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. 2018 Mar 2;8(1):3953.
doi: 10.1038/s41598-018-22318-5.

Metabolic Reprogramming in Amyotrophic Lateral Sclerosis

M Szelechowski  1   2 , N Amoedo  2   3 , E Obre  4 , C Léger  1   2 , L Allard  1   2 , M Bonneu  2   5 , S Claverol  2   5 , D Lacombe  2   3 , S Oliet  1   2 , S Chevallier  1   2 , G Le Masson  6   7 , R Rossignol  8   9
Affiliations

Metabolic Reprogramming in Amyotrophic Lateral Sclerosis

M Szelechowski et al. Sci Rep. .

Abstract

Mitochondrial dysfunction in the spinal cord is a hallmark of amyotrophic lateral sclerosis (ALS), but the neurometabolic alterations during early stages of the disease remain unknown. Here, we investigated the bioenergetic and proteomic changes in ALS mouse motor neurons and patients' skin fibroblasts. We first observed that SODG93A mice presymptomatic motor neurons display alterations in the coupling efficiency of oxidative phosphorylation, along with fragmentation of the mitochondrial network. The proteome of presymptomatic ALS mice motor neurons also revealed a peculiar metabolic signature with upregulation of most energy-transducing enzymes, including the fatty acid oxidation (FAO) and the ketogenic components HADHA and ACAT2, respectively. Accordingly, FAO inhibition altered cell viability specifically in ALS mice motor neurons, while uncoupling protein 2 (UCP2) inhibition recovered cellular ATP levels and mitochondrial network morphology. These findings suggest a novel hypothesis of ALS bioenergetics linking FAO and UCP2. Lastly, we provide a unique set of data comparing the molecular alterations found in human ALS patients' skin fibroblasts and SODG93A mouse motor neurons, revealing conserved changes in protein translation, folding and assembly, tRNA aminoacylation and cell adhesion processes.

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Conflict of interest statement

The authors declare no competing interests.

Figures

Figure 1
Figure 1
Bioenergetics of SOD1G93A ALS motor neurons. (A) Schematic workflow of the biochemical and cell biology investigations on presymptomatic mouse motor neurons at DIV3-5. (B) Oxygen consumption rate (OCR determined on the Seahorse XF96 extracellular flux analyzer) on SOD1G93A ALS mouse motor neurons (SOD) or wild-type mouse motor neurons (WT). Mitochondrial respiration was measured in routine, oligo or FCCP conditions as explained in the methods. (C) Total and mitochondrial steady-state ATP content in SOD or WT motor neurons. (D) Representative image of the TMRM signal for the measurement of mitochondrial transmembrane electric potential (Δψ) in WT or SOD mice motor neurons and (E) related quantification. (F) Mitochondrial network total area (TOM20 labelling) in mouse motor neurons normalized to the cellular area (SMI32 labeling) and corresponding images (G). (H) Mitochondrial network morphometric analysis in mouse motor neurons.
Figure 2
Figure 2
Role of UCP2 in ALS motor neurons bioenergetics and H2O2-scavenging. (A) Effect of UCP2 inhibition with genipin on motor neurons total ATP level, (B) mitochondrial network morphology, (C) H2O2 steady-state level (as measured by CM-H2DCFDA fluorescence) and (D) gluthathione redox state. (E) Westernblot analysis of the expression level of SOD1, (F) SOD2 and (G) relative quantification of SOD1 and SOD2 protein content (normalized to actin). (H) Westernblot analysis of the expression level of catalase and (I) relative quantification of catalase protein content (normalized to actin).
Figure 3
Figure 3
SODG93A ALS mouse motor neurons proteome. (A) Over-represented GO terms in the proteome of ALS mouse motor neurons as compared to the mouse reference transcriptome. (B) Comparison of the proteome of SODG93A ALS mice motor neurons and that of wild-type mice motor neurons. The Ingenuity Pathways Analysis was performed to identify the pathway significantly different (p < 0.05) between the two proteomes. The −log p value is shown in the orange line and in the bottom abscissa. The percentage of proteins from the pathway found in the differential proteome is given in the top abscissa. (C) Table of the predicted main regulators of the ALS mouse motor neurons proteome remodeling.
Figure 4
Figure 4
Proteome remodeling in SODG93A ALS motor neurons. Chart of the metabolic proteome remodeling in SODG93A ALS motor neurons. Over represented proteins appear in red and the corresponding fold change (SOD/WT) is given after the name of each protein.
Figure 5
Figure 5
Bioenergetic alterations and proteome remodeling in ALS patient skin fibroblasts. (A) Respiration of skin fibroblasts in routine conditions and calculation of the respiratory control ratio (routine/oligo respiration) and of the uncoupling ration (FCCP/oligomycin respiration). (B) ADP/ATP ratio determined in skin fibroblasts. (C) Proteome analysis (over-representation test) of ALS skin patients fibroblasts. (D) Comparison of the proteome between ALS skin patients fibroblasts and fibroblasts from healthy individuals. Ingenuity Pathway Analysis (IPA) was performed and the pathway with a difference of −log pvalue > 5 are shown. (E) Bioenergetic model of ALS adaptive metabolism showing the role of HADHA, ACAT2, UCP2 and HMGCS1. In this hypothetical model, ALS motor neurons shift to FAO and amino-acids degradation to transduce energy but in turn generate acetyl-CoA which can then be used for fatty-acid synthesis, ketogenesis and cholesterol synthesis. Therefore, a fine tuning between the use of FAO and the elimination of acetyl-CoA must be maintained to promote cell survival, and UCP2 could play a central role in this balance. In our model, the use of acetyl-coA produced by FAO for either oxidative phosphorylation (route 1) or ketogenesis and cholesterol synthesis (route 2) depends on the export of oxaloacetate by UCP2 (route 2), as proposed by Vozza et al.. In line with this hypothesis, (i) the inhibition of UCP2 with genipin leads to an increase in cellular ATP levels (blockade of route 1; Fig. 2A), while ii) the blockade of FAO using trimetazidine (HADHB inhibitor) reduces cell viability in SOD motor neurons (blockade of route 1), with minor effects on wild-type cells (Fig. 5E). This model considers previous findings indicating that VDAC is blocked by mutant-SOD in ALS, forcing cells to depend on outer membrane-diffusive energy sources such as fatty acids. It also considers recent findings showing the accumulation of cholesterol in the cerebrospinal fluid of ALS patients.
Figure 6
Figure 6
Common pathogenic proteomic features of Amyotrohpic Lateral Sclerosis between SOD1G93A mice motor neurons and skin fibroblasts. The KEGG pathway significantly enriched (FDR < 0.05) in the dataset of proteins over-expressed in SODG93A mice motor neurons (versus WTs) are illustrated on top of the figure and the corresponding proteins are also shown. Similar analysis performed in ALS patients skin fibroblasts is shown on the bottom of the figure. The proteins over-expressed in both models are shown in bold.
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