DLK initiates a transcriptional program that couples apoptotic and regenerative responses to axonal injury Trent A. Watkins a,1 , Bei Wang a,1 , Sarah Huntwork-Rodriguez a , Jing Yang a,2 , Zhiyu Jiang a , Jeffrey Eastham-Anderson b , Zora Modrusan c , Joshua S. Kaminker d , Marc Tessier-Lavigne a,2 , and Joseph W. Lewcock a,3 a Neurodegeneration Laboratories, Department of Neuroscience, b Department of Pathology, c Department of Molecular Biology, and d Department of Bioinformatics and Computational Biology, Genentech, Inc., South San Francisco, CA 94080 Edited* by Jeremy Nathans, Johns Hopkins University, Baltimore, MD, and approved January 22, 2013 (received for review June 28, 2012) The cell intrinsic factors that determine whether a neuron regen- erates or undergoes apoptosis in response to axonal injury are not well deﬁned. Here we show that the mixed-lineage dual leucine zipper kinase (DLK) is an essential upstream mediator of both of these divergent outcomes in the same cell type. Optic nerve crush injury leads to rapid elevation of DLK protein, ﬁrst in the axons of retinal ganglion cells (RGCs) and then in their cell bodies. DLK is required for the majority of gene expression changes in RGCs ini- tiated by injury, including induction of both proapoptotic and re- generation-associated genes. Deletion of DLK in retina results in robust and sustained protection of RGCs from degeneration after optic nerve injury. Despite this improved survival, the number of axons that regrow beyond the injury site is substantially re- duced, even when the tumor suppressor phosphatase and tensin homolog (PTEN) is deleted to enhance intrinsic growth potential. These ﬁndings demonstrate that these seemingly con- tradictory responses to injury are mechanistically coupled through a DLK-based damage detection mechanism. A xonal damage results in signiﬁcant neuronal cell death and axon degeneration, often leading to permanent functional deﬁcits. For example, optic nerve crush rapidly induces a stress response in retinal ganglion cells (RGCs) that includes profound alterations in gene expression patterns (1) and ultimately leads to apoptosis of these neurons (2). As axon injury may occur a signif- icant distance from the cell body, it has been proposed that ret- rograde molecular motors play a critical role in conveying damage signals to the nucleus, allowing the cell to respond to damage (3). Attenuation of this transport mechanism has been shown to reduce degeneration, suggesting that the ability of the nucleus to detect an insult is an essential component of the injury response (4). Recent data suggest that dual leucine zipper kinase (DLK) is an essential component of the neuronal response to axon dam- age. DLK protein is present in axons, and protein levels are in- creased in response to axonal injury (5). Loss of DLK has been shown to protect distal axons from Wallerian degeneration (6) and to abrogate stress-induced retrograde c-Jun N-terminal ki- nase (JNK) signaling through interaction with the scaffolding protein JNK-interacting protein 3 (JIP3) (7-9). In many instan- ces, injury-induced JNK activation in neurons results in apoptosis through phosphorylation of activator protein 1 (AP-1) tran- scription factors such as c-Jun, which initiates a proapoptotic gene expression program (10, 11). Consistent with this, genetic deletion of JNK2 and/or JNK3 is sufﬁcient to protect neurons from degeneration in a range of CNS injury models, including axotomy (12–14), although the role of DLK in these contexts is not known. In contrast, DLK has been shown to regulate axon regeneration after axonal injury in adult peripheral nerves (9) and invertebrate systems (5, 15). The mechanism underlying the divergence be- tween these apoptotic and regenerative phenotypes is unclear, but it could reﬂect distinct signaling pathways downstream of DLK in each system. Alternatively, this disparity may be a result of dif- ferences in the intrinsic or extrinsic factors that govern the potential for regrowth in the CNS and peripheral nerves. In the current study, we use the optic nerve crush model in DLK- inducible knockout mice to investigate the role of this kinase after CNS axonal injury. Our results demonstrate that although neuro- degeneration takes place during a period of weeks after nerve crush, initiation of a transcriptional stress response occurs rapidly in RGCs and includes both proapoptotic and proregenerative gene expression changes. DLK deletion broadly attenuates this re- sponse, provides substantial protection of RGCs from apoptosis, and eliminates the modest but reproducible axon regrowth ob- served after injury. These observations suggest a model in which optic nerve crush induces prolonged DLK-dependent stress