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

•The same enzyme, ZDHHC17, palmitoylates DLK and NMNAT2•DLK palmitoylation by ZDHHC17 is essential for somal responses to axonal injury•Prolonged loss of ZDHHC17 causes NMNAT-dependent fragmentation of distal axons•Conserved motifs in NMNAT2 and DLK govern their ZDHHC17-dependent regulation 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. 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. Retinal ganglion cells (RGCs) are thought to undergo compartmentalized cell death after axonal injury, with different pathways controlling degeneration of the RGC soma and distal axons. This model emerged from findings that knockout (KO) of the pro-apoptotic protein Bax strongly protects RGC somas, but not distal axons after optic nerve crush (ONC), whereas the WldS (Wallerian degeneration slow) spontaneous mutation protects distal axons, but not RGC somas (Howell et al., 2013Howell G.R. Soto I. Libby R.T. John S.W. Intrinsic axonal degeneration pathways are critical for glaucomatous damage.Exp. Neurol. 2013; 246: 54-61Crossref PubMed Scopus (69) Google Scholar; Li et al., 1999Li Y. Schlamp C.L. Nickells R.W. Experimental induction of retinal ganglion cell death in adult mice.Invest. Ophthalmol. Vis. Sci. 1999; 40: 1004-1008PubMed Google Scholar, Li et al., 2000Li Y. Schlamp C.L. Poulsen K.P. Nickells R.W. Bax-dependent and independent pathways of retinal ganglion cell death induced by different damaging stimuli.Exp. Eye Res. 2000; 71: 209-213Crossref PubMed Scopus (85) Google Scholar; Libby et al., 2005Libby R.T. Li Y. Savinova O.V. Barter J. Smith R.S. Nickells R.W. John S.W. Susceptibility to neurodegeneration in a glaucoma is modified by Bax gene dosage.PLoS Genet. 2005; 1: 17-26Crossref PubMed Scopus (295) Google Scholar). Since those initial findings, more is now known about pathways that trigger somal Bax activation and how WldS protects axons. Bax lies downstream of a mitogen-activated protein kinase (MAPK) pathway involving dual leucine-zipper kinase (DLK) and DLK’s direct targets MKK4/MKK7, which in turn activate c-Jun N-terminal kinase (JNK) (Fernandes et al., 2012Fernandes K.A. Harder J.M. Fornarola L.B. Freeman R.S. Clark A.F. Pang I.H. John S.W. Libby R.T. JNK2 and JNK3 are major regulators of axonal injury-induced retinal ganglion cell death.Neurobiol. Dis. 2012; 46: 393-401Crossref PubMed Scopus (109) Google Scholar, Fernandes et al., 2014Fernandes K.A. Harder J.M. John S.W. Shrager P. Libby R.T. DLK-dependent signaling is important for somal but not axonal degeneration of retinal ganglion cells following axonal injury.Neurobiol. Dis. 2014; 69: 108-116Crossref PubMed Scopus (59) Google Scholar; Maes et al., 2017Maes M.E. Schlamp C.L. Nickells R.W. BAX to basics: how the BCL2 gene family controls the death of retinal ganglion cells.Prog. Retin. Eye Res. 2017; 57: 1-25Crossref PubMed Scopus (119) Google Scholar; Watkins et al., 2013Watkins T.A. Wang B. Huntwork-Rodriguez S. Yang J. Jiang Z. Eastham-Anderson J. Modrusan Z. Kaminker J.S. Tessier-Lavigne M. Lewcock J.W. DLK initiates a transcriptional program that couples apoptotic and regenerative responses to axonal injury.Proc. Natl. Acad. Sci. USA. 2013; 110: 4039-4044Crossref PubMed Scopus (196) Google Scholar; Welsbie et al., 2013Welsbie D.S. Yang Z. Ge Y. Mitchell K.L. Zhou X. Martin S.E. Berlinicke C.A. Hackler Jr., L. Fuller J. Fu J. et al.Functional genomic screening identifies dual leucine zipper kinase as a key mediator of retinal ganglion cell death.Proc. Natl. Acad. Sci. USA. 2013; 110: 4045-4050Crossref PubMed Scopus (160) Google Scholar, Welsbie et al., 2017Welsbie D.S. Mitchell K.L. Jaskula-Ranga V. Sluch V.M. Yang Z. Kim J. Buehler E. Patel A. Martin S.E. Zhang P.