Michael J. Richards

Lipidomic Analysis of the Retina in a Rat Model of Smith-Lemli-Opitz Syndrome: Alterations in Docosahexaenoic Acid Content of Phospholipid Molecular Species

Smith–Lemli–Opitz syndrome (SLOS) is a complex hereditary disease caused by an enzymatic defect in the last step of cholesterol biosynthesis. Progressive retinal degeneration occurs in an AY9944‐induced rat model of SLOS, with biochemical and electroretinographic hallmarks comparable with the human disease. We evaluated alterations in the non‐sterol lipid components of the retina in this model, compared with age‐matched controls, using lipidomic analysis. The levels of 16:0–22:6 and 18:0–22:6 phosphatidylcholine molecular species in retinas were less by > 50% and > 33%, respectively, in rats treated for either 2 or 3 months with AY9944. Relative to controls, AY9944 treatment resulted in > 60% less di‐22:6 and > 15% less 18:0–22:6 phosphatidylethanolamine molecular species. The predominant phosphatidylserine (PS) molecular species in control retinas were 18:0–22:6 and di‐22:6; notably, AY9944 treatment resulted in > 80% less di‐22:6 PS, relative to controls. Remarkably, these changes occurred in the absence of n3 fatty acid deficiency in plasma or liver. Thus, the retinal lipidome is globally altered in the SLOS rat model, relative to control rats, with the most profound changes being less phosphatidylcholine, phosphatidylethanolamine, and PS molecular species containing docosahexaenoic acid (22:6). These findings suggest that SLOS may involve additional metabolic compromise beyond the primary enzymatic defect in the cholesterol pathway.

Disturbed cholesterol homeostasis in a peroxisome-deficient PEX2 knockout mouse model.

ABSTRACT We evaluated the major pathways of cholesterol regulation in the peroxisome-deficient PEX2−/− mouse, a model for Zellweger syndrome. Zellweger syndrome is a lethal inherited disorder characterized by severe defects in peroxisome biogenesis and peroxisomal protein import. Compared with wild-type mice, PEX2 −/− mice have decreased total and high-density lipoprotein cholesterol levels in plasma. Hepatic expression of the SREBP-2 gene is increased 2.5-fold in PEX2 −/− mice and is associated with increased activities and increased protein and expression levels of SREBP-2-regulated cholesterol biosynthetic enzymes. However, the upregulated cholesterogenic enzymes appear to function with altered efficiency, associated with the loss of peroxisomal compartmentalization. The rate of cholesterol biosynthesis in 7- to 9-day-old PEX2 −/− mice is markedly increased in most tissues, except in the brain and kidneys, where it is reduced. While the cholesterol content of most tissues is normal in PEX2 −/− mice, in the knockout mouse liver it is decreased by 40% relative to that in control mice. The classic pathway of bile acid biosynthesis is downregulated in PEX2 −/− mice. However, expression of CYP27A1, the rate-determining enzyme in the alternate pathway of bile acid synthesis, is upregulated threefold in the PEX2 −/− mouse liver. The expression of hepatic ATP-binding cassette (ABC) transporters (ABCA1 and ABCG1) involved in cholesterol efflux is not affected in PEX2 −/− mice. These data illustrate the diversity in cholesterol regulatory responses among different organs in postnatal peroxisome-deficient mice and demonstrate that peroxisomes are critical for maintaining cholesterol homeostasis in the neonatal mouse.