sig- naling that coordinately primes RGCs for both apoptosis and regrowth but ultimately leads to cell death resulting from the ab- sence of regenerative potential in the optic nerve. In this model, DLK-deﬁcient neurons do not display either outcome, as they are largely unable to detect axonal injury. Results To determine whether DLK is activated in RGC axons after optic nerve crush, we stained sections of nerves and retinas for DLK and other markers 1–7 d after injury. DLK levels increased within 1 d in RGC axons, but not other cells (Fig. S1A), of the injured nerve (Fig. 1 A and B), and within 3 d in the ganglion cell layer (GCL) and nerve ﬁber layer of the retina (Fig. S1 B and C), implying that DLK signaling initiates in RGC axons after crush and remains limited to RGCs. Similarly, despite a high basal level of stress-independent phosphorylated JNK (p-JNK) in retinal neu- rons (16), injury-dependent p-JNK is increased in retina within 1 d (Fig. 1C), with elevated phosphorylation of its downstream AP-1 transcription factor c-Jun in the GCL during the ﬁrst 3 d after nerve crush serving as a more sensitive readout of this stress-mediated JNK signaling (Fig. 1C and Fig. S1D). In contrast, expression of brain-speciﬁc homeobox/POU domain protein 3 (Brn3), a well- characterized RGC marker (17), was greatly reduced during this time (Fig. S1E). Only a small fraction of cells was actively undergoing apoptosis during this period, with the ﬁrst detectable staining for active caspase 3 appearing 3 d after crush (Fig. S1F), which is consistent with previous studies (14). Thus, DLK up-regulation, JNK activation, and Brn3 down-regulation occur rapidly after nerve crush injury and precede neuronal apoptosis. To investigate the role of DLK after optic nerve crush, we generated mice with a tamoxifen-inducible Cre recombinase- Author contributions: T.A.W., B.W., S.H.-R., Z.M., J.S.K., and J.W.L. designed research; T.A.W., B.W., S.H.-R., Z.J., J.E.-A., Z.M., and J.S.K. performed research; J.Y. and M.T.-L. contributed new reagents/analytic tools; T.A.W., B.W., S.H.-R., J.E.-A., J.S.K., M.T.-L., and J.W.L. analyzed data; and T.A.W., B.W., S.H.-R., M.T.-L., and J.W.L. wrote the paper. The authors declare no conﬂict of interest. *This Direct Submission article had a prearranged editor. 1 T.A.W. and B.W. contributed equally to this work. 2 Present address: Laboratory of Brain Development and Repair, The Rockefeller Univer- sity, New York, NY 10065. 3 To whom correspondence should be addressed. E-mail: email@example.com. This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. 1073/pnas.1211074110/-/DCSupplemental. www.pnas.org/cgi/doi/10.1073/pnas.1211074110 PNAS | March 5, 2013 | vol. 110 | no. 10 | 4039–4044 NEUROSCIENCE
DLK initiates a transcriptional program that couples ... · Optic nerve crush injury leads to rapid elevation of DLK protein, ﬁrst in the axons of retinal ganglion cells (RGCs)
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DLK initiates a transcriptional program that couplesapoptotic and regenerative responses to axonal injuryTrent A. Watkinsa,1, Bei Wanga,1, Sarah Huntwork-Rodrigueza, Jing Yanga,2, Zhiyu Jianga, Jeffrey Eastham-Andersonb,Zora Modrusanc, Joshua S. Kaminkerd, Marc Tessier-Lavignea,2, and Joseph W. Lewcocka,3
aNeurodegeneration Laboratories, Department of Neuroscience, bDepartment of Pathology, cDepartment of Molecular Biology, and dDepartment ofBioinformatics and Computational Biology, Genentech, Inc., South San Francisco, CA 94080
Edited* by Jeremy Nathans, Johns Hopkins University, Baltimore, MD, and approved January 22, 2013 (received for review June 28, 2012)
The cell intrinsic factors that determine whether a neuron regen-erates or undergoes apoptosis in response to axonal injury are notwell defined. Here we show that the mixed-lineage dual leucinezipper kinase (DLK) is an essential upstream mediator of both ofthese divergent outcomes in the same cell type. Optic nerve crushinjury leads to rapid elevation of DLK protein, first in the axons ofretinal ganglion cells (RGCs) and then in their cell bodies. DLK isrequired for the majority of gene expression changes in RGCs ini-tiated by injury, including induction of both proapoptotic and re-generation-associated genes. Deletion of DLK in retina results inrobust and sustained protection of RGCs from degeneration afteroptic nerve injury. Despite this improved survival, the number ofaxons that regrow beyond the injury site is substantially re-duced, even when the tumor suppressor phosphatase andtensin homolog (PTEN) is deleted to enhance intrinsic growthpotential. These findings demonstrate that these seemingly con-tradictory responses to injury are mechanistically coupledthrough a DLK-based damage detection mechanism.
