InteractiveFly: GeneBrief

Sterile alpha and Armadillo motif: Biological Overview | References


Gene name - Sterile alpha and Armadillo motif

Synonyms -

Cytological map position - 66B5-66B6

Function - enzyme

Keywords - NAD(+) hydrolase activity - required for activation of an injury-induced axon death pathway - Axundead is a mediator of axon death downstream of Sarm - required cell autonomously for these changes in neuronal cell biology in bystander neurons following axotomy

Symbol - Sarm

FlyBase ID: FBgn0262579

Genetic map position - chr3L:8,063,829-8,108,905

NCBI classification - SAM_SARM1-like_repeat1: SAM domain ot SARM1-like proteins, repeat 1; TIR: Toll - interleukin 1 - resistance

Cellular location - cytoplasmic



NCBI links: EntrezGene, Nucleotide, Protein
BIOLOGICAL OVERVIEW

Nervous system injury and disease have broad effects on the functional connectivity of the nervous system, but how injury signals are spread across neural circuits remains unclear. This study explored how axotomy changes the physiology of severed axons and adjacent uninjured 'bystander' neurons in a simple in vivo nerve preparation. Within hours after injury, suppression of axon transport was observed in all axons, whether injured or not, and decreased mechano- and chemosensory signal transduction was observed in uninjured bystander neurons. Unexpectedly, it was found the axon death molecule Sterile alpha and Armadillo motif (dSarm), but not its NAD(+) hydrolase activity, was required cell autonomously for these early changes in neuronal cell biology in bystander neurons, as were the voltage-gated calcium channel Cacophony (Cac) and the mitogen-activated protein kinase (MAPK) signaling cascade. Bystander neurons functionally recovered at later time points, while severed axons degenerated via α/Armadillo/Toll-interleukin receptor homology domain (dSarm)/Axundead signaling, and independently of Cac/MAPK. Interestingly, suppression of bystander neuron function required Draper/MEGF10 signaling in glia, indicating glial cells spread injury signals and actively suppress bystander neuron function. This work identifies a new role for dSarm and glia in suppression of bystander neuron function after injury and defines two genetically and temporally separable phases of dSarm signaling in the injured nervous system (Hsu, 2020).

Nervous system injury or neurodegenerative disease can lead to profound alterations in neural circuit function. The precise cellular basis is poorly defined in any context, but disruption of circuit signaling is generally thought to occur as a result of a loss of physical connectivity between damaged neurons. Indeed, axon and synapse degeneration are among the best correlates of functional loss in patients with a variety of brain injuries or neurological diseases. But whether, and the extent to which, an injured or diseased neuron might also alter the functional properties of neighboring healthy 'bystander' neurons (i.e., those not damaged or expressing disease-associated molecules) is an important and open question. If the physiology of bystander neurons is radically altered by their damaged neighbors, this would force reconsideration of the simple loss-of-physical-connectivity model as the appropriate explanation for functional loss in neural circuits after trauma (Hsu, 2020).

It is well documented that bystander neurons can change their physiology in response to their neighbors being injured. For instance, mouse L5 spinal nerve transection results in the degeneration of distal L5 afferents in sciatic nerve alongside intact L4 C fiber afferents. Within 1 day after L5 lesion, L4 C fibers develop spontaneous activity that lasts for at least 1 week and appears to mediate injury-induced pain and hyperalgesia behaviors. Bystander effects have also been observed in the central nervous system (CNS). In a mouse model of mild traumatic brain injury (TBI), 1 day after injury, pyramidal neurons with severed axons and intact bystander neurons both exhibited injury-induced changes in action potential firing and afterhyperpolarization. Injured neurons failed to recover, while bystander neurons ultimately exhibited a return to normal firing properties. How injured neurons or surrounding glia signal to bystander neurons, or how bystander neurons receive this signal, is not known, but the similar electrophysiological changes observed in axotomized and intact dorsal root ganglion neurons have been proposed to be associated with Wallerian degeneration (Hsu, 2020).

Recent work has begun to illuminate the mechanisms by which damaged axons autonomously drive their own degeneration during Wallerian degeneration. A forward genetic screen in Drosophila identified the sterile α/Armadillo/Toll-interleukin receptor homology domain (dSarm) molecule as essential for axon auto-destruction, as loss of dSarm completely blocked Wallerian degeneration (Osterloh, 2012). All known dSarm pro-degenerative function requires the BTB and BACK domain molecule Axundead (Axed), another powerful regulator of axon degeneration (Neukomm, 2017). dSarm function in axon degeneration after injury is conserved in mouse: Sarm1-/- mutants block Wallerian degeneration, and loss of Sarm1 also suppresses axon degeneration in mouse models of TBI and peripheral neuropathy. Sarm1 inhibition is thus an exciting potential approach for blocking axon loss and neuroinflammation in human disease (Hsu, 2020).

dSarm/Sarm1 has been studied primarily in the nervous system as a positive regulator of axonal degeneration. In mammals, axotomy leads to the depletion of the labile NAD+ biosynthetic enzyme Nmnat2 and a decrease in NAD+ in severed axons. Nmnat2 loss somehow activates Sarm1 (Gilley, 2015), which is proposed to lead to further NAD+ depletion and metabolic catastrophe in the severed axon (Gerdts, 2015) through a Sarm1-intrinsic NAD+ hydrolase activity (Essuman, 2017). The Sarm1 NAD+ hydrolase activity appears to be activated directly by the NAD+ precursor, NMN, presumably through allosteric conformational changes in Sarm1 upon NMN binding (Bratkowski, 2020; Di Stefano, 2015; Zhao, 2019). This NAD+ depletion model has been proposed as the primary mechanism by which Sarm1 drives axon loss, and to explain the mechanistic basis of protection by several other neuroprotective molecules (Gerdts, 2016). For instance, the slow Wallerian degeneration molecule (WldS), which includes the highly stable NAD+ biosynthetic enzyme Nmnat1, is thought to protect axons by substituting for the labile Nmnat2 molecule, thereby reducing NMN levels and avoiding NAD+ depletion. Similarly, the protective effects of loss of the E3 ubiquitin ligase Highwire/Phr1 is thought to result from blockade of its direct role in degrading Nmnat2, such that in hiw/phr1 mutants Nmnat2 is stabilized and continues to maintain NAD+ levels (Hsu, 2020).

Elegant genetic studies in C. elegans demonstrated that TIR-1 (the worm homolog of dSarm/Sarm1) is part of a signaling cascade downstream of the voltage-gated calcium channel UNC-36 and CamK-II and signals via the mitogen-activated protein kinase (MAPK) signaling cascade. Based on this work, MAPK signaling was examined for roles in Wallerian degeneration but met with mixed results. Changes in MAPK signaling (i.e., phosphorylation of MAPK pathway members) were found in axons within 15-30 min after axotomy, were Sarm1 dependent and suppressed by Nmnat overexpression, and partial suppression of axon degeneration was observed after simultaneous blockade of multiple MAPK components. But how MAPK signaling modulates axon degeneration, particularly in the context of Sarm1 signaling, remains controversial, as one study proposed MAPK signals downstream of Sarm1, while another argued Sarm1 was upstream of MAPK signaling, and the neuroprotective phenotypes resulting from MAPK blockade do not approach levels afforded by loss of Sarm1 in vivo (Hsu, 2020).

This study used a partial nerve injury model to examine early changes in the physiology of severed axons and neighboring uninjured bystander neurons. Axotomy of even a small subset of neurons was shown to leads to inhibition of cargo transport in all axons within the nerve and suppression of sensory signal transduction in bystander neurons. Surprisingly, this early blockade of axon transport and sensory signal transduction required dSarm in both severed and uninjured bystander neurons, where it signaled via the conserved UNC-36/MAPK signaling pathway. Early suppression of axon transport and bystander neuron function did not require dSarm NAD+ hydrolase function or Axed, was not modulated by NMN, and was not induced by depletion of dNmnat. This suggests it is mechanistically different from later events in axon death, where dSarm drives axon degeneration with Axed. Intriguingly, this study found that this early spreading of injury signals to bystander neurons required the Draper receptor in surrounding glia, indicating that glial cells actively signal to inhibit the function of bystander neurons in vivo. This work identifies new roles for dSarm and glia in modifying neurophysiology early after injury, assigns the NAD+ hydrolase function exclusively to later axon degenerative events, and reveals a new role for UNC-36/MAPK signaling in promoting these dSarm-dependent changes early after an injury has occurred in the nervous system. It is proposed that two temporally and genetically separable phases of dSarm signaling exist that mediate these distinct injury-induced changes in neurophysiology and axon degeneration (Hsu, 2020).

This study shows that relatively small injuries can lead to the rapid and efficient spreading of injury signals across nerves that potently suppress axon transport throughout the nerve, and broadly inhibit neurophysiology in uninjured bystander neurons. Surprisingly, the same molecule was found to be required to drive explosive axon degeneration in severed axons at later stages, dSarm/Sarm1, is required for this early suppression of neuronal function, although the signaling mechanisms at each stage appear to be different. The data support a model whereby early (i.e., 1–3 h after injury) dSarm signals with Cac and MAPK components, but independent of its NAD+ hydrolase activity, to suppress axon transport and neurophysiology, while at later stages (8–12 h), dSarm signals with Axed to promote explosive axon degeneration. This significantly expands the role for dSarm/Sarm1 in regulating nervous system responses to injury to include even uninjured bystander neurons. Furthermore, a critical role was discoverd for glial cells, through Draper, in signaling to bystander neurons to inhibit their axon transport and neurophysiology. Together, this work suggests that a significant amount of functional loss after neural trauma is a result of not only frank degeneration but also more widespread changes in neuronal function, and it occurs in uninjured neurons through glial spreading of injury signals (Hsu, 2020).

The data support the notion that widespread signaling occurs between cells in injured neural tissues immediately after injury and that injury signals can radically alter neuronal function. Severing even a small number of axons led to a suppression of axon transport within hours in all axons in the adult wing nerve, even in uninjured bystander neurons. Beyond axon transport, local uninjured bystander sensory neurons also exhibited a disruption of mechano- and chemo-sensory signal transduction, which was partially reversible within a few hours. These observations suggest that beyond simple breakage of connectivity, a significant part of functional loss after brain injury or in neurodegenerative disease may also be occurring in healthy, intact neurons that have received function-suppressing signals from nearby damaged neurons (Hsu, 2020).