Peroxisome Deficiency Causes a Complex Phenotype because of Hepatic SREBP/Insig Dysregulation Associated with Endoplasmic Reticulum Stress

Regulation of hepatic cholesterol biosynthesis, lipogenesis, and insulin signaling intersect at the transcriptional level by control of SREBP and Insig genes. We previously demonstrated that peroxisome-deficient PEX2-/- mice activate SREBP-2 pathways but are unable to maintain normal cholesterol homeostasis. In this study, we demonstrate that oral bile acid treatment normalized hepatic and plasma cholesterol levels and hepatic cholesterol synthesis in early postnatal PEX2 mutants, but SREBP-2 and its target gene expressions remained increased. SREBP-2 pathway induction was also observed in neonatal and longer surviving PEX2 mutants, where hepatic cholesterol levels were normal. Abnormal expression patterns for SREBP-1c and Insig-2a, and novel regulation of Insig-2b, further demonstrate that peroxisome deficiency widely affects the regulation of related metabolic pathways. We have provided the first demonstration that peroxisome deficiency activates hepatic endoplasmic reticulum (ER) stress pathways, especially the integrated stress response mediated by PERK and ATF4 signaling. Our studies suggest a mechanism whereby ER stress leads to dysregulation of the endogenous sterol response mechanism and concordantly activates oxidative stress pathways. Several metabolic derangements in peroxisome-deficient PEX2-/- liver are likely to trigger ER stress, including perturbed flux of mevalonate metabolites, altered bile acid homeostasis, changes in fatty acid levels and composition, and oxidative stress.

Recruitment of peripheral mononuclear cells by mammalian collagenase digests of type I collagen.

Type I collagen is highly susceptible to proteolytic cleavage by neutral mammalian collagenase. Following an initial site specific cleavage of the substrate, two characteristic products are generated, TCA and TCB. These two products then spontaneously denature and are degraded into multiple smaller molecular weight peptides. We prepared TCA and TCB from native type I collagen by the action of rat uterine fibroblast neutral collagenase. In addition we prepared denatured type I alpha chains and exposed them to the action of collagenase under controlled conditions in order to generate small molecular weight peptides. We then examined intact type I collagen, TCA and TCB and type I gelatin peptides for chemotactic activity in a Boyden chamber assay using both human peripheral monocytes and polymorphonuclear leucocytes as target cells. Intact type I collagen, while chemotactic for neutrophils, failed to elicit any chemotactic response in mononuclear cells. In addition, the results demonstrate an absence of any detectable chemotactic activity for either TCA or TCB when human peripheral monocytes were used as the target cells. However, type I collagen peptides demonstrated chemotactic activity for peripheral monocytes. Maximum cell migration was found with digests which had been exposed to neutral mammalian collagenase for three to four hours. No chemotactic activity was found using the same peptides, when neutrophils were used as the target cells. The data strongly suggest that chemotactic activity for mononuclear cells, normally suppressed in intact type I collagen, is revealed and/or activated by neutral collagenase digestion. Conversely, chemotactic activity for neutrophils is lost when intact type I collagen is digested into smaller molecular weight fragments.(ABSTRACT TRUNCATED AT 250 WORDS)

Partial Rescue of Retinal Function and Sterol Steady-State in a Rat Model of Smith-Lemli-Opitz Syndrome

The Smith-Lemli-Opitz syndrome (SLOS) is the first-described in a growing family of hereditary defects in cholesterol biosynthesis, and presents with a spectrum of serious abnormalities, including multiple dysmorphologies, failure to thrive, cognitive and behavioral impairments, and retinopathy. Using a pharmacologically induced rat model of SLOS that exhibits key hallmarks of the disease, including progressive retinal degeneration and dysfunction, we show that a high-cholesterol diet can substantially correct abnormalities in retinal sterol composition, with concomitant improvement of visual function, particularly within the cone pathway. Although histologic degeneration still occurred, a high-cholesterol diet reduced the number of pyknotic photoreceptor nuclei, relative to animals on a cholesterol-free diet. These findings demonstrate that cholesterol readily crosses the blood-retina barrier (unlike the blood-brain barrier) and suggest that cholesterol supplementation may be efficacious in treating SLOS-associated retinopathy.

Cholesterol synthesis in the vertebrate retina: effects of U18666A on rat retinal structure, photoreceptor membrane assembly, and sterol metabolism and composition.