Axonal damage results in significant neuronal cell death andaxon degeneration, often leading to permanent functional
deficits. For example, optic nerve crush rapidly induces a stressresponse in retinal ganglion cells (RGCs) that includes profoundalterations in gene expression patterns (1) and ultimately leads toapoptosis of these neurons (2). As axon injury may occur a signif-icant distance from the cell body, it has been proposed that ret-rograde molecular motors play a critical role in conveying damagesignals to the nucleus, allowing the cell to respond to damage (3).Attenuation of this transport mechanism has been shown to reducedegeneration, suggesting that the ability of the nucleus to detect aninsult is an essential component of the injury response (4).Recent data suggest that dual leucine zipper kinase (DLK) is
an essential component of the neuronal response to axon dam-age. DLK protein is present in axons, and protein levels are in-creased in response to axonal injury (5). Loss of DLK has beenshown to protect distal axons from Wallerian degeneration (6)and to abrogate stress-induced retrograde c-Jun N-terminal ki-nase (JNK) signaling through interaction with the scaffoldingprotein JNK-interacting protein 3 (JIP3) (7-9). In many instan-ces, injury-induced JNK activation in neurons results in apoptosisthrough phosphorylation of activator protein 1 (AP-1) tran-scription factors such as c-Jun, which initiates a proapoptoticgene expression program (10, 11). Consistent with this, geneticdeletion of JNK2 and/or JNK3 is sufficient to protect neuronsfrom degeneration in a range of CNS injury models, includingaxotomy (12–14), although the role of DLK in these contexts isnot known.In contrast, DLK has been shown to regulate axon regeneration
after axonal injury in adult peripheral nerves (9) and invertebratesystems (5, 15). The mechanism underlying the divergence be-tween these apoptotic and regenerative phenotypes is unclear, butit could reflect distinct signaling pathways downstream of DLKin each system. Alternatively, this disparity may be a result of dif-ferences in the intrinsic or extrinsic factors that govern thepotential for regrowth in the CNS and peripheral nerves.
In the current study, we use the optic nerve crushmodel inDLK-inducible knockout mice to investigate the role of this kinase afterCNS axonal injury. Our results demonstrate that although neuro-degeneration takes place during a period of weeks after nervecrush, initiation of a transcriptional stress response occurs rapidlyinRGCs and includes both proapoptotic and proregenerative geneexpression changes. DLK deletion broadly attenuates this re-sponse, provides substantial protection of RGCs from apoptosis,and eliminates the modest but reproducible axon regrowth ob-served after injury. These observations suggest a model in whichoptic nerve crush induces prolonged DLK-dependent stress sig-naling that coordinately primes RGCs for both apoptosis andregrowth but ultimately leads to cell death resulting from the ab-sence of regenerative potential in the optic nerve. In this model,DLK-deficient neurons do not display either outcome, as they arelargely unable to detect axonal injury.
ResultsTo determine whether DLK is activated in RGC axons afteroptic nerve crush, we stained sections of nerves and retinas forDLK and other markers 1–7 d after injury. DLK levels increasedwithin 1 d in RGC axons, but not other cells (Fig. S1A), of theinjured nerve (Fig. 1 A and B), and within 3 d in the ganglion celllayer (GCL) and nerve fiber layer of the retina (Fig. S1 B and C),implying thatDLK signaling initiates inRGCaxons after crush andremains limited to RGCs. Similarly, despite a high basal level ofstress-independent phosphorylated JNK (p-JNK) in retinal neu-rons (16), injury-dependent p-JNK is increased in retina within 1 d(Fig. 1C), with elevated phosphorylation of its downstream AP-1transcription factor c-Jun in theGCLduring thefirst 3 d after nervecrush serving as a more sensitive readout of this stress-mediatedJNK signaling (Fig. 1C and Fig. S1D). In contrast, expression ofbrain-specific homeobox/POU domain protein 3 (Brn3), a well-characterized RGC marker (17), was greatly reduced during thistime (Fig. S1E).Only a small fraction of cells was actively undergoingapoptosis during this period, with the first detectable staining foractive caspase 3 appearing 3 d after crush (Fig. S1F), which isconsistent with previous studies (14). Thus, DLK up-regulation,JNK activation, and Brn3 down-regulation occur rapidly afternerve crush injury and precede neuronal apoptosis.To investigate the role of DLK after optic nerve crush, we
generated mice with a tamoxifen-inducible Cre recombinase-
Author contributions: T.A.W., B.W., S.H.-R., Z.M., J.S.K., and J.W.L. designed research; T.A.W.,B.W., S.H.-R., Z.J., J.E.-A., Z.M., and J.S.K. performed research; J.Y. and M.T.-L. contributednew reagents/analytic tools; T.A.W., B.W., S.H.-R., J.E.-A., J.S.K., M.T.-L., and J.W.L. analyzeddata; and T.A.W., B.W., S.H.-R., M.T.-L., and J.W.L. wrote the paper.
The authors declare no conflict of interest.
*This Direct Submission article had a prearranged editor.1T.A.W. and B.W. contributed equally to this work.2Present address: Laboratory of Brain Development and Repair, The Rockefeller Univer-sity, New York, NY 10065.
3To whom correspondence should be addressed. E-mail: firstname.lastname@example.org.
