Desmosterol-driven atypical macrophage polarization regulates podocyte dynamics in diabetic nephropathy.

BACKGROUND: Diabetic nephropathy (DN) stands as a leading diabetes complication, with macrophages intricately involved in its evolution. While glucose metabolism's impact on macrophage activity is well-established, cholesterol metabolism's contributions remain less explored. Our study seeks to elucidate this association. METHODS AND RESULTS: Methods and Results: Gene expression analysis of monocytes from the blood of both normal and diabetic patients was conducted using public databases, showing that cholesterol metabolism pathways, especially Bloch and Kandutsch-Russell, were more altered in diabetic monocytes/macrophages than glucose-responsive pathways. When bone marrow-derived macrophages (BMDMs) were subjected to desmosterol, they exhibited an unconventional polarization. These BMDMs displayed heightened levels of both M1-related pro-inflammatory cytokines and M2-linked anti-inflammatory factors. Further, in co-culture, desmosterol-conditioned BMDMs paralleled M2 macrophages in augmenting Ki-67 + podocyte populations while mimicking M1 macrophages in elevating TUNEL + apoptotic podocytes. Comparable outcomes on podocytes were obtained using conditioned media from the respective BMDMs. CONCLUSIONS: Our data underscores the pivotal role of cholesterol metabolism, particularly via desmosterol, in steering macrophages toward an unconventional polarization marked by both inflammatory and regulatory traits. Such unique macrophage behavior concurrently impacts podocyte proliferation and apoptosis, shedding fresh light on DN pathogenesis and hinting at potential therapeutic interventions.

Effects of long-term sex steroid hormones (estradiol and testosterone)-supplemented feeds on the growth performance of Chinese tongue sole (Cynoglossus semilaevis).

The phenomenon of sexual size dimorphism (SSD), existing in mammals, birds, reptiles, spiders, amphibians, insects, and fishes, is generally related to feeding efficiency, energy allocation, sex steroids, and somatotropic and reproductive endocrine axes. Recently, positive and negative regulations of sex steroids have been reported on SSD in various species. Chinese tongue soles (Cynoglossus semilaevis) at 4 months were fed with 17ß-estradiol (E2) and testosterone (T) supplemented feeds for 8 months to assess the effect of sex steroids on growth traits in different sexes. The potential genetic regulation was examined using several growth-related genes. The results showed that two sex steroid hormones had inhibitory effects on the growth performance of different sexes of C. semilaevis. At the age of 8 months, the expression of insulin-like growth factor 2 gene (igf2), 24-dehydrocholesterol reductase (dhcr24), leptin, and estrogen receptor 2 (esr2) in the liver showed an overall downward trend. The expression of insulin-like growth factor 1 (igf1) was reduced, while thyroid hormone receptor-associated protein 3 (thrap3) expression tended to increase in the gonad after T and E2 treatments. In the brain, somatostatin 1, tandem duplicate 2 (sst1.2) expression increased with the treatment of T and E2 (P < 0.05), while growth hormone-releasing hormone (ghrh) expression decreased. E2 and T had different effects on growth differentiation factor 8 (gdf8) and insulin-like growth factor-binding protein 7 (igfbp7) expression in the muscle. Expression of gdf8 increased in the treated fishes in contrast to the reduction expression of igfbp7. This study provided important clues for understanding the role of sex steroids in flatfish SSD.

Squalene through Its Post-Squalene Metabolites Is a Modulator of Hepatic Transcriptome in Rabbits.

