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The current study aimed to determine the protective effect of AY9944 related to Caveolin-1 and Claudin-5 role in lipid raft, which can rescue the blood-brain barrier from enhanced permeability. Therefore, in vivo analyses were performed following ischemia in normal, ischemic, and AY9944-treated animal groups. The results revealed that AY9944 reduced the infarct size, edema, and brain water content. The extravasation of Alb-Alexa 594 and biocytin-TMR was minimum in the AY9944-treated animals. The results showed a significant decrease in the expression level of Caveolin-1 over 8 h and 48 h and a remarkable increase in the level of Claudin-5 over 48 h following ischemia in AY9944-treated animals. Molecular docking simulation demonstrated that AY9944 exerts a possible protective role via attenuating the interaction of the Caveolin-1 and cholesterol in lipid raft. These findings point out that AY9944 plays a protective role in stroke by means of blood-brain barrier preservation. Proper neural function essentially needs a constant homeostatic brain environment which is provided by the blood-brain barrier. Rescuing blood-brain barrier from enhanced permeability via inducing the protective effect of AY9944 related to caveolin-1 and claudin-5 role in lipid raft was the aim of the current study., (© 2020. Springer Science+Business Media, LLC, part of Springer Nature.)

Neuronal regeneration in the injured central nervous system is hampered by multiple extracellular proteins. These proteins exert their inhibitory action through interactions with receptors that are located in cholesterol rich compartments of the membrane termed lipid rafts. Here we show that cholesterol-synthesis inhibition prevents the association of the Neogenin receptor with lipid rafts. Furthermore, we show that cholesterol-synthesis inhibition enhances axonal growth both on inhibitory -myelin and -RGMa substrates. Following optic nerve injury, lowering cholesterol synthesis with both drugs and siRNA-strategies allows for robust axonal regeneration and promotes neuronal survival. Cholesterol inhibition also enhanced photoreceptor survival in a model of Retinitis Pigmentosa. Our data reveal that Lovastatin leads to several opposing effects on regenerating axons: cholesterol synthesis inhibition promotes regeneration whereas altered prenylation impairs regeneration. We also show that the lactone prodrug form of lovastatin has differing effects on regeneration when compared to the ring-open hydroxy-acid form. Thus the association of cell surface receptors with lipid rafts contributes to axonal regeneration inhibition, and blocking cholesterol synthesis provides a potential therapeutic approach to promote neuronal regeneration and survival in the diseased Central Nervous System. SIGNIFICANCE STATEMENT: Statins have been intensively used to treat high levels of cholesterol in humans. However, the effect of cholesterol inhibition in both the healthy and the diseased brain remains controversial. In particular, it is unclear whether cholesterol inhibition with statins can promote regeneration and survival following injuries. Here we show that late stage cholesterol inhibition promotes robust axonal regeneration following optic nerve injury. We identified distinct mechanisms of action for activated vs non-activated Lovastatin that may account for discrepancies found in the literature. We show that late stage cholesterol synthesis inhibition alters Neogenin association with lipid rafts, thereby i) neutralizing the inhibitory function of its ligand and ii) offering a novel opportunity to promote CNS regeneration and survival following injuries., (Copyright © 2021 The Author(s). Published by Elsevier Inc. All rights reserved.)

Follicular fluid meiosis-activating sterol (FF-MAS) exerts beneficial effects on the meiotic resumption of mammalian oocytes and their subsequent early embryonic development, but the signaling pathway underlying these effects has not been elucidated. Therefore, the objective of the present study was to investigate whether the mitogen-activated protein kinase (MAPK) pathway is involved in the FF-MAS-induced in vitro resumption of meiosis in porcine oocytes. Porcine cumulus oocyte complexes (COCs) were allocated in several groups cultured in TCM-199 medium with different concentration of AY 9944-A-7 (20, 30, 40 μmol/L) or ketoconazole (20 μmol/L) to increase or decrease endogenous accumulation of FF-MAS. Each experimental condition was repeated at least six times. After maturation for 44 h, the resumption of meiosis was assessed by the frequency of germinal vesicle breakdown (GVBD) and the first polar body (PBI) extrusion, The relative expressions of related genes in MAPK pathway [c-mos, mitogen-activated protein kinase kinase (MEK) and extracellular signal-regulated kinase 1/2 (ERK1/2)] at both transcriptional and translational levels were detected to investigate the kinetic trend of expression throughout oocyte maturation in vitro in response to the addition of AY 9944-A-7 or ketoconazole to the maturation medium. Results indicated that AY 9944-A-7 promoted, while ketoconazole inhibited, the in vitro maturation (IVM) of porcine oocytes. Relative expression of meiosis related genes was upregulated by AY 9944-A-7 and downregulated by ketoconazole. With extended culturing time, c-mos mRNA expression levels reached their peak at 12 h of maturation and decreased gradually thereafter, while MEK, ERK1 and ERK2 expression increased after an initial decrease peaking at 44 h of culture in the AY 9944-A-7-group. And the trend of the protein expression of c-mos, MEK, ERK1/2 was basically consistent with the mRNA expression of these genes. These results imply that the endogenous accumulation of FF-MAS is beneficial to resumption of meiosis in porcine oocytes and that MAPK signaling is involved in FF-MAS-induced meiotic resumption., Competing Interests: Declaration of competing interest All the authors involved in this work declare that there are no financial and personal relationships with other people or organizations that can inappropriately influence our work, and there are no financial or other contractual agreements that might cause a conflict of interest., (Copyright © 2019 Elsevier Inc. All rights reserved.)

Melanin in the epidermis is known to ultimately regulate human skin pigmentation. Recently, we exploited a phenotypic-based screening system composed of ex vivo human skin cultures to search for effective materials to regulate skin pigmentation. Since a previous study reported the potent inhibitory effect of metformin on melanogenesis, we evaluated several biguanide compounds. The unexpected effect of phenformin, once used as an oral anti-diabetic drug, on cutaneous darkening motivated us to investigate its underlying mechanism utilizing a chemical genetics approach, and especially to identify alternatives to phenformin because of its risk of severe lactic acidosis. Chemical pull-down assays with phenformin-immobilized beads were performed on lysates of human epidermal keratinocytes, and subsequent mass spectrometry identified 7-dehydrocholesterol reductase (DHCR7). Consistent with this, AY9944, an inhibitor of DHCR7, was found to decrease autophagic melanosome degradation in keratinocytes and to intensely darken skin in ex vivo cultures, suggesting the involvement of cholesterol biosynthesis in the metabolism of melanosomes. Thus, our results validated the combined utilization of the phenotypic screening system and chemical genetics as a new approach to develop promising materials for brightening/lightening and/or tanning technologies.

Absence-epileptic seizures appear in the EEG as Spike and Wave Discharges (SWDs). Typical SWDs develop spontaneously in WAG/Rij rats, an inbred Wistar strain. Atypical SWDs however were reported in studies in which the cholesterol synthesis inhibitor AY-9944 was administered to neonatal Wistar rats, causing absence-like seizures later in life. Atypical SWDs seemed to differ from typical SWDs in 3 aspects: lower peak frequency, longer duration, and involvement of the hippocampus. The aim of the present study was to investigate the effect of AY-9944 on typical SWDs. Male Wistar and WAG/Rij rats were injected with 7.5 mg/kg AY-9944 or saline postnatally. After 6 months, EEGs were recorded from the cortex and the hippocampus. Incidence, duration and peak frequency of the SWDs were determined. The SWD stopping probability was estimated by hazard rate analysis. Hippocampal involvement was assessed by cross correlation analysis of the hippocampus and cortex channels. The Wistar rats unexpectedly showed a high incidence of spontaneous SWDs. The AY-treatment increased the total SWD duration in both Wistar and WAG/Rij rats: the incidence was 1.6 times higher and the mean SWD duration was 1.4 times longer than in the saline-treated rats. The peak frequency of the SWDs did not change. The hazard rates were lower in the AY-treated rats, so some very long SWDs were observed. Cross correlations of spiky activity in the hippocampus pointed to volume conduction rather than to genuine SWD activity in this area. In summary, we found no indication that SWDs in AY-treated animals differ from typical SWDs. However, since saline-treated rats had many spontaneous SWDs, other rat strains might respond differently. With respect to the mechanism, the appearance of long SWDs suggests that the SWD stopping mechanism is affected by the treatment. We speculate that this effect is due to changes in the distribution of GABA-ergic and glutamatergic receptors in lipid rafts., (Copyright © 2019 Elsevier B.V. All rights reserved.)