-W. et al.Enhanced functional genomic screening identifies novel mediators of dual leucine zipper kinase-dependent injury signaling in neurons.Neuron. 2017; 94: 1142-1154.e6Abstract Full Text Full Text PDF PubMed Scopus (74) Google Scholar). The DLK-JNK pathway is thought to convey an axon-to-soma signal, resulting in phosphorylation of transcription factors, most notably c-Jun, and upregulation of pro-apoptotic genes that then activate Bax (Watkins et al., 2013Watkins T.A. Wang B. Huntwork-Rodriguez S. Yang J. Jiang Z. Eastham-Anderson J. Modrusan Z. Kaminker J.S. Tessier-Lavigne M. Lewcock J.W. DLK initiates a transcriptional program that couples apoptotic and regenerative responses to axonal injury.Proc. Natl. Acad. Sci. USA. 2013; 110: 4039-4044Crossref PubMed Scopus (196) Google Scholar). Like Bax KO, DLK KO strongly protects RGC somas post-ONC but protects distal RGC axons minimally, if at all (Fernandes et al., 2014Fernandes K.A. Harder J.M. John S.W. Shrager P. Libby R.T. DLK-dependent signaling is important for somal but not axonal degeneration of retinal ganglion cells following axonal injury.Neurobiol. Dis. 2014; 69: 108-116Crossref PubMed Scopus (59) Google Scholar; Yang et al., 2015Yang J. Wu Z. Renier N. Simon D.J. Uryu K. Park D.S. Greer P.A. Tournier C. Davis R.J. Tessier-Lavigne M. Pathological axonal death through a MAPK cascade that triggers a local energy deficit.Cell. 2015; 160: 161-176Abstract Full Text Full Text PDF PubMed Scopus (203) Google Scholar). In diverse neuron types, continuous anterograde transport of the labile biosynthetic enzyme nicotinamide mononucleotide adenylyltransferase-2 (NMNAT2) maintains axon integrity (Coleman and Freeman, 2010Coleman M.P. Freeman M.R. Wallerian degeneration, wld(s), and nmnat.Annu. Rev. Neurosci. 2010; 33: 245-267Crossref PubMed Scopus (364) Google Scholar; Gilley and Coleman, 2010Gilley J. Coleman M.P. Endogenous Nmnat2 is an essential survival factor for maintenance of healthy axons.PLoS Biol. 2010; 8: e1000300Crossref PubMed Scopus (331) Google Scholar; Yamagishi and Tessier-Lavigne, 2016Yamagishi Y. Tessier-Lavigne M. An atypical SCF-like ubiquitin ligase complex promotes Wallerian degeneration through regulation of axonal Nmnat2.Cell Rep. 2016; 17: 774-782Abstract Full Text Full Text PDF PubMed Scopus (31) Google Scholar). Axonal injury interrupts this transport, and rapid loss of remaining NMNAT2 in the severed distal axon triggers Wallerian degeneration (Gerdts et al., 2016Gerdts J. Summers D.W. Milbrandt J. DiAntonio A. Axon self-destruction: new links among SARM1, MAPKs, and NAD+ metabolism.Neuron. 2016; 89: 449-460Abstract Full Text Full Text PDF PubMed Scopus (220) Google Scholar). The WldS protein (an unnatural, stable form of NMNAT2’s paralog NMNAT1) or a cytosolic form of NMNAT1 (cytoNMNAT1) (Yang et al., 2015Yang J. Wu Z. Renier N. Simon D.J. Uryu K. Park D.S. Greer P.A. Tournier C. Davis R.J. Tessier-Lavigne M. Pathological axonal death through a MAPK cascade that triggers a local energy deficit.Cell. 2015; 160: 161-176Abstract Full Text Full Text PDF PubMed Scopus (203) Google Scholar) strongly protects RGC distal axons post-injury, likely by substituting for NMNAT2 (Fernandes et al., 2014Fernandes K.A. Harder J.M. John S.W. Shrager P. Libby R.T. DLK-dependent signaling is important for somal but not axonal degeneration of retinal ganglion cells following axonal injury.Neurobiol. Dis. 2014; 69: 108-116Crossref PubMed Scopus (59) Google Scholar; Lorber et al., 2012Lorber B. Tassoni A. Bull N.D. Moschos M.M. Martin K.R. Retinal ganglion cell survival and axon regeneration in WldS transgenic rats after optic nerve crush and lens injury.BMC Neurosci. 