The CCL2-CCR2 axis drives neuromuscular denervation in amyotrophic lateral sclerosis

Abstract

Systemic immune changes have been implicated in amyotrophic lateral sclerosis (ALS), but precise mechanisms and cellular targets remain unknown. Neuromuscular junction (NMJ) denervation is another major pathophysiological event in ALS, but it remains unclear whether immune system dysregulation contributes to this process. Here, we report leukocyte and macrophage infiltration in ALS patient-derived skeletal muscle biopsies. Immune cell infiltration was replicated across the hTDP-43, TDP-43^A315T (male only) and TDP-43^M337V mouse models, occurring from pre-symptomatic stages and targeted to NMJ-enriched muscle regions. Proteomic analysis implicated the CCL2-CCR2 axis as a driving factor. CCL2⁺ cells were enriched around NMJs in hTDP-43 mice, and in ALS patient skeletal muscle. Local treatment with CCL2-neutralising antibodies or normal IgG antibodies in hTDP-43 mice reduced leukocyte infiltration and ameliorated NMJ denervation. These results demonstrate that the CCL2-CCR2 axis drives immune cell infiltration targeting NMJs in ALS, identifying a potential avenue for therapeutic intervention to prevent NMJ denervation.

Fig. 1. Immune cell infiltration in skeletal muscle of ALS patients.

A CD45⁺ leukocytes in skeletal muscle (cross section) of control individuals (“control”) and ALS patients (“ALS”). Dystrophin co-staining also revealed notable changes in skeletal muscle architecture in ALS samples, confirming the presence of neuromuscular pathology. Scale bar = 40 µm. B Quantification of CD45⁺ cells in control (n = 4) and ALS (n = 4) patient samples. Each data point represents an average of 10 fields of view per patient. C CD68⁺ macrophage cell count in skeletal muscle (cross section) of control individuals and ALS patients. Laminin co-staining also revealed notable changes in skeletal muscle architecture in ALS samples, confirming the presence of neuromuscular pathology. Scale bar = 40 µm. D Quantification of CD68⁺ cells in control (n = 4) and ALS (n = 4) patient samples. Each data point represents an average of 10 fields of view per patient. Data is presented as mean ± sem. Both datasets were analysed using unpaired, two-tailed t-tests, (B) p = 0.0009, (D) p = 0.0388. * = p < 0.05; *** = p < 0.001.

Fig. 2. Immune cell infiltration is a common feature across animal models of ALS.

A Immunohistochemical analysis of CD45⁺ cells infiltrating into the innervation zone (NMJs visualised with α-bungarotoxin, BTX) of gastrocnemius (GC) muscle from late-symptomatic (postnatal day 19) hTDP43^Tg/Tg mice (Tg/Tg; n = 4) versus wild-type littermate controls (WT; n = 4). B Immunohistochemical analysis of CD45⁺ cells infiltrating into the innervation zone (NMJs visualised with α-bungarotoxin, BTX) of GC muscle from late-symptomatic TDP-43^A315T mice (postnatal day 90; Tg/0; n = 5) versus WT littermate controls (n = 5). C Immunohistochemical analysis of CD45⁺ cells infiltrating the innervation zone (NMJs visualised with α-bungarotoxin, BTX) of GC muscle from early-symptomatic TDP-43^M337V mice (12 months old; Tg/Tg; n = 3) versus WT littermate controls (n = 3). Data points represent an average from 10 fields of view from 1 mouse. Data is presented as mean ± sem. Scale bars = 40 µm. Data analysis: (A) two-tailed Mann-Whitney test [Shapiro-Wilk normality test, WT group: p = 0.0328, Tg/Tg group: p = 0.6879], p = 0.0286, (B) unpaired, two-tailed t-test, p = 0.0004, (C) unpaired, two-tailed t-test, p = 0.0427. * = p < 0.05; *** = p < 0.001.

Fig. 3. Degree of macrophage infiltration correlates with vulnerability to NMJ denervation.

A–C Immunohistochemical analysis of CD68⁺ macrophages in the innervation zone of the gastrocnemius (GC; A), tibialis anterior (TA; B) and extensor digitorum longus (EDL; C) muscle (longitudinal section) of control wild-type (WT) and hTDP-43^Tg/Tg mice (postnatal day 19; Tg/Tg). NMJs were visualised with α-bungarotoxin (BTX). Scale bar = 40 µm. D–F Quantitative analysis of CD68⁺ cells in the innervation zone of GC, TA and EDL muscles. Data is presented as mean ± sem. Data analysis: (D) gastrocnemius: n = 4 mice (WT, Tg/Tg), unpaired, two-tailed t-test, p = 0.0034; (E) tibialis anterior: n = 3 (WT), n = 4 (Tg/Tg), unpaired, two-tailed Mann-Whitney test [Shapiro-Wilk normality test, WT group: p = 0.1555, Tg/Tg group: p = 0.0459]; (F) extensor digitorum longus: n = 3 (WT, Tg/Tg), unpaired, two-tailed t-test. n.s. = non-significant; ** = p < 0.01.