Squalene is a natural bioactive triterpene and an important intermediate in the biosynthesis of sterols. To assess the effect of this compound on the hepatic transcriptome, RNA-sequencing was carried out in two groups of male New Zealand rabbits fed either a diet enriched with 1% sunflower oil or the same diet with 0.5% squalene for 4 weeks. Hepatic lipids, lipid droplet area, squalene, and sterols were also monitored. The Squalene administration downregulated 9 transcripts and upregulated 13 transcripts. The gene ontology of transcripts fitted into the following main categories: transporter of proteins and sterols, lipid metabolism, lipogenesis, anti-inflammatory and anti-cancer properties. When the results were confirmed by RT-qPCR, rabbits receiving squalene displayed significant hepatic expression changes of LOC100344884 (PNPLA3), GCK, TFCP2L1, ASCL1, ACSS2, OST4, FAM91A1, MYH6, LRRC39, LOC108176846, GLT1D1 and TREH. A squalene-enriched diet increased hepatic levels of squalene, lanosterol, dihydrolanosterol, lathosterol, zymostenol and desmosterol. Strong correlations were found among specific sterols and some squalene-changed transcripts. Incubation of the murine AML12 hepatic cell line in the presence of lanosterol, dihydrolanosterol, zymostenol and desmosterol reproduced the observed changes in the expressions of Acss2, Fam91a1 and Pnpla3. In conclusion, these findings indicate that the squalene and post-squalene metabolites play important roles in hepatic transcriptional changes required to protect the liver against malfunction.

Desmosterol: A natural product derived from macroalgae modulates inflammatory response and oxidative stress pathways in intestinal epithelial cells.

The serum level of cholesterol and its biosynthetic intermediates are critical indicators to access metabolism-related disorders in humans and animals. However, the molecular actions of these intermediates on gene functions and regulation remained elusive. Here, we show that desmosterol (DES) is the most abundant intermediate involved in cholesterol biosynthesis and is highly enriched in red/brown algae. It exerts a pivotal role in modulating core genes involved in oxidative stress and inflammatory response processes in the ileum epithelial cells (IPI-2I). We observed that the DES extracted from red algae did not affect IPI-2I cell growth or survival. A transcriptomic measurement revealed that the genes enrolled in the oxidative process and cholesterol homeostasis pathway were significantly down-regulated by DES treatment. Consistent with this notion, cellular reactive oxygen species (ROS) levels were markedly decreased in response to DES treatment. In contrast, key inflammatory genes including IL-6, TNF-α, and IFN-γ were remarkably upregulated in the RNA-seq analysis, as further confirmed by qRT-PCR. Given that DES is a specific agonist of nuclear receptor RORγ, we also found that DES caused the elevated expression of RORγ at mRNA and protein levels, suggesting it is a potential mediator under DES administration. Together, these results underscore the vital physiological actions of DES in inflammatory and oxidative processes possibly via RORγ, and may be helpful in anti-oxidation treatment and immunotherapy in the future.

Dehydrocholesterol Reductase 24 (DHCR24): Medicinal Chemistry, Pharmacology and Novel Therapeutic Options.

During the last decade, the understanding of the biological functions of cholesterol biosynthesis intermediates has changed significantly. Particularly, the enzyme sterol dehydrocholesterol reductase 24 (DHCR24) has taken center stage as a potential drug target. Inhibition of DHCR24 leads to accumulation of the endogenous, biologically active metabolite cholesta-5,24-dien-3ß-ol (desmosterol). Desmosterol is an endogenous agonist of the liver X receptor (LXR). LXR is a master regulator of lipid metabolism and, as such, is involved in numerous pathophysiological processes such as inflammation, atherosclerosis, cancer, diabetes mellitus (DM), multiple sclerosis (MS), nonalcoholic steatohepatitis (NASH), and the progression of viral infections. Up to now, selective pharmacological targeting of LXR without activating the sterol-response element binding proteins (SREBP) and thereby boosting endogenous lipid biosynthesis has not been achieved. In turn, no selective LXR receptor agonists leveraging its beneficial activation have yet reached the clinic. Therefore, using potent and selective inhibitors of DHCR24 leading to an accumulation of endogenous desmosterol is a promising alternative strategy for the selective activation of LXR. Here we summarize the present landscape of novel lead structures for targeting DHCR24, covering steroidal enzyme inhibitors (e.g., 20,25-diazacholesterol, SH42) as well as nonsteroidal scaffolds (e.g., amiodarone, triparanol). Further, we explain the molecular mechanisms of DHCR24 inhibition/LXR activation, discuss possible therapeutic applications, and underpin why DHCR24 is an upcoming promising drug target.

Microglia facilitate repair of demyelinated lesions via post-squalene sterol synthesis.