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 β-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. βA3/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., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

Smith-Lemli-Opitz syndrome (SLOS) is an autosomal recessive human disease caused by mutations in the gene encoding 7-dehydrocholesterol (7DHC) reductase (DHCR7), resulting in abnormal accumulation of 7DHC and reduced levels of cholesterol in bodily tissues and fluids. A rat model of the disease has been created by treating normal rats with the DHCR7 inhibitor, AY9944, which causes progressive, irreversible retinal degeneration. Herein, we review the features of this disease model and the evidence linking 7DHC-derived oxysterols to the pathobiology of the disease, with particular emphasis on the associated retinal degeneration. A recent study has shown that treating the rat model with cholesterol plus suitable antioxidants completely prevents the retinal degeneration. These findings are discussed with regard to their translational implications for developing an improved therapeutic intervention for SLOS over the current standard of care.

Colorectal cancer is one of the most common causes of cancer death worldwide. In patients with metastatic colorectal cancer, combination treatment with several anti-cancer drugs is employed and improves overall survival in some patients. Nevertheless, most patients with metastatic disease are not cured owing to the drug resistance. Cancer stem cells are known to regulate resistance to chemotherapy. In the previous study, we established a novel three-dimensional organoid culture model from tumor colorectal tissues of human patients using an air-liquid interface (ALI) method, which contained numerous cancer stem cells and showed resistance to 5-fluorouracil (5-FU) and Irinotecan. Here, we investigate which inhibitor for stem cell-related signal improves the sensitivity for anti-cancer drug treatment in tumor ALI organoids. Treatment with Hedgehog signal inhibitors (AY9944, GANT61) decreases the cell viability of organoids compared with Notch (YO-01027, DAPT) and Wnt (WAV939, Wnt-C59) signal inhibitors. Combination treatment of AY9944 or GANT61 with 5-FU, Irinotecan or Oxaliplatin decreases the cell viability of tumor organoids compared with each anti-cancer drug alone treatment. Treatment with AY9944 or GANT61 inhibits expression of stem cell markers c-Myc, CD44 and Nanog, likely through the decrease of their transcription factor, GLI-1 expression. Combination treatment of AY9944 or GANT61 with 5-FU or Irinotecan also prevents colony formation of colorectal cancer cell lines HCT116 and SW480. These findings suggest that Hedgehog signals mediate anti-cancer drug resistance in colorectal tumor patient-derived ALI organoids and that the inhibitors are useful as a combinational therapeutic strategy against colorectal cancer., Competing Interests: The authors declare no conflict of interest.

Sterol intermediates of the cholesterol biosynthetic pathway have drawn attention for novel biological activities. Follicular fluid meiosis activating sterol (FF-MAS) is a LXRα ligand and a potential modulator of physiologic processes regulated by nuclear receptors, such as lipid homeostasis and cell proliferation. In this work, we established a model to selectively accumulate FF-MAS in HepG2 cells, by using a combination of the inhibitors AY9944 and 17-hydroxyprogesterone to block C14-sterol reductases and the downstream C4-demethylase complex. We investigated the effects produced by altered levels of cholesterol biosynthesis intermediates, in order to dissect their influence on LXRα signaling. In particular, endogenously accumulated FF-MAS was able to modulate the expression of key genes in cholesterol metabolism, to activate LXRα nuclear signaling resulting in increased lipogenesis, and to inhibit HepG2 cells proliferation. Moreover, a fluorescent ester derivative of FF-MAS localized in nuclear lipid droplets, suggesting a role for these organelles in the storage of signaling lipids interacting with nuclear partners., (Copyright © 2017 Elsevier B.V. All rights reserved.)

In this study, we aim to identify environmental molecules that can inhibit cholesterol biosynthesis, potentially leading to the same biochemical defects as observed in cholesterol biosynthesis disorders, which are often characterized by congenital malformations and developmental delay. Using the Distributed Structure-Searchable Toxicity (DSSTox) Database Network developed by EPA, we first carried out in silico screening of environmental molecules that display structures similar to AY9944, a known potent inhibitor of 3β-hydroxysterol-Δ(7)-reductase (DHCR7)-the last step of cholesterol biosynthesis. Molecules that display high similarity to AY9944 were subjected to test in mouse and human neuroblastoma cells for their effectiveness in inhibiting cholesterol biosynthesis by analyzing cholesterol and its precursor using gas chromatography-mass spectrometry. We found that a common disinfectant mixture, benzalkonium chlorides (BACs), exhibits high potency in inhibiting DHCR7, as suggested by greatly elevated levels of the cholesterol precursor, 7-dehydrocholesterol (7-DHC). Subsequent structure-activity studies suggested that the potency of BACs as Dhcr7 inhibitors decrease with the length of their hydrocarbon chain: C10 > C12 ≫ C14 > C16. Real-time qPCR analysis revealed upregulation of the genes related to cholesterol biosynthesis and downregulation of the genes related to cholesterol efflux, suggesting a feedback response to the inhibition. Furthermore, an oxidative metabolite of 7-DHC that was previously identified as a biomarker in vivo was also found in cells exposed to BACs by liquid chromatography-mass spectrometry. Our findings suggest that certain environmental molecules could potently inhibit cholesterol biosynthesis, which could be a new link between environment and developmental disorders., (© The Author 2016. Published by Oxford University Press on behalf of the Society of Toxicology. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.)

Amyloid-β (Aβ) is one of major molecules contributing to the pathogenesis of Alzheimer's disease (AD). Aβ is derived from amyloid-β protein precursor (AβPP) through sequential cleavages by β- and γ-secretases. Regulation of these components is thought to be an important factor in Aβ generation during the pathogenesis of AD. AβPP, β-secretase, and γ-secretase reside in lipid rafts, where cholesterol regulates the integrity and flexibility of membrane proteins and Aβ is generated. However, the relationship between cholesterol and Aβ generation is controversial. In this study, we aimed to elucidate the direct effects of cholesterol depletion on AβPP processing using AY9944, which blocks the last step of cholesterol biosynthesis and thus minimizes the unknown side effects of upstream inhibitors, such as HMG-CoA reductase inhibitors. Treatment with AY9944 decreased γ-secretase activity and Aβ generation. These results suggested that changes in membrane composition by lowering cholesterol with AY9944 affected γ-secretase activity and Aβ generation, which is associated with AD pathogenesis.

Follicular fluid meiosis-activating sterol (FF-MAS), an intermediate in the cholesterol biosynthetic pathway, has been identified as a compound that induces the resumption of meiosis in mammalian oocyte. Follicular fluid meiosis-activating sterol is converted to testis meiosis-activating sterol by a sterol Δ14-reductase. An inhibitor of Δ14-reductase and Δ7-reductase, AY9944 A-7, causes accumulation of FF-MAS by inhibiting its metabolism. The objective of this research was to investigate the specific contribution of AY9944 A-7 on gonadotropin-induced meiotic resumption and its interactive effects with FSH or LH on meiotic maturation of oocytes and preimplantation development of parthenogenetic embryo in sheep by addition of AY9944 A-7 during IVM to cause accumulation of FF-MAS. First, ovine cumulus-oocyte complexes (COCs) were cultured in the presence of FSH (10 μg/mL), LH (10 μg/mL), AY9944 A-7 (20 μmol/L), FSH (10 μg/mL)+AY9944 A-7 (20 μmol/L), or LH (10 μg/ml) + AY9944 A-7 (20 μmol/L) with an inhibitor hypoxanthine (Hx) to prevent spontaneous meiosis of oocytes. The resumption of meiosis was assessed by the frequency of germinal vesicle breakdown and the first polar body (PBI) extrusion. The kinetics of gonadotropin and AY9944 A-7-induced meiotic resumption in vitro was also evaluated in the study. The numbers of oocytes resuming meiosis and undergoing germinal vesicle breakdown were counted after the COCs were cultured for 2, 4, 8, 12, 16, 20, and 24 hours. Matured oocytes extruding PBI were selected for parthenogenetic activation, and the percentages developing to the two-cell stage and blastocyst stage were recorded as indicators of parthenogenetic embryo developmental competence. It was observed that FSH could induce the resumption of meiosis of ovine COCs cultured in the presence of Hx, but LH could not. AY9944 A-7 had a synergistic effect with FSH on nuclear maturation and developmental competence of embryos produced by parthenogenetic activation, whereas it had no added advantage on LH action. However, the kinetics of meiotic resumption after AY9944 A-7 stimulation was remarkably delayed when compared with FSH-induced maturation. In conclusion, the current study suggested that AY9944 A-7 supplementation in IVM medium optimized the beneficial effects of FSH on meiotic maturation of ovine oocytes and subsequent developmental competence of embryos produced by parthenogenetic activation. This work had important potential for developing a novel technique in IVM of ovine oocytes., (Copyright © 2015 Elsevier Inc. All rights reserved.)