Fig. 4. Leukocyte infiltration at presymptomatic and early symptomatic stages in the innervation zone of the severely affected gastrocnemius (GC) muscle.

A, B Immunohistochemical analysis of CD45⁺ leukocytes in the innervation zone of the GC muscle in presymptomatic (A) and early symptomatic (B) hTDP-43^Tg/Tg mice (Tg/Tg) and age-matched wild-type (WT) littermates. NMJs were visualised with α-bungarotoxin (BTX), nuclei were labeled with DAPI (blue). Scale bar = 40 µm. C Violin plots showing quantitative analysis of CD45⁺ in the innervation zone of the GC muscle of presymptomatic (postnatal day 7; WT n = 4; Tg/Tg n = 3) and early symptomatic mice (postnatal day 15; WT n = 3; Tg/Tg n = 3), compared to late symptomatic mice (postnatal day 19 – data from Fig. 2). Each time point represents a separate cohort of mice. Bold dashed line represents median, thin dashed lines represent quartiles. D Violin plots showing quantitative analysis of CD68⁺ in the innervation zone of the GC muscle of presymptomatic (postnatal day 7; WT n = 4; Tg/Tg n = 3) and early symptomatic mice (postnatal day 15; WT n = 3; Tg/Tg n = 3), compared to late symptomatic mice (postnatal day 19 – data from Fig. 3). Each time point represents a separate cohort of mice. Bold dashed line represents median, thin dashed lines represent quartiles. Data analysis: (C) P7: unpaired, two-tailed t-test, p = 0.0003; (C) P15: unpaired, two-tailed t-test, p < 0.0001; (C) P19: two-tailed Mann-Whitney test [Shapiro-Wilk normality test, WT group: p = 0.0328, Tg/Tg group: p = 0.6879], p = 0.0286; (D) P7: unpaired, two-tailed t-test, (D) P15: unpaired, two-tailed t-test, (D) P19: unpaired, two-tailed t-test, p = 0.0034. n.s. = non-significant; * = p < 0.05; ** = p < 0.01; *** = p < 0.001.

Fig. 5. Proteomic characterisation of inflammatory responses in hTDP-43^Tg/Tg mice.

A Proteome profiler analysis of 111 inflammatory proteins in gastrocnemius (GC), tibialis anterior (TA) and extensor digitorum longus (EDL) muscles and spinal cord (SC) samples from late symptomatic (postnatal day 19) hTDP-43^Tg/Tg mice. Fold change in protein levels is presented relative to wild-type (WT) littermate controls. In the GC muscle, CCL2, CCL3, CCL4, CCL5 and CCL6 chemokines showed the greatest increase in protein levels (magnified and highlighted in the top right panel). Exact fold changes are available in the Source Data file. B–E Quantitative analysis of CCL2 (B), CCL3 (C), CCL4 (D) and CCL5 (E) proteins in the GC and TA muscles and SC using Legendplex immunoassay. Data is presented as mean ± sem. The Legendplex multiplex immunoassay and statistical analysis was performed with n = 4 mice in the GC, TA and SC of the WT group and TA and SC of the Tg/Tg group. The GC of the Tg/Tg group was quantified in n = 3 mice. Data analysis: In each case (B–E), unpaired, one-tailed t-test was performed for direct comparison of WT vs Tg/Tg groups; (B) gastrocnemius: p = 0.0350; (C) gastrocnemius: p = 0.0174; (C) tibialis anterior: p = 0.0318; (C) spinal cord: p = 0.0083; (D) gastrocnemius: p = 0.0209; (E) gastrocnemius: p = 0.0031; (E) tibialis anterior: p = 0.0456. * = p < 0.05; ** = p < 0.01.

Fig. 6. CCL2 expression in the gastrocnemius (GC) muscle across different disease stages.

A–C CCL2 staining (green) in the GC muscle of wild-type (WT; A) and late symptomatic postnatal day 19 (P19) hTDP-43^Tg/Tg (Tg/Tg; B, C) mice. Arrows indicate CCL2⁺ cells in the close vicinity of neuromuscular junctions (NMJs), visualised with α-bungarotoxin (BTX; magenta). C High magnification image of CCL2⁺ cells around a single NMJ in the GC muscle of late stage hTDP-43^Tg/Tg mice. Innervation is shown through NEFH/SV2a (gray) staining. Asterisks label individual nuclei of CCL2⁺ cells. 3D-reconstructed version of this image is available as Supplementary movie 1. D–F Quantitative analysis of the ratio of NMJs with perisynaptic CCL2-expressing cells in presymptomatic (D), early symptomatic (E) and late symptomatic (F) mice. Data is presented as mean ± sem. Scale bar represents 50 µm on (A, B) and 10 µm on (C). Data analysis: (D) Presymptomatic stage (postnatal day 7): n = 4 (WT), n = 3 (Tg/Tg), unpaired, two-tailed t-test, p = 0.0466; (E) Early symptomatic stage (postnatal day 15): n = 3 (WT, Tg/Tg), unpaired, two-tailed t-test, p = 0.0088; (F) Late symptomatic stage: n = 4 (WT, Tg/Tg), unpaired, two-tailed t-test, p = 0.0011. * = p < 0.05; ** = p < 0.01.