Surprisingly, dSarm was found to be required cell autonomously in bystander neurons to alter axon transport and nerve function in response to injury, and this role did not require its NAD+ hydrolase activity. Reception of this injury signal in the bystander neuron (and severed axons) requires the VGCC Cac and the MAPK signaling cascade, similar to Tir-1 signaling in C. elegans, but not Axed. Reciprocally, Cac and MAPK components are not required for Wallerian degeneration at later stages. Explosive axon degeneration requires dSarm, its NAD+ hydrolase activity, and Axed. Based on the timing of these different events (i.e., changes in neuronal function versus frank degeneration) with the genetic studies indicating they are separable, a two-phase model is proposed for dSarm signaling in injured neural tissues: early dSarm-dependent changes in axon biology and neurophysiology that occur within hours after injury are mediated by the Cac/dSarm/MAPK signaling cascade (phase I), while late-stage axon degeneration is driven by dSarm signaling through Axed (phase II). The existence of these temporally distinct phases of dSarm signaling likely explain previous results that seemed in conflict, where MAPK signaling was proposed to act both upstream (Yang, 2015) and downstream (Walker, 2017) of Sarm1 after axotomy. According to the current model, both of these assertions would be correct, with dSarm/Sarm1 acting upstream of MAPK early (phase I) and independent but ultimately downstream of MAPK later to drive dSarm/Axed-dependent axon degeneration (phase II)(Hsu, 2020).

To date, dSarm/Sarm1 has been thought of primarily as a cell-autonomous regulator of explosive axon degeneration, but the current work shows that dSarm can also drive important changes in circuit function through altering neuronal cell biology and neurophysiology. That bystander neurons recover and remain viable also demonstrates that activation of dSarm after injury does not necessarily lead to axon death. It is suspected that recovery occurs in large part because bystander neurons have not been severed, which is an extreme injury, and depends on their connection to the cell body, which is a source of axon survival factors like Nmnat2. Connection to the cell body may also explain why axon transport was less severely suppressed in the bystander neurons; additional transport factors can still be continuously supplied to the distal axon from the soma. Defining how dSarm activity is regulated in each of these contexts to interact with Cac/MAPKs versus Axed, and why the first phase does not require NAD+ hydrolase function, are key questions for the future (Hsu, 2020).

A compelling case exists for the NAD+ depletion hypothesis for dSarm/Sarm1 function in axon degeneration (Essuman, 2017; Gerdts, 2015, 2016), although arguments have been made this dSarm/Sarm1 signaling is likely more complex (Neukomm, 2017). In this model, depletion of Nmnat2 via Hiw/Phr1 results in the accumulation of NMN, which functions as an activator of Sarm1, with Sarm1 NAD+ hydrolase activity driving metabolic catastrophe. This study provides several lines of evidence that the above, newly described early dSarm signaling events (i.e., suppression of axon transport and neurophysiology) are mechanistically distinct but are nevertheless also regulated by some axon-death-associated molecules. First, while NMNd can suppress axon degeneration in flies and other species, it cannot block early suppression of axon transport or changes in bystander neuron function. This argues that NMN is not a driving force for dSarm activation in the early phase. Second, although limited to tagged versions of dNmnat for this analysis, no depletion of dNmnat was observed within the time frame of 6 h after injury. Previous studies in SCG or DRG cultures in vitro suggest Nmnat2 depletion takes 4-6 h and NAD+ depletion begins ~2-3 h after axotomy, which is slightly later than the bystander effect was observed in vivo. Because full axon degeneration is prolonged in vivo compared to in vitro studies, the timing of Nmnat2 loss and NAD+ depletion is likely also prolonged in vivo, further suggesting this likely happens after cessation of axon transport. Third, Axed, which is genetically downstream of dSarm during axon degeneration (Neukomm, 2017), is not required for early suppression of nerve responses to injury in either severed or intact neurons, only later axon degenerative events in the severed axons. Finally, this study shows that while the NAD+ hydrolase function of dSarm is required in vivo for efficient axon degeneration, it is dispensable for early suppression of axon transport (Hsu, 2020).

Despite these clear molecular and genetic differences between early- and late-phase signaling events, WldS or dNmant expression or hiw mutants are capable of suppressing early changes in axon transport and neurophysiology, even in bystander neurons. This could be interpreted as evidence for similarity in signaling mechanisms at early and late stages of dSarm signaling (i.e., that they act by maintaining NAD+). However, the alternative possibility is favored that these data point to an important role for dNmnat in mediating early dSarm signaling events during suppression of bystander neuron function. Loss of Axed does not affect the bystander effect, and axon transport is suppressed. However, this study found that loss of dNmnat in axed null backgrounds (which allows for preservation of neuronal integrity despite loss of dNmnat) blocked the ability of injury to induce the bystander effect. This result reveals a paradoxical, positive role for dNmnat in promoting the bystander effect early. It is suspected that dNmnat exerts this effect through modulating MAPK signaling, whose interactions are complex: loss of Nmant has been shown to suppress MAPK signaling, while increased Nmnat activity can also potently block the activation of MAPK signaling within the first few hours after axotomy. It is proposed that dNmnat activity is required early for the bystander effect and that dNmnat levels need to be precisely tuned for proper signaling at each phase (Hsu, 2020).

Glial cells are well positioned to rapidly spread signals to all axons in the wing nerve. Much like Remak bundles in mammals, the Drosophila L1 wing nerve has glial cells that appear to wrap axons individually, which would imply that axon-to-axon signals must pass through glia. The observation that selective elimination of Draper signaling in glia is sufficient to inhibit the spreading of injury signals to bystander neurons is consistent with an axon->glia->bystander neuron signaling event, although it is also possible that glia are directly injured by the axotomy and signal to bystander neurons without input from the severed axons. Given the similarities in the response of severed axons and those of bystanders (i.e., both block axon transport on the same timescale), and the selective effects of Draper on the bystander neuron axons, the former model is favored rather than the latter (Hsu, 2020).

Draper signals to bystander neurons through a transcriptional JNK/dAP-1 cascade, likely through activating MMP-1. Nerve injury also rapidly activates JNK/c-Jun signaling in mammalian Schwann cells, where JNK/c-Jun mediate most aspects of Schwann cell injury responses and reprogramming events. This conserved glial response likely occurs in Schwann-cell-like wrapping glia present in the Drosophila L1 wing nerve, although it may be activated in the subperineurial glia, which can act in a partially redundant fashion with wrapping glia. The involvement of Mmp-1 is intriguing given its well-known role in neuroinflammatory responses to brain injury in mammals, where it functions to break down the extracellular matrix and has been proposed to promote diffuse axon injury. Other key components of the Draper signaling pathway (dCed-6 in particular, which is required for Draper signaling in all other known contexts) were not required for suppression of bystander neuron neurophysiology (Hsu, 2020).

How bystander neurons receive injury signals and respond has remained unclear, although injury- or disease-induced effects on bystander neurons is well documented. In most cases, this has been explored in the context of bystander neuron cell death driven by neuroinflammatory cells. For instance, release of C1q, interleukin-1α (IL-1α), tumor necrosis factor (TNF) from microglia following brain injury drives the formation of neurotoxic astrocytes, which can promote the death of neurons through release of yet-to-be-identified toxins. Bystander neuronal cell death is also driven by brain-infiltrating inflammatory monocytes in viral encephalitis, in a way that is mediated by calpains, which are also important regulators of axon degeneration. Secondary axon degeneration (i.e., that occurring in neurons not damaged by the initial injury) can be driven in a way that requires intracellular Ca2+ release through IP3Rs and ryanodine receptors. These represent extreme cases of bystander effects, where cells undergo apoptosis or their axons degeneration. Whether dSarm/Sarm1 is involved in these effects is an open question. The model employed by this study is likely most relevant to partial nerve injury, where non-autonomous changes in bystander neurons have been well documented. Uninjured bystander neurons in mild TBI models are certainly altered physiologically in a reversible way. The molecular basis of any of these signaling events remains unknown, but this study points to dSarm/Sarm1 as a candidate mediator. It is interesting to note that in contrast to control mice, which show significant behavioral defects for hours after mild TBI, Sarm1-/- animals exhibited almost immediate recovery, and this was at a time point long before diffuse axon injury is observed in TBI models. It is plausible that this early loss of function is mediated in part by the bystander effect (Hsu, 2020).

In summary, this study defines two genetically separable phases of dSarm signaling, places dSarm/Sarm1 at the heart of neuronal injury signaling throughout neural tissues, identifies new signaling partners for dSarm, and expands its role to regulating the responses of uninjured neurons to local tissue injury (Hsu, 2020).

Transcription factor Pebbled/RREB1 regulates injury-induced axon degeneration

Genetic studies of Wallerian degeneration have led to the identification of signaling molecules (e.g., dSarm/Sarm1, Axundead, and Highwire) that function locally in axons to drive degeneration. This study identified a role for the Drosophila C2H2 zinc finger transcription factor Pebbled [Peb, Ras-responsive element binding protein 1 (RREB1) in mammals] in axon death. Loss of Peb in Drosophila glutamatergic sensory neurons results in either complete preservation of severed axons, or an axon death phenotype where axons fragment into large, continuous segments, rather than completely disintegrate. Peb is expressed in developing and mature sensory neurons, suggesting it is required to establish or maintain their competence to undergo axon death. peb mutant phenotypes can be rescued by human RREB1, and they exhibit dominant genetic interactions with dsarm mutants, linking peb/RREB1 to the axon death signaling cascade. Surprisingly, Peb is only able to fully block axon death signaling in glutamatergic, but not cholinergic sensory neurons, arguing for genetic diversity in axon death signaling programs in different neuronal subtypes. These findings identify a transcription factor that regulates axon death signaling, and peb mutant phenotypes of partial fragmentation reveal a genetically accessible step in axon death signaling (Farley, 2018).

Neurons are connected over long distances by their axons, which can extend over more than a meter in humans. Maintenance of axon integrity is essential for sustained neural circuit function because axon breakage can block nervous system signal propagation. Axon loss is a hallmark of nervous system injuries, such as traumatic brain injury and spinal cord injury, is a unifying feature of neurodegenerative diseases, and is strongly correlated with functional loss in patients (Farley, 2018).

Wallerian degeneration (WD; axotomy) serves as a useful model to study basic aspects of axon biology, and to identify axon death signaling molecules. Severed axons, after a defined latent phase, undergo explosive fragmentation and are ultimately cleared by surrounding phagocytes. The discovery of the slow WD (WldS) mutant mouse, where severed distal axons survived for weeks after axotomy, radically changed our view of axonal biology. The observed long-term survival of distal severed axon fibers in WldS animals demonstrated that axon degeneration is a controlled process, and that under some conditions, axons could survive for weeks without a cell body. A growing number of studies across several species support the notion that the competence to undergo degeneration is likely a genetically programmed event: in the rock lobster, distal severed axons have been found to survive for a year after transection in vivo, and remain capable of evoked release at neuromuscular junctions; fragments of Aplysia axons can survive in vitro for extended periods of time without degeneration; and in Caenorhabditis elegans, most distal severed axons never degenerate. Despite these surprising observations, nothing is known about transcriptional mechanisms that regulate the competence of axons to degenerate (Farley, 2018).

