Laura Conforti

Wallerian degeneration of injured axons and synapses is delayed by a Ube4b/Nmnat chimeric gene

Axons and their synapses distal to an injury undergo rapid Wallerian degeneration, but axons in the C57BL/WldS mouse are protected. The degenerative and protective mechanisms are unknown. We identified the protective gene, which encodes an N-terminal fragment of ubiquitination factor E4B (Ube4b) fused to nicotinamide mononucleotide adenylyltransferase (Nmnat), and showed that it confers a dose-dependent block of Wallerian degeneration. Transected distal axons survived for two weeks, and neuromuscular junctions were also protected. Surprisingly, the Wld protein was located predominantly in the nucleus, indicating an indirect protective mechanism. Nmnat enzyme activity, but not NAD+ content, was increased fourfold in WldS tissues. Thus, axon protection is likely to be mediated by altered ubiquitination or pyridine nucleotide metabolism.

dSarm/Sarm1 Is Required for Activation of an Injury-Induced Axon Death Pathway

Sarm-Assisted Suicide Neurodegenerative disease or nerve lesions cause axons and synapses to disintegrate through a process known as Wallerian degeneration, which may involve an active “axon death program.” Osterloh et al. (p. 481, published online 7 June; see the Perspective by Yu and Luo) identify loss-of-function mutations in Drosophila dSarm that are capable of blocking the degeneration of severed axons for the fly life span. Deletion of mouse Sarm1 provides similar protection to severed axons for weeks after injury, which suggests that Sarm is part of an ancient axonal death signaling cascade. Mutations in a scaffold protein block the Wallerian degeneration of axons in flies and mice. Axonal and synaptic degeneration is a hallmark of peripheral neuropathy, brain injury, and neurodegenerative disease. Axonal degeneration has been proposed to be mediated by an active autodestruction program, akin to apoptotic cell death; however, loss-of-function mutations capable of potently blocking axon self-destruction have not been described. Here, we show that loss of the Drosophila Toll receptor adaptor dSarm (sterile α/Armadillo/Toll-Interleukin receptor homology domain protein) cell-autonomously suppresses Wallerian degeneration for weeks after axotomy. Severed mouse Sarm1 null axons exhibit remarkable long-term survival both in vivo and in vitro, indicating that Sarm1 prodegenerative signaling is conserved in mammals. Our results provide direct evidence that axons actively promote their own destruction after injury and identify dSarm/Sarm1 as a member of an ancient axon death signaling pathway.

Wallerian degeneration: an emerging axon death pathway linking injury and disease

Axon degeneration is a prominent early feature of most neurodegenerative disorders and can also be induced directly by nerve injury in a process known as Wallerian degeneration. The discovery of genetic mutations that delay Wallerian degeneration has provided insight into mechanisms underlying axon degeneration in disease. Rapid Wallerian degeneration requires the pro-degenerative molecules SARM1 and PHR1. Nicotinamide mononucleotide adenylyltransferase 2 (NMNAT2) is essential for axon growth and survival. Its loss from injured axons may activate Wallerian degeneration, whereas NMNAT overexpression rescues axons from degeneration. Here, we discuss the roles of these and other proposed regulators of Wallerian degeneration, new opportunities for understanding disease mechanisms and intriguing links between Wallerian degeneration, innate immunity, synaptic growth and cell death.

Neuronal death: where does the end begin?

Neurodegenerative disorders involve death of cell bodies, axons, dendrites and synapses, but it is surprisingly difficult to determine the spatiotemporal sequence of events and the causal relationships among these events. Neuronal compartments often crucially depend upon one another for survival, and molecular defects in one compartment can trigger cellular degeneration in distant parts of the neuron. Here, we consider the novel approaches used to understand these biologically complex and technically challenging questions in amyotrophic lateral sclerosis, spinal muscular atrophy, glaucoma, Alzheimers disease, Parkinsons disease and polyglutamine disorders. We conclude that there is partial understanding of what degenerates first and why, but that controversy remains the rule not the exception. Finally, we highlight strategies for resolving these fundamental issues.

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

The slow Wallerian degeneration protein (WldS), 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 WldS mice. Nmnat1 overexpression in five lines of transgenic mice failed to delay Wallerian degeneration in transected sciatic nerves in contrast to WldS 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 WldS, and lentiviral overexpressed enzyme-dead WldS still displayed residual neurite protection. Thus, Nmnat1 is significantly weaker than WldS at protecting axons against traumatic or toxic injury in vitro, and has no detectable effect in vivo. The full protective effect of WldS requires more N-terminal sequences of the protein.

A rise in NAD precursor nicotinamide mononucleotide (NMN) after injury promotes axon degeneration.

