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BAX-Depleted Retinal Ganglion Cells Survive and Become Quiescent Following Optic Nerve Damage

  • Ryan J. Donahue
  • Margaret E. Maes
  • Joshua A. Grosser
  • Robert W. NickellsEmail author
Article

Abstract

Removal of the Bax gene from mice completely protects the somas of retinal ganglion cells (RGCs) from apoptosis following optic nerve injury. This makes BAX a promising therapeutic target to prevent neurodegeneration. In this study, Bax+/− mice were used to test the hypothesis that lowering the quantity of BAX in RGCs would delay apoptosis following optic nerve injury. RGCs were damaged by performing optic nerve crush (ONC) and then immunostaining for phospho-cJUN, and quantitative PCR were used to monitor the status of the BAX activation mechanism in the months following injury. The apoptotic susceptibility of injured cells was directly tested by virally introducing GFP-BAX into Bax−/− RGCs after injury. The competency of quiescent RGCs to reactivate their BAX activation mechanism was tested by intravitreal injection of the JNK pathway agonist, anisomycin. Twenty-four weeks after ONC, Bax+/− mice had significantly less cell loss in their RGC layer than Bax+/+ mice 3 weeks after ONC. Bax+/− and Bax+/+ RGCs exhibited similar patterns of nuclear phospho-cJUN accumulation immediately after ONC, which persisted in Bax+/− RGCs for up to 7 weeks before abating. The transcriptional activation of BAX-activating genes was similar in Bax+/− and Bax+/+ RGCs following ONC. Intriguingly, cells deactivated their BAX activation mechanism between 7 and 12 weeks after crush. Introduction of GFP-BAX into Bax−/− cells at 4 weeks after ONC showed that these cells had a nearly normal capacity to activate this protein, but this capacity was lost 8 weeks after crush. Collectively, these data suggest that 8–12 weeks after crush, damaged cells no longer displayed increased susceptibility to BAX activation relative to their naïve counterparts. In this same timeframe, retinal glial activation and the signaling of the pro-apoptotic JNK pathway also abated. Quiescent RGCs did not show a timely reactivation of their JNK pathway following intravitreal injection with anisomycin. These findings demonstrate that lowering the quantity of BAX in RGCs is neuroprotective after acute injury. Damaged RGCs enter a quiescent state months after injury and are no longer responsive to an apoptotic stimulus. Quiescent RGCs will require rejuvenation to reacquire functionality.

Keywords

BAX Retinal ganglion cells Optic nerve crush Intrinsic apoptosis cJun Glia Neuroprotection 

Abbreviations

RGC

retinal ganglion cell

ONC

optic nerve crush

MOM

mitochondrial outer membrane

MOMP

mitochondrial outer membrane permeabilization

BH3

BCL2-homology domain, 3

BAX

BCL2 associated X, apoptosis regulator

BAM

BAX activation mechanism

JNK

c-JUN N-terminal kinase

DAPI

4′,6-diamidino-2-phenylindole

qRGC

quiescent retinal ganglion cell

Notes

Acknowledgements

We would like to thank Satoshi Kinoshita at the Translational Research Initiative in Pathology (TRIP) lab at the University of Wisconsin–Madison for cutting all retinal sections analyzed in this manuscript.

Author’s Contributions

RWN conceived the study. RJD and MEM contributed intellectually to the study design. JAG imaged and counted whole-mounted retinas. MEM conducted some of the viral injections, immunofluorescence, and imaging pertaining to GFP-BAX colocalization with TOM20. RJD performed all other experiments including all surgical procedures, immunofluorescence, qPCR, and counting of whole-mounted retinas and retinal sections. RWN prepared all the figures for the manuscript and processed images for figures. RJD and RWN wrote the manuscript. All authors contributed to editing the manuscript and approved the final manuscript.

Funding Information

This work was supported by National Eye Institute grants R01 EY012223 (RWN), R01 EY030123 (RWN), T32 EY027721 (Department of Ophthalmology and Visual Sciences, University of Wisconsin-Madison), and a Vision Science Core grant P30 EY016665 (Department of Ophthalmology and Visual Sciences, University of Wisconsin-Madison), an unrestricted funding grant from Research to Prevent Blindness (Department of Ophthalmology and Visual Sciences, University of Wisconsin-Madison), the Frederick A. Davis Endowment (RWN), and the Mr. and Mrs. George Taylor Foundation (RWN).

Compliance with Ethical Standards

Ethics Approval

Adult mice were used for this study and handled according to the Association for Research in Vision and Ophthalmology statement for the use of animals in research and all experiments were approved by the Animal Care and Use Committee at the University of Wisconsin–Madison.

Competing Interests

The authors declare that they have no competing interests.

Supplementary material

12035_2019_1783_MOESM1_ESM.jpg (759 kb)
Supplemental Figure 1 All qPCR data from Bax+/+ mice after optic nerve crush. qPCR data for wild type animals 1, 3 and 7 days after optic nerve crush (ONC). Bars represent the fold change in the retinas of experimental eyes relative to the contralateral control eyes. The data for the groups collected 1 and 3 days after ONC are the average of 2 cohorts of 3 mice each, whose eyes were pooled for analysis. The data for the group collected 7 days after ONC is the average of 4 cohorts of 3 mice each, whose eyes were pooled for analysis. Error bars represent the standard deviation from the mean. P values were calculated using a one sided t test, assuming equal variance. *P < 0.05, **P < 0.01, ***P < 0.001. (JPG 758 kb)
12035_2019_1783_MOESM2_ESM.jpg (567 kb)
Supplemental Figure 2 The average number of cells labeled per field in each cohort of mice that was transduced with AAV2/2-Pgk-GFP-Bax. Bars represent the mean number of GFP-BAX positive cells labeled in each image for each group. Error bars represent the standard deviation from the mean. P values were calculated using a one-sided test, assuming equal variance between groups. Number of mice per group is as follows: 10 for Bax+/+ transduced before ONC, 5 for Bax+/− transduced before ONC, 9 for Bax−/− transduced before ONC, 7 for Bax−/− transduced 4 weeks after ONC and 7 for Bax−/− transduced 8 weeks after ONC. *P < 0.05, **P < 0.01, ***P < 0.001. (JPG 567 kb)

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Copyright information

© Springer Science+Business Media, LLC, part of Springer Nature 2019

Authors and Affiliations

  1. 1.Department of Ophthalmology and Visual SciencesUniversity of WisconsinMadisonUSA
  2. 2.Cellular and Molecular Pathology Graduate ProgramUniversity of WisconsinMadisonUSA
  3. 3.Department of Life SciencesInstitute of Science and TechnologyKlosterneuburgAustria

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