Structural basis of SARM1 activation, substrate recognition, and inhibition by small molecules.

The NADase SARM1 (sterile alpha and TIR motif containing 1) is a key executioner of axon degeneration and a therapeutic target for several neurodegenerative conditions. We show that a potent SARM1 inhibitor undergoes base exchange with the nicotinamide moiety of nicotinamide adenine dinucleotide (NAD+) to produce the bona fide inhibitor 1AD. We report structures of SARM1 in complex with 1AD, NAD+ mimetics and the allosteric activator nicotinamide mononucleotide (NMN). NMN binding triggers reorientation of the armadillo repeat (ARM) domains, which disrupts ARM:TIR interactions and leads to formation of a two-stranded TIR domain assembly. The active site spans two molecules in these assemblies, explaining the requirement of TIR domain self-association for NADase activity and axon degeneration. Our results reveal the mechanisms of SARM1 activation and substrate binding, providing rational avenues for the design of new therapeutics targeting SARM1.

SIK3 and Wnk converge on Fray to regulate glial K+ buffering and seizure susceptibility.

Glial cells play a critical role in maintaining homeostatic ion concentration gradients. Salt-inducible kinase 3 (SIK3) regulates a gene expression program that controls K+ buffering in glia, and upregulation of this pathway suppresses seizure behavior in the eag, Shaker hyperexcitability mutant. Here we show that boosting the glial SIK3 K+ buffering pathway suppresses seizures in three additional molecularly diverse hyperexcitable mutants, highlighting the therapeutic potential of upregulating glial K+ buffering. We then explore additional mechanisms regulating glial K+ buffering. Fray, a transcriptional target of the SIK3 K+ buffering program, is a kinase that promotes K+ uptake by activating the Na+/K+/Cl- co-transporter, Ncc69. We show that the Wnk kinase phosphorylates Fray in Drosophila glia and that this activity is required to promote K+ buffering. This identifies Fray as a convergence point between the SIK3-dependent transcriptional program and Wnk-dependent post-translational regulation. Bypassing both regulatory mechanisms via overexpression of a constitutively active Fray in glia is sufficient to robustly suppress seizure behavior in multiple Drosophila models of hyperexcitability. Finally, we identify cortex glia as a critical cell type for regulation of seizure susceptibility, as boosting K+ buffering via expression of activated Fray exclusively in these cells is sufficient to suppress seizure behavior. These findings highlight Fray as a key convergence point for distinct K+ buffering regulatory mechanisms and cortex glia as an important locus for control of neuronal excitability.

Distinct developmental and degenerative functions of SARM1 require NAD+ hydrolase activity.

SARM1 is the founding member of the TIR-domain family of NAD+ hydrolases and the central executioner of pathological axon degeneration. SARM1-dependent degeneration requires NAD+ hydrolysis. Prior to the discovery that SARM1 is an enzyme, SARM1 was studied as a TIR-domain adaptor protein with non-degenerative signaling roles in innate immunity and invertebrate neurodevelopment, including at the Drosophila neuromuscular junction (NMJ). Here we explore whether the NADase activity of SARM1 also contributes to developmental signaling. We developed transgenic Drosophila lines that express SARM1 variants with normal, deficient, and enhanced NADase activity and tested their function in NMJ development. We find that NMJ overgrowth scales with the amount of NADase activity, suggesting an instructive role for NAD+ hydrolysis in this developmental signaling pathway. While degenerative and developmental SARM1 signaling share a requirement for NAD+ hydrolysis, we demonstrate that these signals use distinct upstream and downstream mechanisms. These results identify SARM1-dependent NAD+ hydrolysis as a heretofore unappreciated component of developmental signaling. SARM1 now joins sirtuins and Parps as enzymes that regulate signal transduction pathways via mechanisms that involve NAD+ cleavage, greatly expanding the potential scope of SARM1 TIR NADase functions.

The structure of NAD+ consuming protein Acinetobacter baumannii TIR domain shows unique kinetics and conformations.