In axons that do undergo WD, the execution of degeneration is driven by axon death signaling molecules. Drosophila dSarm (sterile α, ARM, and TIR domain protein) was the first endogenous molecule shown to actively promote axon death. Sarm1 functions in a conserved role in mammals, where it has been proposed to act as an NAD+ hydrolase that drives axonal degeneration through promoting metabolic catastrophe. Drosophila dSarm is similarly capable of NAD+ hydrolysis, but requires signaling downstream through the BTB/BACK domain molecule Axundead to execute axon death in vivo (Neukomm, 2017). The E3 ubiquitin ligase Highwire/Phr1 also modulates axon death signaling through a mechanism that appears to involve regulating levels of the NAD+ biosynthetic molecule dNmnat/Nmnat2. In both Drosophila and mammals, all neurons tested thus far have been strongly protected by loss-of-function mutations in dSarm/Sarm1, which suggests that axon death signaling molecules are engaged to drive destruction in a wide array of, or perhaps all, neuronal subtypes (Farley, 2018).

This study presents the identification and characterization of a role for Pebbled (Peb), a transcription factor, in axon degeneration. peb mutants show two predominant axon death-defective phenotypes: (i) severed distal axons are fully preserved morphologically, or (ii) the axon shaft breaks into large fragments (partially fragmented axons, PFAs) that fail to disintegrate further, lingering in the nervous system for weeks. The PFA phenotype in peb mutants is not observed in control or axon death mutants, and therefore defines a genetically accessible step in axon death signaling. Surprisingly, while PFAs form in all neurons, the ability of peb mutations to completely suppress axon degeneration was only observed in glutamatergic neurons, and not in cholinergic neurons, arguing that Peb functions to differentially modulate competence to undergo axon degeneration in distinct subsets of neurons (Farley, 2018).

Distal axons separated from their cell bodies can survive in a functionally competent state for days to weeks after axotomy in multiple species. It therefore seems plausible that some axons are programmed to degenerate while others are not. This study identified the transcription factor Pebbled (Peb)/RREB1 as an essential modulator of axon death in Drosophila. Through epistatic analysis, peb is placed upstream of dSarm or in a separate, parallel pathway, which ultimately converges on axon death. The simplest interpretation of the data is that Peb regulates axon death signaling in glutamatergic axons at the transcriptional level. Human RREB1 can rescue axon death phenotypes associated with loss of peb, arguing for strong conservation of the binding properties of Peb and hRREB1, and implying that RREB1 may play similar roles in axon biology in mammals (Farley, 2018).

Pebbled appears to identify a step in the axon death signaling cascade. Loss of Pebbled function resulted in the appearance of three axon phenotypes after injury: (i) full morphological preservation with a slightly delayed loss of mitochondria; (ii) the generation of PFAs that linger in the nervous system for weeks, but which also lose mitochondria after a short delay; or (iii) apparently normal axon death signaling and clearance. The cell biology of axon preservation in peb mutants is unique, and implies that peb mutants identify a genetically accessible step in axon death signaling. PFAs have not been observed in other axon death mutants (i.e., dsarm, hiw, or axed), as these mutants all fully block axon degeneration after axotomy. Understanding the nature of PFA production compared with normal axon degeneration is an important goal for future study. In the case of dsarm mutants, in addition to the axon shaft maintaining complete integrity, mitochondria also appeared well-preserved. That was not the case in peb mutants, where mitochondria degenerated after only a short delay, and preserved axons were severely depleted of mitochondria for the duration of their extended survival. Interestingly, the phenotype of individual peb mutant axons is consistent along the entirety of the axon shaft: an axon that generated PFAs in one portion, but was fully protected elsewhere, is never observed. Unraveling the molecular basis of this all-or-none type of phenotypic expression is of great interest for the future. Finally, while some PFAs are observed in control animals immediately after the initiation of axon fragmentation, they quickly undergo explosive degeneration and are cleared. From these data, it is concluded that Peb functions both at the initial phase of axon breakage into smaller fragments, and subsequently during the phase of explosive degeneration (Farley, 2018).

To date all known axon death signaling molecules -- dSarm, Hiw, and Axed -- have been proposed to function locally in the axon to drive destruction, and their neuroprotective effects extend to both glutamatergic and cholinergic neurons. Based on its expression in the nuclei of wing sensory neuron precursors and mature neurons, and the fact it is a C2H2 zinc finger transcription factor, it is proposed that Peb functions at the transcriptional level to help establish and maintain competence to undergo axon degeneration. While Peb appears to be expressed broadly in wing sensory neurons, surprisingly, the ability of peb mutants to fully block axon fragmentation is restricted to glutamatergic neurons. How Peb selectively protects glutamatergic axons is unclear, but could modify axonal phenotypes through the JNK signaling cascade. During embryogenesis, in AS peb mutants show increased levels of AP-1 transcriptional activity downstream of the JNK signaling cascade, which in turn inhibits cytoskeletal rearrangements that allow for cell migration. This study found a lack of evidence to support a role for JNK signaling in axon death, but some data support a neuroprotective role for this pathway by controlling baseline levels of Nmnat. Peb has also been shown to negatively regulate nervy, the Drosophila homolog of mammalian MTG8 proto-oncogene; however, this study observed no alterations in axon death when nervy was overexpressed in glutamatergic neurons (Farley, 2018).

The nuclear localization requirement of the carboxy terminal DNA binding zinc finger domains of Peb for rescue suggests that Peb is regulating injury-induced axon degeneration at the transcriptional level. Attempts were made to identify direct transcriptional targets of Peb by expressing a tagged version of Peb in the Drosophila embryonic cell line, GM2, and performing ChIP with antibodies specific to tagged Peb and subsequent deep sequencing (ChIP-seq). This approach successfully identified the one known target for Peb, the transcriptional regulator Nervy, whose overexpression did not block axon death, and several potential Peb targets. No evidence was found for direct binding of Peb to regions containing known axon death signaling genes (i.e., dsarm, axed, or hiw). This could indicate that Peb does not directly modulate axon death genes in vivo to exert its effects, although it remains unclear how similar Peb transcriptional activity in GM2 cells might be compared with neurons. Finally, it remains unclear why dsarm, but not axed mutations, modify peb mutant phenotypes, given that Axed signals downstream of dSarm in axon death. A deeper understanding of the molecular basis of axon degeneration, and the nature of PFAs, will likely be required to answer this question (Farley, 2018).

This analysis of peb mutant phenotypes reveals features of the cell biology of axon death. The discovery of PFAs in peb mutants implies that axon degeneration can be genetically dissected into distinct activation and execution phases, and it is proposed that PFAs represent activation, but failure to execute axon death. Either increased or decreased Peb levels could lead to the production of PFAs, arguing that fine-tuning of Peb levels is essential for appropriate execution of axon death. Furthermore, Peb modulation of PFA production is not limited to glutamatergic neurons, since PFAs were found in cholinergic neurons under both loss- and gain-of-function Peb conditions. Interestingly, Peb allows genetic separation of mitochondrial loss from axon degeneration. Mitochondrial degeneration was observed even in fully protected peb mutant axons, indicating that mitochondrial destruction must occur through a Peb-independent signaling pathway (Farley, 2018).

Axon death pathways converge on Axundead to promote functional and structural axon disassembly

Axon degeneration is a hallmark of neurodegenerative disease and neural injury. Axotomy activates an intrinsic pro-degenerative axon death signaling cascade involving loss of the NAD+ biosynthetic enzyme Nmnat/Nmnat2 in axons, activation of dSarm/Sarm1, and subsequent Sarm-dependent depletion of NAD+. This study has identified Axundead (Axed) as a mediator of axon death. axed mutants suppress axon death in several types of axons for the lifespan of the fly and block the pro-degenerative effects of activated dSarm in vivo. Neurodegeneration induced by loss of the sole fly Nmnat ortholog is also fully blocked by axed, but not dsarm, mutants. Thus, pro-degenerative pathways activated by dSarm signaling or Nmnat elimination ultimately converge on Axed. Remarkably, severed axons morphologically preserved by axon death pathway mutations remain integrated in circuits and able to elicit complex behaviors after stimulation, indicating that blockade of axon death signaling results in long-term functional preservation of axons (Neukomm, 2017).

Maintenance of the morphological integrity of neurons is essential for sustained nervous system function throughout an animal's lifespan. Nervous system injury or neurological disease leads to axonal and synaptic degeneration and, in turn, loss of neural circuit connectivity and function. Molecular pathways driving axonal degeneration remain poorly defined in any context; however, recent work on Wallerian degeneration (WD) has revealed that axon injury activates an intrinsic, conserved, pro-degenerative (axon death) signaling pathway. dSarm/Sarm1 (sterile α/Armadillo/Toll-Interleukin receptor homology domain protein) was previously identified as a key mediator of axon death signaling. Loss of dSarm in Drosophila, or Sarm1 in mouse, resulted in severed distal axons remaining morphologically preserved for weeks after injury (Gerdts, 2013, Osterloh, 2012), indicating that dSarm/Sarm1 pro-degenerative signaling is an ancient mechanism used by axons to drive self-destruction. How dSarm/Sarm1 signals to execute axon death remains unclear, but dSarm/Sarm1 has recently been linked to the NAD+ metabolic pathway, which based on extensive evidence appears to be a central mediator of axonal integrity (Neukomm, 2017).

The first evidence supporting a role for NAD+ in axon maintenance came from the identification and characterization of the slow Wallerian degeneration (WldS) mouse, where severed axons fibers survived for weeks when detached from their cell bodies. This remarkable neuroprotective phenotype effect was due to a chromosomal rearrangement that led to the generation of the novel WldS molecule, a fusion protein consisting of the NAD+ biosynthetic enzyme nicotinamide mononucleotide adenylyltransferase 1 (Nmnat1) and a short fragment of Ube4b. Consistent with a positive role for NAD+ in sustaining axons, NAD+ levels were found to plummet in axons immediately prior to granular fragmentation. Axon degeneration could be rescued by exogenous NAD+ or its precursors, and axotomy-induced NAD+ depletion was blocked by WldS. Numerous studies have demonstrated neuroprotective roles for NAD+-related metabolites. The current proposed mechanism for activation of axon degeneration after injury is the depletion of Nmnat2, a labile form of Nmnat found in mammalian axons that is normally transported down axons from the soma. Nmnat2 is seen as a critical regulator of axon survival: its half-life approximates the latent phase prior to explosive axon fragmentation, depletion of Nmnat2 from axons induces spontaneous degeneration, and stabilization of Nmnat2 can phenocopy the effects of WldS (Neukomm, 2017).

