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Lessons from Injury: How Nerve Injury Studies Reveal Basic Biological Mechanisms and Therapeutic Opportunities for Peripheral Nerve Diseases.
Arthur-Farraj P, Coleman MP

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.

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Neurotherapeutics : the journal of the American Society for Experimental NeuroTherapeutics, 1, 1, , 30 Sep 2021


A Novel NAD Signaling Mechanism in Axon Degeneration and its Relationship to Innate Immunity.
Hopkins EL, Gu W, Kobe B, Coleman MP

Axon degeneration represents a pathological feature of many neurodegenerative diseases, including Alzheimer's disease and Parkinson's disease where axons die before the neuronal soma, and axonopathies, such as Charcot-Marie-Tooth disease and hereditary spastic paraplegia. Over the last two decades, it has slowly emerged that a central signaling pathway forms the basis of this process in many circumstances. This is an axonal NAD-related signaling mechanism mainly regulated by the two key proteins with opposing roles: the NAD-synthesizing enzyme NMNAT2, and SARM1, a protein with NADase and related activities. The crosstalk between the axon survival factor NMNAT2 and pro-degenerative factor SARM1 has been extensively characterized and plays an essential role in maintaining the axon integrity. This pathway can be activated in necroptosis and in genetic, toxic or metabolic disorders, physical injury and neuroinflammation, all leading to axon pathology. SARM1 is also known to be involved in regulating innate immunity, potentially linking axon degeneration to the response to pathogens and intercellular signaling. Understanding this NAD-related signaling mechanism enhances our understanding of the process of axon degeneration and enables a path to the development of drugs for a wide range of neurodegenerative diseases.

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Frontiers in molecular biosciences, 8, 1, , 2021


A Novel NAD Signaling Mechanism in Axon Degeneration and its Relationship to Innate Immunity.
Hopkins EL, Gu W, Kobe B, Coleman MP

Axon degeneration represents a pathological feature of many neurodegenerative diseases, including Alzheimer's disease and Parkinson's disease where axons die before the neuronal soma, and axonopathies, such as Charcot-Marie-Tooth disease and hereditary spastic paraplegia. Over the last two decades, it has slowly emerged that a central signaling pathway forms the basis of this process in many circumstances. This is an axonal NAD-related signaling mechanism mainly regulated by the two key proteins with opposing roles: the NAD-synthesizing enzyme NMNAT2, and SARM1, a protein with NADase and related activities. The crosstalk between the axon survival factor NMNAT2 and pro-degenerative factor SARM1 has been extensively characterized and plays an essential role in maintaining the axon integrity. This pathway can be activated in necroptosis and in genetic, toxic or metabolic disorders, physical injury and neuroinflammation, all leading to axon pathology. SARM1 is also known to be involved in regulating innate immunity, potentially linking axon degeneration to the response to pathogens and intercellular signaling. Understanding this NAD-related signaling mechanism enhances our understanding of the process of axon degeneration and enables a path to the development of drugs for a wide range of neurodegenerative diseases.

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Frontiers in molecular biosciences, 8, 1, , 2021


Open Access

Protection against oxaliplatin-induced mechanical and thermal hypersensitivity in Sarm1 mice.
Gould SA, White M, Wilbrey AL, Pór E, Coleman MP, Adalbert R

Chemotherapy-induced peripheral neuropathy (CIPN) is a common dose-limiting side effect of cancer treatment, often associated with degeneration of sensory axons or their terminal regions. Presence of the slow Wallerian degeneration protein (WLD), or genetic deletion of sterile alpha and TIR motif containing protein 1 (SARM1), which strongly protect axons from degeneration after injury or axonal transport block, alleviate pain in several CIPN models. However, oxaliplatin can cause an acute pain response, suggesting a different mechanism of pain generation. Here, we tested whether the presence of WLD or absence of SARM1 protects against acute oxaliplatin-induced pain in mice after a single oxaliplatin injection. In BL/6 and Wld mice, oxaliplatin induced significant mechanical and cold hypersensitivities which were absent in Sarm1 mice. Despite the presence of hypersensitivity there was no significant loss of intraepidermal nerve fibers (IENFs) in the footpads of any mice after oxaliplatin treatment, suggesting that early stages of pain hypersensitivity could be independent of axon degeneration. To identify other changes that could underlie the pain response, RNA sequencing was carried out in DRGs from treated and control mice of each genotype. Sarm1 mice had fewer gene expression changes than either BL/6 or Wld mice. This is consistent with the pain measurements in demonstrating that Sarm1DRGs remain relatively unchanged after oxaliplatin treatment, unlike those in BL/6 and Wld mice. Changes in levels of four transcripts - Alas2, Hba-a1, Hba-a2, and Tfrc - correlated with oxaliplatin-induced pain, or absence thereof, across the three genotypes. Our findings suggest that targeting SARM1 could be a viable therapeutic approach to prevent oxaliplatin-induced acute neuropathic pain.

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Experimental neurology, 1, 1, , 15 Jan 2021


Structural basis for RING-Cys-Relay E3 ligase activity and its role in axon integrity.
Mabbitt PD, Loreto A, Déry MA, Fletcher AJ, Stanley M, Pao KC, Wood NT, Coleman MP, Virdee S

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.

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Nature chemical biology, 1, 1, , 03 Aug 2020


Axon Degeneration: Which Method to Choose?
Coleman MP

Axons are diverse. They have different lengths, different branching patterns, and different biological roles. Methods to study axon degeneration are also diverse. The result is a bewildering range of experimental systems in which to study mechanisms of axon degeneration, and it is difficult to extrapolate from one neuron type and one method to another. The purpose of this chapter is to help readers to do this and to choose the methods most appropriate for answering their particular research question.

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Methods in molecular biology (Clifton, N.J.), 2143, 1, , 2020


Loss of Protects Against the Deleterious Effects of Traumatic Brain Injury in .
Hill CS, Sreedharan J, Loreto A, Menon DK, Coleman MP

Traumatic brain injury is a major global cause of death and disability. Axonal injury is a major underlying mechanism of TBI and could represent a major therapeutic target. We provide evidence that targeting the axonal death pathway known as Wallerian degeneration improves outcome in a model of high impact trauma. This cell-autonomous neurodegenerative pathway is initiated following axon injury, and in Drosophila, involves activity of the E3 ubiquitin ligase . We demonstrate that a loss-of-function mutation in the gene rescues deleterious effects of a traumatic injury, including-improved functional outcomes, lifespan, survival of dopaminergic neurons, and retention of synaptic proteins. This data suggests that represents a potential therapeutic target in traumatic injury.

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Frontiers in neurology, 11, 1, , 2020


Human endogenous retrovirus HERV-K(HML-2) RNA causes neurodegeneration through Toll-like receptors.
Dembny P, Newman AG, Singh M, Hinz M, Szczepek M, Krüger C, Adalbert R, Dzaye O, Trimbuch T, Wallach T, Kleinau G, Derkow K, Richard BC, Schipke C, Scheidereit C, Stachelscheid H, Golenbock D, Peters O, Coleman M, Heppner FL, Scheerer P, Tarabykin V, Ruprecht K, Izsvák Z, Mayer J, Lehnardt S

Although human endogenous retroviruses (HERVs) represent a substantial proportion of the human genome and some HERVs, such as HERV-K(HML-2), are reported to be involved in neurological disorders, little is known about their biological function. We report that RNA from an HERV-K(HML-2) envelope gene region binds to and activates human Toll-like receptor (TLR) 8, as well as murine Tlr7, expressed in neurons and microglia, thereby causing neurodegeneration. HERV-K(HML-2) RNA introduced into the cerebrospinal fluid (CSF) of either C57BL/6 wild-type mice or APPPS1 mice, a mouse model for Alzheimer's disease (AD), resulted in neurodegeneration and microglia accumulation. Tlr7-deficient mice were protected against neurodegenerative effects but were resensitized toward HERV-K(HML-2) RNA when neurons ectopically expressed murine Tlr7 or human TLR8. Transcriptome data sets of human AD brain samples revealed a distinct correlation of upregulated HERV-K(HML-2) and TLR8 RNA expression. HERV-K(HML-2) RNA was detectable more frequently in CSF from individuals with AD compared with controls. Our data establish HERV-K(HML-2) RNA as an endogenous ligand for species-specific TLRs 7/8 and imply a functional contribution of human endogenous retroviral transcripts to neurodegenerative processes, such as AD.

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JCI insight, 5, 7, , 09 Apr 2020


Programmed axon degeneration: from mouse to mechanism to medicine.
Coleman MP, Höke A

Wallerian degeneration is a widespread mechanism of programmed axon degeneration. In the three decades since the discovery of the Wallerian degeneration slow (Wld) 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.

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Nature reviews. Neuroscience, 1, 1, , 09 Mar 2020


Beta secretase 1-dependent amyloid precursor protein processing promotes excessive vascular sprouting through NOTCH3 signalling.
Durrant CS, Ruscher K, Sheppard O, Coleman MP, Özen I

Amyloid beta peptides (Aβ) proteins play a key role in vascular pathology in Alzheimer's Disease (AD) including impairment of the blood-brain barrier and aberrant angiogenesis. Although previous work has demonstrated a pro-angiogenic role of Aβ, the exact mechanisms by which amyloid precursor protein (APP) processing and endothelial angiogenic signalling cascades interact in AD remain a largely unsolved problem. Here, we report that increased endothelial sprouting in human-APP transgenic mouse (TgCRND8) tissue is dependent on β-secretase (BACE1) processing of APP. Higher levels of Aβ processing in TgCRND8 tissue coincides with decreased NOTCH3/JAG1 signalling, overproduction of endothelial filopodia and increased numbers of vascular pericytes. Using a novel in vitro approach to study sprouting angiogenesis in TgCRND8 organotypic brain slice cultures (OBSCs), we find that BACE1 inhibition normalises excessive endothelial filopodia formation and restores NOTCH3 signalling. These data present the first evidence for the potential of BACE1 inhibition as an effective therapeutic target for aberrant angiogenesis in AD.

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Cell death & disease, 11, 2, , 06 Feb 2020

DOI: 10.1038/s41419-020-2288-4

Novel HDAC6 Inhibitors Increase Tubulin Acetylation and Rescue Axonal Transport of Mitochondria in a Model of Charcot-Marie-Tooth Type 2F.
Adalbert R, Kaieda A, Antoniou C, Loreto A, Yang X, Gilley J, Hoshino T, Uga K, Makhija MT, Coleman MP

Disruption of axonal transport causes a number of rare, inherited axonopathies and is heavily implicated in a wide range of more common neurodegenerative disorders, many of them age-related. Acetylation of α-tubulin is one important regulatory mechanism, influencing microtubule stability and motor protein attachment. Of several strategies so far used to enhance axonal transport, increasing microtubule acetylation through inhibition of the deacetylase enzyme histone deacetylase 6 (HDAC6) has been one of the most effective. Several inhibitors have been developed and tested in animal and cellular models, but better drug candidates are still needed. Here we report the development and characterization of two highly potent HDAC6 inhibitors, which show low toxicity, promising pharmacokinetic properties, and enhance microtubule acetylation in the nanomolar range. We demonstrate their capacity to rescue axonal transport of mitochondria in a primary neuronal culture model of the inherited axonopathy Charcot-Marie-Tooth Type 2F, caused by a dominantly acting mutation in heat shock protein beta 1.

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ACS chemical neuroscience, 1, 1, , 08 Jan 2020

DOI: 10.1021/acschemneuro.9b00338

Mitochondrial impairment activates the Wallerian pathway through depletion of NMNAT2 leading to SARM1-dependent axon degeneration.
Loreto A, Hill CS, Hewitt VL, Orsomando G, Angeletti C, Gilley J, Lucci C, Sanchez-Martinez A, Whitworth AJ, Conforti L, Dajas-Bailador F, Coleman MP

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

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Neurobiology of disease, 134, 1, , 15 Nov 2019

DOI: 10.1016/j.nbd.2019.104678

Sarm1 deletion suppresses TDP-43-linked motor neuron degeneration and cortical spine loss.
White MA, Lin Z, Kim E, Henstridge CM, Pena Altamira E, Hunt CK, Burchill E, Callaghan I, Loreto A, Brown-Wright H, Mead R, Simmons C, Cash D, Coleman MP, Sreedharan J

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

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Acta neuropathologica communications, 7, 2051-5960, , 2019


Open Access

NAD cleavage activity by animal and plant TIR domains in cell death pathways.
Horsefield S, Burdett H, Zhang X, Manik MK, Shi Y, Chen J, Qi T, Gilley J, Lai JS, Rank MX, Casey LW, Gu W, Ericsson DJ, Foley G, Hughes RO, Bosanac T, von Itzstein M, Rathjen JP, Nanson JD, Boden M, Dry IB, Williams SJ, Staskawicz BJ, Coleman MP, Ve T, Dodds PN, Kobe B

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.