2012; 13: 56Crossref PubMed Scopus (17) Google Scholar). KO of SARM1 (Fernandes et al., 2018Fernandes K.A. Mitchell K.L. Patel A. Marola O.J. Shrager P. Zack D.J. Libby R.T. Welsbie D.S. Role of SARM1 and DR6 in retinal ganglion cell axonal and somal degeneration following axonal injury.Exp. Eye Res. 2018; 171: 54-61Crossref PubMed Scopus (43) Google Scholar; Yang et al., 2015Yang J. Wu Z. Renier N. Simon D.J. Uryu K. Park D.S. Greer P.A. Tournier C. Davis R.J. Tessier-Lavigne M. Pathological axonal death through a MAPK cascade that triggers a local energy deficit.Cell. 2015; 160: 161-176Abstract Full Text Full Text PDF PubMed Scopus (203) Google Scholar), an “axon executioner” enzyme that is activated downstream of NMNAT2 loss (Gilley et al., 2015Gilley J. Orsomando G. Nascimento-Ferreira I. Coleman M.P. Absence of SARM1 rescues development and survival of NMNAT2-deficient axons.Cell Rep. 2015; 10: 1974-1981Abstract Full Text Full Text PDF PubMed Scopus (115) Google Scholar, Gilley et al., 2017Gilley J. Ribchester R.R. Coleman M.P. Sarm1 deletion, but not WldS, confers lifelong rescue in a mouse model of severe axonopathy.Cell Rep. 2017; 21: 10-16Abstract Full Text Full Text PDF PubMed Scopus (68) Google Scholar) also blocks ONC-induced axon degeneration, but neither WldS nor SARM1 KO prevent ONC-induced RGC somal loss (Beirowski et al., 2008Beirowski B. Babetto E. Coleman M.P. Martin K.R. The WldS gene delays axonal but not somatic degeneration in a rat glaucoma model.Eur. J. Neurosci. 2008; 28: 1166-1179Crossref PubMed Scopus (113) Google Scholar; Fernandes et al., 2014Fernandes K.A. Harder J.M. John S.W. Shrager P. Libby R.T. DLK-dependent signaling is important for somal but not axonal degeneration of retinal ganglion cells following axonal injury.Neurobiol. Dis. 2014; 69: 108-116Crossref PubMed Scopus (59) Google Scholar, Fernandes et al., 2018Fernandes K.A. Mitchell K.L. Patel A. Marola O.J. Shrager P. Zack D.J. Libby R.T. Welsbie D.S. Role of SARM1 and DR6 in retinal ganglion cell axonal and somal degeneration following axonal injury.Exp. Eye Res. 2018; 171: 54-61Crossref PubMed Scopus (43) Google Scholar; Lorber et al., 2012Lorber B. Tassoni A. Bull N.D. Moschos M.M. Martin K.R. Retinal ganglion cell survival and axon regeneration in WldS transgenic rats after optic nerve crush and lens injury.BMC Neurosci. 2012; 13: 56Crossref PubMed Scopus (17) Google Scholar). The above DLK-JNK and NMNAT2-SARM1 studies support the compartmentalized RGC death model. However, we hypothesized that these pathways may be coordinated, based on findings that both DLK and NMNAT2 localize to axonal vesicles and that their vesicle targeting involves the same mechanism—palmitoylation. This protein-lipid modification is essential for vesicular trafficking of both DLK and NMNAT2 in cultured neurons (Holland et al., 2016Holland S.M. Collura K.M. Ketschek A. Noma K. Ferguson T.A. Jin Y. Gallo G. Thomas G.M. Palmitoylation controls DLK localization, interactions and activity to ensure