Treatment of neonatal rats with U18666A, an inhibitor of desmosterol Δ24-reductase, results in accumulation of desmosterol (Δ5,24) and depletion of cholesterol (Δ5) in various bodily tissues and also causes cataracts. We evaluated the effects of U18666A on the sterol composition, de novo sterol synthesis, and histological structure of the retina. Neonatal Sprague-Dawley rats were injected subcutaneously with U18666A (15 mg/kg, in olive oil) every other day from birth through 3 wk of age; in parallel, control rats received olive oil alone. At 21 d, treated and control groups each were subdivided into two groups: one group of each was injected intravitreally with [3H]acetate; retinas were removed 20 h later and non-saponifiable lipids (NSL) were analyzed by radio-high-performance liquid chromatography. The other group was injected intravitreally with [3H]leucine; 4 d later, one eye of each animal was evaluated by light and electron microscopy and light microscopic autoradiography, while contralateral retinas and rod outer segment (ROS) membranes prepared thereform were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis/fluorography. In the treated group, the Δ5/Δ5,24 mole ratio of retinas was ca. 1.0, and >88% of the NSL radioactivity was in Δ5,24; in contrast, control retinas had Δ5/Δ5,24 >170, with >80% of the NSL radioactivity in Δ5. Retinal histology, ultrastructure, ROS renewal rates, and rhodopsin synthesis and intracellular trafficking were comparable in both treated and control animals. These results suggest that desmosterol can either substitute functionally for cholesterol in the retina or it can complement subthreshold levels of cholesterol by sterol synergism.

Retinal structure and function in an animal model that replicates the biochemical hallmarks of desmosterolosis

Desmosterolosis is a rare, autosomal recessive, human disease characterized by multiple congenital anomalies in conjunction with grossly elevated levels of desmosterol and markedly reduced levels of cholesterol in all bodily tissues. Herein, we evaluated retinal sterol composition, histology, and electrophysiological function in an animal model that exhibited the biochemical features of desmosterolosis, produced by treating pregnant rats and their progeny with U18666A, an inhibitor of desmosterol reductase. Treated rats had cataracts, were substantially smaller, and had markedly high levels of desmosterol and profoundly low levels of cholesterol in their retinas and other tissues compared to age-matched controls. However, their retinas were histologically normal and electrophysiologically functional. These results suggest that desmosterol may be able to replace cholesterol in the retina, both structurally and functionally. These findings are discussed in the context of “sterol synergism.”

Peroxisome deficiency-induced ER stress and SREBP-2 pathway activation in the liver of newborn PEX2 knock-out mice

Disruption of the Pex2 gene leads to peroxisome deficiency and widespread metabolic dysfunction. We previously demonstrated that peroxisomes are critical for maintaining cholesterol homeostasis, using peroxisome-deficient Pex2(-/-) mice on a hybrid Swiss Webster×129S6/SvEv (SW/129) genetic background. Peroxisome deficiency activates hepatic endoplasmic reticulum (ER) stress pathways, leading to dysregulation of the endogenous sterol response mechanism. Herein, we demonstrate a more profound dysregulation of cholesterol homeostasis in newborn Pex2(-/-) mice congenic on a 129S6/SvEv (129) genetic background, and substantial differences between newborn versus postnatal Pex2(-/-) mice in factors that activate ER stress. These differences extend to relationships between activation of genes regulated by SREBP-2 versus PPARα. The SREBP-2 pathway is induced in neonatal Pex2(-/-) livers from 129 and SW/129 strains, despite normal hepatic cholesterol levels. ER stress markers are increased in newborn 129 Pex2(-/-) livers, which occurs in the absence of hepatic steatosis or accumulation of peroxins in the ER. Moreover, the induction of SREBP-2 and ER stress pathways is independent of PPARα activation in livers of newborn 129 and SW/129 Pex2(-/-) mice. Two-week-old wild-type mice treated with the peroxisome proliferator WY-14,643 show strong induction of PPARα-regulated genes and decreased expression of SREBP-2 and its target genes, further demonstrating that SREBP-2 pathway induction is not dependent on PPARα activation. Lastly, there is no activation of either SREBP-2 or ER stress pathways in kidney and lung of newborn Pex2(-/-) mice, suggesting a parallel induction of these pathways in peroxisome-deficient mice. These findings establish novel associations between SREBP-2, ER stress and PPARα pathway inductions.

