Repurposing of Trimetazidine for amyotrophic lateral sclerosis: A study in SOD1G93A mice

Abstract

Background and purpose: Amyotrophic lateral sclerosis (ALS), a neurodegenerative disease characterized by the degeneration of upper and lower motor neurons, progressive wasting and paralysis of voluntary muscles and is currently incurable. Although considered to be a pure motor neuron disease, increasing evidence indicates that the sole protection of motor neurons by a single targeted drug is not sufficient to improve the pathological phenotype. We therefore evaluated the therapeutic potential of the multi-target drug used to treatment of coronary artery disease, trimetazidine, in SOD1^G93A mice.

Experimental approach: As a metabolic modulator, trimetazidine improves glucose metabolism. Furthermore, trimetazidine enhances mitochondrial metabolism and promotes nerve regeneration, exerting an anti-inflammatory and antioxidant effect. We orally treated SOD1^G93A mice with trimetazidine, solubilized in drinking water at a dose of 20 mg kg^-1 , from disease onset. We assessed the impact of trimetazidine on disease progression by studying metabolic parameters, grip strength and histological alterations in skeletal muscle, peripheral nerves and the spinal cord.

Key results: Trimetazidine administration delays motor function decline, improves muscle performance and metabolism, and significantly extends overall survival of SOD1^G93A mice (increased median survival of 16 days and 12.5 days for male and female respectively). Moreover, trimetazidine prevents the degeneration of neuromuscular junctions, attenuates motor neuron loss and reduces neuroinflammation in the spinal cord and in peripheral nerves.

Conclusion and implications: In SOD1^G93A mice, therapeutic effect of trimetazidine is underpinned by its action on mitochondrial function in skeletal muscle and spinal cord.

Keywords: SOD1G93A mice; Trimetazidine; amyotrophic lateral sclerosis; hypermetabolism; mitochondria; neurodegeneration.

FIGURE 1

Trimetazidine (TMZ) improves muscle strength and extends lifespan of SOD1^G93A mice. (a and b) Maximal grip strength of SOD1^G93A mice (males in (a) and females in (b)) treated with (G93A TMZ) or without (G93A) TMZ (20 mg kg⁻¹ in drinking water) from the onset of the disease (70 days of age) through to disease end point (when mice were no longer able to perform the test). Data presented as means ± SEM, ^# P < 0.05 when compared with untreated littermates SOD1^G93A mice, n ≥ 12 per group. Numbers under data points indicate the number of animals remaining in the study from 154 days for males and 147 days for females. (c–e) The midpoint (left panels) and the humane endpoint (middle panels) of disease in TMZ treated (G93A TMZ) and littermates untreated SOD1^G93A mice (G93A) are defined by a 25% and 50% decrease in grip strength respectively, starting from the onset of disease; males in (c) and females in (e). Data are presented as means ± SEM, #P < 0.05 when compared with untreated age‐matched SOD1^G93A mice, n ≥ 12 per group. (d–f) Disease duration in TMZ treated (G93A TMZ) and untreated (G93A) littermates SOD1^G93A mice; males in (d) and females in (f). Data are presented as means ± SEM. ^# P < 0.05, n ≥ 12 per group. (g and h) Kaplan–Meier survival curves of treated (G93A TMZ) and untreated (G93A) littermates SOD1^G93A mice; males in (g) and females in (g), n ≥ 12 per group. P values were obtained using parametric two‐way ANOVA with Bonferroni post hoc test (a, b) or t‐test (e–h)

FIGURE 2

Trimetazidine (TMZ) prevents metabolic alterations in SOD1^G93A mice. (a) Data traces representing mean oxygen consumption (a proxy for energy expenditure) over 24 h in 120‐day old male (a) and female (b) wild‐type and SOD1^G93A mice untreated (WT and G93A) and treated with TMZ (WT TMZ and G93A TMZ) from 70 days of age (disease onset). Dotted lines represent SEM. (c and d) Cumulative oxygen consumption in mice over a 24‐h period and during the light and dark cycle. (e) Glucose tolerance test in 120‐day old wild‐type and SOD1^G93A mice untreated (WT and G93A) and treated with TMZ (WT TMZ and G93A TMZ) from 70 days of age (disease onset). Blood glucose was assessed before glucose injection (0 min), and 20, 40 and 60 min following glucose injection. (f) Body mass evolution of SOD1^G93A and wild‐type littermate mice (males left panel, females right panel) with (G93A TMZ and WT TMZ) or without (G93A and WT) TMZ. Starting body weight at 70 days was assigned as 100%, and the decline in body weight over time was calculated as percentage loss relative to the starting body weight. Data are presented as means ± SEM ^# P < .05, compared with untreated G93A, n ≥ 6 per group. P values were obtained using one‐way ANOVA with Bonferroni post hoc test

