PSMC5 regulates microglial polarization and activation in LPS-induced cognitive deficits and motor impairments by interacting with TLR4

Abstract

Luteolin is a flavonoid found in high concentrations in celery and green pepper, and acts as a neuroprotectant. PSMC5 (proteasome 26S subunit, ATPase 5) protein levels were reduced after luteolin stimulation in activated microglia. We aimed to determine whether regulating PSMC5 expression could inhibit neuroinflammation, and investigate the underlying mechanisms.BV2 microglia were transfected with siRNA PSMC5 before the addition of LPS (lipopolysaccharide, 1.0 µg/ml) for 24 h in serum free DMEM. A mouse model of LPS-induced cognitive and motor impairment was established to evaluate the neuroprotective effects of shRNA PSMC5. Intracerebroventricular administration of shRNA PSMC5 was commenced 7 days prior to i.p. injection of LPS (750 μg/kg). Treatments and behavioral experiments were performed once daily for 7 consecutive days. Behavioral tests and pathological/biochemical assays were performed to evaluate LPS-induced hippocampal damage. Molecular dynamics simulation was used to confirm the interaction between PSMC5 and TLR4 (Toll-like receptor 4) in LPS-stimulated BV2 microglia. SiRNA PSMC5 inhibited BV2 microglial activation, and suppressed the release of inflammatory factors (IL-1β, COX-2, PGE₂, TNF-α, and iNOS) upon after LPS stimulation in BV2 microglia. LPS increased IκB-α and p65 phosphorylation, which was attenuated by siRNA PSMC5. Behavioral tests and pathological/biochemical assays showed that shRNA PSMC5 attenuated LPS-induced cognitive and motor impairments, and restored synaptic ultrastructure and protein levels in mice. ShRNA PSMC5 reduced pro-inflammatory cytokine (TNF-α, IL-1β, PGE₂, and NO) levels in the serum and brain, and relevant protein factors (iNOS and COX-2) in the brain. Furthermore, shRNA PSMC5 upregulated the anti-inflammatory mediators interleukin IL-4 and IL-10 in the serum and brain, and promoted a pro-inflammation-to-anti-inflammation phenotype shift in microglial polarization. Mechanistically, shRNA PSMC5 significantly alleviated LPS-induced TLR4 expression. The polarization of LPS-induced microglial pro-inflammation phenotype was abolished by TLR4 inhibitor and in the TLR-4^-/- mouse, as in shRNA PSMC5 treatment. PSMC5 interacted with TLR4 via the amino sites Glu284, Met139, Leu127, and Phe283. PSMC5 site mutations attenuated neuroinflammation and reduced pro-inflammatory factors by reducing TLR4-related effects, thereby reducing TLR4-mediated MyD88 (myeloid differentiation factor 88)-dependent activation of NF-κB. PSMC5 could be an important therapeutic target for treatment of neurodegenerative diseases involving neuroinflammation-associated cognitive deficits and motor impairments induced by microglial activation.

Keywords: Luteolin; Microglia; Neuroinflammation; Proteasome 26S subunit (PSMC5), ATPase 5; Synapse; TLR4.

Fig. 1

Schematic representation of experimental procedure. I.c.v. shRNA PSMC5 injection was administrated 7 days before LPS injection. Behavioral training tests were delivered to mice at days -7, -5, and -3 respectively. At day 0, behavioral tests were performed

Fig. 2

PSMC5 expression in microglia upon after luteolin treatment. A, B Protein spots identified using 2-DE gels between LPS and luteolin (Lut) treatment groups in BV2 cells, and differences in spot intensity shown by MALDI–TOF-MS to identify PSMC5. C Lut treatment downregulated PSMC5 protein levels in BV2 cells. *p < 0.05 and **p < 0.01 versus the LPS group. D PSMC5 protein levels in mouse brain at different time points in mice treated with LPS. *p < 0.05 and **p < 0.01 versus the control group. E PSMC5 protein levels in the mouse brain after i.c.v. shRNA PSMC5 injection at different times and volumes. F PSMC5 protein levels in the mouse brain after i.c.v. shRNA PSMC5 injection. n = 3–4, data are expressed as means ± SEM, *p < 0.05 and **p < 0.01 versus the control, saline, shRNA PSMC5, and NC groups; ^#P < 0.05, ^##P < 0.01 versus the shRNA PSMC5 + LPS group

Fig. 3

ShRNA PSMC5 suppressed neuroinflammation in BV2 cells. A The effect of shRNA PSMC5 on the pro-inflammatory cytokines IL-1α, COX-2, PGE₂, and TNF-α in LPS-stimulated BV2 microglia. Q-PCR analysis of IL-1β, COX-2, and TNF-α mRNA expression. ELISA measuring the production of IL-1β, PGE₂, and TNF-α. B Effect of shRNA PSMC5 on iNOS and COX-2 levels in BV2 microglia after LPS-induced neuroinflammation. C Signaling components of MyD88-dependent signaling pathway analyzed in BV2 using western blot with the indicated antibodies. D Cellular distribution of NF-κB p65 subunit (Green). Hoechst 33,258 (blue) was used for visualizing nuclei. Scale bar = 100 μm