Fig. 7. Activation of the CCR2 pathway in ALS patients and in the hTDP-43^Tg/Tg mice across different disease stages.

A–C CCR2 staining in the GC muscle of wild-type (WT; A) and late symptomatic (postnatal day 19) hTDP-43^Tg/Tg mice (Tg/Tg; B, C). C High magnification image reveals co-expression of CCR2 and CD45 markers, however, not all of the CD45⁺ cells show CCR2 staining. D–F Quantitative analysis of CCR2⁺ cells in the GC muscle of presymptomatic (D), early symptomatic (E) and late symptomatic (F) hTDP-43^Tg/Tg mice. G–H CCR2 staining in the skeletal muscle of control individuals (control; G) and ALS patients (ALS; H). I Quantification of CCR2⁺ cells in the skeletal muscle of healthy control (CTRL-2 in Table 1) and ALS patient (ALS-1 in Table 1) muscle samples. Data is presented as mean ± sem. Scale bar represents 50 µm on (A, B), 20 µm on (C) and 40 µm on (G, H). Data analysis: (D) Presymptomatic stage: n = 4 (WT), n = 3 (Tg/Tg), unpaired, two-tailed t-test; (E) Early symptomatic stage: n = 3 (WT, Tg/Tg), unpaired, two-tailed t-test, p = 0.0234; (F) Late symptomatic stage: n = 4 (WT, Tg/Tg), unpaired, two-tailed t-test, p = 0.0012; (I) Human muscle samples: n = 4 (control, ALS), unpaired, two-tailed t-test, p = 0.0038. * = p < 0.05; ** = p < 0.01.

Fig. 8. Local intramuscular CCL2 neutralisation or IgG treatment ameliorates NMJ pathology and reduces immune cell infiltration in the GC muscle of hTDP-43^Tg/Tg mice.

A Schematic of experimental design. B–D Representative images of NMJ denervation in the gastrocnemius (GC) muscle of untreated (B), IgG-treated (C) and CCL2-neutralising antibody-treated (D) late symptomatic postnatal day 19 (P19) hTDP-43^Tg/Tg mice. Red arrows indicate fully innervated NMJs, as revealed by the presynaptic NEFH/SV2a staining. Scale bar represents 40 µm. E–G Representative images of the CD45⁺ leukocyte infiltration in the innervation zone of the GC muscle in untreated (E), IgG-treated (F) and CCL2-neutralising antibody-treated (G) late symptomatic hTDP-43^Tg/Tg mice. Scale bar represents 40 µm. H Qualitative analysis of NMJ innervation, representing the ratio of fully innervated NMJs in the GC muscle. Analysis was performed with n = 7 (untreated) and n = 4 (IgG, CCL2) mice/group and one-way ANOVA with Fisher’s LSD post-hoc, no treatment vs. IgG: p < 0.0001, no treatment vs. CCL2: p < 0.0001. On average 30-40 NMJs were analysed per muscle. I–K Quantitative morphometric analysis of the NMJs revealed improvement in axonal diameter (I), nerve terminal area (J) and overlap (K) variables. The exact p values and statistical analyses are detailed in Table 2. L, M Quantitative analysis of CD45⁺ (L) and CCR2⁺ (M) cell count in the late symptomatic GC muscle of hTDP-43^Tg/Tg mice. Analysis was performed on n = 8 (untreated) and n = 4 (IgG, CCL2) mice/group. Data analysis: (L) CD45 analysis: Kruskal-Wallis test and uncorrected Dunn’s post-hoc [Shapiro-Wilk normality test, ALS untreated group: p = 0.0091, ALS IgG group: p = 0.7802, ALS CCL2 group p = 0.3820], no treatment vs. IgG: p = 0.0114, no treatment vs. CCL2: p = 0.0053; (M) CCR2 analysis: one-way ANOVA with Fisher’s LSD post-hoc, no treatment vs. IgG: p < 0.0001, no treatment vs. CCL2: p = 0.0002. * = p < 0.05; ** = p < 0.01; *** = p < 0.001. Data is presented as mean ± sem.