ALS muscle changes emerge early, but FAAH treatment falls short

Skeletal muscle atrophy in amyotrophic lateral sclerosis (ALS) drives loss of muscle strength, function and quality of life in ALS patients. The endocannabinoid system (ECS) regulates muscle homeostasis via regenerative and metabolic processes, and although ECS alterations have been reported in ALS neural tissues, ECS remodelling within ALS skeletal muscle has never been studied. This study investigated temporal and muscle type-specific ECS changes in ALS. Female hSOD1G93A transgenic mice and nontransgenic littermates were studied at presymptomatic and symptomatic ages (56-138&#x2009;days of age; n&#x2009;=&#x2009;7-8/group). Endocannabinoids, N-acyl-ethanolamine congeners and inflammatory lipid mediators were quantified using targeted LC-MS/MS in the tibialis anterior (TA) and soleus (SOL) muscles. ECS-related enzymes and receptors were assessed by immunoblotting and integrated with transcriptomic analyses of skeletal muscle biopsies from ALS patients (n&#x2009;=&#x2009;5/group; ~63&#x2009;years). To evaluate therapeutic relevance, ALS mice were treated with the fatty acid amide hydrolase (FAAH) inhibitor URB937 or vehicle (n&#x2009;=&#x2009;10-11/group), and survival, body weight, welfare and motor function were assessed longitudinally. ALS caused severe atrophy in the predominantly fast-twitch TA muscle (-76.5%; p&#x2009;<&#x2009;0.01), while the slow-twitch soleus was largely preserved (-14.4%; p&#x2009;<&#x2009;0.01). Accordingly, the lipid perturbation due to ALS was more pronounced in the TA, reflected by extensive alterations in unsaturated fatty acids, hydroxy- and epoxy-fatty acids (TA: 63% and SOL: 22% of lipid mediators different between ALS vs. NTG) and marked ECS remodelling, including elevated anandamide (+37.3%; p&#x2009;=&#x2009;0.03) and multiple N-acyl-ethanolamine congeners (+76-102%; p&#x2009;<&#x2009;0.05), reduced 2-arachidonoylglycerol (-28%; p&#x2009;=&#x2009;0.06), increased CB1 receptor expression (+93%; p&#x2009;<&#x2009;0.01) and dynamic, age-dependent regulation of FAAH (presymptomatic: -68%; p&#x2009;=&#x2009;0.04, symptomatic: +21%; p&#x2009;=&#x2009;0.02). In contrast, the SOL showed modest or opposite changes, consistent with its relative resistance to atrophy. Notably, ECS remodelling in the TA was already evident at presymptomatic age (e.g., CB1: +76%; p&#x2009;=&#x2009;0.01) and the same ECS enzymes were affected in human ALS skeletal muscle transcriptomes (e.g., twofold decrease in FAAH; pFDR&#x2009;=&#x2009;0.010). Despite evidence for a therapeutic potential, chronic peripheral FAAH inhibition with URB937 did not improve weight loss, motor functions and survival of ALS mice (all p&#x2009;>&#x2009;0.05). Muscle type-specific endocannabinoid system remodelling in ALS precedes overt neurological decline and might relate to degenerative features such as metabolic disturbance and inflammation. Although peripheral FAAH inhibition alone was insufficient to modify disease outcomes, these findings identify the endocannabinoid system as an integral component of ALS muscle pathology and support skeletal muscle lipid signalling as a potentially relevant early target for adjunctive therapeutic strategies.