Toll-like and interleukin-1/18 receptor/resistance (TIR) domain-containing proteins function as important signaling and immune regulatory molecules. TIR domain-containing proteins identified in eukaryotic and prokaryotic species also exhibit NAD+ hydrolase activity in select bacteria, plants, and mammalian cells. We report the crystal structure of the Acinetobacter baumannii TIR domain protein (AbTir-TIR) with confirmed NAD+ hydrolysis and map the conformational effects of its interaction with NAD+ using hydrogen-deuterium exchange-mass spectrometry. NAD+ results in mild decreases in deuterium uptake at the dimeric interface. In addition, AbTir-TIR exhibits EX1 kinetics indicative of large cooperative conformational changes, which are slowed down upon substrate binding. Additionally, we have developed label-free imaging using the minimally invasive spectroscopic method 2-photon excitation with fluorescence lifetime imaging, which shows differences in bacteria expressing native and mutant NAD+ hydrolase-inactivated AbTir-TIRE208A protein. Our observations are consistent with substrate-induced conformational changes reported in other TIR model systems with NAD+ hydrolase activity. These studies provide further insight into bacterial TIR protein mechanisms and their varying roles in biology.

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. Here we discovered multiple intramolecular and intermolecular domain interfaces required for SARM1 autoinhibition using peptide mapping and cryo-electron microscopy (cryo-EM). We identified a candidate autoinhibitory region 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.

FK506-binding protein-like and FK506-binding protein 8 regulate dual leucine zipper kinase degradation and neuronal responses to axon injury.

The dual leucine zipper kinase (DLK) is a key regulator of axon regeneration and degeneration in response to neuronal injury; however, regulatory mechanisms of the DLK function via its interacting proteins are largely unknown. To better understand the molecular mechanism of DLK function, we performed yeast two-hybrid screening analysis and identified FK506-binding protein-like (FKBPL, also known as WAF-1/CIP1 stabilizing protein 39) as a DLK-binding protein. FKBPL binds to the kinase domain of DLK and inhibits its kinase activity. In addition, FKBPL induces DLK protein degradation through ubiquitin-dependent pathways. We further assessed other members in the FKBP protein family and found that FK506-binding protein 8 (FKBP8) also induced DLK degradation. We identified the lysine 271 residue in the kinase domain as a major site of DLK ubiquitination and SUMO3 conjugation and was thus responsible for regulating FKBP8-mediated proteasomal degradation that was inhibited by the substitution of the lysine 271 to arginine. FKBP8-mediated degradation of DLK is mediated by autophagy pathway because knockdown of Atg5 inhibited DLK destabilization. We show that in vivo overexpression of FKBP8 delayed the progression of axon degeneration and suppressed neuronal death after axotomy in sciatic and optic nerves. Taken together, this study identified FKBPL and FKBP8 as novel DLK-interacting proteins that regulate DLK stability via the ubiquitin-proteasome and lysosomal protein degradation pathways.

The human cognition-enhancing CORD7 mutation increases active zone number and synaptic release.

Humans carrying the CORD7 (cone-rod dystrophy 7) mutation possess increased verbal IQ and working memory. This autosomal dominant syndrome is caused by the single-amino acid R844H exchange (human numbering) located in the 310 helix of the C2A domain of RIMS1/RIM1 (Rab3-interacting molecule 1). RIM is an evolutionarily conserved multi-domain protein and essential component of presynaptic active zones, which is centrally involved in fast, Ca2+-triggered neurotransmitter release. How the CORD7 mutation affects synaptic function has remained unclear thus far. Here, we established Drosophila melanogaster as a disease model for clarifying the effects of the CORD7 mutation on RIM function and synaptic vesicle release. To this end, using protein expression and X-ray crystallography, we solved the molecular structure of the Drosophila C2A domain at 1.92 Šresolution and by comparison to its mammalian homologue ascertained that the location of the CORD7 mutation is structurally conserved in fly RIM. Further, CRISPR/Cas9-assisted genomic engineering was employed for the generation of rim alleles encoding the R915H CORD7 exchange or R915E, R916E substitutions (fly numbering) to effect local charge reversal at the 310 helix. Through electrophysiological characterization by two-electrode voltage clamp and focal recordings we determined that the CORD7 mutation exerts a semi-dominant rather than a dominant effect on synaptic transmission resulting in faster, more efficient synaptic release and increased size of the readily releasable pool but decreased sensitivity for the fast calcium chelator BAPTA. In addition, the rim CORD7 allele increased the number of presynaptic active zones but left their nanoscopic organization unperturbed as revealed by super-resolution microscopy of the presynaptic scaffold protein Bruchpilot/ELKS/CAST. We conclude that the CORD7 mutation leads to tighter release coupling, an increased readily releasable pool size and more release sites thereby promoting more efficient synaptic transmitter release. These results strongly suggest that similar mechanisms may underlie the CORD7 disease phenotype in patients and that enhanced synaptic transmission may contribute to their increased cognitive abilities.

