Selectively knocking down hexokinase 2 in rods leads to retinal degeneration

Abstract

Purpose : The retina is a metabolically active tissue and its energy demands are normally met through aerobic glycolysis. However, the cell types that carry out glycolysis in the mammalian retina are unclear. The aim of this project was to use a cell-specific approach to study the consequences of selectively knocking down hexokinase 2 (HK2), the first rate-limiting enzyme in the glycolytic pathway, in rod photoreceptor cells of the mouse retina.

Methods : We crossed rhodopsin (RHO)-Cre mice (Li et al. Genesis 2005) with Hk2 flox mice (HK2^fl/fl) to create RHO-Cre:HK2^+/fl and RHO-Cre:HK2^fl/fl mice, with RHO-Cre mice crossed with Z-EG and Rosa-LacZ Cre reporter mice serving as controls. Retinal changes were studied by immunostaining and Western blots using antibodies against Hk2, lactate dehydrogenase A (LDH-A), pyruvate dehydrogenase E1a (PDH-E1a), recoverin, blue- and red-green opsin. Fluorescence-labelled peanut agglutinin (PNA) was also used to study changes in cone photoreceptor apical processes.

Results : The rod-specific gene targeting in RHO-Cre mice was evidenced by reporter gene expression in rod photoreceptors after crossing them with Cre-reporter mice. Hk2 was expressed in ganglion cells, the inner nuclear layer, the outer plexform layer and the photoreceptor inner segments in the normal retina. Western blot analysis indicated that HK2 was knocked down by ~37% in the retina of RHO-Cre mice crossed with Hk2-floxed mice at 8-10 weeks of age, with no significant difference between RHO-Cre:HK2^+/fl and RHO-Cre:HK2^fl/fl mice. Findings from Western blots were confirmed by immunostaining for Hk2. Selectively knocking down Hk2 in rods led to photoreceptor degeneration, as evidenced by the loss of PNA-stained photoreceptor apical processes and reduced expression of recoverin, blue- and red-green opsin. Hk2 knockdown also led to reduced expression of LDH-A, but with little effect on PDH-E1α expression.

Conclusions : Our data provide evidence that the activity of HK2 in rods is important for the maintenance of photoreceptor health.

This is an abstract that was submitted for the 2018 ARVO Annual Meeting, held in Honolulu, Hawaii, April 29 - May 3, 2018.