FIGURE 3

Trimetazidine (TMZ) restores the glycolytic phenotype and reduces atrophic/fibrotic markers in the tibialis anterior (TA) muscle of SOD1^G93A mice. (a) Representative western blots (left) and quantification (right) of sarcolipin (SLN) and CPT1 protein expression in the TA of 120‐day old wild‐type (WT) and SOD1^G93A (G93A) mice untreated (−) and treated (+) with TMZ. Data are presented as means ± SEM, *P < 0.05 compared with WT, ^# P < 0.05 compared with G93A, n ≥ 5 per group. (b) Expression level of mRNAs encoding Myosin Heavy Chain isoforms MyHCIIa (top row) and MyHCIIb (bottom row) in TA obtained from 90‐, 120‐ and 150‐day old wild‐type and SOD1^G93A and WT mice untreated (WT and G93A) and treated with TMZ (WT TMZ and G93A TMZ). Data are presented as means ± SEM, * P < 0.05, compared with WT (arbitrarily set at 1), #P < 0.05, compared with G93A, n ≥ 5 per group. (c) Expression level of mRNAs encoding myoglobin in TA obtained from 120‐day old wild‐type and SOD1^G93A mice untreated (WT and G93A) and treated with TMZ (WT TMZ and G93A TMZ). Data are presented as means ± SEM, *P < 0.05 compared with WT (arbitrarily set at 1), #P < 0.05 compared with G93A, n ≥ 4 per group. (d) Representative images (left) of in situ NADH‐tetrazolium reductase activity staining in cross sections of TA muscle obtained from 120‐day old wild‐type and SOD1^G93A littermates untreated (WT and G93A) and treated with TMZ (WT TMZ and G93A TMZ). Scale bar, 200 μm. Graphical representation (right) of the percentage NADH‐positive area of whole transverse sections of TA muscle. Data are presented as means ± SEM, *P < 0.05, compared with WT, ^# P < 0.05 compared with G93A, n ≥ 5 per group. (e) Expression level of mRNAs encoding atrogin and Col3a1 in TA obtained from 120‐day old wild‐type and SOD1^G93A mice untreated (WT and G93A) and treated with TMZ (WT TMZ and G93A TMZ). Data are presented as means ± SEM, *P < 0.05 compared with WT, ^# P < 0.05 compared with G93A, n ≥ 5 per group. P values were obtained using parametric one‐way ANOVA with Bonferroni post hoc test

FIGURE 4

Trimetazidine (TMZ) preserves the integrity of neuromuscular junctions (NMJs). (a) Representative maximum projection images of EDL NMJs from 120‐ and 150‐day old wild‐type and SOD1^G93A mice untreated (WT and G93A) and treated with TMZ (WT TMZ and G93A TMZ), stained with synaptophysin (Syn), α‐bungarotoxin (nACh receptor), and Neurofilament (NF). (b–f) Quantitation of NMJ morphometry in EDL muscles of WT, WT TMZ, G93A and G93A TMZ mice at 120 and 150 days of age, highlighting the number of primary (b) and secondary (c) ramifications, the number of fragments per NMJ (d), the area of the nACh receptor clusters (e) and the percentage of colocalized voxels (f). Data are presented as means ± SEM. *P < 0.05, compared with WT mice and #P < 0.05 compared to SOD1^G93A mice, n ≥ 4 per group (n = 4 for WT, n = 6 for WT TMZ, G93A, G93A TMZ). The trend towards a reduced neuromuscular endplate fragmentation in TMZ treated SOD1G93A mice (d) is shown as percentage. (g and h) Representative western blots (left) and quantification (right) of Dystrophin (Dys), lipoprotein receptor‐related protein 4 (LRP4), agrin, and phosphorylated Protein Kinase C theta/Protein Kinase C theta (pPKCθ/PKCθ) protein expression in the tibialis anterior of 120‐day old (g) and 150‐day old (h) wild‐type (WT) and SOD1^G93A (G93A) mice untreated (−) and treated (+) with TMZ from disease onset (70 days of age). Data are presented as means ± SEM, *P < 0.05, compared with WT mice and ^# P < 0.05, compared with G93A mice, n ≥ 4 per group (n = 4 for WT, n = 6 for WT TMZ, G93A, G93A TMZ). P value was obtained using parametric one‐way ANOVA with Bonferroni post hoc test

