BAX is a proapoptotic protein, which is critical for the execution of intrinsic apoptosis in retinal ganglion cells (RGCs) after optic nerve damage, suggesting that targeting BAX may be an effective therapeutic strategy for neuroprotection. It has been reported, however, that reducing BAX levels only provides a transient protection to axons in a mouse model of glaucoma. Whether or not this transient protection is sufficient to allow axons to recover after elimination of the original stressor, has not been tested.
Aim 1 will test the protective effect of BAX reduction on the ability of axons to survive and/or recover in models that mimic the IOP-lowering therapy experienced by patients with ocular hypertension (OHT). Two models of inducible OHT in mice, microbead injection and steroid induced OHT, will be employed on both wild type and Bax+/- mice (where reduced BAX more likely mimics a human treatment). In both models IOP levels can be returned to normal after a desired interval. We will test if reducing BAX protein levels both reduces soma and axon pathology, and either protects axon function, or allows functional recovery. We will also examine the effects of controlled experimental IOP (CEI). The advantage of CEI is that it normalizes the IOP insult, which eliminates the confounding factors of variable IOP associated with other models of OHT. Dorsal Root Ganglion cells contain a BAX-dependent degenerative pathway that is activated early and before other more well-characterized catabolic reactions involving calcium influx. This pathway is activated by a BH3- only protein produced in the cell soma and then transported to the axon. A similar mechanism in RGC axons may explain the partial protective effect of BAX reduction.
Aim 2 will evaluate the presence of this pathway in RGC axons where degeneration will be induced in optic nerves ex vivo. We will also interrogate axons for the presence and localization of key molecules that activate BAX, and molecules that act down-stream of BAX activation. Preliminary studies suggest that the BH3-only protein NOXA may be an important regulator of BAX activation in axons. We will explore this in detail using Noxa-deficient mice subjected to induced OHT. Protected RGCs silence transcription of genes required for function. New studies show that the committed process of dying in Bax-deficient cells, is shut down after 8 weeks. Thus cells can be classified into two categories, those actively dying (AD-mode) and those that have gone quiescent (Q-mode).
Aim 3 will investigate if cells in Q-mode respond differently to 3 attempts to reactivate them, including using HDAC inhibitors to attenuate the transcriptional silencing of RGC-specific gene expression, the addition of Zymosan and CPT-cAMP to induce regeneration, or the up-regulation of transcription factors that can specify retinal precursor cells as RGCs. These experiments will be conducted on RGCs after acute damage to the optic nerve, which serves as a model where regeneration has a high priority for a therapeutic outcome.
Optic neuropathies, including glaucoma, optic neuritis, and optic nerve trauma combined, present a serious health burden to a growing aged population. While there are therapies for glaucoma that are targeted at reducing the damaging effects of ocular hypertension (the principal risk factor for this disease), a protective therapy for optic nerve damage and retinal ganglion cell loss is lacking. Additionally, there is no recourse for individuals suffering from acute optic nerve trauma. A promising therapeutic target exists, however, in blocking the function of the pro-death protein BAX. Blocking BAX function completely abrogates ganglion cell death, and provides substantial protection to the optic nerve. There are many gaps in our knowledge base, however, that prevent us from moving forward with the development of a viable therapy targeting BAX. Not the least of which is a lack of understanding how BAX functions in the process of optic nerve degeneration, or if inhibiting BAX will allow nerves to recover function in cases where ocular hypertension is relieved. The experiments in this proposal aim to fill some of these knowledge gaps, which is a precursor to developing a BAX therapeutic. We will test if BAX-depletion in mice, makes the optic nerve more resistant to ocular hypertension in models that more closely mimic how a glaucoma patient is treated - by lowering intraocular pressure. We will also investigate the molecular mechanism by which reduced BAX levels conveys a transient protective effect to axons in the optic nerve. Lastly, building from a new discovery in the lab, we will test if BAX-depleted ganglion cells have a greater potential to regain function when they enter a phase where they stop directing cellular resources to executing the apoptotic death pathway.
