LRP1 modulates the microglial immune response via regulation of JNK and NF-κB signaling pathways

Abstract

Background: Neuroinflammation is characterized by microglial activation and the increased levels of cytokines and chemokines in the central nervous system (CNS). Recent evidence has implicated both beneficial and toxic roles of microglia when over-activated upon nerve injury or in neurodegenerative diseases, including Alzheimer's disease (AD). The low-density lipoprotein receptor-related protein 1 (LRP1) is a major receptor for apolipoprotein E (apoE) and amyloid-β (Aβ), which play critical roles in AD pathogenesis. LRP1 regulates inflammatory responses in peripheral tissues by modulating the release of inflammatory cytokines and phagocytosis. However, the roles of LRP1 in brain innate immunity and neuroinflammation remain unclear.

Methods: In this study, we determined whether LRP1 modulates microglial activation by knocking down Lrp1 in mouse primary microglia. LRP1-related functions in microglia were also assessed in the presence of LRP1 antagonist, the receptor-associated protein (RAP). The effects on the production of inflammatory cytokines were measured by quantitative real-time PCR (qRT-PCR) and enzyme-linked immunosorbent assay (ELISA). Potential involvement of specific signaling pathways in LRP1-regulated functions including mitogen-activated protein kinases (MAPKs) and nuclear factor-κB (NF-κB) were assessed using specific inhibitors.

Results: We found that knocking down of Lrp1 in mouse primary microglia led to the activation of both c-Jun N-terminal kinase (JNK) and NF-κB pathways with corresponding enhanced sensitivity to lipopolysaccharide (LPS) in the production of pro-inflammatory cytokines. Similar effects were observed when microglia were treated with LRP1 antagonist RAP. In addition, treatment with pro-inflammatory stimuli suppressed Lrp1 expression in microglia. Interestingly, NF-κB inhibitor not only suppressed the production of cytokines induced by the knockdown of Lrp1 but also restored the down-regulated expression of Lrp1 by LPS.

Conclusions: Our study uncovers that LRP1 suppresses microglial activation by modulating JNK and NF-κB signaling pathways. Given that dysregulation of LRP1 has been associated with AD pathogenesis, our work reveals a critical regulatory mechanism of microglial activation by LRP1 that could be associated with other AD-related pathways thus further nominating LRP1 as a potential disease-modifying target for the treatment of AD.

Keywords: AD; Inflammation; JNK; LRP1; Microglia; NF-κB; RAP.

Fig. 1

Lrp1 knockdown exacerbates the production of pro-inflammatory cytokines. a–d Primary microglia were transiently transfected with non-targeting siRNA (NT) or two independent Lrp1-specific siRNAs for 48 h, then incubated with serum-free medium in the presence or absence of LPS (100 ng/ml) for 4 h (for qRT-PCR) or 24 h (for ELISA). RNA was extracted and the relative mRNA levels of Lrp1 (a), Il-1β (b), Tnf-α (c), and Il-10 (d) were determined by qRT-PCR and shown as bar graph (n = 3). β-Actin was used as an internal control. IL-1β (e) and TNF-α (f) in conditioned media were determined by ELISA (n = 3). Data were plotted as mean ± SEM and normalized to the corresponding control group. *p < 0.05; **p < 0.01; ***p < 0.001 (one-way ANOVA with post hoc Tukey’s t test)

Fig. 2

Both JNK and NF-κB pathways are activated in Lrp1-deficient primary microglia. a Primary microglia were transiently transfected with non-targeting siRNA (NT) or Lrp1-specific siRNAs for 48 h. Cells were stimulated with 100 ng/mL LPS for the indicated time (min). Cell lysates were prepared and analyzed by Western blotting using antibodies specific for LRP1, NF-κB, JNK, p38, ERK, or the phosphorylated forms of these proteins. β-Actin served as a loading control. b–f The quantification of Western blots of LRP1 (b), ratios of phospho-JNK/total JNK (c), phospho-NF-κB/total NF-κB (d), phospho-ERK/total ERK (e), and phospho-p38/total p38 (f). The relative signal intensities of each pathway at various time points were normalized to NT-treated microglia in the absence of LPS stimulation (0 min time point) (n = 3). Data were plotted as mean ± SEM *p < 0.05; **p < 0.01; ***p < 0.001 (one-way ANOVA with post hoc Tukey’s t test)

