Nmnat1 Modulates Mitochondrial Oxidative Stress by Inhibiting Caspase-3 Signaling in Alzheimer's Disease.

Nigrostriatal pathway disturbance is one of the major pathogenic factors in Alzheimer's disease (AD). Dopaminergic neuron dysfunction results in bradykinesia and akinesia (inability to initiate movement), indicating a significant risk factor for substantia nigra pars compacta lesions. Furthermore, the nicotinamide adenine dinucleotide (NAD+) is associated with Aß toxicity decline in AD therapy. Nicotinamide mononucleotide adenylyltransferase 1 (Nmnat1) is an essential enzyme that preserves normal neuronal function and protects neurons from insult. This study aimed to investigate the potential therapeutic effects of Nmnat1 and its underlying mechanisms in a triple-transgenic mouse model of AD (3xTgAD). Results showed that Nmnat1 improved the substantial behavioral measures of cognitive impairments compared with the 3xTgAD control. Additionally, Nmnat1 overexpression significantly increased tyrosine hydroxylase-positive neurons and anti-apoptotic protein Bcl2 and caspase-3 expression levels in 3xTgAD mice. Nmnat1 also effectively controlled SOD1 activation. In conclusion, Nmnat1 substantially decreases multiple AD-associated pathological characteristics at least partially by the increase of caspase-3 activation.

Nicotinamide mononucleotide adenylyltransferase uses its NAD+ substrate-binding site to chaperone phosphorylated Tau.

Tau hyper-phosphorylation and deposition into neurofibrillary tangles have been found in brains of patients with Alzheimer's disease (AD) and other tauopathies. Molecular chaperones are involved in regulating the pathological aggregation of phosphorylated Tau (pTau) and modulating disease progression. Here, we report that nicotinamide mononucleotide adenylyltransferase (NMNAT), a well-known NAD+ synthase, serves as a chaperone of pTau to prevent its amyloid aggregation in vitro as well as mitigate its pathology in a fly tauopathy model. By combining NMR spectroscopy, crystallography, single-molecule and computational approaches, we revealed that NMNAT adopts its enzymatic pocket to specifically bind the phosphorylated sites of pTau, which can be competitively disrupted by the enzymatic substrates of NMNAT. Moreover, we found that NMNAT serves as a co-chaperone of Hsp90 for the specific recognition of pTau over Tau. Our work uncovers a dedicated chaperone of pTau and suggests NMNAT as a key node between NAD+ metabolism and Tau homeostasis in aging and neurodegeneration.

The SARM1 axon degeneration pathway: control of the NAD+ metabolome regulates axon survival in health and disease.

Axons are essential for nervous system function and axonal pathology is a common hallmark of many neurodegenerative diseases. Over a century and a half after the original description of Wallerian axon degeneration, advances over the past five years have heralded the emergence of a comprehensive, mechanistic model of an endogenous axon degenerative process that can be activated by both injury and disease. Axonal integrity is maintained by the opposing actions of the survival factors NMNAT2 and STMN2 and pro-degenerative molecules DLK and SARM1. The balance between axon survival and self-destruction is intimately tied to axonal NAD+ metabolism. These mechanistic insights may enable axon-protective therapies for a variety of human neurodegenerative diseases including peripheral neuropathy, traumatic brain injury and potentially ALS and Parkinson's.

Effect of NMDAR-NMNAT1/2 pathway on neuronal cell damage and cognitive impairment of sevoflurane-induced aged rats.