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Science (New York, N.Y.), 365, 1095-9203, , 2019


Severe biallelic loss-of-function mutations in nicotinamide mononucleotide adenylyltransferase 2 (NMNAT2) in two fetuses with fetal akinesia deformation sequence.
Lukacs M, Gilley J, Zhu Y, Orsomando G, Angeletti C, Liu J, Yang X, Park J, Hopkin RJ, Coleman MP, Zhai RG, Stottmann RW

The three nicotinamide mononucleotide adenylyltransferase (NMNAT) family members synthesize the electron carrier nicotinamide adenine dinucleotide (NAD) and are essential for cellular metabolism. In mammalian axons, NMNAT activity appears to be required for axon survival and is predominantly provided by NMNAT2. NMNAT2 has recently been shown to also function as a chaperone to aid in the refolding of misfolded proteins. Nmnat2 deficiency in mice, or in its ortholog dNmnat in Drosophila, results in axon outgrowth and survival defects. Peripheral nerve axons in NMNAT2-deficient mice fail to extend and innervate targets, and skeletal muscle is severely underdeveloped. In addition, removing NMNAT2 from established axons initiates axon death by Wallerian degeneration. We report here on two stillborn siblings with fetal akinesia deformation sequence (FADS), severely reduced skeletal muscle mass and hydrops fetalis. Clinical exome sequencing identified compound heterozygous NMNAT2 variant alleles in both cases. Both protein variants are incapable of supporting axon survival in mouse primary neuron cultures when overexpressed. In vitro assays demonstrate altered protein stability and/or defects in NAD synthesis and chaperone functions. Thus, both patient NMNAT2 alleles are null or severely hypo-morphic. These data indicate a previously unknown role for NMNAT2 in human neurological development and provide the first direct molecular evidence to support the involvement of Wallerian degeneration in a human axonal disorder. SIGNIFICANCE: Nicotinamide Mononucleotide Adenylyltransferase 2 (NMNAT2) both synthesizes the electron carrier Nicotinamide Adenine Dinucleotide (NAD) and acts a protein chaperone. NMNAT2 has emerged as a major neuron survival factor. Overexpression of NMNAT2 protects neurons from Wallerian degeneration after injury and declining levels of NMNAT2 have been implicated in neurodegeneration. While the role of NMNAT2 in neurodegeneration has been extensively studied, the role of NMNAT2 in human development remains unclear. In this work, we present the first human variants in NMNAT2 identified in two fetuses with severe skeletal muscle hypoplasia and fetal akinesia. Functional studies in vitro showed that the mutations impair both NMNAT2 NAD synthase and chaperone functions. This work identifies the critical role of NMNAT2 in human development.

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Experimental neurology, 320, 1090-2430, , 2019


Homozygous NMNAT2 mutation in sisters with polyneuropathy and erythromelalgia.
Huppke P, Wegener E, Gilley J, Angeletti C, Kurth I, Drenth JPH, Stadelmann C, Barrantes-Freer A, Brück W, Thiele H, Nürnberg P, Gärtner J, Orsomando G, Coleman MP

We identified a homozygous missense mutation in the gene encoding NAD synthesizing enzyme NMNAT2 in two siblings with childhood onset polyneuropathy with erythromelalgia. No additional homozygotes for this rare allele, which leads to amino acid substitution T94M, were present among the unaffected relatives tested or in the 60,000 exomes of the ExAC database. For axons to survive, axonal NMNAT2 activity has to be maintained above a threshold level but the T94M mutation confers a partial loss of function both in the ability of NMNAT2 to support axon survival and in its enzymatic properties. Electrophysiological tests and histological analysis of sural nerve biopsies in the patients were consistent with loss of distal sensory and motor axons. Thus, it is likely that NMNAT2 mutation causes this pain and axon loss phenotype making this the first disorder associated with mutation of a key regulator of Wallerian-like axon degeneration in humans. This supports indications from numerous animal studies that the Wallerian degeneration pathway is important in human disease and raises important questions about which other human phenotypes could be linked to this gene.

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Experimental neurology, 320, 1090-2430, , 2019


Lipopolysaccharide-induced neuroinflammation induces presynaptic disruption through a direct action on brain tissue involving microglia-derived interleukin 1 beta.
Sheppard O, Coleman MP, Durrant CS

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.

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Journal of neuroinflammation, 16, 1742-2094, , 2019


Open Access

A closer look at neuron interaction with track-etched microporous membranes.
George JH, Nagel D, Waller S, Hill E, Parri HR, Coleman MD, Cui Z, Ye H

Microporous membranes support the growth of neurites into and through micro-channels, providing a different type of neural growth platform to conventional dish cultures. Microporous membranes are used to support various types of culture, however, the role of pore diameter in relation to neurite growth through the membrane has not been well characterised. In this study, the human cell line (SH-SY5Y) was differentiated into neuron-like cells and cultured on track-etched microporous membranes with pore and channel diameters selected to accommodate neurite width (0.8 µm to 5 µm). Whilst neurites extended through all pore diameters, the extent of neurite coverage on the non-seeded side of the membranes after 5 days in culture was found to be directly proportional to channel diameter. Neurite growth through membrane pores reduced significantly when neural cultures were non-confluent. Scanning electron microscopy revealed that neurites bridged pores and circumnavigated pore edges - such that the overall likelihood of a neurite entering a pore channel was decreased. These findings highlight the role of pore diameter, cell sheet confluence and contact guidance in directing neurite growth through pores and may be useful in applications that seek to use physical substrates to maintain separate neural populations whilst permitting neurite contact between cultures.

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Scientific reports, 8, 2045-2322, , 2018


Open Access

P7C3-A20 neuroprotection is independent of Wallerian degeneration in primary neuronal culture.
Hill CS, Menon DK, Coleman MP

The antiapoptotic, neuroprotective compound P7C3-A20 reduces neurological deficits when administered to murine in-vivo models of traumatic brain injury. P7C3-A20 is thought to exert its activity through small-molecule activation of the enzyme nicotinamide phosphoribosyltransferase. This enzyme converts nicotinamide to nicotinamide mononucleotide, the precursor to nicotinamide adenine dinucleotide synthesis. Alterations to this bioenergetic pathway have been shown to induce Wallerian degeneration (WD) of the distal neurite following injury. This study aimed to establish whether P7C3-A20, through induction of nicotinamide phosphoribosyltransferase activity, would affect the rate of WD. The model systems used were dissociated primary cortical neurons, dissociated superior cervical ganglion neurons and superior cervical ganglion explants. P7C3-A20 failed to show any protection against WD induced by neurite transection or vincristine administration. Furthermore, there was a concentration-dependent neurotoxicity. These findings are important in understanding the mechanism by which P7C3-A20 mediates its effects - a key step before moving to human clinical trials.This is an open access article distributed under the Creative Commons Attribution License 4.0 (CCBY), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

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Neuroreport, , 1473-558X, , 2018


Open Access

Low levels of NMNAT2 compromise axon development and survival.
Gilley J, Mayer P, Yu G, Coleman MP

NMNAT2 is an endogenous axon maintenance factor that preserves axon health by blocking Wallerian-like axon degeneration. Mice lacking NMNAT2 die at birth with severe axon defects in both the PNS and CNS so a complete absence of NMNAT2 in humans is likely to be similarly harmful, but probably rare. However, there is evidence of widespread natural variation in human NMNAT2 mRNA expression so it is important to establish whether reduced levels of NMNAT2 have consequences that impact health. Whilst mice that express reduced levels of NMNAT2, either those heterozygous for a silenced Nmnat2 allele, or compound heterozygous for one silenced and one partially silenced Nmnat2 allele, remain overtly normal into old age, we now report that Nmnat2 compound heterozygote mice present with early and age-dependent peripheral nerve axon defects. Compound heterozygote mice already have reduced numbers of myelinated sensory axons at 1.5 months and lose more axons, likely motor axons, between 18 and 24 months and, crucially, these changes correlate with early temperature insensitivity and a later-onset decline in motor performance. Slower neurite outgrowth and increased sensitivity to axonal stress are also evident in primary cultures of Nmnat2 compound heterozygote superior cervical ganglion neurons. These data reveal that reducing NMNAT2 levels below a particular threshold compromises the development of peripheral axons and increases their vulnerability to stresses. We discuss the implications for human neurological phenotypes where axons are longer and have to be maintained over a much longer lifespan.

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Human molecular genetics, , 1460-2083, , 2018


Publisher Correction: TDP-43 gains function due to perturbed autoregulation in a Tardbp knock-in mouse model of ALS-FTD.
White MA, Kim E, Duffy A, Adalbert R, Phillips BU, Peters OM, Stephenson J, Yang S, Massenzio F, Lin Z, Andrews S, Segonds-Pichon A, Metterville J, Saksida LM, Mead R, Ribchester RR, Barhomi Y, Serre T, Coleman MP, Fallon JR, Bussey TJ, Brown RH, Sreedharan J

In the version of this article initially published, the footnote number 17 was missing from the author list for the two authors who contributed equally. Also, the authors have added a middle initial for author Justin R. Fallon and an acknowledgement to the Babraham Institute Imaging Facility and Sequencing Core Facility. The errors have been corrected in the HTML and PDF versions of the article.

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Nature neuroscience, , 1546-1726, , 2018


Interaction between a MAPT variant causing frontotemporal dementia and mutant APP affects axonal transport.
Adalbert R, Milde S, Durrant C, Ando K, Stygelbout V, Yilmaz Z, Gould S, Brion JP, Coleman MP

In Alzheimer's disease, many indicators point to a central role for poor axonal transport, but the potential for stimulating axonal transport to alleviate the disease remains largely untested. Previously, we reported enhanced anterograde axonal transport of mitochondria in 8- to 11-month-old MAPT knockin mice, a genetic model of frontotemporal dementia with parkinsonism-17T. In this study, we further characterized the axonal transport of mitochondria in younger MAPT mice crossed with the familial Alzheimer's disease model, TgCRND8, aiming to test whether boosting axonal transport in young TgCRND8 mice can alleviate axonal swelling. We successfully replicated the enhancement of anterograde axonal transport in young MAPT knockin animals. Surprisingly, we found that in the presence of the amyloid precursor protein mutations, MAPT impaired anterograde axonal transport. The numbers of plaque-associated axonal swellings or amyloid plaques in TgCRND8 brains were unaltered. These findings suggest that amyloid-β promotes an action of mutant tau that impairs axonal transport. As amyloid-β levels increase with age even without amyloid precursor protein mutation, we suggest that this rise could contribute to age-related decline in frontotemporal dementia.

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Neurobiology of aging, 68, 1558-1497, , 2018


Open Access

TDP-43 gains function due to perturbed autoregulation in a Tardbp knock-in mouse model of ALS-FTD.
White MA, Kim E, Duffy A, Adalbert R, Phillips BU, Peters OM, Stephenson J, Yang S, Massenzio F, Lin Z, Andrews S, Segonds-Pichon A, Metterville J, Saksida LM, Mead R, Ribchester RR, Barhomi Y, Serre T, Coleman MP, Fallon J, Bussey TJ, Brown RH, Sreedharan J

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-43mice 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.

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Nature neuroscience, , 1546-1726, , 2018


Neuronal Cell Death.
Fricker M, Tolkovsky AM, Borutaite V, Coleman M, Brown GC

Neuronal cell death occurs extensively during development and pathology, where it is especially important because of the limited capacity of adult neurons to proliferate or be replaced. The concept of cell death used to be simple as there were just two or three types, so we just had to work out which type was involved in our particular pathology and then block it. However, we now know that there are at least a dozen ways for neurons to die, that blocking a particular mechanism of cell death may not prevent the cell from dying, and that non-neuronal cells also contribute to neuronal death. We review here the mechanisms of neuronal death by intrinsic and extrinsic apoptosis, oncosis, necroptosis, parthanatos, ferroptosis, sarmoptosis, autophagic cell death, autosis, autolysis, paraptosis, pyroptosis, phagoptosis, and mitochondrial permeability transition. We next explore the mechanisms of neuronal death during development, and those induced by axotomy, aberrant cell-cycle reentry, glutamate (excitoxicity and oxytosis), loss of connected neurons, aggregated proteins and the unfolded protein response, oxidants, inflammation, and microglia. We then reassess which forms of cell death occur in stroke and Alzheimer's disease, two of the most important pathologies involving neuronal cell death. We also discuss why it has been so difficult to pinpoint the type of neuronal death involved, if and why the mechanism of neuronal death matters, the molecular overlap and interplay between death subroutines, and the therapeutic implications of these multiple overlapping forms of neuronal death.

