Vision Core Publications

Featured Core Publication:

Donahue RJ, Maes ME, Grosser JA, Nickells RW. (2019) Bax-depleted retinal ganglion cells survive and become quiescent following optic nerve damage. Mol Neurobiol. doi: 10.1007/s12035-019-01783-7

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.


Figure 2 (click on image to enlarge)

AAV2/2-Pgk-GFP-Bax transduction of RGCs to monitor BAX activity in vivo. a Tiled image of a retina that has been transduced with AAV2/2-Pgk-GFP-Bax. Side bar = 75 μm. bd The GFP-BAX transgene is expressed in cells in the RGC layer, the majority of which are positive for the RGC marker BRN3A (asterisks indicate examples of colocalization). GFP-BAX expressing cells that are not BRN3A positive (arrows) may represent displaced amacrines or some of the 15% of RGCs that do not express this marker [26]. Side bar = 10 μm. eg Immunostained sections of a retina from a control eye. e In healthy RGCs, GFP-BAX is localized throughout the cytosol. TOM20 staining shows the distribution of mitochondria in these cells. hj Following optic nerve crush, GFP-BAX translocates to the mitochondrial outer membrane, as evidenced by colocalization with TOM20 and exhibits a punctate pattern. g, j Side bars = 5 μm


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