Botulinum toxin type A inhibits microglia pyroptosis by suppressing Cblb-mediated degradation of Pdlim1 to attenuate neuropathic pain

Abstract

Background: Microglia pyroptosis, a newly identified form of inflammatory cell death, is involved in the development of neuropathic pain (NP). Botulinum toxin type A (BTX-A) has been shown to be effective in relieving NP, but the mechanisms involved have not been clarified.

Methods: A mice model of NP was established with chronic constriction injury (CCI) method. The expression levels of key molecules and the extent of microglia pyroptosis were assessed using RT-qPCR, western blot, ELISA and immunofluorescence. Moreover, lipopolysaccharide (LPS) was used in vitro to induce pyroptosis of microglia to explore the potential molecular mechanisms of BTX-A.

Result: In a mice model of NP, BTX-A administration increased the pain threshold and decreased the Cblb protein expression level, consistent with the results of in vitro experiments. Functional experiments and mouse models were respectively used to evaluate the severity of microglia pyroptosis. The results showed that BTX-A inhibited microglia pyroptosis through Cblb protein. Subsequently, mass spectrometry (MS) analysis and immunoprecipitation were conducted to identify proteins interacting with Cblb. The results identified Pdlim1 was a potential interacting partner of Cblb, which regulats the ubiquitination of Pdlim1. Mechanically, Cblb binds to the PDZ and LIM domains of Pdlim1 and then targets Pdlim1 at K244 for ubiquitination and proteasome-mediated degradation. Pdlim1 knockdown lentiviral plasmid was constructed and stable Pdlim1 knockdown microglial cell lines were established for rescue experiments. The results demonstrated that BTX-A suppresses microglia pyroptosis via Pdlim1/NF-κB signaling axis. Finally, intrathecal injection of adeno-associated virus overexpressing Cblb was used in rescue experiments. The results confirmed that BTX-A attenuates neuropathic pain via the Cblb/Pdlim1/NF-kB signaling axis.

Conclusions: This study demonstrates that BTX-A suppresses the activity of Cblb, thereby reducing Pdlim1 protein degradation, inhibiting the NF-kB pathway, and ultimately mitigating microglia pyroptosis. Our findings suggest that Cblb could serve as a novel therapeutic target for BTX-A in the treatment of NP.

Keywords: BTX-A; Cblb; Microglia pyroptosis; Neuropathic pain.

Fig. 1

Screening and identification of the regulatory effect of BTX-A on Cblb protein. A The MWT was measured at 0, 3, 6, 9, 12, 14 days after induction of CCI. B The TWL was measured at 0, 3, 6, 9, 12, 14 days after induction of CCI. C-D Heatmap of different protein at the proteomics from spinal cord tissue in different groups. E qRT-PCR analysis was used to determine the expression levels of differential proteins in the spinal cord tissue. F qRT-PCR analysis was used to determine the expression levels of differential proteins in the microglia. G-H Western blotting was used to determine the expression levels of Cblb in the spinal cord tissue. I-J Western blotting was used to determine the expression levels of Cblb in the microglia. K-L Immunofluorescence was performed to clarify the co-location of Cblb and microglia. All data in the figure are shown as mean ± SEM. *** P < 0.001, ** P < 0.01, *P < 0.05; ### P < 0.001, ## P < 0.01, # P < 0.05

Fig. 2

BTX-A inhibits microglia pyroptosis through downgrading the expression of Cblb. A-C The protein expression of NLRP3, ASC, Caspase-1, GSDMD-N in spinal cord tissue under different treatments was evaluated by western blotting. D-E Serum concentrations of IL-1β and IL-18 were measured by ELISA. F-G The protein expression of NLRP3, ASC, Caspase-1, GSDMD-N in microglia under different treatments was evaluated by western blotting. H-I The protein expression of NLRP3, ASC, Caspase-1, GSDMD-N in rescue experiments was evaluated by western blotting. J-K Concentrations of IL-1β and IL-18 in the supernatant were measured by ELISA. L-M Microglia activity was measured using flow cytometry. All data in the figure are shown as mean ± SEM.*** P < 0.001, ** P < 0.01, *P < 0.05; ### P < 0.001, ## P < 0.01, # P < 0.05