Benefits from incorporation of combined cycle propulsion

Abstract The X-33 program was initiated to develop a testbed for integrated RLV technologies that pave the way for a full scale development of a launch vehicle (Venture Star). Within the Nasa Future X Trailblazer program there is an Upgrade X-33 that focuses on materials and upgrades. The authors propose that the most significant gains can be realized by changing the propulsion cycle, not materials. The cycles examined are rocket cycles, with the combustion in the rocket motor. Specifically, these rocket cycles are: turbopump, topping, expander, air augmented, air augmented ram, LACE and deeply cooled. The vehicle size, volume, structural weight remain constant. The system and propellant tank weights vary with the propulsion system cycle. A reduction in dry weight, made possible by a reduced propellant tank volume, was converted into payload weight provided sufficient volume was made available by the propellant reduction. This analysis was extended to Venture Star for selected engine cycles. The results show that the X-33 test bed could carry a significant payload to LEO (10,000 Ib) and be a valuable test bed in developing a frequent flight to LEO capability. From X-33 published information the maximum speed is about 15,000 ft/sec. With a LACE rocket propulsion system Venture Star vehicle could be sized to a smaller vehicle with greater payload than the Venture Star baseline. Vehicle layout and characteristics were obtained from: http:// www.venturestar.com .

Lipid-derived and other oxidative modifications of retinal proteins in a rat model of Smith-Lemli-Opitz syndrome

ABSTRACT Oxidative modification of proteins can perturb their structure and function, often compromising cellular viability. Such modifications include lipid‐derived adducts (e.g., 4‐hydroxynonenal (HNE) and carboxyethylpyrrole (CEP)) as well as nitrotyrosine (NTyr). We compared the retinal proteome and levels of such modifications in the AY9944‐treated rat model of Smith‐Lemli‐Opitz syndrome (SLOS), in comparison to age‐matched controls. Retinas harvested at 3 months of age were either subjected to proteomic analysis or to immuno‐slot blot analysis, the latter probing blots with antibodies raised against HNE, CEP, and NTyr, followed by quantitative densitometry. HNE modification of retinal proteins was markedly (>9‐fold) higher in AY9944‐treated rats compared to controls, whereas CEP modification was only modestly (≤2‐fold) greater, and NTyr modification was minimal and exhibited no difference as a function of AY9944 treatment. Anti‐HNE immunoreactivity was greatest in the plexiform and ganglion cell layers, but also present in the RPE, choroid, and photoreceptor outer segment layer in AY9944‐treated rats; control retinas showed minimal HNE labeling. 1D‐PAGE/Western blot analysis of rod outer segment (ROS) membranes revealed HNE modification of both opsin and &bgr;‐transducin. Proteomic analysis revealed the differential expression of several retinal proteins as a consequence of AY9944 treatment. Upregulated proteins included those involved in chaperone/protein folding, oxidative and cellular stress responses, transcriptional regulation, and energy production. &bgr;A3/A1 Crystallin, which has a role in regulation of lysosomal acidification, was down‐regulated. Hence, oxidative modification of retinal proteins occurs in the SLOS rat model, in addition to the previously described oxidation of lipids. The results are discussed in the context of the histological and physiological changes that occur in the retina in the SLOS rat model. HighlightsOxidative modification of retinal proteins (e.g., opsin, &bgr;‐transducin) occurs in the AY9944‐induced rat model of SLOS.4‐Hydroxy‐2‐nonenal (HNE) protein adducts are significantly elevated in this rat model of SLOS.Expression levels of multiple proteins were altered by AY9944 treatment, notably stress‐related proteins.