Objective: The possible effect of NMDAR (N-methyl-D-aspartate receptor)-NMNAT1/2 (nicotinamide/nicotinic acid mono-nucleotide adenylyltransferase) signaling pathway on the neuronal cell damage and cognitive impairment of aged rats anesthetized by sevoflurane was explored.Methods: Adult male Wistar rats were selected and divided into Control, Sevo (Sevoflurane), Sevo+DCS (NMDAR agonist D-cycloserine) 30 mg/kg, Sevo+DCS 100 mg/kg, and Sevo+DCS 200 mg/kg groups. Morris water maze and fear conditioning text were used to observe cognitive function changes of rats. The inflammatory cytokines were determined by quantitative real-time polymerase chain reaction (qRT-PCR) and enzyme-linked immunosorbent assay (ELISA) assay, neuronal apoptosis by terminal deoxynucleotidyl transferase (TdT)-mediated dUTP nick-end labelling (TUNEL) staining and MDAR-NMNAT1/2 pathway-related proteins by Western blotting.Results: The longer escape latency, decreased platform crossing times and reduced staying time spent in platform quadrant were found in rats from Sevo group, with decreased percentage of freezing time in contextual test and tone cued test; and meanwhile, these rats had increased inflammatory cytokines (interleukin (IL)-1ß, tumor necrosis factor (TNF-α), IL-6, and IL-8) and neuronal apoptosis, but declined expressions of MDAR-NMNAT1/2 pathway-related proteins. However, the above changes were exhibited an opposite tendency in those Sevo rats treated with different concentrations of DCS (including 30, 100, and 200 mg/kg, respectively). Particularly, the improving effect of low-dose DCS on each aspect in aged rats was better than high-dose ones.Conclusion: Activation of NMDAR-NMNAT1/2 signaling pathway could not only reduce neuronal apoptosis, but also alleviate sevoflurane-induced neuronal inflammation and cognitive impairment in aged rats.

Axonal transport declines with age in two distinct phases separated by a period of relative stability.

Axonal transport is critical for supplying newly synthesized proteins, organelles, mRNAs, and other cargoes from neuronal cell bodies into axons. Its impairment in many neurodegenerative conditions appears likely to contribute to pathogenesis. Axonal transport also declines during normal aging, but little is known about the timing of these changes, or about the effect of aging on specific cargoes in individual axons. This is important for understanding mechanisms of age-related axon loss and age-related axonal disorders. Here we use fluorescence live imaging of peripheral nerve and central nervous system tissue explants to investigate vesicular and mitochondrial axonal transport. Interestingly, we identify 2 distinct periods of change, 1 period during young adulthood and the other in old age, separated by a relatively stable plateau during most of adult life. We also find that after tibial nerve regeneration, even in old animals, neurons are able to support higher transport rates of each cargo for a prolonged period. Thus, the age-related decline in axonal transport is not an inevitable consequence of either aging neurons or an aging systemic milieu.

Novel assay for simultaneous measurement of pyridine mononucleotides synthesizing activities allows dissection of the NAD(+) biosynthetic machinery in mammalian cells.

The redox coenzyme NAD(+) is also a rate-limiting co-substrate for several enzymes that consume the molecule, thus rendering its continuous re-synthesis indispensable. NAD(+) biosynthesis has emerged as a therapeutic target due to the relevance of NAD(+) -consuming reactions in complex intracellular signaling networks whose alteration leads to many neurologic and metabolic disorders. Distinct metabolic routes, starting from various precursors, are known to support NAD(+) biosynthesis with tissue/cell-specific efficiencies, probably reflecting differential expression of the corresponding rate-limiting enzymes, i.e. nicotinamide phosphoribosyltransferase, quinolinate phosphoribosyltransferase, nicotinate phosphoribosyltransferase and nicotinamide riboside kinase. Understanding the contribution of these enzymes to NAD(+) levels depending on the tissue/cell type and metabolic status is necessary for the rational design of therapeutic strategies aimed at modulating NAD(+) availability. Here we report a simple, fast and sensitive coupled fluorometric assay that enables simultaneous determination of the four activities in whole-cell extracts and biological fluids. Its application to extracts from various mouse tissues, human cell lines and plasma yielded for the first time an overall picture of the tissue/cell-specific distribution of the activities of the various enzymes. The screening enabled us to gather novel findings, including (a) the presence of quinolinate phosphoribosyltransferase and nicotinamide riboside kinase in all examined tissues/cell lines, indicating that quinolinate and nicotinamide riboside are relevant NAD(+) precursors, and (b) the unexpected occurrence of nicotinate phosphoribosyltransferase in human plasma.

Axonal trafficking of NMNAT2 and its roles in axon growth and survival in vivo.

