Therapeutic blockade of HMGB1 reduces early motor deficits, but not survival in the SOD1G93A mouse model of amyotrophic lateral sclerosis

Abstract

Background: Amyotrophic lateral sclerosis (ALS) is a fatal and rapidly progressing neurodegenerative disease without effective treatment. The receptor for advanced glycation end products (RAGE) and the toll-like receptor (TLR) system are major components of the innate immune system, which have been implicated in ALS pathology. Extracellularly released high-mobility group box 1 (HMGB1) is a pleiotropic danger-associated molecular pattern (DAMP), and is an endogenous ligand for both RAGE and TLR4.

Methods: The present study examined the effect of HMGB1 inhibition on disease progression in the preclinical SOD1^G93A transgenic mouse model of ALS using a potent anti-HMGB1 antibody (2G7), which targets the extracellular DAMP form of HMGB1.

Results: We found that chronic intraperitoneal dosing of the anti-HMGB1 antibody to SOD1^G93A mice transiently improved hind-limb grip strength early in the disease, but did not extend survival. Anti-HMGB1 treatment also reduced tumour necrosis factor α and complement C5a receptor 1 gene expression in the spinal cord, but did not affect overall glial activation.

Conclusions: In summary, our results indicate that therapeutic targeting of an extracellular DAMP, HMGB1, improves early motor dysfunction, but overall has limited efficacy in the SOD1^G93A mouse model of ALS.

Keywords: Innate immune system; Neuroinflammation; RAGE; TLR4.

Fig. 1

Pre-onset anti-HMGB1 2G7 treatment improves early hind-limb grip strength deficit, but does not extend survival in SOD1^G93A transgenic mice. SOD1^G93A mice were intraperitoneally injected weekly with the anti-HMGB1 antibody at 35 days of age (red line). a Left panel shows a Kaplan-Meier plot of ages (in days) in which SOD1^G93A mice treated with isotype control (vehicle, 100 μg; orange line) or anti-HMGB1 antibody (100 μg; blue line) reached end-stage of disease (complete hind-limb paralysis and an inability to right itself once placed on its back; n = 13, P = 0.362, log-rank test). Anti-HMGB1 treatment at 35 days of age (100 μg) resulted in no extension in survival time compared with vehicle treatment. a Right panel shows the end-stage survival age for each litter-matched pair of vehicle- and anti-HMGB1-treated SOD1^G93A mice. b, c Shows no differences in body weight and motor score between vehicle (orange line) and anti-HMGB1 (blue line) treated SOD1^G93A mice (n = 13, P > 0.05, two-way ANOVA). d Shows an early transient improvement in hind-limb grip strength for anti-HMGB1-treated versus vehicle-treated SOD1^G93A mice at 56 and 63 days of age (n = 13, *p < 0.05, +p < 0.0001, two-way ANOVA with post-hoc Bonferroni’s multiple comparisons test). Data are expressed as mean ± SEM

Fig. 2

Post-onset anti-HMGB1 2G7 treatment has no effect on disease in SOD1^G93A transgenic mice. SOD1^G93A mice were intraperitoneally injected weekly with the anti-HMGB1 antibody at 70 days of age (red line). a Left panel shows a Kaplan-Meier plot of ages (in days) in which SOD1^G93A mice treated with isotype control (vehicle, 100 μg; orange line) or anti-HMGB1 antibody (100 μg; blue line) reached end-stage of disease (complete hind-limb paralysis and an inability to right itself once placed on its back; n = 12, p = 0.6384, log-rank test). a Right panel shows the end-stage survival age for each litter-matched pair of vehicle- and anti-HMGB1-treated SOD1^G93A mice, demonstrating no differences in survival time between the groups. b–d Panels show no difference in body weight (b), motor score (c) and hind-limb grip strength (d) between vehicle (orange line) and anti-HMGB1 (blue line) treated SOD1^G93A mice (n = 12, p > 0.05, two-way ANOVA). Data are expressed as mean ± SEM

