Dysregulation of muscle cholesterol transport in amyotrophic lateral sclerosis

Abstract

Amyotrophic lateral sclerosis (ALS) is a progressive neurodegenerative disorder affecting motor neurons, with a typical lifespan of 3-5 years. Altered metabolism is a key feature of ALS that strongly influences prognosis, with an increase in whole body energy expenditure and changes in skeletal muscle metabolism, including greater reliance on fat oxidation. Dyslipidaemia has been described in ALS as part of the metabolic dysregulation, but its role in the pathophysiology of the disease remains controversial. Among the lipids, cholesterol is of particular interest as a vital component of cell membranes, playing a key role in signal transduction and mitochondrial function in muscle. The aim of this study was to investigate whether motor dysfunction in ALS might be associated with dysregulation of muscle cholesterol metabolism. We determined cholesterol content and analysed the expression of key determinants of the cholesterol metabolism pathway in muscle biopsies from 13 ALS patients and 10 asymptomatic ALS-mutation gene carriers compared to 16 control subjects. Using human control primary myotubes, we investigated the potential contribution of cholesterol dyshomeostasis to reliance on mitochondrial fatty acid. We found that cholesterol accumulates in the skeletal muscle of ALS patients and that cholesterol overload significantly correlates with disease severity evaluated by the Revised ALS Functional Rating Scale. These defects are associated with overexpression of the genes of the lysosomal cholesterol transporters Niemann-Pick type C1 (NPC1) and 2 (NPC2), which are required for cholesterol transfer from late endosomes/lysosomes to cellular membranes. Most notably, a significant increase in NPC2 mRNA levels could be detected in muscle samples from asymptomatic ALS-mutation carriers, long before disease onset. We found that filipin-stained unesterified cholesterol accumulated in the lysosomal compartment in ALS muscle samples, suggesting dysfunction of the NPC1/2 system. Accordingly, we report here that experimental NPC1 inhibition or lysosomal pH alteration in human primary myotubes was sufficient to induce the overexpression of NPC1 and NPC2 mRNA. Finally, acute NPC1 inhibition in human control myotubes induced a shift towards a preferential use of fatty acids, thus reproducing the metabolic defect characteristic of ALS muscle. We conclude that cholesterol homeostasis is dysregulated in ALS muscle from the presymptomatic stage. Targeting NPC1/2 dysfunction may be a new therapeutic strategy for ALS to restore muscle energy metabolism and slow motor symptom progression.

Keywords: disease progression; metabolism; presymptomatic stage.

Figure 1

Cholesterol accumulation in muscles of ALS patients correlates with the ALSFRS-R score. (A) Cholesterol concentration (μmol/g) in deltoid muscle biopsies from 10 control subjects and 13 amyotrophic lateral sclerosis (ALS) participants. ***P < 0.001 (Mann-Whitney U-test). Data represent mean + standard deviation (SD) and each dot represents an individual patient. Orange triangles, blue circles and green squares represent ALS patients with bulbar onset, spinal upper limb onset and spinal lower limb onset, respectively. (B) Correlation analysis between revised ALS Functional Rating Scale (ALSFRS-R) total score and cholesterol concentration measured in the deltoid muscle of ALS participants. Non-parametric Spearman rank correlation test (n = 9).

Figure 2

Cholesterol metabolism is dysregulated in muscles of ALS patients. (A–E) mRNA expression analysis by RT-qPCR of HMGCR (A), LDLR (B), ABCA1 (C), NPC1 (D) and NPC2 (E). mRNA expressions were normalized by SDHA and RPL13A. Data represent mean + standard deviation (SD), and each dot represents an individual patient. *P < 0.05, **P < 0.01, ***P < 0.001 (Mann-Whitney U-test, n = 11 control and 12 ALS). ALS = amyotrophic lateral sclerosis; CTRL = control; HMGCR = 3-hydroxy-3-methyl-glutaryl-coenzyme A reductase; LDLR = low-density lipoprotein receptor.

