Title: Axon Self-Destruction: New Links among SARM1, MAPKs, and NAD+ Metabolism
Abstract: Wallerian axon degeneration is a form of programmed subcellular death that promotes axon breakdown in disease and injury. Active degeneration requires SARM1 and MAP kinases, including DLK, while the NAD+ synthetic enzyme NMNAT2 prevents degeneration. New studies reveal that these pathways cooperate in a locally mediated axon destruction program, with NAD+ metabolism playing a central role. Here, we review the biology of Wallerian-type axon degeneration and discuss the most recent findings, with special emphasis on critical signaling events and their potential as therapeutic targets for axonopathy. Wallerian axon degeneration is a form of programmed subcellular death that promotes axon breakdown in disease and injury. Active degeneration requires SARM1 and MAP kinases, including DLK, while the NAD+ synthetic enzyme NMNAT2 prevents degeneration. New studies reveal that these pathways cooperate in a locally mediated axon destruction program, with NAD+ metabolism playing a central role. Here, we review the biology of Wallerian-type axon degeneration and discuss the most recent findings, with special emphasis on critical signaling events and their potential as therapeutic targets for axonopathy. Injury-induced axonal degeneration is a genetically encoded program of subcellular self-destruction. Recent genetic studies have identified essential molecular components of this axon degeneration program. In this review, we focus on three key players: (1) the axonal maintenance factor NMNAT2, whose regulation helps explain the potent axoprotective activity of the “Wallerian degeneration slow” protein; (2) dual leucine zipper kinase (DLK) and associated MAP kinase components, which promote both axon degeneration and axon regeneration; and (3) SARM1, which has emerged as the central executioner in the axonal degeneration program. Recent exciting work is uncovering mechanistic links among these proteins, suggesting that this field is on the cusp of a unified model for the mechanism of axonal degeneration. In this review, we summarize the current understanding of axon degeneration, with particular emphasis on the integration of these components into a single pathway, highlighting biochemical and metabolic steps within the degeneration program that represent therapeutic targets to block axon loss in disease. Axons can extend to great lengths of more than 1 m in humans, making them uniquely susceptible to damage that often results in irreversible disability. Axon loss is a prominent feature of many important neurological disorders, including neuropathies, traumatic injury, and multiple neurodegenerative disorders. Peripheral neuropathy is the most common condition in which axon dysfunction and degeneration are the central abnormalities, and it may be either acquired or hereditary. Acquired neuropathies include diabetic and chemotherapy-induced neuropathy (Albers and Pop-Busui, 2014Albers J.W. Pop-Busui R. Diabetic neuropathy: mechanisms, emerging treatments, and subtypes.Curr. Neurol. Neurosci. Rep. 2014; 14: 473Crossref PubMed Scopus (0) Google Scholar, Cashman and Höke, 2015Cashman C.R. Höke A. Mechanisms of distal axonal degeneration in peripheral neuropathies.Neurosci. Lett. 2015; 596: 33-50Crossref PubMed Scopus (10) Google Scholar, Grisold et al., 2012Grisold W. Cavaletti G. Windebank A.J. Peripheral neuropathies from chemotherapeutics and targeted agents: diagnosis, treatment, and prevention.Neuro-oncol. 2012; 14: iv45-iv54Crossref PubMed Scopus (0) Google Scholar), which are increasingly common because of the growing prevalence of diabetes and improving rates of cancer survivorship. Traumatic brain injury also involves prominent axon damage, resulting in diffuse axonal injury in the brain and spinal cord that directly impairs neuronal function and accelerates neurodegeneration (Johnson et al., 2013Johnson V.E. Stewart W. Smith D.H. Axonal pathology in traumatic brain injury.Exp. Neurol. 2013; 246: 35-43Crossref PubMed Scopus (146) Google Scholar). The full contribution of axon degeneration to human morbidity is difficult to estimate because no pharmacologic tools currently exist that slow or halt axon degeneration; however, histologic studies have revealed early and prominent axon loss in Alzheimer’s disease, Parkinson’s disease, multiple sclerosis, amyotrophic lateral sclerosis, and others (Benarroch, 2015Benarroch E.E. Acquired axonal degeneration and regeneration: Recent insights and clinical correlations.Neurology. 