July 2018
Volume 59, Issue 9
Open Access
ARVO Annual Meeting Abstract  |   July 2018
Amacrine cells control retinal Zn2+ levels, ganglion cell survival, and axon regeneration after optic nerve injury via two opposing effects of nitric oxide
Author Affiliations & Notes
  • Yiqing Li
    Departments of Neurosurgery and Neurology and F.M. Kirby Neurobiology Center, Boston Children’s Hospital and Harvard Medical School, Boston, Massachusetts, United States
    State Key Laboratory of Ophthalmology, Zhongshan Ophthalmic Center, Sun Yat-sen University, Guangzhou, Guangdong, China
  • Kenya Yuki
    Departments of Neurosurgery and Neurology and F.M. Kirby Neurobiology Center, Boston Children’s Hospital and Harvard Medical School, Boston, Massachusetts, United States
  • Kumiko Omura
    Departments of Neurosurgery and Neurology and F.M. Kirby Neurobiology Center, Boston Children’s Hospital and Harvard Medical School, Boston, Massachusetts, United States
  • Hui-ya Gilbert
    Departments of Neurosurgery and Neurology and F.M. Kirby Neurobiology Center, Boston Children’s Hospital and Harvard Medical School, Boston, Massachusetts, United States
  • Yuqin Yin
    Departments of Neurosurgery and Neurology and F.M. Kirby Neurobiology Center, Boston Children’s Hospital and Harvard Medical School, Boston, Massachusetts, United States
  • Silmara de Lima
    Departments of Neurosurgery and Neurology and F.M. Kirby Neurobiology Center, Boston Children’s Hospital and Harvard Medical School, Boston, Massachusetts, United States
  • Melody Walter
    Department of Chemistry, MIT, Cambridge, Massachusetts, United States
  • Paul Huang
    Department of Medicine, MGH and Harvard Medical School, Boston, Massachusetts, United States
  • Stephen Lippard
    Department of Chemistry, MIT, Cambridge, Massachusetts, United States
  • Yehong Zhuo
    State Key Laboratory of Ophthalmology, Zhongshan Ophthalmic Center, Sun Yat-sen University, Guangzhou, Guangdong, China
  • Paul A. Rosenberg
    Departments of Neurosurgery and Neurology and F.M. Kirby Neurobiology Center, Boston Children’s Hospital and Harvard Medical School, Boston, Massachusetts, United States
  • Larry I. Benowitz
    Departments of Neurosurgery and Neurology and F.M. Kirby Neurobiology Center, Boston Children’s Hospital and Harvard Medical School, Boston, Massachusetts, United States
  • Footnotes
    Commercial Relationships   Yiqing Li, None; Kenya Yuki, None; Kumiko Omura, None; Hui-ya Gilbert, None; Yuqin Yin, None; Silmara de Lima, None; Melody Walter, None; Paul Huang, None; Stephen Lippard, None; Yehong Zhuo, None; Paul Rosenberg, None; Larry Benowitz, None
  • Footnotes
    Support  We are grateful for the support of the NIH (EY024481 to P.A.R. and L.B.; NS066019 to P.A.R.; MH104318 to P.A.R.; GM 065519 to S.J.L), U.S. Department of Defense/CDMRP (DM102446 to L.B.), Dr. Miriam and Sheldon G. Adelson Medical Research Foundation (to L.B.), and the China Scholarship Council 2010638086 (Y.L.).
Investigative Ophthalmology & Visual Science July 2018, Vol.59, 2012. doi:
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      Yiqing Li, Kenya Yuki, Kumiko Omura, Hui-ya Gilbert, Yuqin Yin, Silmara de Lima, Melody Walter, Paul Huang, Stephen Lippard, Yehong Zhuo, Paul A. Rosenberg, Larry I. Benowitz; Amacrine cells control retinal Zn2+ levels, ganglion cell survival, and axon regeneration after optic nerve injury via two opposing effects of nitric oxide. Invest. Ophthalmol. Vis. Sci. 2018;59(9):2012.

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      © ARVO (1962-2015); The Authors (2016-present)

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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;nos1flx/flx, VGLUT2-Cre;nos1flx/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;nos1flx/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: nos1flx/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;nos1flx/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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