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Physiological reviews, 98, 1522-1210, , 2018


Sarm1 Deletion, but Not Wld(S), Confers Lifelong Rescue in a Mouse Model of Severe Axonopathy.
Gilley J, Ribchester RR, Coleman MP

Studies with the Wld(S) mutant mouse have shown that axon and synapse pathology in several models of neurodegenerative diseases are mechanistically related to injury-induced axon degeneration (Wallerian degeneration). Crucially, an absence of SARM1 delays Wallerian degeneration as robustly as Wld(S), but their relative capacities to confer long-term protection against related, non-injury axonopathy and/or synaptopathy have not been directly compared. While Sarm1 deletion or Wld(S) can rescue perinatal lethality and widespread Wallerian-like axonopathy in young NMNAT2-deficient mice, we report that an absence of SARM1 enables these mice to survive into old age with no overt phenotype, whereas those rescued by Wld(S) invariantly develop a progressive neuromuscular defect in their hindlimbs from around 3 months of age. We therefore propose Sarm1 deletion as a more reliable tool than Wld(S) for investigating Wallerian-like mechanisms in disease models and suggest that SARM1 blockade may have greater therapeutic potential than WLD(S)-related strategies.

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Cell reports, 21, 2211-1247, , 2017


Open Access

NMN Deamidase Delays Wallerian Degeneration and Rescues Axonal Defects Caused by NMNAT2 Deficiency In Vivo.
Di Stefano M, Loreto A, Orsomando G, Mori V, Zamporlini F, Hulse RP, Webster J, Donaldson LF, Gering M, Raffaelli N, Coleman MP, Gilley J, Conforti L

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

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Current biology : CB, , 1879-0445, , 2017


Open Access

KIF1A mediates axonal transport of BACE1 and identification of independently moving cargoes in living SCG neurons.
Hung CO, Coleman MP

Neurons rely heavily on axonal transport to deliver materials from the sites of synthesis to the axon terminals over distances that can be many centimetres long. KIF1A is the neuron-specific kinesin with the fastest reported anterograde motor activity. Previous studies have shown that KIF1A transports a subset of synaptic proteins, neurofilaments and dense-core vesicles. Using two-colour live imaging, we showed that BACE1-mCherry moves together with KIF1A-GFP in both the anterograde and retrograde directions in SCG neurons. We confirmed that KIF1A is functionally required for BACE1 transport by using KIF1A siRNA and a KIF1A mutant construct (KIF1A-T312M) to impair its motor activity. We further identified several cargoes that have little or no co-migration with KIF1A-GFP and also move independently from BACE1-mCherry. Together, these findings support a primary role for KIF1A in the anterograde transport of BACE1 and suggest that axonally transported cargoes are sorted into different classes of carrier vesicles in the cell body and are transported by cargo-specific motor proteins through the axon.

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Traffic (Copenhagen, Denmark), , 1600-0854, , 2016


Open Access

Synaptophysin depletion and intraneuronal Aβ in organotypic hippocampal slice cultures from huAPP transgenic mice.
Harwell CS, Coleman MP

To date, there are no effective disease-modifying treatments for Alzheimer's disease (AD). In order to develop new therapeutics for stages where they are most likely to be effective, it is important to identify the first pathological alterations in the disease cascade. Changes in Aβ concentration have long been reported as one of the first steps, but understanding the source, and earliest consequences, of pathology requires a model system that represents all major CNS cell types, is amenable to repeated observation and sampling, and can be readily manipulated. In this regard, long term organotypic hippocampal slice cultures (OHSCs) from neonatal amyloid mice offer an excellent compromise between in vivo and primary culture studies, largely retaining the cellular composition and neuronal architecture of the in vivo hippocampus, but with the in vitro advantages of accessibility to live imaging, sampling and intervention.

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Molecular neurodegeneration, 11, 1750-1326, , 2016


Open Access

Application of virtual screening to the discovery of novel nicotinamide phosphoribosyltransferase (NAMPT) inhibitors with potential for the treatment of cancer and axonopathies.
Clark DE, Waszkowycz B, Wong M, Lockey PM, Adalbert R, Gilley J, Clark J, Coleman MP

NAMPT may represent a novel target for drug discovery in various therapeutic areas, including oncology and inflammation. Additionally, recent work has suggested that targeting NAMPT has potential in treating axon degeneration. In this work, publicly available X-ray co-crystal structures of NAMPT and the structures of two known NAMPT inhibitors were used as the basis for a structure- and ligand-based virtual screening campaign. From this, two novel series of NAMPT inhibitors were identified, one of which showed a statistically significant protective effect when tested in a cellular model of axon degeneration.

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Bioorganic & medicinal chemistry letters, , 1464-3405, , 2016


Open Access

Traumatic Axonal Injury: Mechanisms and Translational Opportunities.
Hill CS, Coleman MP, Menon DK

Traumatic axonal injury (TAI) is an important pathoanatomical subgroup of traumatic brain injury (TBI) and a major driver of mortality and functional impairment. Experimental models have provided insights into the effects of mechanical deformation on the neuronal cytoskeleton and the subsequent processes that drive axonal injury. There is also increasing recognition that axonal or white matter loss may progress for years post-injury and represent one mechanistic framework for progressive neurodegeneration after TBI. Previous trials of novel therapies have failed to make an impact on clinical outcome, in both TBI in general and TAI in particular. Recent advances in understanding the cellular and molecular mechanisms of injury have the potential to translate into novel therapeutic targets.

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Trends in neurosciences, 39, 1878-108X, , 2016


Open Access

Mislocalization of neuronal tau in the absence of tangle pathology in phosphomutant tau knockin mice.
Gilley J, Ando K, Seereeram A, Rodríguez-Martín T, Pooler AM, Sturdee L, Anderton BH, Brion JP, Hanger DP, Coleman MP

Hyperphosphorylation and fibrillar aggregation of the microtubule-associated protein tau are key features of Alzheimer's disease and other tauopathies. To investigate the involvement of tau phosphorylation in the pathological process, we generated a pair of complementary phosphomutant tau knockin mouse lines. One exclusively expresses phosphomimetic tau with 18 glutamate substitutions at serine and/or threonine residues in the proline-rich and first microtubule-binding domains to model hyperphosphorylation, whereas its phosphodefective counterpart has matched alanine substitutions. Consistent with expected effects of genuine phosphorylation, association of the phosphomimetic tau with microtubules and neuronal membranes is severely disrupted in vivo, whereas the phosphodefective mutations have more limited or no effect. Surprisingly, however, age-related mislocalization of tau is evident in both lines, although redistribution appears more widespread and more pronounced in the phosphomimetic tau knockin. Despite these changes, we found no biochemical or immunohistological evidence of pathological tau aggregation in mice of either line up to at least 2 years of age. These findings raise important questions about the role of tau phosphorylation in driving pathology in human tauopathies.

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Neurobiology of aging, 39, 1558-1497, , 2016


Open Access

Reduced number of axonal mitochondria and tau hypophosphorylation in mouse P301L tau knockin neurons.
Rodríguez-Martín T, Pooler AM, Lau DH, Mórotz GM, De Vos KJ, Gilley J, Coleman MP, Hanger DP

Expression of the frontotemporal dementia-related tau mutation, P301L, at physiological levels in adult mouse brain (KI-P301L mice) results in overt hypophosphorylation of tau and age-dependent alterations in axonal mitochondrial transport in peripheral nerves. To determine the effects of P301L tau expression in the central nervous system, we examined the kinetics of mitochondrial axonal transport and tau phosphorylation in primary cortical neurons from P301L knock-in (KI-P301L) mice. We observed a significant 50% reduction in the number of mitochondria in the axons of cortical neurons cultured from KI-P301L mice compared to wild-type neurons. Expression of murine P301L tau did not change the speed, direction of travel or likelihood of movement of mitochondria. Notably, the angle that defines the orientation of the mitochondria in the axon, and the volume of individual moving mitochondria, were significantly increased in neurons expressing P301L tau. We found that murine tau phosphorylation in KI-P301L mouse neurons was diminished and the ability of P301L tau to bind to microtubules was also reduced compared to tau in wild-type neurons. The P301L mutation did not influence the ability of murine tau to associate with membranes in cortical neurons or in adult mouse brain. We conclude that P301L tau is associated with mitochondrial changes and causes an early reduction in murine tau phosphorylation in neurons coupled with impaired microtubule binding of tau. These results support the association of mutant tau with detrimental effects on mitochondria and will be of significance for the pathogenesis of tauopathies.

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Neurobiology of disease, 85, 1095-953X, , 2015


Open Access

Short-term diabetic hyperglycemia suppresses celiac ganglia neurotransmission, thereby impairing sympathetically mediated glucagon responses.
Mundinger TO, Cooper E, Coleman MP, Taborsky GJ

Short-term hyperglycemia suppresses superior cervical ganglia neurotransmission. If this ganglionic dysfunction also occurs in the islet sympathetic pathway, sympathetically mediated glucagon responses could be impaired. Our objectives were 1) to test for a suppressive effect of 7 days of streptozotocin (STZ) diabetes on celiac ganglia (CG) activation and on neurotransmitter and glucagon responses to preganglionic nerve stimulation, 2) to isolate the defect in the islet sympathetic pathway to the CG itself, and 3) to test for a protective effect of the WLD(S) mutation. We injected saline or nicotine in nondiabetic and STZ-diabetic rats and measured fos mRNA levels in whole CG. We electrically stimulated the preganglionic or postganglionic nerve trunk of the CG in nondiabetic and STZ-diabetic rats and measured portal venous norepinephrine and glucagon responses. We repeated the nicotine and preganglionic nerve stimulation studies in nondiabetic and STZ-diabetic WLD(S) rats. In STZ-diabetic rats, the CG fos response to nicotine was suppressed, and the norepinephrine and glucagon responses to preganglionic nerve stimulation were impaired. In contrast, the norepinephrine and glucagon responses to postganglionic nerve stimulation were normal. The CG fos response to nicotine, and the norepinephrine and glucagon responses to preganglionic nerve stimulation, were normal in STZ-diabetic WLD(S) rats. In conclusion, short-term hyperglycemia's suppressive effect on nicotinic acetylcholine receptors of the CG impairs sympathetically mediated glucagon responses. WLD(S) rats are protected from this dysfunction. The implication is that this CG dysfunction may contribute to the impaired glucagon response to insulin-induced hypoglycemia seen early in type 1 diabetes.

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American journal of physiology. Endocrinology and metabolism, 309, 1522-1555, , 2015


Absence of SARM1 rescues development and survival of NMNAT2-deficient axons.
Gilley J, Orsomando G, Nascimento-Ferreira I, Coleman MP

SARM1 function and nicotinamide mononucleotide adenylyltransferase 2 (NMNAT2) loss both promote axon degeneration, but their relative relationship in the process is unknown. Here, we show that NMNAT2 loss and resultant changes to NMNAT metabolites occur in injured SARM1-deficient axons despite their delayed degeneration and that axon degeneration specifically induced by NMNAT2 depletion requires SARM1. Strikingly, SARM1 deficiency also corrects axon outgrowth in mice lacking NMNAT2, independently of NMNAT metabolites, preventing perinatal lethality. Furthermore, NAMPT inhibition partially restores outgrowth of NMNAT2-deficient axons, suggesting that the NMNAT substrate, NMN, contributes to this phenotype. NMNAT2-depletion-dependent degeneration of established axons and restricted extension of developing axons are thus both SARM1 dependent, and SARM1 acts either downstream of NMNAT2 loss and NMN accumulation in a linear pathway or in a parallel branch of a convergent pathway. Understanding the pathway will help establish relationships with other modulators of axon survival and facilitate the development of effective therapies for axonopathies.

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Cell reports, 10, 2211-1247, , 2015


Open Access

Axonal transport declines with age in two distinct phases separated by a period of relative stability.
Milde S, Adalbert R, Elaman MH, Coleman MP

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.