This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.1073/pnas.1211074110/-/DCSupplemental.
estrogen receptor (Cre-ERT) transgene driven by the chickenbeta-actin-CMV hybrid (CAG) promoter, resulting in high levelsof Cre-ERT in many tissues, including retina. CAG Cre-ERT:DLKlox/lox (referred to as DLKlox:Crepos) mice survive to adult-hood with no evidence of the developmental abnormalities seenin DLK-null animals (8, 18). Dosing of DLKlox:Crepos mice at10–12 wk of age with tamoxifen (Fig. S2A) results in eliminationof the majority of DLK expression in retina (Fig. 1 C and L), withthe small amount of remaining DLK protein varying slightly fromanimal to animal (Fig. 1C and Fig. S2B). No differences in healthor behavior were observed between tamoxifen-treated DLKlox:Crepos mice and their tamoxifen-treated DLKlox:Creneg controllittermates.To examine the effect of DLK deletion on the stress response
after optic nerve crush, DLKlox:Crepos and control animals wereanalyzed 3 d after injury in retina whole mounts. c-Jun phos-phorylation and caspase 3 activation were significantly increasedafter nerve crush in control animals (compared with contralat-eral uncrushed retinas), accompanied by an increase in DLK anda decrease in Brn3-positive cells (Fig. 1 D–K). Evaluation ofDLKlox:Crepos retinas revealed attenuation of all these injury-induced changes. Up-regulation of DLK protein after crush wasabsent in all but a small fraction of GCL neurons (Fig. 1L). Thenumber of brightly p-c-Jun positive cells was also reduced to13% of that seen in littermate controls (Fig. 1 E, I, M, and P).
The persistence of some p-c-Jun labeled nuclei in DLK-deficientretinas likely reflects incomplete knockout, given that p-c-Jun-positive cell number is variable from animal to animal and levelsof p-c-Jun correlate with the amount of DLK protein remaining(Fig. S2 B and C). Brn3 expression in DLKlox:Crepos retinas wasmaintained in 84% of RGCs after injury, whereas only 13%retained expression in DLKlox:Creneg retinas (Fig. 1 F, J, N, andQ), and caspase 3 activation in DLKlox:Crepos retinas was nearlyeliminated (5% of littermate controls; Fig. 1 G, K, O, and R).Intravitreal injection of DLKlox:Creneg retinas with a low-titeradeno-associated viral vector (AAV) driving 2A peptide-medi-ated bicistronic neuronal expression of cytoplasmic GFP andcodon-improved nuclear Cre (AAV-GFP-2A-iCre) resulted insimilar attenuation of these injury-induced changes, specificallyin transduced RGCs, demonstrating that DLK acts cell-autono-mously to direct the neuronal injury response after nerve crush(Fig. 1 S–V).We next addressed whether the reduced caspase activation ob-
served at early points after optic nerve crush in DLKlox:Crepos
retinas results in persistent protection of RGC axons and cellbodies. As Brn3 is rapidly down-regulated upon injury, we evalu-ated γ-synuclein as a marker based on the specific RGC expressionobserved by in situ hybridization (19, 20). An antibody againstγ-synuclein displayed strong nuclear staining that colabeled, butwas not limited to, all Brn3-positive nuclei (Fig. S3A). Further
Fig. 1. DLK is required for c-Jun phosphorylation and RGC apoptosis after nerve crush. Staining of proximal optic nerve 24 h after sham surgery (A) or nervecrush (B) reveals a crush-dependent increase in DLK protein. (Scale bar, 100 μm.) (C) Western blot from DLKlox:Creneg and DLKlox:Crepos retinas 24 h after nervecrush from three animals of each genotype. Nerve crush results in elevated p-JNK and p-c-Jun levels in DLKlox:Creneg retinas (lanes 1–6). DLK expression issignificantly reduced in DLKlox:Crepos retinas, which attenuates the crush-induced increase in p-JNK and p-c-Jun (lanes 7–12). (D–G) Staining of whole-mountretinas from eyes with uncrushed optic nerves reveals many Brn3-positive RGCs (F) and no detectable DLK (D), p-c-Jun (E), or activated caspase 3 (G). (H–K)Retinas from DLKlox:Creneg mice 3 d after nerve crush. DLK is visible in many RGCs (H). A large number of RGCs are p-c-Jun positive (I), whereas the number ofBrn3-positive RGCs is greatly reduced (J). A small fraction of cells show activation of caspase 3 (K). (L–O) Retinas from DLKlox:Crepos mice 3 d after nerve crush.Expression of DLK is visible in a small fraction of RGCs (L). A similarly small number of RGCs are brightly p-c-Jun positive, whereas the remainder show onlyvery low levels of p-c-Jun staining (M). The number of Brn3-positive RGCs is comparable to uncrushed retinas (N), and only minimal activation of caspase 3 isobserved (O). (Scale bar, 200 μm.) (P–R) Quantification of p-c-Jun staining shown in I and M. (P; n = 3/genotype), Brn3 staining shown in F, J, and N. (Q; n = 5/genotype), and caspase 3 staining shown in K and O. (R; n = 5/genotype; all error bars, SEM; ***P < 0.001). (S–U) Low-titer transduction of DLKlox:Creneg
retinas with AAV-GFP-2A-iCre vector demonstrates cell-autonomous regulation of p-c-Jun by DLK. GFP-expressing DLK-null RGCs (arrows) exhibit greatlyreduced p-c-Jun staining (red) compared with adjacent uninfected neurons 3 d after nerve crush. (Scale bar, 50 μm.) (V) Quantification of the proportion ofAAV-GFP-2A-iCre–transduced RGCs displaying strong staining for p-c-Jun, Brn3, and activated caspase-3 3 d after nerve crush in DLKwild-type and DLKlox mice(**P < 0.01, ***P < 0.001; error bars, SEM; n = 3/genotype).
