N-acetylaspartate promotes glycolytic-to-oxidative fiber-type switch and resistance to atrophic stimuli in myotubes

The study investigates whether N-acetylaspartate (NAA), a metabolite synthesized predominantly in neurons and elevated in blood during neurodegenerative diseases such as ALS, modulates skeletal muscle metabolism and phenotype. Skeletal muscle fibers differ in contractile speed and metabolic profile (oxidative slow-twitch type I vs glycolytic fast-twitch type IIB/X, with type IIA intermediate). In ALS and other motor neuron diseases, a glycolytic-to-oxidative fiber-type switch is observed. Given broad peripheral expression of ASPA (the NAA-degrading enzyme) in muscle and increased circulating NAA in ALS, the authors hypothesize that NAA catabolism in muscle drives a metabolic shift toward oxidative metabolism, influencing fiber identity and resistance to atrophic stimuli. They aim to characterize NAA’s effects on differentiated C2C12 myotubes, the dependence on ASPA, and relevant changes in ALS mouse muscles and related tissues.

Background literature establishes: (1) NAA is synthesized by mitochondrial NAT8L in neurons and catabolized by ASPA predominantly in oligodendrocytes; NAA can be released to extracellular fluids and is elevated in blood/CSF in neurodegeneration. (2) Outside the brain, brown adipose tissue (BAT) expresses both NAT8L and ASPA; NAA synthesis/catabolism in BAT supports a futile lipid cycle fueling mitochondrial oxidation and thermogenesis. (3) Skeletal muscle fiber types have distinct metabolic programs; ALS muscles exhibit a glycolytic-to-oxidative switch with increased lipid oxidation and MHC isoform changes. These data motivate exploring NAA’s role and ASPA-mediated catabolism in peripheral muscle metabolic remodeling.

- Cell model: Murine C2C12 myoblasts (ATCC) differentiated into myotubes for 7 days in DMEM (4.5 g/L glucose) with 2% horse serum; medium changed daily. NAA (Sigma Aldrich) was added at day 5 of differentiation for 48 h. - Inhibitor treatments: ATGListatin (lipolysis inhibitor), etomoxir (CPT1 inhibitor, blocks mitochondrial FA import), oligomycin (ATP synthase inhibitor), 2-deoxyglucose (glycolysis inhibitor), NH4Cl/leupeptin (autophagy inhibition), MG132 (proteasome inhibition). Oleic acid pre-loading used to boost lipid droplet (LD) accumulation in some assays. - Gene editing: CRISPR/Cas9 knockouts in C2C12 for ASPA and ATGL using specified gRNAs; Cas9-alone transfection as control. KO validation by Western blot. - Biochemical assays: - Western blot for acetyl-lysine, ASPA, ATGL, HSL, PGC1α, NRF1, ACO2, TFAM, p-mTOR, mTOR, p-p70S6K (Thr389), ubiquitinated proteins, BNIP3, LC3-II, β-Actin loading control. - ATP quantification (colorimetric kit based on phosphocreatine formation). - Extracellular lactate assay (TCA precipitation; LDH-based NADH absorbance at 340 nm; normalized to protein). - Complex I activity (NADH oxidation at 340 nm). - HPLC quantification of NAA in serum, spinal cord, and BAT. - Imaging: - Oil Red O staining for LD content; quantification by mean fluorescence intensity ratio (± ATGListatin). - JC-1 dye for mitochondrial membrane potential (red/green ratio). - Bright-field imaging for myotube diameter; ImageJ quantification; fusion index assessment. - Puromycin incorporation assay to assess protein synthesis; Ponceau S as loading control. - Gene expression: RT-qPCR for fiber-type markers (MyH4/MHC IIb, MyH2/MHC IIa, MyH7b), myoglobin, ASPA; ACTB reference. - Animal model: SOD1-G93A ALS mice (B6.Cg-Tg(SOD1 G93A)1Gur/J); symptomatic defined by hanging grid test at 20–22 weeks. Tissues analyzed: quadriceps, gastrocnemius, spinal cord, BAT; ASPA, HSL, ATGL protein levels by Western blot; NAA by HPLC. Additional model: wild-type sciatic nerve-crush. - Bioinformatics: Analysis of ASPA expression in oxidative (soleus) vs glycolytic (EDL) muscles from GEO dataset (GSE23244); ASPA expression in gastrocnemius of SOD1-G93A and nerve-crushed mice (GSE16362). GO enrichment of genes co-expressed with ASPA using DAVID. GTEx used for tissue-wide ASPA expression. - Statistics: Data presented as mean ± SD; significance thresholds indicated (p < 0.05, p < 0.01, p < 0.001) across n=3 independent experiments unless specified.

