Amacrine cells control retinal Zn²⁺ levels, ganglion cell survival, and axon regeneration after optic nerve injury via two opposing effects of nitric oxide

Abstract

Purpose : Mobile zinc (Zn²⁺) contributes to the death of retinal ganglion cells (RGCs) after optic nerve injury (ONI) and to RGCs’ inability to regenerate axons (Li et al. PNAS 2017). Zn²⁺ accumulates in amacrine cell (AC) terminals within an hour of ONI and is then slowly exocytosed and accumulates in RGCs. Chelating Zn²⁺ provides long-term protection to RGCs and considerable axon regeneration. Here we examined the role of nitric oxide (NO) generated by a small group of ACs on Zn²⁺ elevation, RGC survival and optic nerve regeneration.

Methods : ONI and/or intraocular injections were performed on 8 week old male C57 wild-type (wt) mice or mice with a general, or AC-specific, or RGC-specific deletion of nos1 (nos1^-/-, VGAT-Cre;nos1^flx/flx, VGLUT2-Cre;nos1^flx/flx respectively; founders from JAX, N ≥ 6/group). Zn²⁺ was detected with autometallography (AMG). NO was detected with the novel fluorescent sensor Cu₂FL2. Other agents included the Zn²⁺ chelator TPEN, nNOS-selective inhibitor L-NPA, protein kinase G inhibitor Rp-8-Br-PET-cGMPS (all 3 at 100 μM); the NO donor DETA-NONOate (0.1-100 μM) and NO scavenger PTIO (300 μM). Cell-type specific and synaptic markers were visualized by immunohistochemistry.

Results : Zn²⁺ levels rose continuously in the inner plexiform layer (IPL) 1-24 hours post-NC and in RGCs at 2-3 days. Zn²⁺ was nearly eliminated in nos1^-/- and VGAT-Cre;nos1^flx/flx mice (P < 0.001, Fig. 1a-c) and in wt mice treated with PTIO or L-NPA. Conversely, DETA-NONOate increased retinal Zn²⁺ even without NC. NOS1is only expressed in a small population of ACs, and was absent in VGAT-Cre: nos1^flx/flx mice (Fig. 1d). Following ONI, retinal NO increased in ≤ 1 h and remained elevated for ~ 3 days (both P < 0.02, Fig. 2a,b). The validity of the signal was confirmed by its suppression by L-NPA and in VGAT-Cre;nos1^flx/flx mice (Fig. 2a,b). Surprisingly, eliminating NOS1 in ACs (but not RGCs) eliminated the regeneration that was otherwise seen with Zn²⁺ chelation (Fig. 2c). Conversely, DETA-NONOate (10 μM) promoted optic nerve regeneration (P <0.01), and this effect was eliminated with Rp-8-Br-PET-cGMPS (P <0.001, Fig. 2d).

Conclusions : NO generated in a small group of NOS1-positive ACs leads to the deleterious elevation of Zn²⁺ after optic nerve injury but also exerts positive effects on optic nerve regeneration via cGMP signaling.

This is an abstract that was submitted for the 2018 ARVO Annual Meeting, held in Honolulu, Hawaii, April 29 - May 3, 2018.

 

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