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analysis revealed a strong correlation between γ-synuclein and theneuronal nuclei antigen (NeuN) (Fig. S3A), a pan-neuronalmarker that labelsRGCs and displaced amacrine cells, whichmakeup∼50%of total GCL neurons (21). Despite the lack of specificityfor RGCs, the nuclear staining and uniform intensity enabled re-liable quantification of GCL neurons and complemented assess-ment of RGC axons with an antineurofilament antibody. UnlikeBrn3, neither of these markers displayed obvious changes instaining 3 d after nerve crush (Fig. S3B).Six weeks after optic nerve crush, a substantial reduction in the
number of neurofilament-labeled axons and a 44% reduction inGCL neurons were observed in DLKlox:Creneg retinas, whereasDLKlox:Crepos retained their axons and lost only 11% of GCLneurons (Fig. 2 A, B, D, E, G, H, and J). Brn3 was largely absentfrom DLKlox:Creneg retinas, but 65% of RGCs still maintainedexpression in DLKlox:Crepos retinas, even at this later time (Fig. 2C, F, I, and K). Consistent with this finding, DLKlox:Crepos retinasdisplayed only 3% of the p-c-Jun labeled cells seen in littermatecontrols 3 wk after nerve crush (Fig. 2 L–N), indicating that DLKdeficiency does not simply delay the injury response. Robust pro-tection of RGC axons and GCL neurons, as well as Brn3 staining,persisted even 18 wk after injury, a time by which neurofilamentlabeled axons were nearly absent in control animals (Fig. 2O and Pand Fig. S3C).To better understand the mechanisms underlying DLK-
dependent cell death, we next evaluated injury-induced geneexpression changes by microarray in whole retina from DLKlox:Crepos mice and littermate controls 3 d after nerve crush. Acuteisolation of RNA from whole retina avoids stimulation of a stressresponse during retinal dissociation yet still detects many of theexpression changes found in purified RGCs after nerve crush (1,22). In agreement with these previous studies, optic nerve injuryin DLKlox:Creneg controls induced widespread changes in theretinal expression profile, with 342 genes up- or down-regulated
(P < 0.01; Fig. 3A and Dataset S1). Unbiased pathway analysisrevealed that the most highly significant genes up-regulated byinjury were involved in biological processes enriched for termssuch as stress response, cell death, and inflammation (DatasetS2). These include the proapoptotic genes Harakiri (Hrk) and C/EBP homologous protein (CHOP), both of which are required forneuronal cell death after axonal injury (23, 24), and suggest thatmany of the genes that regulate the apoptosis of RGCs are inducedwithin 3 d of nerve crush.Interestingly, the expression-profiling data also provided evi-
dence of a proregenerative response to nerve crush in DLKlox:Creneg retinas. Among the up-regulated genes were a number thatare also induced after sciatic nerve lesion (25) and have demon-strated roles in promoting axon regeneration in other systems(Dataset S3). These include small proline-rich repeat protein 1A(Sprr1a), fibroblast growth factor-inducible 14 (Fn14), and heatshock protein beta-1 (Hspb1) (26-28), as well as the transcriptionfactors activating transcription factor 3 (ATF3) and Kruppel-likefactor 6 (Klf6) (29, 30). Gene ontology and pathway analysis ofthe most significantly down-regulated genes revealed enrichmentfor terms relevant for the maturation of neurons (Dataset S2),including Brn3a and Brn3b, which may reflect a broader patternof RGC dedifferentiation that could enhance regenerative ca-pacity (31).To identify which gene expression changes depend on DLK,
expression profiles after injury were compared between DLKlox:Crepos and littermate DLKlox:Creneg retinas. Of 342 highly sig-nificant injury-induced changes, 201 (59%) displayed significantdifferences (P < 0.05) and 151 (44%) displayed highly significantdifferences between genotypes (P < 0.01). These values may rep-resent an underestimation resulting from variance and incompleteexcision (95%–99%) of DLK (Fig. 1L and Fig. S4 A and B), asevaluation of the top 100 most unambiguous injury-inducedchanges revealed that 92 of them exhibited dependence onDLK at
Fig. 2. Loss of DLK results in sustained protection ofRGC axons and cell bodies from degeneration. (A–C)Retinas from DLKlox:Creneg mice with uncrushed op-tic nerves. Neurofilament staining labels RGC axons(A). GCL neurons (B) and Brn3-positive RGCs (C) fromthe same retina are shown in higher magnification.(D–F) Retinas from DLKlox:Creneg mice 6 wk afternerve crush. The majority of neurofilament-positiveRGC axons have degenerated at this time (D). Aconcordant reduction in the number of GCL neuronsis observed (E), and very few Brn3-positive cells arevisible (F). (G–I) Retinas from DLKlox:Crepos mice 6 wkafter nerve crush. Neurofilament-positive RGC axonsremain largely intact (G). Only a small reduction inthe number of GCL neurons is observed (H), andBrn3 expression is retained in many RGCs (I). (Scalebars, 200 μm for NF and 100 μm for Brn3 and GCL.)(J–L) Quantification of GCL neuron staining shown inE and H relative to contralateral control eye (J; n = 5/genotype), Brn3 staining shown in F and I relative tocontrol (K; n = 5/genotype), and p-c-Jun stainingshown in M and N (L; error bars, SEM; n = 3/geno-type; ***P < 0.001). (M and N) p-c-Jun staining ofretinas 3 wk after nerve crush. DLKlox:Creneg retinashave many p-c-Jun positive cells, whereas there arevery few positive cells in DLKlox:Crepos retinas. (O andP) Neurofilament staining of retinas from DLKlox:Creneg and DLKlox:Crepos mice 18 wk after nervecrush. RGC axons are almost completely degen-erated in DLKlox:Creneg but are still present in DLKlox:Crepos retinas. Most labeling in DLKlox:Creneg retinasreflects nonspecific staining of the retinal vascula-ture and a few remaining RGC axons in the lowerleft corner (n = 4 DLKlox:Creneg; n = 2 DLKlox:Crepos).
