REVIEWS

Programmed axon degeneration: from mouse to mechanism to medicine

Michael P. Coleman 1,2 ✉ and Ahmet Höke 3 ✉ Abstract | Wallerian degeneration is a widespread mechanism of programmed axon degeneration. In the three decades since the discovery of the Wallerian degeneration slow (WldS) mouse, research has generated extensive knowledge of the molecular mechanisms underlying Wallerian degeneration, demonstrated its involvement in non-injury disorders and found multiple ways to block it. Recent developments have included: the detection of NMNAT2 mutations that implicate Wallerian degeneration in rare human diseases; the capacity for lifelong rescue of a lethal condition related to Wallerian degeneration in mice; the discovery of ‘druggable’ enzymes, including SARM1 and MYCBP2 (also known as PHR1), in Wallerian pathways; and the elucidation of protein structures to drive further understanding of the underlying mechanisms and drug development. Additionally, new data have indicated the potential of these advances to alleviate a number of common disorders, including chemotherapy-​induced and diabetic peripheral neuropathies, traumatic brain injury, and amyotrophic lateral sclerosis.

Wallerian degeneration Wallerian degeneration was originally defined as the result from a lack of ‘nourishment’ of the distal axon by slow (WldS) mice degeneration of an axon that takes place distal to an the soma in transected axons1. Alternatively, Lubinska5 A mutant strain of mouse injury, characterized by granular disintegration of the had proposed that one specific, essential substance, showing a tenfold delay cytoskeleton, mitochondrial swelling and axon frag- delivered by axonal transport, inhibits Wallerian degen- in the onset of Wallerian 1 degeneration after axotomy mentation ; however, the mechanism is now known eration. Lubinska had no means of identifying this sub- as well as axon protection to also occur in many non-​injury disorders (Table 1, stance; the existence of a mutant mouse has now made in many disease models. Fig. 1). Wallerian degeneration has gained impor- this possible. tance in the fields of neurodegenerative and axonal Structure–function analysis of the protective WldS Axonal transport disorders because of its widespread occurrence, its gene product, a unique fusion of nicotinamide mono- The ATP-dependent,​ bidirectional trafficking of well-​characterized and ‘druggable’ mechanism and nucleotide adenylyltransferase 1 (NMNAT1; an enzyme axonal proteins, organelles, the ability to alleviate — and sometimes completely involved in the synthesis of the redox and signalling mRNAs and other cargoes prevent — axon loss by blocking it2. Our deep mech- cofactor nicotinamide adenine dinucleotide (NAD)), delivering axonal constituents anistic understanding of Wallerian degeneration stems with part of the ubiquitin ligase UBE4B6, suggested a key to where they are required, 7 often over large distances. largely from axon injury studies, but the regulatory role for axonal NAD metabolism in axon maintenance . genes that have been identified in these studies are now Indeed, most subsequent studies have suggested that known to also influence axon survival in many other the NMNAT enzyme activity of the WldS gene product, circumstances3. Recent advances (highlighted below) localized to axons by the fused protein, is required for 1 John van Geest Centre for include the identification of a genetic association the axon protection phenotype8–10. NMNAT2, a related Brain Repair, University of Cambridge, Cambridge, between Wallerian degeneration and human disease, protein that is normally found in the axon and for which United Kingdom. new rescue effects in animal models, and the discovery WLDS can substitute, was subsequently found to be 11–13 2Signalling Programme, of new drug targets with unexpected enzyme activities essential for axon growth and survival and remains The Babraham Institute, associated with Wallerian degeneration and progress the best fit for Lubinska’s natural ‘inhibitor’ of Wallerian Cambridge, United Kingdom. towards the elucidation of their protein structures. degeneration. A key property of NMNAT2 is its short 3Neuromuscular Division, The discovery, three decades ago, of a mouse strain half-​life12,14, which necessitates constant resupply by Johns Hopkins School of with an unusual phenotype began the research that new protein synthesis and axonal transport. When Medicine, Baltimore, MD, USA. has led us to today’s detailed molecular understand- NMNAT2 is lost from axons, whether owing to injury, (Fig. 2) Wallerian ✉e-mail:​ [email protected]; ing of Wallerian degeneration . In these mutation, silencing, axonal transport disruption, or S [email protected] degeneration slow (Wld ) mice, transected axons sur- other causes, the resulting degeneration can be blocked 4 S https://doi.org/10.1038/ vive tenfold longer than normal . Until this finding, by WLD , indicating that it involves a Wallerian-​like s41583-020-0269-3 Wallerian degeneration was generally considered to mechanism3 (Fig. 1).

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Table 1 | Human diseases associated with the Wallerian degeneration pathway Human disease Findings Comment Refs Alzheimer disease NMNAT2 protein and mRNA are reduced in This finding suggests that reduced 95 brains of individuals with Alzheimer disease NMNAT2 is associated with, and may be a risk factor for, the development of Alzheimer disease Erythromelalgia A homozygous NMNAT2 partial loss-​of- Loss-of-function​ mutation in NMNAT2 89 and peripheral function mutation in two individuals with results in development of peripheral neuropathy erythromelalgia and peripheral neuropathy neuropathy Fetal akinesia Compound heterozygous NMNAT2 severe Severe loss-of-function​ mutation in 90 deformation loss-​of-function mutations in two fetuses NMNAT2 results in stillbirth, mimicking syndrome with fetal akinesia deformation syndrome observations in NMNAT2 null mice ALS GWAS show association of the SARM1 locus A pathogenic role of SARM1 in ALS must 101,102 with ALS involve gain-of-function​ variants that increase the risk of axonal degeneration ALS An ALS-causing​ mutation in TARDBP reduces Low levels of stathmin 2 increase the 96,97 levels of stathmin 2 in iPSC-derived​ motor likelihood of Wallerian degeneration and neurons; a similar decrease is common in could therefore be an important modifier sporadic ALS or risk factor for ALS ALS, amyotrophic lateral sclerosis; GWAS, genome-wide​ association studies; iPSC, induced pluripotent stem cell; NMNAT2, nicotinamide mononucleotide adenylyltransferase 2; SARM1, sterile-α​ and Toll/interleukin 1 receptor (TIR) motif containing protein 1; TDP43, transactive response DNA binding protein 43 kDa.