2015; 84: 2076-2085Crossref PubMed Google Scholar, Burke and O’Malley, 2013Burke R.E. O’Malley K. Axon degeneration in Parkinson’s disease.Exp. 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Neurol. 2002; 52: 442-447Crossref PubMed Scopus (78) Google Scholar), suggesting that the mechanism of Wallerian-type axon degeneration is engaged in many neurological disorders involving axon loss. Throughout this review we use the term “axon degeneration” to refer exclusively to the Wallerian axon destruction pathway that promotes pathologic axon degeneration in the settings of injury, transport failure, and poisoning with chemotherapeutic agents. There is a distinct caspase- and BAX-dependent pathway that promotes degeneration in the setting of developmental axon pruning and growth factor deprivation (Nikolaev et al., 2009Nikolaev A. McLaughlin T. O’Leary D.D.M. Tessier-Lavigne M. APP binds DR6 to trigger axon pruning and neuron death via distinct caspases.Nature. 2009; 457: 981-989Crossref PubMed Scopus (559) Google Scholar, Pease and Segal, 2014Pease S.E. Segal R.A. 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In this review, we focus solely on the Wallerian degeneration pathway and do not address developmental axon loss or the phagocytic clearance of damaged axons, which have been reviewed elsewhere (Luo and O’Leary, 2005Luo L. O’Leary D.D.M. Axon retraction and degeneration in development and disease.Annu. Rev. Neurosci. 2005; 28: 127-156Crossref PubMed Scopus (433) Google Scholar, Schuldiner and Yaron, 2015Schuldiner O. Yaron A. Mechanisms of developmental neurite pruning.Cell. Mol. Life Sci. 2015; 72: 101-119Crossref PubMed Scopus (14) Google Scholar). However, there is some molecular commonality between injury-induced and developmental axon loss (Gerdts et al., 2013Gerdts J. Summers D.W. Sasaki Y. DiAntonio A. Milbrandt J. Sarm1-mediated axon degeneration requires both SAM and TIR interactions.J. Neurosci. 2013; 33: 13569-13580Crossref PubMed Scopus (33) Google Scholar, Schoenmann et al., 2010Schoenmann Z. Assa-Kunik E. Tiomny S. Minis A. Haklai-Topper L. Arama E. Yaron A. Axonal degeneration is regulated by the apoptotic machinery or a NAD+-sensitive pathway in insects and mammals.J. Neurosci. 2010; 30: 6375-6386Crossref PubMed Scopus (76) Google Scholar, Vohra et al., 2010Vohra B.P.S. Sasaki Y. Miller B.R. Chang J. DiAntonio A. Milbrandt J. Amyloid precursor protein cleavage-dependent and -independent axonal degeneration programs share a common nicotinamide mononucleotide adenylyltransferase 1-sensitive pathway.J. Neurosci. 2010; 30: 13729-13738Crossref PubMed Scopus (53) Google Scholar), so processes described below may also play a currently unappreciated role in the development and plasticity of neural circuits. Axon degeneration signaling is intrinsic to the axon. After injury, pro-destructive signaling takes place within the distal axon segment independent of de novo transcription or translation or external cues. The temporal progression of axon destruction following axotomy involves an early “latent” period lasting ∼4–6 hr in vitro (Figure 1) and ∼36 hr in adult nerves in vivo. During this phase the distal axon remains physically and metabolically intact (Coleman, 2005Coleman M. Axon degeneration mechanisms: commonality amid diversity.Nat. Rev. Neurosci. 2005; 6: 889-898Crossref PubMed Scopus (426) Google Scholar). Critical steps in axon degeneration signaling take place early during the latent period, long before axon degeneration is morphologically evident. This early latent phase thus appears to be an ideal window for therapeutic intervention. In contrast, late steps in axon degeneration, such as energetic failure, influx of calcium and resultant calpain-mediated proteolysis of neurofilaments and other structural proteins, axon fragmentation, and engulfment by phagocytic cells (Kurant, 2011Kurant E. Keeping the CNS clear: glial phagocytic functions in Drosophila.Glia. 2011; 59: 1304-1311Crossref PubMed Scopus (14) Google Scholar, Wang et al., 2012Wang J.T. Medress Z.A. Barres B.A. Axon degeneration: molecular mechanisms of a self-destruction pathway.J. Cell Biol. 2012; 196: 7-18Crossref PubMed Scopus (112) Google Scholar, Yang et al., 2013Yang J. Weimer R.M. Kallop D. Olsen O. Wu Z. Renier N. Uryu K. Tessier-Lavigne M. Regulation of axon degeneration after injury and in development by the endogenous calpain inhibitor calpastatin.Neuron. 