Fig. 3

RAP increases the expression of pro-inflammatory cytokines and induces both JNK and NF-κB activation in microglia. a–c Primary microglia were treated with control, LPS (100 ng/ml) or RAP (25 or 50 nM) for 4 h (for qRT-PCR) or 30 min (for Western blotting). The qRT-PCR was performed to determine mRNA levels for Il-1β (a), Tnf-α (b), and Lrp1 (c) (n = 3). d–h The protein levels of phospho-IκBα, total IκBα, phospho-NF-κB, total NF-κB, phospho-c-Jun, total c-Jun, and LRP1 in cell lysates were examined by Western blot analysis (d) and quantified (e–h) (n = 3). β-Actin served as a loading control. Data were plotted as mean ± SEM and normalized to the corresponding control group. *p < 0.05; **p < 0.01; ***p < 0.001; N.S. not significant (one-way ANOVA with post hoc Tukey’s t test)

Fig. 4

JNK and NF-κB inhibitors suppress the production of pro-inflammatory cytokines induced by LRP1 knockdown. a, b Mouse primary microglia were transiently transfected with non-targeting siRNA (NT) or LRP1-specific siRNA#1 for 48 h. Cells were stimulated with 100 ng/mL LPS or vehicle for 4 h in the presence or absence of 10 μM SP600125 (pretreated for 30 min). The qRT-PCR analysis was then performed to detect the expression levels of IL-1β (a) and TNF-α (b) (n = 3). β-Actin was used as an internal control. c, d Mouse primary microglia were transiently transfected with non-targeting siRNA (NT) or Lrp1-specific siRNA#1 for 48 h. The cells were then pretreated with 10 μM Bay 11-7082 for 30 min, followed by treatment with 100 ng/mL LPS or vehicle for 4 h. RNA was extracted and the relative mRNA levels of IL-1β (c) and TNF-α (d) were determined by qRT-PCR (n = 3). β-Actin was used as an internal control. Data were plotted as mean ± SEM and normalized to the corresponding control group. e, f Reduction of Lrp1 mRNA on siRNA-mediated knockdown was verified by qRT-PCR. *p < 0.05; **p < 0.01; ***p < 0.001; N.S. not significant (one-way ANOVA with post hoc Tukey’s t test)

Fig. 5

Pro-inflammatory cytokines and Aβ oligomers suppress the expression of LRP1 in microglia. a–c Primary microglia were cultured in the presence of either LPS (100 ng/ml), mouse TNF-α (100 ng/ml), or oligomeric Aβ (10 μM) for 24 h. RNA was extracted and the relative mRNA levels of Il-1β (a), Lrp1 (b), and Apoe (c) were determined and quantified by qRT-PCR (n = 3). β-Actin was used as an internal control. d–f Cell lysates from the same treatments were collected, and the protein levels of LRP1 and apoE were analyzed by Western blot (d) and quantified (e, f). Data represent mean ± SEM and normalized to the corresponding control group. *p < 0.05; **p < 0.01; ***p < 0.001 (one-way ANOVA with post hoc Tukey’s t test)

Fig. 6

NF-κB inhibitor restores LRP1 expression suppressed by LPS. a–b Primary microglia were pretreated with 10 μM SP600125 or Bay 11-7082 for 30 min, followed by stimulation with LPS (100 ng/mL) or vehicle for 24 h. RNA was extracted, and the relative mRNA levels of Lrp1 in microglia treated with SP600125 (a) and BAY11-7082 (b) were determined by qRT-PCR (n = 3). β-Actin was used as an internal control. Data were plotted as mean ± SEM ***p < 0.001; N.S. not significant (two-tailed Student’s t test). c–f The protein levels of LRP1, phospho-NF-κB, phospho-c-Jun, phospho-IκBα, total NF-κB, total c-Jun, and β-actin in cell lysates were examined by Western blot analysis (c) and quantified (d–f) (n = 3). Data represent mean ± SEM. ***p < 0.001; N.S. not significant (one-way ANOVA with post hoc Tukey’s t test)