The second line of evidence supporting a role for NAD+ in axonal protection came from drug screens for molecules that promoted neurogenesis (or neuroprotection) in vivo. P7C3 was identified as an activity-enhancing compound of nicotinamide phosphoribosyltransferase (Nampt), a rate-limiting enzyme in the NAD+ salvage pathway, which likely leads to increased levels of NAD+ in injured axons to help sustain integrity. Derivatives of the P7C3 series have since been shown to be neuroprotective in many models of neurodegenerative disease, and in some models of neural injury . However, whether P7C3 harbors the ability to attenuate axon death remains to be determined (Neukomm, 2017).

Finally, recent work has demonstrated that NAD+ depletion after axotomy is blocked in Sarm1-/- mutants, and dimerization of the Sarm1 TIR domain can drive rapid depletion of NAD+ from cells and axons (Gerdts, 2015). Surprisingly, it appears that the Sarm1 TIR domain harbors endogenous NAD+ hydrolase activity (Essuman, 2017). Thus an emerging stepwise model of WD is that axotomy leads to depletion of labile pools of Nmnat2 in axons detached from their cell bodies; this in turn results in an initial depletion of NAD+ levels due to lack of new NAD+ synthesis and/or salvage, dSarm/Sarm1 signaling is then activated followed by rapid depletion of NAD+ levels below a threshold needed to maintain axonal integrity, and finally explosive degeneration of the axon ensues. NAD+ depletion from axons is a compelling model for WD, although alternative mechanisms cannot be excluded, due to limited understanding of the genetics of axon death signaling. Besides dSarm/Sarm1, only one other molecule, the E3 ubiquitin ligase Highwire/Phr1, has been shown to be required for axon death in vivo: loss-of-function mutations in highwire/Phr1 potently suppress axon death in fly nerve injury models and mammalian sciatic nerve lesion experiments. Highwire/Phr1 likely acts upstream of dSarm/Sarm1, and is required for the normal turnover of Nmnat/Nmnat2. Loss of Highwire/Phr1 is therefore proposed to stabilize Nmnat/Nmnat2 and maintain axonal pools of NAD+. Are there other signaling molecules acting downstream of dSarm/Sarm1 to execute axonal death, or is NAD+ depletion the final step? Sarm1 has also been reported to drive axon death through a downstream MAPK signaling cascade (Yang, 2015), similar to the signaling mechanism used by C. elegans TIR-1 during regulation of odorant receptor expression; however, this remains controversial. A recent contradictory study argues that MAPKs exert their effect by fine-tuning levels of Nmnat2 upstream of Sarm1 signaling (Neukomm, 2017).

This paper reports the identification and characterization of Axundead (Axed), an axonal BTB and BACK domain protein that signals downstream of dSarm. Axed is essential for injury-induced axon death signaling, and axed mutations can fully suppress degeneration induced by activated dSarm, or complete elimination of Nmnat activity from axons. Thus, Axed is a novel pro-degenerative signaling molecule, and the neuroprotective effects of loss of Axed exceed those of dsarm null mutants (Neukomm, 2017).

This study presents the identification and initial characterization of the BTB and BACK domain molecule Axed. Loss of Axed function was sufficient to block axon death for the lifespan of the fly, and axed mutants were neuroprotective in all neurons tested. Axed, like dSarm, appears to function selectively in axon death during WD, as axed mutants blocked neither cell death nor developmental pruning of axons or dendrites, supporting the notion that the Nmnat-depletion/dSarm/Axed signaling pathway is engaged specifically in response to axonal injury. However, it remains possible that while Axed or dSarm elimination is not sufficient to block cell death, redundant genetic pathways might work together with dSarm and Axed in the context of apoptotic cell death (Neukomm, 2017).

Axed encodes a previously uncharacterized BTB and BACK domain protein. The best-characterized role for BTB-containing proteins is the recruitment of substrates to Cullin Ring Ubiquitin Ligase (CRL) complexes for ubiquitin tagging and proteasome degradation, where BTB domains function in homo- or hetero-dimerization while the BACK domain (often with the BTB domain) interacts directly with Cullins. Typically, the C-terminal regions in BTB domains molecules function within CRL complexes to bind target substrates designated either for ubiquitination by the CRL and subsequent degradation by the proteasome or for signaling. The BTB and BACK domains of Axed are required for optimal Axed function in vivo based on their partial abilities to rescue axon death phenotypes in axed mutant backgrounds. In contrast, the C terminus appears to be absolutely essential for Axed function as AxedδCterm completely fails to rescue axon death. This is not likely due to destabilization of the molecule lacking the C terminus, since AxedδCterm can be stably expressed in Drosophila S2 cells. Despite extensive attempts to implicate the wide array of CRLs in Drosophila in axon death and Axed function, no evidence was found supporting a role for CRLs in axon degeneration. Given that there are five Cullin-like molecules in the Drosophila genome, it seems possible that genetic redundancy between Cullins might explain the lack of phenotype. Alternatively, Axed could be functioning to promote axon death in a completely novel Cullin-independent manner. Future studies aimed at identifying direct binding partners for Axed will be essential to resolve these issues. If Axed functions as a bridging molecule for CRL ubiquitination activity, of particular interest will be identifying molecules that bind to the Axed C-terminal putative substrate binding domain (Neukomm, 2017).

Sarm1 functions in axons after injury (Gerdts, 2015). Given that Axed functions genetically downstream of Sarm1, it is proposed that Axed also functions in axons. Using a functional, endogenously enhanced GFP-tagged version of Axed (AxedeGFP), it was found that Axed protein is enriched in the neuropil in both the larva and the adult nervous system. The Drosophila larval neuropil is highly enriched for dendrites and axons and is the site of all CNS synapse formation. AxedeGFP signals overlapped extensively with axons, dendrites, and synapses, further supporting a cell-autonomous role for Axed in neurons during axon death. AxedeGFP is also present in the adult neuropil, and AxedeGFP signals transiently increased at 4 and 6 hr in the antennal lobe after injury but returned to baseline levels by 24 hr, a time point at which most severed axons have fragmented. The return to baseline levels likely represents AxedeGFP staining in local and projection interneurons in the antennal lobe that were not severed by antennal ablation. Why AxedeGFP signals might transiently increase remains unclear. Perhaps AxedeGFP is relocalized in a way that increases GFP exposure, or new AxedeGFP protein may be locally synthesized. Regardless of the precise mechanisms, the observations indicate that AxedeGFP dynamics in vivo are sensitive to axonal injury (Neukomm, 2017).

axed mutant phenotypes are indistinguishable from dsarm mutants with respect to axon preservation after injury. In addition, axed mutants completely blocked the pro-degenerative activity of the gain-of-function dSarm molecule dSarmΔdsarm in vivo. These genetic data strongly support a model whereby Axed functions genetically downstream of dSarm, and argue that dSarm and Axed drive axon death through the same genetic pathway (Neukomm, 2017).

dSarmΔdsarm appears to induce a Wallerian-like program in vivo based on several observations. First, the explosive fragmentation observed is morphologically similar to WD. Second, dSarmΔdsarm-induced degeneration can be suppressed partially by WldS, whose activity is highly selective to WD. Third, dSarmΔdsarm pro-degenerative activity can be fully suppressed in axed mutants. Finally, all structural and biochemical studies to date support the notion that elimination of the N-terminal ARM domain of dSarm (Drosophila), TIR-1(C. elegans), or Sarm1 (mammals) leads to the production of a gain-of-function molecule that activates signaling. That dSarmΔdsarm signaling in the absence of injury is potently suppressed by axed mutants provides a compelling argument for Axed to act downstream of dSarm. The nature of the genetic program that drives death of neuronal cell bodies after dSarm/Sarm1 activation remains mysterious. It could not be blocked with the broad caspase inhibitor P35. Related studies in mouse found a lack of evidence for apoptotic signaling molecules, necroptosis, and parthanatos in death induced by activated Sarm1. The observation that it was possible to induce robust degeneration of axons, dendrites, and cell bodies by adult-specific induction of dSarmΔdsarm in adult PDF+ neurons, even in the absence of injury, suggests that dSarmΔdsarm could provide a useful tool for conditional removal of selected neurons from adult circuits for functional studies (Neukomm, 2017).

In the larger context of axon death signaling, the finding that mutations in hiw do not suppress dSarmΔdsarm in vivo agrees with the proposed model that Hiw/Phr1 acts genetically upstream of dSarm/Sarm1 and Nmnat degradation. The position of the MAPK signaling cascade relative to dSarm/Sarm1 is still debated in the field and remains to be determined. The fact that the Drosophila genome houses only a single JNK family member, and the observation that null alleles had no axon death phenotype, argues strongly against a central role for JNK signaling in axon death (Neukomm, 2017).

With respect to their roles in the functional disassembly of axons, this study has demonstrated that blocking axon death with mutations in dsarm, axed, or highwire is sufficient to preserve axons in a functional state in neural circuits where they can elicit complex grooming behaviors for weeks after axotomy. Therefore, axon death mutants, like the WldS molecule, exert their neuroprotective effects very early in the axon death genetic program, and their therapeutic blockade is likely to lead to the preservation of functional axons in the context of neurological disease (Neukomm, 2017).

A number of recent studies have led to the following model for axon death signaling: (1) axotomy results in the degradation of pools of labile Nmnat/Nmnat2 in distal severed axons, (2) depletion of Nmnat/Nmnat2 in turn leads to decreases in axonal NAD+, (3) loss of Nmnat/Nmnat2 or decreased NAD+ somehow activates dSarm/Sarm1 signaling, and (4) Sarm1 NAD+ hydrolase activity then drives pathological depletion of axonal NAD+ pools, causing catastrophic energy failure that ultimately drives axon degeneration. A number of observations were made that are difficult to rectify with a simple Nmnat2/Sarm1-dependent NAD+ depletion model. The first is that expression of dSarmΔdsarm, which based on previous reports should rapidly degrade axonal NAD+ and normally leads to rapid axonal degeneration (Essuman, 2017), can be completely suppressed by loss of Axed. If the terminal step in axonal death were Nmnat2/Sarm1-dependent depletion of NAD+ pools, it is hard to imagine how the NAD+ hydrolase activity of the Sarm1 TIR domain, once unleashed, could be suppressed by the loss of a BTB and BACK domain protein (i.e., Axed). The alternative hypothesis is favored that Sarm1 signaling, perhaps through its TIR domain NAD+ hydrolase activity, activates an Axed-dependent downstream signaling pathway essential for axon death. ADP-ribosylation of targets is one mechanism that allows certain E3 ubiquitin ligases to bind substrates. There is certainly a tight association between NAD+ metabolism and axon death signaling, and the NAD+ hydrolase activity associated with the Sarm1 TIR domain can lead to the production of ADPr. Perhaps BTB and BACK domain proteins like Axed also require ADP-ribosylation of selected targets during axon death. In this model, the degradation of NAD+ would be a byproduct of the reaction, not a driving force (Neukomm, 2017).

