A bioenergetic crisis in the retinal pigment epithelium contributes to photoreceptor degeneration

Abstract

Purpose : Recent in vitro studies indicate that the retinal pigment epithelium (RPE) and photoreceptors form a “metabolic ecosystem” and a bioenergetic crisis in the RPE is a potential cause of the atrophic form of age-related macular degeneration. This project aimed to study the effect of selectively knocking down key metabolic genes in the RPE on the health of the mouse retina.

Methods : Immunohistochemistry (IHC) was performed to study expression of insulin receptor (IR), glucose transporter (GLUT)-1, hexokinase (HK)-1 and 2, lactate dehydrogenase (LDH)-A and B and pyruvate dehydrogenase E1α (PDH-E1α) in the mouse retina. RPE-Cre mice were crossed with transgenic mice carrying a floxed metabolic gene including IR, HK2, LDH-A or PDH-E1α. Changes in target gene expression, GLUT1 and mitochondrial proteins including succinate dehydrogenase complex flavoprotein subunit A (SDHA), voltage dependent anion channel (VDAC) and heat-shock protein (HSP)-60, were studied by Western blots using proteins extracted from RPE-choroid-sclera complexes. We also studied changes in RPE morphonology, photoreceptor health and retinal function in the transgenic mice we generated.

Results : The RPE expressed IR, GLUT1, LDH-B and PDH-E1α strongly but very little HK1, HK2 and LDH-A. IHC and Western blots confirmed successful knockdown of IR and PDH-E1α in the RPE after crossing RPE-Cre mice with floxed mice. Knocking down IR in the RPE led to reduced expression of GLUT1, PDH-E1α and SDHA but not VDAC and HSP60, while knocking down PDH-E1α resulted in reduced expression of IR, GLUT1 and all mitochondrial proteins including SDHA, VDAC and HSP60 in the RPE. Flatmount immunostaining revealed that knocking down IR- or PDH-E1α resulted in an irregular morphology of the RPE and disruption of cone photoreceptor apical processes. These changes were accompanied by impaired electroretinographic responses. We did not observe any retinal abnormalities in RPE-Cre mice crossed with HK2- or LDH-A floxed mice despite IHC confirming strong Cre expression in the RPE.

Conclusions : Our study provides in vivo evidence that a bioenergetic crisis in the RPE leads to photoreceptor degeneration. Future research is warranted to study changes in RPE mitochondrial density and function and alterations in the metabolic symbiosis between the RPE and photoreceptors after selectively knocking down IR and PDH-E1α.

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