Protein phosphatase 2A restrains DLK signaling to promote proper Drosophila synaptic development and mammalian cortical neuron survival.

Protein phosphatase 2A (PP2A) is a major cellular phosphatase with many protein substrates. As expected for a signaling molecule with many targets, inhibition of PP2A disrupts fundamental aspects of cellular physiology including cell division and survival. In post-mitotic neurons, the microtubule associated protein Tau is a particularly well-studied PP2A substrate as hyperphosphorylation of Tau is a hallmark of Alzheimer's disease. Although many cellular targets are likely altered by loss of PP2A, here we find that activation of a single pathway can explain important aspects of the PP2A loss-of-function phenotype in neurons. We demonstrate that PP2A inhibits activation of the neuronal stress kinase DLK and its Drosophila ortholog Wallenda. In the fly, PP2A inhibition activates a DLK/Wallenda-regulated transcriptional program that induces synaptic terminal overgrowth at the neuromuscular junction. In cultured mammalian neurons, PP2A inhibition activates a DLK-dependent apoptotic program that induces cell death. Since hyperphosphorylated Tau is toxic, we wished to test the hypothesis that dephosphorylation of Tau by PP2A is required for neuronal survival. Contrary to expectations, in the absence of Tau PP2A inhibition still activates DLK and induces neuronal cell death, demonstrating that hyperphosphorylated Tau is not required for cell death in this model. Moreover, hyperphosphorylation of Tau following PP2A inhibition does not require DLK. Hence, loss of PP2A function in cortical neurons triggers two independent neuropathologies: 1) Tau hyperphosphorylation and 2) DLK activation and subsequent neuronal cell death. These findings demonstrate that inhibition of the DLK pathway is an essential function of PP2A required for normal Drosophila synaptic terminal development and mammalian cortical neuron survival.

A phytobacterial TIR domain effector manipulates NAD+ to promote virulence.

The Pseudomonas syringae DC3000 type III effector HopAM1 suppresses plant immunity and contains a Toll/interleukin-1 receptor (TIR) domain homologous to immunity-related TIR domains of plant nucleotide-binding leucine-rich repeat receptors that hydrolyze nicotinamide adenine dinucleotide (NAD+ ) and activate immunity. In vitro and in vivo assays were conducted to determine if HopAM1 hydrolyzes NAD+ and if the activity is essential for HopAM1's suppression of plant immunity and contribution to virulence. HPLC and LC-MS were utilized to analyze metabolites produced from NAD+ by HopAM1 in vitro and in both yeast and plants. Agrobacterium-mediated transient expression and in planta inoculation assays were performed to determine HopAM1's intrinsic enzymatic activity and virulence contribution. HopAM1 is catalytically active and hydrolyzes NAD+ to produce nicotinamide and a novel cADPR variant (v2-cADPR). Expression of HopAM1 triggers cell death in yeast and plants dependent on the putative catalytic residue glutamic acid 191 (E191) within the TIR domain. Furthermore, HopAM1's E191 residue is required to suppress both pattern-triggered immunity and effector-triggered immunity and promote P. syringae virulence. HopAM1 manipulates endogenous NAD+ to produce v2-cADPR and promote pathogenesis. This work suggests that HopAM1's TIR domain possesses different catalytic specificity than other TIR domain-containing NAD+ hydrolases and that pathogens exploit this activity to sabotage NAD+ metabolism for immune suppression and virulence.

The E3 ligase Highwire promotes synaptic transmission by targeting the NAD-synthesizing enzyme dNmnat.