- NAA enhances lipid turnover in myotubes: - Increased acetyl-lysine levels following NAA treatment, consistent with elevated acetylation capacity. - Oil Red O staining decreased after NAA, but LDs accumulated when lipolysis was blocked (ATGListatin) or in ATGL KO cells, indicating concurrent upregulated lipogenesis and lipolysis. ACC1 dephosphorylation/activation supports increased lipogenesis. ATGL and HSL lipases were upregulated. - NAA shifts metabolism toward oxidative phosphorylation: - ATP levels increased after 2 mM NAA; increase was abolished by etomoxir and oligomycin, but not reduced by 2-DG, indicating ATP rise is primarily from mitochondrial FAO-driven OXPHOS (Fig. 1E; **p < 0.01, ***p < 0.001 vs controls). - Elevated mitochondrial biogenesis and activity: increased PGC1α, NRF1, ACO2, TFAM protein levels; increased JC-1 red/green ratio; elevated complex I activity. - Decreased extracellular lactate following NAA indicates reduced glycolytic flux. Blocking lipolysis with ATGListatin prevented the NAA-induced reduction in lactate, linking the shift to enhanced lipid use. - Fiber-type remodeling: - Decreased MyH4 (fast glycolytic, MHC IIb) and increased MyH2 (fast oxidative/intermediate, MHC IIa) and MyH7b (slow oxidative) mRNAs after NAA; myoglobin mRNA increased (RT-qPCR; *p < 0.05, **p < 0.01 vs CTRL). - Morphology and protein turnover: - Myotube diameter decreased with NAA (consistent with oxidative fiber phenotype) without altered differentiation (fusion index unchanged) and with unaltered or reduced atrogene expression (TRIM63/Murf1, FBXO32/Atrogin1). - Protein synthesis increased: higher phosphorylation of mTOR/p70S6K and greater puromycin incorporation. - Protein degradation pathways activated: slightly increased ubiquitinated proteins; robust autophagy activation evidenced by increased BNIP3 and LC3-II upon autophagy inhibition. Inhibition of proteasome (MG132) or autophagy (NH4Cl/leupeptin) abrogated the NAA-induced diameter reduction. - ASPA dependence: - ASPA expression higher in oxidative soleus vs glycolytic EDL; genes co-expressed with ASPA enriched for oxidative phosphorylation, FA β-oxidation, TCA cycle, and respiratory chain terms. - NAA upregulated ASPA expression in C2C12 myotubes. - ASPA KO abolished NAA effects on myotube diameter, LD dynamics (Oil Red O), and lactate extrusion, demonstrating necessity of NAA catabolism. - Resistance to atrophic stimuli: - NAA-treated myotubes resisted diameter reduction induced by dexamethasone; similar protection observed against TNFα and cisplatin. - In vivo/biological context (ALS and denervation): - ASPA levels increased in gastrocnemius of WT sciatic nerve-crushed mice and asymptomatic SOD1-G93A mice (significant in nerve-crush), and significantly upregulated in quadriceps and gastrocnemius of symptomatic SOD1-G93A mice, along with increased ATGL and HSL. - ALS mice displayed higher serum NAA, decreased spinal cord NAA, increased BAT NAA with decreased BAT ASPA, suggesting sources and altered handling of NAA during disease.

The findings demonstrate that NAA drives a metabolic reprogramming of skeletal muscle cells toward an oxidative phenotype through its catabolism by ASPA. NAA increases acetyl-group availability (acetate/acetyl-CoA), stimulating lipogenesis (ACC1 activation) while concurrently enhancing lipolysis (ATGL/HSL upregulation), culminating in elevated fatty acid oxidation that fuels mitochondrial ATP production. The oxidative shift is confirmed by increased mitochondrial biogenesis (PGC1α/NRF1/TFAM), activity (membrane potential, complex I), and reduced glycolysis (lower extracellular lactate). Phenotypically, NAA-treated myotubes adopt oxidative fiber characteristics: smaller diameters, increased oxidative MHC markers (MyH2, MyH7b), and higher myoglobin. Elevated protein turnover (increased protein synthesis and autophagy/proteasomal activity) supports remodeling, and blocking degradation pathways prevents diameter changes, indicating their role. ASPA is essential: genetic ablation of ASPA abolishes NAA’s metabolic and morphological effects, linking outcomes to NAA catabolism rather than direct signaling alone. In disease-relevant contexts, muscles undergoing a glycolytic-to-oxidative switch (ALS, denervation) upregulate ASPA and lipases, aligning with the in vitro mechanism. Elevated serum NAA in ALS, coupled with decreased spinal cord NAA and increased BAT NAA (with reduced BAT ASPA), suggests that neuronal degeneration and BAT may contribute to circulating NAA that muscles can catabolize. The oxidative shift and enhanced resistance to multiple atrophic stimuli imply NAA catabolism functions as a compensatory mechanism promoting a more resilient muscle phenotype in neuromuscular disease.

NAA, via ASPA-mediated catabolism, promotes lipid turnover, mitochondrial biogenesis, and a glycolytic-to-oxidative fiber-type switch in C2C12 myotubes, enhancing resistance to atrophic stimuli. ASPA is upregulated in oxidative muscles and in ALS-affected muscles undergoing metabolic reprogramming, and ASPA loss abrogates NAA’s effects, establishing causality. These data identify NAA catabolism as a key regulator of muscle metabolic identity with potential protective roles in neuromuscular conditions. Future directions include: testing in vivo NAA administration or modulation of ASPA activity on muscle function and atrophy; dissecting signaling pathways linking acetate/NAA to lipase activation; determining cell sources and kinetics of NAA delivery to muscle in disease; and exploring therapeutic strategies that harness NAA-ASPA pathways to promote oxidative resilience in skeletal muscle.

The study primarily uses an in vitro C2C12 differentiation model to establish mechanistic links, complemented by associative analyses in mouse tissues and public datasets. Direct in vivo intervention studies modulating NAA or ASPA in muscle were not performed, and the precise contribution of different tissue sources to circulating NAA in disease remains to be fully quantified.