2013; 80: 1175-1189Abstract Full Text Full Text PDF PubMed Scopus (30) Google Scholar), may be beyond the point of no return from a therapeutic perspective. Axon destruction is unique among cellular destruction programs because it is spatially restricted. With classic Wallerian degeneration of a peripheral nerve, the axon segment distal to a point of injury undergoes selective breakdown, while the proximal axon segment and cell soma remain intact. Hence, axon destruction signaling must distinguish between the portions of axon to be destroyed and those to be spared. Two potential mechanisms could explain the differential sensitivity of proximal and distal axons to injury-induced destruction. The loss of communication with the cell body could deprive the distal axon of an axonal maintenance factor required for axon survival. Alternatively, a pro-degenerative signal could be selectively activated in the distal axon following injury. As we shall see, both mechanisms are at play following axon injury and likely work together to trigger axon loss. The coordinated activity of both positive and negative axon stability mechanisms, exemplified by NMNAT2 and SARM1, may help ensure that in healthy axons, degeneration signaling is tightly maintained in an “off” state in order to prevent spurious axon degeneration. A strong yet mysterious link between axon degeneration and nicotinamide adenine dinucleotide (NAD+) metabolism emerged from studies of the Wlds mouse. Cloning of the Wlds gene revealed it to encode a chimeric fusion protein comprised of the NAD biosynthetic enzyme nicotinamide mononucleotide adenyltransferase (NMNAT1), which forms NAD+ from nicotinamide mononucleotide (NMN) and ATP (Figure 2), and a fragment of the ubiquitination factor UBE4B (Conforti et al., 2000Conforti L. Tarlton A. Mack T.G. Mi W. Buckmaster E.A. Wagner D. Perry V.H. Coleman M.P. A Ufd2/D4Cole1e chimeric protein and overexpression of Rbp7 in the slow Wallerian degeneration (WldS) mouse.Proc. Natl. Acad. Sci. U S A. 2000; 97: 11377-11382Crossref PubMed Google Scholar). Although there was initially controversy as to the functional domains of the Wlds protein, it is now clear that NMNAT1 is the axoprotective component (Araki et al., 2004Araki T. Sasaki Y. Milbrandt J. Increased nuclear NAD biosynthesis and SIRT1 activation prevent axonal degeneration.Science. 2004; 305: 1010-1013Crossref PubMed Scopus (676) Google Scholar). The Wlds fusion protein confers aberrant localization of the nuclear enzyme NMNAT1 to the axon, where it functions autonomously. Accordingly, manipulations that increase axonal localization of NMNAT1 confer Wlds-like axon protection (Babetto et al., 2010Babetto E. Beirowski B. Janeckova L. Brown R. Gilley J. Thomson D. Ribchester R.R. Coleman M.P. Targeting NMNAT1 to axons and synapses transforms its neuroprotective potency in vivo.J. Neurosci. 2010; 30: 13291-13304Crossref PubMed Scopus (45) Google Scholar, Sasaki et al., 2009aSasaki Y. Vohra B.P.S. Baloh R.H. Milbrandt J. Transgenic mice expressing the Nmnat1 protein manifest robust delay in axonal degeneration in vivo.J. Neurosci. 2009; 29: 6526-6534Crossref PubMed Scopus (68) Google Scholar). Direct transduction of NMNAT1 protein into severed axons in vitro within 4 hr after axon transection (see Figure 1) is sufficient to prevent later fragmentation of the axons (Sasaki and Milbrandt, 2010Sasaki Y. Milbrandt J. Axonal degeneration is blocked by nicotinamide mononucleotide adenylyltransferase (Nmnat) protein transduction into transected axons.J. Biol. Chem. 2010; 285: 41211-41215Crossref PubMed Scopus (28) Google Scholar), definitively demonstrating that NMNAT1 exerts its protective effect locally within the axonal compartment. Moreover, Wlds-expressing axons rapidly degenerate when Wlds is depleted after injury by protein destabilization, demonstrating a continuous local requirement for NMNAT activity in isolated axons (Wang et al., 2015Wang J.T. Medress Z.A. Vargas M.E. Barres B.A. Local axonal protection by WldS as revealed by conditional regulation of protein stability.Proc. Natl. Acad. Sci. U S A. 2015; 112: 10093-10100Crossref PubMed Scopus (0) Google Scholar). This axon protective activity is not a specific property of NMNAT1 but is shared with divergent NMNAT proteins, including the three mammalian NMNAT paralogs (1–3) and structurally dissimilar enzymes from archaebacteria (Sasaki et al., 2009bSasaki Y. Vohra B.P.S. Lund F.E. Milbrandt J. Nicotinamide mononucleotide adenylyl transferase-mediated axonal protection requires enzymatic activity but not increased levels of neuronal nicotinamide adenine dinucleotide.J. Neurosci. 