The NAD-synthesizing enzyme NMNAT2 is critical for axon survival in primary culture and its depletion may contribute to axon degeneration in a variety of neurodegenerative disorders. Here we discuss several recent reports from our laboratory that establish a critical role for NMNAT2 in axon growth in vivo in mice and shed light on the delivery and turnover of this survival factor in axons. In the absence of NMNAT2, axons fail to extend more than a short distance beyond the cell body during embryonic development, implying a requirement for NMNAT2 in axon maintenance even during development. Furthermore, we highlight findings regarding the bidirectional trafficking of NMNAT2 in axons on a vesicle population that undergoes fast axonal transport in primary culture neurites and in mouse sciatic nerve axons in vivo. Surprisingly, loss of vesicle association boosts the axon protective capacity of NMNAT2, an effect that is at least partially mediated by a longer protein half-life of cytosolic NMNAT2 variants. Analysis of wild-type and variant NMNAT2 in mouse sciatic nerves and Drosophila olfactory receptor neuron axons supports the existence of a similar mechanism in vivo, highlighting the potential for regulation of NMNAT2 stability and turnover as a mechanism to modulate axon degeneration in vivo.

Subcellular localization determines the stability and axon protective capacity of axon survival factor Nmnat2.

Axons require a constant supply of the labile axon survival factor Nmnat2 from their cell bodies to avoid spontaneous axon degeneration. Here we investigate the mechanism of fast axonal transport of Nmnat2 and its site of action for axon maintenance. Using dual-colour live-cell imaging of axonal transport in SCG primary culture neurons, we find that Nmnat2 is bidirectionally trafficked in axons together with markers of the trans-Golgi network and synaptic vesicles. In contrast, there is little co-migration with mitochondria, lysosomes, and active zone precursor vesicles. Residues encoded by the small, centrally located exon 6 are necessary and sufficient for stable membrane association and vesicular axonal transport of Nmnat2. Within this sequence, a double cysteine palmitoylation motif shared with GAP43 and surrounding basic residues are all required for efficient palmitoylation and stable association with axonal transport vesicles. Interestingly, however, disrupting this membrane association increases the ability of axonally localized Nmnat2 to preserve transected neurites in primary culture, while re-targeting the strongly protective cytosolic mutants back to membranes abolishes this increase. Larger deletions within the central domain including exon 6 further enhance Nmnat2 axon protective capacity to levels that exceed that of the slow Wallerian degeneration protein, Wld(S). The mechanism underlying the increase in axon protection appears to involve an increased half-life of the cytosolic forms, suggesting a role for palmitoylation and membrane attachment in Nmnat2 turnover. We conclude that Nmnat2 activity supports axon survival through a site of action distinct from Nmnat2 transport vesicles and that protein stability, a key determinant of axon protection, is enhanced by mutations that disrupt palmitoylation and dissociate Nmnat2 from these vesicles.

Nmnat2 attenuates Tau phosphorylation through activation of PP2A.

The activity of protein phosptase-2A (PP2A) is significantly decreased in the brains of Alzheimer's disease (AD) patients, but the upstream effectors for regulating PP2A activity are not fully understood. Nicotinamide mononucleotide adenylyltransferase 2 (Nmnat2) is a key enzyme involved in energy metabolism and its gene expression level is reduced in AD brain specimens. Whether Nmnat2 can activate PP2A deserves to be explored. Here, we first measured the level of Nmnat2, Tyr307-phosphorylation of PP2A, and tau phosphorylation in Tg2576 mice. We observed that the mRNA and protein levels of Nmnat2 were significantly decreased with a simultaneous elevation of p-Tyr307-PP2A and tau phosphorylation in Tg2576 mice. Further studies in HEK293 cells with stable expression of human tau441 (HEK293/tau) demonstrated that simultaneous inhibition of PP2A by okadaic acid abolished the Nmnat2-induced tau dephosphorylation. Moreover, we further demonstrated that overexpression of Nmnat2 could activate PP2A with attenuation of tau phosphorylation, whereas downregulation of Nmnat2 by shRNA inhibited PP2A with tau hyperphosphorylation at multiple AD-associated sites. Our data provide the first evidence that Nmnat2 affects tau phosphorylation by regulating PP2A activity, suggesting that Nmnat2 may serve as a potential target in arresting AD-like tau pathologies.

NMNAT1 mutations cause Leber congenital amaurosis.