Follicular fluid meiosis-activating sterol (FF-MAS), an intermediate in the cholesterol biosynthetic pathway, has been identified as a compound that induces the resumption of meiosis in mammalian oocyte. FF-MAS is converted to testis meiosis-activating sterol by a sterol Δ14-reductase. An inhibitor of Δ14-reductase and Δ7-reductase, AY9944 A-7, causes accumulation of FF-MAS by inhibiting its metabolism. The objective of this study was to determine the effects of AY9944 A-7 supplementation to oocyte maturation media on prepubertal sheep oocyte meiotic resumption and subsequent preimplantation development of embryos. Prepubertal sheep oocytes isolated at the germinal vesicle stage from their follicles were cultured with 0, 10, 20, 30, and 40 μM AY9944 A-7 for 24 hours in media with or without a meiotic inhibitor hypoxanthine (Hx, 4 mM). The resumption of meiosis was assessed by the frequency of germinal vesicle breakdown and the first polar body (PBI) extrusion. After maturation for 24 hours, oocytes with PBI were inseminated in vitro, and the percentages developing to the two-cell stage and blastocyst stage were measured as indicators of early embryonic developmental competence. AY9944 A-7 induced maturation of sheep cumulus-oocyte complexes with optimal concentrations of 10 and 20 μM both in Hx-inhibited meiotic maturation and spontaneous maturation, whereas AY9944 A-7 with any concentrations had no significant effect on that of denuded oocytes and split cumulus-oocyte complexes. Furthermore, maturing oocytes treated with either 10 or 20 μM AY9944 A-7 dramatically increased the percentages of ovine embryos developing to the two-cell stage and blastocyst stage. Higher concentrations of AY9944 A-7, 30 and 40 μM, were detrimental to oocytes and led to their degeneration. The present findings indicated for the first time that AY9944 A-7 was not only able to promote meiotic maturation, both Hx-inhibited and spontaneous, but also enhanced preimplantation developmental competence of prepubertal sheep oocytes maturing in vitro., (Copyright © 2013 Elsevier Inc. All rights reserved.)

Treatment of Sprague-Dawley rats with AY9944, an inhibitor of 3β-hydroxysterol-Δ(7)-reductase (Dhcr7), leads to elevated levels of 7-dehydrocholesterol (7-DHC) and reduced levels of cholesterol in all biological tissues, mimicking the key biochemical hallmark of Smith-Lemli-Opitz syndrome (SLOS). Fourteen 7-DHC-derived oxysterols previously have been identified as products of free radical oxidation in vitro; one of these oxysterols, 3β,5α-dihydroxycholest-7-en-6-one (DHCEO), was recently identified in Dhcr7-deficient cells and in brain tissues of Dhcr7-null mouse. We report here the isolation and characterization of three novel 7-DHC-derived oxysterols (4α- and 4β-hydroxy-7-DHC and 24-hydroxy-7-DHC) in addition to DHCEO and 7-ketocholesterol (7-kChol) from the brain tissues of AY9944-treated rats. The identities of these five oxysterols were elucidated by HPLC-ultraviolet (UV), HPLC-MS, and 1D- and 2D-NMR. Quantification of 4α- and 4β-hydroxy-7-DHC, DHCEO, and 7-kChol in rat brain, liver, and serum were carried out by HPLC-MS using d(7)-DHCEO as an internal standard. With the exception of 7-kChol, these oxysterols were present only in tissues of AY9944-treated, but not control rats, and 7-kChol levels were markedly (>10-fold) higher in treated versus control rats. These findings are discussed in the context of the potential involvement of 7-DHC-derived oxysterols in the pathogenesis of SLOS.

Recently we identified GANT61, a small-molecule antagonist of Gli transcription factors, which are the final effectors of the mammalian Hedgehog (HH) signaling pathway. Here we describe a diamine substructure of GANT61 that carries the biological activity and show that this part of the molecule is structurally related to trans-1,4-bis(2-chlorobenzaminomethyl)cyclohexane dihydrochloride (AY9944), an inhibitor of the enzymatic activity and transcriptional inducer of 7-dehydrocholesterol-reductase (Dhcr7, EC 1.3.1.21). Treatment of cells with the GANT61 diamine, AY9944, or overexpression of DHCR7 results in the attenuation of Smoothened-dependent and -independent HH signaling. Whereas GANT61 function is independent of Dhcr7, AY9944 does require up-regulation of endogenous Dhcr7. In line with these findings, Dhcr7-modulating antipsychotic (clozapine, chlorpromazine, haloperidol) and antidepressant (imipramine) drugs regulate HH signaling in vitro and in vivo. Modulation of HH signaling may represent a hitherto undiscovered biological (side) effect of therapeutics used to treat schizophrenia and depression.

Lipid rafts are plasma membrane microdomains that are highly enriched with cholesterol and sphingolipids and in which various receptors and other proteins involved in signal transduction reside. In the present work, we analyzed the effect of cholesterol biosynthesis inhibition on lipid raft/caveolae composition and functionality and assessed whether sterol precursors of cholesterol could substitute for cholesterol in lipid rafts/caveolae. 3T3-L1 preadipocytes were treated with distal inhibitors of cholesterol biosynthesis or vehicle (control) and then membrane rafts were isolated by sucrose density gradient centrifugation. Inhibition of cholesterol biosynthesis with either SKF 104976, AY 9944, 5,22-cholestadien-3beta-ol or triparanol, which inhibit different enzymes on the pathway, led to a marked reduction in cholesterol content and accumulation of different sterol intermediates in both lipid rafts and non-raft domains. These changes in sterol composition were accompanied by disruption of lipid rafts, with redistribution of caveolin-1 and Fyn, impairment of insulin-Akt signaling and the inhibition of insulin-stimulated glucose transport. Cholesterol repletion abrogated the effects of cholesterol biosynthesis inhibitors, reflecting they were specific. Our results show that cholesterol is required for functional raft-dependent insulin signaling.

In this study, we evaluate the effects of (3beta)-3-[2-(diethylamino)ethoxy]androst-5-en-17-one dihydrochloride (U18666A), a cholesterol synthesis/transporter inhibitor, on selected human neuronal nicotinic acetylcholine receptors (nAChRs) heterologously expressed in the SH-EP1 cell line using whole-cell patch-clamp recordings. The results indicate that with 2-min pretreatment, U18666A inhibited different nAChR subtypes with a rank-order of potency (IC(50) of whole-cell peak current): alpha4beta2 (8.0 +/- 3.0 nM) > alpha3beta2 (1.7 +/- 0.4 microM) > alpha4beta4 (26 +/- 7.2 microM) > alpha7 (> 100 microM), suggesting this compound is more selective to alpha4beta2-nAChRs. Thus, the pharmacological profiles and mechanisms of U18666A acting on alpha4beta2-nAChRs were investigated in detail. U18666A suppresses both peak and steady state components of whole-cell currents mediated by human alpha4beta2-nAChRs in response to nicotine. In nicotine-induced concentration-response curves, U18666A reduces nicotine-induced current at maximally effective agonist concentrations without influencing nicotine's EC(50) value, suggesting a non-competitive inhibition. U18666A-induced inhibition of nAChR function is concentration-, voltage-, and use-dependent, suggesting an open channel block. Taken into consideration of approximately 10 000-fold enhancement of the potency of U18666A after 2-min pre-treatment, this compound also likely inhibits alpha4beta2-nAChRs through a close channel block. In addition, the U18666A-induced inhibition in alpha4beta2-nAChRs is not mediated by either increased receptor endocytosis or altered cell cholesterol. These data indicate that U18666A is a potent antagonist of alpha4beta2-nAChRs and may be useful as a tool in the functional characterization and pharmacological profiling of nAChRs, as well as a potential candidate for smoking cessation.