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Neurobiology of aging, , 1558-1497, , 2014


Open Access

The Axon-Protective WLD(S) Protein Partially Rescues Mitochondrial Respiration and Glycolysis After Axonal Injury.
Godzik K, Coleman MP

The axon-protective Wallerian degeneration slow (WLD(S)) protein can ameliorate the decline in axonal ATP levels after neurite transection. Here, we tested the hypothesis that this effect is associated with maintenance of mitochondrial respiration and/or glycolysis. We used isolated neurites of superior cervical ganglion (SCG) cultures in the Seahorse XF-24 Metabolic Flux Analyser to determine mitochondrial respiration and glycolysis under different conditions. We observed that both mitochondrial respiration and glycolysis declined significantly during the latent phase of Wallerian degeneration. WLD(S) partially reduced the decline both in glycolysis and in mitochondrial respiration. In addition, we found that depleting NAD levels in uncut cultures led to changes in mitochondrial respiration and glycolysis similar to those rescued by WLD(S) after cut, suggesting that the maintenance of NAD levels in Wld (S) neurites after axonal injury at least partially underlies the maintenance of ATP levels. However, by using another axon-protective mutation (Sarm1 (-/-)), we could demonstrate that rescue of basal ECAR (and hence probably glycolysis) rather than basal OCR (mitochondrial respiration) may be part of the protective phenotype to delay Wallerian degeneration. These findings open new routes to study glycolysis and the connection between NAD and ATP levels in axon degeneration, which may help to eventually develop therapeutic strategies to treat neurodegenerative diseases.

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Journal of molecular neuroscience : MN, , 1559-1166, , 2014


Open Access

A rise in NAD precursor nicotinamide mononucleotide (NMN) after injury promotes axon degeneration.
Di Stefano M, Nascimento-Ferreira I, Orsomando G, Mori V, Gilley J, Brown R, Janeckova L, Vargas ME, Worrell LA, Loreto A, Tickle J, Patrick J, Webster JR, Marangoni M, Carpi FM, Pucciarelli S, Ro.ssi F, Meng W, Sagasti A, Ribchester RR, Magni G, Coleman MP, Conforti L

NAD metabolism regulates diverse biological processes, including ageing, circadian rhythm and axon survival. Axons depend on the activity of the central enzyme in NAD biosynthesis, nicotinamide mononucleotide adenylyltransferase 2 (NMNAT2), for their maintenance and degenerate rapidly when this activity is lost. However, whether axon survival is regulated by the supply of NAD or by another action of this enzyme remains unclear. Here we show that the nucleotide precursor of NAD, nicotinamide mononucleotide (NMN), accumulates after nerve injury and promotes axon degeneration. Inhibitors of NMN-synthesising enzyme NAMPT confer robust morphological and functional protection of injured axons and synapses despite lowering NAD. Exogenous NMN abolishes this protection, suggesting that NMN accumulation within axons after NMNAT2 degradation could promote degeneration. Ectopic expression of NMN deamidase, a bacterial NMN-scavenging enzyme, prolongs survival of injured axons, providing genetic evidence to support such a mechanism. NMN rises prior to degeneration and both the NAMPT inhibitor FK866 and the axon protective protein Wld(S) prevent this rise. These data indicate that the mechanism by which NMNAT and the related Wld(S) protein promote axon survival is by limiting NMN accumulation. They indicate a novel physiological function for NMN in mammals and reveal an unexpected link between new strategies for cancer chemotherapy and the treatment of axonopathies.Cell Death and Differentiation advance online publication, 17 October 2014; doi:10.1038/cdd.2014.164.

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Cell death and differentiation, , 1476-5403, , 2014


Open Access

Identification of palmitoyltransferase and thioesterase enzymes that control the subcellular localization of axon survival factor nicotinamide mononucleotide adenylyltransferase 2 (NMNAT2).
Milde S, Coleman MP

The NAD-synthesizing enzyme nicotinamide mononucleotide adenylyltransferase 2 (NMNAT2) is a critical survival factor for axons and its constant supply from neuronal cell bodies into axons is required for axon survival in primary culture neurites and axon extension in vivo. Recently, we showed that palmitoylation is necessary to target NMNAT2 to post-Golgi vesicles, thereby influencing its protein turnover and axon protective capacity. Here we find that NMNAT2 is a substrate for cytosolic thioesterases APT1 and APT2 and that palmitoylation/depalmitoylation dynamics are on a time scale similar to its short half-life. Interestingly, however, depalmitoylation does not release NMNAT2 from membranes. The mechanism of palmitoylation-independent membrane attachment appears to be mediated by the same minimal domain required for palmitoylation itself. Furthermore, we identify several zDHHC palmitoyltransferases that influence NMNAT2 palmitoylation and subcellular localization, among which a role for zDHHC17 (HIP14) in neuronal NMNAT2 palmitoylation is best supported by our data. These findings shed light on the enzymatic regulation of NMNAT2 palmitoylation and highlight individual thioesterases and palmitoyltransferases as potential targets to modulate NMNAT2-dependent axon survival.

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The Journal of biological chemistry, 289, 1083-351X, , 2014


Open Access

Age-related axonal swellings precede other neuropathological hallmarks in a knock-in mouse model of Huntington's disease.
Marangoni M, Adalbert R, Janeckova L, Patrick J, Kohli J, Coleman MP, Conforti L

Axon degeneration precedes cell body death in many age-related neurodegenerative disorders, often determining symptom onset and progression. A sensitive method for revealing axon pathology could indicate whether this is the case also in Huntington's disease (HD), a fatal, devastating neurodegenerative disorder causing progressive deterioration of both physical and mental abilities, and which brain region is affected first. We studied the spatio-temporal relationship between axon pathology, neuronal loss, and mutant Huntingtin aggregate formation in HD mouse models by crossing R6/2 transgenic and HdhQ140 knock-in mice with YFP-H mice expressing the yellow fluorescent protein in a subset of neurons. We found large axonal swellings developing age-dependently first in stria terminalis and then in corticostriatal axons of HdhQ140 mice, whereas alterations of other neuronal compartments could not be detected. Although mutant Huntingtin accumulated with age in several brain areas, inclusions in the soma did not correlate with swelling of the corresponding axons. Axon abnormalities were not a prominent feature of the rapid progressive pathology of R6/2 mice. Our findings in mice genetically similar to HD patients suggest that axon pathology is an early event in HD and indicate the importance of further studies of stria terminalis axons in man.

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Neurobiology of aging, 35, 1558-1497, , 2014


Open Access

Wallerian degeneration: an emerging axon death pathway linking injury and disease.
Conforti L, Gilley J, Coleman MP

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.

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Nature reviews. Neuroscience, 15, 1471-0048, , 2014


Open Access

Axonal trafficking of NMNAT2 and its roles in axon growth and survival in vivo.
Milde S, Gilley J, Coleman MP

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.

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Bioarchitecture, 3, 1949-100X, , 0


Open Access

MEK Inhibitor U0126 Reverses Protection of Axons from Wallerian Degeneration Independently of MEK-ERK Signaling.
C Evans, SJ Cook, MP Coleman, J Gilley

Wallerian degeneration is delayed when sufficient levels of proteins with NMNAT activity are maintained within axons after injury. This has been proposed to form the basis of 'slow Wallerian degeneration' (Wld (S)), a neuroprotective phenotype conferred by an aberrant fusion protein, Wld(S). Proteasome inhibition also delays Wallerian degeneration, although much less robustly, with stabilization of NMNAT2 likely to play a key role in this mechanism. The pan-MEK inhibitor U0126 has previously been shown to reverse the axon-protective effects of proteasome inhibition, suggesting that MEK-ERK signaling plays a role in delayed Wallerian degeneration, in addition to its established role in promoting neuronal survival. Here we show that whilst U0126 can also reverse Wld(S)-mediated axon protection, more specific inhibitors of MEK1/2 and MEK5, PD184352 and BIX02189, have no significant effect on the delay to Wallerian degeneration in either situation, whether used alone or in combination. This suggests that an off-target effect of U0126 is responsible for reversion of the axon protective effects of Wld(S) expression or proteasome inhibition, rather than inhibition of MEK1/2-ERK1/2 or MEK5-ERK5 signaling. Importantly, this off-target effect does not appear to result in alterations in the stabilities of either Wld(S) or NMNAT2.

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PloS one, 8, 10, , 2013

DOI: 10.1371/journal.pone.0076505

Open Access

Deletions within its subcellular targeting domain enhance the axon protective capacity of Nmnat2 in vivo.
S Milde, AN Fox, MR Freeman, MP Coleman

The NAD-synthesising enzyme Nmnat2 is a critical survival factor for axons in vitro and in vivo. We recently reported that loss of axonal transport vesicle association through mutations in its isoform-specific targeting and interaction domain (ISTID) reduces Nmnat2 ubiquitination, prolongs its half-life and boosts its axon protective capacity in primary culture neurons. Here, we report evidence for a role of ISTID sequences in tuning Nmnat2 localisation, stability and protective capacity in vivo. Deletion of central ISTID sequences abolishes vesicle association and increases protein stability of fluorescently tagged, transgenic Nmnat2 in mouse peripheral axons in vivo. Overexpression of fluorescently tagged Nmnat2 significantly delays Wallerian degeneration in these mice. Furthermore, while mammalian Nmnat2 is unable to protect transected Drosophila olfactory receptor neuron axons in vivo, mutant Nmnat2s lacking ISTID regions substantially delay Wallerian degeneration. Together, our results establish Nmnat2 localisation and turnover as a valuable target for modulating axon degeneration in vivo.

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Scientific reports, 3, , , 2013

DOI: 10.1038/srep02567

Open Access

Rescue of Peripheral and CNS Axon Defects in Mice Lacking NMNAT2.
J Gilley, R Adalbert, G Yu, MP Coleman

NMNAT2 is an NAD(+)-synthesizing enzyme with an essential axon maintenance role in primary culture neurons. We have generated an Nmnat2 gene trap mouse to examine the role of NMNAT2 in vivo. Homozygotes die perinatally with a severe peripheral nerve/axon defect and truncated axons in the optic nerve and other CNS regions. The cause appears to be limited axon extension, rather than dying-back degeneration of existing axons, which was previously proposed for the NMNAT2-deficient Blad mutant mouse. Neurite outgrowth in both PNS and CNS neuronal cultures consistently stalls at 1-2 mm, similar to the length of truncated axons in the embryos. Crucially, this suggests an essential role for NMNAT2 during axon growth. In addition, we show that the Wallerian degeneration slow protein (Wld(S)), a more stable, aberrant NMNAT that can substitute the axon maintenance function of NMNAT2 in primary cultures, can also correct developmental defects associated with NMNAT2 deficiency. This is dose-dependent, with extension of life span to at least 3 months by homozygous levels of Wld(S) the most obvious manifestation. Finally, we propose that endogenous mechanisms also compensate for otherwise limiting levels of NMNAT2. This could explain our finding that conditional silencing of a single Nmnat2 allele triggers substantial degeneration of established neurites, whereas similar, or greater, reduction of NMNAT2 in constitutively depleted neurons is compatible with normal axon growth and survival. A requirement for NMNAT2 for both axon growth and maintenance suggests that reduced levels could impair axon regeneration as well as axon survival in aging and disease.

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The Journal of neuroscience : the official journal of the Society for Neuroscience, 33, 33, , 2013

DOI: 10.1523/JNEUROSCI.1534-13.2013

Open Access

The challenges of axon survival: introduction to the special issue on axonal degeneration.
MP Coleman

Early axon loss is a common feature of many neurodegenerative disorders. It renders neurons functionally inactive, or less active if axon branches are lost, in a manner that is often irreversible. In the CNS, there is no long-range axon regeneration and even peripheral nerve axons are unlikely to reinnervate their targets while the cause of the problem persists. In most disorders, axon degeneration precedes cell death so it is not simply a consequence of it, and it is now clear that axons have at least one degeneration mechanism that differs from that of the soma. It is important to understand these degeneration mechanisms and their contribution to axon loss in neurodegenerative disorders. In this way, it should become possible to prevent axon loss as well as cell death. This special edition considers the roles and mechanisms of axon degeneration in amyotrophic lateral sclerosis, Charcot-Marie-Tooth disease, hereditary spastic paraplegia, ischemic injury, traumatic brain injury, Alzheimer's disease, glaucoma, Huntington's disease and Parkinson's disease. Using examples from these and other disorders, this introduction considers some of the reasons for axon vulnerability. It also illustrates how molecular genetics and studies of Wallerian degeneration have contributed to our understanding of axon degeneration mechanisms.

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Experimental neurology, 246, , , 2013

DOI: 10.1016/j.expneurol.2013.06.007

Open Access

Autophagy in axonal and dendritic degeneration.
Y Yang, M Coleman, L Zhang, X Zheng, Z Yue

Degeneration of axons and dendrites is a common and early pathological feature of many neurodegenerative disorders, and is thought to be regulated by mechanisms distinct from those determining death of the cell body. The unique structures of axons and dendrites (collectively neurites) may cause them to be particularly vulnerable to the accumulation of protein aggregates and damaged organelles. Autophagy is a catabolic mechanism in which cells clear protein aggregates and damaged organelles. Basal autophagy occurs continuously as a housekeeping function, and can be acutely expanded in response to stress or injury. Emerging evidence shows that insufficient or excessive autophagy contributes to neuritic degeneration. Here, we review the recent progress that has begun to reveal the role of autophagy in neurite function and degeneration.