The discovery of the mouse WldS gene also led to degeneration18. It has an ‘execution’ role in Wallerian degen­ the finding that the pathway is conserved in Drosophila eration, analogous to the roles of caspases as executors of melanogaster15,16 (Fig. 2). Powerful D. melanogaster genetic apoptosis, although the molecular mechanism is distinct. studies subsequently revealed a set of additional positive This function was discovered in D. melanogaster, rep- and negative regulators of Wallerian degener­ation17–19 licated using Sarm1–/– mice18 and later confirmed in a that are largely conserved in mammals and have recently separate mammalian RNA interference study24. However, provided a strong basis for drug development efforts the mechanism by which it kills axons was not immedi- (Fig. 3). The key roles of D. melanogaster in elucidating ately apparent. In 2015, experiments employing forced this pathway, and the similarities and differences between dimerization of the SARM1 TIR signalling domain the pathway in mammals and flies, have recently been demonstrated an unknown NADase activity that gen- reviewed elsewhere20; therefore, they are not the main erates ADP-​ribose (ADPR) and cyclic ADPR (cADPR) focus of this article. from NAD25. It was subsequently shown that murine This Review describes the major recent advances in Sarm1 is required for extensive NAD loss after injury25 our understanding of the Wallerian degeneration mech- and that a mutated human SARM1 that is unable to anism and distinguishes areas of agreement and ongo- degrade NAD cannot, unlike wild-type​ human SARM1, ing debate. It reviews what we have learned from animal support fast Wallerian degeneration when introduced models about the roles of Wallerian degeneration in dis- to Sarm1–/– mouse neurons23. Isotopic labelling of NAD ease and explains why complementary human studies are in mouse dorsal root ganglion (DRG) neuron cultures vital. Finally, it discusses which proteins in the pathway showed that there is both NAD syn­thesis failure at around might be targeted for therapeutic intervention and con- 2 h after axon transection (likely owing to NMNAT2 siders outstanding questions that remain to be addressed loss) and a SARM1-dependent, fourfold acceleration in order to move the field towards clinical application. of NAD degradation26. The intrinsic NADase activity of SARM1’s own TIR domain was then demonstrated by Wallerian mechanisms: recent advances experiments with cell-​free transcribed and translated Mechanisms driving NAD loss. NAD levels are known TIR protein23. to decline in injured axons and nerves21,22. Part of the SARM1 is known to act downstream of NMNAT2 explanation for this decrease is the rapid loss of its normal loss in the Wallerian degeneration pathway as its synthetic axonal enzyme, NMNAT2, which has a short removal in mice protects NMNAT2 null axons without half-life​ and cannot be replenished by axon transport from fully preserving NAD levels27. SARM1 activation exac- the soma after injury. Indeed, the presence of a more stable erbates the effects of slowing NAD generation caused by axonally targeted NMNAT, such as WLDS, stabilizes NAD NMNAT2 loss through an increase in NAD degradation, 22 (Fig. 3) Toll-like​ receptor levels after injury . However, an important recent discov- causing a precipitous decline in NAD levels . The A family of receptors on ery was the identification of the NAD-degrading​ activity discovery of this enzyme activity has also changed plasma and endosomal of another axonal protein, sterile-​α and Toll/interleukin 1 the perception of the suitability of SARM1 as a drug membranes that detect receptor (TIR) motif containing protein 1 (SARM1), as target (discussed below). molecular patterns associated well as its activation as part of the molecular pathway that with infection or cell damage, 23 (Fig. 3) activating signalling pathways induces Wallerian degeneration . The contribution of NMN to Wallerian degeneration. leading to inflammation or SARM1 is a Toll-​like receptor adapter protein that Given the well-known​ roles of NAD in ATP synthesis and cell death. is required for the normal, rapid rate of Wallerian many other cellular activities28, its loss is an obvious

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and attractive candidate mechanism to explain the nicotinamide phosphoribosyl transferase (NAMPT), the axon degeneration that occurs after injury. However, it synthetic enzyme that generates NMN, such as FK866, remains unclear whether the loss of NAD offers a full lower NAD by up to 90%; however, rather than killing explanation. Manipulating NAD levels through methods axons as expected, they partially phenocopy the effects that do not involve altering NMNATs (or their substrates of WLDS, protecting injured axons22,29–31. To explain and precursors) has been repeatedly found not to alter this paradox, a harmful role for the NAD precursor Wallerian degeneration22,29,30. Moreover, inhibitors of nicotinamide mononucleotide (NMN) was proposed22.

a Wallerian degeneration

Axotomy blocks delivery of all soma-derived cargoes including NMNAT2

Local loss of NMNAT2 activates SARM1-dependent Wallerian degeneration b Axonal transport impairment

Axonal transport impairment reduces deliverryy of all soma-derived cargoes, including NMNAAT2. Distal regions are most affected

Local loss of NMNAT2 Nmnat2 null mutation triggers SARM1- c dependent ‘dying back’

Nmnat2 mutation prevents its gene product reaching axons

Without NMNAT2, Wallerian degeneration is constitutively activated and axons fail to grow

Fig. 1 | Activation of Wallerian degeneration in injury and disease. The figure shows three ways in which the Wallerian degeneration mechanism can be activated. a | In classical Wallerian degeneration paradigms, axon injury prevents the delivery of nicotinamide mononucleotide adenylyltransferase 2 (NMNAT2) from the soma along with all other cargoes12. Because of its short half-​life, NMNAT2 is quickly lost in the distal stump, which triggers sterile-α​ and Toll/interleukin 1 receptor (TIR) motif containing protein 1 (SARM1)-dependent degeneration27. b | Axonal transport impairment (for example, due to a toxin, a protein defect or ageing) limits the supply of all cargoes to distal axons. Delivery of NMNAT2 to distal ends of the axon is particularly affected because of its short half-life.​ When NMNAT2 falls below the threshold for survival, the distal terminal dies back. c | Mutation of the Nmnat2 gene leads to loss or dysfunction of the protein13,89,90. Without it, the axon cannot grow, causing a developmental disorder (unless rescued through a Sarm1 deletion, for example). If the mutation causes only a partial loss of function, the axon grows but is more susceptible to degeneration.

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Mechanism studies Waller transects nerves 1850 Cajal proposes ‘neuron Disease studies and concludes the soma doctrine’ to explain ‘nourishes’ the axon... Wallerian degeneration Rat ...proposes that Wallerian 1906 observations degeneration is likely to Mouse be important in disease 1948 Weiss and Hiscoe propose axonal Fly transport to explain human nerve injury observations Human First electron 1958 microscopy studies of Other Wallerian degeneration 1962

1964 Calcium is shown to be required for Wallerian degeneration 1973 Cavanagh proposes ‘chemical transection’ model for toxic 1977 ‘dying back’ neurons Lubinska proposes that a soma-derived ‘trophic substance’ inhibits 1979 Wallerian degeneration 1982

S 1989 Wld mouse discovered WldS gene mapped 1993 Soma and axon death 1994 are shown to be distinctly regulated S Wld mutation identified 1998 Vincristine-induced WldS gene identified 2001 axon degeneration blocked by WldS in vitro Axon loss in pmn and 2003 Mpz–/– mice blocked by WldS in vivo Wallerian degeneration 2004 shown to be a sudden event WldS does not rescue after a long latent phase transgenic SOD1 ALS mice 2005

WldS delays Wallerian 2006 degeneration in flies WldS structure–function studies 2007 show that axonal NMNAT activity WldS alleviates glaucoma determines axon survival in animal models 2008 DLK inhibition or deletion Loss of NMNAT2, a labile, 2009 rapidly transported axonal shown to modestly delay protein, shown to trigger Wallerian degeneration Wallerian degeneration 2010