The repair of inflamed, demyelinated lesions as in multiple sclerosis (MS) necessitates the clearance of cholesterol-rich myelin debris by microglia/macrophages and the switch from a pro-inflammatory to an anti-inflammatory lesion environment. Subsequently, oligodendrocytes increase cholesterol levels as a prerequisite for synthesizing new myelin membranes. We hypothesized that lesion resolution is regulated by the fate of cholesterol from damaged myelin and oligodendroglial sterol synthesis. By integrating gene expression profiling, genetics and comprehensive phenotyping, we found that, paradoxically, sterol synthesis in myelin-phagocytosing microglia/macrophages determines the repair of acutely demyelinated lesions. Rather than producing cholesterol, microglia/macrophages synthesized desmosterol, the immediate cholesterol precursor. Desmosterol activated liver X receptor (LXR) signaling to resolve inflammation, creating a permissive environment for oligodendrocyte differentiation. Moreover, LXR target gene products facilitated the efflux of lipid and cholesterol from lipid-laden microglia/macrophages to support remyelination by oligodendrocytes. Consequently, pharmacological stimulation of sterol synthesis boosted the repair of demyelinated lesions, suggesting novel therapeutic strategies for myelin repair in MS.

Structural Stringency and Optimal Nature of Cholesterol Requirement in the Function of the Serotonin1A Receptor.

The role of membrane cholesterol in modulating G protein-coupled receptor (GPCR) structure and function has emerged as a powerful theme in contemporary biology. In this paper, we report the subtlety and stringency involved in the interaction of sterols with the serotonin1A receptor. For this, we utilized two immediate biosynthetic precursors of cholesterol, 7-dehydrocholesterol (7-DHC) and desmosterol, which differ with cholesterol merely in a double bond in their chemical structures in a position-dependent manner. We show that whereas 7-DHC could not support the ligand binding function of the serotonin1A receptor in live cells, desmosterol could partially support it. Importantly, depletion and enrichment of membrane cholesterol over basal level resulted in an increase and reduction of the basal receptor activity, respectively. These results demonstrate the relevance of optimal membrane cholesterol in maintaining the activity of the serotonin1A receptor, thereby elucidating the relevance of cellular cholesterol homeostasis.

The coronary artery calcium score is linked to plasma cholesterol synthesis and absorption markers: Brazilian Longitudinal Study of Adult Health.

It is controversial whether atherosclerosis is linked to increased intestinal cholesterol absorption or synthesis in humans. The aim of the present study was to relate atherosclerosis to the measurements of plasma markers of cholesterol synthesis (desmosterol, lathosterol) and absorption (campesterol, sitosterol). In healthy male (n=344), non-obese, non-diabetics, belonging to the city of São Paulo branch of the Brazilian Longitudinal Study of Adult Health (ELSA-Brasil), we measured in plasma these non-cholesterol sterol markers, together with their anthropometric, dietary parameters, traditional atherosclerotic risk factors, and blood chemistry, coronary arterial calcium score (CAC), and ultrasonographically measured common carotid artery intima-media thickness (CCA-IMT). Cases with CAC>zero had the following parameters higher than cases with CAC = zero: age, waist circumference (WC), plasma total cholesterol (TC), low-density lipoprotein-cholesterol (LDL-C), and non-high density lipoprotein-cholesterol (non HDL-C). Plasma desmosterol and campesterol, duly corrected for TC, age, body mass index (BMI), waist circumference (WC), hypertension, smoking, and the homeostasis model assessment-insulin resistance (HOMA-IR) correlated with CAC, but not with CCA-IMT. The latter related to increased age, BMI, waist circumference (WC), and systolic blood pressure (SBP). Plasma HDL-C concentrations did not define CAC or CCA-IMT degrees, although in relation to the lower tertile of HDL-C in plasma the higher tertile of HDL-C had lower HOMA-IR and concentration of a cholesterol synthesis marker (desmosterol). Present work indicated that increased cholesterol synthesis and absorption represent primary causes of CAD, but not of the common carotid artery atherosclerosis.

Desmosterolosis and desmosterol homeostasis in the developing mouse brain.