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Trends in neurosciences, 36, 7, , 2013

DOI: 10.1016/j.tins.2013.04.001

Open Access

Subcellular localization determines the stability and axon protective capacity of axon survival factor Nmnat2.
S Milde, J Gilley, MP Coleman

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.

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PLoS biology, 11, 4, , 2013

DOI: 10.1371/journal.pbio.1001539

Open Access

Simultaneous single-sample determination of NMNAT isozyme activities in mouse tissues.
G Orsomando, L Cialabrini, A Amici, F Mazzola, S Rugieri, L Conforti, L Janeckova, MP Coleman, G Magni

A novel assay procedure has been developed to allow simultaneous activity discrimination in crude tissue extracts of the three known mammalian nicotinamide mononucleotide adenylyltransferase (NMNAT, EC isozymes. These enzymes catalyse the same key reaction for NAD biosynthesis in different cellular compartments. The present method has been optimized for NMNAT isozymes derived from Mus musculus, a species often used as a model for NAD-biosynthesis-related physiology and disorders, such as peripheral neuropathies. Suitable assay conditions were initially assessed by exploiting the metal-ion dependence of each isozyme recombinantly expressed in bacteria, and further tested after mixing them in vitro. The variable contributions of the three individual isozymes to total NAD synthesis in the complex mixture was calculated by measuring reaction rates under three selected assay conditions, generating three linear simultaneous equations that can be solved by a substitution matrix calculation. Final assay validation was achieved in a tissue extract by comparing the activity and expression levels of individual isozymes, considering their distinctive catalytic efficiencies. Furthermore, considering the key role played by NMNAT activity in preserving axon integrity and physiological function, this assay procedure was applied to both liver and brain extracts from wild-type and Wallerian degeneration slow (Wld(S)) mouse. Wld(S) is a spontaneous mutation causing overexpression of NMNAT1 as a fusion protein, which protects injured axons through a gain-of-function. The results validate our method as a reliable determination of the contributions of the three isozymes to cellular NAD synthesis in different organelles and tissues, and in mutant animals such as Wld(S).

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PloS one, 7, 12, , 2012

DOI: 10.1371/journal.pone.0053271

Open Access

Axon pathology in age-related neurodegenerative disorders.
R Adalbert, MP Coleman

'Dying back' axon degeneration is a prominent feature of many age-related neurodegenerative disorders and is widespread in normal ageing. Although the mechanisms of disease- and age-related losses may differ, both contribute to symptoms. Here, we review recent advances in understanding axon pathology in age-related neurodegenerative disorders such as Alzheimer's disease, Parkinson's disease, amyotrophic lateral sclerosis and glaucoma. In particular, we highlight the importance of axonal transport, autophagy, traumatic brain injury, and mitochondrial quality control. We then place these disease mechanisms in the context of changes to axons and dendrites that occur during normal ageing. We discuss what makes ageing such an important risk factor for many neurodegenerative disorders and conclude that the processes of normal ageing and disease combine at the molecular, cellular or systems levels in a range of disorders to produce symptoms. Pathology identical to disease also occurs at the cellular level in most elderly individuals. Thus, normal ageing and age-related disease are inextricably linked and the term 'healthy ageing' downplays the important contributions of cellular pathology. For a full understanding of normal ageing or age-related disease we must study both processes.

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Neuropathology and applied neurobiology, , , , 2012

DOI: 10.1111/j.1365-2990.2012.01308.x

Intra-axonal calcium changes after axotomy in wild-type and slow Wallerian degeneration axons.
R Adalbert, G Morreale, M Paizs, L Conforti, SA Walker, HL Roderick, MD Bootman, L Siklós, MP Coleman

Calcium accumulation induces the breakdown of cytoskeleton and axonal fragmentation in the late stages of Wallerian degeneration. In the early stages there is no evidence for any long-lasting, extensive increase in intra-axonal calcium but there does appear to be some redistribution. We hypothesized that changes in calcium distribution could have an early regulatory role in axonal degeneration in addition to the late executionary role of calcium. Schmidt-Lanterman clefts (SLCs), which allow exchange of metabolites and ions between the periaxonal and extracellular space, are likely to have an increased role when axon segments are separated from the cell body, so we used the oxalate-pyroantimonate method to study calcium at SLCs in distal stumps of transected wild-type and slow Wallerian degeneration (Wld(S)) mutant sciatic nerves, in which Wallerian degeneration is greatly delayed. In wild-type nerves most SLCs show a step gradient of calcium distribution, which is lost at around 20% of SLCs within 3mm of the lesion site by 4-24h after nerve transection. To investigate further the association with Wallerian degeneration, we studied nerves from Wld(S) rats. The step gradient of calcium distribution in Wld(S) is absent in around 20% of the intact nerves beneath SLCs but 4-24h following injury, calcium distribution in transected axons remained similar to that in uninjured nerves. We then used calcium indicators to study influx and buffering of calcium in injured neurites in primary culture. Calcium penetration and the early calcium increase in this system were indistinguishable between Wld(S) and wild-type axons. However, a significant difference was observed during the following hours, when calcium increased in wild-type neurites but not in Wld(S) neurites. We conclude that there is little relationship between calcium distribution and the early stages of Wallerian degeneration at the time points studied in vivo or in vitro but that Wld(S) neurites fail to show a later calcium rise that could be a cause or consequence of the later stages of Wallerian degeneration.

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Neuroscience, 225, , , 2012

DOI: 10.1016/j.neuroscience.2012.08.056

Open Access

dSarm/Sarm1 is required for activation of an injury-induced axon death pathway.
JM Osterloh, J Yang, TM Rooney, AN Fox, R Adalbert, EH Powell, AE Sheehan, MA Avery, R Hackett, MA Logan, JM MacDonald, JS Ziegenfuss, S Milde, YJ Hou, C Nathan, A Ding, RH Brown, L Conforti, M Coleman, M Tessier-Lavigne, S Züchner, MR Freeman

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.

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Science (New York, N.Y.), 337, 6093, , 2012

DOI: 10.1126/science.1223899

Open Access

Mitochondria as a central sensor for axonal degenerative stimuli.
FA Court, MP Coleman

Axonal degeneration is a major contributor to neuronal dysfunction in many neurological conditions and has additional roles in development. It can be triggered by divergent stimuli including mechanical, metabolic, infectious, toxic, hereditary and inflammatory stresses. Axonal mitochondria are an important convergence point as regulators of bioenergetic metabolism, reactive oxygen species (ROS), Ca²⁺ homeostasis and protease activation. The challenges likely to render axonal mitochondria more vulnerable than their cellular counterparts are reviewed, including axonal transport, replenishing nuclear-encoded proteins and maintenance of quality control, fusion and fission in locations remote from the cell body. The potential for mitochondria to act as a decision node in axon loss is considered, highlighting the need to understand the biology of axonal mitochondria and their contributions to degenerative mechanisms for novel therapeutic strategies.

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Trends in neurosciences, 35, 6, , 2012

DOI: 10.1016/j.tins.2012.04.001

Open Access

Modelling early responses to neurodegenerative mutations in mice.
J Gilley, R Adalbert, MP Coleman

Considering the many differences between mice and humans, it is perhaps surprising how well mice model late-onset human neurodegenerative disease. Models of Alzheimer's disease, frontotemporal dementia, Parkinson's disease and Huntington's disease show some striking similarities to the corresponding human pathologies in terms of axonal transport disruption, protein aggregation, synapse loss and some behavioural phenotypes. However, there are also major differences. To extrapolate from mouse models to human disease, we need to understand how these differences relate to intrinsic limitations of the mouse system and to the effects of transgene overexpression. In the present paper, we use examples from an amyloid-overexpression model and a mutant-tau-knockin model to illustrate what we learn from each type of approach and what the limitations are. Finally, we discuss the further contributions that knockin and similar approaches can make to understanding pathogenesis and how best to model disorders of aging in a short-lived mammal.

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Biochemical Society transactions, 39, 4, , 2011

DOI: 10.1042/BST0390933

Reducing expression of NAD+ synthesizing enzyme NMNAT1 does not affect the rate of Wallerian degeneration.
L Conforti, L Janeckova, D Wagner, F Mazzola, L Cialabrini, M Di Stefano, G Orsomando, G Magni, C Bendotti, N Smyth, M Coleman

NAD(+) synthesizing enzyme NMNAT1 constitutes most of the sequence of neuroprotective protein Wld(S), which delays axon degeneration by 10-fold. NMNAT1 activity is necessary but not sufficient for Wld(S) neuroprotection in mice and 70 amino acids at the N-terminus of Wld(S), derived from polyubiquitination factor Ube4b, enhance axon protection by NMNAT1. NMNAT1 activity can confer neuroprotection when redistributed outside the nucleus or when highly overexpressed in vitro 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.

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The FEBS journal, 278, 15, , 2011

DOI: 10.1111/j.1742-4658.2011.08193.x

Age-dependent axonal transport and locomotor changes and tau hypophosphorylation in a "P301L" tau knockin mouse.
J Gilley, A Seereeram, K Ando, S Mosely, S Andrews, M Kerschensteiner, T Misgeld, JP Brion, B Anderton, DP Hanger, MP Coleman

Tauopathies are characterized by hyperphosphorylation of the microtubule-associated protein tau and its accumulation into fibrillar aggregates. Toxic effects of aggregated tau and/or dysfunction of soluble tau could both contribute to neural defects in these neurodegenerative diseases. We have generated a novel knockin mouse model of an inherited tauopathy, frontotemporal dementia with parkinsonism linked to tau mutations on chromosome 17 (FTDP-17T). We incorporated a single mutation, homologous to the common FTDP-17T P301L mutation, directly into the endogenous mouse gene, mimicking the human disease situation. These mice express P301L-equivalent mutant tau at normal physiological levels from the knockin allele. Importantly, in contrast to existing transgenic mouse models that overexpress human P301L mutant tau, no overt tau pathology developed during the normal lifespan of the knockin mice. In fact, overall phosphorylation of tau was reduced, perhaps due to reduced microtubule binding. However, homozygous knockin mice did display intriguing age-dependent changes in axonal transport of mitochondria, and increased spontaneous locomotor activity in old age. These could represent early consequences of the tau dysfunction that eventually precipitates pathogenesis in humans.

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Neurobiology of aging, 33, 3, , 2012

DOI: 10.1016/j.neurobiolaging.2011.02.014

Molecular signaling how do axons die?
M Coleman

Axons depend critically on axonal transport both for supplying materials and for communicating with cell bodies. This chapter looks at each activity, asking what aspects are essential for axon survival. Axonal transport declines in neurodegenerative disorders, such as Alzheimer's disease, amyotrophic lateral sclerosis, and multiple sclerosis, and in normal ageing, but whether all cargoes are equally affected and what limits axon survival remains unclear. Cargoes can be differentially blocked in some disorders, either individually or in groups. Each missing protein cargo results in localized loss-of-function that can be partially modeled by disrupting the corresponding gene, sometimes with surprising results. The axonal response to losing specific proteins also depends on the rates of protein turnover and on whether the protein can be locally synthesized. Among cargoes with important axonal roles are components of the PI3 kinase, Mek/Erk, and Jnk signaling pathways, which help to communicate with cell bodies and to regulate axonal transport itself. Bidirectional trafficking of Bdnf, NT-3, and other neurotrophic factors contribute to intra- and intercellular signaling, affecting the axon's cellular environment and survival. Finally, several adhesion molecules and gangliosides are key determinants of axon survival, probably by mediating axon-glia interactions. Thus, failure of long-distance intracellular transport can deprive axons of one, few, or many cargoes. This can lead to axon degeneration either directly, through the absence of essential axonal proteins, or indirectly, through failures in communication with cell bodies and nonneuronal cells.