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 WldS prevent this rise. These data indicate that the mechanism by which NMNAT and the related WldS 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.

Reducing expression of NAD+ synthesizing enzyme NMNAT1 does not affect the rate of Wallerian degeneration

NAD+ synthesizing enzyme NMNAT1 constitutes most of the sequence of neuroprotective protein WldS, which delays axon degeneration by 10‐fold. NMNAT1 activity is necessary but not sufficient for WldS neuroprotection in mice and 70 amino acids at the N‐terminus of WldS, derived from polyubiquitination factor Ube4b, enhance axon protection by NMNAT1. NMNAT1 activity can confer neuroprotection when redistributed outside the nucleus or when highly overexpressed inu2003vitro and partially in Drosophila. However, the role of endogenous NMNAT1 in normal axon maintenance and in Wallerian degeneration has not been elucidated yet. To address this question we disrupted the Nmnat1 locus by gene targeting. Homozygous Nmnat1 knockout mice do not survive to birth, indicating that extranuclear NMNAT isoforms cannot compensate for its loss. Heterozygous Nmnat1 knockout mice develop normally and do not show spontaneous neurodegeneration or axon pathology. Wallerian degeneration after sciatic nerve lesion is neither accelerated nor delayed in these mice, consistent with the proposal that other endogenous NMNAT isoforms play a principal role in Wallerian degeneration.

Evolutionary divergence of valosin‐containing protein/cell division cycle protein 48 binding interactions among endoplasmic reticulum‐associated degradation proteins

Endoplasmic reticulum (ER)‐associated degradation (ERAD) is a cell‐autonomous process that eliminates large quantities of misfolded, newly synthesized protein, and is thus essential for the survival of any basic eukaryotic cell. Accordingly, the proteins involved and their interaction partners are well conserved from yeast to mammals, and Saccharomyces cerevisiae is widely used as a model system with which to investigate this fundamental cellular process. For example, valosin‐containing protein (VCP) and its yeast homologue cell division cycle proteinu200348 (Cdc48p), which help to direct polyubiquitinated proteins for proteasome‐mediated degradation, interact with an equivalent group of ubiquitin ligases in mouse and in S.u2003cerevisiae. A conserved structural motif for cofactor binding would therefore be expected. We report a VCP‐binding motif (VBM) shared by mammalian ubiquitin ligaseu2003E4b (Ube4b)–ubiquitin fusion degradation proteinu20032a (Ufd2a), hydroxymethylglutaryl reductase degradation protein 1 (Hrd1)–synoviolin and ataxinu20033, and a related sequence in Mru200378u2003000 glycoprotein–Amfr with slightly different binding properties, and show that Ube4b and Hrd1 compete for binding to the N‐terminal domain of VCP. Each of these proteins is involved in ERAD, but none has an S.u2003cerevisiae homologue containing the VBM. Some other invertebrate model organisms also lack the VBM in one or more of these proteins, in contrast to vertebrates, where the VBM is widely conserved. Thus, consistent with their importance in ERAD, evolution has developed at least two ways to bring these proteins together with VCP–Cdc48p. However, the differing molecular architecture of VCP–Cdc48p complexes indicates a key point of divergence in the molecular details of ERAD mechanisms.

Diversification of NAD biological role : the importance of location

Over 100 years after its first discovery, several new aspects of the biology of the redox co‐factor NAD are rapidly emerging. NAD, as well as its precursors, its derivatives, and its metabolic enzymes, have been recently shown to play a determinant role in a variety of biological functions, from the classical role in oxidative phosphorylation and redox reactions to a role in regulation of gene transcription, lifespan and cell death, from a role in neurotransmission to a role in axon degeneration, and from a function in regulation of glucose homeostasis to that of control of circadian rhythm. It is also becoming clear that this variety of specialized functions is regulated by the fine subcellular localization of NAD, its related nucleotides and its metabolic enzymatic machinery. Here we describe the known NAD biosynthetic and catabolic pathways, and review evidence supporting a specialized role for NAD metabolism in a subcellular compartment‐dependent manner.

Axonopathy in Huntington's disease

Personality changes, psychiatric disturbances and cognitive abnormalities frequently characterise the prodromal phase in Huntingtons disease (HD), a devastating monogenic neurodegenerative disorder manifesting with abnormal motor movements and early death. Selective loss of medium-sized spiny striatal neurons has been related to the onset of motor symptoms but it does not completely explain the psychiatric and cognitive changes that often precede motor abnormalities. Here we review the evidence of synaptic and axonal dysfunction and neurite dystrophy preceding neuronal loss in HD patients and models. We discuss possible mechanisms leading to dysfunction of the axonal and synaptic compartments and identify potential novel targets for effective therapeutic intervention.