effective axonal injury signaling.Proc. Natl. Acad. Sci. USA. 2016; 113: 763-768Crossref PubMed Scopus (58) Google Scholar; Milde et al., 2013Milde S. Gilley J. Coleman M.P. Subcellular localization determines the stability and axon protective capacity of axon survival factor Nmnat2.PLoS Biol. 2013; 11: e1001539Crossref PubMed Scopus (84) Google Scholar). However, the importance of palmitoylation of DLK and NMNAT2 in the optic nerve in vivo and the palmitoyl acyltransferase (PAT) responsible are unknown. Here, we use adeno-associated virus (AAV)-mediated molecular replacement to define the importance of DLK and NMNAT2 palmitoylation for RGC somal and distal axon integrity in vivo. We first show that DLK palmitoylation is critical for ONC-induced axon-to-soma signaling and subsequent RGC somal degeneration. We then identify the PAT ZDHHC17 as an evolutionarily conserved regulator of DLK palmitoylation and signaling. We further show that DLK and NMNAT2 palmitoyl sites are homologous and provide evidence that ZDHHC17-dependent palmitoylation of NMNAT2 is essential for distal axon integrity in cultured neurons and in vivo. These results provide unexpected insights into the control of DLK and NMNAT2 signaling and increase our understanding of how somal and distal axon degeneration is coupled and controlled. We first used genetic manipulation, mediated by intravitreal AAV delivery (Martin et al., 2002Martin K.R. Klein R.L. Quigley H.A. Gene delivery to the eye using adeno-associated viral vectors.Methods. 2002; 28: 267-275Crossref PubMed Scopus (133) Google Scholar; Figure 1A), to ask whether DLK palmitoylation is necessary for RGC somal responses to ONC. In uninfected wild-type (WT) mice, ONC greatly increased phosphorylation of c-Jun (P-c-Jun) in RGCs and decreased levels of Brn3a, a marker of healthy RGCs, as in prior studies (Figure S1A; Fernandes et al., 2014Fernandes K.A. Harder J.M. John S.W. Shrager P. Libby R.T. DLK-dependent signaling is important for somal but not axonal degeneration of retinal ganglion cells following axonal injury.Neurobiol. Dis. 2014; 69: 108-116Crossref PubMed Scopus (59) Google Scholar; Watkins et al., 2013Watkins T.A. Wang B. Huntwork-Rodriguez S. Yang J. Jiang Z. Eastham-Anderson J. Modrusan Z. Kaminker J.S. Tessier-Lavigne M. Lewcock J.W. DLK initiates a transcriptional program that couples apoptotic and regenerative responses to axonal injury.Proc. Natl. Acad. Sci. USA. 2013; 110: 4039-4044Crossref PubMed Scopus (196) Google Scholar; Welsbie et al., 2013Welsbie D.S. Yang Z. Ge Y. Mitchell K.L. Zhou X. Martin S.E. Berlinicke C.A. Hackler Jr., L. Fuller J. Fu J. et al.Functional genomic screening identifies dual leucine zipper kinase as a key mediator of retinal ganglion cell death.Proc. Natl. Acad. Sci. USA. 2013; 110: 4045-4050Crossref PubMed Scopus (160) Google Scholar, Welsbie et al., 2017Welsbie D.S. Mitchell K.L. Jaskula-Ranga V. Sluch V.M. Yang Z. Kim J. Buehler E. Patel A. Martin S.E. Zhang P.-W. et al.Enhanced functional genomic screening identifies novel mediators of dual leucine zipper kinase-dependent injury signaling in neurons.Neuron. 