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Advances in genetics, 73, , , 2011

DOI: 10.1016/B978-0-12-380860-8.00005-7

The Wlds transgene reduces axon loss in a Charcot-Marie-Tooth disease 1A rat model and nicotinamide delays post-traumatic axonal degeneration.
G Meyer zu Horste, TA Miesbach, JI Muller, R Fledrich, RM Stassart, BC Kieseier, MP Coleman, MW Sereda

Charcot-Marie-Tooth disease (CMT) is the most common inherited neuropathy and a duplication of the peripheral myelin protein of 22 kDa (PMP22) gene causes the most frequent subform CMT1A. Clinical impairments are determined by the amount of axonal loss. Axons of the spontaneous mouse mutant Wallerian degeneration slow (Wlds) show markedly reduced degeneration following various types of injuries. Protection is conferred by a chimeric Wlds gene encoding an N-terminal part of ubiquitination factor Ube4b and full length nicotinamide mononucleotide adenylyl transferase 1 (Nmnat1). Nmnat1 enzyme generates nicotinamide adenine dinucleotide (NAD) from nicotinamide mononucleotide. Here, in a Pmp22 transgenic animal model of Charcot-Marie-Tooth disease type 1A (CMT rat), the Wlds transgene reduced axonal loss and clinical impairments without altering demyelination. Furthermore, nicotinamide - substrate precursor of the Nmnat1 enzyme - transiently delayed posttraumatic axonal degeneration in an in vivo model of acute peripheral nerve injury, but to a lower extent than Wlds. In contrast, 8 weeks of nicotinamide treatment did not influence axonal loss or clinical manifestations in the CMT rat. Therefore, nicotinamide can partially substitute for the protective Wlds effect in acute traumatic, but not in chronic secondary axonal injury. Future studies are needed to develop axon protective therapy in CMT1A which may be combined with therapeutic strategies aimed at downregulation of toxic PMP22 overexpression.

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Neurobiology of disease, 42, 1, , 2011

DOI: 10.1016/j.nbd.2010.12.006

Targeting NMNAT1 to axons and synapses transforms its neuroprotective potency in vivo.
E Babetto, B Beirowski, L Janeckova, R Brown, J Gilley, D Thomson, RR Ribchester, MP Coleman

Axon and synapse degeneration are common components of many neurodegenerative diseases, and their rescue is essential for effective neuroprotection. The chimeric Wallerian degeneration slow protein (Wld(S)) protects axons dose dependently, but its mechanism is still elusive. We recently showed that Wld(S) acts at a non-nuclear location and is present in axons. This and other recent reports support a model in which Wld(S) protects by extranuclear redistribution of its nuclear NMNAT1 portion. However, it remains unclear whether cytoplasmic NMNAT1 acts locally in axons and synapses or at a non-nuclear site within cell bodies. The potency of axon protection by non-nuclear NMNAT1 relative to Wld(S) also needs to be established in vivo. Because the N-terminal portion of Wld(S) (N70) localized to axons, we hypothesized that it mediates the trafficking of the NMNAT1 portion. To test this, we substituted N70 with an axonal targeting peptide derived from amyloid precursor protein, and fused this to NMNAT1 with disrupted nuclear targeting. In transgenic mice, this transformed NMNAT1 from a molecule unable to inhibit Wallerian degeneration, even at high expression levels, into a protein more potent than Wld(S), able to preserve injured axons for several weeks at undetectable expression levels. Preventing NMNAT1 axonal delivery abolished its protective effect. Axonally targeted NMNAT1 localized to vesicular structures, colocalizing with extranuclear Wld(S), and was cotransported at least partially with mitochondria. We conclude that axonal targeting of NMNAT activity is both necessary and sufficient to delay Wallerian degeneration, and that promoting axonal and synaptic delivery greatly enhances the effectiveness.

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The Journal of neuroscience : the official journal of the Society for Neuroscience, 30, 40, , 2010

DOI: 10.1523/JNEUROSCI.1189-10.2010

Open Access

Difference Tracker: ImageJ plugins for fully automated analysis of multiple axonal transport parameters.
S Andrews, J Gilley, MP Coleman

Studies of axonal transport are critical, not only to understand its normal regulation, but also to determine the roles of transport impairment in disease. Exciting new resources have recently become available allowing live imaging of axonal transport in physiologically relevant settings, such as mammalian nerves. Thus the effects of disease, ageing and therapies can now be assessed directly in nervous system tissue. However, these imaging studies present new challenges. Manual or semi-automated analysis of the range of transport parameters required for a suitably complete evaluation is very time-consuming and can be subjective due to the complexity of the particle movements in axons in ex vivo explants or in vivo. We have developed Difference Tracker, a program combining two new plugins for the ImageJ image-analysis freeware, to provide fast, fully automated and objective analysis of a number of relevant measures of trafficking of fluorescently labeled particles so that axonal transport in different situations can be easily compared. We confirm that Difference Tracker can accurately track moving particles in highly simplified, artificial simulations. It can also identify and track multiple motile fluorescently labeled mitochondria simultaneously in time-lapse image stacks from live imaging of tibial nerve axons, reporting values for a number of parameters that are comparable to those obtained through manual analysis of the same axons. Difference Tracker therefore represents a useful free resource for the comparative analysis of axonal transport under different conditions, and could potentially be used and developed further in many other studies requiring quantification of particle movements.

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Journal of neuroscience methods, 193, 2, , 2010

DOI: 10.1016/j.jneumeth.2010.09.007

Mechanisms of axonal spheroid formation in central nervous system Wallerian degeneration.
B Beirowski, A Nógrádi, E Babetto, G Garcia-Alias, MP Coleman

Wallerian degeneration of the CNS is accompanied by axonal dystrophy or swelling. To understand the mechanisms by which swellings arise, we studied their spatiotemporal dynamics, ultrastructure, composition, and the conditions that affect their formation in vivo and ex vivo. In contrast to peripheral nerve axons, lesioned optic nerve (ON) axons in vivo developed focal swellings asynchronously within 6 hours, long before there is any axon fragmentation. Axons in ON, spinal cord dorsal column, and corpus callosum all showed marked gradients with more swellings in proximal regions of their distal stumps early after lesion. Time-lapse imaging of a validated ex vivo system showed that multiple focal swellings arise after around 1 hour close to the injury site, followed by anterograde wave-like progression on continuous ON axon stumps. Swellings were largely stable but occasionally seemed to fuse with neighboring swellings. Their ultrastructural appearances resembled disease-associated spheroids. Although accumulation of axonal markers suggested transport deficits, large accumulations of mitochondria were not observed. Early swelling formation was decreased in Wld gene-expressing rodents and by removing extracellular calcium. Several pharmacologic agents that inhibit axon loss in vitro and/or in vivo also prevented early formation of axonal spheroids in acute ON explants. Because axonal swellings are hallmarks of many neurodegenerative conditions, these data suggest that they are a manifestation of Wallerian-like degeneration in some cases. Thus, Wallerian-like degeneration may be a more common component mechanism in CNS diseases than previously thought.

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Journal of neuropathology and experimental neurology, 69, 5, , 2010

DOI: 10.1097/NEN.0b013e3181da84db

Wallerian degeneration, wld(s), and nmnat.
MP Coleman, MR Freeman

Traditionally, researchers have believed that axons are highly dependent on their cell bodies for long-term survival. However, recent studies point to the existence of axon-autonomous mechanism(s) that regulate rapid axon degeneration after axotomy. Here, we review the cellular and molecular events that underlie this process, termed Wallerian degeneration. We describe the biphasic nature of axon degeneration after axotomy and our current understanding of how Wld(S)--an extraordinary protein formed by fusing a Ube4b sequence to Nmnat1--acts to protect severed axons. Interestingly, the neuroprotective effects of Wld(S) span all species tested, which suggests that there is an ancient, Wld(S)-sensitive axon destruction program. Recent studies with Wld(S) also reveal that Wallerian degeneration is genetically related to several dying back axonopathies, thus arguing that Wallerian degeneration can serve as a useful model to understand, and potentially treat, axon degeneration in diverse traumatic or disease contexts.

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Annual review of neuroscience, 33, , , 2010

DOI: 10.1146/annurev-neuro-060909-153248

Endogenous Nmnat2 is an essential survival factor for maintenance of healthy axons.
J Gilley, MP Coleman

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.

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PLoS biology, 8, 1, , 2010

DOI: 10.1371/journal.pbio.1000300

Open Access

WldS can delay Wallerian degeneration in mice when interaction with valosin-containing protein is weakened.
B Beirowski, G Morreale, L Conforti, F Mazzola, M Di Stefano, A Wilbrey, E Babetto, L Janeckova, G Magni, MP Coleman

Axon degeneration is an early event in many neurodegenerative disorders. In some, the mechanism is related to injury-induced Wallerian degeneration, a proactive death program that can be strongly delayed by the neuroprotective slow Wallerian degeneration protein (Wld(S)) protein. Thus, it is important to understand the Wallerian degeneration mechanism and how Wld(S) blocks it. Wld(S) location is influenced by binding to valosin-containing protein (VCP), an essential protein for many cellular processes including membrane fusion and endoplasmic reticulum-associated degradation. In mice, the N-terminal 16 amino acids (N16), which mediate VCP binding, are essential for Wld(S) to protect axons, a role which another VCP binding sequence can substitute. In Drosophila, the Wld(S) phenotype is weakened by a similar N-terminal truncation and by knocking down the VCP homologue ter94. Neither null nor floxed VCP mice are viable so it is difficult to confirm the requirement for VCP binding in mammals in vivo. However, the hypothesis can be tested further by introducing a Wld(S) missense mutation, altering its affinity for VCP but minimizing the risk of disturbing other aspects of its structure or function. We introduced the R10A mutation, which weakens VCP binding in vitro, and expressed it in transgenic mice. R10AWld(S) fails to co-immunoprecipitate VCP from mouse brain, and only occasionally and faintly accumulates in nuclear foci for which VCP binding is necessary but not sufficient. Surprisingly however, axon protection remains robust and indistinguishable from that in spontaneous Wld(S) mice. We suggest that either N16 has an additional, VCP-independent function in mammals, or that the phenotype requires only weak VCP binding which may be driven forwards in vivo by the high VCP concentration.

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Neuroscience, 166, 1, , 2010

DOI: 10.1016/j.neuroscience.2009.12.024

Axonal and neuromuscular synaptic phenotypes in Wld(S), SOD1(G93A) and ostes mutant mice identified by fiber-optic confocal microendoscopy.
F Wong, L Fan, S Wells, R Hartley, FE Mackenzie, O Oyebode, R Brown, D Thomson, MP Coleman, G Blanco, RR Ribchester

We used live imaging by fiber-optic confocal microendoscopy (CME) of yellow fluorescent protein (YFP) expression in motor neurons to observe and monitor axonal and neuromuscular synaptic phenotypes in mutant mice. First, we visualized slow degeneration of axons and motor nerve terminals at neuromuscular junctions following sciatic nerve injury in Wld(S) mice with slow Wallerian degeneration. Protection of axotomized motor nerve terminals was much weaker in Wld(S) heterozygotes than in homozygotes. We then induced covert modifiers of axonal and synaptic degeneration in heterozygous Wld(S) mice, by N-ethyl-N-nitrosourea (ENU) mutagenesis, and used CME to identify candidate mutants that either enhanced or suppressed axonal or synaptic degeneration. From 219 of the F1 progeny of ENU-mutagenized BALB/c mice and thy1.2-YFP16/Wld(S) mice, CME revealed six phenodeviants with suppression of synaptic degeneration. Inheritance of synaptic protection was confirmed in three of these founders, with evidence of Mendelian inheritance of a dominant mutation in one of them (designated CEMOP_S5). We next applied CME repeatedly to living Wld(S) mice and to SOD1(G93A) mice, an animal model of motor neuron disease, and observed degeneration of identified neuromuscular synapses over a 1-4day period in both of these mutant lines. Finally, we used CME to observe slow axonal regeneration in the ENU-mutant ostes mouse strain. The data show that CME can be used to monitor covert axonal and neuromuscular synaptic pathology and, when combined with mutagenesis, to identify genetic modifiers of its progression in vivo.

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Molecular and cellular neurosciences, 42, 4, , 2009

DOI: 10.1016/j.mcn.2009.08.002

Tau inclusions in retinal ganglion cells of human P301S tau transgenic mice: effects on axonal viability.
L Gasparini, RA Crowther, KR Martin, N Berg, M Coleman, M Goedert, MG Spillantini

Tau inclusions play a key role in the pathogenesis of tauopathies. Altered tau levels have been detected in retina and optic nerve of patients with glaucoma, suggesting the possibility of shared pathogenic mechanisms with tauopathies. Here we report that hyperphosphorylated transgenic tau accumulates in the nerve fibre layer and, from 2 months of age, aggregates into filamentous inclusions in retinal ganglion cells of human P301S tau transgenic mice. Axonopathy and accumulation of hyperphosphorylated tau in the nerve fibre layer preceded inclusion formation. Hyperphosphorylated tau and tau inclusions were also detected in cultured retinal explants from 5-month-old transgenic mice. Axonal outgrowth was similar in transgenic and wild-type retinal explants under basal conditions. However, when exposed to growth-promoting stimuli, axon elongation was enhanced in explants from wild-type but not transgenic mice, indicating that the presence of abnormal tau can impair stimulated axonal outgrowth. These findings suggest that the retina is a good model system for investigating tau-driven neurodegeneration and for assessing potential pharmacological modifiers for tauopathies.