Although Caenorhabditis elegans lacks several components of the de novo sterol biosynthetic pathway, it requires sterols as essential nutrients. Supplemental cholesterol undergoes extensive enzymatic modification in C. elegans to form certain sterols of unknown function. Since sterol metabolism in C. elegans differs from that in other species, such as mammals and yeast, it is important to examine how sterols regulate worm physiology. To examine the functions of sterols in C. elegans, a sterol-feeding experiment was carried out and several critical parameters, such as brood size, growth rate, and life span, were measured. In addition, the change in lipid distribution in C. elegans can be both qualitatively and quantitatively determined by various methods, including staining and chromatographic techniques. Taken together, the effects of sterols on C. elegans are very prominent and can be easily assessed using the techniques described here.

Gonadotropic stimulation of meiotic resumption in mice is dependent upon mitogen-activated protein kinase (MAPK) activation in the somatic compartment of the follicle. By contrast, spontaneous resumption of meiosis is independent of MAPK activation. In view of the suggested role of meiosis-activating sterol (MAS) in oocyte maturation we have (i) compared MAPK activation in rat preovulatory follicles stimulated by LH or by accumulation of endogenous MAS by using an inhibitor of MAS conversion, AY9944; (ii) examined whether stimulation of meiosis by MAS is dependent upon MAPK activation using denuded oocytes (DO) of Mos- null mice (hereafter Mos(-/-)) with oocytes unable to activate MAPK. Rat preovulatory follicles responded to LH or AY9944 stimulation by MAPK activation. Inhibition of MAPK phosphorylation blocked both LH- and AY9944 triggered resumption of meiosis. In mouse cumulus-enclosed oocytes (CEOs) and DOs AY9944 stimulated GVB in wild-type and Mos(-/-) mouse CEOs cultured with hypoxanthine (Hx). Addition of MAS or AY9944 to mouse DOs cultured with Hx induced resumption of meiosis only in wild-type and Mos(+/-) oocytes, but they were ineffective in Mos(-/-) oocytes. The observed sluggish activation of MAPK induced by AY9944 in rat follicle-enclosed oocytes (FEO) may cause the delay in meiotic resumption in response to MAS and AY9944 stimulation. Further, it is incompatible with the suggested role of MAS as an obligatory mediator of LH in the induction of meiotic maturation. MAPK/MOS activation, whether in the somatic compartment or in denuded oocytes, is required for MAS- like LH-, FSH-, or EGF-induced resumption of meiosis.

Smith-Lemli-Opitz syndrome (SLOS) is caused by an inherited defect in the last step in cholesterol (Chol) biosynthesis, leading to abnormal accumulation of 7-dehydrocholesterol and decreased Chol levels. Progressive retinal degeneration occurs in an animal model of SLOS, induced by treating rats with AY9944, a selective inhibitor of the enzyme affected in SLOS. Here we evaluated alterations in the biochemical and physical properties of retinal rod outer segment (ROS) membranes in this animal model. At 1 month of AY9944 treatment, there were modest alterations in fatty acid composition, but no significant differences in cis-parinaric acid (cPA) spectroscopic parameters in ROS membranes from treated versus control rats. However, at 3 months, ROS docosahexaenoic acid (DHA) content was dramatically reduced, and cPA fluorescence anisotropy values were decreased, relative to controls. Also, 1,6-diphenyl-1,3,5-hexatriene exhibited decreased rotational motion and increased orientational order in ROS membranes from 3 month-old AY9944-treated rats, relative to controls. No significant changes in protein:lipid ratios were observed; however, rhodopsin regenerability was compromised by 3 months of treatment. These findings are consistent with reduced ROS membrane fluidity in the SLOS rat model, relative to controls, primarily due to the dramatic reduction in membrane DHA levels, rather than altered sterol composition.

The Smith-Lemli-Opitz Syndrome (SLOS) is a congenital and developmental malformation syndrome associated with defective cholesterol biosynthesis. SLOS is clinically diagnosed by reduced plasma levels of cholesterol along with elevated levels of 7-dehydrocholesterol (and its positional isomer 8-dehydrocholesterol) and the ratio of their concentrations to that of cholesterol. Since SLOS is associated with neurological deformities and malfunction, exploring the function of neuronal receptors and their interaction with membrane cholesterol under these conditions assumes significance. We have earlier shown the requirement of membrane cholesterol for the ligand binding function of an important neurotransmitter G-protein coupled receptor, the serotonin(1A) receptor. In the present work, we have generated a cellular model of SLOS using CHO cells stably expressing the human serotonin(1A) receptor. This was achieved by metabolically inhibiting the biosynthesis of cholesterol, utilizing a specific inhibitor (AY 9944) of the enzyme required in the final step of cholesterol biosynthesis. We utilized this cellular model to monitor the function of the human serotonin(1A) receptor under SLOS-like condition. Our results show that ligand binding activity, G-protein coupling and downstream signaling of serotonin(1A) receptors are impaired in SLOS-like condition, although the membrane receptor level does not exhibit any reduction. Importantly, metabolic replenishment of cholesterol using serum partially restored the ligand binding activity of the serotonin(1A) receptor. These results are potentially useful in developing strategies for the future treatment of the disease since intake of dietary cholesterol is the only feasible treatment for SLOS patients.

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.

Background and Purpose: Excess brain extracellular glutamate induced by cerebral ischemia leads to neuronal death, mainly through overactivation of N-methyl-D-aspartate (NMDA) receptors. The cholesterol-lowering drugs statins have been reported to protect from NMDA-induced neuronal death but, so far, the mechanism underlying this protection remains unclear. Because NMDA receptors have been reported to be associated with the cholesterol-rich membrane domains known as lipid rafts, we have investigated the effect of treatments that deplete cholesterol levels on excitotoxicity and on association of NMDA receptors to lipid rafts., Methods: Primary neuronal cultures were pretreated with inhibitors of cholesterol synthesis and cholesterol, and NMDA-induced cell death was determined by measuring release of lactate dehydrogenase. Lipid raft fractions were isolated and Western blots were performed., Results: Treatment with the inhibitors of cholesterol synthesis simvastatin, which inhibits the first step of cholesterol synthesis, or AY9944, which inhibits the last step of cholesterol synthesis, protected neurons from NMDA-induced neuronal death by 70% and 54%, respectively. Treatment with these compounds reduced neuronal cholesterol levels by 35% and 13%, respectively. Simvastatin and AY9944 reduced the association of the subunit 1 of NMDA receptors (NMDAR1) to lipid rafts by 42% and 21%, respectively, and did not change total expression of NMDAR1. Addition of cholesterol reduced neuroprotection by statins and AY9944, and partially reverted the effect of simvastatin on the association of NMDAR1 to lipid rafts., Conclusions: These data demonstrate that reduction of cholesterol levels protects from NMDA-induced neuronal damage probably by reducing the association of NMDA receptors to lipid rafts.

Purpose: Typical absence seizures differ from atypical absence seizures in terms of semiology, EEG morphology, network circuitry, and cognitive outcome, yet have the same pharmacological profile. We have compared typical to atypical absence seizures, in terms of the recruitment of different brain areas. Our initial question was whether brain areas that do not display apparent paroxysmal discharges during typical absence seizures, are affected during the ictal event in terms of synchronized activity, by other, distant areas where seizure activity is evident. Because the spike-and-wave paroxysms in atypical absence seizures invade limbic areas, we then asked whether an alteration in inhibitory processes in hippocampi may be related to the spread seizure activity beyond thalamocortical networks, in atypical seizures., Methods: We used two models of absence seizures in rats: one of typical and the other of atypical absence seizures. We estimated phase synchronization, and evaluated inhibitory transmission using a paired-pulse paradigm., Results: In typical absence seizures, we observed an increase in synchronization between hippocampal recordings when spike-and-wave discharges occurred in the cortex and thalamus. This indicates that seizure activity in the thalamocortical circuitry enhances the propensity of limbic areas to synchronize, but is not sufficient to drive hippocampal circuitry into a full paroxysmal discharge. Lower paired-pulse depression was then found in hippocampus of rats that displayed atypical absence seizures., Conclusions: These observations suggest that circuitries in brain areas that do not display apparent seizure activity become synchronized as seizures occur within thalamocortical circuitry, and that a weakened hippocampal inhibition may predispose to develop synchronization into full paroxysms during atypical absence seizures.

