April 2014
Volume 55, Issue 13
Free
ARVO Annual Meeting Abstract  |   April 2014
Direct and indirect mechanisms of RGC neuroprotection and axon regeneration induced by siRNA-mediated knockdown of the mTOR regulator RTP801 after optic nerve crush
Author Affiliations & Notes
  • Peter J Morgan-Warren
    Neurotrauma & Neurodegeneration, University of Birmingham, Birmingham, United Kingdom
  • Jenna ONeill
    Neurotrauma & Neurodegeneration, University of Birmingham, Birmingham, United Kingdom
  • Zubair Ahmed
    Neurotrauma & Neurodegeneration, University of Birmingham, Birmingham, United Kingdom
  • Martin Berry
    Neurotrauma & Neurodegeneration, University of Birmingham, Birmingham, United Kingdom
  • Robert A H Scott
    Neurotrauma & Neurodegeneration, University of Birmingham, Birmingham, United Kingdom
    Royal Centre for Defence Medicine, Birmingham, United Kingdom
  • Ann Logan
    Neurotrauma & Neurodegeneration, University of Birmingham, Birmingham, United Kingdom
  • Footnotes
    Commercial Relationships Peter Morgan-Warren, None; Jenna ONeill, None; Zubair Ahmed, None; Martin Berry, None; Robert A H Scott, None; Ann Logan, Quark Pharmaceuticals (F)
  • Footnotes
    Support None
Investigative Ophthalmology & Visual Science April 2014, Vol.55, 5734. doi:https://doi.org/
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      Peter J Morgan-Warren, Jenna ONeill, Zubair Ahmed, Martin Berry, Robert A H Scott, Ann Logan; Direct and indirect mechanisms of RGC neuroprotection and axon regeneration induced by siRNA-mediated knockdown of the mTOR regulator RTP801 after optic nerve crush. Invest. Ophthalmol. Vis. Sci. 2014;55(13):5734. doi: https://doi.org/.

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

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Abstract

Purpose: The mammalian Target of Rapamycin (mTOR) is implicated as a determinant of neuronal survival and axon regeneration after optic nerve (ON) injury. RTP801 is a negative regulator of mTOR signalling, activated by cellular stress and hypoxia. We investigated the effects of RTP801 knockdown with a targeted siRNA, siRTP801 (PF-04523655), on retinal ganglion cell (RGC) survival, axon regeneration, mTOR activity and glial activation after optic nerve crush (ONC).

Methods: Adult rats underwent bilateral ONC followed by intravitreal injections of siRTP801 (right eye) and control siRNA (siEGFP, left eye) on days 0, 8 and 16 post-lesion, and tissues harvested on day 24. Retinal cultures were obtained from intact rats, transfected with siRTP801 or siEGFP in the presence or absence of rapamycin, and incubated for 3 days. Retinal and ON sections, and cultured cells were labelled for RGC and glial markers, regenerating axons, and phospho-S6 (pS6).

Results: siRTP801 treatment was RGC neuroprotective compared to control after ONC (16.9±0.6 vs 9.2±0.6 RGC/250µm linear sample, p<0.001) and after transfection of retinal cells in vitro (577±17 vs 302±14 RGC/well, p<0.001). RTP801 knockdown increased the number of GAP43+ regenerating axons in the distal ON up to 1200µm beyond the lesion (55.8±11.5 vs 15.4±9.9, p=0.002), but did not significantly increase neuritogenesis in surviving RGC in vitro (27.5±3.4 vs 21.4±4.1 %RGC with neurites, p=0.192). siRTP801 prevented the ONC-induced downregulation of RGC pS6 expression (intact 15.4±1.7, siRTP801 13.7±1.4, siEGFP 5.9±0.5% pS6+ RGC, p=0.001), although most surviving RGC were pS6-, and enhanced glial activation after ONC (14.9±0.7 vs 10.2±0.5 GFAP+ processes/250µm inner plexiform layer, p<0.001). RGC survival in vitro was attenuated by rapamycin, whereas neuritogenesis was unaffected by rapamycin and was not correlated with pS6 expression.

Conclusions: RTP801 knockdown is neuroprotective and stimulates RGC axonal regeneration, a response associated with enhanced gliosis after ONC. siRTP801 transfection enhances RGC survival in vitro, although does not initiate neuritogenesis. Despite maintained RGC pS6 expression, pS6 activity is not a requirement for RGC survival or neuritogenesis. In conclusion, both direct and indirect mechanisms contribute to neuroprotection and axon regeneration after RTP801 knockdown.

Keywords: 615 neuroprotection • 687 regeneration • 531 ganglion cells  
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