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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)
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).
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).
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.
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.
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