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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.

Since Waller and Cajal in the nineteenth and early twentieth centuries, laboratory traumatic peripheral nerve injury studies have provided great insight into cellular and molecular mechanisms governing axon degeneration and the responses of Schwann cells, the major glial cell type of peripheral nerves. It is now evident that pathways underlying injury-induced axon degeneration and the Schwann cell injury-specific state, the repair Schwann cell, are relevant to many inherited and acquired disorders of peripheral nerves. This review provides a timely update on the molecular understanding of axon degeneration and formation of the repair Schwann cell. We discuss how nicotinamide mononucleotide adenylyltransferase 2 (NMNAT2) and sterile alpha TIR motif containing protein 1 (SARM1) are required for axon survival and degeneration, respectively, how transcription factor c-JUN is essential for the Schwann cell response to nerve injury and what each tells us about disease mechanisms and potential therapies. Human genetic association with NMNAT2 and SARM1 strongly suggests aberrant activation of programmed axon death in polyneuropathies and motor neuron disorders, respectively, and animal studies suggest wider involvement including in chemotherapy-induced and diabetic neuropathies. In repair Schwann cells, cJUN is aberrantly expressed in a wide variety of human acquired and inherited neuropathies. Animal models suggest it limits axon loss in both genetic and traumatic neuropathies, whereas in contrast, Schwann cell secreted Neuregulin-1 type 1 drives onion bulb pathology in CMT1A. Finally, we discuss opportunities for drug-based and gene therapies to prevent axon loss or manipulate the repair Schwann cell state to treat acquired and inherited neuropathies and neuronopathies.

Background Systemic inflammation has been linked to synapse loss and cognitive decline in human patients and animal models. A role for microglial release of pro-inflammatory cytokines has been proposed based on in vivo and primary culture studies. However, mechanisms are hard to study in vivo as specific microglial ablation is challenging and the extracellular fluid cannot be sampled without invasive methods. Primary cultures have different limitations as the intricate multicellular architecture in the brain is not fully reproduced. It is essential to confirm proposed brain-specific mechanisms of inflammatory synapse loss directly in brain tissue. Organotypic hippocampal slice cultures (OHSCs) retain much of the in vivo neuronal architecture, synaptic connections and diversity of cell types whilst providing convenient access to manipulate and sample the culture medium and observe cellular reactions. Methods OHSCs were generated from P6-P9 C57BL/6 mice. Inflammation was induced via addition of lipopolysaccharide (LPS), and cultures were analysed for changes in synaptic proteins, gene expression and protein secretion. Microglia were selectively depleted using clodronate, and the effect of IL1β was assessed using a specific neutralising monoclonal antibody. Results LPS treatment induced loss of the presynaptic protein synaptophysin without altering PSD95 or Aβ protein levels. Depletion of microglia prior to LPS application prevented the loss of synaptophysin, whilst microglia depletion after the inflammatory insult was partially effective, although less so than pre-emptive treatment, indicating a time-critical window in which microglia can induce synaptic damage. IL1β protein and mRNA were increased after LPS addition, with these effects also prevented by microglia depletion. Direct application of IL1β to OHSCs resulted in synaptophysin loss whilst pre-treatment with IL1β neutralising antibody prior to LPS addition prevented a significant loss of synaptophysin but may also impact basal synaptic levels. Conclusions The loss of synaptophysin in this system confirms LPS can act directly within brain tissue to disrupt synapses, and we show that microglia are the relevant cellular target when all major CNS cell types are present. By overcoming limitations of primary culture and in vivo work, our study strengthens the evidence for a key role of microglia-derived IL1β in synaptic dysfunction after inflammatory insult.

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.

SARM1 (sterile alpha and TIR motif containing 1) is responsible for depletion of nicotinamide adenine dinucleotide in its oxidized form (NAD⁺) during Wallerian degeneration associated with neuropathies. Plant nucleotide-binding leucine-rich repeat (NLR) immune receptors recognize pathogen effector proteins and trigger localized cell death to restrict pathogen infection. Both processes depend on closely related Toll/interleukin-1 receptor (TIR) domains in these proteins, which, as we show, feature self-association–dependent NAD⁺ cleavage activity associated with cell death signaling. We further show that SARM1 SAM (sterile alpha motif) domains form an octamer essential for axon degeneration that contributes to TIR domain enzymatic activity. The crystal structures of ribose and NADP⁺ (the oxidized form of nicotinamide adenine dinucleotide phosphate) complexes of SARM1 and plant NLR RUN1 TIR domains, respectively, reveal a conserved substrate binding site. NAD⁺ cleavage by TIR domains is therefore a conserved feature of animal and plant cell death signaling pathways.

Maintenance of NAD pools is critical for neuronal survival. The capacity to maintain NAD pools declines in neurodegenerative disease. We identify that low NMNAT2, the critical neuronal NAD producing enzyme, drives retinal susceptibility to neurodegenerative insults. As proof of concept, gene therapy over-expressing full length human NMNAT2 is neuroprotective. To pharmacologically target NMNAT2, we identify that epigallocatechin gallate (EGCG) can drive NAD production in neurons through an NMNAT2 and NMN dependent mechanism. We confirm this by pharmacological and genetic inhibition of the NAD-salvage pathway. EGCG is neuroprotective in rodent (mixed sex) and human models of retinal neurodegeneration. As EGCG has poor drug-like qualities, we use it as a tool compound to generate novel small molecules which drive neuronal NAD production and provide neuroprotection. This class of NMNAT2 targeted small molecules could have an important therapeutic impact for neurodegenerative disease following further drug development.Neurodegenerative disease is a significant global health and economic burden. NAD homoeostasis is a critical factor that influences neurodegeneration and neuroprotection. Increasing levels of NAD provide neuroprotection in multiple cell and animal models of disease and in human clinical trials 1 . Glaucoma is one of the most prevalent neurodegenerations which affects ~80 million people worldwide 2 . In glaucoma, the progressive dysfunction and loss of retinal ganglion cells (RGCs; the output neuron of the retina whose axons make up the optic nerve) results in irreversible blindness. There are no clinically available neuroprotective strategies.Recent animal and human studies have uncovered metabolic dysfunction occurring early in RGCs in glaucoma, in particular the critical dependency of RGCs on sufficient levels of NAD 3-5 . In neurons, NAD levels are maintained predominantly through the NAD-salvage pathway's two terminal enzymes; NMNAT1 (localized to the nucleus) and NMNAT2 (localized in the cytoplasm 6 ). Protein expression of