Fig. 3

No change in microglia and astrocyte markers in lumbar spinal cord between isotype control and anti-HMGB1 antibody treated SOD1^G93A mice. SOD1^G93A mice were intraperitoneally injected weekly with the anti-HMGB1 antibody at 35 days of age (100 μg). Major non-neuronal cell populations (microglia/monocytes and astrocytes) in vehicle and anti-HMGB1-treated SOD1^G93A mice were investigated at mid-symptomatic stage of disease (133 days) using quantitative PCR and immunohistochemistry. a–c Shows anti-HMGB1 treatment had no effect on microglia (Itgam, Cd68 and Aif1) mRNA transcript levels (n = 6, p > 0.05, Student’s t test). d Shows a reduction in monocyte (Ly6c) mRNA transcript levels in anti-HMGB1-treated SOD1^G93A mice when compared to isotype control-treated SOD1^G93A mice (n = 6, * P < 0.05, Student’s t test). e Shows representative images of CD11b-positive microglia in the lumbar spinal cord of isotype control and anti-HMGB1-treated SOD1^G93A mice at 133 days of age. Dashed line shows the outline of the ventral horn with higher magnification of the white square. Scale bar = 100 μm. f, g Shows no change in microglia expression and activated microglia (amoeboid) in anti-HMGB1-treated SOD1^G93A mice compared with isotype control-treated SOD1^G93A mice (n = 4, p > 0.05, Student’s t test). h Shows no change in astrocyte (Gfap) mRNA transcript levels between isotype control and anti-HMGB1-treated SOD1^G93A mice (n = 6, p > 0.05, Student’s t test). i Show representative images of GFAP-positive astrocytes in the lumbar spinal cord of isotype control and anti-HMGB1-treated SOD1^G93A mice at 133 days of age. Dashed line shows the outline of the ventral horn with higher magnification of the white squares. Scale bars = 100 μm. j Shows no change in astrocyte expression in anti-HMGB1 treated-SOD1^G93A mice compared with isotype control-treated SOD1^G93A mice (n = 4, p > 0.05, Student’s t test). Data are presented as mean ± SEM

Fig. 4

Tnf and C5ar1 transcripts are reduced in lumbar spinal cord of anti-HMGB1 antibody-treated SOD1^G93A mice. SOD1^G93A mice were intraperitoneally injected weekly with the anti-HMGB1 antibody at 35 days of age (100 μg). Pro-inflammatory cytokines (TNFα and IL-1β) and major innate immune receptors (RAGE, C5aR1 and TLR4) in vehicle and anti-HMGB1-treated SOD1^G93A mice was investigated at mid-symptomatic stage of disease (133 days) using quantitative PCR. a Show anti-HMGB1 treatment reduces pro-inflammatory cytokine Tnf in the spinal cord of SOD1^G93A mice (n = 6, **p < 0.01, Student’s t test), while no change in Il1β was evident between isotype control and anti-HMGB1-treated SOD1^G93A mice (b; n = 6, p > 0.05, Student’s t test). Anti-HMGB1 treatment showed slight reduction in C5ar1 mRNA transcript levels while no change was observed for Ager and Tlr4 (c–e; n = 6, *p < 0.05, Student’s t test). Data are presented as mean ± SEM

Fig. 5

Monocyte and macrophage markers in tibialis anterior and gastrocnemius muscle are not altered between isotype control and anti-HMGB1 antibody-treated SOD1^G93A mice. SOD1^G93A mice were intraperitoneally injected weekly with the anti-HMGB1 antibody at 35 days of age (100 μg). Monocytes and macrophage markers in tibialis anterior (TA) and gastrocnemius (GN) muscle of vehicle and anti-HMGB1-treated SOD1^G93A mice was investigated at mid-symptomatic stage (133 days) using quantitative PCR. a–c Shows anti-HMGB1 treatment had no effect on macrophage (Itgam, Cd68 and Aif1) mRNA transcript levels in both TA and GN muscles (n = 6, p > 0.05, Student’s t test). d Shows a reduction in monocyte (Ly6c) mRNA transcript levels in TA muscle of anti-HMGB1-treated SOD1^G93A mice, while no change was evident in GN muscle when compared to isotype control-treated SOD1^G93A mice (n = 6, *p < 0.05, Student’s t test). Data are presented as mean ± SEM

Fig. 6

Immune and inflammatory markers are not altered in tibialis anterior and gastrocnemius muscle of anti-HMGB1 antibody-treated SOD1^G93A mice. SOD1^G93A mice were intraperitoneally injected weekly with the anti-HMGB1 antibody at 35 days of age (100 μg). Pro-inflammatory cytokines (TNFα and IL-1β) and major innate immune receptors (RAGE, C5aR1 and TLR4) in tibialis anterior (TA) and gastrocnemius (GN) muscle of vehicle and anti-HMGB1-treated SOD1^G93A mice was investigated at mid-symptomatic stage of disease (133 days) using quantitative PCR. a, b Shows no change in pro-inflammatory cytokines Tnf and Il1β in both TA and GN muscle of anti-HMGB1-treated SOD1^G93A mice when compared to isotype control-treated SOD1^G93A mice (n = 6, p > 0.05, Student’s t test). Ager and C5ar1 mRNA transcript levels were not different in isotype control and anti-HMGB1-treated SOD1^G93A mice in both TA and GN muscle (c, d; n = 6, p > 0.05, Student’s t test). While anti-HMGB1 treatment showed a slight reduction in Tlr4 transcript levels in TA muscle, no change was observed in GN muscle of SOD1^G93A mice (e; n = 6 *p < 0.05, Student’s t test). Data are presented as mean ± SEM