Retinal ganglion cell survival and axon regeneration after optic nerve injury: crosstalk among early injury signals

Abstract

Purpose : Our limited understanding of the signals that regulate retinal ganglion cell (RGC) death and axon regeneration after optic nerve injury has prevented 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/LZK). Inhibition of individual events is partially neuroprotective, but effects are incomplete, transient, or counteract axonal regenerative capacity, respectively. The objective of our studies was to investigate possible crosstalk among these signals and determine whether synergistic inhibition may enhance neuroprotection and axon regeneration.

Methods : In the mouse optic nerve injury (ONI) injury model, Zn²⁺-selenite autometallography was used to visualize Zn²⁺ in retinal sections. Microglial activation was assayed morphologically and transcriptionally after ONI. DLK/LZK signaling was assessed via downstream transcription factors (TFs), c-JUN, ATF2, MEF2A, and SOX11. Loss-of-function for each signal was utilized to assess crosstalk: reduction of retinal Zn²⁺ by intraocular chelator (ZX1) injections or genetic deletion of Zinc Transporter 3 (ZnT3: Slc30a3); systemic microglia ablation (CSF-1R inhibitor, PLX5662); and conditional deletion of DLK/LZK in RGCs (DLK^fl/fl;LZK^fl/fl).

Results : Within 1 dpi, we observed simultaneous elevation of mobile zinc (Zn²⁺), activation of DLK/LZK-dependent TFs, and increased microglial proliferation and activation in the retina. DLK/LZK activation and microglia activation persisted, peaking at 5 dpi concomitant with the onset of RGC death. ZX1 treatment or ZnT3 knockout partially repressed the activation of both DLK and microglia. PLX5622 treatment reduced microglial density in the retina by 96% and reduced expression of inflammatory markers, but did not repress Zn²⁺ elevation or DLK signaling. DLK/LZK deletion blocked injury-induced transcriptional activity to prevent RGC death and partially inhibited microglia activation.

Conclusions : Our data suggest that Zn²⁺ elevation contributes to the activation of microglia and DLK/LZK signaling after ONI. Thus, crosstalk exists between the early injury signaling pathways to regulate RGC cell death after axon injury.

This is a 2021 ARVO Annual Meeting abstract.

 

Proposed signaling schematic