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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.

Overexpression of the NAD+ biosynthetic enzyme NMNAT1 leads to preservation of injured axons. While increased NAD+ or decreased NMN levels are thought to be critical to this process, the mechanism(s) of this axon protection remain obscure. Using steady-state and flux analysis of NAD+ metabolites in healthy and injured mouse dorsal root ganglion axons, we find that rather than altering NAD+ synthesis, NMNAT1 instead blocks the injury-induced, SARM1-dependent NAD+ consumption that is central to axon degeneration.

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. We report that SARM1 initiates a local destruction program involving rapid breakdown of nicotinamide adenine dinucleotide (NAD+) after injury. We used an engineered protease-sensitized SARM1 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 (Wlds) mutant mice.

SARM1 is an inducible NAD + hydrolase that triggers axon loss and neuronal cell death in the injured and diseased nervous system. While SARM1 activation and enzyme function are well defined, the cellular events downstream of SARM1 activity but prior to axonal demise are much less well understood. Defects in calcium, mitochondria, ATP, and membrane homeostasis occur in injured axons, but the relationships among these events have been difficult to disentangle because prior studies analyzed large collections of axons in which cellular events occur asynchronously. Here, we used live imaging of mouse sensory neurons with single axon resolution to investigate the cellular events downstream of SARM1 activity. Our studies support a model in which SARM1 NADase activity leads to an ordered sequence of events from loss of cellular ATP, to defects in mitochondrial movement and depolarization, followed by calcium influx, externalization of phosphatidylserine, and loss of membrane permeability prior to catastrophic axonal self-destruction.

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.

Charcot-Marie-Tooth disease type 2A (CMT2A) is an axonal neuropathy caused by mutations in the mitofusin 2 (MFN2) gene. MFN2 mutations result in profound mitochondrial abnormalities, but the mechanism underlying the axonal pathology is unknown. Sterile α and Toll/IL-1 receptor motif-containing 1 (SARM1), the central executioner of axon degeneration, can induce neuropathy and is activated by dysfunctional mitochondria. We tested the role of SARM1 in a rat model carrying a dominant CMT2A mutation (Mfn2H361Y) that exhibits progressive dying-back axonal degeneration, neuromuscular junction (NMJ) abnormalities, muscle atrophy, and mitochondrial abnormalities - all hallmarks of the human disease. We generated Sarm1-KO (Sarm1-/-) and Mfn2H361Y Sarm1 double-mutant rats and found that deletion of Sarm1 rescued axonal, synaptic, muscle, and functional phenotypes, demonstrating that SARM1 was responsible for much of the neuropathology in this model. Despite the presence of mutant MFN2 protein in these double-mutant rats, loss of SARM1 also dramatically suppressed many mitochondrial defects, including the number, size, and cristae density defects of synaptic mitochondria. This surprising finding indicates that dysfunctional mitochondria activated SARM1 and that activated SARM1 fed back on mitochondria to exacerbate the mitochondrial pathology. As such, this work identifies SARM1 inhibition as a therapeutic candidate for the treatment of CMT2A and other neurodegenerative diseases with prominent mitochondrial pathology.

Axon degeneration is a prominent component of many neurological disorders. Identifying cellular pathways that contribute to axon vulnerability may identify new therapeutic strategies for maintenance of neural circuits. Dual leucine zipper kinase (DLK) is an axonal stress response MAP3K that is chronically activated in several neurodegenerative diseases. Activated DLK transmits an axon injury signal to the neuronal cell body to provoke transcriptional adaptations. However, the consequence of enhanced DLK signaling to axon vulnerability is unknown. We find that stimulating DLK activity predisposes axons to SARM1-dependent degeneration. Activating DLK reduces levels of the axon survival factors NMNAT2 and SCG10, accelerating their loss from severed axons. Moreover, mitochondrial dysfunction independently decreases the levels of NMNAT2 and SCG10 in axons, and in conjunction with DLK activation, leads to a dramatic loss of axonal NMNAT2 and SCG10 and evokes spontaneous axon degeneration. Hence, enhanced DLK activity reduces axon survival factor abundance and renders axons more susceptible to trauma and metabolic insult.

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.

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. Here we investigate the mechanism by which MAPK signaling facilitates axonal degeneration. We show that MAPK signaling promotes 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.

Glia modulate neuronal excitability and seizure sensitivity by maintaining potassium and water homeostasis. A salt inducible kinase 3 (SIK3)-regulated gene expression program controls the glial capacity to buffer K + and water in Drosophila , however upstream regulatory mechanisms are unknown. Here, we identify an octopaminergic circuit linking neuronal activity to glial ion and water buffering. Under basal conditions, octopamine functions through the inhibitory octopaminergic G-protein-coupled receptor (GPCR) OctβR to upregulate glial buffering capacity, while under pathological K + stress, octopamine signals through the stimulatory octopaminergic GPCR OAMB1 to downregulate the glial buffering program. Failure to downregulate this program leads to intracellular glia swelling and stress signaling, suggesting that turning down this pathway is glioprotective. In the eag shaker Drosophila seizure model, the SIK3-mediated buffering pathway is inactivated. Reactivation of the glial buffering program dramatically suppresses neuronal hyperactivity, seizures, and shortened life span in this mutant. These findings highlight the therapeutic potential of a glial-centric therapeutic strategy for diseases of hyperexcitability.