Diabetes and overexpression of proNGF cause retinal neurodegeneration via activation of RhoA pathway.
Our previous studies showed positive correlation between accumulation of proNGF, activation of RhoA and neuronal death in diabetic models. Here, we examined the neuroprotective effects of selective inhibition of RhoA kinase in the diabetic rat retina and in a model that stably overexpressed the cleavage-resistance proNGF plasmid in the retina. Male Sprague-Dawley rats were rendered diabetic using streptozotocin or stably express cleavage-resistant proNGF plasmid. The neuroprotective effects of the intravitreal injection of RhoA kinase inhibitor Y27632 were examined in vivo. Effects of proNGF were examined in freshly isolated primary retinal ganglion cell (RGC) cultures and RGC-5 cell line. Retinal neurodegeneration was assessed by counting TUNEL-positive and Brn-3a positive retinal ganglion cells. Expression of proNGF, p75(NTR), cleaved-PARP, caspase-3 and p38MAPK/JNK were examined by Western-blot. Activation of RhoA was assessed by pull-down assay and G-LISA. Diabetes and overexpression of proNGF resulted in retinal neurodegeneration as indicated by 9- and 6-fold increase in TUNEL-positive cells, respectively. In vitro, proNGF induced 5-fold cell death in RGC-5 cell line, and it induced >10-fold cell death in primary RGC cultures. These effects were associated with significant upregulation of p75(NTR) and activation of RhoA. While proNGF induced TNF-α expression in vivo, it selectively activated RhoA in primary RGC cultures and RGC-5 cell line. Inhibiting RhoA kinase with Y27632 significantly reduced diabetes- and proNGF-induced activation of proapoptotic p38MAPK/JNK, expression of cleaved-PARP and caspase-3 and prevented retinal neurodegeneration in vivo and in vitro. Taken together, these results provide compelling evidence for a causal role of proNGF in diabetes-induced retinal neurodegeneration through enhancing p75(NTR) expression and direct activation of RhoA and p38MAPK/JNK apoptotic pathways.
Program in Clinical and Experimental Therapeutics. College of Pharmacy, University of Georgia, Athens, Georgia, United States of America.
Mohammed M H
MM
Al-Gayyar
Barbara A
BA
Mysona
Suraporn
S
Matragoon
Mohammed A
MA
Abdelsaid
Mona F
MF
El-Azab
Ahmed Y
AY
Shanab
Yonju
Y
Ha
Sylvia B
SB
Smith
Kathryn E
KE
Bollinger
Azza B
AB
El-Remessy
D000577:Amides / Q000494:pharmacology; D000818:Animals; D017209:Apoptosis / Q000187:drug effects; D053148:Caspase 3 / Q000235:genetics / Q000378:metabolism; D003921:Diabetes Mellitus, Experimental / Q000235:genetics* / Q000378:metabolism / Q000473:pathology; D005786:Gene Expression Regulation / Q000187:drug effects; D058449:Intravitreal Injections; D008297:Male; D020932:Nerve Growth Factor / Q000235:genetics* / Q000378:metabolism; D009419:Nerve Tissue Proteins; D011065:Poly(ADP-ribose) Polymerases; D061251:Primary Cell Culture; D047428:Protein Kinase Inhibitors / Q000494:pharmacology; D011498:Protein Precursors / Q000235:genetics* / Q000378:metabolism; D059748:Proteolysis / Q000187:drug effects; D011725:Pyridines / Q000494:pharmacology; D051381:Rats; D017207:Rats, Sprague-Dawley; D017978:Receptors, Growth Factor; D017475:Receptors, Nerve Growth Factor / Q000235:genetics / Q000378:metabolism; D012162:Retinal Degeneration / Q000235:genetics* / Q000378:metabolism / Q000473:pathology; D012165:Retinal Ganglion Cells / Q000187:drug effects / Q000378:metabolism* / Q000473:pathology; D015398:Signal Transduction / Q000187:drug effects; D013311:Streptozocin; D048051:p38 Mitogen-Activated Protein Kinases / Q000235:genetics / Q000378:metabolism; D020742:rhoA GTP-Binding Protein / Q000235:genetics* / Q000378:metabolism
D000577:Amides; D009419:Nerve Tissue Proteins; D047428:Protein Kinase Inhibitors; D011498:Protein Precursors; D011725:Pyridines; D017978:Receptors, Growth Factor; D017475:Receptors, Nerve Growth Factor; C061397:pro-nerve growth factor, human; C068313:Ngfr protein, rat; C108830:Y 27632; D013311:Streptozocin; D020932:Nerve Growth Factor; D011065:Poly(ADP-ribose) Polymerases; D048051:p38 Mitogen-Activated Protein Kinases; D053148:Caspase 3; D020742:rhoA GTP-Binding Protein