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Neurobiology of aging, 32, 3, , 2011

DOI: 10.1016/j.neurobiolaging.2009.03.002

Wld S protein requires Nmnat activity and a short N-terminal sequence to protect axons in mice.
L Conforti, A Wilbrey, G Morreale, L Janeckova, B Beirowski, R Adalbert, F Mazzola, M Di Stefano, R Hartley, E Babetto, T Smith, J Gilley, RA Billington, AA Genazzani, RR Ribchester, G Magni, M Coleman

The slow Wallerian degeneration (Wld(S)) protein protects injured axons from degeneration. This unusual chimeric protein fuses a 70-amino acid N-terminal sequence from the Ube4b multiubiquitination factor with the nicotinamide adenine dinucleotide-synthesizing enzyme nicotinamide mononucleotide adenylyl transferase 1. The requirement for these components and the mechanism of Wld(S)-mediated neuroprotection remain highly controversial. The Ube4b domain is necessary for the protective phenotype in mice, but precisely which sequence is essential and why are unclear. Binding to the AAA adenosine triphosphatase valosin-containing protein (VCP)/p97 is the only known biochemical property of the Ube4b domain. Using an in vivo approach, we show that removing the VCP-binding sequence abolishes axon protection. Replacing the Wld(S) VCP-binding domain with an alternative ataxin-3-derived VCP-binding sequence restores its protective function. Enzyme-dead Wld(S) is unable to delay Wallerian degeneration in mice. Thus, neither domain is effective without the function of the other. Wld(S) requires both of its components to protect axons from degeneration.

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The Journal of cell biology, 184, 4, , 2009

DOI: 10.1083/jcb.200807175

Open Access

Evolutionary divergence of valosin-containing protein/cell division cycle protein 48 binding interactions among endoplasmic reticulum-associated degradation proteins.
G Morreale, L Conforti, J Coadwell, AL Wilbrey, MP Coleman

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 protein 48 (Cdc48p), which help to direct polyubiquitinated proteins for proteasome-mediated degradation, interact with an equivalent group of ubiquitin ligases in mouse and in S. cerevisiae. A conserved structural motif for cofactor binding would therefore be expected. We report a VCP-binding motif (VBM) shared by mammalian ubiquitin ligase E4b (Ube4b)-ubiquitin fusion degradation protein 2a (Ufd2a), hydroxymethylglutaryl reductase degradation protein 1 (Hrd1)-synoviolin and ataxin 3, and a related sequence in M(r) 78,000 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. cerevisiae 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.

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The FEBS journal, 276, 5, , 2009

DOI: 10.1111/j.1742-4658.2008.06858.x

Non-nuclear Wld(S) determines its neuroprotective efficacy for axons and synapses in vivo.
B Beirowski, E Babetto, J Gilley, F Mazzola, L Conforti, L Janeckova, G Magni, RR Ribchester, MP Coleman

Axon degeneration contributes widely to neurodegenerative disease but its regulation is poorly understood. The Wallerian degeneration slow (Wld(S)) protein protects axons dose-dependently in many circumstances but is paradoxically abundant in nuclei. To test the hypothesis that Wld(S) acts within nuclei in vivo, we redistributed it from nucleus to cytoplasm in transgenic mice. Surprisingly, instead of weakening the phenotype as expected, extranuclear Wld(S) significantly enhanced structural and functional preservation of transected distal axons and their synapses. In contrast to native Wld(S) mutants, distal axon stumps remained continuous and ultrastructurally intact up to 7 weeks after injury and motor nerve terminals were robustly preserved even in older mice, remaining functional for 6 d. Moreover, we detect extranuclear Wld(S) for the first time in vivo, and higher axoplasmic levels in transgenic mice with Wld(S) redistribution. Cytoplasmic Wld(S) fractionated predominantly with mitochondria and microsomes. We conclude that Wld(S) can act in one or more non-nuclear compartments to protect axons and synapses, and that molecular changes can enhance its therapeutic potential.

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The Journal of neuroscience : the official journal of the Society for Neuroscience, 29, 3, , 2009

DOI: 10.1523/JNEUROSCI.3814-08.2009

Open Access

Severely dystrophic axons at amyloid plaques remain continuous and connected to viable cell bodies.
R Adalbert, A Nogradi, E Babetto, L Janeckova, SA Walker, M Kerschensteiner, T Misgeld, MP Coleman

Synapse loss precedes cell death in Alzheimer's disease, but the timing of axon degeneration relative to these events, and the causal relationships remain unclear. Axons become so severely dystrophic near amyloid plaques that their interruption, causing permanent loss of function, extensive synapse loss, and potentially cell death appears imminent. However, it remains unclear whether axons are truly interrupted at plaques and whether cell bodies fail to support their axons and dendrites. We traced TgCRND8 mouse axons longitudinally through, distal to, and proximal from dystrophic regions. The corresponding neurons not only survived but remained morphologically unaltered, indicating absence of axonal damage signalling or a failure to respond to it. Axons, no matter how dystrophic, remained continuous and initially morphologically normal outside the plaque region, reflecting support by metabolically active cell bodies and continued axonal transport. Immunochemical and ultrastructural studies showed dystrophic axons were tightly associated with disruption of presynaptic transmission machinery, suggesting local functional impairment. Thus, we rule out long-range degeneration axons or dendrites as major contributors to early synapse loss in this model, raising the prospect of a therapeutic window for functional rescue of individual neurons lasting months or even years after their axons become highly dystrophic. We propose that multi-focal pathology has an important role in the human disease in bringing about the switch from local, and potentially recoverable, synapse loss into permanent loss of neuronal processes and eventually their cell bodies.

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Brain : a journal of neurology, 132, Pt 2, , 2009

DOI: 10.1093/brain/awn312

Open Access

The WldS gene delays axonal but not somatic degeneration in a rat glaucoma model.
B Beirowski, E Babetto, MP Coleman, KR Martin

Glaucoma is a leading cause of blindness caused by progressive degeneration of retinal ganglion cells (RGCs) and their axons. The pathogenesis of glaucoma remains incompletely understood, but optic nerve (ON) axonal injury appears to be an important trigger of RGC axonal and cell body degeneration. Rat models are widely used in glaucoma research to explore pathogenic mechanisms and to test novel neuroprotective approaches. Here we investigated the mechanism of axon loss in glaucoma, studying axon degeneration in slow Wallerian degeneration (Wld(S)) rats after increasing intraocular pressure. Wld(S) delays degeneration of experimentally transected axons for several weeks, so it can provide genetic evidence for Wallerian-like degeneration in disease. As apoptosis is unaffected, Wld(S) also provides information on whether cell death results from axon degeneration or arises independently, an important question yet to be resolved in glaucoma. Having confirmed expression of Wld(S) protein, we found that Wld(S) delayed ON axonal degeneration in experimental rat glaucoma for at least 2 weeks, especially in proximal ON where wild-type axons are most severely affected. The duration of axonal protection is similar to that after ON transection and crush, suggesting that axonal degeneration in glaucoma follows a Wallerian-like mechanism. Axonal degeneration must be prevented for RGCs to remain functional, so pharmacologically mimicking and enhancing the protective mechanism of Wld(S) could offer an important route towards therapy. However, Wld(S) did not protect RGC bodies in glaucoma or after ON lesion, suggesting that combination treatments protecting both axons and cell bodies offer the best therapeutic prospects.

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The European journal of neuroscience, 28, 6, , 2008

DOI: 10.1111/j.1460-9568.2008.06426.x

VCP binding influences intracellular distribution of the slow Wallerian degeneration protein, Wld(S).
AL Wilbrey, JE Haley, TM Wishart, L Conforti, G Morreale, B Beirowski, E Babetto, R Adalbert, TH Gillingwater, T Smith, DJ Wyllie, RR Ribchester, MP Coleman

Wallerian degeneration slow (Wld(S)) mice express a chimeric protein that delays axonal degeneration. The N-terminal domain (N70), which is essential for axonal protection in vivo, binds valosin-containing protein (VCP) and targets both Wld(S) and VCP to discrete nuclear foci. We characterized the formation, composition and localization of these potentially important foci. Missense mutations show that the N-terminal sixteen residues (N16) of Wld(S) are essential for both VCP binding and targeting Wld(S) to nuclear foci. Removing N16 abolishes foci, and VCP binding sequences from ataxin-3 or HrdI restore them. In vitro, these puncta co-localize with proteasome subunits. In vivo, Wld(S) assumes a range of nuclear distribution patterns, including puncta, and its neuronal expression and intranuclear distribution is region-specific and varies between spontaneous and transgenic Wld(S) models. We conclude that VCP influences Wld(S) intracellular distribution, and thus potentially its function, by binding within the N70 domain required for axon protection.

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Molecular and cellular neurosciences, 38, 3, , 2008

DOI: 10.1016/j.mcn.2008.03.004

Design of a novel quantitative PCR (QPCR)-based protocol for genotyping mice carrying the neuroprotective Wallerian degeneration slow (Wlds) gene.
TM Wishart, SH Macdonald, PE Chen, MJ Shipston, MP Coleman, TH Gillingwater, RR Ribchester

Mice carrying the spontaneous genetic mutation known as Wallerian degeneration slow (Wlds) have a unique neuroprotective phenotype, where axonal and synaptic compartments of neurons are protected from degeneration following a wide variety of physical, toxic and inherited disease-inducing stimuli. This remarkable phenotype has been shown to delay onset and progression in several mouse models of neurodegenerative disease, suggesting that Wlds-mediated neuroprotection may assist in the identification of novel therapeutic targets. As a result, cross-breeding of Wlds mice with mouse models of neurodegenerative diseases is used increasingly to understand the roles of axon and synapse degeneration in disease. However, the phenotype shows strong gene-dose dependence so it is important to distinguish offspring that are homozygous or heterozygous for the mutation. Since the Wlds mutation comprises a triplication of a region already present in the mouse genome, the most stringent way to quantify the number of mutant Wlds alleles is using copy number. Current approaches to genotype Wlds mice are based on either Southern blots or pulsed field gel electrophoresis, neither of which are as rapid or efficient as quantitative PCR (QPCR).

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Molecular neurodegeneration, 2, , , 2007

DOI: 10.1186/1750-1326-2-21

Open Access

Late onset distal axonal swelling in YFP-H transgenic mice.
KE Bridge, N Berg, R Adalbert, E Babetto, T Dias, MG Spillantini, RR Ribchester, MP Coleman

Axonal swellings, or spheroids, are a feature of central nervous system (CNS) axon degeneration during normal aging and in many disorders. The direct cause and mechanism are unknown. The use of transgenic mouse line YFP-H, which expresses yellow-fluorescent protein (YFP) in a subset of neurons, greatly facilitates longitudinal imaging and live imaging of axonal swellings, but it has not been established whether long-term expression of YFP itself contributes to axonal swelling. Using conventional methods to compare YFP-H mice with their YFP negative littermates, we found an age-related increase in swellings in discrete CNS regions in both genotypes, but the presence of YFP caused significantly more swellings in mice aged 8 months or over. Increased swelling was found in gracile tract, gracile nucleus and dorsal roots but not in lateral columns, olfactory bulb, motor cortex, ventral roots or peripheral nerve. Thus, long-term expression of YFP accelerates age-related axonal swelling in some axons and data reliant on the presence of YFP in these CNS regions in older animals needs to be interpreted carefully. The ability of a foreign protein to exacerbate age-related axon pathology is an important clue to the mechanisms by which such pathology can arise.

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Neurobiology of aging, 30, 2, , 2009

DOI: 10.1016/j.neurobiolaging.2007.06.002

Abeta, tau and ApoE4 in Alzheimer's disease: the axonal connection.
R Adalbert, J Gilley, MP Coleman

Mutations in amyloid precursor protein (APP), tau and apolipoprotein E4 (ApoE4) lead to Alzheimer's disease (AD) or related pathologies. Pathogenesis and interactions between these pathways have been studied in mouse models. Here, we highlight the fact that axons are important sites of cellular pathology in each pathway and propose that pathway convergence at the molecular level might occur in axons. Recent developments suggest that axonal transport of APP influences beta-amyloid deposition and that tau regulates axonal transport. ApoE4 influences both axonal tau phosphorylation and amyloid-induced neurite pathology. Thus, a better understanding of axonal events in AD might help connect the pathogenic mechanisms of beta-amyloid, ApoE4 and tau, indicating the most important steps for therapeutic targeting.

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Trends in molecular medicine, 13, 4, , 2007

DOI: 10.1016/j.molmed.2007.02.004

Neuronal death: where does the end begin?
L Conforti, R Adalbert, MP Coleman

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, Alzheimer's disease, Parkinson's 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.

