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. 2017 Oct 2;12(1):71.
doi: 10.1186/s13024-017-0214-8.

Together JUN and DDIT3 (CHOP) control retinal ganglion cell death after axonal injury

Stephanie B Syc-Mazurek  1   2 , Kimberly A Fernandes  1 , Michael P Wilson  1 , Peter Shrager  3 , Richard T Libby  4   5   6
Affiliations

Together JUN and DDIT3 (CHOP) control retinal ganglion cell death after axonal injury

Stephanie B Syc-Mazurek et al. Mol Neurodegener. .

Abstract

Background: Optic nerve injury is an important pathological component in neurodegenerative diseases such as traumatic optic neuropathies and glaucoma. The molecular signaling pathway(s) critical for retinal ganglion cell (RGC) death after axonal insult, however, is/are not fully defined. RGC death after axonal injury is known to occur by BAX-dependent apoptosis. Two transcription factors JUN (the canonical target of JNK) and DDIT3 (CHOP; a key mediator of the endoplasmic reticulum stress response) are known to be important apoptotic signaling molecules after axonal injury, including in RGCs. However, neither Jun nor Ddit3 deficiency provide complete protection to RGCs after injury. Since Jun and Ddit3 are important apoptotic signaling molecules, we sought to determine if their combined deficiency might provide additive protection to RGCs after axonal injury.

Methods: To determine if DDIT3 regulated the expression of JUN after an axonal insult, mice deficient for Ddit3 were examined after optic nerve crush (ONC). In order to critically test the importance of these genes in RGC death after axonal injury, RGC survival was assessed at multiple time-points after ONC (14, 35, 60, and 120 days after injury) in Jun, Ddit3, and combined Jun/Ddit3 deficient mice. Finally, to directly assess the role of JUN and DDIT3 in axonal degeneration, compound actions potentials were recorded from Jun, Ddit3, and Jun/Ddit3 deficient mice after ONC.

Results: Single and combined deficiency of Jun and Ddit3 did not appear to alter gross retinal morphology. Ddit3 deficiency did not alter expression of JUN after axonal injury. Deletion of both Jun and Ddit3 provided significantly greater long-term protection to RGCs as compared to Jun or Ddit3 deficiency alone. Finally, despite the profound protection to RGC somas provided by the deficiency of Jun plus Ddit3, their combined loss did not lessen axonal degeneration.

Conclusions: These results suggest JUN and DDIT3 are independently regulated pro-death signaling molecules in RGCs and together account for the vast majority of apoptotic signaling in RGCs after axonal injury. Thus, JUN and DDIT3 may represent key molecular hubs that integrate upstream signaling events triggered by axonal injury with downstream transcriptional events that ultimately culminate in RGC apoptosis.

Keywords: Axonopathy; Endoplasmic reticulum stress; Mitogen-activated protein kinase; Neurodegeneration; Retinal ganglion cell.

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Conflict of interest statement

Ethics approval and consent to participate

All experiments were conducted in adherence with the Association for Research in Vision and Ophthalmology’s statement on the use of animals in ophthalmic and vision research and were approved by the University of Rochester’s University Committee on Animal Resources.

Consent for publication

Not applicable.

Competing interests

The authors declare that they have no competing interests.