In prion diseases, the normal cellular form of prion protein (PrP(C)) is converted into the disease-associated isoforms (PrP(Sc)) which accumulate in the infected tissues. Although the precise mechanism of this conversion remains unsolved, drugs of various categories have been reported to reduce the accumulation of PrP(Sc) in prion-infected cultured cells. We here show that AY-9944 (a 7-dehydrocholesterol reductase inhibitor) and U18666A (a 24-dehydrocholesterol reductase inhibitor) prevent PrP(Sc) from accumulating in prion-infected mouse neuroblastoma cells (ScN2a), with an ED50 of about 0.5 microM and 10 nM, respectively. In order to evaluate the efficacy of these two inhibitors in vivo, C57BL/6J mice inoculated with mouse-adapted scrapie-prion received repetitive intraperitoneal injections of U18666A (10 mg/kg) or a mixture of U18666A (10 mg/kg) and AY-9944 (12 mg/kg). By contrast to the potent anti-prion effects observed in ScN2a cells, the in vivo trial was abortive with neither drug halting the progression of the disease.

Neonatal treatment of Long-Evans Hooded rats with the cholesterol synthesis inhibitor (CSI) AY9944 has been shown to increase occurrence of spike-waves in EEG recordings and decrease benzodiazepines sensitivity of GABA(A) receptor-mediated responses in neurons from the thalamic reticular nuclei (nRt, Wu et al., 2004). The present experiments were designed to investigate the changes in the gamma2 and alpha1 subunits of the GABA(A) receptor in CSI model rats as possible mechanisms of these changes. Western blot, immunohistochemistry and real-time PCR techniques were performed to measure the levels of GABA(A) receptor gamma2 and alpha1 subunit transcripts and protein in the nRt and ventrobasal (VB) relay nuclei of thalamus and in somatosensory cortex. In CSI model animals, Western blot results showed that gamma2 subunit expression significantly decreased in thalamus (control, n=6: 0.17+/-0.02 relative to actin vs. CSI model, n=6: 0.11+/-0.01, P<0.05) but neither in cortex nor in hippocampal tissues. Conversely, alpha1 subunit expression decreased in CSI model somatosensory cortex, but not in nRt and VB. The present results demonstrate that neonatal block of cholesterol synthesis produces region- and subunit-specific decreases in GABA(A) receptor subunits in thalamus and cortex. Selective reductions in GABA(A) receptor subunits in thalamus may play a role in pathophysiology of absence epilepsy.

Meiosis activating sterol (MAS) have been found to be able to promote oocytes meiotic maturation of small animals in vitro, such as mouse, rat and rabbit. But in large animals, whether MAS play the same function, especially the physiological mechanisms of MAS on oocytes maturation are not clear. To our knowledge, this is the first time to investigate the role and signal pathway of MAS on FSH-induced porcine oocytes meiotic resumption. Porcine cumulus-enclosed oocytes (CEOs) isolated from 3 to 5mm follicles were cultured in the FSH-medium for 24h supplemented with 0-50 microM RS21745 or 0-100 microM RS21607 (two specific inhibitors of lanosterol 14alpha-demethylase that converts lanosterol to FF-MAS), or cultured in FSH-medium with 25 microM RS21745 for 0-24h firstly, then transferred into a new FSH-medium (the total culture time is 24h). The results revealed that RS21745 or RS21607 could inhibit FSH-induced porcine CEOs meiotic resumption in a dose and time-dependent manner. Meanwhile, FSH-induced cumulus expansion could also be inhibited dose-dependently by RS21745 or RS21607. Otherwise, AY9944-A-7, an inhibitor of Delta14-reductase which promotes cholesterol accumulation from FF-MAS, had no effect on both denuded oocytes (DOs) cultured for 24 or 44 h and CEOs cultured for 24h meiotic resumption, but it could promote CEOs meiotic resumption after 44 h culture. In addition, we got that 10(-8) to 10(-6)M PMA, an activator of PKC pathway, could reverse the inhibiting effect of RS21745 on FSH-induced CEOs meiotic resumption and enhance the rate of germinal vesicle breakdown (GVBD) of CEOs cultured in medium with hypoxanthine (HX). Moreover, 5-10 microM chelerythrine chloride, an inhibitor of PKC, could enhance the inhibitory effect of RS21745 on FSH-induced porcine oocytes resumption of meiosis. All the data of this study support that endogenous FF-MAS takes part in the FSH-induced porcine oocytes meiotic resumption and might play an active role via PKC signal pathway.

Progesterone is produced from cholesterol in cumulus cells during meiotic resumption of porcine oocytes. In follicular cells, it has been shown that exogenous lipoprotein-bound cholesterol ester can be used for steroid hormone production. However, in serum-free medium, progesterone is also secreted by FSH- and LH-stimulated cumulus-oocyte complexes, suggesting that progesterone could be produced from de novo synthesized cholesterol in cumulus cells. In the present study, we investigated the expression of Delta14-reductase and Delta7-reductase, which are the members of the superfamily that converts acetyl-CoA to cholesterol in cumulus cells. The expression of both genes was analyzed by RT-PCR. Both Delta14-reductase mRNA and Delta7-reductase mRNA in cumulus cells, cultured until 4 h, were under the level of detection limit. In response to gonadotropins, both mRNA levels were dramatically up-regulated, reaching a maximum at 20 h. To clarify the role of induced enzymes in cumulus cells, cumulus-oocyte complexes were cultured with either Delta14-reductase inhibitor, AY9944-A-7, or Delta7-reductase inhibitor, BM15.766. The results indicated that these inhibitors significantly suppressed the progesterone production in cumulus cells and meiotic progression of oocytes. The inhibitory effects reached a maximum at 1 microM AY9944-A-7 or 20 microM BM15.766. The addition of 20 ng/ml progesterone overcame the inhibitory effects of both drugs on meiotic resumption of oocytes. These results imply that gonadotropin-induced expression and function of Delta14-reductase and Delta7-reductase in cumulus cells contribute to oocyte meiotic resumption via a progesterone-dependent pathway.

Meiosis-activating sterol (MAS) was shown to overcome the inhibitory effect of hypoxanthine on spontaneous maturation of mouse oocytes and was suggested to mediate the stimulation of meiosis by gonadotropins. Follicular fluid (FF)-MAS is synthesized by cytochrome P450 lanosterol 14alpha-demethylase (LDM). Follicular LDM was preferentially localized in oocytes by immunohistochemistry. Using [3H]acetate or R-[5-3H]mevalonate as precursors as well as high-performance liquid chromatographic and thin-layer chromatographic separation, we have measured the concentrations of de novo-synthesized lanosterol, FF-MAS, and cholesterol in rat graafian follicles, cumulus-oocyte complexes (COCs), and denuded oocytes (DOs) treated with LH, AY-9944 (an inhibitor of Delta14-reductase, which was anticipated to increase FF-MAS levels by inhibiting its metabolism), or both after 8 h of culture. In follicles, both LH and AY-9944 increased the accumulation of FF-MAS as compared to controls. In COCs, AY-9944 caused a marked increase in FF-MAS, but we were unable to detect accumulation of FF-MAS in DOs. Neither the endogenous increases in FF-MAS accumulation nor the addition of FF-MAS to the culture medium could overcome the inhibition on resumption of meiosis by phosphodiesterase inhibitors. Compared to LH-induced resumption of meiosis in follicles, that induced by AY-9944 was much delayed. These results call into question any role of FF-MAS as an obligatory mediator of LH activity on germinal vesicle breakdown. The discrepancy between the positive staining for LDM in oocytes and our inability to detect de novo synthesized FF-MAS in DOs may relate to the sensitivity of the methodology employed and either the number of oocytes used or a deficiency in LDM synthetic activity in such oocytes. Further studies are required to confirm any of these alternatives.

AY-9944 (AY) exacerbates chronic recurrent seizures in rats that are analogous to atypical absence epilepsy in humans. The mechanism by which AY affects the slow spike-and-wave discharges associated with these seizures is not known, but is thought to involve inhibition of cholesterol synthesis. We tested the hypothesis that seizures seen with AY are due to significant reduction in brain cholesterol and/or elevated brain 7-dehydrocholesterol by assessing whether three other cholesterol synthesis inhibitors mimic AY seizures in rats. Effects of AY on brain sterols and spike-and-wave discharge duration were compared with those of two other late-stage cholesterol inhibitors [BM 15.766 (BM) and U18666A (UA)] and to an HMG-CoA reductase (early-stage cholesterol) inhibitor, lovastatin. With BM or UA, prolongation of seizure duration and brain sterol changes was similar to that caused by AY. AY effects on both brain sterols and seizure duration were dose-related. Lovastatin, with or without concurrent AY, mimicked AY seizures but reduced brain cholesterol by <10% and did not significantly change brain 7-dehydrocholesterol. Either lovastatin has a different mechanism of action than these late-stage cholesterol inhibitors or the brain sterol changes are not directly responsible for seizures in this model.

