Spatial Distribution of the Pathways of Cholesterol Homeostasis in Human Retina

The retina is a light-sensitive tissue lining the inner surface of the eye and one of the few human organs whose cholesterol maintenance is still poorly understood. Challenges in studies of the retina include its complex multicellular and multilayered structure; unique cell types and functions; and specific physico-chemical environment.We isolated specimens of the neural retina (NR) and underlying retinal pigment epithelium (RPE)/choroid from six deceased human donors and evaluated them for expression of genes and proteins representing the major pathways of cholesterol input, output and regulation. Eighty-four genes were studied by PCR array, 16 genes were assessed by quantitative real time PCR, and 13 proteins were characterized by immunohistochemistry. Cholesterol distribution among different retinal layers was analyzed as well by histochemical staining with filipin. Our major findings pertain to two adjacent retinal layers: the photoreceptor outer segments of NR and the RPE. We demonstrate that in the photoreceptor outer segments, cholesterol biosynthesis, catabolism and regulation via LXR and SREBP are weak or absent and cholesterol content is the lowest of all retinal layers. Cholesterol maintenance in the RPE is different, yet the gene expression also does not appear to be regulated by the SREBPs and varies significantly among different individuals.This comprehensive investigation provides important insights into the relationship and spatial distribution of different pathways of cholesterol input, output and regulation in the NR-RPE region. The data obtained are important for deciphering the putative link between cholesterol and age-related macular degeneration, a major cause of irreversible vision loss in the elderly

RETRACTED ARTICLE: Age-dependent Increase in Desmosterol Restores DRM Formation and Membrane-related Functions in Cholesterol-free DHCR24−/− Mice

Cholesterol is a prominent modulator of the integrity and functional activity of physiological membranes and the most abundant sterol in the mammalian brain. DHCR24-knock-out mice lack cholesterol and accumulate desmosterol with age. Here we demonstrate that brain cholesterol deficiency in 3-week-old DHCR24−/− mice was associated with altered membrane composition including disrupted detergent-resistant membrane domain (DRM) structure. Furthermore, membrane-related functions differed extensively in the brains of these mice, resulting in lower plasmin activity, decreased β-secretase activity and diminished Aβ generation. Age-dependent accumulation and integration of desmosterol in brain membranes of 16-week-old DHCR24−/− mice led to the formation of desmosterol-containing DRMs and rescued the observed membrane-related functional deficits. Our data provide evidence that an alternate sterol, desmosterol, can facilitate processes that are normally cholesterol-dependent including formation of DRMs from mouse brain extracts, membrane receptor ligand binding and activation, and regulation of membrane protein proteolytic activity. These data indicate that desmosterol can replace cholesterol in membrane-related functions in the DHCR24−/− mouse

Analyses of Cholesterol and Derivatives in Ocular Tissues Using LC-MS/MS Methods

Cholesterol is considered one of the most abundant sterols present in mammals, amphipathic in nature, and a key constituent of the cell membrane. Its unique chemical structure consisting of four linked hydrocarbon rings, with an aliphatic chain on one end and a hydroxyl group on the other, confers it the ability to form hydrogen bonds with other lipid classes, like phospholipids or sphingolipids head groups. However, due to its hydrophobic moiety, the fatty acid chain, free cholesterol (FC) it is difficult to be analyzed via electrospray ionization (ESI). The method presented in this chapter to identify and quantify free cholesterol and cholesteryl esters (CE) is based on the chemical derivatization of the sample, strategy devised to avoid the problematic of ESI. However, relevant mention should be made to an alternative separation protocol, which uses ultrahigh performance liquid chromatography and in-source collision-induced dissociation to achieve a simultaneous quantification of FC, CE, and triglycerides (TG)