Fig. 4

ShRNA PSMC5 alleviated cognitive impairment and motor disjunction, and improved synaptic structure in LPS-induced mice. A Results of the MWM test for shRNA PSMC5-treated mice, n = 15. B Results of the PAT test for shRNA PSMC5-treated mice, n = 15. C, D Motor coordination scores for shRNA PSMC5-treated mice. C Pole test score, n = 15. D Traction test score, n = 15. Data are presented as mean ± SEM. *P < 0.05, **P < 0.01 compared to the control, saline, shRNA PSMC5, and VIPER groups; ^#P < 0.05, ^##P < 0.01 compared to the shRNA PSMC5 + LPS, VIPER + LPS, and shRNA PSMC5 + VIPER + LPS groups, analyzed by one-way ANOVA. Error bars indicate SEM. E Hematoxylin and eosin staining shows effect of shRNA PSMC5 on hippocampus neurons in LPS-induced mice. Representative photomicrographs of the hippocampus CA3 and CA4 area showing the histological changes of each group. n = 5 mice/group, n = 30 felds/group. F Transmission electron microscopy staining shows effects of shRNA PSMC5 treatment on synapses in the hippocampus. The green arrow shows the synaptic structure. n = 5 mice/group, n = 30 felds/group. G Effect of shRNA PSMC5 treatment on SYP and PSD95 levels in the mice brain. n = 3–4 mice/group

Fig. 5

ShRNA PSMC5 treatment shifted microglial polarization from pro-inflammatory phenotypes toward to anti-inflammatory phenotypes after LPS-induced neuroinflammation. A ShRNA PSMC5 decreased the number of TNF-α-positive microglia (M1) and increased the number of YM-1-positive microglia (M2) in the hippocampus. Representative images of triple-staining immunofluorescence for TNF-α (red), YM-1 (green), and IBA-1 (purple) with DAPI nuclear counterstain in the hippocampus. B ShRNA PSMC5 treatment decreased LPS-induced pro-inflammatory cytokines in the serum and brain. Pro-inflammatory cytokines were detected by ELISA and Griess assay. C ShRNA PSMC5 treatment increased LPS-induced anti-inflammatory cytokine production in the serum and brain. Anti-inflammatory cytokines were detected by ELISA. D Effect of shRNA PSMC5 treatment on iNOS and COX-2 levels in the brain after LPS-induced neuroinflammation. ShRNA PSMC5 treatment alleviated the expression of iNOS and COX-2 protein. n = 3–4 mice/group.Data are presented as mean ± SEM. *P < 0.05, **P < 0.01 compared to the control, saline, shRNA PSMC5, and VIPER groups; ^#P < 0.05, ^##P < 0.01 compared to the shRNA PSMC5 + LPS, VIPER + LPS, and shRNA PSMC5 + VIPER + LPS groups, analyzed by one-way ANOVA. Error bars indicate SEM

Fig. 6

ShRNA PSMC5 inhibited MyD88-dependent signaling pathway activation and suppressed transition of microglial polarization from M2 to M1 phenotype under neuroinflammatory conditions. Protein expression of signaling components of the MyD88-dependent pathway were analyzed using the indicated antibodies by western blot. n = 3–4 mice/group.Data are presented as mean ± SEM. *P < 0.05, **P < 0.01 compared to the control, saline, shRNA PSMC5, and VIPER groups; ^#P < 0.05, ^##P < 0.01 compared to the shRNA PSMC5 + LPS, VIPER + LPS, and shRNA PSMC5 + VIPER + LPS groups, analyzed by one-way ANOVA. Error bars indicate SEM

Fig. 7

TLR4 knockout in mice protected against LPS-induced neuroinflammation and cognitive and motor impairments. A–D Morris water maze (MWM), passive avoidance test (PAT), pole test, and traction test were performed to test the memory ability and motor coordination in mice that received the indicated treatments. n = 15. E The expression of the pro-inflammatory cytokines TNF-α, IL-1β, and PGE₂ in mouse serum and brain were determined using ELISA kits. F Expression of the anti-inflammatory cytokines IL-4 and IL-10 in mouse serum and brain were studied using ELISA kits. G ShRNA PSMC5 targeted TLR4 to ameliorate LPS-induced neuroinflammation. The expression levels of the signaling components of the TLR4-pathway in mouse hippocampus were determined using western blot with the indicated antibodies. E–G n = 3–4 mice/group,data are presented as mean ± SEM. *P < 0.05, **P < 0.01 compared to control group; ^#P < 0.05, ^##P < 0.01 compared to LPS (TLR4^−/−) group, analyzed by one-way ANOVA. Error bars indicate SEM

Fig. 8

PSMC5 interacts with TLR4. A Colocalization of PSMC5 (red) and TLR4 (green), with DAPI nuclear counterstain in the BV2 microglia. Scale bar = 10 µm. B In silico modeling of PSMC5 interactions with TLR4. C Coimmunoprecipitation of endogenous PSMC5 and TLR4 in BV2 cells; precipitation was carried out with TLR4 antibody or IgG followed by WB analysis with antibodies against PSMC5 and TLR4. D GST-pull down to verify the computer molecular docking results. E PSMC5 expression after PSMC5 knockdown in the PSMC5 wild-type and PSMC5 mutants. F Western blot analysis of protein expression of the inflammatory proteins iNOS and COX-2, and MyD88-dependent signaling pathway components in the PSMC5 wild-type and mutants. G Results of the Griess assay showing NO content in the PSMC5 mutants. H ELISA analysis showing the PGE₂ content in the PSMC5 mutants