Up-regulation of low-density lipoprotein receptor (LDLr) is a key mechanism to control elevated plasma LDL-cholesterol levels. In the present paper, we compare the ability of four distinct pharmacological drugs to up-regulate LDLr expression in human hepatocytes. HepG2 cells were treated with the steroidal analog GW707, the oxidosqualene cyclase inhibitor U18666A, the 3beta-hydroxysterol Delta(7)-reductase inhibitor AY-9944 and the vacuolar-type ATPase inhibitor bafilomycin A1. We found that the four compounds induced sequestration of free cholesterol in the endosomal/lysosomal compartment leading to a positive filipin staining pattern and a complete inhibition of cholesteryl ester synthesis. As a consequence of the sequestration of cholesterol, the expression and the activity of LDLr were strongly induced resulting from a transcriptional effect which was measured by a reporter gene assay. These effects were fully abolished when an exogenous water soluble cholesterol analog was added to the cells. These findings have led to the identification of a common mechanism to up-regulate LDLr expression in human hepatocytes and may represent an interesting alternative approach to identify new hypolipidemic drugs.

This study describes a model for short-term culture of intact mouse follicles under serum-free conditions. Follicles were either obtained from immature mice receiving no ovarian stimulation (i.e. no eCG-primed protocol, group I) or from mice undergoing ovarian stimulation (i.e. eCG-primed protocol, group II). Follicles were grouped according to size (100-170, 180-200, 210-250, 260-350 and 360-400 microm, respectively) and cultured for 24h (group I) or for only 6h (group II). Induced meiotic resumption of follicle-enclosed oocytes were evaluated following stimulation with gonadotropins (i.e. FSH and hCG), AY9944-A-7, an inhibitor of Delta14-reductase, and RS-21745, an inhibitor of lanosterol 14alpha-demethylase; both enzymes affect synthesis of the meiosis activating sterols (MAS) that induce oocyte maturation. The frequency of oocyte degeneration was also recorded. In group I, FSH (10-200 IUl-1) and AY9944-A-7 (5, 25 and 50 microM) separately induced resumption of meiosis in oocytes derived from follicles with a diameter of 180-400 microm. hCG (1.0 and 10 IUml-1) exhibited a similar but weaker effect on oocytes present in follicles with a diameter of 260-400 microm. Irrespective of follicular diameter oocytes obtained from follicles in group II responded to hCG and FSH by resuming meiosis. FSH (50 IUl-1) alone or hCG (10 IUml-1) alone both increased the GVBD percentage of oocytes enclosed in follicles with a diameter 260-400 microm, but the response to hCG was not significant compared to control. FSH (50 IUl-1) combination with hCG (10 IUml-1) showed an additive effect raising the rate of GVBD after 6h culture. Addition of 50 or 100 microM RS-21745 was able to attenuate gonadotropins-induced resumption of meiosis to below background levels. In conclusion, the ability of FSH to induce meiotic resumption of follicle-enclosed mouse oocytes is correlated to follicle size, being most pronounced in larger follicles. hCG caused a similar but less pronounced effect. The ability of RS-21745 to inhibit and the ability of AY9944-A-7 to enhance oocyte maturation of follicle-enclosed oocytes support the concept of FSH employing MAS as a downstream signal transduction molecule for initiation of oocyte maturation in mice.

Inborn errors of cholesterol synthesis are associated with multiple systemic abnormalities, including skeletal malformations. The regulatory role of cholesterol during embryogenesis appears to be mediated by Shh, a signaling molecule in which activity depends on molecular events involving cholesterol. Based on this evidence, we hypothesized that cholesterol, by modifying the activity of Ihh (another of the Hedgehog family proteins) in the growth plate, regulates longitudinal bone growth. To test this hypothesis, we treated rats with AY 9944, an inhibitor of the final reaction of cholesterol synthesis. After 3 weeks, AY 9944 reduced the cumulative growth, tibial growth, and the tibial growth plate height of the rats. To determine whether cholesterol deficiency affects bone growth directly at the growth plate, we then cultured fetal rat metatarsal bones in the presence of AY 9944. After 4 days, AY 9944 suppressed metatarsal growth and growth plate chondrocyte proliferation and hypertrophy. The inhibitory effect on chondrocyte hypertrophy was confirmed by the AY 9944-mediated decreased expression of collagen X. Lastly, AY 9944 decreased the expression of Ihh in the metatarsal growth plate. We conclude that reduced cholesterol synthesis in the growth plate, possibly by altering the normal activity of Ihh, results in suppressed longitudinal bone growth and growth plate chondrogenesis.

The ketogenic diet (KD), a treatment for drug-resistant epilepsy, elevates brain acetone. Acetone has been shown to suppress experimental seizures. Whether elevation of acetone is the basis of the anticonvulsant effects of the KD and whether acetone, like the KD, antagonizes many different types of seizures, however, is unknown. This study investigated the spectrum of the anticonvulsant effects of acetone in animal seizure models. Rats were injected with acetone intraperitoneally. Dose-response effects were measured in four different models: (1) the maximal electroshock test, which models human tonic-clonic seizures; (2) the subcutaneous pentylenetetrazole test, which models human typical absence seizures; (3) the amygdala kindling test, which models human complex partial seizures with secondary generalization; and (4) the AY-9944 test, which models chronic atypical absence seizures, a component of the Lennox-Gastaut syndrome. Acetone suppressed seizures in all of the models, with the following ED(50)'s (expressed in mmol/kg): maximal electroshock, 6.6; pentylenetetrazole, 9.7; generalized kindled seizures, 13.1; focal kindled seizures, 26.5; AY-9944, 4.0. Acetone appears to have a broad spectrum of anticonvulsant effects. These effects parallel the effects of the KD. Elevation of brain acetone therefore may account for the efficacy of the KD in intractable epilepsy.

Patients with Smith-Lemli-Opitz syndrome, a genetic disorder associated with severe mental retardation, are unable to convert 7-dehydrocholesterol to cholesterol. Treatment of rats with agents that block cholesterol synthesis produces a sterol profile reminiscent of Smith-Lemli-Opitz patients i.e., low levels of cholesterol accompanied by the appearance of its immediate precursor 7-dehydrocholesterol. In previous work, chronic inhibition of cholesterol synthesis in just-weaned rats impaired acquisition of the classically conditioned eyeblink response. The present study had two primary goals--(1) to determine whether the learning impairment depended on the age in which treatment was initiated; and (2) to determine whether the deficit was associative or due to performance factors. Consistent with earlier work, acquisition of the eyeblink conditioned response was impaired when the 30-day treatment was initiated on postnatal day (PND) 21. Reactivity to acoustic stimuli and to eyelid stimulation were normal, suggesting that the learning impairment was associative in nature. The learning impairment was transitory; acquisition was normal when evaluated 30 days after the cessation of treatment. When treatment was initiated 30 days after weaning (PND 51), acquisition of the eyeblink response was normal. However, brain sterols of young adult rats were less affected than those of just-weaned rats. Thus, there is a developmental sensitivity to cholesterol synthesis blocking agents both in terms of their effects on brain sterols and new motor learning.

Holoprosencephaly is a common developmental anomaly of the forebrain and midface, that has been associated with mutations in the Sonic Hedgehog gene, and with perturbations of cholesterol synthesis and metabolism in mammalian embryos. The study presented here was aimed to evaluate the functional relationship between these two causal agents in the genesis of the phenotype. Therefore, we used rat embryos exposed in utero to a distal inhibitor of cholesterol biosynthesis (AY9944) in which we analyzed different Shh-dependent processes, as evaluated by the expression of eight target genes. In addition, to delineate between the impact of cholesterol shortage and/or sterol precursors accumulation on the Shh signaling cascade we exposed rat embryos to AY9944 and we provided complementary diets rich in cholesterol and 7-DHC. At the early-somite stage we observed a reduction of Shh signaling in AY9944 treated embryos, resulting in the definition of a narrower ventral domain. Later in development this reduction of Shh signaling led to a complete interruption of the pathway in the rostral hindbrain and caudal midbrain. Other regions such as the forebrain and the spinal cord appeared less sensitive to the reduction of Shh signaling and interruption of the pathway was only observed in a subset of embryos. Finally, we did provide evidence that 7-DHC accumulation is compatible with normal activity of Shh, as long as cholesterol levels in embryonic tissue is sufficient., (Copyright 2001 Wiley-Liss, Inc.)

