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Treatment of rats with the cholesterol pathway inhibitor AY9944 produces an animal model of Smith-Lemli-Opitz syndrome (SLOS), an autosomal recessive disease caused by defective cholesterol synthesis. This SLOS rat model undergoes progressive and irreversible degeneration of the neural retina, with associated pathological features of the retinal pigmented epithelium (RPE). Here, we provide further insights into the mechanism involved in the RPE pathology. In the SLOS rat model, markedly increased RPE apical autofluorescence is observed, compared to untreated animals, which correlates with increased levels of A2E and other bisretinoids. Utilizing cultured human induced pluripotent stem cell (iPSC)- derived SLOS RPE cells, we found significantly elevated steady-state levels of 7-dehydrocholesterol (7DHC) and decreased cholesterol levels (key biochemical hallmarks of SLOS). Western blot analysis revealed altered levels of the macroautophagy/autophagy markers MAP1LC3B-II and SQSTM1/p62, and build-up of ubiquitinated proteins. Accumulation of immature autophagosomes was accompanied by inefficient degradation of phagocytized, exogenously supplied retinal rod outer segments (as evidenced by persistence of the C-terminal 1D4 epitope of RHO [rhodopsin]) in SLOS RPE compared to iPSC-derived normal human control. SLOS RPE cells exhibited lysosomal pH levels and CTSD activity within normal physiological limits, thus discounting the involvement of perturbed lysosomal function. Furthermore, 1D4-positive phagosomes that accumulated in the RPE in both pharmacological and genetic rodent models of SLOS failed to fuse with lysosomes. Taken together, these observations suggest that defective phagosome maturation underlies the observed RPE pathology. The potential relevance of these findings to SLOS and the requirement of cholesterol for phagosome maturation are discussed.

Rhodopsin, the G-protein coupled receptor in retinal rod photoreceptors, is a highly conserved protein that undergoes several types of post-translational modifications. These modifications are essential to maintain the protein’s structure as well as its proper function in the visual transduction cycle. Rhodopsin is N-glycosylated at Asn-2 and Asn-15 in its extracellular N-terminal domain. Mutations within the glycosylation consensus sequences of rhodopsin cause autosomal dominant retinitis pigmentosa, a disease that leads to blindness. Several groups have studied the role of rhodopsin’s N-linked glycan chains in protein structure and function using a variety of approaches. These include the generation of a transgenic mouse model, study of a naturally occurring mutant animal model, in vivo pharmacological inhibition of glycosylation, and in vitro analyses using transfected COS-1 cells. These studies have provided insights into the possible role of rhodopsin glycosylation, but have yielded conflicting results.