2011 SARM1 shown to Drosophila proteins be required for Highwire and MYCBP2 2012 Wallerian shown to be required for degeneration WldS delays Wallerian degeneration Nmnat2 null mice found to Wallerian 2013 die at birth with axon growth degeneration in failure; rescued by WldS human axons GWAS links SARM1 shown to 2014 SARM1 locus to act downstream sporadic ALS of NMNAT2 loss 2015 SARM1 shown to have intrinsic NADase activity NMN accumulation shown to that can be activated by TIR dimerization promote Wallerian degeneration 2016 Rescue of CIPN and HFD in vivo models 2017 Rescue of Nmnat2 null mice by by Sarm1 deletion –/– Sarm1 is shown to be lifelong NMNAT2 mutations Drosophila Axundead is SARM1 domain 2019 identified in human shown to be required for structures neurological disorders Wallerian degeneration determined NMN shown to activate SARM1

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◀ Fig. 2 | Wallerian degeneration timeline. Prior to discovery of the Wallerian of degeneration or as a negative regulator of a power- S degeneration slow (Wld ) mouse, there was wide acceptance that axonal transport ful compensatory mechanism. Axed is a BTB/BACK-​ was required for axon survival and that axon injury could serve as a model for containing protein with no obvious role in calcium or disease, albeit with no molecular understanding of the process5. The identification NAD metabolism, although its ability to protect either of the axon-protective gene encoded by WldS made it possible to address this question and led to the identification of nicotinamide mononucleotide adenylyl- in the presence of activated dSarm or in the absence 19 transferase 2 (NMNAT2) as the best match for the previously proposed ‘Wallerian of dNmnat (the only known NAD synthetic enzyme degeneration inhibitor’12. Conservation of the Wallerian mechanism in Drosophila in D. melanogaster) strongly suggests that it influences melanogaster has enabled the discovery of other major players in the pathway, NAD in some way. Elucidation of its function, as well as including sterile-α​ and TIR motif containing protein 1 (SARM1)18. More recently, the identification and characterization of any mamma- human genetic studies have started to identify the clinical disorders involving this lian orthologue will be crucial steps in understanding preventable degeneration pathway, with promising leads in the quest to develop and preventing Wallerian degeneration. drugs to block it89,90. ALS, amyotrophic lateral sclerosis; CIPN, chemotherapy-​ induced peripheral neuropathy; DLK, dual leucine zipper kinase; GWAS, genome-​ Regulating NMNAT2 stability. Understanding the wide association studies; HFD, high-​fat diet; NMN, nicotinamide mononucleotide; regulation of NMNAT2 stability is another rapidly SOD1, superoxide dismutase 1; TIR, Toll/interleukin 1 receptor. developing area. NMNAT2 had a half-​life of less than 40 min in human embryonic kidney cell experiments14 NMN accumulates in the axon distal to the injury, at and was greatly depleted in mouse neurites 4 h after least transiently, during Wallerian degeneration22,26 transection12. How such a labile protein can reach dis- and both WLDS and FK866 prevent this accumula- tal axons before being degraded, a journey that takes tion — WLDS maintains NMN removal from axons by 2 days in the longest human axons, remains unclear37. metabolizing it to produce NAD, whereas FK866 stops The existence of two distinct NMNAT2 pools — NMN synthesis22 (Fig. 4). Supporting the idea of NMN vesicular and non-​vesicular — is probably part of the toxicity, ectopically expressed bacterial NMN deami- explanation14. It has been shown that, in mice, the dis- dase, which removes NMN without altering NAD (at tribution between vesicular and non-vesicular​ Nmnat2 least after axon injury), strongly protects cut axons in is regulated in part by palmitoylation14,38. However, stud- mice22,26,32 and exogenous NMN or ectopically expressed ies that hypothesized that the moving, vesicular form NMN synthase both restore degeneration to cut murine of the enzyme may be more stable than non-​vesicular neurites that have been protected by FK866 (refs30,33,34). NMNAT2 found that the opposite was true: disrupting However, in conditions in which NAD levels are also vesicle targeting lengthened NMNAT2’s half-​life and high, NMN does not activate axon degeneration26. greatly strengthened its capacity to protect axons14,39. Since neither NAD loss nor NMN accumulation by Although candidate palmitoylation and depalmitoy- themselves explain all available data, it can be hypoth­ lation enzymes were identified40, these are probably esized that both may be part of the mechanism. For not good drug targets as they have many substrates. example, the ratio between NAD and NMN levels may It has also been shown that mitogen-​activated protein be important. Alternatively, the recent finding that kinase (MAPK) signalling promotes NMNAT2 turn- NMN, or a synthetic membrane-​permeable analogue over as depletion of MKK4 (also known as MAP2K4) (CZ-48), can activate SARM1 suggests that NMN and MKK7 or blockage of their upstream regulators could drive further NAD loss35 (Fig. 3). However, such dual leucine zipper kinase (DLK) and leucine zipper-​ NMN-​dependent SARM1 activation has not yet been bearing kinase (LZK) extends the half-​life of the palmi- demonstrated in axons. toylated form of NMNAT2 and increases the survival of transected murine neurites38,41. MAPK signalling is Downstream executors of degeneration. Even once also influenced by SARM1, but whether this influences SARM1 is activated, it is still unclear whether the cause axon survival remains unclear38,42. of the axon degeneration is NAD loss, an increase in Turnover of the soluble, more stable NMNAT2 the level of a SARM1 enzyme product or the increased pool14 is influenced by the MYC-​binding protein 2 metabolism of an alternative substrate35. If it is NAD (MYCBP2)–S-phase​ kinase-associated​ protein 1 (SKP1)– loss, it is clear that levels must become very low to have F-​box protein 45 (FBXO45) ubiquitin ligase complex38, an effect: NAD declines by 50% in transected sciatic each member of which has been separately implicated in nerves in Sarm1–/– mice within 5 days of injury27 and by Wallerian degeneration17,43–46. Ubiquitylation determines 80% in severed DRG neurites in Sarm1–/– mice within NMNAT2 turnover rate14, but the newly discovered ser- 36 h of injury36; however, in both cases, the neurons ine/threonine monoubiquitylation activity of MYCBP2 survive for a considerably longer period. Changes (ref.47) raises important questions about the role of in calcium mobilization and/or influx provide one alter- classical lysine polyubiquitylation45 in this process. Like native mechanism. The SARM1 products ADPR and SARM1 and MAPK signalling, this is another promising Calcium mobilization 28 The release of calcium from cADPR are powerful calcium mobilizers and, in mice, area for drug development, especially as the structural 47 intracellular stores such as NMN activates a Sarm1-dependent rise in intra-​ basis of serine/threonine ubiquitylation is known . endoplasmic reticulum and axonal calcium that immediately precedes the demise mitochondria, potentially of the axon34. Interestingly, in D. melanogaster, the Next steps. The recent rapid progress in our under- activating calcium-activated​ protein Axundead (Axed), which (like SARM1 and its standing of Wallerian degeneration mechanisms has proteases, under the control of second messenger molecules D. melanogaster orthologue dSarm) is required for rapid raised many new questions. Prominent among them (many of which are NAD Wallerian degeneration, appears to act downstream of is ‘how is SARM1 activated?’ The recently discovered metabolites). dSarm19. Axed is likely to act either as an executioner activation of SARM1 by NMN appears to provide one