Watkins et al. PNAS | March 5, 2013 | vol. 110 | no. 10 | 4041
P < 0.05, and assessment across a range of P values yields similarresults (Datasets S1 and S3). Furthermore, hierarchical clusteringof all samples across the top 342 genes indicated that crushedDLKlox:Crepos retinas are more similar to uninjured than injuredcontrol retinas (Fig. 3A). The list of DLK-dependent expression
changes includes many genes with AP-1 transcription factor bindingsites (Dataset S3) and genes implicated in both neuronal cell deathand axon regeneration (Fig. 3B and Dataset S3).To validate these results, we performed quantitative RT-PCR
(qPCR) on independent samples for a number of prominent in-jury-induced genes. The results were consistent with those from themicroarray, with the expression of both proapoptotic [CHOP, p53upregulated modulator of apoptosis (Puma), Bcl-2 interactingmediator of cell death (Bim)] and regeneration-associated genes(ATF3, Sprr1a, Klf6) displaying clear DLK-dependence after in-jury (Fig. 3C). ForATF3,DLK-dependence was further confirmedby immunostaining of whole-mount retinas, which showed that up-regulation in DLKlox:Crepos retinas is restricted to a few scatteredRGCs (Fig. 3 D–F). Low-titer transduction of DLKlox:Creneg
RGCs with AAV-GFP-2A-iCre confirmed that injury-induced up-regulation of nuclear ATF3 is cell-autonomous (Fig. S4 C–F).The lesion-induced elevation of regeneration-associated genes
led us to investigate the extent to whichRGC axons regrow past theinjury site and whether this regeneration is dependent on DLK.Because axon regrowth after optic nerve crush is sparse (32), weused two-photon microscopy for optical sectioning through theentire depth of whole-mounted nerves to observe all of the axonspast the lesion site (Fig. S5 A and B). Maximum projections of theresulting Z-stacks revealed that RGCs mount a modest but re-producible regenerative response (Fig. 4A). To determine whetherthe DLK-mediated transcriptional response is necessary for thisregrowth, we evaluated axon regeneration 2 wk after optic nervecrush in DLKlox mice and c-Junlox mice injected intravitreally withAAV-Cre (32) (Fig. S5C). Disruption of either DLK or c-Jun re-duced the regrowth of axons by greater than 85% compared to thatobserved in littermates injected with a control AAV-GFP vector(Fig. 4 A–F). Although the regrowth of RGCs axons after crushinjury is limited, these findings show that they do initiate a re-generative response that requires both DLK and one of its down-stream transcription factors, c-Jun. Together, these results arguethat the DLK-mediated transcriptional response is necessary forthe regrowth of injured RGC axons, but it is not certain whetheractivation of c-Jun alone would be sufficient to rescue the re-generative defects observed in the absence of DLK.DLK transiently delays Wallerian degeneration of distal axons
after sciatic nerve injury (6), so we next assessed whether failureof distal axon degeneration might contribute to the lack of axonregrowth in DLK-deficient optic nerves. We evaluated axon re-generation in an nicotinamide mononucleotide adenylyltransfer-ase 1 (NMNAT1)-overexpressing transgenic mouse line in whichdistal RGC axons display strong protection 7 d after crush (Fig.S5 D and E). Despite this protection, we observed no significantdifference in regrowth past the lesion (Fig. S5 F–H).To determine whether manipulations that relieve the cell-
intrinsic limitations on axon regeneration in the CNS are able tooverride the requirement for DLK, we next evaluated regrowthafter deletion of the tumor suppressor phosphatase and tensinhomolog (PTEN), a negative regulator of the progrowth mam-malian target of rapamycin (mTOR) pathway (32). As has beenobserved in previous studies (32), enhanced axon regenerationwas enabled by AAV-Cre–mediated or AAV-GFP-2A-iCre–medi-ated knockout of PTEN in RGCs (Fig. 4D and Fig. S4I). However,this regeneration was reduced by more than 90%, but not elimi-nated, in mice harboring conditional alleles of both PTEN andDLK(Fig. 4 E and F and Fig. S4 J and K). Although differentialknockout of these genes in a small number of RGCs cannot beentirely excluded, these results suggest that deletion of PTEN mayprovide a small DLK-independent enhancement of axon growth,but that substantial regeneration of growth-enabled RGCs requiresthe DLK-mediated neuronal stress response.