2009; 29: 5525-5535Crossref PubMed Scopus (88) Google Scholar, Yahata et al., 2009Yahata N. Yuasa S. Araki T. Nicotinamide mononucleotide adenylyltransferase expression in mitochondrial matrix delays Wallerian degeneration.J. Neurosci. 2009; 29: 6276-6284Crossref PubMed Scopus (82) Google Scholar, Yan et al., 2010Yan T. Feng Y. Zheng J. Ge X. Zhang Y. Wu D. Zhao J. Zhai Q. Nmnat2 delays axon degeneration in superior cervical ganglia dependent on its NAD synthesis activity.Neurochem. 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NAD synthase NMNAT acts as a chaperone to protect against neurodegeneration.Nature. 2008; 452: 887-891Crossref PubMed Scopus (94) Google Scholar). Hence, the axoprotective mechanism of NMNAT enzymes is evolutionarily conserved. Although NMNAT1 is the active moiety of the Wlds protein, axonal mis-localization of this nuclear protein is an unnatural consequence of mutation, and endogenous NMNAT1 is not believed to have a role in axon destruction or maintenance in wild-type animals. Instead, it is likely that NMNAT1 protects axons by substituting for its axonal paralog, NMNAT2. Gilley and colleagues demonstrated that NMNAT2 is trafficked anterogradely in the axoplasm, and unlike NMNAT1 and NMNAT3, NMNAT2 is labile because of constitutive proteasomal degradation. NMNAT2 turnover in the setting of disrupted axon transport thus leads to depletion of axonal NMNAT2. NMNAT2 may represent a “survival factor” whose depletion can trigger the axon destruction cascade, as knockdown of NMNAT2 in cultured neurons is sufficient to cause axon degeneration in the absence of injury (Gilley and Coleman, 2010Gilley J. Coleman M.P. Endogenous Nmnat2 is an essential survival factor for maintenance of healthy axons.PLoS Biol. 2010; 8: e1000300Crossref PubMed Scopus (116) Google Scholar); however, this model has yet to be tested in in vivo injury models. Thus, it is likely that NMNAT2 loss following axon injury is an initiating event in the axon destruction pathway, and axon protection by other NMNAT proteins, including NMNAT1 and Wlds, occurs because they provide continuous NMNAT activity within the axon. Although at one level this explains why Wlds and NMNAT1 are axoprotective—they substitute for the labile NMNAT2—at a more mechanistic level it leaves open the question of how NMNAT activity blocks axon degeneration. Indeed, despite the discovery of axon protection by NMNAT more than a decade ago, the role of its product NAD+ in axon protection and destruction remains unclear. NAD+ is a ubiquitous metabolite with critical roles in energy metabolism and cell signaling (Belenky et al., 2007Belenky P. Bogan K.L. Brenner C. NAD+ metabolism in health and disease.Trends Biochem. Sci. 2007; 32: 12-19Abstract Full Text Full Text PDF PubMed Scopus (331) Google Scholar, Chiarugi et al., 2012Chiarugi A. Dölle C. Felici R. Ziegler M. The NAD metabolome--a key determinant of cancer cell biology.Nat. Rev. Cancer. 2012; 12: 741-752Crossref PubMed Scopus (116) Google Scholar). Surprisingly, a series of results suggest that increased axonal NAD+ levels alone cannot account for the protective activity of NMNAT. First, NAD+ steady-state levels are unchanged by NMNAT1 overexpression (Mack et al., 2001Mack T.G. Reiner M. Beirowski B. Mi W. Emanuelli M. Wagner D. Thomson D. Gillingwater T. Court F. 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NMNAT expression delayed both NAD+ loss and axon degeneration, consistent with an upstream role for NAD+ synthesis in axonal preservation; however, it was unclear whether NAD+ depletion was a cause or consequence of the degenerative process. As discussed in the section on SARM1 below, recent findings demonstrate that activation of this axodestructive molecule triggers the rapid consumption of NAD+, supporting the model that NMNAT protects axons at least in part by countering SARM1-dependent NAD+ loss. Overexpression of Wlds and NMNAT1 demonstrate that genetic manipulations can regulate axon degeneration; however, these are gain-of-function manipulations and so do not prove whether or not an endogenous pro-degenerative program exists. If gene products function to promote axon degeneration, then loss of function mutations in these components should delay or block axon degeneration. 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DLK works through the downstream MAPK JNK (c-jun n-terminal kinase), as pharmacological inhibitors of the JNK, but not P38 MAPKs, lead to axon preservation comparable with DLK ablation (Miller et al., 2009Miller B.R. Press C. Daniels R.W. Sasaki Y. Milbrandt J. DiAntonio A. A dual leucine kinase-dependent axon s