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NMNAT2 turnover Disrupted delivery of NMNAT2 NMNAT2 turnover this is altered by some viruses and Toll-​like receptor via ubiquitylation to axons resulting from axon promotion via ligands in neuronal and/or non-​neuronal cells51,52. injury, axon transport failure, MAPK signalling MYCBP2 A full understanding of SARM1 regulation also requires a NMNAT2 mutation or NMNAT2 LZK FBXO45 SKP1A inhibition DLK better understanding of the roles of post-translational​ modifications, such as the reported JNK-dependent phosphorylation53 and of mitochondrial targeting, NMNAT2 loss reported by some to be important for the prodegener- ative function of SARM1 (refs53,54) (but not by others24). The normal subcellular location of SARM1 also remains ↑ NMN to be resolved. While overexpressed and fluorescent Other signal? protein-tagged​ SARM1 colocalizes with mitochondria under basal conditions55,56, the endogenous protein SARM1 activation does not colocalize with mitochondria18, or at least not completely24,57. Other questions arise about the evolution of SARM1 and its relationship to NAD hydrolysis by other,

↑cADPR ↓NAD (and other 49,58,59 ↓NAD evolutionary-​divergent TIR domains . The wider ↑ ADPR substrates?) involvement of this newly discovered enzyme activity in innate immunity suggests that there may be an ancient Calcium mobilization/influx mechanism of pathogen defence involving NAD mod- 49,59 Axed ulation . It could be hypothesized that axon injury may have co-opted​ this mechanism later in evolution to Calpain activation remove damaged axons. However, the ultimate effector of this mechanism remains unknown. Axon degeneration Questions also remain about other regulators of NMNAT2. Could local translation of NMNAT2 (ref.60) | Fig. 3 A working model of the Wallerian degeneration pathway. The figure shows as well as the existence of different NMNAT2 pools14,38 current knowledge of the factors that determine nicotinamide mononucleotide adenylyl- help explain how this unstable protein reaches distal transferase 2 (NMNAT2) activity in axons, how NMNAT2 loss activates sterile-α​ and Toll/interleukin 1 receptor (TIR) motif containing protein 1 (SARM1) and events downstream axons without being degraded? It is not known precisely of SARM1 leading to axon degeneration. Various factors disrupt the delivery of functional how MYCBP2, DLK and LZK influence the steady state NMNAT2 to axons. NMNAT2 turnover is mediated by the mitogen-activated​ protein level of each NMNAT2 pool; specifically, it is unclear kinases (MAPK) dual leucine zipper kinase (DLK) and leucine zipper-bearing​ kinase (LZK), which NMNAT2 residues they may phosphorylate or as well as a ubiquitin ligase complex consisting of MYC-binding​ protein 2 (MYCBP2), ubiquitylate and whether they are responsible for its s-​phase kinase-associated​ protein 1A (SKP1A) and F-box​ protein 45 (FBXO45)38. polyubiquitylation or monoubiquitylation. Stabilizing, In combination, this reduces NMNAT2 levels and causes downstream activation of boosting the synthesis of 61 and activating NMNAT2 SARM1, probably as a result of increased levels of nicotinamide mononucleotide (NMN) have all been proposed as important therapeutic strat- 35 but potentially also in other ways . The combined effect of decreased NMNAT2 and egies for axonopathies, making these key questions for increased activation of SARM1 leads to a great decrease in axonal nicotinamide adenine dinucleotide (NAD), which may itself cause axon degeneration through ATP synthesis advancement in this area. Finally, identifying proteins failure25. Alternatively, other SARM1 substrates or its calcium mobilizing products could that control NMNAT2 expression level, enzyme activ- be important for the later stages of Wallerian degeneration. In Drosophila melanogaster, ity and axonal transport is also important. Given the there is also an as-​yet undefined role for Axundead (Axed) in Wallerian axon absolute requirement for NMNAT2 for axon survival degeneration19. ADPR, ADP-ribose;​ cADPR, cyclic ADP-ribose.​ and the enhanced level of protection provided by the soluble form of this enzyme39, such modulators are likely to influence axon survival; however, very few of these possible answer to this question35 and, if confirmed in factors have yet been identified62,63. axons, could explain why NMN promotes Wallerian degeneration22,32,34 (Fig. 4); however, whether NMN binds Linking degeneration to disease directly to SARM1 (and if so where), whether other Animal models of disease. The notion that Wallerian proteins are involved and whether NAD can block this degeneration mechanisms are important in disease dates activation (and if so, what level of NAD is required to back to the work of Waller1 (Fig. 2) but could only be do so)26 remain unclear. Emerging information about tested after mutant WldS mice were discovered. Axon the structure of SARM1 (refs48,49) will be important in degeneration in many animal models of disease can answering these questions. be alleviated by the presence of WLDS (as previously Both in axons and in non-​neuronal cells there are summarized3), which has prompted a number of studies indications of other SARM1 activation mechanisms that investigating the effects of manipulating the Wallerian need to be brought together for a full under­standing degeneration pathway in other ways in animal models of how this protein is regulated. On the proximal side of of disease. an axon injury, SARM1 is required for proin­flammatory Several studies have attempted to prevent distal axonal Local translation responses50; however, in this location, there is no degeneration in mouse models of peripheral neuropa- The synthesis of proteins 12 directly within axons using NMNAT2 loss nor a rise in NMN, so there must be thy by Sarm1 deletion. Peripheral neuropathy caused by mRNAs delivered by axonal another activation mechanism at work. SARM1 expres- the commonly used chemotherapy drugs vincristine or transport. sion level appears to be another point of regulation as bortezomib was prevented in Sarm1–/– mice64,65. Similarly,