Nmnat2-/- mutant mice die perinatally with neurons containing short axons, but these embryos can be rescued to adulthood by Sarm1-/- null mutations. It is likely that Nmnat2-/- animals and axons can survive in Sarm1-/- backgrounds because Nmnat1 and Nmnat3, the additional isoforms of Nmnat found in mice, could partially compensate. An nmnatRNAi experiment may therefore more closely resemble conditions in the Nmnat2-/-, Sarm1-/- mouse, with respect to the partial, but not complete elimination of Nmnat function. dsarm null alleles could only partially protect axons from Nmnat depletion with nmnatRNAi, as far fewer axons and cell bodies were observed compared to controls. Consistent with a requirement for Nmnat activity in cell survival, this study found null clones of the sole fly Nmnat molecule die within 1 day after eclosion. Remarkably, this phenotype can be completely overcome by loss of Axed, since axed, nmnat double mutant clones survive for weeks. However, it was surprising to find that loss of dSarm fails to block this degeneration. Whether Sarm1-/- animals could suppress the degeneration of mammalian neurons completely lacking Nmnat function would require the simultaneous elimination of all Nmnat isoforms (i.e., Nmnat1,2,3 triple mutants), which has not been explored. In the absence of Nmnat activity, invertebrate cells should not be able to autonomously synthesize NAD+. How these cells survive remains a mystery, but a number of possibilities might explain this phenotype. Perhaps yet-to-be-identified NAD+ biosynthetic pathways exist that can function without Nmnat. Alternatively, axons in vivo could be supplemented with NAD+ or key intermediates by surrounding glia and thereby sustain their integrity in axed, nmnat double mutant axons. Finally, it is possible that in the absence of Axed, Nmnat protein may not be turned over appropriately in axons and that perdurance of small amounts of Nmnat may generate sufficient levels of NAD+ for axon survival. Direct, in vivo measurement of NAD+ levels in axons in Drosophila will be essential to answer this question, but current NAD+ sensors have not yet provided sufficient sensitivity for this analysis(Neukomm, 2017).

Axed appears to sit genetically at a convergence point in axon death signaling. There are three ways to activate axon death: axotomy, Nmnat/Nmnat2 depletion, or expression of gain-of-function dSarm/Sarm1. Axed mutants, but not dsarm, can suppress each of these treatments, and, amazingly, axed mutants can even survive the combination of all three insults at once. The neuroprotective effects of axed therefore exceed those of dsarm mutants. It remains to be determined whether any of the four putative mammalian paralogs (BTBD1, BTBD2, BTBD3, and BTBD6) play a role in axon death, but this seems likely based on the strong conservation of dSarm/Sarm1 function in axon degeneration. If so, these would represent important new therapeutic targets for blocking axon death in neurological diseases such as traumatic brain injury, peripheral neuropathy, or nerve injury, where dSarm/Sarm1 signaling is known to drive axon loss (Neukomm, 2017).

Three-tier regulation of cell number plasticity by neurotrophins and Tolls in Drosophila

Cell number plasticity is coupled to circuitry in the nervous system, adjusting cell mass to functional requirements. In mammals, this is achieved by neurotrophin (NT) ligands, which promote cell survival via their Trk and p75NTR receptors and cell death via p75NTR and Sortilin. Drosophila NTs (DNTs; see NT1) bind Toll receptors (see Toll-6 & Toll-7) instead to promote neuronal survival, but whether they can also regulate cell death is unknown. This study show that DNTs and Tolls can switch from promoting cell survival to death in the central nervous system (CNS) via a three-tier mechanism. First, DNT cleavage patterns result in alternative signaling outcomes. Second, different Tolls can preferentially promote cell survival or death. Third, distinct adaptors downstream of Tolls can drive either apoptosis or cell survival. Toll-6 promotes cell survival via MyD88-NF-κB and cell death via Wek-Sarm-JNK. The distribution of adaptors changes in space and time and may segregate to distinct neural circuits. This novel mechanism for CNS cell plasticity may operate in wider contexts (Foldi, 2017).

Balancing cell death and cell survival enables structural plasticity and homeostasis, regeneration, and repair and fails in cancer and neurodegeneration. In the nervous system, cell number plasticity is linked to neural circuit formation, adjusting neuronal number to functional requirements. In mammals, the neurotrophin (NT) protein family [NGF, brain-derived neurotrophic factor (BDNF), NT3, and NT4] regulates neuronal number through two mechanisms. First, full-length pro-NTs, comprised of a disordered prodomain and a cystine-knot (CK) domain, induce cell death; in contrast, mature NTs formed of CK dimers promote cell survival. Second, pro-NTs bind p75NTR and Sortilin receptors, inducing apoptosis via JNK signaling, whereas mature NTs bind p75NTR, promoting cell survival via NF-κB and TrkA, B, and C, promoting cell survival via PI3K/AKT and MAPK/ERK. As the NTs also regulate connectivity and synaptic transmission, they couple the regulation of cell number to neural circuitry and function, enabling structural brain plasticity. There is abundant evidence that cell number plasticity occurs in Drosophila melanogaster central nervous system (CNS) development, with neurotrophic factors including NTs and mesencephalic astrocyte-derived neurotrophic factor (MANF), but fruit flies lack p75NTR and Trk receptors, raising the question of how this is achieved in the fly. Finding this out is important, as it could lead to novel mechanisms of structural plasticity for both flies and humans (Foldi, 2017).

The Drosophila NTs (DNTs) Spätzle (Spz), DNT1, and DNT2 share with mammalian NTs the characteristic structure of a prodomain and a conserved CK of 13-15 kD, which forms a disulfide-linked dimer. Spz resembles NGF biochemically and structurally, and the binding of its Toll-1 receptor resembles that of NGF to p75NTR. DNT1 (also known as spz2) was discovered by homology to BDNF, and DNT2 (also known as spz5) as a paralogue of spz and DNT1. DNT1 and 2 promote neuronal survival, and DNT1 and 2, Spz, and Spz3 are required for connectivity and synaptogenesis. Spz, DNT1, and DNT2 are ligands for Toll-1, -7, and -6, respectively, which function as NT receptors and promote neuronal survival, circuit connectivity, and structural synaptic plasticity. Tolls belong to the Toll receptor superfamily, which underlies innate immunity. There are nine Toll paralogues in flies, of which only Toll-1, -5, -7, and -9 are involved in immunity. Tolls are also involved in morphogenesis, cell competition, and epidermal repair. Whether DNTs and Tolls can balance cell number plasticity is unknown (Foldi, 2017).

Like the p75NTR receptor, Toll-1 activates NF-κB (a potent neuronal prosurvival factor with evolutionarily conserved functions also in structural and synaptic plasticity) signaling downstream. Toll-1 signaling involves the downstream adaptor MyD88, which forms a complex with Tube and Pelle. Activation of Toll-1 triggers the degradation of the NF-κB inhibitor Cactus, enabling the nuclear translocation of the NF-κB homologues Dorsal and Dorsal-related immunity factor (Dif), which function as transcription factors. Other Tolls have also been suggested to activate NF-κB. However, only Toll-1 has been shown to bind MyD88, raising the question of how the other Tolls signal in flies (Foldi, 2017).

Whether Tolls regulate cell death is also obscure. Toll-1 activates JNK, causing apoptosis, but its expression can also be activated by JNK to induce nonapoptotic cell death. Toll-2, -3, -8, and -9 can induce apoptosis via NF-κB and dSarm independently of MyD88 and JNK. However, in the CNS, dSarm induces axonal degeneration, but there is no evidence that it can promote apoptosis in flies. In other animals, Sarm orthologues are inhibitors of Toll signaling and MyD88, but there is no evidence that dSarm is an inhibitor of MyD88 in Drosophila. Thus, whether or how Tolls may regulate apoptosis in flies is unclear (Foldi, 2017).

In the mammalian brain, Toll-like receptors (TLRs) are expressed in neurons, where they regulate neurogenesis, apoptosis, and neurite growth and collapse in the absence of any insult. However, their neuronal functions have been little explored, and their endogenous ligands in neurons remain unknown (Foldi, 2017).

Because Toll-1 and p75NTR share common downstream signaling pathways and p75NTR can activate NF-κB to promote cell survival and JNK to promote cell death, this study asked whether the DNTs and their Toll receptors could have dual roles controlling cell survival and death in the Drosophila CNS (Foldi, 2017).

In the first regulatory tier, each DNT has unique features conducive to distinctive functions. Spz, DNT1, and DNT2 share with the mammalian NTs the unequivocal structure of the CK domain unique to this protein family. However, DNT1, DNT2, and Spz have distinct prodomain features and are processed differently, leading to distinct cellular outcomes. Spz is only secreted full length and cleaved by serine proteases. DNT1 and 2 are cleaved intracellularly by conserved furins. In cell culture, DNT1 was predominantly secreted with a truncated prodomain (pro-DNT1), whereas DNT2 was secreted mature. In vivo, both pro- and mature DNTs were produced from neurons. Interestingly, DNT1 also has an isoform lacking the CK domain, and Spz has multiple isoforms with truncated prodomains. Thus, in vivo, whether DNT1 and 2 are secreted full length or cleaved and whether Spz is activated will depend on the proteases that each cell type may express. Pro-DNT1 activates apoptotic JNK signaling, whereas mature DNT1 and 2 activate the prosurvival NF-κB (Dorsal and Dif) and ERK signaling pathways. Mature Spz does not activate ERK. This first tier is evolutionarily conserved, as mammalian pro-NTs can promote cell death, whereas furin-cleaved mature NTs promote cell survival. NF-κB, JNK, and ERK are downstream targets shared with the mammalian NTs, downstream of p75NTR (NF-κB and JNK) and Trks (ERK), to regulate neuronal survival and death. Thus, whether a cell lives or dies will depend on the available proteases, the ligand type, and the ligand cleavage product it receives (Foldi, 2017).

In a second regulatory tier, this study showed that the specific Toll family receptor activated by a DNT matters. Toll-6 and -7 could maintain neuronal survival, whereas Toll-1 had a predominant proapoptotic effect. Because there are nine Tolls in Drosophila, some Tolls could have prosurvival functions, whereas others could have proapoptotic functions. Different Tolls also lead to different cellular outcomes in immunity and development. Thus, the life or death of a neuron will depend on the Toll or combination of Tolls it expresses. Binding of Spz to Toll-1 is most likely unique, but DNT1 and 2 bind Toll-6 and -7 promiscuously, and, additionally, DNT1 and 2 with Toll-6 and -7 activate NF-κB and ERK, whereas pro-DNT1 activates JNK. This suggests that ligand prodomains might alter the affinity for Toll receptors and/or facilitate the formation of heterodimers between different Tolls and/or with other coreceptors to induce cell death. A 'DNT-Toll code' may regulate neuronal numbers (Foldi, 2017).

