Novel inhibitors of acute, axonal DLK palmitoylation are neuroprotective and avoid the deleterious side effects of cell-wide DLK inhibition.

Dual leucine-zipper kinase (DLK) drives acute and chronic forms of neurodegeneration, suggesting that inhibiting DLK signaling could ameliorate diverse neuropathological conditions. However, direct inhibition of DLK's kinase domain in human patients and conditional knockout of DLK in mice both cause unintended side effects, including elevated plasma neurofilament levels, indicative of neuronal cytoskeletal disruption. Indeed, we found that a DLK kinase domain inhibitor acutely disrupted the axonal cytoskeleton and caused vesicle aggregation in cultured dorsal root ganglion (DRG) neurons, further cautioning against this therapeutic strategy. In seeking a more precise intervention, we found that retrograde (axon-to-soma) pro-degenerative signaling requires acute, axonal palmitoylation of DLK and hypothesized that modulating this post-translational modification might be more specifically neuroprotective than cell-wide DLK inhibition. To address this possibility, we screened >28,000 compounds using a high-content imaging assay that quantitatively evaluates DLK's palmitoylation-dependent subcellular localization. Of the 33 hits that significantly altered DLK localization in non-neuronal cells, several reduced DLK retrograde signaling and protected cultured DRG neurons from DLK-dependent neurodegeneration. Mechanistically, the two most neuroprotective compounds selectively prevent stimulus-dependent palmitoylation of axonal pools of DLK, a process crucial for DLK's recruitment to axonal vesicles. In contrast, these compounds minimally impact DLK localization and signaling in healthy neurons and avoid the cytoskeletal disruption associated with direct DLK inhibition. Importantly, our hit compounds also reduce pro-degenerative retrograde signaling in vivo, suggesting that modulating DLK's palmitoylation-dependent localization could be a novel neuroprotective strategy.

Actin cytoskeletal dynamics do not impose an energy drain on growth cone bioenergetics.

The regulation of the intracellular level of ATP is a fundamental aspect of bioenergetics. Actin cytoskeletal dynamics have been reported to be an energetic drain in developing neurons and platelets. We addressed the role of actin dynamics in primary embryonic chicken neurons using luciferase assays, and by measurement of the ATP/ADP ratio using the ratiometric reporter PercevalHR and the ATP level using the ratiometric reporter mRuby-iATPSnFR. None of the methods revealed an effect of suppressing actin dynamics on the decline in the neuronal ATP level or the ATP/ADP ratio following shutdown of ATP production. Similarly, we find that treatments that elevate or suppress actin dynamics do not alter the ATP/ADP ratio in growth cones, the subcellular domain with the highest actin dynamics in developing neurons. Collectively, the data indicate that actin cytoskeletal dynamics are not a significant energy drain in developing neurons and that the ATP/ADP ratio is maintained when energy utilization varies. Discrepancies between prior work and the current data are discussed with emphasis on methodology and interpretation of the data.

SARM1 Suppresses Axon Branching Through Attenuation of Axonal Cytoskeletal Dynamics.

Axon branching is a fundamental aspect of neuronal morphogenesis, neuronal circuit formation, and response of the nervous system to injury. Sterile alpha and TIR motif containing 1 (SARM1) was initially identified as promoting Wallerian degeneration of axons. We now report a novel function of SARM1 in postnatal sensory neurons; the suppression of axon branching. Axon collateral branches develop from axonal filopodia precursors through the coordination of the actin and microtubule cytoskeleton. In vitro analysis revealed that cultured P0-2 dorsal root ganglion sensory neurons from a SARM1 knockout (KO) mouse exhibit increased numbers of collateral branches and axonal filopodia relative to wild-type neurons. In SARM1 KO mice, cutaneous sensory endings exhibit increased branching in the skin in vivo with normal density of innervation. Transient axonal actin patches serve as cytoskeletal platforms from which axonal filopodia emerge. Live imaging analysis of axonal actin dynamics showed that SARM1 KO neurons exhibit increased rates of axonal actin patch formation and increased probability that individual patches will give rise to a filopodium before dissipating. SARM1 KO axons contain elevated levels of drebrin and cortactin, two actin regulatory proteins that are positive regulators of actin patches, filopodia formation, and branching. Live imaging of microtubule plus tip dynamics revealed an increase in the rate of formation and velocity of polymerizing tips along the axons of SARM1 KO neurons. Stationary mitochondria define sites along the axon where branches may arise, and the axons of SARM1 KO sensory neurons exhibit an increase in stationary mitochondria. These data reveal SARM1 to be a negative regulator of axonal cytoskeletal dynamics and collateral branching.