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–/– peripheral neuropathy was prevented in Sarm1 mice CZ-48 given either paclitaxel, another common chemotherapy drug that causes neuropathy, or a high-​fat diet (HFD) NMN deamidase (a model of metabolic syndrome that results in neurop- FK866 athy)66. In each study, the primary end point was the NAMN loss of intraepidermal nerve fibres, the distal endings NMNAT1, NMNAT2, of unmyelinated sensory axons that tend to degenerate NAMPT NMNAT3 or WLDS first in most axonal peripheral neuropathies. Thus, it Nm NMN NAD is possible that the distal axonal de­generation seen in these toxic and metabolic syndromes involves molecu- Activation lar mechanisms that are shared with those of Wallerian degeneration. SARM1 What about the other types of axonal injury seen in human diseases? For example, traumatic brain cADPR ADPR injury (TBI) causes stretching and shearing of axons. One would expect that axons in WldS and Sarm1–/– mice would be protected from axon degeneration following Fig. 4 | Activation of SARM1 by NMN. The figure TBI, as the mechanism may be similar to traumatic summarizes evidence supporting activation of sterile-α​ and axotomy. Indeed, in two separate studies, Sarm1–/– mice Toll/interleukin 1 receptor (TIR) motif containing protein 1 showed decreased axonal damage in TBI models67,68, (SARM1) by nicotinamide mononucleotide (NMN). NMN accumulates after axon injury22 because the enzyme that with functional preservation also observed in the milder 67 normally removes it, nicotinamide mononucleotide adenyl- model . Furthermore, blocking NAMPT with FK866 yltransferase 2 (NMNAT2), is quickly degraded12. If left –/– partially phenocopied Sarm1 , mirroring findings in unchecked, this activates SARM1, leading to axon peripheral nerves68. Obviously, traumatic injuries pose a degeneration27. However, NMN accumulation can be therapeutic challenge as any clinical intervention has to prevented in three ways (dark blue): through inhibition happen after injury. It is unclear how effective a delayed of its synthetic enzyme, nicotinamide phosphoribosyl blockade of SARM1 will be in these models; neverthe- transferase (NAMPT)22,29–31; by overexpression, or increased less, interventions such as the application of FK866, axonal targeting, of any isoform of NMNAT3,10, its normal cleavage of SARM1, or transduction of NMNAT1 pro- processing enzyme in mammals; or by ectopic expression 22,32 tein have been found to be effective several hours after of bacterial NMN deamidase . Conversely, its membrane-​ permeable analogue CZ-48 activates SARM1 directly35. injury in animal models22,25,69. Combined with the obser- S Thus, NMN, or a close analogue, appears to be necessary vation that delayed overexpression of WLD in axons up and sufficient for SARM1 activation. ADPR, ADP-ribose;​ to 4–5 h after axotomy can prevent Wallerian degenera- cADPR, cyclic ADP-ribose;​ NAD, nicotinamide adenine tion70, these results suggest that intervention early after dinucleotide; NAMN, nicotinic acid mononucleotide; TBI could be feasible. Nm, nicotinamide; WLDS, Wallerian degeneration slow. Secondary or primary progressive multiple sclerosis, in which gradual axonal degeneration is important in the progressive loss of neurological function, may models is much higher than equivalent doses feasible be another potential therapeutic target for Wallerian in humans: the maximum tested dose of nicotinamide in degeneration-​blocking drugs. Axonal injury has been humans is 1,000 mg twice a day, which showed no effi- shown to be present in several rodent models of multiple cacy against several metabolomic parameters in obese sclerosis, with studies showing that behavioural deficits men despite raising blood NAD levels76. Perhaps com- and axonal loss are attenuated in an experimental auto- bining NR or nicotinic acid riboside with the manipu- immune encephalitis (EAE) model of multiple sclerosis lation of enzymes in the NAD synthesis pathway (using in WldS mice71,72. No studies have been published to date NAMPT inhibitors, for example) may be more effective, investigating the effects of EAE in Sarm1–/– mice. as observed in a recent vincristine-​induced neuropathy Manipulation of the Wallerian degeneration pathway model in murine primary cultures30 (Fig. 4). Similarly, through treatment with nicotinamide, a precursor of in models of increased intraocular pressure glaucoma, NAD, has been shown to be effective in a genetic mouse both axons77,78 and NAD levels73 are preserved in WldS model of glaucoma in which there is an age-​associated mice and, when supplemented with additional nicotina- decline in retinal NAD levels and axonal degeneration mide, axon protection is further increased. A similar of retinal ganglion cells73,74. Furthermore, the same treatment also further abrogated the disease phenotype approach has been used successfully in murine models and axonal loss in EAE in mice71. of diabetic peripheral neuropathy75. Dietary supple- The molecular mechanism by which Sarm1 deletion mentation of nicotinamide riboside (NR) prevented prevents distal axonal degeneration is likely to be neuro­pathy both in a mouse model of HFD-​induced cell-​autonomous within axons, but the role of non-​ metabolic syndrome and in mice that received both neuronal cells in peripheral neuropathies needs to be HFD and low-dose​ streptozotocin to model type 2 dia- considered. Examples of sites of non-neuronal​ actions of betes75. However, in these studies, it is unclear whether SARM1 include macrophages79 and in D. melanogaster, NR truly acts by blocking the Wallerian degeneration glia80. Overexpressed SARM1 was found to be pres- pathway as no mechanistic studies were carried out. ent in both the cytosol and the mitochondrial matrix Furthermore, the dose required for effect in these of human embryonic kidney cells and endogenous

