Abstract
Purpose :
Peroxisomes are multifaceted yet underappreciated organelles central to lipid metabolism, reactive oxygen species detoxification, and cholesterol homeostasis. Peroxisomal biogenesis impairment results in Zellweger Spectrum Disorder (ZSD), a spectrum of retinopathies caused by loss of function of any one of 13 pex genes. Despite its causal role in ZSDs, whether peroxisomes function to maintain retinal homeostasis in more complex retinal diseases such as AMD remains unclear. Both cholesterol ester (CE) and free cholesterol (FC) are elevated in Bruch’s membrane in the aging macula (Curcio et al., 2001), raising the possibility of cholesterol mis-trafficking in the retinal pigment epithelium (RPE)/choroid tissue. This study explores that possibility in the context of peroxisomal dysfunction in cell and animal models.
Methods :
Immunohistochemistry was conducted in AMD1 (N=4) and AMD3 (N=4) donor eye sections. LC/MS was conducted in zebrafish posterior eye cup (PEC) (N=5). Zebrafish eyes were enucleated and sectioned for immunofluorescence. iPS-RPE cells were obtained from Cellular Dynamics, Inc and cultured according to standard protocol.
Results :
Markers of peroxisomes are enriched in RPE cells of both human and animal models. CATALASE punctae in AMD3 donor RPE are mis-localized. pex1 mutant zebrafish retina shows neutral lipid accumulation in the RPE layer via lipid stains and reduced photoreceptor (PR) markers indicating PR degeneration. Both FC and CE are increased in the PEC of pex1 -/- zebrafish. Lysosome volume measured via LAMP1 staining is also increased in pex1 -/- zebrafish RPE flat mount. Finally, inhibition of intra-lysosomal catabolism via chloroquine (CQ) treatment leads to cholesterol accumulation in iPS-RPE lysosomes.
Conclusions :
Our results are consistent with our hypothesis that peroxisomes are required in RPE cells for proper lipid handling. With the observations of cholesterol mis-localization to lysosomes via CQ treatment, we are currently exploring whether defective peroxisomes impair intracellular cholesterol trafficking through lysosomes and thus causes abnormal build-up of toxic materials in the RPE. Our research will potentially provide molecular underpinning of intermediate AMD mechanisms.
This abstract was presented at the 2023 ARVO Annual Meeting, held in New Orleans, LA, April 23-27, 2023.