Cholesterol serves as a building material for cellular membranes and plays an important role in cellular metabolism. The brain relies on its own cholesterol biosynthesis, which starts during embryonic development. Cholesterol is synthesized from two immediate precursors, desmosterol and 7-dehydrocholesterol (7-DHC). Mutations in the DHCR24 enzyme, which converts desmosterol into cholesterol, lead to desmosterolosis, an autosomal recessive developmental disorder. In this study, we assessed the brain content of desmosterol, 7-DHC, and cholesterol from development to adulthood, and analyzed the biochemical, molecular, and anatomical consequences of Dhcr24 mutations on the sterol profile in a mouse model of desmosterolosis and heterozygous Dhcr24+/- carriers. Our HPLC-MS/MS studies revealed that by P0 desmosterol almost entirely replaced cholesterol in the Dhcr24-KO brain. The greatly elevated desmosterol levels were also present in the Dhcr24-Het brains irrespective of maternal genotype, persisting into adulthood. Furthermore, Dhcr24-KO mice brains showed complex changes in expression of lipid and sterol transcripts, nuclear receptors, and synaptic plasticity transcripts. Cultured Dhcr24-KO neurons showed increased arborization, which was also present in the Dhcr24-KO mouse brains. Finally, we observed a shared pathophysiological mechanism between the mouse models of desmosterolosis and Smith-Lemli-Opitz syndrome (a genetic disorder of conversion of 7-DHC to cholesterol).

Identification and characterization of prescription drugs that change levels of 7-dehydrocholesterol and desmosterol.

Regulating blood cholesterol (Chol) levels by pharmacotherapy has successfully improved cardiovascular health. There is growing interest in the role of Chol precursors in the treatment of diseases. One sterol precursor, desmosterol (Des), is a potential pharmacological target for inflammatory and neurodegenerative disorders. However, elevating levels of the precursor 7-dehydrocholesterol (7-DHC) by inhibiting the enzyme 7-dehydrocholesterol reductase is linked to teratogenic outcomes. Thus, altering the sterol profile may either increase risk toward an adverse outcome or confer therapeutic benefit depending on the metabolite affected by the pharmacophore. In order to characterize any unknown activity of drugs on Chol biosynthesis, a chemical library of Food and Drug Administration-approved drugs was screened for the potential to modulate 7-DHC or Des levels in a neural cell line. Over 20% of the collection was shown to impact Chol biosynthesis, including 75 compounds that alter 7-DHC levels and 49 that modulate Des levels. Evidence is provided that three tyrosine kinase inhibitors, imatinib, ponatinib, and masitinib, elevate Des levels as well as other substrates of 24-dehydrocholesterol reductase, the enzyme responsible for converting Des to Chol. Additionally, the mechanism of action for ponatinib and masitinib was explored, demonstrating that protein levels are decreased as a result of treatment with these drugs.

Modification of membrane cholesterol and desmosterol in chicken spermatozoa improves post-thaw survival and prevents impairment of sperm function after cryopreservation.

During cryopreservation, spermatozoa are subjected to cryodamage that leads to a decline in fertilisation ability. Due to the complex nature of this process, the initial trigger for cryodamage remains unknown. Recently, we demonstrated that cryopreservation induces early apoptotic changes characterised by phosphatidylserine (PS) translocation via sterol loss from the plasma membrane of chicken spermatozoa. This led us to hypothesise that sterol incorporation into membranes minimises cryodamage, thereby improving the quality of cryopreserved chicken spermatozoa. In the present study, treating spermatozoa with 1.5mgmL-1 cholesterol- and 3mgmL-1 desmosterol-loaded cyclodextrin (CLC and DLC respectively) increased post-thaw survival and motility. These effects appeared to be highly dependent the amount of sterol loaded into the spermatozoa. Localisation experiments confirmed the incorporation of exogenous cholesterol into the sperm head region. Detection of PS translocation showed that elevation of these sterols inhibited early apoptotic changes, thereby enhancing post-thaw survival. Furthermore, CLC and DLC treatment suppressed spontaneous acrosome reaction after cryopreservation, preserving the ability of spermatozoa to undergo acrosome reactions in response to physiological stimulation. These results demonstrate that loading sterols into chicken spermatozoa before cryopreservation enhances their quality by inhibiting early apoptotic changes and spontaneous acrosome reactions. The present study provides new mechanistic insight into cryodamage in chicken spermatozoa.