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SARM1 was associated with the matrix proapoptotic is likely to be particularly effective. One is in disor- Nod-​like receptor, NLRX1 (ref.57). Although apoptosis ders caused or exacerbated by specific disruption of in non-​neuronal cells was dependent on both SARM1 NMNAT2, for example, those driven by its genetic and NLRX1, axonal degeneration in murine primary mutation89,90 or inhibition91. Another is in disorders in neurons induced by vinblastine was dependent only on which there is an ongoing modest impairment of the SARM1, indicating perhaps a different role for SARM1 delivery of many cargoes to distal axons but in which in non-​neuronal cells. only NMNAT2 falls below the threshold for survival; Despite these promising results, the findings of this could include age-​related disorders of long or animal studies suggest that manipulating SARM1 or highly branched axons, where the age-​related decline other components of the Wallerian degeneration path- in axonal transport62 and disease-​specific mechanisms way may not prevent all axonal degeneration. Although together impair the delivery of cargoes to axon termi- one of the earliest events in a mouse model of amyo- nals. A third scenario is in disorders in which there is trophic lateral sclerosis (ALS) driven by mutations in a temporary impairment of transport or synthesis that superoxide dismutase (SOD1) is distal axonal degen- is resolved before the loss of other more stable pro- eration and denervation at the neuromuscular junc- teins becomes limiting. For example, the treatment of tions81, when the mutant Sod1 mice were crossed to DRG primary cultures with vincristine for 24 h, fol- WldS mice, there was minimal impact on disease onset lowed by washout, causes the degeneration of wild-​ or survival82. Similarly, when Sod1G93A mutant mice were type neurites from which WLDS provides permanent bred with Sarm1–/– mice, there was no rescue83. Thus, protection92. A similar outcome was seen when protein neuro­degeneration in this particular model of ALS is synthesis was transiently blocked and then restored in unlikely to depend on the molecular players involved superior cervical ganglion neurons12. The treatment in Wallerian degeneration82, although there is evidence of chemotherapy-​induced peripheral neuropathy that Wallerian degeneration influences axon survival in (CIPN) is an obvious application of this knowledge, as a TDP-43 model of ALS84. chemotherapy is temporary, the timing predictable, and The role that SARM1 plays in axon degeneration rescue has been very widely replicated and extended to versus neuronal cell death has also been shown to vary in vivo studies9,42,64–66,93. significantly across cell populations and disease models. There are many other ways in which axons may be For example, in an optic nerve crush-​induced retinal depleted of functional NMNAT2 in disease, which we ganglion cell (RGC) death model of glaucoma, Sarm1–/– suggest may explain why blocking Wallerian degen- mice showed preservation of distal optic nerve axons but eration alleviates symptoms in some disease mod- no prevention of DLK–JNK pathway activation in the els that are not obviously caused by axonal transport somanor of RGC death85. Similarly, in a prion disease impairment. Axonal NMNAT2 activity is likely to be model, Sarm1–/– mice had an exacerbated neuronal death lowered if there is impaired synthesis at the transcrip- upon infection, independent of any role of SARM1 in tional, post-​transcriptional or translational level, or by immune cells86. This observation mirrors the findings reduced protein stability, enzyme inhibition or post-​ of the initial study that described the development of translational modifications that influence any of the a Sarm1–/– mouse line, in which infection with West above. NMNAT2 transcription is regulated in part by Nile virus led to increased neuronal death in Sarm1–/– cAMP response element-​binding protein (CREB)63 and mice due to impairment of microglial activation87. Ras-​responsive element-​binding protein 1 (RREB1; Finally, the expression of mutant proteins that cause the mammalian orthologue of D. melanogaster zinc ALS in Caenorhabditis elegans motor neurons, induces transcription factor Pebbled) is another candidate reg- an innate immune response that propagates neuronal ulator of NMNAT2 transcription given its influence death88; this process requires the SARM1 orthologue. on Wallerian degeneration94. There is huge variation Therefore, these studies point to a potentially complex between individuals in NMNAT2 mRNA expression in role for SARM1 in neuronal and non-neuronal​ cells and human post mortem brains, suggesting that there are their interactions in disease states. multiple transcriptional and epigenetic regulators of this protein95. Acute protein synthesis blockade causes Mechanistic basis of disease-​modifying effects. As out- axon degeneration through the Wallerian pathway12, lined above, axotomy studies have revealed Wallerian suggesting that disorders of protein synthesis may acti- Stathmin 2 degeneration to be a pathway that can be activated in vate it. Normal ageing lowers NMNAT2 axonal trans- A protein that, like NMNAT2, is palmitoylated, has a short multiple ways, the most specific of which is the removal of port by 40–50%, potentially contri­buting to axon loss in 62 half-life​ and negatively just one protein, NMNAT2. Under these circumstances, age-​related diseases . Depletion of stathmin 2 (SCG10), regulates Wallerian SARM1 is absolutely required for degeneration as axons a protein that co-​migrates with NMNAT2 in axons and degeneration. Its loss is are rescued permanently in Nmnat2::Sarm1 double null shares several properties with it14, potentiates Wallerian insufficient to activate 2 96,97 Wallerian degeneration but mutant mice . Less specific lesions, such as axotomy or degeneration and is frequently seen in sporadic ALS , accelerates it after injury and general axonal transport impairment, prevent the deliv- raising the prospect of a role in that disease. Finally, its overexpression delays ery of all cargoes from the soma. Eventually, the levels of environmental inhibitors of NMNAT2 enzyme activity91 degeneration. The same other proteins must fall below the threshold for survival, are likely to activate a Wallerian mechanism and have protein is depleted in many which we believe may explain why rescue through Sarm1 been associated with peripheral neuropathy98. These induced pluripotent stem cell-​ derived motor neurons from knockout is only temporary in these models. many influences on axonal NMNAT2 help to explain sporadic amyotrophic lateral With this in mind, there are several scenarios in how diverse primary causes could activate a common sclerosis patients. which blocking Wallerian degeneration in the clinic Wallerian degeneration response99.

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Wallerian degeneration in human disease. Identifying high doses of chemotherapy carry more loss-of-function​ the pathogenic or disease-​modifying effects of mutations in SARM1. Wallerian-​regulating proteins directly in human A genome-​wide association study (GWAS) showed disorders promises to be a powerful indicator of where linkage to the SARM1 locus in sporadic ALS101, an obser- best to apply Wallerian-blocking​ drugs (Table 1). Animal vation that was later replicated in a separate GWAS studies are extremely useful but also have unavoidable study102. Although the authors in the second GWAS sug- caveats, making it essential to complement them with gested that a cis-​expression quantitative trait loci clinical data. For example, EAE is a very limited model of (eQTL) effect on Polymerase delta-interacting​ protein 2 human MS and extrapolating directly from effectiveness (POLDIP2) might be the driver of linkage at this locus, in this model to the human disease is risky. Conversely, a role for SARM1 has not been ruled out. For exam- the failure of WLDS, or SARM1 ablation, to rescue SOD1 ple, SARM1 expression might respond in a genetically ALS mouse models by no means rules out the potential determined way to environmental ALS risk factors for therapy in other inherited forms of human ALS or in that are not represented in the expression quantitative the much more prevalent sporadic forms. trait loci data. Two recent studies provide a further link Although no major neurological disease has so far between Wallerian degeneration and ALS. ALS-causing​ been associated with mutations in genes involved in mutations in TDP43 reduce levels of the JNK substrate Wallerian degeneration, levels of NMNAT2 mRNA SCG10 in patient fibroblast-derived​ motor neurons and and protein are reduced in brains of individuals with this is associated with reduced axonal growth96,97. This Alzheimer disease95. Whether this is a cause or con- phenotype could be rescued by overexpression of SCG10 sequence of axonal and neuronal degeneration in in human induced pluripotent stem cell-​derived motor Alzheimer disease remains unknown. Nevertheless, neurons. Previously, it had been shown that axotomy in two recent studies have identified potentially patho- mouse DRG neurons leads to a rapid reduction in SCG10 genic mutations in NMNAT2 in two rare neurological levels and that axonal degeneration could be delayed by conditions89,90. The first of these describes two sisters its overexpression, indicating that SCG10 is a moder- who developed childhood onset peripheral neuropathy ately strong modifier of Wallerian degeneration103. These and erythromelalgia and were found to be homozygous observations do not imply that SCG10 depletion by itself for a T94M mutation in the NMNAT2 gene, likely indi- is sufficient to cause axonal degeneration but that, when cating an autosomal recessive pattern of inheritance89. SCG10 is lowered as part of another pathogenic mecha- The T94M mutation confers partial loss of function for nism, Wallerian-​like axon degeneration becomes more NMNAT2 (full loss of function of NMNAT2 is likely to likely. Moreover, upregulation of SCG10 levels may be be lethal). A companion report describes two fetuses sufficient to protect axons from degeneration. with severe fetal akinesia deformation sequence and Following the discovery of NMNAT2 loss-of-function​ multiple developmental neurological abnormalities mutations in rare disorders89,90, it will be important to leading to stillbirth. These two siblings carried biallelic see what other pathogenic mutations emerge in these, loss-​of-function mutations in the NMNAT2 gene, with or related, disorders. The human population exome and one (NMNAT2R232Q) disrupting both NAD+ synthase and genome sequence database, the Genome Aggregation chaperone activities and the other (NMNAT2Q135Pfs*44) Database (gnomAD)104, compares the observed and greatly lowering protein expression. Each variant expected frequencies of nonsense, frameshift and splic- NMNAT2 was unable to prevent Wallerian degeneration ing mutations to estimate a ‘loss-of-function​ intolerance’ when overexpressed in neurons90. score, likely related to pathogenicity. Within this data- In another study, a 72-year-old​ patient with a 2-year base, NMNAT2 has a 0.99 probability of loss-​of-func- history of progressive brainstem atrophy was shown to tion intolerance, one of the highest scores, suggesting have a heterozygous mutation in NMNAT2 that had that many heterozygous alleles can also cause lethality or the potential to result in haploinsufficiency, although disease, at least in combination with other genes and/or no functional analysis was presented100. Given the nor- environmental factors. SARM1 gain of function (for malcy of the heterozygous parents and siblings of the example, resulting in a mutant that is overexpressed, individuals carrying biallelic loss-​of-function mutations more stable or more easily activated) would also be of NMNAT2 (refs89,90), it is unclear whether NMNAT2 expected to cause or increase disease risk. haploinsufficiency necessarily leads to axonopathies and Wallerian gene variants are likely to have wider which other environmental and genetic factors influence clinical significance through disease-​modifying effects: this. Furthermore, in the report on the patient with pro- that is, by influencing risk and severity in both common gressive brainstem atrophy, whole exome sequencing and rare disorders. Evidence is emerging of a spectrum identified other potential candidate genes100. of axon vulnerability in the human population (Fig. 5) Loss-​of-function mutations of SARM1 would be caused by both coding and gene expression variation of expected to be protective, meaning that exome sequencing Wallerian pathway genes89,90,95. Data from mouse exper- of disease cases will not find these mutations. However, iments already indicate that gene expression alterations evidence that SARM1 loss of function reduces disease risk have disease-​modifying effects105. With reports that or severity in humans would indicate involvement of the NMNAT2 expression level varies widely in post mortem Wallerian degeneration mechanism in the disease. An brains95, it will be important to determine the conse- ideal cohort in which to evaluate this possibility would quences of this variation for disease and which genetic be patients undergoing chemotherapy, assessing whether and environmental factors bring this about, especially those who do not develop peripheral neuropathy despite as low Nmnat2 expression is associated with sensory

