PKA inhibition protects optic nerve head astrocytes by modulating Akt/Bax phosphorylation and Mfn1/2 oligomerization against oxidative stress

Abstract

Purpose : The goal of this study is to investigate the impact of activation of cyclic adenosine 3′,5′-monophosphate (cAMP)/protein kinase A (PKA) pathway on mitochondrial dynamics in oxidative stress-induced optic nerve head (ONH) astrocytes.

Methods : Primary ONH astrocytes were prepared from postnatal day 5 Sprague-Dawley rats. Structural changes and mitochondrial morphology were assessed by serial block-face scanning electron microscopy (SBEM). ONH astrocyte cultures were treated with hydrogen peroxide (H₂O₂, 50 μM), dibutyryl-cAMP (dbcAMP, 100 μM), isobutylmethylxanthine (IBMX, 500 μM) and/or H89. Mitochondrial morphology and respiration were assessed by staining with MitoTracker Red CMXRos and by measuring oxygen consumption rate, respectively. Mitochondrial dynamics alteration and Akt/Bax phosphorylation were assessed by Western blot analysis using antibodies for dynamin-related protein 1 (Drp1), phopho-Drp1 at Ser 616, phopho-Drp1 at Ser 637, optic atrophy type 1 (OPA1), and Mitofusin (Mfn) 1 and 2, as well as Akt, Bax, phopho-Akt at Ser 473 and phopho-Bax at Ser 184.

Results : SBEM data analysis demonstrated that numbers of total and branched mitochondria were significantly increased in glaucomatous ONH astrocytes, while mitochondrial lengths and volume density were significantly decreased. Using ONH astrocyte cultures, we found that H₂O₂-induced oxidative stress compromised not only mitochondrial bioenergetics by reducing the basal and maximal respiration, but also mitochondrial dynamics by decreasing Drp1 protein expression in ONH astrocytes. In contrast, elevated cAMP by dbcAMP and IBMX treatment significantly increased Drp1 protein expression in ONH astrocytes. Intriguingly, elevated cAMP exacerbated impairment of mitochondrial dynamics and reduction of cell viability to oxidative stress in ONH astrocytes by decreasing Mfn1/2 protein expression. Following treatment with both H₂O₂ and dbcAMP, PKA inhibition preserved mitochondrial dynamics by increasing mitochondrial length and decreasing mitochondrial number, as well as promoted cell viability in ONH astrocytes by Akt/Bax phosphorylation and Mfn1/2 oligomerization.

Conclusions : These results suggest that modulation of cAMP/PKA signaling pathway may have therapeutic potential by activating Akt/Bax phosphorylation and promoting Mfn1/2 oligomerization in glaucomatous ONH astrocytes.

This abstract was presented at the 2019 ARVO Annual Meeting, held in Vancouver, Canada, April 28 - May 2, 2019.