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Trends in neurosciences, 30, 4, , 2007

DOI: 10.1016/j.tins.2007.02.004

The slow Wallerian degeneration gene in vivo protects motor axons but not their cell bodies after avulsion and neonatal axotomy.
R Adalbert, A Nógrádi, A Szabó, MP Coleman

The slow Wallerian degeneration gene (Wld(S)) delays Wallerian degeneration and axon pathology for several weeks in mice and rats. Interestingly, neuronal cell death is also delayed in some in vivo models, most strikingly in the progressive motoneuronopathy mouse. Here, we tested the hypothesis that Wld(S) has a direct protective effect on motoneurone cell bodies in vivo. Cell death was induced in rat L4 motoneurones by intravertebral avulsion of the corresponding ventral roots. This simultaneously removed most of the motor axon, minimizing the possibility that the protective effect toward axons could rescue cell bodies secondarily. There was no significant difference between the survival of motoneurones in control and Wld(S) rats, suggesting that the Wld(S) gene has no direct protective effect on cell bodies. We also tested for any delay in apoptotic motoneurone death following neonatal nerve injury in Wld(S) rats and found that, unlike Wld(S) mice, Wld(S) rats show no delay in cell death. However, the corresponding distal axons were preserved, confirming that motoneurone cell bodies and motor axons die by different mechanisms. Thus, Wld(S) does not directly prevent death of motoneurone cell bodies. It follows that the protection of neuronal cell bodies observed in several disease and injury models where axons or significant axonal stumps remain is most probably secondary to axonal protection.

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The European journal of neuroscience, 24, 8, , 2006

DOI: 10.1111/j.1460-9568.2006.05103.x

Geminin is essential to prevent endoreduplication and to form pluripotent cells during mammalian development.
Gonzalez MA, Tachibana KE, Adams DJ, van der Weyden L, Hemberger M, Coleman N, Bradley A, Laskey RA

In multicellular eukaryotes, geminin prevents overreplication of DNA in proliferating cells. Here, we show that genetic ablation of geminin in the mouse prevents formation of inner cell mass (ICM) and causes premature endoreduplication at eight cells, rather than 32 cells. All cells in geminin-deficient embryos commit to the trophoblast cell lineage and consist of trophoblast giant cells (TGCs) only. Geminin is also down-regulated in TGCs of wild-type blastocysts during S and gap-like phases by proteasome-mediated degradation, suggesting that loss of geminin is part of the mechanism regulating endoreduplication.

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Genes & development, 20, 0890-9369, , 2006


Open Access

NAD(+) and axon degeneration revisited: Nmnat1 cannot substitute for Wld(S) to delay Wallerian degeneration.
L Conforti, G Fang, B Beirowski, MS Wang, L Sorci, S Asress, R Adalbert, A Silva, K Bridge, XP Huang, G Magni, JD Glass, MP Coleman

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.

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Cell death and differentiation, 14, 1, , 2007

DOI: 10.1038/sj.cdd.4401944

Open Access

The neuroprotective WldS gene regulates expression of PTTG1 and erythroid differentiation regulator 1-like gene in mice and human cells.
Gillingwater TH, Wishart TM, Chen PE, Haley JE, Robertson K, MacDonald SH, Middleton S, Wawrowski K, Shipston MJ, Melmed S, Wyllie DJ, Skehel PA, Coleman MP, Ribchester RR

Wallerian degeneration of injured neuronal axons and synapses is blocked in Wld(S) mutant mice by expression of an nicotinamide mononucleotide adenylyl transferase 1 (Nmnat-1)/truncated-Ube4b chimeric gene. The protein product of the Wld(S) gene localizes to neuronal nuclei. Here we show that Wld(S) protein expression selectively alters mRNA levels of other genes in Wld(S) mouse cerebellum in vivo and following transfection of human embryonic kidney (HEK293) cells in vitro. The largest changes, identified by microarray analysis and quantitative real-time polymerase chain reaction of cerebellar mRNA, were an approximate 10-fold down-regulation of pituitary tumour-transforming gene-1 (pttg1) and an approximate 5-fold up-regulation of a structural homologue of erythroid differentiation regulator-1 (edr1l-EST). Transfection of HEK293 cells with a Wld(S)-eGFP construct produced similar changes in mRNA levels for these and seven other genes, suggesting that regulation of gene expression by Wld(S) is conserved across different species, including humans. Similar modifications in mRNA levels were mimicked for some of the genes (including pttg1) by 1 mm nicotinamide adenine dinucleotide (NAD). However, expression levels of most other genes (including edr1l-EST) were insensitive to NAD. Pttg1(-/-) mutant mice showed no neuroprotective phenotype. Transfection of HEK293 cells with constructs comprising either full-length Nmnat-1 or the truncated Ube4b fragment (N70-Ube4b) demonstrated selective effects of Nmnat-1 (down-regulated pttg1) and N70-Ube4b (up-regulated edr1l-EST) on mRNA levels. Similar changes in pttg1 and edr1l-EST were observed in the mouse NSC34 motor neuron-like cell line following stable transfection with Wld(S). Together, the data suggest that the Wld(S) protein co-regulates expression of a consistent subset of genes in both mouse neurons and human cells. Targeting Wld(S)-induced gene expression may lead to novel therapies for neurodegeneration induced by trauma or by disease in humans.

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Human molecular genetics, 15, 0964-6906, , 2006


Open Access

The slow Wallerian degeneration protein, WldS, binds directly to VCP/p97 and partially redistributes it within the nucleus.
H Laser, L Conforti, G Morreale, TG Mack, M Heyer, JE Haley, TM Wishart, B Beirowski, SA Walker, G Haase, A Celik, R Adalbert, D Wagner, D Grumme, RR Ribchester, M Plomann, MP Coleman

Slow Wallerian degeneration (Wld(S)) mutant mice express a chimeric nuclear protein that protects sick or injured axons from degeneration. The C-terminal region, derived from NAD(+) synthesizing enzyme Nmnat1, is reported to confer neuroprotection in vitro. However, an additional role for the N-terminal 70 amino acids (N70), derived from multiubiquitination factor Ube4b, has not been excluded. In wild-type Ube4b, N70 is part of a sequence essential for ubiquitination activity but its role is not understood. We report direct binding of N70 to valosin-containing protein (VCP; p97/Cdc48), a protein with diverse cellular roles including a pivotal role in the ubiquitin proteasome system. Interaction with Wld(S) targets VCP to discrete intranuclear foci where ubiquitin epitopes can also accumulate. Wld(S) lacking its N-terminal 16 amino acids (N16) neither binds nor redistributes VCP, but continues to accumulate in intranuclear foci, targeting its intrinsic NAD(+) synthesis activity to these same foci. Wild-type Ube4b also requires N16 to bind VCP, despite a more C-terminal binding site in invertebrate orthologues. We conclude that N-terminal sequences of Wld(S) protein influence the intranuclear location of both ubiquitin proteasome and NAD(+) synthesis machinery and that an evolutionary recent sequence mediates binding of mammalian Ube4b to VCP.

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Molecular biology of the cell, 17, 3, , 2006

DOI: 10.1091/mbc.E05-04-0375

Open Access

Axon degeneration mechanisms: commonality amid diversity.
M Coleman

A wide range of insults can trigger axon degeneration, and axons respond with diverse morphology, topology and speed. However, recent genetic, immunochemical, morphological and pharmacological investigations point to convergent degeneration mechanisms. The principal convergence points - poor axonal transport, mitochondrial dysfunction and an increase in intra-axonal calcium - have been identified by rescuing axons with the slow Wallerian degeneration gene (Wld(S)) and studies with blockers of sodium or calcium influx. By understanding how the pathways fit together, we can combine our knowledge of mechanisms, and potentially also treatment strategies, from different axonal disorders.

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Nature reviews. Neuroscience, 6, 11, , 2005

DOI: 10.1038/nrn1788

The progressive nature of Wallerian degeneration in wild-type and slow Wallerian degeneration (WldS) nerves.
Beirowski B, Adalbert R, Wagner D, Grumme DS, Addicks K, Ribchester RR, Coleman MP

The progressive nature of Wallerian degeneration has long been controversial. Conflicting reports that distal stumps of injured axons degenerate anterogradely, retrogradely, or simultaneously are based on statistical observations at discontinuous locations within the nerve, without observing any single axon at two distant points. As axon degeneration is asynchronous, there are clear advantages to longitudinal studies of individual degenerating axons. We recently validated the study of Wallerian degeneration using yellow fluorescent protein (YFP) in a small, representative population of axons, which greatly improves longitudinal imaging. Here, we apply this method to study the progressive nature of Wallerian degeneration in both wild-type and slow Wallerian degeneration (WldS) mutant mice.

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BMC neuroscience, 6, 1471-2202, , 2005


Open Access

A rat model of slow Wallerian degeneration (WldS) with improved preservation of neuromuscular synapses.
R Adalbert, TH Gillingwater, JE Haley, K Bridge, B Beirowski, L Berek, D Wagner, D Grumme, D Thomson, A Celik, K Addicks, RR Ribchester, MP Coleman

The slow Wallerian degeneration phenotype, Wld(S), which delays Wallerian degeneration and axon pathology for several weeks, has so far been studied only in mice. A rat model would have several advantages. First, rats model some human disorders better than mice. Second, the larger body size of rats facilitates more complex surgical manipulations. Third, rats provide a greater yield of tissue for primary culture and biochemical investigations. We generated transgenic Wld(S) rats expressing the Ube4b/Nmnat1 chimeric gene in the central and peripheral nervous system. As in Wld(S) mice, their axons survive up to 3 weeks after transection and remain functional for at least 1 week. Protection of axotomized nerve terminals is stronger than in mice, particularly in one line, where 95-100% of neuromuscular junctions remained intact and functional after 5 days. Furthermore, the loss of synaptic phenotype with age was much less in rats than in mice. Thus, the slow Wallerian degeneration phenotype can be transferred to another mammalian species and synapses may be more effectively preserved after axotomy in species with longer axons.

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The European journal of neuroscience, 21, 1, , 2005

DOI: 10.1111/j.1460-9568.2004.03833.x

The slow Wallerian degeneration gene, WldS, inhibits axonal spheroid pathology in gracile axonal dystrophy mice.
W Mi, B Beirowski, TH Gillingwater, R Adalbert, D Wagner, D Grumme, H Osaka, L Conforti, S Arnhold, K Addicks, K Wada, RR Ribchester, MP Coleman

Axonal dystrophy is the hallmark of axon pathology in many neurodegenerative disorders of the CNS, including Alzheimer's disease, Parkinson's disease and stroke. Axons can also form larger swellings, or spheroids, as in multiple sclerosis and traumatic brain injury. Some spheroids are terminal endbulbs of axon stumps, but swellings may also occur on unbroken axons and their role in axon loss remains uncertain. Similarly, it is not known whether spheroids and axonal dystrophy in so many different CNS disorders arise by a common mechanism. These surprising gaps in current knowledge result largely from the lack of experimental methods to manipulate axon pathology. The slow Wallerian degeneration gene, Wld(S), delays Wallerian degeneration after injury, and also delays 'dying-back' in peripheral nervous system disorders, revealing a mechanistic link between two forms of axon degeneration traditionally considered distinct. We now report that Wld(S) also inhibits axonal spheroid pathology in gracile axonal dystrophy (gad) mice. Both gracile nucleus (P < 0.001) and cervical gracile fascicle (P = 0.001) contained significantly fewer spheroids in gad/Wld(S) mice, and secondary signs of axon pathology such as myelin loss were also reduced. Motor nerve terminals at neuromuscular junctions continued to degenerate in gad/Wld(S) mice, consistent with previous observations that Wld(S) has a weaker effect on synapses than on axons, and probably contributing to the fact that Wld(S) did not alleviate gad symptoms. Wld(S) acts downstream of the initial pathogenic events to block gad pathology, suggesting that its effect on axonal swelling need not be specific to this disease. We conclude that axon degeneration mechanisms are more closely related than previously thought and that a link exists in gad between spheroid pathology and Wallerian degeneration that could hold for other disorders.

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Brain : a journal of neurology, 128, Pt 2, , 2005

DOI: 10.1093/brain/awh368

Open Access

Wallerian degeneration of injured axons and synapses is delayed by a Ube4b/Nmnat chimeric gene.
TG Mack, M Reiner, B Beirowski, W Mi, M Emanuelli, D Wagner, D Thomson, T Gillingwater, F Court, L Conforti, FS Fernando, A Tarlton, C Andressen, K Addicks, G Magni, RR Ribchester, VH Perry, MP Coleman

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.

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Nature neuroscience, 4, 12, , 2001

DOI: 10.1038/nn770