The retina and optic nerve have been widely studied for insights into factors that suppress or promote cell survival and axon regeneration after CNS injury. Following injury to the optic nerve, retinal ganglion cells (RGCs), the pro- jection neurons of the eye, cannot regenerate their axons and begin to die after a few days. Despite the development of treatments that improve RGC survival and/or axon regeneration, levels of visual recovery achieved to date remain modest, underscoring the need to better understand the mechanisms that produce cell death and regenerative failure. We recently reported (Li et al., PNAS, 2017, ref. 1) that optic nerve injury leads to a rapid elevation of mobile/free zinc (Zn2+) in synaptic terminals of amacrine cells, followed by exocytosis and Zn2+ accumulation within RGCs; and that Zn2+ chelation leads to long-term survival of many RGCs and considerable axon regeneration. Our preliminary data indicate that the mechanisms underlying Zn2+ elevation involve a previously unknown, multi-cellular network that utilizes non-classical signaling mechanisms and that ultimately determines the fate of RGCs. This network ap- pears to involve phosphorylation of a K+ channel in RGCs by a signal conveyed up the injured axons; retrograde sig- naling between injured RGCs and interneurons (or Muller glia) via elevation of extracellular K+, causing reversal of the glutamate transporter GLT-1 and glutamate efflux; this in turn activates NMDA receptors, leading to Ca2+ entry, activation of neuronal nitric oxide (NO) synthase-1 (NOS1), and NO-mediated liberation of Zn2+ from metal- lothionein(s). This sequence is based on preliminary results using pharmacological inhibitors and immunohistochem- istry, but precise knowledge of the specific cell types and signals involved remains to be established. Based on the observation that NOS1- mediated NO generation lies directly upstream of Zn2+ liberation, Aims 1 and 2 will work back from this point to identify the cellular populations and signals that link optic nerve damage to RGC death.
Aim 1 will test the hypothesis that glutamate efflux from bipolar or Mueller cells (via reversal of the glutamate transporter GLT-1) and activation of NMDA receptors on NOS1-positive amacrine cells lie directly upstream of NO generation.
Aim 2 will test the hypothesis that the further upstream steps involve activation of a MAP kinase cascade and/or Ca2+ signaling in injured RGCs, leading to phosphorylation and activation of potassium channels (and possibly other channels) in RGCs, causing an elevation of extracellular K+ that leads to a reversal of the normal operation of glu- tamate transporters in bipolar or Mueller cells.
Aim 3 will test the hypothesis that this pathway contributes to RGC death in a mouse model of glaucoma. In keeping with NIH guidelines, the proposed studies will use both male and female mice in a key experiment to determine whether sex differences exist in the signaling pathway we have un- covered that might provide further insights into the mechanisms underlying cell death and regenerative failure. These studies will define a novel multi-cellular signaling network in the retina that regulates the viability and regenerative capacity of RGCs after optic nerve injury and perhaps in glaucoma.
Retinal ganglion cells (RGCs), the neurons that convey information from the eye to the brain, cannot regenerate their axons and soon begin to die if the optic nerve is injured, precluding visual recovery after traumatic or ischemic optic nerve damage or in glaucoma. The proposed studies will define a previously unrecognized, retrograde signaling pathway between RGCs and cells in the inner plexiform layer of the retina (amacrine cells, bipolar cells, Mueller cells) that ultimately controls the fate of injured RGCs. These studies will provide insights that may enable us to improve the survival of RGCs and their capacity to regenerate axons.