The ubiquitin ligase Highwire restrains synaptic growth and promotes evoked neurotransmission at NMJ synapses in Drosophila Highwire regulates synaptic morphology by downregulating the MAP3K Wallenda, but excess Wallenda signaling does not account for the decreased presynaptic release observed in highwire mutants. Hence, Highwire likely has a second substrate that inhibits neurotransmission. Highwire targets the NAD+ biosynthetic and axoprotective enzyme dNmnat to regulate axonal injury responses. dNmnat localizes to synapses and interacts with the active zone protein Bruchpilot, leading us to hypothesize that Highwire promotes evoked release by downregulating dNmnat. Here, we show that excess dNmnat is necessary in highwire mutants and sufficient in wild-type larvae to reduce quantal content, likely via disruption of active zone ultrastructure. Catalytically active dNmnat is required to drive defects in evoked release, and depletion of a second NAD+ synthesizing enzyme is sufficient to suppress these defects in highwire mutants, suggesting that excess NAD+ biosynthesis is the mechanism inhibiting neurotransmission. Thus, Highwire downregulates dNmnat to promote evoked synaptic release, suggesting that Highwire balances the axoprotective and synapse-inhibitory functions of dNmnat.

Palmitoylation enables MAPK-dependent proteostasis of axon survival factors.

Axon degeneration is a prominent event in many neurodegenerative disorders. Axon injury stimulates an intrinsic self-destruction program that culminates in activation of the prodegeneration factor SARM1 and local dismantling of damaged axon segments. In healthy axons, SARM1 activity is restrained by constant delivery of the axon survival factor NMNAT2. Elevating NMNAT2 is neuroprotective, while loss of NMNAT2 evokes SARM1-dependent axon degeneration. As a gatekeeper of axon survival, NMNAT2 abundance is an important regulatory node in neuronal health, highlighting the need to understand the mechanisms behind NMNAT2 protein homeostasis. We demonstrate that pharmacological inhibition of the MAP3Ks dual leucine zipper kinase (DLK) and leucine zipper kinase (LZK) elevates NMNAT2 abundance and strongly protects axons from injury-induced degeneration. We discover that MAPK signaling selectively promotes degradation of palmitoylated NMNAT2, as well as palmitoylated SCG10. Conversely, nonpalmitoylated NMNAT2 is degraded by the Phr1/Skp1a/Fbxo45 ligase complex. Combined inactivation of both pathways leads to synergistic accumulation of NMNAT2 in axons and dramatically enhanced protection against pathological axon degeneration. Hence, the subcellular localization of distinct pools of NMNAT2 enables differential regulation of NMNAT2 abundance to control axon survival.

HSP90 is a chaperone for DLK and is required for axon injury signaling.

Peripheral nerve injury induces a robust proregenerative program that drives axon regeneration. While many regeneration-associated genes are known, the mechanisms by which injury activates them are less well-understood. To identify such mechanisms, we performed a loss-of-function pharmacological screen in cultured adult mouse sensory neurons for proteins required to activate this program. Well-characterized inhibitors were present as injury signaling was induced but were removed before axon outgrowth to identify molecules that block induction of the program. Of 480 compounds, 35 prevented injury-induced neurite regrowth. The top hits were inhibitors to heat shock protein 90 (HSP90), a chaperone with no known role in axon injury. HSP90 inhibition blocks injury-induced activation of the proregenerative transcription factor cJun and several regeneration-associated genes. These phenotypes mimic loss of the proregenerative kinase, dual leucine zipper kinase (DLK), a critical neuronal stress sensor that drives axon degeneration, axon regeneration, and cell death. HSP90 is an atypical chaperone that promotes the stability of signaling molecules. HSP90 and DLK show two hallmarks of HSP90-client relationships: (i) HSP90 binds DLK, and (ii) HSP90 inhibition leads to rapid degradation of existing DLK protein. Moreover, HSP90 is required for DLK stability in vivo, where HSP90 inhibitor reduces DLK protein in the sciatic nerve. This phenomenon is evolutionarily conserved in Drosophila Genetic knockdown of Drosophila HSP90, Hsp83, decreases levels of Drosophila DLK, Wallenda, and blocks Wallenda-dependent synaptic terminal overgrowth and injury signaling. Our findings support the hypothesis that HSP90 chaperones DLK and is required for DLK functions, including proregenerative axon injury signaling.

DLK regulates a distinctive transcriptional regeneration program after peripheral nerve injury.