2017; 94: 1142-1154.e6Abstract Full Text Full Text PDF PubMed Scopus (74) Google Scholar). ONC also increased levels of P-c-Jun and DLK in retinas that had been intravitreally infected with control (GFP-expressing) AAV (Figures 1B, 1C, S1B, and S1C). However, ONC-induced P-c-Jun and DLK increases were prevented in retinas infected with AAV expressing GFP plus DLK short hairpin RNA (shRNA) (AAV-GFP-DLKsh; Figures 1B, 1C, S1B, and S1C), similar to prior work (Watkins et al., 2013Watkins T.A. Wang B. Huntwork-Rodriguez S. Yang J. Jiang Z. Eastham-Anderson J. Modrusan Z. Kaminker J.S. Tessier-Lavigne M. Lewcock J.W. DLK initiates a transcriptional program that couples apoptotic and regenerative responses to axonal injury.Proc. Natl. Acad. Sci. USA. 2013; 110: 4039-4044Crossref PubMed Scopus (196) Google Scholar; Welsbie et al., 2013Welsbie D.S. Yang Z. Ge Y. Mitchell K.L. Zhou X. Martin S.E. Berlinicke C.A. Hackler Jr., L. Fuller J. Fu J. et al.Functional genomic screening identifies dual leucine zipper kinase as a key mediator of retinal ganglion cell death.Proc. Natl. Acad. Sci. USA. 2013; 110: 4045-4050Crossref PubMed Scopus (160) Google Scholar). DLK’s Cys127 palmitoylation site is critical for DLK localization and function in cultured neurons (Holland et al., 2016Holland S.M. Collura K.M. Ketschek A. Noma K. Ferguson T.A. Jin Y. Gallo G. Thomas G.M. Palmitoylation controls DLK localization, interactions and activity to ensure effective axonal injury signaling.Proc. Natl. Acad. Sci. USA. 2016; 113: 763-768Crossref PubMed Scopus (58) Google Scholar). To address the importance of DLK palmitoylation in somal responses to ONC, we coinfected retinas with AAV-GFP-DLKsh and a second AAV expressing hemagglutinin (HA)-tagged shRNA-resistant forms of either WT or palmitoyl mutant (Cys127Ser: “CS”) DLK (AAV-WT-DLK∗-HA, AAV-DLK-CS∗-HA; “WT∗” and “CS∗” in Figure 1B). WT-DLK∗-HA, but not DLK-CS∗-HA, restored ONC-induced P-c-Jun to WT levels (Figures 1B and 1C). WT-DLK∗-HA and DLK-CS∗-HA expressed at similar levels (Figure 1B) and in a similar fraction of GFP-positive neurons (Figure S1D). We next used RNA-binding protein with multiple splicing (RBPMS), an RGC marker whose expression is unaffected by ONC (Kwong et al., 2011Kwong J.M. Quan A. Kyung H. Piri N. Caprioli J. Quantitative analysis of retinal ganglion cell survival with Rbpms immunolabeling in animal models of optic neuropathies.Invest. Ophthalmol. Vis. Sci. 2011; 52: 9694-9702Crossref PubMed Scopus (49) Google Scholar), to assess RGC somal viability at 3 weeks post-ONC (Figures 1A and 1D). ONC greatly reduced RGC counts in AAV-GFP-infected, but not AAV-GFP-DLKsh-infected, retinas (Figures 1D and 1E), similar to prior studies (Fernandes et al., 2014Fernandes K.A. Harder J.M. John S.W. Shrager P. Libby R.T. DLK-dependent signaling is important for somal but not axonal degeneration of retinal ganglion cells following axonal injury.Neurobiol. Dis. 2014; 69: 108-116Crossref PubMed Scopus (59) Google Scholar; Watkins et al., 2013Watkins T.A. Wang B. Huntwork-Rodriguez S. Yang J. Jiang Z. Eastham-Anderson J. Modrusan Z. Kaminker J.S. Tessier-Lavigne M. Lewcock J.W. DLK initiates a transcriptional program that couples apoptotic and regenerative responses to axonal injury.Proc. Natl. Acad. Sci. USA. 2013; 110: 4039-4044Crossref PubMed Scopus (196) Google Scholar; Welsbie et al., 2013Welsbie D.S. Yang Z. Ge Y. Mitchell K.L. Zhou X. Martin S.E. Berlinicke C.A. Hackler Jr., L. Fuller J. Fu J. et al.Functional genomic screening identifies dual leucine zipper kinase as a key mediator of retinal ganglion cell death.Proc. Natl. Acad. Sci. USA. 2013; 110: 4045-4050Crossref PubMed Scopus (160) Google Scholar). AAV-WT-DLK∗-HA, but not AAV-DLK-CS∗-HA, rescued ONC-induced RGC loss (WT∗ and CS∗; Figures 1D and 1E). In uninjured retinas, WT-DLK∗-HA slightly increased P-c-Jun but did not reduce RGC viability (Figures 1C and 1E). AAV also sparsely infected RBPMS-negative cells (Figure 1D), likely displaced amacrine cells (Perry, 1981Perry V.H. Evidence for an amacrine cell system in the ganglion cell layer of the rat retina.Neuroscience. 