Spermidine treatment affects skeletal muscle gene expression in a mouse model of Amyotrophic Lateral Sclerosis

Neurodegenerative diseases, including Alzheimer's, Parkinson's, Huntington's, and Amyotrophic Lateral Sclerosis (ALS), pose serious challenges to human health, particularly as the aging population increases. These diseases exhibit a range of symptoms, from memory and cognitive impairments to motor deficits affecting movement, speech, and breathing. ALS leads to the progressive degeneration of upper and lower motor neurons. This results in skeletal muscle atrophy, severe functional decline, and eventual death. ALS is a complex and multifactorial disease, with several mechanisms contributing to its pathology. These mechanisms include disruptions in RNA metabolism, oxidative stress, neuroinflammation, autophagic dysfunction, mitochondrial abnormalities, and defects in protein homeostasis. Mitochondrial dysfunction, in particular, has been heavily implicated in ALS, affecting energy production and contributing to oxidative stress. Alterations in lipid metabolism and increased energy consumption are also observed early in disease progression, often correlating with a poor prognosis. Despite growing knowledge of ALS pathology, effective therapies to halt or reverse disease progression remain elusive. Spermidine (SPD) is a polyamine with well-documented roles in promoting health and longevity. Studies have demonstrated its neuroprotective effects in various models of neurodegenerative diseases, including Parkinson's, Huntington's, and multiple sclerosis. Spermidine enhances autophagy, a critical cellular process that helps eliminate damaged proteins and organelles, thereby reducing cellular stress. In the context of neurodegeneration, spermidine has shown promise in mitigating neurotoxicity and rescuing motor dysfunction in animal models. Given spermidine’s neuroprotective and anti-atrophic properties, we explored its potential therapeutic effects in ALS. The SOD1-G93A mouse model, which carries a mutation linked to familial ALS, was chosen for this study. These mice are transgenic for the systemic expression of human SOD1-G93A, mutation linked to ALS that presents ALS-like symptoms, including motor neuron loss and muscle weakness. SPD was administered at the onset of symptoms from day 70 to day 120, after which gene expression analysis was performed to evaluate its effects on neurodegeneration and muscle atrophy. RNA was extracted from gastrocnemius (GNM) muscle from untreated wild-type (WT) and SOD1-G93A mice at 120 days of age, untreated and SPD-treated. RNA sequencing (RNAseq) revealed a total of 5826 differentially expressed genes (DEGs) between ALS and control mice. In the comparison between ALS + SPD and untreated ALS mice, 2894 DEGs were identified, 7 with 2159 genes common to both comparisons. Gene enrichment analysis, using the WebGestalt tool, categorized DEGs into and pathways. The most represented processes included inflammation and Cytoplastmic Ribosomal Proteins, while Oxidative Phosphorylation and Electron Transport Chain dysfunction were prominent in downregulated pathways. Among the pathways analysed, genes involved in mitochondrial function were of particular interest. Mitochondrial dysfunction is a hallmark of ALS, and SPD treatment appeared to mitigate some of these defects. Notably, the expression of PGC1-α, a critical regulator of mitochondrial biogenesis, was downregulated in ALS mice but restored following SPD treatment. This gene plays a central role in regulating energy metabolism, and its rescue suggests that SPD could improve mitochondrial health in ALS. The metabolism of polyamines, including spermidine, is intricately linked to cellular functions such as protein synthesis and DNA stability. In ALS, polyamine levels are dysregulated, contributing to disease pathology. In this study, we measured the levels of putrescine (PUT), spermidine (SPD), and spermine (SPM) in the gastrocnemius muscle of ALS mice. Higher levels of all three polyamines were detected in ALS mice compared to controls, indicating a clear dysregulation of polyamine metabolism. SPD treatment reduced the levels of spermine but did not significantly alter putrescine or spermidine concentrations. Additionally, the expression of key polyamine-related genes, such as SMOX (spermine oxidase) and ODC1 (ornithine decarboxylase), was assessed. While SPD reduced ODC1 expression, other polyamine-related genes are not affected by the treatment. To confirm the RNAseq results, we performed quantitative PCR (qPCR) on selected genes from key pathways. Genes involved in ribosomal protein synthesis, such as Rpl3 and Rps14, were validated as being downregulated in ALS and reversed by SPD treatment. For the oxidative phosphorylation pathway, mt-Nd1, mt-Nd2, and mt-Nd6, which encode components of the mitochondrial electron transport chain, were downregulated in ALS and restored by SPD. This was consistent with the RNAseq data, highlighting the impact of SPD on mitochondrial gene expression. In addition to molecular analyses, we assessed the functional effects of SPD treatment on ALS progression. Body weight and muscle strength were monitored in treated and untreated mice. As expected, ALS mice exhibited significant weight loss and decreased muscle strength compared to WT mice. Although SPD did not fully recover the weight loss, it appeared to delay the decline in muscle strength, suggesting a partial protective effect. We also evaluated 8 mitochondrial function using NSC34 (Neuroblastoma x Spinal Cord -34) cellular model carrying Q331K mutation for TDP-43 protein, to measure oxygen consumption rates (OCR). SPD treatment significantly improved mitochondrial respiration, increasing ATP production, spare respiratory capacity, and proton leak. These improvements in bioenergetic profiles suggest that SPD enhances mitochondrial function in this cellular model, which could contribute to the delay in muscle strength loss observed in vivo. This research highlights spermidine’s potential as a therapeutic agent for ALS. SPD treatment showed promising effects in improving mitochondrial function, modulating polyamine metabolism, and partially reversing ribosomal and mitochondrial dysregulations in SOD1-G93A mice. While SPD did not fully restore muscle strength or weight, its ability to delay functional decline and improve mitochondrial bioenergetics warrants further investigation. These findings contribute to our understanding of ALS pathology in skeletal muscle and open avenues for exploring SPD as a potential treatment for neurodegenerative diseases like ALS.