Fig. 3. DLK broadly regulates the transcriptional response to axonal injury.(A) Heat map of injury-induced gene expression changes (P < 0.01) betweenuncrushed and crushed control and DLK-deficient retinas observed 3 d afteroptic nerve crush (n = 342). Groups are clustered on the basis of similarityanalysis. (B) Selected genes showing significant up- or down-regulation afternerve crush. The increases in ATF3, CHOP, Klf6, and Sprr1a expression andthe decrease in Brn3b were attenuated in DLKlox:Crepos retinas. The increasein GFAP was genotype-independent. (C) Quantitative RT-PCR of selectedexpression changes observed in microarray. Numbers represent fold increasecompared with uncrushed eyes of same genotype (error bars, SEM; n = 3/genotype; *P < 0.05, **P < 0.01). (D–F) Retinas stained for ATF3. Expressionis negligible in uncrushed retinas (D) but is significantly increased through-out the GCL after nerve crush in DLKlox:Creneg mice (E). DLKlox:Crepos retinasdisplay ATF3 in only a few scattered cells after crush (F). (Scale bar, 200 μm.)
4042 | www.pnas.org/cgi/doi/10.1073/pnas.1211074110 Watkins et al.
DiscussionIn this study, we demonstrate that DLK is required for both theproapoptotic and proregenerative responses that occur after opticnerve lesion through broad regulation of injury-induced alterationsin gene expression. Our results demonstrate that DLK is an es-sential component of the initial injury response apparatus in RGCaxons. Four lines of evidence support this conclusion: (i) DLKprotein is elevated before themajority of injury-dependent changesin RGC cell bodies; (ii) DLK is required for JNK activation, whichis known to initiate in axons (7, 8); (iii) the diversity of DLK-dependent gene expression changes is consistent with a function asa common upstream signal for multiple stress-induced pathways,as suggested by its role in enabling retrograde signaling of bothphosphorylated STAT3 and JNK after sciatic nerve lesion (9); and(iv) the persistence of Brn3 expression and absence of c-Junphosphorylation in DLK-deficient RGCs several weeks after crushsuggests that DLK cannot be circumvented by alternative signals.Taken together, these observations suggest that DLK acts at anearly stage of the injury response and is essential for nuclear de-tection of axonal damage.DLK-dependent induction of a number of proapoptotic genes
occurs within 3 d of injury, including the unfolded protein re-sponse transcription factor CHOP and the BH3-only Bcl-2 familymember Bim, both of which contribute to the death of RGCs (23,33). Despite this early priming for apoptosis, many RGCs havenot yet degenerated 3 wk after injury and remain p-c-Jun positive.These observations suggest that RGCs do not undergo a rapiddegeneration after insult but, rather, enter a prolonged period inwhich stress signals are elevated before their eventual apoptosis.The type of slow, progressive degeneration may more accuratelyreflect the mechanism of RGC loss that occurs in the context ofneurodegenerative diseases such as glaucoma, and this study andothers suggest that DLK inhibition may represent an attractivetherapeutic target in this indication (34).Injury to peripheral nerves not only promotes apoptosis but also
initiates a transcriptional program for axon regeneration in sensoryneurons, whereas regeneration is poorly activated by lesions of thecentral branches of the same axons (35). This observation, com-bined with the lack of regeneration in the CNS, has led to a modelin which CNS injury fails to robustly stimulate the intrinsic re-growth program. However, our results reveal that the DLK-mediated stress response involves a substantial proregenerativecomponent in injured RGCs, including activation of c-Jun (36).Our whole-nerve evaluation of regeneration after injury un-
covered modest but significant DLK-dependent axon regrowththat remains limited because of the barriers on CNS regeneration.Moreover, we found that DLK is required for RGC axon re-generation even in the context of PTEN deletion, indicating thatimproved mTOR signaling is not sufficient for regeneration in theabsence of this endogenous DLK-mediated stress response. To-gether these findings indicate that DLK directs a previously un-derappreciated proregenerative response after optic nerve injury,just as it does for the more familiar regeneration stimulated byperipheral nerve lesion (9) (Fig. 4J).Previous studies indicate that neuronal stress can promote cell
death in some contexts or axon regrowth in others (36, 37).However, the control of both responses in RGCs revealed by ourcurrent work demonstrates that DLK mechanistically couplesthese disparate reactions at an early stage of the injury response.It is possible that DLK may in fact coordinately prime for bothresponses across many systems, with the primary outcome de-termined by distinct features of each context that control re-generative potential. This unexpected coupling of seemingly op-posing pathways is reminiscent of cell growth regulation, in whicha number of potential oncogenes not only stimulate proliferationbut, paradoxically, also predispose cells to apoptosis. This con-nection is thought to act as a safeguard against malignancy, such