In vitro studies on mouse oocytes have shown that two closely related sterols, subsequently named meiosis-activating sterols (MAS), can overcome the inhibitory effect of hypoxanthine on the resumption of meiosis. These sterols are synthesized by cytochrome P(450) lanosterol 14alpha-demethylase (LDM), a key enzyme in cholesterol biosynthesis. We have used specific inhibitors of LDM, azalanstat (RS-21607) and RS-21745, to test whether MAS is an obligatory mediator in the resumption of meiosis in the rat. Addition of azalanstat and RS-21745 (1-200 microM) to culture medium of rat isolated cumulus-enclosed oocyte and preovulatory follicle-enclosed oocyte stimulated by LH/hCG did not allow separation between their inhibition of the resumption of meiosis and the degeneration of oocytes. In both models, doses of the drug that inhibited oocyte maturation also increased oocyte degeneration. The inhibitors only partially suppressed follicular progesterone production. We have examined by reverse transcriptase-polymerase chain reaction, Western blotting, and immunocytochemistry the ovarian expression of LDM mRNA and protein during the preovulatory period. We did not find evidence for the stimulation of this enzyme by LH/hCG. The strongest staining by LDM antiserum was obtained in primordial and primary oocytes, and the staining was reduced with oocyte growth. In addition, strong LDM staining could be observed in some of the granulosa cells, especially of the corona radiata localized in close proximity to the oocyte. In conclusion, our results with specific inhibitors and molecular approaches do not reveal evidence to support the hypothesis that MAS is an obligatory step in the stimulation of the resumption of meiosis. Specific inhibitors of MAS synthesis did not prevent spontaneous or LH-stimulated meiosis at doses that have previously been shown to effectively suppress LDM activity. Much higher concentrations of the inhibitors, which affected meiosis, were detrimental to oocytes, leading to their degeneration. The timing of LDM expression in the ovary was incompatible with a role for MAS in meiosis. Finally, the preferential localization of LDM protein to the oocytes suggests MAS production in oocytes rather than its transport from the somatic compartment as implied by the proposed role of MAS as a cumulus-oocyte signal molecule.

Holoprosencephaly, or an undivided forebrain, is a complex brain malformation associated with Sonic hedgehog (Shh) mutations. Other causes of holoprosencephaly have converged upon the Shh signaling pathway: genetic and pharmacologic impairment of cholesterol synthesis, and the action of the steroidal alkaloid cyclopamine. This review focuses on recent studies aimed at determining how Shh signaling is affected by these causes of holoprosencephaly, whether they involve a common mechanism and the role played by cholesterol. Cholesterol is potentially important for both biogenesis of Shh and in signal transduction in Shh-responsive cells. Teratogens that induce holoprosencephaly appear to affect Shh signal transduction rather than Shh biogenesis. Analysis of these agents and other compounds that affect various aspects of cellular cholesterol distribution indicates that the role of cholesterol in Shh signal transduction is novel and complicated. The similarity of the Shh receptor, Patched (Ptc), to the Niemann-Pick Cl protein, which is involved in the vesicular trafficking of cholesterol, provides insight into the role of cholesterol and the action of compounds like cyclopamine.

Low cholesterol and high 7-dehydrocholesterol (7DHC) levels are associated with a blockade of Delta7-reductase in the Smith-Lemli-Opitz syndrome (SLOS) and in the animals treated with the inhibitor AY9944. The impact of the cholesterol deficit and of the accumulation of 7DHC on the embryo were investigated in AY9944-treated pregnant rats receiving an enriched cholesterol or 7DHC diet. Sterol profiling was performed under the various nutritional conditions. AY9944 caused a severe decrease in the maternal and embryo cholesterol. The deficit in the embryo was sustained by the embryonic uptake of the inhibitor. A cholesterol-rich diet was efficient in restoring the maternal and embryonic cholesterol and phenotype but a 7DHC-rich diet did not modify the sterol status compared with dams treated with only AY9944. The offspring phenotype remained deleterious whether or not the dams received 7DHC-rich diet. Over 80% of the 7DHC was absorbed, as was cholesterol, which was not quantitatively influenced by AY9944. When cholesterol and 7DHC were simultaneously administered, a competition for intestinal absorption enhanced the lowering cholesterol effect of AY9944. Whether or not the dams received a 7DHC dietary supplement, the offspring's phenotype became normal when the diet was supplemented with cholesterol. Under conditions in which the ratio of cholesterol/7DHC is substantially varied, the normal development of embryos can be achieved as long as the cholesterol is sufficient. The phenotype is reversed in vivo by cholesterol which contrasts with the irreversible effects manifested in vitro by oxidized 7DHC by-products.

Two sterols of the cholesterol biosynthetic pathway induce resumption of meiosis in mouse oocytes in vitro. The sterols, termed meiosis-activating sterols (MAS), have been isolated from human follicular fluid (FF-MAS, 4,4-dimethyl-5 alpha-cholest-8,14,24-triene-3 beta-ol) and from bull testicular tissue (T-MAS, 4,4-dimethyl-5 alpha-cholest-8,24-diene-3 beta-ol). FF-MAS is the first intermediate in the cholesterol biosynthesis from lanosterol and is converted to T-MAS by sterol delta 14-reductase. An inhibitor of delta 7-reductase and delta 14 reductase, AY9944-A-7, causes cells with a constitutive cholesterol biosynthesis to accumulate FF-MAS and possibly other intermediates between lanosterol and cholesterol. The aim of the present study was to evaluate whether AY9944-A-7 added to cultures of cumulus-oocyte complexes (COC) from mice resulted in accumulation of MAS and meiotic maturation. AY9944-A-7 stimulated dose dependently (5-25 mumol l-1) COC to resume meiosis when cultured for 22 h in alpha minimal essential medium (alpha-MEM) containing 4 mmol hypoxanthine l-1, a natural inhibitor of meiotic maturation. In contrast, naked oocytes were not induced to resume meiosis by AY9944-A-7. When cumulus cells were separated from their oocytes and co-cultured, AY9944-A-7 did not affect resumption of meiosis, indicating that intact oocyte-cumulus cell connections are important for AY9944-A-7 to exert its effect on meiosis. Cultures of COC with 10 mumol AY9944-A-7 l-1 in the presence of [3H]mevalonic acid, a natural precursor for steroid synthesis, resulted in accumulation of labelled FF-MAS, which had an 11-fold greater amount of radioactivity incorporated per COC compared with the control culture without AY9944-A-7. In contrast, incorporation of radioactivity into the cholesterol fraction was reduced 30-fold in extracts from the same oocytes. The present findings demonstrate for the first time that COC can synthesize cholesterol from mevalonate and accumulate FF-MAS in the presence of AY9944-A-7. Furthermore, AY9944-A-7 stimulated meiotic maturation dose dependently, indicating that FF-MAS, and possibly other sterol intermediates of the cholesterol synthesis pathway, play a central role in stimulating mouse oocytes to resume meiosis. The results also indicate that oocytes may not synthesize steroids from mevalonate.

Purpose: To evaluate the consequences of altering retinal sterol metabolism and composition on the development, histologic organization, and electrophysiological function of the retina, under conditions that mimic the biochemical hallmarks of the Smith-Lemli-Opitz (SLO) syndrome., Methods: Pregnant Sprague-Dawley rats were fed cholesterol-free chow containing AY9944 (treated group), an inhibitor of 3beta-hydroxysterol delta7-reductase, from gestational day 6 through postnatal day (P)28. Control animals were fed the same chow, but without AY9944. In addition, progeny in the treated group were injected subcutaneously every other day from birth to P28 with an olive oil emulsion containing AY9944; control animals received olive oil emulsion alone. At various postnatal times, tissues from treated and control animals were harvested, and their sterol profiles were analyzed by reversed-phase high-performance liquid chromatography. Companion eyes from animals of both groups were examined histologically at P1. At P28, animals were evaluated by electroretinography; tissues were then harvested for biochemical analysis and companion eyes were subjected to histologic and ultrastructural analyses., Results: Treatment of developing rats with AY9944 caused markedly abnormal accumulation of 7-dehydrosterols and severely reduced cholesterol levels in all tissues examined, relative to control animals. Despite this, treated animals exhibited normal retinal development and had no overt ocular defects or decrease in electroretinographic function, up to P28., Conclusions: These results were unexpected, given the known biophysical effects of such sterol alterations on membrane properties and the profound dysmorphic and cognitive abnormalities associated with genetic defects in 3beta-hydroxysterol delta7-reductase that have been linked to the SLO syndrome. The results suggest that 7-dehydrosterols can substitute functionally for cholesterol in the retina or perhaps can act synergistically with subthreshold levels of residual cholesterol to allow normal cellular structure and function to be achieved.

