Retinal ganglion cell survival after optic nerve injury: crosstalk among early injury responses

Abstract

Purpose : Following optic nerve injury, retinal ganglion cells (RGCs) cannot regenerate their axons and soon undergo apoptosis, precluding visual recovery. Our limited understanding of the signals that trigger RGC death after injury or disease has prevented the development of curative therapies. Recently, three phenomena have been identified that contribute strongly to RGC death after injury: elevation of mobile zinc (Zn²⁺) in the inner retina, activation of microglia and neurotoxic astrocytes, and activation of transcriptional networks via MAP3K kinase cascades (DLK and LZK). Individually inhibiting each pathway was found to be partially neuroprotective, but effects are either incomplete or transient. The objective of our studies was to investigate possible crosstalk among these three injury responses, whether they synergize to initiate RGC death, and whether combined treatments enhance neuroprotection.

Methods : Using a mouse optic nerve crush (ONC) injury model, we assessed microglial activation by qualitative scoring and qPCR. Zn²⁺-selenite autometallography was used to visualize Zn²⁺ in retinal sections. DLK activation was assessed by immunofluorescent staining of retinas and optic nerves for DLK and its downstream effector, phospho-c-JUN. Loss-of-function analysis was conducted for each signaling cascade to assess crosstalk, i.e., Zn²⁺ chelation with intraocular ZX1, systemic microglia ablation (CSF-1R inhibitor, PLX5662, oral), and conditional deletion of DLK and LZK in RGCs (DLK^fl/fl;LZK^fl/fl+AAV2-Sncg-CRE).

Results : As noted previously, Zn²⁺ was elevated in the inner retina by 6 hr and peaked at 1 day post-injury (dpi). Intraocular Zn²⁺ chelation (ZX1) at the time of ONC repressed Zn²⁺ elevation and increased RGC survival and axon regeneration. We observed a simultaneous increase in DLK signaling (via p-c-JUN) in RGCs within 1 dpi, and an increase in microglial proliferation and activation between 1 and 3 dpi. Zn²⁺ chelation partially repressed the activation of both DLK and microglia. PLX5622 treatment led to a 96% decrease in microglial density in the retina and repression of inflammatory signaling cascades but did not repress Zn²⁺ elevation or DLK accumulation.

Conclusions : Zn²⁺ chelation at the time of optic nerve injury partially prevents the activation of DLK-dependent signaling pathways and microglial activation. Thus, there is some crosstalk among the pathways that regulate RGC cell death.

This is a 2020 ARVO Annual Meeting abstract.