TY - JOUR
T1 - Virally delivered, constitutively active NFκB improves survival of injured retinal ganglion cells
AU - Dvoriantchikova, Galina
AU - Pappas, Steve
AU - Luo, Xueting
AU - Ribeiro, Marcio
AU - Danek, Dagmara
AU - Pelaez, Daniel
AU - Park, Kevin K.
AU - Ivanov, Dmitry
N1 - Funding Information:
This work was supported in part by National Eye Institute/National Institutes of Health (NIH) Grant R01 EY022348 (DI), Center Core Grant P30 EY014801 to the University of Miami Department of Ophthalmology, Dr. Nasser Al-Rashid Orbital Vision Research Center Grant (DP), GemCon Family Foundation Research Grant (DP), NIH/NEI R01 EY022961 (KKP), the Ziegler Foundation (KKP), the Pew Charitable Trust (KKP), the Craig H. Neilsen Foundation (KKP) and the Buoniconti Fund (KKP). We thank the Analytic Imaging Facility (Gabriel Gaidosh) at the Bascom Palmer Eye Institute (BPEI) and the Viral Vector Core at the Miami Project to Cure Paralysis. Abbreviations AAV2 adeno-associated virus serotype 2 FBS fetal bovine serum MCS multiple cloning site NFκB nuclear factor kappa B ONC optic nerve crush model p65mut p65 S276DS536A double mutant PFA paraformaldehyde PVDF membrane polyvinylidene difluoride membrane RGCs retinal ganglion cells RIPA buffer radioimmunoprecipitation assay buffer SDS-PAGE sodium dodecyl sulfate polyacrylamide gel electrophoresis TBS tris-buffered saline Tubb3 beta III Tubulin
Publisher Copyright:
© 2016 Federation of European Neuroscience Societies and John Wiley & Sons Ltd
PY - 2016/12/1
Y1 - 2016/12/1
N2 - As axon damage and retinal ganglion cell (RGC) loss lead to blindness, therapies that increase RGC survival and axon regrowth have direct clinical relevance. Given that NFκB signaling is critical for neuronal survival and may regulate neurite growth, we investigated the therapeutic potential of NFκB signaling in RGC survival and axon regeneration. Although both NFκB subunits (p65 and p50) are present in RGCs, p65 exists in an inactive (unphosphorylated) state when RGCs are subjected to neurotoxic conditions. In this study, we used a phosphomimetic approach to generate DNA coding for an activated (phosphorylated) p65 (p65mut), then employed an adeno-associated virus serotype 2 (AAV2) to deliver the DNA into RGCs. We tested whether constitutive p65mut expression prevents death and facilitates neurite outgrowth in RGCs subjected to transient retinal ischemia or optic nerve crush (ONC), two models of neurotoxicity. Our data indicate that RGCs treated with AAV2-p65mut displayed a significant increase in survival compared to controls in ONC model (77 ± 7% vs. 25 ± 3%, P-value = 0.0001). We also found protective effect of modified p65 in RGCs of ischemic retinas (55 ± 12% vs. 35 ± 6%), but not to a statistically significant degree (P-value = 0.14). We did not detect a difference in axon regeneration between experimental and control animals after ONC. These findings suggest that increased NFκB signaling in RGCs attenuates retinal damage in animal models of neurodegeneration, but insignificantly impacts axon regeneration.
AB - As axon damage and retinal ganglion cell (RGC) loss lead to blindness, therapies that increase RGC survival and axon regrowth have direct clinical relevance. Given that NFκB signaling is critical for neuronal survival and may regulate neurite growth, we investigated the therapeutic potential of NFκB signaling in RGC survival and axon regeneration. Although both NFκB subunits (p65 and p50) are present in RGCs, p65 exists in an inactive (unphosphorylated) state when RGCs are subjected to neurotoxic conditions. In this study, we used a phosphomimetic approach to generate DNA coding for an activated (phosphorylated) p65 (p65mut), then employed an adeno-associated virus serotype 2 (AAV2) to deliver the DNA into RGCs. We tested whether constitutive p65mut expression prevents death and facilitates neurite outgrowth in RGCs subjected to transient retinal ischemia or optic nerve crush (ONC), two models of neurotoxicity. Our data indicate that RGCs treated with AAV2-p65mut displayed a significant increase in survival compared to controls in ONC model (77 ± 7% vs. 25 ± 3%, P-value = 0.0001). We also found protective effect of modified p65 in RGCs of ischemic retinas (55 ± 12% vs. 35 ± 6%), but not to a statistically significant degree (P-value = 0.14). We did not detect a difference in axon regeneration between experimental and control animals after ONC. These findings suggest that increased NFκB signaling in RGCs attenuates retinal damage in animal models of neurodegeneration, but insignificantly impacts axon regeneration.
KW - adeno-associated virus serotype 2
KW - optic nerve crush model
KW - phosphomimetic
KW - retinal ganglion cells
KW - transient retinal ischemia model
UR - http://www.scopus.com/inward/record.url?scp=84987653323&partnerID=8YFLogxK
U2 - 10.1111/ejn.13383
DO - 10.1111/ejn.13383
M3 - Article
C2 - 27564592
AN - SCOPUS:84987653323
VL - 44
SP - 2935
EP - 2943
JO - European Journal of Neuroscience
JF - European Journal of Neuroscience
SN - 0953-816X
IS - 11
ER -