In a third tier, available downstream adaptors determine the outcome between cell survival and death. Toll-6 and -7 activate cell survival by binding MyD88 and activating NF-κB and ERK (whether ERK activation depends on MyD88 is not known), and Toll-6 can activate cell death via Wek, dSarm, and JNK signaling. Toll-6 was shown to bind MyD88 and Wek, which binds dSarm, and dSarm binds MyD88 and promotes apoptosis by inhibiting MyD88 and activating JNK. Wek also binds MyD88 and Toll-1. So, evidence suggests that Wek recruits MyD88 and dSarm downstream of Tolls. Because Toll-6 binds both MyD88 and Wek and Wek binds both MyD88 and dSarm, Wek functions like a hinge downstream of Toll-6 to facilitate signaling via MyD88 or dSarm, resulting in alternative outcomes. Remarkably, adaptor expression profiles change over time, switching the response to Toll-6 from cell survival to cell death. In the embryo, when both MyD88 and dSarm are abundant, there is virtually no Wek, and Toll-6 can only bind MyD88 to promote cell survival. As Wek levels rise, Toll-6 signaling can also induce cell death. If the Wek-Sarm-JNK route prevails, Toll-6 induces apoptosis; if the Wek-MyD88-NF-κB route prevails, Toll-6 signaling induces cell survival (Foldi, 2017).

Thus, the cellular outcome downstream of DNTs and Tolls is context and time dependent. Whether a cell survives or dies downstream of DNTs and Tolls will depend on which proteases are expressed nearby, which ligand it receives and in which form, which Toll or combination of Tolls it expresses, and which adaptors are available for signaling (Foldi, 2017).

How adaptor profiles come about or change is not understood. A neuronal type may be born with a specific adaptor gene expression profile, or Toll receptor activation may influence their expression. In fact, MyD88 reinforces its own signaling pathway, as Toll-6 and -7 up-regulate Dorsal, Dif, and Cactus protein levels and TLR activation increases Sarm levels. This study showed that apoptosis caused by MyD88 excess depends on JNK signaling. Because JNK functions downstream of Wek and dSarm, this suggests that MyD88, presumably via NF-κB, can activate the expression of JNK, wek, or dsarm. By positively regulating wek expression, MyD88 and dSarm could establish positive feedback loops reinforcing their alternative pathways. Because dSarm inhibits MyD88, mutual regulation between them could drive negative feedback. Positive and negative feedback loops underlie pattern formation and structural homeostasis and could regulate neuronal number in the CNS as well. Whether cell-autonomous or -nonautonomous mechanisms result in the diversification of adaptor profiles, either in time or cell type, remains to be investigated (Foldi, 2017).

Either way, over time the Toll adaptors segregate to distinct neural circuits, where they exert further functions in the CNS. Toll-1, -6, and -8 regulate synaptogenesis and structural synaptic plasticity. Sarm regulates neurite degeneration, and in the worm, it functions at the synapse to determine neuronal identity. The reporters used in this study revealed a potential segregation of MyD88 to the motor circuit and dSarm to the sensory circuit, but this is unlikely to reflect the endogenous complexity of Toll-signaling circuitry, as dsarmMIMIC- has a GFP insertion into one of eight potential isoforms, and dsarm also functions in the motor system (McLaughlin, 2016). Importantly, cell death in the normal CNS occurs mostly in late embryogenesis and in pupae, coinciding with neural circuit formation and remodeling, when neuronal number is actively regulated. Thus, the link by DNTs and Tolls from cell number to circuitry offers a complex matrix of possible ways to regulate structural plasticity in the CNS (Foldi, 2017).

This study has uncovered remarkable similarities between Drosophila Toll-6 and mammalian TLR signaling involving MyD88 and Sarm. All TLRs except TLR3 signal via MyD88 and activate NF-κB . Neuronal apoptosis downstream of TLRs is independent of NF-κB and instead depends on TRIF and Sarm1. Sarm1 is a negative regulator of TLR signaling, an inhibitor of MyD88 and TRIF. sarm1 is expressed in neurons, where it activates JNK and promotes apoptosis. However, the endogenous ligands for TLRs in the normal undamaged brains are not known. Preliminary analysis has revealed the intriguing possibility that NTs either can bind TLRs or induce interactions between Trks, p75NTR, and TLRs. It is compelling to find out whether TLRs regulate structural plasticity in the mammalian brain in concert with NTs (Foldi, 2017).

To conclude, DNTs with Tolls constitute a novel molecular system for structural plasticity in the Drosophila CNS. This could be a general mechanism to be found also in the mammalian brain and in other contexts as well, such as epithelial cell competition and regeneration, and altered in cancer and neurodegeneration (Foldi, 2017).

MAPK signaling promotes axonal degeneration by speeding the turnover of the axonal maintenance factor NMNAT2

Injury-induced (Wallerian) axonal degeneration is regulated via the opposing actions of pro-degenerative factors such as SARM1 and a MAPK signal and pro-survival factors, the most important of which is the NAD+ biosynthetic enzyme NMNAT2 that inhibits activation of the SARM1 pathway. This study investigated the mechanism by which MAPK signaling facilitates axonal degeneration. MAPK signaling was shown to promote the turnover of the axonal survival factor NMNAT2 in cultured mammalian neurons as well as the Drosophila ortholog dNMNAT in motoneurons. The increased levels of NMNAT2 are required for the axonal protection caused by loss of MAPK signaling. Regulation of NMNAT2 by MAPK signaling does not require SARM1, and so cannot be downstream of SARM1. Hence, pro-degenerative MAPK signaling functions upstream of SARM1 by limiting the levels of the essential axonal survival factor NMNAT2 to promote injury-dependent SARM1 activation. These findings are consistent with a linear molecular pathway for the axonal degeneration program (Walker, 2017)

A Toll receptor-FoxO pathway represses Pavarotti/MKLP1 to promote microtubule dynamics in motoneurons

FoxO proteins are evolutionarily conserved regulators of neuronal structure and function, yet the neuron-specific pathways within which they act are poorly understood. To elucidate neuronal FoxO function in Drosophila melanogaster, a screen was performed for FoxO's upstream regulators and downstream effectors. On the upstream side, genetic and molecular pathway analyses is presented indicating that the Toll-6 receptor, the Toll/interleukin-1 receptor domain adaptor dSARM, and FoxO function in a linear pathway. On the downstream side, it was found that Toll-6-FoxO signaling represses the mitotic kinesin Pavarotti/MKLP1 (Pav-KLP), which itself attenuates microtubule (MT) dynamics. In vivo functions were probed for this novel pathway, and it was found to be essential for axon transport and structural plasticity in motoneurons. Elevated expression of Pav-KLP underlies transport and plasticity phenotypes in pathway mutants, indicating that Toll-6-FoxO signaling promotes MT dynamics by limiting Pav-KLP expression. In addition to uncovering a novel molecular pathway, this work reveals an unexpected function for dynamic MTs in enabling rapid activity-dependent structural plasticity (McLaughlin, 2016).

dSarm/Sarm1 is required for activation of an injury-induced axon death pathway

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. This study shows that loss of the Drosophila Toll receptor adaptor dSarm (sterile alpha/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. The 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 (Osterloh, 2012).

When axons are severed, the portion of the axon distal to the injury site undergoes extensive fragmentation. This process, termed Wallerian degeneration, was traditionally thought to result from passive wasting away of axons due to a lack of cell body-derived nutrients (1). However, this notion was challenged by the slow Wallerian degeneration ( WldS) mutant mouse in which the distal portion of severed axons remained morphologically intact for weeks after axotomy. The long-term survival of severed WldS+ axons raised the intriguing possibility that Wallerian degeneration might be driven by an active molecular program akin to apoptotic cell death. However, the WldS phenotype results from a gain of function, likely neomorphic mutation that leads to neuronal overexpression of a chimeric fusion protein containing the nicotinamide adenine dinucleotide (NAD+) biosynthetic enzyme Nmnat1. Despite its ability to inhibit axonal degeneration, the gain-of-function nature of the WldS phenotype does not provide direct evidence supporting the existence of an axon death signaling pathway and may be unrelated to normal Nmnat1 function. Importantly, mutants reported to suppress Wallerian degeneration, such as wnd/DLK, delay the clearance of degenerating axons in Drosophila for only ~1 to 2 days, and mouse axons for several hours. This is quite weak suppression when compared with WldS. Thus, the existence of axon death pathways in Wallerian degeneration has remained largely speculative (Osterloh, 2012).

Wallerian degeneration appears to be molecularly distinct from apoptosis, because potent genetic or chemical inhibitors of cell death do not block axonal disintegration. This question was revisited and a comprehensive screen of existing mutants and dominant negative constructs was performed for Drosophila genes affecting apoptosis, autophagy, or other defined cell degradative pathways, but these failed to suppress Wallerian degeneration (Osterloh, 2012).

If Wallerian degeneration is indeed an active process, then loss-of-function mutants that exhibit-WldS-like protection of severed axons should exist. An F2 forward genetic screen was performed in Drosophila for mutants that exhibited long-term survival of severed axons. Because genes required for Wallerian degeneration may be lethal when mutated, the screen was designed to allow for characterization of both viable and lethal mutants through mosaic analysis with a repressible cell marker (MARCM) clonal analysis. In control animals, severed olfactory receptor neuron (ORN) axons degenerated and were completely cleared from the antennal lobe 7 days after axotomy. Three lines, l(3)896, l(3)4621, and l(3)4705, were identified in which severed homozygous mutant axons generated by MARCM remained intact 1 week after axotomy. Although the number of uninjured axons is slightly reduced in each mutant, 100% of green fluorescent protein (GFP)-labeled axons exhibited long-term preservation after injury. Mutant axons remained fully intact 30 days after injury, and a significant but reduced number remained even 50 days after injury. l(3)896, l(3)4621, and l(3)4705 therefore provide axonal preservation that rivals that of WldS in Drosophila, and lasts throughout the life span of the fly. Neuroprotection in these mutants extended to synapses: Synaptobrevin punctae localized to synaptic terminals even 30 days after axotomy. Neuronal morphology appeared normal in mutant animals, indicating that these mutations do not grossly affect neuronal development (Osterloh, 2012).

It was next asked whether l(3)896 was broadly required for neuron pruning or apoptotic cell death. Dendritic and axonal pruning were examined in MARCM clones in Drosophila mushroom body γ neurons during metamorphosis. In both control and l(3)896 animals, mushroom body γ neuron axons and dendrites were pruned normally. During early embryogenesis, dMP2 neurons are present in each segment, but by late embryogenesis, all but the posterior three pairs undergo developmentally programmed apoptosis. dMP2 neurons were generated normally in l(3)4621 animals, and the appropriate subset of neurons underwent apoptosis. Finally, the proapoptotic gene hid was expressed in the Drosophila visual system to induce widespread apoptotic death in cells of the developing eye disc. It as found that l(3)896 mutant clones failed to suppress activation of cell death (Osterloh, 2012).

