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.

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.

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.

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.

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.

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.

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.

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.