Palmitoylation couples the kinases DLK and JNK3 to facilitate prodegenerative axon-to-soma signaling.

Dual leucine-zipper kinase (DLK; a MAP3K) mediates neuronal responses to diverse injuries and insults through the c-Jun N-terminal kinase (JNK) family of mitogen-activated protein kinases (MAPKs). Here, we identified two ways through which DLK is coupled to the neural-specific isoform JNK3 to control prodegenerative signaling. JNK3 catalyzed positive feedback phosphorylation of DLK that further activated DLK and locked the DLK-JNK3 module in a highly active state. Neither homologous MAP3Ks nor a homologous MAPK could support this positive feedback loop. Unlike the related JNK1 isoform JNK2 and JNK3 promote prodegenerative axon-to-soma signaling and were endogenously palmitoylated. Moreover, palmitoylation targeted both DLK and JNK3 to the same axonal vesicles, and JNK3 palmitoylation was essential for axonal retrograde signaling in response to optic nerve crush injury in vivo. These findings provide previously unappreciated insights into DLK-JNK signaling relevant to neuropathological conditions and answer long-standing questions regarding the selective prodegenerative roles of JNK2 and JNK3.

The Axonal Glycolytic Pathway Contributes to Sensory Axon Extension and Growth Cone Dynamics.

Understanding the bioenergetics of axon extension and maintenance has wide ranging implications for neurodevelopment and disease states. Glycolysis is a pathway consisting of 10 enzymes and separated into preparatory and payoff phases, the latter producing ATP. Using embryonic chicken sensory neurons, we report that glycolytic enzymes are found through the axon and the growth cone. Pharmacological inhibition of glycolysis in the presence of NGF impairs axon extension and growth cone dynamics within minutes without affecting axon maintenance. Experiments using microfluidic chambers show that the effect of inhibiting glycolysis on axon extension is local along distal axons and can be reversed by promoting mitochondrial respiration. Knockdown of GAPDH simplifies growth cone morphology and is rescued by shRNA-resistant GAPDH expression. Rescue of GAPDH using KillerRed fused to GAPDH followed by localized chromophore-assisted light inactivation of KillerRed-GAPDH in distal axons halts growth cone dynamics. Considering filament polymerization requires ATP, inhibition of glycolysis results in a paradoxical increase in axonal actin filament levels. The effect on actin filaments is because of enzymes before GAPDH, the first enzyme in the payoff phase. In the absence of NGF, inhibition of glycolysis along distal axons results in axon degeneration independent of cell death. These data indicate that the glycolytic pathway is operative in distal axons and contributes to the rate of axon extension and growth cone dynamics in the presence of NGF and that, in the absence of NGF, the axonal glycolytic pathway is required for axon maintenance.SIGNIFICANCE STATEMENT Elucidation of the sources of ATP required for axon extension and maintenance has implications for understanding the mechanism of neuronal development and diseases of the nervous system. While recent work has emphasized the importance of mitochondrial oxidative phosphorylation, the role of the glycolytic pathway in axon morphogenesis and maintenance remains minimally understood. The data reveal that the glycolytic pathway is required for normal sensory axon extension in the presence of NGF, while in the absence of NGF the glycolytic pathway is required for axon maintenance. The results have implications for the understanding of the bioenergetics of axon morphogenesis and plasticity and indicate that NGF has protective effects on sensory axon maintenance in hypoglycemic states.

Coupled Control of Distal Axon Integrity and Somal Responses to Axonal Damage by the Palmitoyl Acyltransferase ZDHHC17.