1981; 6: 931-944Crossref PubMed Scopus (369) Google Scholar), but these were not included in RGC viability analyses. These data suggest that RGC somal responses to ONC require palmitoyl-DLK. The expression of >20 of the 24 mammalian PATs in RGCs (Sajgo et al., 2017Sajgo S. Ghinia M.G. Brooks M. Kretschmer F. Chuang K. Hiriyanna S. Wu Z. Popescu O. Badea T.C. Molecular codes for cell type specification in Brn3 retinal ganglion cells.Proc. Natl. Acad. Sci. USA. 2017; 114: E3974-E3983Crossref PubMed Scopus (44) Google Scholar; Tran et al., 2019Tran N.M. Shekhar K. Whitney I.E. Jacobi A. Benhar I. Hong G. Yan W. Adiconis X. Arnold M.E. Lee J.M. et al.Single-cell profiles of retinal ganglion cells differing in resilience to injury reveal neuroprotective genes.Neuron. 2019; 104: 1039-1055.e12Abstract Full Text Full Text PDF PubMed Scopus (206) Google Scholar) potentially complicates identification of the PAT(s) that palmitoylates DLK. As CS mutation shifts localization of DLK orthologs from membrane associated to diffuse in HEK293T cells and also in rodent and C. elegans sensory neurons (Holland et al., 2016Holland S.M. Collura K.M. Ketschek A. Noma K. Ferguson T.A. Jin Y. Gallo G. Thomas G.M. Palmitoylation controls DLK localization, interactions and activity to ensure effective axonal injury signaling.Proc. Natl. Acad. Sci. USA. 2016; 113: 763-768Crossref PubMed Scopus (58) Google Scholar; Martin et al., 2019Martin D.D.O. Kanuparthi P.S. Holland S.M. Sanders S.S. Jeong H.K. Einarson M.B. Jacobson M.A. Thomas G.M. Identification of novel inhibitors of DLK palmitoylation and signaling by high content screening.Sci. Rep. 2019; 9: 3632Crossref PubMed Scopus (11) Google Scholar), we reasoned that the DLK PAT should be evolutionarily conserved. The most conserved PAT is ZDHHC17, which, unlike most PATs, has six rather than four predicted transmembrane domains (TMDs), plus an N-terminal ankyrin repeat (AnkR) region, in addition to the enzymatic DHHC (Asp-His-His-Cys) domain (Figure 2A; Roth et al., 2002Roth A.F. Feng Y. Chen L. Davis N.G. The yeast DHHC cysteine-rich domain protein Akr1p is a palmitoyl transferase.J. Cell Biol. 2002; 159: 23-28Crossref PubMed Scopus (375) Google Scholar; Young et al., 2012Young F.B. Butland S.L. Sanders S.S. Sutton L.M. Hayden M.R. Putting proteins in their place: palmitoylation in Huntington disease and other neuropsychiatric diseases.Prog. Neurobiol. 2012; 97: 220-238Crossref PubMed Scopus (98) Google Scholar). Two C. elegans PATs, DHHC-13 and DHHC-14 (Edmonds and Morgan, 2014Edmonds M.J. Morgan A. A systematic analysis of protein palmitoylation in Caenorhabditis elegans.BMC Genomics. 2014; 15: 841Crossref PubMed Scopus (16) Google Scholar), are presumptive ZDHHC17 orthologs (Figure 2A). Double KO of dhhc-13 and dhhc-14 shifted localization of a GFP-tagged C. elegans DLK ortholog (GFP-CeDLK-1) from punctate to diffuse (Figures 2B, 2C, and S2A), suggesting that ZDHHC17 orthologs regulate CeDLK-1. We next asked whether ZDHHC17 is a PAT for mammalian DLK. In cotransfected HEK293T cells, ZDHHC17 coimmunoprecipitated GFP-tagged WT-DLK (WT-DLK-GFP) more effectively than did six other candidate DLK PATs (Holland et al., 2016Holland