Leber congenital amaurosis (LCA) is an infantile-onset form of inherited retinal degeneration characterized by severe vision loss(1,2). Two-thirds of LCA cases are caused by mutations in 17 known disease-associated genes(3) (Retinal Information Network (RetNet)). Using exome sequencing we identified a homozygous missense mutation (c.25G>A, p.Val9Met) in NMNAT1 that is likely to be disease causing in two siblings of a consanguineous Pakistani kindred affected by LCA. This mutation segregated with disease in the kindred, including in three other children with LCA. NMNAT1 resides in the previously identified LCA9 locus and encodes the nuclear isoform of nicotinamide mononucleotide adenylyltransferase, a rate-limiting enzyme in nicotinamide adenine dinucleotide (NAD(+)) biosynthesis(4,5). Functional studies showed that the p.Val9Met alteration decreased NMNAT1 enzyme activity. Sequencing NMNAT1 in 284 unrelated families with LCA identified 14 rare mutations in 13 additional affected individuals. These results are the first to link an NMNAT isoform to disease in humans and indicate that NMNAT1 mutations cause LCA.

Mutations in NMNAT1 cause Leber congenital amaurosis and identify a new disease pathway for retinal degeneration.

Leber congenital amaurosis (LCA) is a blinding retinal disease that presents within the first year after birth. Using exome sequencing, we identified mutations in the nicotinamide adenine dinucleotide (NAD) synthase gene NMNAT1 encoding nicotinamide mononucleotide adenylyltransferase 1 in eight families with LCA, including the family in which LCA was originally linked to the LCA9 locus. Notably, all individuals with NMNAT1 mutations also have macular colobomas, which are severe degenerative entities of the central retina (fovea) devoid of tissue and photoreceptors. Functional assays of the proteins encoded by the mutant alleles identified in our study showed that the mutations reduce the enzymatic activity of NMNAT1 in NAD biosynthesis and affect protein folding. Of note, recent characterization of the slow Wallerian degeneration (Wld(s)) mouse model, in which prolonged axonal survival after injury is observed, identified NMNAT1 as a neuroprotective protein when ectopically expressed. Our findings identify a new disease mechanism underlying LCA and provide the first link between endogenous NMNAT1 dysfunction and a human nervous system disorder.

WldS but not Nmnat1 protects dopaminergic neurites from MPP+ neurotoxicity.

BACKGROUND: The WldS mouse mutant ("Wallerian degeneration-slow") delays axonal degeneration in a variety of disorders including in vivo models of Parkinson's disease. The mechanisms underlying WldS -mediated axonal protection are unclear, although many studies have attributed WldS neuroprotection to the NAD+-synthesizing Nmnat1 portion of the fusion protein. Here, we used dissociated dopaminergic cultures to test the hypothesis that catalytically active Nmnat1 protects dopaminergic neurons from toxin-mediated axonal injury. RESULTS: Using mutant mice and lentiviral transduction of dopaminergic neurons, the present findings demonstrate that WldS but not Nmnat1, Nmnat3, or cytoplasmically-targeted Nmnat1 protects dopamine axons from the parkinsonian mimetic N-methyl-4-phenylpyridinium (MPP+). Moreover, NAD+ synthesis is not required since enzymatically-inactive WldS still protects. In addition, NAD+ by itself is axonally protective and together with WldS is additive in the MPP+ model. CONCLUSIONS: Our data suggest that NAD+ and WldS act through separate and possibly parallel mechanisms to protect dopamine axons. As MPP+ is thought to impair mitochondrial function, these results suggest that WldS might be involved in preserving mitochondrial health or maintaining cellular metabolism.

Regulation of poly(ADP-ribose) polymerase-1-dependent gene expression through promoter-directed recruitment of a nuclear NAD+ synthase.

NMNAT-1 and PARP-1, two key enzymes in the NAD(+) metabolic pathway, localize to the nucleus where integration of their enzymatic activities has the potential to control a variety of nuclear processes. Using a variety of biochemical, molecular, cell-based, and genomic assays, we show that NMNAT-1 and PARP-1 physically and functionally interact at target gene promoters in MCF-7 cells. Specifically, we show that PARP-1 recruits NMNAT-1 to promoters where it produces NAD(+) to support PARP-1 catalytic activity, but also enhances the enzymatic activity of PARP-1 independently of NAD(+) production. Furthermore, using two-photon excitation microscopy, we show that NMNAT-1 catalyzes the production of NAD(+) in a nuclear pool that may be distinct from other cellular compartments. In expression microarray experiments, depletion of NMNAT-1 or PARP-1 alters the expression of about 200 protein-coding genes each, with about 10% overlap between the two gene sets. NMNAT-1 enzymatic activity is required for PARP-1-dependent poly(ADP-ribosyl)ation at the promoters of commonly regulated target genes, as well as the expression of those target genes. Collectively, our studies link the enzymatic activities of NMNAT-1 and PARP-1 to the regulation of a set of common target genes through functional interactions at target gene promoters.