Expression and function of Abcg4 in the mouse blood-brain barrier: role in restricting the brain entry of amyloid-ß peptide.

ABCG4 is an ATP-binding cassette transmembrane protein which has been shown, in vitro, to participate in the cellular efflux of desmosterol and amyloid-ß peptide (Aß). ABCG4 is highly expressed in the brain, but its localization and function at the blood-brain barrier (BBB) level remain unknown. We demonstrate by qRT-PCR and confocal imaging that mouse Abcg4 is expressed in the brain capillary endothelial cells. Modelling studies of the Abcg4 dimer suggested that desmosterol showed thermodynamically favorable binding at the putative sterol-binding site, and this was greater than for cholesterol. Additionally, unbiased docking also showed Aß binding at this site. Using a novel Abcg4-deficient mouse model, we show that Abcg4 was able to export Aß and desmosterol at the BBB level and these processes could be inhibited by probucol and L-thyroxine. Our assay also showed that desmosterol antagonized the export of Aß, presumably as both bind at the sterol-binding site on Abcg4. We show for the first time that Abcg4 may function in vivo to export Aß at the BBB, in a process that can be antagonized by its putative natural ligand, desmosterol (and possibly cholesterol).

Desmosterol Increases Lipid Bilayer Fluidity during Hepatitis C Virus Infection.

Hepatitis C virus (HCV) uniquely affects desmosterol homeostasis by increasing its intracellular abundance and affecting its localization. These effects are important for productive viral replication because the inhibition of desmosterol synthesis has an antiviral effect that can be rescued by the addition of exogenous desmosterol. Here, we use subgenomic replicons to show that desmosterol has a major effect on the replication of HCV JFH1 RNA. Fluorescence recovery after photobleaching (FRAP) experiments performed with synthetic supported lipid bilayers demonstrate that the substitution of desmosterol for cholesterol significantly increases the lipid bilayer fluidity, especially in the presence of saturated phospholipids and ceramides. We demonstrate using LC-MS that desmosterol is abundant in the membranes upon which genome replication takes place and that supported lipid bilayers derived from these specialized membranes also exhibit significantly higher fluidity compared to that of negative control membranes isolated from cells lacking HCV. Together, these data suggest a model in which the fluidity-promoting effects of desmosterol on lipid bilayers play a crucial role in the extensive membrane remodeling that takes place in the endoplasmic reticulum during HCV infection. We anticipate that the supported lipid bilayer system described can provide a useful model system in which to interrogate the effects of lipid structure and composition on the biophysical properties of lipid membranes as well as their function in viral processes such as genome replication.

Lack of insulin results in reduced seladin-1 expression in primary cultured neurons and in cerebral cortex of STZ-induced diabetic rats.

Several studies demonstrated that Diabetes mellitus (DM) enhances the risk for Alzheimer's disease (AD). Although hyperglycemia and perturbed function of insulin signaling have been proposed to contribute to AD pathogenesis, the molecular mechanisms behind this association is not clear yet. Seladin-1 is an enzyme catalyzing the last step in cholesterol biosynthesis converting desmosterol to cholesterol. The neuroprotective function of seladin-1 has gained interest in AD research recently. Seladin-1 has anti-apoptotic properties and regulates the expression of ß-secretase (BACE-1). Here we measured seladin-1 mRNA and protein expressions in rat primary cultured neurons under diabetic conditions and also in the brains of rats with streptozotocine (STZ)-induced diabetes. We show that constant lack of insulin for 5days decreased seladin-1 levels in cultured rat primary neurons. Similarly, a decrease in seladin-1 was found in the brains of rats with STZ-induced diabetes. However, if the lack of insulin and/or high glucose treatment was intermittent, neuronal seladin-1 levels were not affected in vitro. On the other hand, treatment of neurons with metformin resulted in a significant increase in seladin-1. Constant lack of insulin for 5days, as well as high glucose treatment, increased the neuronal expression of BACE-1 in vitro, but not in the in vivo model. Our study defines insulin as a regulator of seladin-1 expression for the first time. The relevance of these findings for the association of DM with AD is discussed.

Hepatitis C Virus Selectively Alters the Intracellular Localization of Desmosterol.