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Non-viable Pathogenic Disease modifier

Stillbirth at Early onset polyneuropathy, Healthy into adulthood, Reduced disease risk Strongly reduced Human 21–27 weeks erythromelalgia normal disease risk or severity disease risk or severity phenotypes

Sensory and motor deficits, Resistance to Wallerian Perinatal Reduced vincristine Mouse enhanced vincristine Wild-type degeneration and lethality sensitivity phenotypes sensitivity multiple disease models

NMNAT2 SARM1

Fig. 5 | An axon vulnerability spectrum in humans and mice. The figure shows the range of known axon survival phenotypes in mice and proposed analogous phenotypes in the human population for which evidence is growing. The line graph depicts the likely prevalence in the human population and the gradients underneath depict protein and enzyme activity levels of nicotinamide mononucleotide adenylyltransferase 2 (NMNAT2) and sterile-α​ and Toll/interleukin 1 receptor (TIR) motif containing protein 1 (SARM1) in axons. The pink and blue boxes show five distinct phenotypes in humans and mice, respectively, all of which have been extensively characterized in mice6,13,105. In humans, the first two phenotypes (non-viable​ and pathogenic) have been linked to NMNAT2 mutation89,90. Causes of the proposed disease-modifying effects indicated towards the right side, corresponding to lower axon vulnerability, may include (1) SARM1 splicing alleles that are present in the Genome Aggregation Database (gnomAD), which are likely to disrupt the function of this pro-degeneration protein and (2) higher than average expression levels of NMNAT295, which are likely to result in gain of function of this pro-survival protein.

and motor deficiencies in mice105. Similarly, increased variation in NMNAT2, SARM1 or other Wallerian-​ SARM1 expression (for example, in viral infections51,52,106 regulating proteins influences neuropathy risk. Carefully or following prolonged low doses of rotenone107) is likely phenotyped patient populations are vital to firmly link to increase disease risk. Interestingly, rotenone also neuropathy to a specific risk factor such as chemotherapy causes SARM1-dependent axon degeneration in vitro108. or diabetes; such cohorts exist115 but await careful geno­ The effect of other pesticides and environmental toxins typing. Genetic disruption of axonal transport is another on the pathway91 is another important area for study. important area for future research. For example, the muta- Factors increasing axonal demand for NMNAT2 tion of tubulin folding cofactor E (TBCE) in early-​onset or reducing the soma’s ability to supply it are another encephalopathy with distal spinal muscular atrophy116 risk; for example, ageing lowers NMNAT2 axonal suggests the involvement of Wallerian degeneration as its transport62 and increases motor unit size (the number mouse orthologue is disrupted in a model that was par- of muscle fibres that each motor neuron innervates) by ticularly strongly protected by WldS, progressive motor up to 2.4-fold109, as surviving axons sprout to innervate neuronopathy (pmn)117. neuromuscular junctions vacated by degenerating axons. Combined with reduced axonal transport, this is likely Criteria for involvement of Wallerian degeneration in to create a ‘double hit’ on the ability to deliver sufficient disease. As the field moves forward to clinical transla- NMNAT2 to keep distal axons alive. Thus, even minor tion, it is important to establish strong criteria for the impairment of the motor neuron soma could under- involvement of Wallerian degeneration in a particular lie the onset of a dying-​back disorder such as ALS or disease (Table 2). For a long time, in animal models, the ageing associated neuromuscular junction denerva- gold standard was evidence for significant alleviation of tion110. Nigrostriatal neurons have even larger axonal axon loss (and, ideally, symptoms) by WldS. To this we arbours than motor neurons, with a combined length can now add alleviation of axon loss and symptoms for a single neuron estimated at up to 4.5 m (refs111,112). through the deletion of Sarm1, which in some models Age-​related compensatory sprouting in this pathway, is more effective than WldS2. Biochemical changes spe- similar to that seen following a partial lesion113, would cifically associated with loss of NMNAT2 (such as put a huge strain on the ability of surviving neurons to changes in the NMN-​to-NAD ratio22) or activation of deliver critical cargoes such as NMNAT2. Thus, it will SARM1 (such as a large increase in cADPR levels35) be important to determine whether such a mechanism may play an increasing role in defining the presence of could contribute to the dying-​back observed in the Wallerian degeneration, both in animal tissue and in striatum in Parkinson disease114. human biopsy material. Meanwhile, significant disease In future studies, it will be important to directly test associations with human genetic variants of Wallerian-​ for Wallerian degeneration mechanisms in human dis- regulating genes will become another strong indicator orders. In axonal transport disorders such as some forms of its presence in human disorders. In these cases, it is of CIPN, it will be important to ask whether genetic expected that increased disease risk should correlate

