DLK initiates a transcriptional program that couples apoptotic and regenerative responses to axonal injury

Abstract

The cell intrinsic factors that determine whether a neuron regenerates 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 initiated by injury, including induction of both proapoptotic and regeneration-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 reduced, even when the tumor suppressor phosphatase and tensin homolog (PTEN) is deleted to enhance intrinsic growth potential. These findings demonstrate that these seemingly contradictory responses to injury are mechanistically coupled through a DLK-based damage detection mechanism.

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 nerve crush (B) reveals a crush-dependent increase in DLK protein. (Scale bar, 100 μm.) (C) Western blot from DLK^lox:Cre^neg and DLK^lox:Cre^pos retinas 24 h after nerve crush from three animals of each genotype. Nerve crush results in elevated p-JNK and p-c-Jun levels in DLK^lox:Cre^neg retinas (lanes 1–6). DLK expression is significantly reduced in DLK^lox:Cre^pos retinas, which attenuates the crush-induced increase in p-JNK and p-c-Jun (lanes 7–12). (D–G) Staining of whole-mount retinas 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 DLK^lox:Cre^neg 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 of Brn3-positive RGCs is greatly reduced (J). A small fraction of cells show activation of caspase 3 (K). (L–O) Retinas from DLK^lox:Cre^pos 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 only very 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 is observed (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 DLK^lox:Cre^neg retinas with AAV-GFP-2A-iCre vector demonstrates cell-autonomous regulation of p-c-Jun by DLK. GFP-expressing DLK-null RGCs (arrows) exhibit greatly reduced p-c-Jun staining (red) compared with adjacent uninfected neurons 3 d after nerve crush. (Scale bar, 50 μm.) (V) Quantification of the proportion of AAV-GFP-2A-iCre–transduced RGCs displaying strong staining for p-c-Jun, Brn3, and activated caspase-3 3 d after nerve crush in DLK^wild-type and DLK^lox mice (**P < 0.01, ***P < 0.001; error bars, SEM; n = 3/genotype).

Fig. 2.

Loss of DLK results in sustained protection of RGC axons and cell bodies from degeneration. (A–C) Retinas from DLK^lox:Cre^neg mice with uncrushed optic nerves. Neurofilament staining labels RGC axons (A). GCL neurons (B) and Brn3-positive RGCs (C) from the same retina are shown in higher magnification. (D–F) Retinas from DLK^lox:Cre^neg mice 6 wk after nerve crush. The majority of neurofilament-positive RGC axons have degenerated at this time (D). A concordant reduction in the number of GCL neurons is observed (E), and very few Brn3-positive cells are visible (F). (G–I) Retinas from DLK^lox:Cre^pos mice 6 wk after nerve crush. Neurofilament-positive RGC axons remain largely intact (G). Only a small reduction in the number of GCL neurons is observed (H), and Brn3 expression is retained in many RGCs (I). (Scale bars, 200 μm for NF and 100 μm for Brn3 and GCL.) (J–L) Quantification of GCL neuron staining shown in E and H relative to contralateral control eye (J; n = 5/genotype), Brn3 staining shown in F and I relative to control (K; n = 5/genotype), and p-c-Jun staining shown in M and N (L; error bars, SEM; n = 3/genotype; ***P < 0.001). (M and N) p-c-Jun staining of retinas 3 wk after nerve crush. DLK^lox:Cre^neg retinas have many p-c-Jun positive cells, whereas there are very few positive cells in DLK^lox:Cre^pos retinas. (O and P) Neurofilament staining of retinas from DLK^lox:Cre^neg and DLK^lox:Cre^pos mice 18 wk after nerve crush. RGC axons are almost completely degenerated in DLK^lox:Cre^neg but are still present in DLK^lox:Cre^pos retinas. Most labeling in DLK^lox:Cre^neg retinas reflects nonspecific staining of the retinal vasculature and a few remaining RGC axons in the lower left corner (n = 4 DLK^lox:Cre^neg; n = 2 DLK^lox:Cre^pos).

Fig. 3.

DLK broadly regulates the transcriptional response to axonal injury. (A) Heat map of injury-induced gene expression changes (P < 0.01) between uncrushed and crushed control and DLK-deficient retinas observed 3 d after optic nerve crush (n = 342). Groups are clustered on the basis of similarity analysis. (B) Selected genes showing significant up- or down-regulation after nerve crush. The increases in ATF3, CHOP, Klf6, and Sprr1a expression and the decrease in Brn3b were attenuated in DLK^lox:Cre^pos retinas. The increase in GFAP was genotype-independent. (C) Quantitative RT-PCR of selected expression changes observed in microarray. Numbers represent fold increase compared with uncrushed eyes of same genotype (error bars, SEM; n = 3/genotype; *P < 0.05, **P < 0.01). (D–F) Retinas stained for ATF3. Expression is negligible in uncrushed retinas (D) but is significantly increased throughout the GCL after nerve crush in DLK^lox:Cre^neg mice (E). DLK^lox:Cre^pos retinas display ATF3 in only a few scattered cells after crush (F). (Scale bar, 200 μm.)

Fig. 4.

DLK is required for axon regeneration after optic nerve crush. (A and B) Cholera toxin β-Alexa 594 (CTB)-labeled axons growing past the injury site in DLK^lox optic nerves 2 wk after crush. A maximum projection of a two-photon Z-series through the whole nerve reveals modest axon regrowth from RGCs previously transduced with a control AAV-GFP vector (A), but this regeneration is reduced after DLK deletion mediated by intravitreal injection of AAV-Cre (B). (Scale bars, 300 μm.) (C) Quantification of labeled axons that have 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 RGC axons growing past the injury site in Z-series maximum projections of c-Jun^lox optic nerves. Intravitreal AAV-Cre–mediated knockout of c-Jun (E) reduces the 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 and E (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 in the presence or absence of DLK 2 wk after optic nerve crush. Prior intravitreal injection of AAV-Cre enables enhanced regeneration in PTEN^lox mice (G) that is reduced in PTEN^lox:DLK^lox mice (H). Triangles mark the injury sites in these maximum projections of whole-nerve two-photon Z-stacks. (I) Quantification of axons growing past the injury site in G and H (error bars, SEM; n = 4/genotype; **P < 0.01, ***P < 0.001). (J) Model for coordinated regulation of apoptosis and axon regeneration by DLK. Axonal injury results in activation of DLK, which engages a transcriptional stress response that primes for both apoptosis and regrowth. DLK signaling combined with progrowth signaling via PTEN deletion results in axon regeneration, whereas DLK activation alone results in neuronal cell death resulting from factors that limit regeneration in the CNS. Active regeneration may suppress apoptosis and thus improve neuronal survival.