After optic nerve crush (ONC), the cell bodies and distal axons of most retinal ganglion cells (RGCs) degenerate. RGC somal and distal axon degenerations were previously thought to be controlled by two parallel pathways, involving activation of the kinase dual leucine-zipper kinase (DLK) and loss of the axon survival factor nicotinamide mononucleotide adenylyltransferase-2 (NMNAT2), respectively. Here, we report that palmitoylation of both DLK and NMNAT2 by the palmitoyl acyltransferase ZDHHC17 couples these signals. ZDHHC17-dependent palmitoylation enables DLK-dependent somal degeneration after ONC and also ensures NMNAT-dependent distal axon integrity in healthy optic nerves. We provide evidence that ZDHHC17 also controls survival-versus-degeneration decisions in dorsal root ganglion (DRG) neurons, and we identify conserved motifs in NMNAT2 and DLK that govern their ZDHHC17-dependent regulation. These findings suggest that the control of somal and distal axon integrity should be considered as a single, holistic process, mediated by the concerted action of two palmitoylation-dependent pathways.

The palmitoyl acyltransferases ZDHHC5 and ZDHHC8 are uniquely present in DRG axons and control retrograde signaling via the Gp130/JAK/STAT3 pathway.

Palmitoylation, the modification of proteins with the lipid palmitate, is a key regulator of protein targeting and trafficking. However, knowledge of the roles of specific palmitoyl acyltransferases (PATs), which catalyze palmitoylation, is incomplete. For example, little is known about which PATs are present in neuronal axons, although long-distance trafficking of palmitoyl-proteins is important for axon integrity and for axon-to-soma retrograde signaling, a process critical for axon development and for responses to injury. Identifying axonally targeted PATs might thus provide insights into multiple aspects of axonal biology. We therefore comprehensively determined the subcellular distribution of mammalian PATs in dorsal root ganglion (DRG) neurons and, strikingly, found that only two PATs, ZDHHC5 and ZDHHC8, were enriched in DRG axons. Signals via the Gp130/JAK/STAT3 and DLK/JNK pathways are important for axonal injury responses, and we found that ZDHHC5 and ZDHHC8 were required for Gp130/JAK/STAT3, but not DLK/JNK, axon-to-soma signaling. ZDHHC5 and ZDHHC8 robustly palmitoylated Gp130 in cotransfected nonneuronal cells, supporting the possibility that Gp130 is a direct ZDHHC5/8 substrate. In DRG neurons, Zdhhc5/8 shRNA knockdown reduced Gp130 palmitoylation and even more markedly reduced Gp130 surface expression, potentially explaining the importance of these PATs for Gp130-dependent signaling. Together, these findings provide new insights into the subcellular distribution and roles of specific PATs and reveal a novel mechanism by which palmitoylation controls axonal retrograde signaling.

Identification of Novel Inhibitors of DLK Palmitoylation and Signaling by High Content Screening.

After axonal insult and injury, Dual leucine-zipper kinase (DLK) conveys retrograde pro-degenerative signals to neuronal cell bodies via its downstream target c-Jun N-terminal kinase (JNK). We recently reported that such signals critically require modification of DLK by the fatty acid palmitate, via a process called palmitoylation. Compounds that inhibit DLK palmitoylation could thus reduce neurodegeneration, but identifying such inhibitors requires a suitable assay. Here we report that DLK subcellular localization in non-neuronal cells is highly palmitoylation-dependent and can thus serve as a proxy readout to identify inhibitors of DLK palmitoylation by High Content Screening (HCS). We optimized an HCS assay based on this readout, which showed highly robust performance in a 96-well format. Using this assay we screened a library of 1200 FDA-approved compounds and found that ketoconazole, the compound that most dramatically affected DLK localization in our primary screen, dose-dependently inhibited DLK palmitoylation in follow-up biochemical assays. Moreover, ketoconazole significantly blunted phosphorylation of c-Jun in primary sensory neurons subjected to trophic deprivation, a well known model of DLK-dependent pro-degenerative signaling. Our HCS platform is thus capable of identifying novel inhibitors of DLK palmitoylation and signalling that may have considerable therapeutic potential.

Roles of palmitoylation in axon growth, degeneration and regeneration.

The protein-lipid modification palmitoylation plays important roles in neurons, but most attention has focused on roles of this modification in the regulation of mature pre- and post-synapses. However, exciting recent findings suggest that palmitoylation is also critical for both the growth and integrity of neuronal axons and plays previously unappreciated roles in conveying axonal anterograde and retrograde signals. Here we review these emerging roles for palmitoylation in the regulation of axons in health and disease. © 2017 Wiley Periodicals, Inc.