Following damage to a peripheral nerve, injury signaling pathways converge in the cell body to generate transcriptional changes that support axon regeneration. Here, we demonstrate that dual leucine zipper kinase (DLK), a central regulator of injury responses including axon regeneration and neuronal apoptosis, is required for the induction of the pro-regenerative transcriptional program in response to peripheral nerve injury. Using a sensory neuron-conditional DLK knockout mouse model, we show a time course for the dependency of gene expression changes on the DLK pathway after sciatic nerve injury. Gene ontology analysis reveals that DLK-dependent gene sets are enriched for specific functional annotations such as ion transport and immune response. A series of comparative analyses shows that the DLK-dependent transcriptional program is distinct from that promoted by the importin-dependent retrograde signaling pathway, while it is partially shared between PNS and CNS injury responses. We suggest that DLK-dependency might provide a selective filter for regeneration-associated genes among the injury-responsive transcriptome.

SARM1-specific motifs in the TIR domain enable NAD+ loss and regulate injury-induced SARM1 activation.

Axon injury in response to trauma or disease stimulates a self-destruction program that promotes the localized clearance of damaged axon segments. Sterile alpha and Toll/interleukin receptor (TIR) motif-containing protein 1 (SARM1) is an evolutionarily conserved executioner of this degeneration cascade, also known as Wallerian degeneration; however, the mechanism of SARM1-dependent neuronal destruction is still obscure. SARM1 possesses a TIR domain that is necessary for SARM1 activity. In other proteins, dimerized TIR domains serve as scaffolds for innate immune signaling. In contrast, dimerization of the SARM1 TIR domain promotes consumption of the essential metabolite NAD+ and induces neuronal destruction. This activity is unique to the SARM1 TIR domain, yet the structural elements that enable this activity are unknown. In this study, we identify fundamental properties of the SARM1 TIR domain that promote NAD+ loss and axon degeneration. Dimerization of the TIR domain from the Caenorhabditis elegans SARM1 ortholog TIR-1 leads to NAD+ loss and neuronal death, indicating these activities are an evolutionarily conserved feature of SARM1 function. Detailed analysis of sequence homology identifies canonical TIR motifs as well as a SARM1-specific (SS) loop that are required for NAD+ loss and axon degeneration. Furthermore, we identify a residue in the SARM1 BB loop that is dispensable for TIR activity yet required for injury-induced activation of full-length SARM1, suggesting that SARM1 function requires multidomain interactions. Indeed, we identify a physical interaction between the autoinhibitory N terminus and the TIR domain of SARM1, revealing a previously unrecognized direct connection between these domains that we propose mediates autoinhibition and activation upon injury.

TMEM184b Promotes Axon Degeneration and Neuromuscular Junction Maintenance.

UNLABELLED: Complex nervous systems achieve proper connectivity during development and must maintain these connections throughout life. The processes of axon and synaptic maintenance and axon degeneration after injury are jointly controlled by a number of proteins within neurons, including ubiquitin ligases and mitogen activated protein kinases. However, our understanding of these molecular cascades is incomplete. Here we describe the phenotype resulting from mutation of TMEM184b, a protein identified in a screen for axon degeneration mediators. TMEM184b is highly expressed in the mouse nervous system and is found in recycling endosomes in neuronal cell bodies and axons. Disruption of TMEM184b expression results in prolonged maintenance of peripheral axons following nerve injury, demonstrating a role for TMEM184b in axon degeneration. In contrast to this protective phenotype in axons, uninjured mutant mice have anatomical and functional impairments in the peripheral nervous system. Loss of TMEM184b causes swellings at neuromuscular junctions that become more numerous with age, demonstrating that TMEM184b is critical for the maintenance of synaptic architecture. These swellings contain abnormal multivesicular structures similar to those seen in patients with neurodegenerative disorders. Mutant animals also show abnormal sensory terminal morphology. TMEM184b mutant animals are deficient on the inverted screen test, illustrating a role for TMEM184b in sensory-motor function. Overall, we have identified an important function for TMEM184b in peripheral nerve terminal structure, function, and the axon degeneration pathway. SIGNIFICANCE STATEMENT: Our work has identified both neuroprotective and neurodegenerative roles for a previously undescribed protein, TMEM184b. TMEM184b mutation causes delayed axon degeneration following peripheral nerve injury, indicating that it participates in the degeneration process. Simultaneously, TMEM184b mutation causes progressive structural abnormalities at neuromuscular synapses and swellings within sensory terminals, and animals with this mutation display profound weakness. Thus, TMEM184b is necessary for normal peripheral nerve terminal morphology and maintenance. Loss of TMEM184b results in accumulation of autophagosomal structures in vivo, fitting with emerging studies that have linked autophagy disruption and neurological disease. Our work recognizes TMEM184b as a new player in the maintenance of the nervous system.