Fig. 4. DLK is required for axon regeneration after optic nerve crush. (A andB) Cholera toxin β-Alexa 594 (CTB)-labeled axons growing past the injury sitein DLKlox optic nerves 2 wk after crush. A maximum projection of a two-photon Z-series through the whole nerve reveals modest axon regrowth fromRGCs previously transduced with a control AAV-GFP vector (A), but this re-generation is reduced after DLK deletion mediated by intravitreal injection ofAAV-Cre (B). (Scale bars, 300 μm.) (C) Quantification of labeled axons thathave grown past the injury site in A and B (error bars, SEM; n = 4 for AAV-GFP, n = 6 for AAV-Cre; **P < 0.01, ***P < 0.001). (D and E) CTB-labeled RGCaxons growing past the injury site in Z-series maximum projections of c-Junlox
optic nerves. Intravitreal AAV-Cre–mediated knockout of c-Jun (E) reducesthe low level of basal regeneration observed 2 wk after crush (D). (F)Quantification of labeled axons that have grown past the injury site in D andE (error bars, SEM; n = 6 for AAV-GFP, n = 5 for AAV-Cre; **P < 0.01, ***P <0.001). (G and H) CTB-labeled regenerating axons of PTEN-deficient RGCs inthe presence or absence of DLK 2 wk after optic nerve crush. Prior intravitrealinjection of AAV-Cre enables enhanced regeneration in PTENlox mice (G) thatis reduced in PTENlox:DLKlox mice (H). Triangles mark the injury sites in thesemaximum projections of whole-nerve two-photon Z-stacks. (I) Quantificationof axons growing past the injury site in G and H (error bars, SEM; n = 4/ge-notype; **P < 0.01, ***P < 0.001). (J) Model for coordinated regulation ofapoptosis and axon regeneration by DLK. Axonal injury results in activation ofDLK, which engages a transcriptional stress response that primes for bothapoptosis and regrowth. DLK signaling combined with progrowth signalingvia PTEN deletion results in axon regeneration, whereas DLK activation aloneresults in neuronal cell death resulting from factors that limit regenerationin the CNS. Active regeneration may suppress apoptosis and thus improveneuronal survival.
Watkins et al. PNAS | March 5, 2013 | vol. 110 | no. 10 | 4043
that the survival of dividing cells is dependent on additional feed-back that affirms that the growth is appropriate for the context (38).The linking of axon regeneration and neurodegeneration by DLKmay represent a similar phenomenon in which stimulation ofgrowth is coupled to increased vulnerability to apoptosis.Given that deletion of PTEN and other manipulations that
enhance optic nerve regeneration also improve RGC survivalafter optic nerve crush (32, 39, 40), it is tempting to speculatethat apoptosis of DLK-primed neurons may in fact ultimately betriggered by inadequate or inappropriate regeneration. In thesimplest form of this model (Fig. 4J), axon injury coordinatelyprimes neurons for both regeneration and apoptosis throughactivation of DLK. In circumstances that permit regeneration(e.g., PTEN knockout), feedback from extrinsic or intrinsicsignals may serve to attenuate the proapoptotic signaling initi-ated by DLK. In cases in which regeneration fails (e.g., wild-typeoptic nerve), the absence of this feedback would lead to ex-tensive cell death over time. As in the “fail-safe” response ofmitotic cells to oncogenic signals, lack of an appropriate growthresponse results in cell death (41). The consequences of re-generation for survival in the PNS appear to further substantiate
this view. Preventing regeneration of injured peripheral nervesreduces long-term sensory neuron survival (42), whereas nerverepair that enables regeneration after transection reduces celldeath (43).Our current study reveals that the susceptibility of RGCs to
apoptosis in the weeks after axonal injury is mechanisticallycoupled through DLK to an attempt to regrow via the activationof proregenerative pathways. The necessity of DLK for the nu-clear detection of axotomy therefore makes it a master integratorof the neuronal stress response, controlling both degenerativeand regenerative reactions to axonal injury.
Materials and MethodsDetails on all procedures used in this study, including mouse lines, optic nervecrush, tissue processing, immunohistochemistry, imaging, quantification,microarray analysis, and qPCR validation can be found in the SI.
ACKNOWLEDGMENTS. T.A.W., B.W., S.H.R, Z.J., J.K., and J.W.L. are employ-ees of Genentech. We thank the Ben A. Barres laboratory at Stanford Uni-versity for assistance with the nerve crush technique and Robby Weimer,Dara Kallop, Jin-Wu Tsai, Hilda Solanoy, Joy Zuchero, and Tiffany Wu fortechnical assistance.
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