Palmitoylation controls DLK localization, interactions and activity to ensure effective axonal injury signaling.

Dual leucine-zipper kinase (DLK) is critical for axon-to-soma retrograde signaling following nerve injury. However, it is unknown how DLK, a predicted soluble kinase, conveys long-distance signals and why homologous kinases cannot compensate for loss of DLK. Here, we report that DLK, but not homologous kinases, is palmitoylated at a conserved site adjacent to its kinase domain. Using short-hairpin RNA knockdown/rescue, we find that palmitoylation is critical for DLK-dependent retrograde signaling in sensory axons. This functional importance is because of three novel cellular and molecular roles of palmitoylation, which targets DLK to trafficking vesicles, is required to assemble DLK signaling complexes and, unexpectedly, is essential for DLK's kinase activity. By simultaneously controlling DLK localization, interactions, and activity, palmitoylation ensures that only vesicle-bound DLK is active in neurons. These findings explain how DLK specifically mediates nerve injury responses and reveal a novel cellular mechanism that ensures the specificity of neuronal kinase signaling.

MYCBP2 Is a Guanosine Exchange Factor for Ran Protein and Determines Its Localization in Neurons of Dorsal Root Ganglia.

The small GTPase Ran coordinates retrograde axonal transport in neurons, spindle assembly during mitosis, and the nucleo-cytoplasmic transport of mRNA. Its localization is tightly regulated by the GTPase-activating protein RanGAP1 and the nuclear guanosine exchange factor (GEF) RCC1. We show that loss of the neuronal E3 ubiquitin ligase MYCBP2 caused the up-regulation of Ran and RanGAP1 in dorsal root ganglia (DRG) under basal conditions and during inflammatory hyperalgesia. SUMOylated RanGAP1 physically interacted with MYCBP2 and inhibited its E3 ubiquitin ligase activity. Stimulation of neurons induced a RanGAP1-dependent translocation of MYCBP2 to the nucleus. In the nucleus of DRG neurons MYCBP2 co-localized with Ran and facilitated through its RCC1-like domain the GDP/GTP exchange of Ran. In accordance with the necessity of a GEF to promote GTP-binding and nuclear export of Ran, the nuclear localization of Ran was strongly increased in MYCBP2-deficient DRGs. The finding that other GEFs for Ran besides RCC1 exist gives new insights in the complexity of the regulation of the Ran signaling pathway.

Regulation of neuronal functions by the E3-ubiquitinligase protein associated with MYC (MYCBP2).

The E3-ubiquitinligase MYCBP2 regulates neuronal growth, synaptogenesis and synaptic plasticity by modulating several signaling pathways including the p38 MAPK signaling cascade. We found that loss of MYCBP2 in peripheral sensory neurons inhibits the internalization of transient receptor potential vanilloid receptor 1 (TRPV1) in a p38 MAPK-dependent manner. This prevented desensitization of activity-induced calcium increases and prolongs formalin-induced thermal hyperalgesia in mice. Besides its function in pain perception TRPV1 is also involved in the regulation of neuronal growth. Therefore, the observed effect of MYCBP2 on TRPV1 internalization could be part of the mechanisms underlying its well documented regulatory role in neuronal growth. The clarification of the mechanism is important for the understanding of the different MYCBP2-functions in diverse neuronal subpopulations and species.

The ubiquitin ligase MYCBP2 regulates transient receptor potential vanilloid receptor 1 (TRPV1) internalization through inhibition of p38 MAPK signaling.

The E3 ubiquitin ligase MYCBP2 negatively regulates neuronal growth, synaptogenesis, and synaptic strength. More recently it was shown that MYCBP2 is also involved in receptor and ion channel internalization. We found that mice with a MYCBP2-deficiency in peripheral sensory neurons show prolonged thermal hyperalgesia. Loss of MYCBP2 constitutively activated p38 MAPK and increased expression of several proteins involved in receptor trafficking. Surprisingly, loss of MYCBP2 inhibited internalization of transient receptor potential vanilloid receptor 1 (TRPV1) and prevented desensitization of capsaicin-induced calcium increases. Lack of desensitization, TRPV internalization and prolonged hyperalgesia were reversed by inhibition of p38 MAPK. The effects were TRPV-specific, since neither mustard oil-induced desensitization nor behavioral responses to mechanical stimuli were affected. In summary, we show here for the first time that p38 MAPK activation can inhibit activity-induced ion channel internalization and that MYCBP2 regulates internalization of TRPV1 in peripheral sensory neurons as well as duration of thermal hyperalgesia through p38 MAPK.

