Inflammasome in ALS Skeletal Muscle: NLRP3 as a Potential Biomarker

Abstract

Since NLRP3 inflammasome plays a pivotal role in several neurodegenerative disorders, we hypothesized that levels of inflammasome components could help in diagnosis or prognosis of amyotrophic lateral sclerosis (ALS). Gene and protein expression was assayed by RT-PCR and Western blot. Spearman's correlation coefficient was used to determine the linear correlation of transcriptional expression levels with longevity throughout disease progression in mice models. Kaplan-Meier analysis was performed to evaluate MCC950 effects (NLRP3 inhibitor) on lifespan of SOD1G93A mice. The results showed significant alterations in NLRP3 inflammasome gene and protein levels in the skeletal muscle of SOD1G93A mice. Spearman's correlation coefficient revealed a positive association between Nlrp3 transcriptional levels in skeletal muscle and longevity of SOD1G93A mice (r = 0.506; p = 0.027). Accordingly, NLRP3 inactivation with MCC950 decreased the lifespan of mice. Furthermore, NLRP3 mRNA levels were significantly elevated in the blood of ALS patients compared to healthy controls (p = 0.03). In conclusion, NLRP3 could be involved in skeletal muscle pathogenesis of ALS, either through inflammasome or independently, and may play a dual role during disease progression. NLRP3 gene expression levels could be used as a biomarker to improve diagnosis and prognosis in skeletal muscle from animal models and also to support diagnosis in clinical practice with the blood of ALS patients.

Keywords: ALS patients; NLRP3; SOD1G93A; amyotrophic lateral sclerosis (ALS); biomarker; blood; inflammasome; neuroinflammation; skeletal muscle.

Figure 1

Expression levels of NLRP3 inflammasome in skeletal muscle of SOD1G93A mice during disease progression (P50: asymptomatic stage; P75: early symptomatic stage; P120: terminal stage). (A) Transcriptional levels of NLRP3 inflammasome components determined by real-time PCR in the quadriceps tissue. Dunnet’s post hoc test correction between groups also yielded statistically significant differences among groups at the asymptomatic stage (Asc, p = 0.037; caspase 1, p = 0.046; Il1β, p = 0.041). (B) Protein expression levels of NLRP3 components in quadriceps muscle determined by Western blotting. Dunnet’s post hoc test correction between groups also yielded statistically significant differences among groups at the terminal stage (NLRP3, p = 0.025; ASC, p = 0.007; pro-caspase 1, p = 0.003; pro-IL1β, p < 0.000). (C) Protein bands of each NLRP3 inflammasome component. Active caspase 1 and mature IL1β were only detected in SOD1G93A mice. The total number of animals used was 24 (8 mice for each stage: WT n = 4 and SOD1G93A n = 4). * p < 0.05, ** p < 0.01; significant differences in comparison to WT mice.

Figure 2

Linear correlation graphs between longevity and transcriptional expression levels of NLRP3 inflammasome components throughout disease progression: (A) Nlrp3, (B) Asc, (C) caspase 1, (D) Il1β. Only Nlrp3 levels showed a significant and positive correlation with longevity. The total number of transgenic SOD1G93A mice used was 20. Three skeletal muscle biopsies were obtained per mouse, at different stages: early symptomatic stage (75 days), symptomatic stage (105 days) and terminal stage (endpoint age).

Figure 3

Kaplan–Meier analysis of the effect of MCC950 drug on survival rate. Non-treated transgenic SOD1G93A mice showed a longer lifespan (128.692 days ± 7.54) than MCC950-treated mice (113.571 days ± 18.48); log-rank test p = 0.025. The total number of animals used was 27 (control n = 13 and MCC950 treatment n = 14).

Figure 4

Transcriptional expression levels of NLRP3 in blood samples from healthy subjects (control), amyotrophic lateral sclerosis (ALS) patients and patients with other myopathies. Kruskal-Wallis tests showed significant differences among ALS patients and the healthy group (p = 0.03). Dunnet’s post hoc test correction between groups also yielded statistically significant differences between these groups (p = 0.04). A total of 42 participants were included in this study: 14 control individuals, 14 patients with other myopathies and 14 ALS patients * p < 0.05.