These mutants were all recessive in axon degenerative phenotypes and fell into a single lethal complementation group; therefore, each line represented an independently isolated lethal mutation in the same gene. To identify the gene mutated in l(3)896, l(3)4621, and l(3)4705, two complementary approaches were taken. First, the lethality of each mutant was mapped using small chromosome deficiencies, and a single gene was identified at cytogenetic region 66B. Next-generation sequencing technology was used to resequence mutant genomes to a read depth of 70x. S single gene affected in all three mutants, which resided in cytogenetic region 66B: ect4, which is refered to as dsarm (Drosophila sterile alpha and Armadillo motif). The dsarm gene encodes a protein with an Armadillo/HEAT (ARM) domain, two sterile alpha motifs (SAM), and a Toll/interleukin-1 receptor homology (TIR) domain. Each identified dsarm allele contained a unique premature stop codon in dsarm open reading frame. From these data it is concluded that l(3)896, l(3)4621, and l(3)4705 are loss-of-function alleles of dsarm. Consistent with this interpretation, expression of full-length dsarm cDNA using the postmitotic OR22a-Gal4 driver in l(3)896 mutant clones was sufficient to fully revert the suppression of axonal degeneration observed in dsarm mutants. In addition, both the lethality and suppression of Wallerian degeneration phenotypes of l(3)896/l(3)4621 and l(3)896/Df(3L)BSC795 trans-heterozygous animals were rescued with a BAC clone containing the dsarm gene. Together, these data indicate that dSarm function is necessary in postmitotic neurons to drive axonal destruction after axotomy (Osterloh, 2012).

Based on RNA in situ hybridizations to embryos, larval brains, and adult brains, reverse transcription polymerase chain reaction from dissected neural tissues, and analysis of a dsarm-Gal4 driver line, dsarm is widely expressed in the Drosophila nervous system. These data raise the possibility that dSarm may be broadly required to promote Wallerian degeneration in the nervous system (Osterloh, 2012).

Next Wallerian degeneration was examined in null mutants for the mouse ortholog of t, Sarm1. We grew 5-day cultures of superior cervical ganglia (SCG) from wild-type, Sarm1-/- and WldS mice, severed axons, and axonal integrity was scored over the next week. Sarm1-/- SCG explants exhibited robust protection from degeneration up to 72 hours after axotomy, similar to what is observed with WldS SCG neurons, whereas wild-type axons degenerated within 8 hours. This robust protection was also seen in cultured Sarm1-/- cortical neuron axonsand dorsal root ganglia (DRG). Notably, Sarm1-/- DRG explants were not protected from nerve growth factor (NGF) deprivation, a model for developmental axon pruning, suggesting that Sarm1 protection is specific to injury-induced axon degeneration (Osterloh, 2012).

To determine whether Sarm1 is required for Wallerian degeneration in vivo, sciatic nerve lesions were performed of the right hind limb of Sarm1-/- mice and controls. Whereas Sarm1+/- controls exhibited a dramatic breakdown of the axon and myelin sheath within 3 days of injury, 61.2% of lesioned Sarm1-/- axons were protected from degeneration at least 14 days after injury. Ultrastructural analysis revealed a remarkable preservation of the Schwann cell and myelin sheath, axonal neurofilaments, and axonal mitochondria at this time point. Western blots of sciatic nerve with antibodies to neurofilament-M (NF-M), α-Internexin, and β-tubulin class III (TUJI) confirmed that the axonal cytoskeleton was robustly preserved in severed Sarm1-/- axons. Preservation of the NF-M signal by immunofluorescent staining nerves was further noted in Sarm1-/- mutants (Osterloh, 2012).

Synaptic integrity was scored by colocalization of presynaptic marker (NF-M/synaptophysin) with the postsynaptic acetylcholine receptor (AChR) in tibialis anterior muscles after sciatic nerve transection. In wild-type animals, motor end plate denervation was complete by 2 days after axotomy. However, in Sarm1-/- animals, most synaptic terminals were partially innervated even at 6 days after injury. Consistent with a strong preservation of nerve integrity, macrophage/monocyte infiltration of lesioned nerves was suppressed in Sarm1 knockout animals. Taken together, our results indicate that Sarm1-/- mutations strongly preserve sciatic nerves in vivo from initial axonal cytoskeletal breakdown to recruitment of macrophages for myelin clearance (Osterloh, 2012).

To help define potential sites of action, localization of dSarm and Sarm1 were assayed in neurons. Expression of dSarm::GFP in Drosophila larval neurons resulted in punctate localization in neuronal cell bodies and broad localization to neurites in vivo. Immunostaining with antibodies to Sarm1 of in vitro cultured mouse neurons showed a similar pattern, and it is noted that endogenous Sarm1 did not show preferential localization with a mitochondrial marker, although mitochondrial localization has been reported for overexpressed Sarm1::GFP. Thus, dSarm/Sarm1 appears to be broadly localized in the axonal compartment (Osterloh, 2012).

This analysis of dSarm/Sarm1 provides evidence that Wallerian degeneration is driven by an ancient, conserved axonal death program. Intriguingly, its Caenorhabditis elegans homolog, Tir-1, was recently implicated in nonapoptotic cell death , suggesting mechanistic links between these nonapoptotic degenerative mechanisms. Influx of extracellular calcium is known to be necessary and sufficient to trigger Wallerian degeneration. Tir-1 functions downstream of the Ca2+-CaM kinase signaling pathway in C. elegans; therefore, dSarm/Sarm1 may respond directly to axonal calcium increases after axotomy. Sarm1-/- neurons in slice cultures exhibit reduced cell death in response to oxygen/glucose deprivation; whether this is an indirect result or reflects axonal preservation remains untested. Pharmacological inhibition of Sarm1 may represent a promising therapeutic avenue for patients with axonal loss, particularly if Sarm1 deletion is shown to benefit animal models of neurodegenerative disease (Osterloh, 2012).


Functions of Sarm orthologs in other species

Multiple domain interfaces mediate SARM1 autoinhibition

Axon degeneration is an active program of self-destruction mediated by the protein SARM1. In healthy neurons, SARM1 is autoinhibited and, upon injury autoinhibition is relieved, activating the SARM1 enzyme to deplete NAD(+) and induce axon degeneration. SARM1 forms a homomultimeric octamer with each monomer composed of an N-terminal autoinhibitory ARM domain, tandem SAM domains that mediate multimerization, and a C-terminal TIR domain encoding the NADase enzyme. This study discovered multiple intramolecular and intermolecular domain interfaces required for SARM1 autoinhibition using peptide mapping and cryo-electron microscopy (cryo-EM). A candidate autoinhibitory region was identified by screening a panel of peptides derived from the SARM1 ARM domain, identifying a peptide mediating high-affinity inhibition of the SARM1 NADase. Mutation of residues in full-length SARM1 within the region encompassed by the peptide led to loss of autoinhibition, rendering SARM1 constitutively active and inducing spontaneous NAD(+) and axon loss. The cryo-EM structure of SARM1 revealed 1) a compact autoinhibited SARM1 octamer in which the TIR domains are isolated and prevented from oligomerization and enzymatic activation and 2) multiple candidate autoinhibitory interfaces among the domains. Mutational analysis demonstrated that five distinct interfaces are required for autoinhibition, including intramolecular and intermolecular ARM-SAM interfaces, an intermolecular ARM-ARM interface, and two ARM-TIR interfaces formed between a single TIR and two distinct ARM domains. These autoinhibitory regions are not redundant, as point mutants in each led to constitutively active SARM1. These studies define the structural basis for SARM1 autoinhibition and may enable the development of SARM1 inhibitors that stabilize the autoinhibited state (Shen, 2021).

Structural and mechanistic regulation of the pro-degenerative NAD hydrolase SARM1

The NADase SARM1 is a central switch in injury-activated axon degeneration, an early hallmark of many neurological diseases. This study presents cryo-electron microscopy (cryo-EM) structures of autoinhibited (3.3 Å) and active SARM1 (6.8 Å) and provide mechanistic insight into the tight regulation of SARM1's function by the local metabolic environment. Although both states retain an octameric core, the defining feature of the autoinhibited state is a lock between the autoinhibitory Armadillo/HEAT motif (ARM) and catalytic Toll/interleukin-1 receptor (TIR) domains, which traps SARM1 in an inactive state. Mutations that break this lock activate SARM1, resulting in catastrophic neuronal death. Notably, the mutants cannot be further activated by the endogenous activator nicotinamide mononucleotide (NMN), and active SARM1 is product inhibited by Nicotinamide (NAM), highlighting SARM1's functional dependence on key metabolites in the NAD salvage pathway. These studies provide a molecular understanding of SARM1's transition from an autoinhibited to an injury-activated state and lay the foundation for future SARM1-based therapies to treat axonopathies (Bratkowski, 2020).

A cell-permeant mimetic of NMN activates SARM1 to produce cyclic ADP-ribose and induce non-apoptotic cell death

SARM1, an NAD-utilizing enzyme, regulates axonal degeneration. CZ-48, a cell-permeant mimetic of NMN, activated SARM1 in vitro and in cellulo to cyclize NAD and produce a Ca(2+) messenger, cADPR, with similar efficiency as NMN. Knockout of NMN-adenylyltransferase elevated cellular NMN and activated SARM1 to produce cADPR, confirming NMN was its endogenous activator. Determinants for the activating effects and cell permeability of CZ-48 were identified. CZ-48 activated SARM1 via a conformational change of the auto-inhibitory domain and dimerization of its catalytic domain. SARM1 catalysis was similar to CD38, despite having no sequence similarity. Both catalyzed similar set of reactions, but SARM1 had much higher NAD-cyclizing activity, making it more efficient in elevating cADPR. CZ-48 acted selectively, activating SARM1 but inhibiting CD38. In SARM1-overexpressing cells, CZ-48 elevated cADPR, depleted NAD and ATP, and induced non-apoptotic death. CZ-48 is a specific modulator of SARM1 functions in cells (Zhao, 2019).

The SARM1 Toll/Interleukin-1 receptor domain possesses intrinsic NAD(+) cleavage activity that promotes pathological axonal degeneration

Axonal degeneration is an early and prominent feature of many neurological disorders. SARM1 is the central executioner of the axonal degeneration pathway that culminates in depletion of axonal NAD(+), yet the identity of the underlying NAD(+)-depleting enzyme(s) is unknown. In a series of experiments using purified proteins from mammalian cells, bacteria, and a cell-free protein translation system, this study shows that the SARM1-TIR domain itself has intrinsic NADase activity-cleaving NAD(+) into ADP-ribose (ADPR), cyclic ADPR, and nicotinamide, with nicotinamide serving as a feedback inhibitor of the enzyme. Using traumatic and vincristine-induced injury models in neurons, this study demonstrates that the NADase activity of full-length SARM1 is required in axons to promote axonal NAD(+) depletion and axonal degeneration after injury. Hence, the SARM1 enzyme represents a novel therapeutic target for axonopathies. Moreover, the widely utilized TIR domain is a protein motif that can possess enzymatic activity (Essuman, 2017).