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with NMNAT2 loss of function or SARM1 gain of func- immune-​modulating therapies120, there are no treat- tion and that protective effects would be associated with ments that prevent secondary axonal degeneration. alleles having the opposite effects. Disease modelling in Given the observations in EAE in WldS mice71, it is human induced pluripotent stem cell lines will also help reasonable to consider axon-​protective strategies that establish roles for Wallerian degeneration as this allows modulate the Wallerian degeneration pathway as a ther- molecular pathways to be manipulated genetically or apeutic target for multiple sclerosis. Clinical trials in this pharmacologically to test for rescue effects. area will, however, be challenging as they will require long-term​ treatment and monitoring. Nevertheless, the Drug development. As the Wallerian degeneration use of biomarkers, such as neurofilament light chain mechanism and its involvement in disease, including levels, may help. human disease, become clearer, it is timely to consider Given that the preclinical data show the efficacy of what will be needed to drive the application of Wallerian targeting several different components of the Wallerian degeneration-​blocking therapies in the clinic. Safe and degeneration pathway, which one is likely to be the most effective drugs will need to be trialled in appropriate suitable for drug development? One of the most attrac- disorders. Where different mechanisms underlie the tive targets is SARM1 as this molecule plays a relatively same or closely related disorders (for example, periph- downstream role in Wallerian degeneration, mean- eral neuropathies), the focus needs to be on the most ing that one drug may be useful in multiple diseases. relevant subtype. Finally, biomarkers are needed to However, it will be challenging to develop a SARM1 measure efficacy. inhibitor that does not affect other NAD-​utilizing One attractive clinical target is CIPN, which allows enzymes in neurons and/or non-neuronal​ cells, in order intervention before significant axonal injury occurs. to avoid unwanted side effects. An inhibitor that targets The possibility of pre-medicating​ patients with a poten- the activation of SARM1 by preventing TIR domain tially neuroprotective drug (such as a SARM1 inhibitor dimerization may be a more suitable target. Recent or an FK866-like drug) before they receive chemo- observations that SARM1 forms an octomeric structure therapy would also simplify clinical trial design. These and that inhibition of oligomerization is able to prevent studies would be relatively short, as most chemotherapy the cell death-promoting​ activity of SARM1, favour this drugs cause peripheral neuropathy within 6 months of approach48,49. Another attractive approach is to develop starting treatment. The presence of validated outcome a dominant negative SARM1 gene therapy121 as this measures for CIPN would also simplify trial design and avoids the side effects of inhibiting other NADases; the recent development of neurofilament light chain however, the delivery of such a therapy to appropriate levels as a potential biomarker of axonal injury make it neuronal populations remains a big challenge. Finally, even simpler118,119. anti-sense​ oligonucleotides have blossomed into FDA-​ In terms of CNS diseases, TBI and primary and/or approved therapies for rare neurodegenerative dis- secondary progressive multiple sclerosis may be good eases such as amyloid neuropathy and spinal muscular initial targets. In TBI, one could potentially inter- atrophy122 and could therefore offer a more suitable vene immediately after an injury and contain the delivery path. consequences of axonal injury. However, variability Other therapeutic interventions may target other in the severity of injury and timely access to patients components of the Wallerian pathway. For example, may make clinical trial design challenging. In mul- drugs may target NMNAT2 expression level61 by pre- tiple sclerosis, despite the development of effective venting its degradation or by using a combined approach

Table 2 | Criteria for defining the contribution of Wallerian degeneration to disease Criterion Established in (disease) Limitations Refs Protection by WLDS, NMNATs or Many (listed in ref.3) as well as Animal and cell culture models 2,3,27, SARM1 ablation in animal or cell metabolic syndrome and/or type II only partially represent 64–68,84 culture models diabetes, CIPN, TBI, nerve growth corresponding human diseases failure and ALS Causation or exacerbation by Nerve growth failure, small Animal and cell culture models 11,13,105 removal and/or depletion of fibre neuropathy (especially only partially represent NMNAT2 in animal or cell culture thermosensitivity and late onset corresponding human diseases models motor impairment) and CIPN Genetic or protein expression Polyneuropathy with Correlation, not causation, 89,90, association in humans with erythromelalgia, FADS, brainstem although some supported by 95–97,100–102 NMNAT2, SARM1 or other Wallerian degeneration, ALS and Alzheimer functional evidence; GWAS link degeneration regulator disease may reflect another nearby gene Chemical markers of Wallerian Glaucoma, CIPN, nerve outgrowth Correlation not causation; 21,27,73–75 degeneration (raised levels of NMN failure and type II diabetes animal and cell culture models or cADPR or lowered levels of NAD) only partially represent the in animal or cell culture models corresponding human diseases ALS, amyotrophic lateral sclerosis; cADPR, cyclic ADP-ribose;​ CIPN, chemotherapy-induced​ peripheral neuropathy; FADS, fetal akinesia deformation sequence; GWAS, genome-wide​ association study; NMN, nicotinamide mononucleotide; NMNATs, nicotinamide mononucleotide adenylyltransferases; SARM1, sterile-α​ and Toll/interleukin 1 receptor (TIR) motif containing protein 1; TBI, traumatic brain injury; WLDs, Wallerian degeneration slow.

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to prevent NMN synthesis (using FK866, for example) see significant advances in translation in this field, as and providing an alternative NAD+ precursor such as well as further developments in the mechanistic under- nicotinic acid riboside30. Alternatively, drugs targeting standing. Genetics — whether in mice, flies or humans pathway components that play a more prominent role in — will continue to provide the major advances that it specific neuronal populations and disease states (such as has done for three decades. The biggest question is per- DLK and/or LZK in RGCs in glaucoma) may lead to the haps whether Wallerian-​blocking drugs will be limited development of disease-specific​ therapies. to rare, inherited disorders, where they are likely to be highly effective, or whether they will find extremely wide Concluding remarks application across the neurodegenerative disease treat- The recent pace of discoveries in Wallerian degener- ment spectrum. The prospects for the next three decades ation research, the choice of suitable drug targets and appear even more exciting than the last three. the attractiveness of CIPN as a primary application, leaves us in little doubt that the next 5–10 years will Published online 9 March 2020

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muscular atrophy. Am. J. Hum. Genet. 99, 974–983 therapy: a randomised controlled phase 3 trial. Lancet Competing interests (2016). 380, 1829–1839 (2012). M.P.C. has an academic collaboration with AstraZeneca and 117. Ferri, A., Sanes, J. R., Coleman, M. P., Cunningham, J. M. 121. Geisler, S. et al. Gene therapy targeting SARM1 blocks is a consultant for Proneurotech. A.H. serves on the scientific & Kato, A. C. Inhibiting axon degeneration and synapse pathological axon degeneration in mice. J. Exp. Med. advisory board of Disarm Therapeutics. loss attenuates apoptosis and disease progression in a 216, 294–303 (2019). mouse model of motoneuron disease. Curr. Biol. 13, 122. Corey, D. R. Nusinersen, an antisense oligonucleotide Publisher’s note Nat. Neurosci. 20 669–673 (2003). drug for spinal muscular atrophy. , Springer Nature remains neutral with regard to jurisdictional 118 . Shahim, P. et al. Serum neurofilament light protein 497–499 (2017). claims in published maps and institutional affiliations. predicts clinical outcome in traumatic brain injury. Sci. Rep. 6, 36791 (2016). Acknowledgements 119. Varhaug, K. N. et al. Neurofilament light chain predicts The authors thank members of the Coleman group for Related links disease activity in relapsing-​remitting MS. Neurol. constructive feedback. gnomAD: https://gnomad.broadinstitute.org/ Neuroimmunol. Neuroinflamm. 5, e422 (2017). 120. Coles, A. J. et al. Alemtuzumab for patients with Author contributions relapsing multiple sclerosis after disease-​modifying The authors contributed equally to all aspects of the article. © Springer Nature Limited 2020

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