Endogenous Nmnat2 is an essential survival factor for maintenance of healthy axons.

The molecular triggers for axon degeneration remain unknown. We identify endogenous Nmnat2 as a labile axon survival factor whose constant replenishment by anterograde axonal transport is a limiting factor for axon survival. Specific depletion of Nmnat2 is sufficient to induce Wallerian-like degeneration of uninjured axons which endogenous Nmnat1 and Nmnat3 cannot prevent. Nmnat2 is by far the most labile Nmnat isoform and is depleted in distal stumps of injured neurites before Wallerian degeneration begins. Nmnat2 turnover is equally rapid in injured Wld(S) neurites, despite delayed neurite degeneration, showing it is not a consequence of degeneration and also that Wld(S) does not stabilize Nmnat2. Depletion of Nmnat2 below a threshold level is necessary for axon degeneration since exogenous Nmnat2 can protect injured neurites when expressed at high enough levels to overcome its short half-life. Furthermore, proteasome inhibition slows both Nmnat2 turnover and neurite degeneration. We conclude that endogenous Nmnat2 prevents spontaneous degeneration of healthy axons and propose that, when present, the more long-lived, functionally related Wld(S) protein substitutes for Nmnat2 loss after axon injury. Endogenous Nmnat2 represents an exciting new therapeutic target for axonal disorders.

Axon degeneration: Mechanisms and implications of a distinct program from cell death.

Axon degeneration has been proposed to be a new therapeutic target for neurodegenerative diseases, because it usually occurs earlier than neuronal cell body death with a distinct active program from apoptosis and necrosis. Overexpression of Wld(S) or Nmnats (nicotinamide mononucleotide adenylytransferase, EC2.7.7.1) has been demonstrated to delay axon degeneration initiated by various insults. NAD synthesis activity of Wld(S) and Nmnats was shown to be responsible for their axon-protective function. The mitochondrial Nmnat3 and cytoplasm-localized mutants of Wld(S) and Nmnat1 have similar or even stronger effect than Wld(S) to delay axon degeneration, which suggest that increased mitochondrial or local NAD synthesis might contribute to the protective function of Wld(S) and Nmnats. Further studies show NAD synthesis pathway and ubiquitin proteasome system play important roles in delaying axon degeneration. Wld(S) mice are resistant to a variety of neurodegenerative diseases, but the role of Nmnats in neurodegenerative diseases are largely unknown. NAD plays key roles in energy metabolism, mitochondrial functions and aging, and is suggested to be involved in neuron degenerative diseases. Future studies to identify the upstream factors inducing NAD depletion and the downstream NAD effectors responsible for the axon-protective function will provide more meaningful insights into the molecular mechanisms of axon degeneration in neurodegenerative diseases.

Wld(S), Nmnats and axon degeneration--progress in the past two decades.

A chimeric protein called Wallerian degeneration slow (Wld(S)) was first discovered in a spontaneous mutant strain of mice that exhibited delayed Wallerian degeneration. This provides a useful tool in elucidating the mechanisms of axon degeneration. Over-expression of Wld(S) attenuates the axon degeneration that is associated with several neurodegenerative disease models, suggesting a new logic for developing a potential protective strategy. At molecular level, although Wld(S) is a fusion protein, the nicotinamide mononucleotide adenylyl transferase 1 (Nmnat1) is required and sufficient for the protective effects of Wld(S), indicating a critical role of NAD biosynthesis and perhaps energy metabolism in axon degeneration. These findings challenge the proposed model in which axon degeneration is operated by an active programmed process and thus may have important implication in understanding the mechanisms of neurodegeneration. In this review, we will summarize these recent findings and discuss their relevance to the mechanisms of axon degeneration.

Transgenic mice expressing the Nmnat1 protein manifest robust delay in axonal degeneration in vivo.

