title: Electroporation-mediated gene delivery of cleavage-resistant pro-nerve growth factor causes retinal neuro- and vascular degeneration.
abstract: OBJECTIVE
  Neurotrophins, including nerve growth factor (NGF), are secreted by glia as a pro-form (proNGF) that is normally cleaved into the mature ligand. Increases of proNGF has been well documented in retinal neurodegenerative diseases. Since systemic overexpression of proNGF exhibits embryonic lethality, we aimed to establish a model that specifically and stably overexpresses a cleavage-resistant mutant of proNGF (proNGF123) plasmid in the retina using electroporation.
  
  
  METHODS
  Male Sprague-Dawley rats were injected intravitreally with pGFP or pGFP-proNGF123 plasmids, then electroporated with various settings for optimization. Retinal cell death and ganglion cell count were assessed by TUNEL and immunostaining with anti-Brn3. Expression of proNGF, NGF, and their receptors was examined by western blot. Retinal vascular permeability was assessed by extravasation of bovine serum albumin-fluorescein. Development of acellular capillaries was assessed by periodic acid-Schiff and hematoxylin staining.
  
  
  RESULTS
  Successful pGFP-proNGF123 gene delivery and expression of proNGF was demonstrated by western blot and extensive proNGF immunostaining in retina sections. Overexpression of proNGF reduced NGF expression while inducing the expression of neurotrophin receptors, including p75(NTR) and tyrosine receptor kinase A, but not sortilin. Overexpression of proNGF resulted in ~50% reduction in ganglion cell count and fivefold increase in TUNEL-positive cells in rat retina. In addition, overexpression of proNGF induced breakdown of the blood-retina barrier evident by twofold increase in extravasation of bovine serum albumin-fluorescein after 1 week and induced the development of acellular capillaries after 4 weeks.
  
  
  CONCLUSIONS
  Electroporation can successfully incorporate and express biologically active cleavage-resistant proNGF locally in rat retinas. Overexpression of cleavage-resistant proNGF can be a useful tool to investigate specific molecular mechanisms by which proNGF causes neurodegeneration and vascular injury in the retina.
authors.affiliation: Program in Clinical and Experimental Therapeutics, College of Pharmacy, University of Georgia, Augusta, GA 30912, USA.
authors.forename: Suraporn
authors.initials: S
authors.lastname: Matragoon
authors.forename: Mohammed M H
authors.initials: MM
authors.lastname: Al-Gayyar
authors.forename: Barbara A
authors.initials: BA
authors.lastname: Mysona
authors.forename: Mohammed A
authors.initials: MA
authors.lastname: Abdelsaid
authors.forename: Bindu A
authors.initials: BA
authors.lastname: Pillai
authors.forename: Kenneth E
authors.initials: KE
authors.lastname: Neet
authors.forename: Susan C
authors.initials: SC
authors.lastname: Fagan
authors.forename: Azza B
authors.initials: AB
authors.lastname: El-Remessy
mesh_terms: D000818:Animals; D001813:Blood-Retinal Barrier / Q000378:metabolism / Q000473:pathology*; D002199:Capillary Permeability; D002470:Cell Survival; D018274:Electroporation; D015870:Gene Expression; D018014:Gene Transfer Techniques; D058449:Intravitreal Injections; D008297:Male; D009410:Nerve Degeneration / Q000378:metabolism / Q000473:pathology; D009414:Nerve Growth Factors / Q000235:genetics / Q000378:metabolism*; D009419:Nerve Tissue Proteins; D011498:Protein Precursors / Q000235:genetics / Q000378:metabolism*; D059748:Proteolysis; D051381:Rats; D017207:Rats, Sprague-Dawley; D020917:Receptor, trkA / Q000235:genetics / Q000378:metabolism; D017978:Receptors, Growth Factor; D017475:Receptors, Nerve Growth Factor / Q000235:genetics / Q000378:metabolism; D012165:Retinal Ganglion Cells / Q000378:metabolism / Q000473:pathology; D055351:Retinal Neurons / Q000378:metabolism / Q000473:pathology*; D012171:Retinal Vessels / Q000378:metabolism / Q000473:pathology*; D050818:Transcription Factor Brn-3 / Q000235:genetics / Q000378:metabolism; D019076:Transgenes
chemical_list: D009414:Nerve Growth Factors; D009419:Nerve Tissue Proteins; D011498:Protein Precursors; D017978:Receptors, Growth Factor; D017475:Receptors, Nerve Growth Factor; D050818:Transcription Factor Brn-3; C514218:pro-nerve growth factor, rat; C068313:Ngfr protein, rat; D020917:Receptor, trkA