite assay as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0030756#s2" target="_blank">Materials and Methods</a>. The amount of nitric oxide is expressed as U/l cell supernatant. B. ELISA was performed on supernatant from culture of BV2 microglia as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0030756#s2" target="_blank">Materials and methods</a>. All data in A and B are means ± s.d. of triplicate samples and are representative of an experimental n of 3 or 4, * P<0.05. C. Cytoplasmic extracts from primary microglia were prepared and immunoblotted with anti-iNOS antibody as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0030756#s2" target="_blank">Materials and Methods</a>. The blot was stripped and reprobed with anti-β-actin antibody to estimate the total amount of protein loaded in gel. Representative blots of iNOS and actin are shown. Bars represent the relative levels of iNOS, compared with β-actin, and were expressed as arbitrary units. Data are the means±S.D of three independent experiments. * P<0.05, significantly different from control cells. 1-PBS, 2–50 µM PrP_106–126, 3- PrP_106–126+irrelevant rabbit IgG (Ab), 4- Prpscr (50 µM), 5- Anti-CD36mAb (1 µg/ml), 6- PrP_106–126+Anti-CD36mAb.</p

Western blot analysis of p-Fyn.

<p>BV2 microglia were treated or not with neurotoxic prion peptides (100 µM) for 24 h after 1 h-preincubation with anti-CD36 mAb. The role of Fyn in CD36 signaling was examined by assessing Fyn phosphorylation. A. Extracts were prepared and immunoblotted with phospho-Fyn (p-Fyn) antibody as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0030756#s2" target="_blank">Materials and Methods</a>. The blot was stripped and reprobed with anti-β-actin antibody to estimate the total amount of protein loaded in gel. Representative blots of p-Fyn and β-actin are shown. B. Bars represent the relative levels of cleaved p-Fyn, compared with β-actin, and were expressed as arbitrary units. Data are the means±S.D of three independent experiments. * P<0.05, significantly different from control cells.</p

Western blot analysis of caspase-1 cleavage.

<p>BV2 microglia were treated or not with two different concentrations of neurotoxic prion peptides (50 µM and 100 µM, respectively) for 24 h. The role of CD36 in caspase1 cleavage was examined by 1 h-preincubation with anti-CD36 antibody. A. Extracts were prepared and immunoblotted with caspase-1 antibody as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0030756#s2" target="_blank">Materials and Methods</a>. The blot was stripped and reprobed with anti-β-actin antibody to estimate the total amount of protein loaded in gel. Representative blots of caspase-1 and β-actin are shown. Extracts of Aβ_1–42-treated cells were used as positive control. B. Bars represent the relative levels of cleaved caspase-1, compared with β-actin, and were expressed as arbitrary units. Data are the means±S.D of three independent experiments. * P<0.05, significantly different from control cells.</p

Quantitative RT-PCR analysis of PrP106–126 effect on mRNA expression of <i>CD36</i>.

<p>A. Electrophoresis of RT-PCR-amplified CD36 (294 bp) and β-actin (223 bp) on 8% agarose gel and stained with ethidium bromide. B. Time course of CD36 mRNA Expression upon exposure to PrP_106–126 in BV2 microglia. Cells were treated for 3, 6, 12 h, or 24 hours with 100 µM PrP_106–126. Total mRNA was isolated and reverse-transcribed. The mRNA level of CD36 were measured by quantitative RT-PCR. Expression of a receptor at each time point is expressed as a percentage of expression in control cells exposed to 100 µM scrambled PrP_106–126 only for the same time. All data are mean ± s.d. of triplicate samples and are representative of an experimental n of 3 or 4. *, P<0.05.</p

The mRNA expression of proinflammatory cytokines IL-1β, IL-6, and TNF-α, in BV2 microglia treated with PrP_106–126 in the presence or not of anti-CD36 antibody.

<p>Cells were first pre-incubated or not with Anti-CD36 antibody (1 µg/ml) or irrelevant rabbit IgG (Ab) and then treated for 12 hours with 50 µM PrP_106–126 (PrP). Total mRNA was isolated and reverse-transcribed. The mRNA levels of pro-inflammatory cytokines were measured by quantitative RT-PCR. The mRNA level of each cytokine is expressed as fold increase over control cells which were exposed to PBS only. Data are means ± s.d. of triplicate samples *P, 0.05.</p

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

<p><b>Abstract</b></p> <p><b>Background</b></p> <p>Prion diseases are neurodegenerative disorders characterized by the accumulation of an abnormal disease-associated prion protein, PrP^Sc. In prion-infected brains, activated microglia are often present in the vicinity of PrP^Sc 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).</p> <p><b>Methods</b></p> <p>The 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.</p> <p><b>Results</b></p> <p>We 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.</p> <p><b>Conclusions</b></p> <p>We 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.</p