Prevention of vincristine-induced peripheral neuropathy by genetic deletion of SARM1 in mice.

Peripheral polyneuropathy is a common and dose-limiting side effect of many important chemotherapeutic agents. Most such neuropathies are characterized by early axonal degeneration, yet therapies that inhibit this axonal destruction process do not currently exist. Recently, we and others discovered that genetic deletion of SARM1 (sterile alpha and TIR motif containing protein 1) dramatically protects axons from degeneration after axotomy in mice. This finding fuels hope that inhibition of SARM1 or its downstream components can be used therapeutically in patients threatened by axonal loss. However, axon loss in most neuropathies, including chemotherapy-induced peripheral neuropathy, is the result of subacute/chronic processes that may be regulated differently than the acute, one time insult of axotomy. Here we evaluate if genetic deletion of SARM1 decreases axonal degeneration in a mouse model of neuropathy induced by the chemotherapeutic agent vincristine. In wild-type mice, 4 weeks of twice-weekly intraperitoneal injections of 1.5 mg/kg vincristine cause pronounced mechanical and heat hyperalgesia, a significant decrease in tail compound nerve action potential amplitude, loss of intraepidermal nerve fibres and significant degeneration of myelinated axons in both the distal sural nerve and nerves of the toe. Neither the proximal sural nerve nor the motor tibial nerve exhibit axon loss. These findings are consistent with the development of a distal, sensory predominant axonal polyneuropathy that mimics vincristine-induced peripheral polyneuropathy in humans. Using the same regimen of vincristine treatment in SARM1 knockout mice, the development of mechanical and heat hyperalgesia is blocked and the loss in tail compound nerve action potential amplitude is prevented. Moreover, SARM1 knockout mice do not lose unmyelinated fibres in the skin or myelinated axons in the sural nerve and toe after vincristine. Hence, genetic deletion of SARM1 blocks the development of vincristine-induced peripheral polyneuropathy in mice. Our results reveal that subacute/chronic axon loss induced by vincristine occurs via a SARM1 mediated axonal destruction pathway, and that blocking this pathway prevents the development of vincristine-induced peripheral polyneuropathy. These findings, in conjunction with previous studies with axotomy and traumatic brain injury, establish SARM1 as the central determinant of a fundamental axonal degeneration pathway that is activated by diverse insults. We suggest that targeting SARM1 or its downstream effectors may be a viable therapeutic option to prevent vincristine-induced peripheral polyneuropathy and possibly other peripheral polyneuropathies.

Functional genomic screening identifies dual leucine zipper kinase as a key mediator of retinal ganglion cell death.

Glaucoma, a major cause of blindness worldwide, is a neurodegenerative optic neuropathy in which vision loss is caused by loss of retinal ganglion cells (RGCs). To better define the pathways mediating RGC death and identify targets for the development of neuroprotective drugs, we developed a high-throughput RNA interference screen with primary RGCs and used it to screen the full mouse kinome. The screen identified dual leucine zipper kinase (DLK) as a key neuroprotective target in RGCs. In cultured RGCs, DLK signaling is both necessary and sufficient for cell death. DLK undergoes robust posttranscriptional up-regulation in response to axonal injury in vitro and in vivo. Using a conditional knockout approach, we confirmed that DLK is required for RGC JNK activation and cell death in a rodent model of optic neuropathy. In addition, tozasertib, a small molecule protein kinase inhibitor with activity against DLK, protects RGCs from cell death in rodent glaucoma and traumatic optic neuropathy models. Together, our results establish a previously undescribed drug/drug target combination in glaucoma, identify an early marker of RGC injury, and provide a starting point for the development of more specific neuroprotective DLK inhibitors for the treatment of glaucoma, nonglaucomatous forms of optic neuropathy, and perhaps other CNS neurodegenerations.

Mitochondrial dysfunction induces Sarm1-dependent cell death in sensory neurons.