FIGURE 5

Trimetazidine (TMZ) preserves sciatic nerve integrity. (a and b) Representative images of sciatic nerve sections and quantifications of fluorescence intensity (histograms) from 120‐ and 150‐day old wild‐type mice and SOD1^G93A treated without (WT and G93A) or with TMZ (WT TMZ and G93A TMZ) from disease onset (70 days of age). Anti‐MBP stains myelin, anti‐NFH stains the axonal cytoskeleton (neurofilament heavy), anti‐CD68 stains activated macrophages. (c and d) Representative images of sciatic nerve sections and quantifications of fluorescence intensity (histograms) from 120‐ and 150‐day old wild‐type mice and SOD1^G93A treated without (WT and G93A) or with TMZ (WT TMZ and G93A TMZ) from disease onset (70 days of age). Anti‐GFAP stains astrocytes. Scale bar 50 μm. Data are presented as means ± SEM, *P < 0.05, compared with WT mice and #P < 0.05, compared with G93A mice, n ≥ 4 per group (n = 4 for WT, n = 6 for WT TMZ, G93A, G93A TMZ). P value was obtained using parametric one‐way ANOVA with Bonferroni post hoc test

FIGURE 6

Trimetazidine (TMZ) protects from motor neuron loss and neuroinflammation. (a and b) Representative lumbar spinal cord sections (L3‐L5) stained with cresyl violet (scale bar 100 μm) and quantification of motor neuron number (histograms) in 120‐ (a) and 150‐day old (b) wild‐type mice treated without TMZ (WT) and SOD1^G93A mice treated without (G93A) or with TMZ (G93A TMZ) from disease onset (70 days of age). Data are presented as means and ± SEM, *P < 0.05, compared with wild‐type; ^# P < 0.05, compared with untreated G93A, n ≥ 4 per group (n = 4 for WT, n = 6 for G93A, G93A TMZ). (c) Representative immunofluorescence images and analysis of astrocytes (GFAP) in lumbar spinal cord sections (L3‐L5) of 120‐ and 150‐day old wild‐type mice treated without TMZ (WT) and SOD1^G93A mice treated without (G93A) or with TMZ (G93A TMZ) from disease onset (70 days of age). (d and e) Representative immunofluorescence images and analysis of activated microglia (CD68) and resting microglia (P2T12) in lumbar spinal cord sections (L3‐L5) of 120‐ and 150‐day old wild‐type mice treated without TMZ (WT) and SOD1^G93A mice treated without (G93A) or with TMZ (G93A TMZ) from disease onset (70 days of age). Scale bar 100 μm. Data are presented as means and ± SEM, *P < 0.05 compared with wild‐type; ^# P < 0.05, compared with untreated G93A, n ≥ 4 per group (n = 4 for WT, n = 6 for G93A, G93A TMZ). P values were obtained using parametric one‐way ANOVA with Bonferroni post hoc test (a–e)

FIGURE 7

Trimetazidine (TMZ) improves mitochondrial coupling state. (a and b) Representative data traces of oxygen consumption rate (OCR) during a coupling assay on isolated mitochondria purified from the tibialis anterior and spinal cord. (c and d) Mitochondrial respiration states (as OCR) are reported as basal respiration (state 2), maximal coupled respiration (state 3), respiration due to proton leak (state 4o) and maximal uncoupled respiration (state 3u). Data are presented as means ± SEM, *P < 0.05 compared with wild‐type; ^# P < 0.05 compared with untreated G93A, n ≥ 6 per group. P values were obtained using parametric one‐way ANOVA with Bonferroni post hoc test

FIGURE 8

Trimetazidine (TMZ) improves electron transport chain complex activity. (a and b) Representative data traces of oxygen consumption rate (OCR) during an electron flow assay on isolated mitochondria purified from tibialis anterior and spinal cord. (c and d) Quantification of respiration (OCR) activity of complex I, complex II/III and complex IV in the presence of rotenone, succinate, antimycin A and ascorbate/N,N,N,N‐tetramethyl‐p‐phenylenediamine (ASC/TMPD), respectively. Data are presented as means ± SEM, *P < 0.05, compared with wild‐type; ^# P < 0.05, compared with untreated G93A, n = ≥6 per group. P values were obtained using parametric one‐way ANOVA with Bonferroni post hoc test