BAX-Depleted Retinal Ganglion Cells Survive and Become Quiescent Following Optic Nerve Damage
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.
KeywordsBAX Retinal ganglion cells Optic nerve crush Intrinsic apoptosis cJun Glia Neuroprotection
retinal ganglion cell
optic nerve crush
mitochondrial outer membrane
mitochondrial outer membrane permeabilization
BCL2-homology domain, 3
BCL2 associated X, apoptosis regulator
BAX activation mechanism
c-JUN N-terminal kinase
quiescent retinal ganglion cell
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.
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.
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
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.
The authors declare that they have no competing interests.
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