Figure 3

Cholesterol metabolism is dysregulated from the presymptomatic phase of ALS. (A) Cholesterol concentration (μmol/g) in deltoid muscle biopsies from 10 control (CTRL) participants and 10 asymptomatic amyotrophic lateral sclerosis (ALS) mutation carriers (Pre-fALS). (B–F) mRNA expression analysis by RT-qPCR of NPC2 (B), NPC1 (C), HMGCR (D), ABCA1 (E) and LDLR (F). mRNA expressions were normalized by SDHA and RPL13A. Data represent mean + standard deviation (SD), and each dot represents an individual patient. *P < 0.05 (Mann-Whitney U-test, n = 13 control and 10 pre-fALS individuals). HMGCR = 3-hydroxy-3-methyl-glutaryl-coenzyme A reductase; LDLR = low-density lipoprotein receptor.

Figure 4

Free cholesterol accumulates in the late endosome/lysosome compartment in ALS muscle. Intracellular distribution of free cholesterol on transversal slices of deltoid muscle biopsies from amyotrophic lateral sclerosis (ALS) and controls (CTRL) (representative image). Lysosomes were labelled by immunofluorescence directed against LAMP3, unesterified cholesterol was stained with filipin and nuclei were stained by propidium iodide (PI). Scale bar = 50 µm. Arrows in the left panels indicate co-localizations between the cholesterol and LAMP3 and arrows in the right panels indicate lysosome without cholesterol co-localization.

Figure 5

Acute muscle denervation does not induce ALS-like alterations in muscle cholesterol metabolism in vivo. (A–F) mRNA expression analysis by RT-qPCR of Chrna1 (A), Chrng (B), Hmgcr (C), Abca1 (D), Npc2 (E) and Npc1(F) in soleus (n = 6) and tibialis anterior (TA, n = 5) without (contralateral) or 7 days after nerve crush (ipsilateral). mRNA expressions were normalized by Rplp0 and ActB. Data represent mean + standard deviation (SD), and each dot represents an individual mouse. *P < 0.05, **P < 0.01 (paired t-tests, n = 3). (G) Histology of tibialis anterior muscles without (contralateral) or 7 days after nerve crush (ipsilateral). Left: Cryo-sections of the tibialis anterior muscle fibres stained by haematoxylin and eosin. Scale bar = 20 μm. Right: Cross-sectional area (CSA) quantification of the contralateral and the ipsilateral tibialis anterior muscle fibres 7 days after never crush. Visual evaluation of haematoxylin and eosin staining of muscle cross-sections and CSA measurement showed significant muscle atrophy in the tibialis anterior and soleus muscles ipsilateral to the nerve crush compared to the opposite limb. Data represent mean + SD and each dot represents an individual mouse (n = 6).

Figure 6

NPC1 inhibition increases dependency on fatty acid β-oxidation in human primary muscle cell cultures. (A–D) Primary muscle cells from control individuals at 6 days of differentiation were treated with U188666A, which inhibits NPC1, or with NH₄Cl to inhibit lysosome activity. (A and B) mRNA expression analysis by RT-qPCR of NPC1 (A) and NPC2 (B). mRNA expressions were normalized by PPIA and RPL13. (C). Left: Western blot of NPC1. Right: Quantification of NPC1 protein expression (n = 3). (D) Analysis of fat oxidation dependency using Seahorse XFe24 FluxPaks (Agilent Technologies). *P < 0.05 (Kruskal-Wallis test, n = 7 in duplicate for the Seahorse experiment). (E and F) mRNA expression analysis by RT-qPCR of SDHB in muscle biopsies from asymptomatic amyotrophic lateral sclerosis (ALS) mutation carriers (pre-fALS) (n = 13 controls and 10 pre-fALS individuals) (E) and from ALS individuals (n = 11 controls and 12 ALS patients) (F). mRNA expressions were normalized by SDHA and RPL13A. Data represent mean + standard deviation (SD) and each dot represents an individual patient. *P < 0.05 (Mann-Whitney U-test). (G) Correlation analysis between revised ALS Functional Rating Scale (ALSFRS-R) total score and SDHB RNA expression measured in the deltoid muscle of ALS participants. Non-parametric Spearman rank correlation test (n = 8). (H) mRNA expression analysis by RT-qPCR of SDHB in primary muscle cells from control individuals at 6 days of differentiation after U188666A or NH₄Cl treatment. mRNA expressions were normalized by PPIA and RPL13A. Data represent mean + SD. *P < 0.05, **P < 0.01, ***P < 0.001 [Kruskal-Wallis test, n = 13 control and 10 pre-fALS individuals (F), n = 11 control and 12 ALS (E)].