Wednesday, February 5, 2025
2/5/2025
ABSTRACT: All eukaryotic cells require cholesterol (Chol) for survival, and regulate their steady-state levels by balancing de novo synthesis, uptake of Chol-containing lipoproteins, and Chol efflux (i.e., “Chol homeostasis”). Our current understanding of Chol homeostasis in the retina remains rudimentary. Hereditary defects in Chol synthesis comprise a family of severe, often lethal, metabolic disorders, e.g., Smith-Lemli-Opitz Syndrome (SLOS). SLOS involves defective conversion of 7-dehydrocholesterol (7DHC) to Chol, which is catalyzed by DHCR7 (7- dehydrocholesterol reductase). Treating rats with a DHCR7 inhibitor (e.g., AY9944) causes progressive, irreversible retinal degeneration: the photoreceptors (PRs) preferentially degenerate and die, and RPE cells also exhibit autophagy/heterophagy defects. However, off-target effects of DHCR7 inhibitors cannot be obviated, and no viable genetic mouse models of SLOS are available. We have generated two novel, viable, conditional allele models of SLOS, allowing targeted ablation of either Dhcr7 exon 8, or separately exon 9, to partially or completely block DHCR7 in selective cell types and tissues. We hypothesize that de novo Chol synthesis by retinal neurons alone is insufficient to maintain their viability and functionality; rather, they rely upon Chol uptake from blood-borne (liver-derived) lipoproteins, Müller glia, and/or the RPE to meet their sterol demands. Also, that disruption of normal Chol homeostasis provokes defective phagolysosomal biology in the RPE and retina. We will test our hypothesis as follows: In Aim 1, we will selectively knock out Dhcr7 in rod PRs and, separately, in Müller glia (or in tandem) and then assess the impact on sterol composition of outer segments and optic nerve, as well as retinal structure/function; in Aim 2, we will generate panretinal knock-out (KO) of Dhcr7 or, separately (and in tandem), in liver (hepatocytes) and then assess the impact on sterol composition retina, liver, and blood. Using these resources, and a stable isotope approach, we will estimate retinal sterol synthesis, uptake and turnover rates; and in Aim 3, we will use RPE and liver-specific Dhcr7 KO mice to assess the effect of de novo 7DHC synthesis and its uptake on RPE phagocytic function. We will model the observed SLOS RPE pathology using patient iPSC-derived RPE cells (multiple clones, with appropriate controls), and screen for candidate drugs to improve the observed EMT and phagocytic defects. We will use novel in vivo assays using a tandem-tagged autophagy reporter mouse model to systematically elucidate the role of Chol synthesis in retinal phagolysosomal biology. The results obtained will significantly advance both our fundamental understanding of Chol homeostasis in the vertebrate retina and mechanisms underlying retinal pathology associated with Chol synthesis defects, and provide new tractable model systems for future testing of more effective therapeutic interventions for SLOS and related orphan diseases.
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