NAD+-related dysregulation in asymptomatic phases of ALS: early biomarkers of the disease?

Background: nowadays, there is a lack of effective therapies for Amyotrophic Lateral Sclerosis (ALS). This is largely attributed to late diagnosis and incomplete understanding of the mechanisms determining the disease. Nicotinamide Adenine Dinucleotide (NAD) is an essential coenzyme in our cells, fundamental for a correct bioenergetic metabolism and homeostasis. Derangement of NAD levels and of NAD-related pathways is emerging in ALS pathogenesis. Specifically, decreased NAD levels were observed both in mice and patients affected by ALS. Astrocytes from human post-mortem ALS spinal cords induced death in co-cultured motor neurons and restoration of NAD levels mitigated ALS astrocyte-mediated toxicity. In an ALS mouse model, supplementation with a NAD precursor delayed motor neuron degeneration and decreased neuroinflammation markers in spinal cords. Aims: the main aim of this study was to investigate whether a dysregulation in NAD synthesis/degradation occurs in asymptomatic phases of ALS in a swine model recently exploited to demonstrate that alteration in astrocytic EV production precedes the onset of disease symptoms. Methods: cervical tracts of spinal cord of hSOD1G93A (SOD) pigs and age-matched WT at asymptomatic (early and late) and symptomatic phases were lysed in PCA or NaOH, for determination of NAD(P) and NAD(P)H levels, respectively. NAD(P)(H), NMN and NAM levels were measured by appropriate and specific enzymatic assays in a fluorimeter plate reader. G6PD activity was evaluated in homogenates of spinal cord by adding glucose-6-P and NADP and measuring NADPH production with a fluorimeter. NADase activity was assessed in homogenates by adding NAD and measuring ADPR production by HPLC. The mRNA levels of CD38, SARM1 and NAMPT were evaluated by qPCR. Results: our data show that NAD and NADH were increased in spinal cords of SOD pigs at early and late asymptomatic phases, whereas at symptomatic stage a depletion in NAD(H) levels was observed. This change in NAD levels is preceded by an increased NAD-glycohydrolase activity, converting NAD into ADPR, occurring already at both asymptomatic stages. The main NADases in mammals are represented by CD38 (mostly expressed on inflammatory cells) and by SARM1 (whose activation has a role in triggering neurodegeneration). We detected increased level of CD38 expression by qPCR in the symptomatic phase of ALS, suggesting that in the asymptomatic phases the upregulated NAD degradation was possibly due to SARM1. Indeed, we observed higher levels of SARM1, especially in late asymptomatic and symptomatic phases of the disease. In addition, the amount of nicotinamide mononucleotide (NMN), a NAD precursor that was reported to activate SARM1, was increased in the late asymptomatic phase of ALS. On the other hand, the content of nicotinamide (NAM) was slightly increased in the symptomatic phases. The expression levels of NAMPT, the rate limiting enzyme involved in NAD synthesis that converts NAM into NMN, were not significantly modified in the different phases. Increased ROS levels were reported to be generated in ALS. In line with these data, we found a decrease in NADPH content in the symptomatic animals. Accordingly, the activity of G6PD, the main cellular source of NADPH and primary defense against oxidative stress, was initially increased in early asymptomatic phase and subsequently decreased in symptomatic animals in comparison to age-matched WT animals. Conclusions: our results suggest that dysregulation in NAD(H), NADP(H) and NAD-degrading enzymes occurs already in asymptomatic phases. Studies are in progress to detect altered levels of NAD-related nucleotides/enzymes in EV, which may be regarded as early biomarkers to identify precociously the disease.