S.M. Collura K.M. Ketschek A. Noma K. Ferguson T.A. Jin Y. Gallo G. Thomas G.M. Palmitoylation controls DLK localization, interactions and activity to ensure effective axonal injury signaling.Proc. Natl. Acad. Sci. USA. 2016; 113: 763-768Crossref PubMed Scopus (58) Google Scholar; Figure S2B). Of these PATs, only ZDHHC17 contains an AnkR, suggesting ZDHHC17 might bind DLK via this region. ZDHHC17’s AnkR recognizes a ZDHHC ankyrin domain binding motif (zDABM; [AIPV][ITV]XX[CMQ][IP]X[KR]) in several ZDHHC17 substrates (Lemonidis et al., 2015Lemonidis K. Sanchez-Perez M.C. Chamberlain L.H. Identification of a novel sequence motif recognized by the ankyrin repeat domain of zDHHC17/13 S-acyltransferases.J. Biol. Chem. 2015; 290: 21939-21950Abstract Full Text Full Text PDF PubMed Scopus (51) Google Scholar, Lemonidis et al., 2017Lemonidis K. MacLeod R. Baillie G.S. Chamberlain L.H. Peptide array-based screening reveals a large number of proteins interacting with the ankyrin-repeat domain of the zDHHC17 S-acyltransferase.J. Biol. Chem. 2017; 292: 17190-17202Abstract Full Text Full Text PDF PubMed Scopus (20) Google Scholar). We identified a matching PVXXMIXK sequence close to DLK’s palmitoylation site (Figure S2C). Mutating this zDABM (PV-AA mutant; Figure S2C) reduced DLK-GFP palmitoylation, assessed by acyl biotin exchange (ABE) assays. We previously reported that Golgi targeting of DLK-GFP in HEK293T cells is palmitoylation dependent (Martin et al., 2019Martin D.D.O. Kanuparthi P.S. Holland S.M. Sanders S.S. Jeong H.K. Einarson M.B. Jacobson M.A. Thomas G.M. Identification of novel inhibitors of DLK palmitoylation and signaling by high content screening.Sci. Rep. 2019; 9: 3632Crossref PubMed Scopus (11) Google Scholar), but the DLK PV-AA mutant showed reduced Golgi targeting and palmitoylation, even when ZDHHC17-HA was cotransfected (Figures S2D–S2G). Neither palmitoylation nor Golgi targeting of WT-DLK-GFP was increased by ZDHHC17-N100A, a mutant that poorly recognizes zDABM-containing substrates (Verardi et al., 2017Verardi R. Kim J.-S. Ghirlando R. Banerjee A. Structural basis for substrate recognition by the ankyrin repeat domain of human DHHC17 palmitoyltransferase.Structure. 2017; 25: 1337-1347.e6Abstract Full Text Full Text PDF PubMed Scopus (36) Google Scholar; Figures S2D–S2G). These findings suggest that ZDHHC17 palmitoylates DLK in an AnkR-zDABM-dependent manner. ZDHHC17 is highly expressed in RGCs (Sajgo et al., 2017Sajgo S. Ghinia M.G. Brooks M. Kretschmer F. Chuang K. Hiriyanna S. Wu Z. Popescu O. Badea T.C. Molecular codes for cell type specification in Brn3 retinal ganglion cells.Proc. Natl. Acad. Sci. USA. 2017; 114: E3974-E3983Crossref PubMed Scopus (44) Google Scholar; Tran et al., 2019Tran N.M. Shekhar K. Whitney I.E. Jacobi A. Benhar I. Hong G. Yan W. Adiconis X. Arnold M.E. Lee J.M. et al.Single-cell profiles of retinal ganglion cells differing in resilience to injury reveal neuroprotective genes.Neuron. 