Fig. 3

Cblb downregulates the protein expression of Pdlim1 by accelerating its ubiquitination. A Identification of proteins interacting with Cblb using IP-MS technology. B Immunoblotting for specific correlation of Cblb with Pdlim1 from Co-IP assays. C Immunofluorescence was performed to clarify the co-location of Cblb and Pdlim1. D The ubiquitination modification status of Pdlim1 was detected by Co-IP and western blotting. E The transfection efficiency of Cblb knockdown was validated by qRT-PCR analysis. F-G Protein stability assay by using cycloheximide to treat cells at the different time (h) was performed to evaluate the effect of Cblb knockdown. H-I The ubiquitination modification assay was performed to evaluate the effect of Cblb knockdown or overexpression. J-K Western blotting analysis of microglia stably transfected with Cblb knockdown and treated with the proteasome inhibitor MG132. All data in the figure are shown as mean ± SEM. *** P < 0.001, ** P < 0.01, *P < 0.05

Fig. 4

BTX-A inhibits microglia pyroptosis through upregulating Pdlim1 and suppressing NF-κB signaling. A-B The transfection efficiency of Pdlim1 knockdown was validated by western blotting. C-D The protein expression of NLRP3, ASC, Caspase-1, GSDMD-N in rescue experiments was evaluated by western blotting. E-F Concentrations of IL-1β and IL-18 in the supernatant were measured by ELISA. G GSEA analysis based on the GEO database was performed to predict that differentially expressed genes was associated with NF-κB pathway and pyroptosis. H-I The protein expression of P-P65, P65 in rescue experiments was evaluated by western blotting. J-L The protein expression of Pdlim1, P-P65, P65 in rescue experiments was evaluated by western blotting. All data in the figure are shown as mean ± SEM. *** P < 0.001, ** P < 0.01, *P < 0.05; ### P < 0.001, ## P < 0.01, # P < 0.05.$$$ P < 0.001, $$ P < 0.01, $ P < 0.05

Fig. 5

Cblb mediates Pdlim1 ubiquitylation at lysine 244. A The diagrams show wild-type Pdlim1 and its truncated variants. B Microglia were transfected with the indicated plasmids, followed by co-IP assays and western blotting to examine the interaction of Cblb with wild-type Pdlim1 or Pdlim1 truncated variants. C Immunofluorescence was performed to clarify the co-location of Cblb and Pdlim1 truncated variants. D-G The protein expression of NLRP3, ASC, Caspase-1, GSDMD-N was measured in wild-type Pdlim1 group and Pdlim1 truncated variants group by western blotting. H Schematic representation of the Pdlim1 protein indicating the lysine residues. I Ubiquitylation assays were performed and the ubiquitylated form of Pdlim1 was detected by immunoblotting. J-K The protein expression of NLRP3, ASC, Caspase-1, GSDMD-N was measured in wild-type Pdlim1 group and individual K-to-R mutant group by western blotting. All data in the figure are shown as mean ± SEM. *** P < 0.001, ** P < 0.01, *P < 0.05

Fig. 6

BTX-A inhibits microglia pyroptosis through Cblb/Pdlim1/NF-κ B signaling axis in vivo. A The infection efficiency of Cblb overexpression was validated by qRT-PCR. B The MWT was measured at 0, 3, 6, 9, 12, 14 days in different groups. C The TWL was measured at 0, 3, 6, 9, 12, 14 days in different groups. D-G The protein expression of NLRP3, ASC, Caspase-1, GSDMD-N and Pdlim1 in rescue experiments was evaluated by western blotting. H-I Serum concentrations of IL-1β and IL-18 were measured by ELISA. J-K The protein expression of P-P65, P65 in rescue experiments was evaluated by western blotting. All data in the figure are shown as mean ± SEM. *** P < 0.001, ** P < 0.01, *P < 0.05; ### P < 0.001, ## P < 0.01, # P < 0.05. $$$ P < 0.001, $$ P < 0.01, $ P < 0.05

Fig. 7

A schematic illustration showing the protective effects of BTX-A on CCI-induced microglia pyroptosis and its underlying mechanisms. Chronic constriction injury of sciatic nerve leads to apparent pain sensitization and activated microglial pyroptosis in the spinal cord of mice. Mechanistically, BTX-A administration inhibits the expression of Cblb, thereby reducing Pdlim1 protein degradation. The upregulation of Pdlim1 suppresses the process of pyroptosis in microglia by reducing the activated inflammasomes, cleaved-GSDMD, IL-18 and IL-1β through NF-κB pathways. In brief, BTX-A could mitigate the neuroinflammation by reducing pro-inflammatory pyropsis of microglia and ultimately protect against pain in CCI-induced NP model