7-Dehydrocholesterol accumulates in fetuses affected by the Smith-Lemli-Opitz syndrome as a result of a deficit in the ultimate step of cholesterol synthesis catalyzed by Delta7 reductase. Rat embryos explanted at gestation day 10 and cultured for 48 h in the presence of the Delta7 reductase inhibitor AY 9944 were used as a model to discriminate between the beneficial effect of supplementation with cholesterol and the deleterious effect of supplementation with 7-dehydrocholesterol. Cholesterol supplementation in the form of mixed cholesterol/lecithin liposomes added to serum serving as the culture medium restores the growth of embryos which is markedly decreased in the presence of the inhibitor. 7-Dehydrocholesterol under identical conditions does not restore growth and impairs the beneficial effect of cholesterol added simultaneously. UV-photooxidation of 7-dehydrocholesterol-supplemented culture medium enhances its embryotoxicity, which suggests uptake by the embryo of toxic by-products formed from 7-dehydrocholesterol. By contrast photooxidation of cholesterol-supplemented culture medium does not induce embryotoxicity. alpha-Tocopherol reduces the toxicity of photooxidized 7-dehydrocholesterol supplementing the culture medium. We conclude that 7-dehydrocholesterol does not fulfill the cholesterol requirement of the developing embryos and exerts an additional embryotoxic effect probably via oxidized by-products. This could explain the antenatal growth retardation of SLOS by a blockage of the maternal compensatory cholesterol influx.

AY 9944 [AY; trans-1,4-bis(chlorobenzylaminomethyl)-cyclohexane dihydrochloride], an inhibitor of sterol synthesis, was found to help restore the normal mitogenic responses and cytokine profiles of peripheral mononuclear cells (PBMCs) from AIDS patients in vitro. Compared to untreated cells, the human immunodeficiency virus type 1 (HIV-1)-infected PBMCs precultured in the presence of AY exhibited a normal rate of either mitogen-induced or recall- and superantigen-induced proliferation. After 2 weeks in the presence of the drug, the percentage of dead CD4(+) cells in HIV-1-infected cultures was comparable to that observed in uninfected cultures, while over the same time interval it increased by three- to fivefold in HIV-1-infected cultures maintained in the absence of AY. AY also stimulated by 2- to 12-fold interleukin-12 (IL-12) and (gamma interferon production. For IL-12, this effect appears to be related to an increase in corresponding IL-12 p35 and IL-12 p40 mRNA levels. Moreover, AY restored the expression of the IL-2 receptor, which was severely impaired in HIV-1-infected PBMCs. Although the drug has no direct antiviral effect (it does not significantly inhibit reverse transcriptase activity measured in vitro), it might be considered a potential therapeutic agent for HIV-infected patients, in that it may correct viral infection-related immune system defects by indirectly enhancing the level of resistance to HIV and opportunistic infections.

The steroidal alkaloid cyclopamine produces cyclopia and holoprosencephaly when administered to gastrulation-stage amniote embryos. Cyclopamine-induced malformations in chick embryos are associated with interruption of Sonic hedgehog (Shh)-mediated dorsoventral patterning of the neural tube and somites. Cell types normally induced in the ventral neural tube by Shh are either absent or appear aberrantly at the ventral midline after cyclopamine treatment, while dorsal cell types normally repressed by Shh appear ventrally. Somites in cyclopamine-treated embryos show Pax7 expression throughout, indicating failure of sclerotome induction. Cyclopamine at concentrations of 20-100 nM blocks the response of neural plate explants to recombinant Shh-N in a dose-dependent manner. Similar concentrations have no effect on the post-translational modification of Shh by cholesterol in transfected COS-1 cells. Comparison of the effects of cyclopamine to those of the holoprosencephaly-inducing cholesterol synthesis inhibitor AY-9944 shows that cyclopamine does not induce malformations by interfering with cholesterol metabolism. Although AY-9944 does not interrupt Shh signaling in ovo, it blocks the response to Shh-N in explants cultured without an exogenous cholesterol source. As predicted by current models of the regulation of cholesterol metabolism, the response to Shh-N in AY-9944-treated explants is restored by providing exogenous cholesterol. However, exogenous cholesterol does not restore Shh signaling in cyclopamine-treated explants. These findings suggest that cyclopamine-induced teratogenesis is due to a more direct antagonism of Shh signal transduction.

Veratrum alkaloids and distal inhibitors of cholesterol biosynthesis have been studied for more than 30 years as potent teratogens capable of inducing cyclopia and other birth defects. Here, it is shown that these compounds specifically block the Sonic hedgehog (Shh) signaling pathway. These teratogens did not prevent the sterol modification of Shh during autoprocessing but rather inhibited the response of target tissues to Shh, possibly acting through the sterol sensing domain within the Patched protein regulator of Shh response.

1. The sigma-drug binding site of guinea-pig liver is carried by a protein which shares significant amino acid sequence similarities with the yeast sterol C8-C7 isomerase (ERG2 protein). Pharmacologically-but not structurally-the sigma 1-site is also related to the emopamil binding protein, the mammalian sterol C8-C7 isomerase. We therefore investigated if sterol C8-C7 isomerase inhibitors are high affinity ligands for the (+)-[3H]-pentazocine labelled sigma 1-binding site. 2. Among the compounds which bound with high affinity to native hepatic and cerebral as well as to yeast expressed sigma 1-binding sites were the agricultural fungicide fenpropimorph (Ki 0.005 nM), the antihypocholesterinaemic drugs triparanol (Ki 7.0 nM), AY-9944 (Ki, 0.46 nM) and MDL28,815 (Ki 0.16 nM), the enantiomers of the ovulation inducer clomiphene (Ki 5.5 and 12 nM, respectively) and the antioestrogene tamoxifen (Ki 26 nM). 3. Except for tamoxifen these affinities are essentially identical with those for the [3H]-ifenprodil labelled sterol C8-C7 isomerase of S. cerevisiae. This demonstrates that sigma 1-binding protein and yeast isomerase are not only structurally but also pharmacologically related. Because of its affiliations with yeast and mammalian sterol isomerases we propose that the sigma 1-binding site is localized on a sterol isomerase related protein, involved in postsqualene sterol biosynthesis.

The Smith-Lemli-Opitz syndrome-affected fetus presents a deficiency in delta7-dehydrocholesterol reductase, the last enzymatic step in the cholesterol biosynthesis pathway. Development of the abnormal human fetus takes place in a normal environment as the heterozygous mother's cholesterolemia remains normal. An animal model for this disease has been obtained from the offspring of pregnant rats treated with "distal" inhibitors of delta7-dehydrocholesterol reductase, AY-9944 or BM15766. In the animal model, embryonic development occurs in a disturbed environment characterized by hypocholesterolemia and accumulation of delta7-dehydrocholesterol and delta8-dehydrocholesterol in the maternal serum. The purpose of the present study was to assess, in cultured rat embryos at early developmental stages, the relative contributions of exogenous and de novo synthesized cholesterol in the total embryonic cholesterol, according to the conditions of normal and altered de novo biosynthesis. Cultured rat embryos are able to synthesize cholesterol as shown by 13C-incorporation into cholesterol from 13C-labeled precursors added to the culture medium. De novo cholesterol biosynthesis is altered by addition to the culture medium of AY-9944 which inhibits the delta7-dehydrocholesterol reductase and the delta8-delta7-sterol isomerase as suggested by the emergence of characteristic aberrant sterols in the embryonic tissues. Cholesterol-rich serum used for embryo culture alters the pattern in a way that confirms that the rat embryos are able to import exogenous cholesterol which down-regulates de novo cholesterol biosynthesis. Exogenous cholesterol substitutes for the deficit in a manner efficient enough to prevent the embryonic abnormalities induced by AY-9944.

The teratogenic action of distal inhibitors of cholesterol synthesis has been known for some time. The induced malformations are of a particular type: they include holoprosencephalies. Recently these observations have solicited increased interest due to: 1/ the discovery in 1993 of a similar form of inhibition of cholesterol synthesis which is responsible for a human malformation syndrome, Smith-Lemli-Opitz; 2/ the demonstration of the involvement of the Sonic Hedgehog gene in normal development of prosencephalon and the description of the mode of action of the protein Shh: autoprocessing followed by "cholesterolisation".