Abstract
Purpose :
Danon disease (DD) is an x-linked disorder caused by loss-of-function mutations in the Lysosome-associated membrane protein 2 (LAMP2) gene leading to the disruption of autophagy and phagocytosis. In addition to cardiomyopathy, mental retardation, and skeletal myopathy, DD results in progressive retinal degeneration.
There are three isoforms of LAMP2 (2A, 2B, 2C) with expression being tissue specific. In this study, we aimed to confirm murine LAMP2 isoform expression in retina and RPE/choroid, before characterizing the phenotype of aged Lamp2 knockout mice (Lamp2y/-) to help clarify the contribution of LAMP2 in retinal degeneration. Finally, we tested LAMP2 isoform expression in a gene therapy construct.
Methods :
Lamp2 isoform expression was quantified by qPCR from fresh retina and RPE/choroid of 4 wildtype (WT) mice. The phenotype of 20–24-month Lamp2y/- mice were compared to age matched WT mice using full-field electroretinography (ffERG, n=3) and M and S-opsin counts of retinal cryosections (n=3).
Plasmids containing all three LAMP2 isoforms with different promoters were tested in hiPSCs derived from a patient with DD.
Results :
The expression of Lamp2B and Lamp2C was 80% and 30% of Lamp2A levels in the retina, respectively. In the RPE, Lamp2C levels were 20% of Lamp2A while Lamp2B was equivalent (fig. 1).
In 20–24-month Lamp2y/- mice, both scotopic and photopic waveforms were severely reduced using ffERG (fig. 1). To investigate if this finding was due to degeneration or dysfunction, cone opsin positive photoreceptors were counted. The number of positive photoreceptors counted per mm of retina was not significantly different between the two groups (fig.1)
Plasmid construct expression in DD hiPSCs including all isoforms demonstrated good expression with the EF1 promoter compared with PGK promoter (fig. 2). Expression of 2A and 2B isoforms was higher than 2C.
Conclusions :
Despite clearly attenuated ffERG responses, aged Lamp2y/- mice retinas still demonstrated similar numbers of cone photoreceptors to age matched WT mice suggesting dysfunction predominated over degeneration. This also supported an approach for using gene therapy constructs, similar to those tested in the present study, to restore retinal function in DD patients.
This abstract was presented at the 2023 ARVO Annual Meeting, held in New Orleans, LA, April 23-27, 2023.