June 2017
Volume 58, Issue 8
Open Access
ARVO Annual Meeting Abstract  |   June 2017
ERK1/2 Induces RGC Degeneration via Necroptosis Following Optic Nerve Axotomy and Crush
Author Affiliations & Notes
  • Philippe M D'Onofrio
    Department of Rehabilitation Science, University of Toronto, Toronto, Ontario, Canada
    Surgery, University of Toronto, Toronto, Ontario, Canada
  • Brian Choi
    Department of Rehabilitation Science, University of Toronto, Toronto, Ontario, Canada
    Surgery, University of Toronto, Toronto, Ontario, Canada
  • Paulo D Koeberle
    Department of Rehabilitation Science, University of Toronto, Toronto, Ontario, Canada
    Surgery, University of Toronto, Toronto, Ontario, Canada
  • Footnotes
    Commercial Relationships   Philippe D'Onofrio, None; Brian Choi, None; Paulo Koeberle, None
  • Footnotes
    Support  CIHR 119309
Investigative Ophthalmology & Visual Science June 2017, Vol.58, 1757. doi:
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      Philippe M D'Onofrio, Brian Choi, Paulo D Koeberle; ERK1/2 Induces RGC Degeneration via Necroptosis Following Optic Nerve Axotomy and Crush. Invest. Ophthalmol. Vis. Sci. 2017;58(8):1757.

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

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Abstract

Purpose : Extracellular signal-related kinases (ERKs) 1 and 2 are highly expressed in the brain and are involved in apoptosis signaling. However, a possible role in necroptosis remains poorly understood. We tested the hypothesis that ERK1/2 is implicated in RGC necroptosis via interactions with receptor interacting protein kinase 3 (RIP3), and that inhibition of ERK1/2 will block necroptotic cell death. This was assessed in vitro in N1E-115 cells and in vivo in retinal ganglion cells (RGCs) using the optic nerve transection model.

Methods : N1E-115 cell death was induced by ODQ toxicity (50μM/100 μM), whereas adult female Sprague-Dawley rats (N=4/treatment) were used for optic nerve crush and transection procedures. In vivo treatments were administered via intra-ocular injection into the vitreous chamber, and results were asessed 14d after axotomy and 21d after crush. Protein levels and interactions were quantified by western blot and immunoprecipitation; localization was assessed via immunohistochemistry on cryosectioned tissue. RGC survival was quantified by retinal flat-mounting followed by immunohistochemistry and visual counting of surviving cells. ANOVA followed by Tukey’s post-hoc test was used for statistical analysis.

Results : Administration of RIP1 inhibitor increased the number of RGCs post-axotomy (1100 RGCs/mm2) and increased the number of axons extending beyond the damage site post-optic nerve crush (2-fold). RIP3 inhibitor significantly increased the number of RGCs post-axotomy (900 RGCs/mm2). ERK1/2 activation was maximal between 12 hours and 2 days post-axotomy and co-immunoprecipitation of ERK1/2 demonstrated RIP3 interaction, suggesting a role in necroptosis initiation. Blocking the ERK1/2 - RIP3 binding site increased survival in vitro and in vivo (1000 RGCs/mm2). Additionally, levels of p-MLKL, a crucial signaling molecule in necroptosis progression, were reduced in the ganglion cell layer following ERK1/2 inhibition.

Conclusions : Our results indicate that ERK1/2 plays a role in the initiation of necroptosis via the activation of RIP3 and leading to MLKL activation, an essential step in necroptosis progression. This supports the hypothesis of necroptosis as an alternative mechanism to apoptosis in RGCs following optic nerve transection and crush damage. It also suggests that it may be an alternative mechanism in other forms of RGC, as well as CNS, neuron death.

This is an abstract that was submitted for the 2017 ARVO Annual Meeting, held in Baltimore, MD, May 7-11, 2017.

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