Amyotrophic lateral sclerosis–frontotemporal dementia (ALS-FTD) constitutes a devastating disease spectrum characterized by 43-kDa TAR DNA-binding protein (TDP-43) pathology. Understanding how TDP-43 contributes to neurodegeneration will help direct therapeutic efforts. Here we have created a TDP-43 knock-in mouse with a human-equivalent mutation in the endogenous mouse Tardbp gene. TDP-43Q331K mice demonstrate cognitive dysfunction and a paucity of parvalbumin interneurons. Critically, TDP-43 autoregulation is perturbed, leading to a gain of TDP-43 function and altered splicing of Mapt, another pivotal dementia-associated gene. Furthermore, a new approach to stratify transcriptomic data by phenotype in differentially affected mutant mice revealed 471 changes linked with improved behavior. These changes included downregulation of two known modifiers of neurodegeneration, Atxn2 and Arid4a, and upregulation of myelination and translation genes. With one base change in murine Tardbp, this study identifies TDP-43 misregulation as a pathogenic mechanism that may underpin ALS-FTD and exploits phenotypic heterogeneity to yield candidate suppressors of neurodegenerative disease.

MYCBP2 is a ubiquitin (Ub) E3 ligase (E3) that is essential for neurodevelopment and regulates axon maintenance. MYCBP2 transfers Ub to nonlysine substrates via a newly discovered RING-Cys-Relay (RCR) mechanism, where Ub is relayed from an upstream cysteine to a downstream substrate esterification site. The molecular bases for E2–E3 Ub transfer and Ub relay are unknown. Whether these activities are linked to the neural phenotypes is also unclear. We describe the crystal structure of a covalently trapped E2~Ub:MYCBP2 transfer intermediate revealing key structural rearrangements upon E2–E3 Ub transfer and Ub relay. Our data suggest that transfer to the dynamic upstream cysteine, whilst mitigating lysine activity, requires a closed-like E2~Ub conjugate with tempered reactivity, and Ub relay is facilitated by a helix–coil transition. Furthermore, neurodevelopmental defects and delayed injury-induced degeneration in RCR-defective knock-in mice suggest its requirement, and that of substrate esterification activity, for normal neural development and programmed axon degeneration. The structural analysis of a covalent E2–E3 ubiquitin transfer intermediate reveals the ubiquitin relay mechanism via two Cys residues of E3 ligase MYCBP2, which is related to neurite growth and neurite protection phenotypes.

SARM1 is a central executioner of programmed axon death, and this role requires intrinsic NAD(P)ase or related enzyme activity. A complete absence of SARM1 robustly blocks axon degeneration in mice, but even a partial depletion confers meaningful protection. Since axon loss contributes substantially to the onset and progression of multiple neurodegenerative disorders, lower inherent SARM1 activity is expected to reduce disease susceptibility in some situations. We, therefore, investigated whether there are naturally occurring SARM1 alleles within the human population that encode SARM1 variants with loss-of-function. Out of the 18 natural SARM1 coding variants we selected as candidates, we found that 10 display loss-of-function in three complimentary assays: they fail to robustly deplete NAD in transfected HEK 293T cells; they lack constitutive and NMN-induced NADase activity; and they fail to promote axon degeneration in primary neuronal cultures. Two of these variants are also able to block axon degeneration in primary culture neurons in the presence of endogenous, wild-type SARM1, indicative of dominant loss-of-function. These results demonstrate that SARM1 loss-of-function variants occur naturally in the human population, and we propose that carriers of these alleles will have different degrees of reduced susceptibility to various neurological conditions.

Here, we report an adapted protocol using the Promega NAD/NADH-Glo™ Assay kit. The assay normally allows quantification of trace amounts of both oxidized and reduced forms of nicotinamide adenine dinucleotide (NAD) by enzymatic cycling, but we now show that the NAD analog 3-acetylpyridine adenine dinucleotide (AcPyrAD) also acts as a substrate for this enzyme-cycling assay. In fact, AcPyrAD generates amplification signals of a larger amplitude than those obtained with NAD. We exploited this finding to devise and validate a novel method for assaying the base-exchange activity of SARM1 in reactions containing NAD and an excess of the free base 3-acetylpyridine (AcPyr), where the product is AcPyrAD. We then used this assay to study competition between AcPyr and other free bases to rank the preference of SARM1 for different base-exchange substrates, identifying isoquinoline as a highly effect substrate that completely outcompetes even AcPyr. This has significant advantages over traditional HPLC methods for assaying SARM1 base exchange as it is rapid, sensitive, cost-effective, and easily scalable. This could represent a useful tool given current interest in the role of SARM1 base exchange in programmed axon death and related human disorders. It may also be applicable to other multifunctional NAD glycohydrolases (EC 3.2.2.6) that possess similar base-exchange activity.