Cholesterol is an essential structural constituent of cellular membranes and a precursor molecule for the synthesis of steroid hormones, bile acids, and regulatory oxysterols. Cholesterol homeostasis is achieved by a series of regulatory steps that control cholesterol biosynthesis, its uptake from plasma lipoproteins, and the conversion of cholesterol to bile acids (27, 45). The prevailing view has been that cholesterol biosynthetic reactions occur in the cytoplasm and endoplasmic reticulum. However, recent studies suggest that the early steps in the isoprenoid/cholesterol biosynthetic pathway occur in peroxisomes, since all of the enzymes required for the conversion of acetyl coenzyme A (acetyl-CoA) to farnesyl diphosphate (FPP) are localized in peroxisomes and the enzymes catalyzing the conversion of mevalonate to FPP appear to be exclusively peroxisomal (30). Proteins imported into the peroxisome matrix use either C-terminal tripeptide (PTS 1) or N-terminal nonapeptide (PTS 2) peroxisome targeting signals that are recognized by the cytosolic receptors Pex5p and Pex7p, respectively (40). Both peroxisomal import pathways are utilized by peroxisomal cholesterol biosynthetic enzymes, and PTS1 and PTS2 are found in an alternating order (30). 3-Hydroxy-3-methylglutaryl (HMG) CoA reductase, localized in the endoplasmic reticulum and peroxisomes, is the only enzyme in the cholesterol biosynthetic pathway required for the conversion of HMG-CoA to mevalonate for which we, as yet, have no peroxisomal targeting information. The importance of peroxisomes for normal cellular functioning is illustrated by the disorders associated with the Zellweger spectrum (Zellweger syndrome [ZS], neonatal adrenoleukodystrophy and infantile Refsum's disease). All of these diseases are characterized by a loss of functional peroxisomes resulting in the presence of matrix enzymes in the cytosol (21). In Zellweger syndrome (ZS), the most severe of these disorders, infants have central nervous system neuronal migration defects associated with neonatal hypotonia, seizures, and hepatic dysfunction. The effect of peroxisomal dysfunction on cholesterol biosynthesis in cells and tissues from patients with the Zellweger spectrum disorders has been examined, with conflicting results (30). Briefly, reduced plasma cholesterol levels have been found in Zellweger spectrum patients (30). Normal enzyme activities for mevalonate kinase (MvK), mevalonate diphosphate decarboxylase (MPD), and isopentenyl diphosphate isomerase (IPPI) have been measured in fibroblasts from ZS patients in one study (54), but decreased MvK activity was found in fibroblasts from ZS and neonatal adrenoleukodystrophy patients in another study (6). While three groups reported significantly decreased cholesterol biosynthesis in fibroblasts from Zellweger spectrum patients (25, 34; J. C. Collins, F. Keyserman, S. C. Rumsey, and R. J. Deckelbaum, Am. Heart Assoc. abstr. 1219, PG I-228, 1993), two other studies showed that cholesterol biosynthesis rates were equivalent to or slightly higher than those in control fibroblasts (33, 37). A study by Krisans et al. (31) analyzing liver samples from Zellweger spectrum patients has shown that enzymatic activities of HMG-CoA reductase, MvK, phosphomevalonate kinase, MPD, IPPI, and FPP synthase were reduced, while another group reported decreased or normal MvK activities in livers of ZS patients (54). In addition, one group reported a decrease in cholesterol biosynthesis in peroxisome-deficient PEX2−/− hamster cells (1), while another group reported increased cholesterol biosynthesis in the same cell lines (52). Recently, peroxisome-deficient mouse models for human ZS have become available for study. In one such model, the PEX5 knockout mouse, the cholesterol biosynthesis rate was normal in immortalized as well as primary PEX5−/− mouse fibroblast cultures (53). Cholesterol levels were also normal in the plasma, brain, and liver of newborn PEX5−/− mice, while hepatic enzyme activities for HMG-CoA reductase and IPP isomerase were slightly elevated (26). These PEX5-deficient mice die within 24 h after birth and thus cannot be used to study postnatal effects (2). The second mouse model for human ZS has a targeted deletion of the PEX2 gene (13), and homozygous mutants survive in the early postnatal period up to 13 days (12, 14). Thus, the PEX2 mouse provides an excellent model to assess the effect of peroxisomal deficiency on cholesterol homeostasis in the early postnatal period. The focus of our study was to characterize the effects of peroxisome deficiency on an array of cholesterol biosynthetic enzymes, bile acid proteins, ATP-binding cassette (ABC) transporters, transcription factors, and nuclear receptors that are involved in maintaining cholesterol homeostasis. Our data illustrate that the loss of peroxisomes leads to an upregulation of the sterol regulatory element-binding protein 2 (SREBP-2) pathway in the liver and a corresponding increase in the activities and the protein and expression levels of SREBP-2-regulated cholesterol biosynthetic enzymes. However, due to the loss of compartmentalization, the cholesterol biosynthetic enzymes function with altered catalytic efficiency. The rate of cholesterol biosynthesis is increased in most tissues, except in the brain and kidneys, where it is reduced. The cholesterol contents of all tissues examined were found to be similar in wild-type and PEX2 knockout mice, with the exception of the liver, where the cholesterol content is significantly reduced in the PEX2 knockout mice. In addition, compared with wild-type mice, PEX2−/− mice have decreased plasma total and high-density lipoprotein (HDL) cholesterol levels. The classic pathway of bile acid biosynthesis is downregulated, while the alternate pathway is unexpectedly increased. Our studies demonstrate that there is a loss of coordinate regulation of cholesterol homeostasis in the PEX2 knockout mice and emphasize the importance of the peroxisome in this process.

We have identified beta-galactosidase activity in purified bovine rod outer segments (ROS), using rho-nitrophenyl-beta-D-galactopyranoside (PNPG) and chlorophenol red-beta-D-galactopyranoside (CPRG) as substrates. This glycosylhydrolase activity did not appear to represent contamination from other retinal subcellular fractions, based upon the relative specific activities of beta-galactosidase vs. other hydrolases (N-acetyl-beta-glucosaminidase, alpha- and beta-mannosidase, alpha-fucosidase, and acid phosphatase) in bovine retina and ROS homogenates. Using PNPG as a substrate, two pH optima were observed (at 3.5 and 5.5), while the hydrolysis of CPRG exhibited a single, broad pH optimum centered at 5.5. In contrast, hydrolysis of PNPG and CPRG by retinal homogenates exhibited single pH optima, at 3.5 and 5.5., respectively. ROS beta-galactosidase activity increased linearly with time, temperature, and protein concentration, and obeyed Michaelis-Menten kinetics with both substrates. For PNPG, Vmax approximately 88 nmol/h/mg protein and the apparent Km approximately 147 microM. For CPRG, Vmax approximately 33 nmol/h/mg protein and the apparent Km approximately 50 microM. ROS beta-galactosidase activity was affected by carbohydrates and their derivatives: glucose, fucose, sucrose, maltose and N-acetyl-galactosamine were found to stimulate the activity, while D-galactono-gamma-lactone and, to a lesser extent, D-galactose were inhibitory. The enzyme activity also was slightly stimulated by [Cl-] and markedly by dithiothreitol (DTT), while rho-chloro-mercuribenzoic acid (PCMB) and rho-hydroxymercuribenzoic acid (PHMB) inactivated the enzyme. In addition, the enzymatic activity was also found to be differentially sensitive to various anionic and nonionic detergents. However, n-octyl-beta-D-glucoside was slightly stimulatory.(ABSTRACT TRUNCATED AT 250 WORDS)