Mitochondrial dysfunction is the underlying cause of many neurological disorders, including peripheral neuropathies. Mitochondria rely on a proton gradient to generate ATP and interfering with electron transport chain function can lead to the deleterious accumulation of reactive oxygen species (ROS). Notably, loss of mitochondrial potential precedes cellular demise in several programmed cell destruction pathways, including axons undergoing Wallerian degeneration. Here, we demonstrate that mitochondrial depolarization triggers axon degeneration and cell death in primary mouse sensory neurons. These degenerative events are not blocked by inhibitors of canonical programmed cell death pathways such as apoptosis, necroptosis, and parthanatos. Instead, the axodestructive factor Sarm1 is required for this axon degeneration and cell death. In the absence of Sarm1, the mitochondrial poison CCCP still induces depolarization of mitochondria, ATP depletion, calcium influx, and the accumulation of ROS, yet cell death and axon degeneration are blocked. The survival of these neurons despite the accumulation of ROS indicates that Sarm1 acts downstream of ROS generation. Indeed, loss of Sarm1 protects sensory neurons and their axons from prolonged exposure to ROS. Therefore, Sarm1 functions downstream of ROS to induce neuronal cell death and axon degeneration during oxidative stress. These findings highlight the central role for Sarm1 in a novel form of programmed cell destruction that we term sarmoptosis.

SkpA restrains synaptic terminal growth during development and promotes axonal degeneration following injury.

The Wallenda (Wnd)/dual leucine zipper kinase (DLK)-Jnk pathway is an evolutionarily conserved MAPK signaling pathway that functions during neuronal development and following axonal injury. Improper pathway activation causes defects in axonal guidance and synaptic growth, whereas loss-of-function mutations in pathway components impairs axonal regeneration and degeneration after injury. Regulation of this pathway is in part through the E3 ubiquitin ligase Highwire (Hiw), which targets Wnd/DLK for degradation to limit MAPK signaling. To explore mechanisms controlling Wnd/DLK signaling, we performed a large-scale genetic screen in Drosophila to identify negative regulators of the pathway. Here we describe the identification and characterization of SkpA, a core component of SCF E3 ubiquitin ligases. Mutants in SkpA display synaptic overgrowth and an increase in Jnk signaling, similar to hiw mutants. The combination of hypomorphic alleles of SkpA and hiw leads to enhanced synaptic growth. Mutants in the Wnd-Jnk pathway suppress the overgrowth of SkpA mutants demonstrating that the synaptic overgrowth is due to increased Jnk signaling. These findings support the model that SkpA and the E3 ligase Hiw function as part of an SCF-like complex that attenuates Wnd/DLK signaling. In addition, SkpA, like Hiw, is required for synaptic and axonal responses to injury. Synapses in SkpA mutants are more stable following genetic or traumatic axonal injury, and axon loss is delayed in SkpA mutants after nerve crush. As in highwire mutants, this axonal protection requires Nmnat. Hence, SkpA is a novel negative regulator of the Wnd-Jnk pathway that functions with Hiw to regulate both synaptic development and axonal maintenance.

Cytoskeletal disruption activates the DLK/JNK pathway, which promotes axonal regeneration and mimics a preconditioning injury.

Nerve injury can lead to axonal regeneration, axonal degeneration, and/or neuronal cell death. Remarkably, the MAP3K dual leucine zipper kinase, DLK, promotes each of these responses, suggesting that DLK is a sensor of axon injury. In Drosophila, mutations in proteins that stabilize the actin and microtubule cytoskeletons activate the DLK pathway, suggesting that DLK may be activated by cytoskeletal disruption. Here we test this model in mammalian sensory neurons. We find that pharmacological agents designed to disrupt either the actin or microtubule cytoskeleton activate the DLK pathway, and that activation is independent of calcium influx or induction of the axon degeneration program. Moreover, activation of the DLK pathway by targeting the cytoskeleton induces a pro-regenerative state, enhancing axon regeneration in response to a subsequent injury in a process akin to preconditioning. This highlights the potential utility of activating the DLK pathway as a method to improve axon regeneration. Moreover, DLK is required for these responses to cytoskeletal perturbations, suggesting that DLK functions as a key neuronal sensor of cytoskeletal damage.