siRNA mediated knockdown of the mTOR inhibitor RTP801 promotes retinal ganglion cell survival and axon elongation by direct and indirect mechanisms.

Research output: Contribution to journalArticle

Authors

  • Peter Morgan-Warren (Contributor)
  • I. Spivak (Contributor)
  • H. Ashush (Contributor)
  • H Kalinski (Contributor)
  • Martin Berry (Contributor)
  • E. Feinstein (Contributor)
  • Robert Scott (Contributor)

Colleges, School and Institutes

Abstract

PURPOSE. To investigate, using in vivo and in vitro models, retinal ganglion cell (RGC) neuroprotective and axon regenerative effects and underlying mechanisms of siRTP801, a translatable small-interfering RNA (siRNA) targeting the mTOR negative regulator RTP801.
METHODS. Adult rats underwent optic nerve (ON) crush (ONC) followed by intravitreal siRTP801 or control siRNA (siEGFP) every 8 days, with Brn3aþ RGC survival, GFAPþ reactive gliosis, and GAP43þ regenerating axons analyzed immunohistochemically 24 days after injury. Retinal cultures, prepared from uninjured animals or 5 days after ONC to activate retinal glia, were treated with siRTP801/controls in the presence/absence of rapamycin and subsequently
assessed for RGC survival and neurite outgrowth, RTP801 expression, glial responses, and mTOR activity. Conditioned medium was analyzed for neurotrophin titers by ELISA.
RESULTS. Intravitreal siRTP801 enabled 82% RGC survival compared to 45% with siEGFP 24 days after ONC, correlated with greater GAP43þ axon regeneration at 400 to 1200 lm beyond the ONC site, and potentiated the reactive GFAPþ Muller glial response. In culture, siRTP801 had a direct RGC neuroprotective effect, but required GFAPþ activated glia to stimulate neurite elongation. The siRTP801-induced neuroprotection was significantly reduced, but not abolished, by rapamycin. The siRTP801 potentiated the production and release of
neurotrophins NGF, NT-3, and BDNF, and prevented downregulation of RGC mTOR activity.
CONCLUSIONS. The RTP801 knockdown promoted RGC survival and axon elongation after ONC, without increasing de novo regenerative sprouting. The neuroprotection was predominantly direct, with mTORC1-dependent and -independent components. Enhanced neurite/axon elongation by siRTP801 required the presence of activated retinal glia and was mediated by potentiated secretion of neurotrophic factors.

Details

Original languageEnglish
Pages (from-to)429-443
JournalInvestigative Ophthalmology & Visual Science (IOVS)
Volume57
Publication statusPublished - 1 Feb 2016