2019; 104: 1039-1055.e12Abstract Full Text Full Text PDF PubMed Scopus (206) Google Scholar), making it a strong candidate to palmitoylate endogenous DLK. However, isolating sufficient amounts of pure RGCs for ABE assays is difficult, so we addressed this question using cultured DRG neurons, in which somal responses to axon insult/injury also require DLK and its palmitoylation (Ghosh et al., 2011Ghosh A.S. Wang B. Pozniak C.D. Chen M. Watts R.J. Lewcock J.W. DLK induces developmental neuronal degeneration via selective regulation of proapoptotic JNK activity.J. Cell Biol. 2011; 194: 751-764Crossref PubMed Scopus (135) Google Scholar; Holland et al., 2016Holland S.M. Collura K.M. Ketschek A. Noma K. Ferguson T.A. Jin Y. Gallo G. Thomas G.M. Palmitoylation controls DLK localization, interactions and activity to ensure effective axonal injury signaling.Proc. Natl. Acad. Sci. USA. 2016; 113: 763-768Crossref PubMed Scopus (58) Google Scholar; Simon et al., 2016Simon D.J. Pitts J. Hertz N.T. Yang J. Yamagishi Y. Olsen O. Tešić Mark M. Molina H. Tessier-Lavigne M. Axon degeneration gated by retrograde activation of somatic pro-apoptotic signaling.Cell. 2016; 164: 1031-1045Abstract Full Text Full Text PDF PubMed Scopus (83) Google Scholar). DLK palmitoylation in DRG neurons (normalized to total DLK expression) was greatly reduced by Zdhhc17 knockdown (Figures 2D–2F, S2H, and S2I). As a control, knockdown of Zdhhc5 and Zdhhc8, two PATs that strongly palmitoylate DLK in non-neuronal cells (Holland et al., 2016Holland S.M. Collura K.M. Ketschek A. Noma K. Ferguson T.A. Jin Y. Gallo G. Thomas G.M. Palmitoylation controls DLK localization, interactions and activity to ensure effective axonal injury signaling.Proc. Natl. Acad. Sci. USA. 2016; 113: 763-768Crossref PubMed Scopus (58) Google Scholar), did not affect DLK palmitoylation (Figure S2J). Zdhhc17 knockdown also reduced palmitoylation of JNK3, DLK’s downstream target (Figure S2K), but did not affect palmitoylation of another axonal palmitoyl protein, GAP43 (Figures 2D–2F). These results suggest that ZDHHC17 is a major PAT for DLK in DRG neurons. Does Zdhhc17 loss prevent somal responses to axon insult or injury through the loss of palmitoyl-DLK? DRG neuron somal responses to trophic factor deprivation (TD) are DLK dependent (Ghosh et al., 2011Ghosh A.S. Wang B. Pozniak C.D. Chen M. Watts R.J. Lewcock J.W. DLK induces developmental neuronal degeneration via selective regulation of proapoptotic JNK activity.J. Cell Biol. 2011; 194: 751-764Crossref PubMed Scopus (135) Google Scholar; Simon et al., 2016Simon D.J. Pitts J. Hertz N.T. Yang J. Yamagishi Y. Olsen O. Tešić Mark M. Molina H. Tessier-Lavigne M. Axon degeneration gated by retrograde activation of somatic pro-apoptotic signaling.Cell. 2016; 164: 1031-1045Abstract Full Text Full Text PDF PubMed Scopus (83) Google Scholar), so we first asked whether such responses also require palmitoyl-DLK. TD increased P-c-Jun in DRG neuron somas and, over prolonged times, activated caspase-3, a key “commitment to die” step, as in prior studies (Deshmukh et al., 2000Deshmukh M. Kuida K. Johnson Jr., E.M. Caspase inhibition extends the commitment to neuronal death beyond cytochrome c release to the point of mitochondrial depolarization.J. Cell Biol. 2000; 150: 131-143Crossref PubMed Scopus (163) Google Scholar; Ghosh et al., 2011Ghosh A.S. Wang B. Pozniak C.D. Chen M. Watts R.J. Lewcock J.W. DLK induces developmental neuronal degeneration via selective regulation of proapoptotic JNK activity.J. Cell Biol. 2011; 194: 751-764Crossref PubMed Scopus (135) Google Scholar; Simon et al., 2016Simon D.J. Pitts J. Hertz N.T. Yang J. Yamagishi Y. Olsen O. Tešić Mark M. Molina H. Tessier-Lavigne M. Axon degeneration gated by retrograde activation of somatic pro-apoptotic signaling.Cell. 2016; 164: 1031-1045Abstract Full Text Full Text PDF PubMed Scopus (83) Google Scholar; Figures S3A–S3D). Both P-c-Jun increases and caspase-3 activation were prevented by DLKs