Abstract
Purpose :
The goal of this study is to determine whether overexpression of A-kinase anchoring protein 1 (AKAP1) protects retinal ganglion cells (RGCs) by modulating mitochondrial activity against oxidative stress.
Methods :
Retinas were prepared from adult DBA/2J and DBA/2J-Gpnmb+mice, as well as AKAP1 knockout and C57BL/6J mice. Primary RGCs were prepared from postnatal day 5 Sprague-Dawley rats. Overexpression of AKAP1 was done by the transduction of adeno-associated virus 2 (AAV2)-AKAP1. Paraquat (PQ) was intravitreally injected to the eyes of C57BL/6J mice and also treated into RGC cultures to induce oxidative stress. Mitochondrial dynamics and related phosphorylation in RGCs were assessed by electron microscope tomography, Western blot analysis and immunohistochemistry using antibodies for calcineurin (CaN), dynamin-related protein 1 (Drp1) as well as phospho-Drp1 at Serine 637 (pDrp1 S637) and phospho-Akt at Serine 473 (pAkt S473). Mitochondria-related cell death pathway was evaluated by Western blot analysis using antibodies for Akt, Bax, and Bim. RGC protection was assessed by whole-mount immunohistochemistry and TUNEL staining.
Results :
Glaucomatous RGCs showed loss of AKAP1, activation of CaN, and reduction of pDrp1 S637 in 10-month-old DBA/2J mice compared with age-matched DBA/2J-Gpnmb+mice. Male mice lacking AKAP1 showed increases of CaN and total Drp1 level, as well as a decrease of pDrp1 S637 in the retina. Loss of AKAP1 triggers mitochondrial fragmentation and loss, as well as mitophagosome formation in RGCs. Loss of AKAP1 deregulated oxidative phosphorylation (OXPHOS) complexes (Cxs) by increasing CxII and decreasing CxIII-V, leading to metabolic and oxidative stress. The loss of AKAP1 decreased pAkt S473 and activated the Bim/Bax signaling pathway in the retina. Overexpression of AKAP1 by AAV transduction promoted RGC survival in the retina treated with PQ. Also, overexpression of AKAP1 significantly increased mitochondrial activity and blocked TUNEL-positive apoptotic cell death in cultured RGCs exposed to PQ.
Conclusions :
These results suggest that loss of AKAP1 may have a critical role in RGC dysfunction by decreasing Drp1 phosphorylation, deregulating the OXPHOS, decreasing Akt phosphorylation and activating the Bim/Bax pathway in glaucomatous neurodegeneration. We proposed that overexpression of AKAP1 has therapeutic potential in RGCs against oxidative stress.
This is a 2020 ARVO Annual Meeting abstract.