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Figures

Fig. 1
Fig. 1
Jun and Ddit3 deficiency does not alter retinal morphology. a Semi-thin retinal cross sections were taken to evaluate the gross structure of the retina in WT, Jun, Ddit3, and Jun/Ddit3 deficient animals. Jun, Ddit3, and combined Jun/Ddit3 deficiency did not appear to alter gross retinal structure. b TUJ1+ cells (a marker of RGCs) were counted in retinal flat mounts in WT, Jun, Ddit3, Jun/Ddit3 deficient animals. No difference was observed across genotypes (P > 0.05 for each comparison; n = 8 per group). c/d JUN accumulation was readily detected in the somas of RGCs of wildtype animals after ONC but was not easily detected in sham retinas. To determine the efficiency of Jun recombination using Six3-cre, the number of JUN+ cells were counted 1 day after controlled optic nerve ONC, a time when JUN is widely expressed prior to RGC death. JUN+ cells are reduced by 77.7% in Jun deficient retinas after ONC (error bars represent standard error of the mean; n = 6 per genotype; P < 0.001; scale bar 50 μm). e/f Western blot analysis was used to determine the level of JUN in wildtype (+/+) and Jun deficient (−/−) animals. JUN was significantly reduced in both Jun deficient unmanipulated retinas (85%, ONC -) and Jun deficient retinas after ONC (92%, ONH +) as compared to wild type animals (* p < 0.01)
Fig. 2
Fig. 2
Jun and Ddit3 are independently regulated after optic nerve injury. To determine if Ddit3 regulates JUN and pJUN expression, retinal flat mounts (RGC side up) were examined from WT and Ddit3 deficient retinas one day after ONC. a JUN (red) accumulates in RGCs (labeled with TUJ1, a marker of RGCs, green) in WT and Ddit3 deficient retinas one day after ONC, but not in sham retinas (n = 4 per genotype and experimental condition). There was no significance difference (ns) in the number of JUN positive RGCs between wildtype and Ddit3 deficient retinas. b Similarly, pJUN (red) accumulates in RGCs (green) in WT and Ddit3 deficient retinas one day after ONC. There was no significance difference (ns) in the number of pJUN positive RGCs between wildtype and Ddit3 deficient retinas. c Western blot analysis was used to determine the level of JUN in wildtype (+/+) and Ddit3 deficient (−/−) animals. There was no significant difference in JUN levels between Ddit3 deficient and wildtype unmanipulated eyes (ONC -) or between Ddit3 deficient and wildtype eyes after ONC (ONC +). Previously, DDIT3 has been shown to be expressed in Jun deficient retinas after ONC [23]. Scale bar: 50 μm
Fig. 3
Fig. 3
Combined Jun/Ddit3 deficiency is more protective after axonal injury than either Jun or Ddit3 deficiency alone. a Example of TUJ1 staining in control and experimental eyes at 120 days after ONC (scale bar = 50 μm). b Ddit3, Jun, and combined Jun and Ddit3 deficient animals had significantly greater RGC survival than WT animals at all time points assessed (p < 0.001). Furthermore, there were significant differences (P < 0.001) found between the Ddit3, Jun, and combined Jun and Ddit3 groups at all time points evaluated expect for between the Jun, and combined Jun and Ddit3 14 days after ONC. Data are plotted as the percentage survival relative to sham animals (n = 8 per condition per genotype for 14, 35, and 60 days after ONC, except Jun deficient ONC n = 7 at 35 days after ONC and n ≥ 6 per condition per genotype for 120 days after ONC; error bars represent SEM). Raw data in RGCs per mm2 and P values for all comparisons are presented in Table 1
Fig. 4
Fig. 4
Combined Jun/Ddit3 deficiency is more protective after axonal injury than Dlk deficiency. a Example images of TUJ1 staining in control and experimental eyes 35 days after ONC (scale bar = 50 μm). b TUJ1+ cell counts showed that Dlk deficient and combined Jun/Ddit3 deficient animals had significantly greater RGC survival than WT mice 35 days after ONC (% given compared to control eyes of same genotype, WT: 20.4% survival, Dlk: 64.3% Jun/Ddit3: 88.5%; *, P < 0.001, n = 8 per condition per genotype except n = 7 for Dlk deficient animals; error bars represent SEM). Importantly, the protection provided by Jun/Ddit3 deficiency was significantly greater than the protection provided by Dlk deficiency alone (P < 0.001)
Fig. 5
Fig. 5
Neither Jun, Ddit3, or combined Jun/Ddit3 deficiency alters axonal degeneration after mechanical optic nerve injury. To assess the role of JUN and DDIT3 in axonal degeneration, compound action potentials (CAPs) were recorded from WT, Jun, Ddit3, and combined Jun/Ddit3 animals 5 days after ONC, a time point when there is significant loss of CAP amplitudes in WT mice [43]. a Representative traces show that sham eyes from all genotypes had normal action potentials, while amplitudes were reduced about 80% in all cases after ONC. b Quantification of CAPs from WT, Jun, Ddit3, and combined Jun/Ddit3 animals showed that there were no differences among the CAP amplitudes of naïve, uninjured eyes of all genotypes (P > 0.05). All genotypes had significantly reduced amplitudes after ONC as compared to naïve animals (P < 0.001 for all comparisons). CAP amplitudes of Jun, Ddit3, and combined Jun/Ddit3 animals were not significantly different after ONC from those of control animals (P > 0.05, n = 4 for each genotype and condition)
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