title: siRNA-Mediated Knockdown of the mTOR Inhibitor RTP801 Promotes Retinal Ganglion Cell Survival and Axon Elongation by Direct and Indirect Mechanisms.
abstract: OBJECTIVE
  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 Brn3a+ RGC survival, GFAP+ reactive gliosis, and GAP43+ 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 GAP43+ axon regeneration at 400 to 1200 μm beyond the ONC site, and potentiated the reactive GFAP+ Müller glial response. In culture, siRTP801 had a direct RGC neuroprotective effect, but required GFAP+ 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.
authors: Neurotrauma Research Group, College of Medical and Dental Sciences, University of Birmingham, Birmingham, United Kingdom.
authors: Peter J
authors: PJ
authors: Morgan-Warren
authors: Neurotrauma Research Group, College of Medical and Dental Sciences, University of Birmingham, Birmingham, United Kingdom 2Neuregenix Ltd., Birmingham, United Kingdom.
authors: Jenna
authors: J
authors: O'Neill
authors: Neurotrauma Research Group, College of Medical and Dental Sciences, University of Birmingham, Birmingham, United Kingdom.
authors: Felicity
authors: F
authors: de Cogan
authors: Research Division, Quark Pharmaceuticals, Ness Ziona, Israel.
authors: Igor
authors: I
authors: Spivak
authors: Research Division, Quark Pharmaceuticals, Ness Ziona, Israel.
authors: Hagit
authors: H
authors: Ashush
authors: Research Division, Quark Pharmaceuticals, Ness Ziona, Israel.
authors: Hagar
authors: H
authors: Kalinski
authors: Neurotrauma Research Group, College of Medical and Dental Sciences, University of Birmingham, Birmingham, United Kingdom.
authors: Zubair
authors: Z
authors: Ahmed
authors: Neurotrauma Research Group, College of Medical and Dental Sciences, University of Birmingham, Birmingham, United Kingdom.
authors: Martin
authors: M
authors: Berry
authors: Research Division, Quark Pharmaceuticals, Ness Ziona, Israel.
authors: Elena
authors: E
authors: Feinstein
authors: Neurotrauma Research Group, College of Medical and Dental Sciences, University of Birmingham, Birmingham, United Kingdom 4Academic Department of Military Surgery and Trauma, Royal Centre for Defence Medicine, Birmingham, United Kingdom 5Birmingham and Mid.
authors: Robert A H
authors: RA
authors: Scott
authors: Neurotrauma Research Group, College of Medical and Dental Sciences, University of Birmingham, Birmingham, United Kingdom 2Neuregenix Ltd., Birmingham, United Kingdom.
authors: Ann
authors: A
authors: Logan
mesh_terms: D000818:Animals; D001369:Axons / Q000502:physiology*; D002470:Cell Survival / Q000502:physiology; D004195:Disease Models, Animal; D004797:Enzyme-Linked Immunosorbent Assay; D005786:Gene Expression Regulation / Q000502:physiology*; D055785:Gene Knockdown Techniques; D007150:Immunohistochemistry; D007166:Immunosuppressive Agents / Q000494:pharmacology; D058449:Intravitreal Injections; D008297:Male; D009409:Nerve Crush; D009414:Nerve Growth Factors / Q000378:metabolism; D009416:Nerve Regeneration / Q000502:physiology*; D020221:Optic Nerve Injuries / Q000209:etiology / Q000517:prevention & control; D034741:RNA, Small Interfering / Q000494:pharmacology*; D051381:Rats; D017208:Rats, Wistar; D012097:Repressor Proteins / Q000235:genetics*; D012165:Retinal Ganglion Cells / Q000166:cytology* / Q000378:metabolism; D020123:Sirolimus / Q000494:pharmacology; D058570:TOR Serine-Threonine Kinases / Q000037:antagonists & inhibitors* / Q000378:metabolism; D014157:Transcription Factors; D014162:Transfection
chemical_list: C000632146:Ddit4 protein, rat; D007166:Immunosuppressive Agents; D009414:Nerve Growth Factors; D034741:RNA, Small Interfering; D012097:Repressor Proteins; D014157:Transcription Factors; C546845:mTOR protein, rat; D058570:TOR Serine-Threonine Kinases; D020123:Sirolimus