Axonal degeneration is a key component of a variety of neurological diseases. Studies using wld(s) mutant mice have demonstrated that delaying axonal degeneration slows disease course and prolongs survival in neurodegenerative disease models. The Wld(s) protein is normally localized to the nucleus, and contains the N terminus of ubiquitination factor Ube4b fused to full-length Nmnat1, an NAD biosynthetic enzyme. While Nmnat enzymatic activity is necessary for Wld(s)-mediated axonal protection, several important questions remain including whether the Ube4b component of Wld(s) also plays a role, and in which cellular compartment (nucleus vs cytosol) the axonal protective effects of Nmnat activity are mediated. While Nmnat alone is clearly sufficient to delay axonal degeneration in cultured neurons, we sought to determine whether it was also sufficient to promote axonal protection in vivo. Using cytNmnat1, an engineered mutant of Nmnat1 localized only to the cytoplasm and axon, that provides more potent axonal protection than that afforded by Wld(s) or Nmnat1, we generated transgenic mice using the prion protein promoter (PrP). The sciatic nerve of these cytNmnat1 transgenic mice was transected, and microscopic analysis of the distal nerve segment 7 d later revealed no evidence of axonal loss or myelin debris, indicating that Nmnat alone, without any other Wld(s) sequences, is all that is required to delay axonal degeneration in vivo. These results highlight the importance of understanding the mechanism of Nmnat-mediated axonal protection for the development of new treatment strategies for neurological disorders.

Nicotinamide mononucleotide adenylyl transferase-mediated axonal protection requires enzymatic activity but not increased levels of neuronal nicotinamide adenine dinucleotide.

Axonal degeneration is a hallmark of many neurological disorders. Studies in animal models of neurodegenerative diseases indicate that axonal degeneration is an early event in the disease process, and delaying this process can lead to decreased progression of the disease and survival extension. Overexpression of the Wallerian degeneration slow (Wld(s)) protein can delay axonal degeneration initiated via axotomy, chemotherapeutic agents, or genetic mutations. The Wld(s) protein consists of the N-terminal portion of the ubiquitination factor Ube4b fused to the nicotinamide adenine dinucleotide (NAD(+)) biosynthetic enzyme nicotinamide mononucleotide adenylyl transferase 1 (Nmnat1). We previously showed that the Nmnat1 portion of this fusion protein was the critical moiety for Wld(s)-mediated axonal protection. Here, we describe the development of an automated quantitative assay for assessing axonal degeneration. This method successfully showed that Nmnat1 enzymatic activity is important for axonal protection as mutants with reduced enzymatic activity lacked axon protective activity. We also found that Nmnat enzymes with diverse sequences and structures from various species, including Drosophila melanogaster, Saccharomyces cerevisiae, and archaebacterium Methanocaldococcus jannaschii, which encodes a protein with no homology to eukaryotic Nmnat enzymes, all mediate robust axonal protection after axotomy. Besides the importance of Nmnat enzymatic activity, we did not observe changes in the steady-state NAD(+) level, and we found that inhibition of nicotinamide phosphoribosyltransferase (Nampt), which synthesizes substrate for Nmnat in mammalian cells, did not affect the protective activity of Nmnat1. These results provide the possibility of a role for new Nmnat enzymatic activity in axonal protection in addition to NAD(+) synthesis.

NAD(+) and axon degeneration revisited: Nmnat1 cannot substitute for Wld(S) to delay Wallerian degeneration.

The slow Wallerian degeneration protein (Wld(S)), a fusion protein incorporating full-length nicotinamide mononucleotide adenylyltransferase 1 (Nmnat1), delays axon degeneration caused by injury, toxins and genetic mutation. Nmnat1 overexpression is reported to protect axons in vitro, but its effect in vivo and its potency remain unclear. We generated Nmnat1-overexpressing transgenic mice whose Nmnat activities closely match that of Wld(S) mice. Nmnat1 overexpression in five lines of transgenic mice failed to delay Wallerian degeneration in transected sciatic nerves in contrast to Wld(S) mice where nearly all axons were protected. Transected neurites in Nmnat1 transgenic dorsal root ganglion explant cultures also degenerated rapidly. The delay in vincristine-induced neurite degeneration following lentiviral overexpression of Nmnat1 was significantly less potent than for Wld(S), and lentiviral overexpressed enzyme-dead Wld(S) still displayed residual neurite protection. Thus, Nmnat1 is significantly weaker than Wld(S) at protecting axons against traumatic or toxic injury in vitro, and has no detectable effect in vivo. The full protective effect of Wld(S) requires more N-terminal sequences of the protein.