NAD metabolism regulates diverse biological processes, including ageing, circadian rhythm and axon survival. Axons depend on the activity of the central enzyme in NAD biosynthesis, nicotinamide mononucleotide adenylyltransferase 2 (NMNAT2), for their maintenance and degenerate rapidly when this activity is lost. However, whether axon survival is regulated by the supply of NAD or by another action of this enzyme remains unclear. Here we show that the nucleotide precursor of NAD, nicotinamide mononucleotide (NMN), accumulates after nerve injury and promotes axon degeneration. Inhibitors of NMN-synthesising enzyme NAMPT confer robust morphological and functional protection of injured axons and synapses despite lowering NAD. Exogenous NMN abolishes this protection, suggesting that NMN accumulation within axons after NMNAT2 degradation could promote degeneration. Ectopic expression of NMN deamidase, a bacterial NMN-scavenging enzyme, prolongs survival of injured axons, providing genetic evidence to support such a mechanism. NMN rises prior to degeneration and both the NAMPT inhibitor FK866 and the axon protective protein Wld^S prevent this rise. These data indicate that the mechanism by which NMNAT and the related Wld^S protein promote axon survival is by limiting NMN accumulation. They indicate a novel physiological function for NMN in mammals and reveal an unexpected link between new strategies for cancer chemotherapy and the treatment of axonopathies.

Axon injury leads to rapid depletion of NAD-biosynthetic enzyme NMNAT2 and high levels of its substrate, NMN. We proposed a key role for NMN in Wallerian degeneration but downstream events and their relationship to other mediators remain unclear. Here, we show, in vitro and in vivo, that axotomy leads to a late increase in intra-axonal Ca(2+), abolished by pharmacological or genetic reduction of NMN levels. NMN requires the pro-degenerative protein SARM1 to stimulate Ca(2+) influx and axon degeneration. While inhibition of NMN synthesis and SARM1 deletion block Ca(2+) rise and preserve axonal integrity, they fail to prevent early mitochondrial dynamic changes. Furthermore, depolarizing mitochondria does not alter the rate of Wallerian degeneration. These data reveal that NMN and SARM1 act in a common pathway culminating in intra-axonal Ca(2+) increase and fragmentation and dissociate mitochondrial dysfunctions from this pathway, elucidating which steps may be most effective as targets for therapy.

Wallerian degeneration of physically injured axons involves a well-defined molecular pathway linking loss of axonal survival factor NMNAT2 to activation of pro-degenerative protein SARM1. Manipulating the pathway through these proteins led to the identification of non-axotomy insults causing axon degeneration by a Wallerian-like mechanism, including several involving mitochondrial impairment. Mitochondrial dysfunction is heavily implicated in Parkinson's disease, Charcot-Marie-Tooth disease, hereditary spastic paraplegia and other axonal disorders. However, whether and how mitochondrial impairment activates Wallerian degeneration has remained unclear. Here, we show that disruption of mitochondrial membrane potential leads to axonal NMNAT2 depletion in mouse sympathetic neurons, increasing the substrate-to-product ratio (NMN/NAD) of this NAD-synthesising enzyme, a metabolic fingerprint of Wallerian degeneration. The mechanism appears to involve both impaired NMNAT2 synthesis and reduced axonal transport. Expression of WLD^S and Sarm1 deletion both protect axons after mitochondrial uncoupling. Blocking the pathway also confers neuroprotection and increases the lifespan of flies with Pink1 loss-of-function mutation, which causes severe mitochondrial defects. These data indicate that mitochondrial impairment replicates all the major steps of Wallerian degeneration, placing it upstream of NMNAT2 loss, with the potential to contribute to axon pathology in mitochondrial disorders.

SARM1 is an NAD(P) glycohydrolase and TLR adapter with an essential, prodegenerative role in programmed axon death (Wallerian degeneration). Like other NAD(P)ases, it catalyzes multiple reactions that need to be fully investigated. Here, we compare these multiple activities for recombinant human SARM1, human CD38, and Aplysia californica ADP ribosyl cyclase. SARM1 has the highest transglycosidation (base exchange) activity at neutral pH and with some bases this dominates NAD(P) hydrolysis and cyclization. All SARM1 activities, including base exchange at neutral pH, are activated by an increased NMN:NAD ratio, at physiological levels of both metabolites. SARM1 base exchange occurs also in DRG neurons and is thus a very likely physiological source of calcium-mobilizing agent NaADP. Finally, we identify regulation by free pyridines, NADP, and nicotinic acid riboside (NaR) on SARM1, all of therapeutic interest. Understanding which specific SARM1 function(s) is responsible for axon degeneration is essential for its targeting in disease.

Axons require the axonal NAD-synthesizing enzyme NMNAT2 to survive. Injury or genetically induced depletion of NMNAT2 triggers axonal degeneration or defective axon growth. We have previously proposed that axonal NMNAT2 primarily promotes axon survival by maintaining low levels of its substrate NMN rather than generating NAD; however, this is still debated. NMN deamidase, a bacterial enzyme, shares NMN-consuming activity with NMNAT2, but not NAD-synthesizing activity, and it delays axon degeneration in primary neuronal cultures. Here we show that NMN deamidase can also delay axon degeneration in zebrafish larvae and in transgenic mice. Like overexpressed NMNATs, NMN deamidase reduces NMN accumulation in injured mouse sciatic nerves and preserves some axons for up to three weeks, even when expressed at a low level. Remarkably, NMN deamidase also rescues axonal outgrowth and perinatal lethality in a dose-dependent manner in mice lacking NMNAT2. These data further support a pro-degenerative effect of accumulating NMN in axons in vivo. The NMN deamidase mouse will be an important tool to further probe the mechanisms underlying Wallerian degeneration and its prevention.

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative condition that primarily affects the motor system and shares many features with frontotemporal dementia (FTD). Evidence suggests that ALS is a ‘dying-back’ disease, with peripheral denervation and axonal degeneration occurring before loss of motor neuron cell bodies. Distal to a nerve injury, a similar pattern of axonal degeneration can be seen, which is mediated by an active axon destruction mechanism called Wallerian degeneration. Sterile alpha and TIR motif-containing 1 (Sarm1) is a key gene in the Wallerian pathway and its deletion provides long-term protection against both Wallerian degeneration and Wallerian-like, non-injury induced axonopathy, a retrograde degenerative process that occurs in many neurodegenerative diseases where axonal transport is impaired. Here, we explored whether Sarm1 signalling could be a therapeutic target for ALS by deletingSarm1from a mouse model of ALS-FTD, a TDP-43^Q331K, YFP-H double transgenic mouse.Sarm1deletion attenuated motor axon degeneration and neuromuscular junction denervation. Motor neuron cell bodies were also significantly protected. Deletion ofSarm1also attenuated loss of layer V pyramidal neuronal dendritic spines in the primary motor cortex. Structural MRI identified the entorhinal cortex as the most significantly atrophic region, and histological studies confirmed a greater loss of neurons in the entorhinal cortex than in the motor cortex, suggesting a prominent FTD-like pattern of neurodegeneration in this transgenic mouse model. Despite the reduction in neuronal degeneration,Sarm1deletion did not attenuate age-related behavioural deficits caused by TDP-43^Q331K. However,Sarm1deletion was associated with a significant increase in the viability of male TDP-43^Q331Kmice, suggesting a detrimental role of Wallerian-like pathways in the earliest stages of TDP-43^Q331K-mediated neurodegeneration. Collectively, these results indicate that anti-SARM1 strategies have therapeutic potential in ALS-FTD.