Absence of SARM1 rescues development and survival of NMNAT2-deficient axons.

SARM1 function and nicotinamide mononucleotide adenylyltransferase 2 (NMNAT2) loss both promote axon degeneration, but their relative relationship in the process is unknown. This study shows 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 (Gilley, 2015).

SARM1 activation triggers axon degeneration locally via NAD(+) destruction

Axon degeneration is an intrinsic self-destruction program that underlies axon loss during injury and disease. Sterile alpha and TIR motif-containing 1 (SARM1) protein is an essential mediator of axon degeneration. This paper reports that SARM1 initiates a local destruction program involving rapid breakdown of nicotinamide adenine dinucleotide (NAD(+)) after injury. An engineered protease-sensitized SARM1 was used to demonstrate that SARM1 activity is required after axon injury to induce axon degeneration. Dimerization of the Toll-interleukin receptor (TIR) domain of SARM1 alone was sufficient to induce locally mediated axon degeneration. Formation of the SARM1 TIR dimer triggered rapid breakdown of NAD(+), whereas SARM1-induced axon destruction could be counteracted by increased NAD(+) synthesis. SARM1-induced depletion of NAD(+) may explain the potent axon protection in Wallerian degeneration slow [Wld(s)] mutant mice (Gerdts, 2015).

Pathological axonal death through a MAPK cascade that triggers a local energy deficit

Axonal death disrupts functional connectivity of neural circuits and is a critical feature of many neurodegenerative disorders. Pathological axon degeneration often occurs independently of known programmed death pathways, but the underlying molecular mechanisms remain largely unknown. Using traumatic injury as a model, this study sytematically investigated mitogen-activated protein kinase (MAPK) families and delineated a MAPK cascade that represents the early degenerative response to axonal injury. The adaptor protein Sarm1 is required for activation of this MAPK cascade, and this Sarm1-MAPK pathway disrupts axonal energy homeostasis, leading to ATP depletion before physical breakdown of damaged axons. The protective cytoNmnat1/Wld(s) protein inhibits activation of this MAPK cascade. Further, MKK4, a key component in the Sarm1-MAPK pathway, is antagonized by AKT signaling, which modulates the degenerative response by limiting activation of downstream JNK signaling. These results reveal a regulatory mechanism that integrates distinct signals to instruct pathological axon degeneration (Yang, 2015).

Sarm1-mediated axon degeneration requires both SAM and TIR interactions

Axon degeneration is an evolutionarily conserved pathway that eliminates damaged or unneeded axons. Manipulation of this poorly understood pathway may allow treatment of a wide range of neurological disorders. In an RNAi-based screen performed in cultured mouse DRG neurons, strong suppression of injury-induced axon degeneration was observed upon knockdown of Sarm1 [SARM (sterile alpha-motif-containing and armadillo-motif containing protein)]. A SARM-dependent degeneration program is engaged by disparate neuronal insults: SARM ablation blocks axon degeneration induced by axotomy or vincristine treatment, while SARM acts in parallel with a soma-derived caspase-dependent pathway following trophic withdrawal. SARM is a multidomain protein that associates with neuronal mitochondria. Deletion of the N-terminal mitochondrial localization sequence disrupts SARM mitochondrial localization in neurons but does not alter its ability to promote axon degeneration. In contrast, mutation of either the SAM (sterile alpha motif) or TIR (Toll-interleukin-1 receptor) domains abolishes the ability of SARM to promote axonal degeneration, while a SARM mutant containing only these domains elicits axon degeneration and nonapoptotic neuronal death even in the absence of injury. Protein-protein interaction studies demonstrate that the SAM domains are necessary and sufficient to mediate SARM-SARM binding. SARM mutants lacking a TIR domain bind full-length SARM and exhibit strong dominant-negative activity. These results indicate that SARM plays an integral role in the dismantling of injured axons and support a model in which SAM-mediated multimerization is necessary for TIR-dependent engagement of a downstream destruction pathway. These findings suggest that inhibitors of SAM and TIR interactions represent therapeutic candidates for blocking pathological axon loss and neuronal cell death (Berdts, 2013).


REFERENCES

Search PubMed for articles about Drosophila Sarm

Bratkowski, M., Xie, T., Thayer, D. A., Lad, S., Mathur, P., Yang, Y. S., Danko, G., Burdett, T. C., Danao, J., Cantor, A., Kozak, J. A., Brown, S. P., Bai, X. and Sambashivan, S. (2020). Structural and mechanistic regulation of the pro-degenerative NAD hydrolase SARM1. Cell Rep 32(5): 107999. PubMed ID: 32755591

Di Stefano, M., Nascimento-Ferreira, I., Orsomando, G., Mori, V., Gilley, J., Brown, R., Janeckova, L., Vargas, M. E., Worrell, L. A., Loreto, A., Tickle, J., Patrick, J., Webster, J. R., Marangoni, M., Carpi, F. M., Pucciarelli, S., Rossi, F., Meng, W., Sagasti, A., Ribchester, R. R., Magni, G., Coleman, M. P. and Conforti, L. (2015). A rise in NAD precursor nicotinamide mononucleotide (NMN) after injury promotes axon degeneration. Cell Death Differ 22(5): 731-742. PubMed ID: 25323584

Essuman, K., Summers, D. W., Sasaki, Y., Mao, X., DiAntonio, A. and Milbrandt, J. (2017). The SARM1 Toll/Interleukin-1 receptor domain possesses intrinsic NAD(+) cleavage activity that promotes pathological axonal degeneration. Neuron 93(6): 1334-1343. PubMed ID: 28334607

Farley, J. E., Burdett, T. C., Barria, R., Neukomm, L. J., Kenna, K. P., Landers, J. E. and Freeman, M. R. (2018). Transcription factor Pebbled/RREB1 regulates injury-induced axon degeneration. Proc Natl Acad Sci U S A. PubMed ID: 29295933

Foldi, I., Anthoney, N., Harrison, N., Gangloff, M., Verstak, B., Nallasivan, M. P., AlAhmed, S., Zhu, B., Phizacklea, M., Losada-Perez, M., Moreira, M., Gay, N. J. and Hidalgo, A. (2017). Three-tier regulation of cell number plasticity by neurotrophins and Tolls in Drosophila. J Cell Biol 216(5):1421-1438. PubMed ID: 28373203

Gerdts, J., Summers, D. W., Sasaki, Y., DiAntonio, A. and Milbrandt, J. (2013). Sarm1-mediated axon degeneration requires both SAM and TIR interactions. J Neurosci 33(33): 13569-13580. PubMed ID: 23946415

Gerdts, J., Brace, E. J., Sasaki, Y., DiAntonio, A. and Milbrandt, J. (2015). SARM1 activation triggers axon degeneration locally via NAD(+) destruction. Science 348(6233): 453-457. PubMed ID: 25908823

Gerdts, J., Summers, D. W., Milbrandt, J. and DiAntonio, A. (2016). Axon self-destruction: new links among SARM1, MAPKs, and NAD+ metabolism. Neuron 89(3): 449-460. PubMed ID: 26844829

Gilley, J., Orsomando, G., Nascimento-Ferreira, I. and Coleman, M. P. (2015). Absence of SARM1 rescues development and survival of NMNAT2-deficient axons. Cell Rep 10(12):1974-81. PubMed ID: 25818290

Hsu, J. M., Kang, Y., Corty, M. M., Mathieson, D., Peters, O. M. and Freeman, M. R. (2020). Injury-induced inhibition of bystander neurons requires dSarm and signaling from glia. Neuron. PubMed ID: 33296670

McLaughlin, C. N., Nechipurenko, I. V., Liu, N. and Broihier, H. T. (2016). A Toll receptor-FoxO pathway represses Pavarotti/MKLP1 to promote microtubule dynamics in motoneurons. J Cell Biol 214: 459-474. PubMed ID: 27502486

Neukomm, L. J., Burdett, T. C., Seeds, A. M., Hampel, S., Coutinho-Budd, J. C., Farley, J. E., Wong, J., Karadeniz, Y. B., Osterloh, J. M., Sheehan, A. E. and Freeman, M. R. (2017). Axon death pathways converge on Axundead to promote functional and structural axon disassembly. Neuron 95(1): 78-91.e75. PubMed ID: 28683272

Osterloh, J. M., Yang, J., Rooney, T. M., Fox, A. N., Adalbert, R., Powell, E. H., Sheehan, A. E., Avery, M. A., Hackett, R., Logan, M. A., MacDonald, J. M., Ziegenfuss, J. S., Milde, S., Hou, Y. J., Nathan, C., Ding, A., Brown, R. H., Jr., Conforti, L., Coleman, M., Tessier-Lavigne, M., Zuchner, S. and Freeman, M. R. (2012). dSarm/Sarm1 is required for activation of an injury-induced axon death pathway. Science 337(6093): 481-484. PubMed ID: 22678360

Shen, C., Vohra, M., Zhang, P., Mao, X., Figley, M. D., Zhu, J., Sasaki, Y., Wu, H., DiAntonio, A. and Milbrandt, J. (2021). Multiple domain interfaces mediate SARM1 autoinhibition. Proc Natl Acad Sci U S A 118(4). PubMed ID: 33468661

Walker, L. J., Summers, D. W., Sasaki, Y., Brace, E. J., Milbrandt, J. and DiAntonio, A. (2017). MAPK signaling promotes axonal degeneration by speeding the turnover of the axonal maintenance factor NMNAT2. Elife 6. PubMed ID: 28095293

Yang, J., Wu, Z., Renier, N., Simon, D. J., Uryu, K., Park, D. S., Greer, P. A., Tournier, C., Davis, R. J. and Tessier-Lavigne, M. (2015). Pathological axonal death through a MAPK cascade that triggers a local energy deficit. Cell 160(1-2): 161-176. PubMed ID: 25594179

Zhao, Z. Y., Xie, X. J., Li, W. H., Liu, J., Chen, Z., Zhang, B., Li, T., Li, S. L., Lu, J. G., Zhang, L., Zhang, L. H., Xu, Z., Lee, H. C. and Zhao, Y. J. (2019). A Cell-Permeant Mimetic of NMN Activates SARM1 to Produce Cyclic ADP-Ribose and Induce Non-apoptotic Cell Death. iScience 15: 452-466. PubMed ID: 31128467


Biological Overview

date revised: 28 February 2021

Home page: The Interactive Fly © 2011 Thomas Brody, Ph.D.