Sphingosine-1-phosphate induced mTOR-activation is mediated by the E3-ubiquitin ligase PAM.

The signaling pathways that are regulated by sphingosine-1-phosphate (S1P) and mammalian target of rapamycin (mTOR) modulate cell growth, mitogenesis and apoptosis in various cell types and are of major interest for the development of new cancer therapeutics. Previous reports show that S1P can cross-activate the mTOR pathway although the mechanisms that connect both pathways are still unknown. We found that S1P-treatment activates mTOR in several cancer cell lines and primary cells. The activation was independent of ERK, Akt and PI3-kinase, but instead was mediated by the E3 ubiquitin ligase Protein Associated with Myc (PAM). Increased intracellular PAM concentrations facilitated S1P- and insulin-induced mTOR activation as well as p70S6K and 4EBP1 phosphorylation while genetic deletion of PAM decreased S1P- and insulin-induced mTOR activation. PAM activated by facilitating the GDP/GTP-exchange of Rheb which is an activator of mTOR. In conclusion we show that PAM is a novel regulator of the mTOR pathway and that PAM may directly activate Rheb as a guanosine exchange factor (GEF).

Toponomics analysis of functional interactions of the ubiquitin ligase PAM (Protein Associated with Myc) during spinal nociceptive processing.

Protein associated with Myc (PAM) is a giant E3 ubiquitin ligase of 510 kDa. Although the role of PAM during neuronal development is well established, very little is known about its function in the regulation of synaptic strength. Here we used multiepitope ligand cartography (MELC) to study protein network profiles associated with PAM during the modulation of synaptic strength. MELC is a novel imaging technology that utilizes biomathematical tools to describe protein networks after consecutive immunohistochemical visualization of up to 100 proteins on the same sample. As an in vivo model to modulate synaptic strength we used the formalin test, a common model for acute and inflammatory pain. MELC analysis was performed with 37 different antibodies or fluorescence tags on spinal cord slices and led to the identification of 1390 PAM-related motifs that distinguish untreated and formalin-treated spinal cords. The majority of these motifs related to ubiquitin-dependent processes and/or the actin cytoskeleton. We detected an intermittent colocalization of PAM and ubiquitin with TSC2, a known substrate of PAM, and the glutamate receptors mGluR5 and GLUR1. Importantly these complexes were detected exclusively in the presence of F-actin. A direct PAM/F-actin interaction was confirmed by colocalization and cosedimentation. The binding of PAM toward F-actin varied strongly between the PAM splice forms found in rat spinal cords. PAM did not ubiquitylate actin or alter actin polymerization and depolymerization. However, F-actin decreased the ubiquitin ligase activity of purified PAM. Because PAM activation is known to involve its translocation, the binding of PAM to F-actin may serve to control its subcellular localization as well as its activity. Taken together we show that defining protein network profiles by topological proteomics analysis is a useful tool to identify previously unknown protein/protein interactions that underlie synaptic processes.

PHI-base update: additions to the pathogen host interaction database.

The pathogen-host interaction database (PHI-base) is a web-accessible database that catalogues experimentally verified pathogenicity, virulence and effector genes from bacterial, fungal and Oomycete pathogens, which infect human, animal, plant, insect, fish and fungal hosts. Plant endophytes are also included. PHI-base is therefore an invaluable resource for the discovery of genes in medically and agronomically important pathogens, which may be potential targets for chemical intervention. The database is freely accessible to both academic and non-academic users. This publication describes recent additions to the database and both current and future applications. The number of fields that characterize PHI-base entries has almost doubled. Important additional fields deal with new experimental methods, strain information, pathogenicity islands and external references that link the database to external resources, for example, gene ontology terms and Locus IDs. Another important addition is the inclusion of anti-infectives and their target genes that makes it possible to predict the compounds, that may interact with newly identified virulence factors. In parallel, the curation process has been improved and now involves several external experts. On the technical side, several new search tools have been provided and the database is also now distributed in XML format. PHI-base is available at: http://www.phi-base.org/.