Hepatitis C virus (HCV) increases intracellular desmosterol without affecting the steady-state abundance of other sterols, and the antiviral activity of inhibitors of desmosterol synthesis is suppressed by the addition of exogenous desmosterol. These observations suggest a model in which desmosterol has a specific function, direct or indirect, in HCV replication and that HCV alters desmosterol homeostasis to promote viral replication. Here, we use stimulated Raman scattering (SRS) microscopy in combination with isotopically labeled sterols to show that HCV causes desmosterol to accumulate in lipid droplets that are closely associated with the viral NS5A protein and that are visually distinct from the broad distribution of desmosterol in mock-infected cells and the more heterogeneous and disperse lipid droplets to which cholesterol traffics. Localization of desmosterol in NS5A-associated lipid droplets suggests that desmosterol may affect HCV replication via a direct mechanism. We anticipate that SRS microscopy and similar approaches can provide much needed tools to study the localization of specific lipid molecules in cellulo and in vivo.

The influence of cholesterol precursor--desmosterol--on artificial lipid membranes.

The disorders in cholesterol biosynthesis pathway and various diseases manifest in the accumulation of cholesterol precursors in the human tissues and cellular membranes. In this paper the effect of desmosterol--one of cholesterol precursors--on model lipid membranes was studied. The investigations were performed for binary SM/desmo and POPC/desmo and ternary SM/POPC/desmo monolayers. Moreover, the experiments based on the gradual substitution of cholesterol by desmosterol in SM/POPC/chol=1:1:1 system were done. The obtained results allowed one to conclude that desmosterol is of lower domains promoting and stabilizing properties and packs less tightly with the lipids in monolayers. Moreover, desmosterol probably could replace cholesterol in model membranes, but only at its low proportion in the system (2%), however, at a higher degree of cholesterol substitution a significant decrease of the monolayer stability and packing and alterations in the film morphology were detected. The results collected in this work together with those from previous experiments allowed one to analyze the effect of a double bond in the sterol side chain as well as its position in the ring system on membrane activity of the molecule and to verify Bloch hypothesis.

Sterol metabolism controls T(H)17 differentiation by generating endogenous RORγ agonists.

Retinoic acid receptor-related orphan receptor γ (RORγt) controls the differentiation of naive CD4(+) T cells into the TH17 lineage, which are critical cells in the pathogenesis of autoimmune diseases. Here we report that during TH17 differentiation, cholesterol biosynthesis and uptake programs are induced, whereas their metabolism and efflux programs are suppressed. These changes result in the accumulation of the cholesterol precursor, desmosterol, which functions as a potent endogenous RORγ agonist. Generation of cholesterol precursors is essential for TH17 differentiation as blocking cholesterol synthesis with chemical inhibitors at steps before the formation of active precursors reduces differentiation. Upon activation, metabolic changes also lead to production of specific sterol-sulfate conjugates that favor activation of RORγ over the TH17-inhibiting sterol receptor LXR. Thus, TH17 differentiation is orchestrated by coordinated sterol synthesis, mobilization and metabolism to selectively activate RORγ.

Protective role of plant sterol and stanol esters in liver inflammation: insights from mice and humans.

The inflammatory component of non-alcoholic steatohepatitis (NASH) can lead to irreversible liver damage. Therefore there is an urgent need to identify novel interventions to combat hepatic inflammation. In mice, omitting cholesterol from the diet reduced hepatic inflammation. Considering the effects of plant sterol/stanol esters on cholesterol metabolism, we hypothesized that plant sterol/stanol esters reduces hepatic inflammation. Indeed, adding plant sterol/stanol esters to a high-fat-diet reduced hepatic inflammation as indicated by immunohistochemical stainings and gene expression for inflammatory markers. Finally, adding sterol/stanol esters lowered hepatic concentrations of cholesterol precursors lathosterol and desmosterol in mice, which were highly elevated in the HFD group similarly as observed in severely obese patients with NASH. In vitro, in isolated LPS stimulated bone marrow derived macrophages desmosterol activated cholesterol efflux whereas sitostanol reduced inflammation. This highly interesting observation that plant sterol/stanol ester consumption leads to complete inhibition of HFD-induced liver inflammation opens new venues in the treatment and prevention of hepatic inflammation.

Molecular requirement for sterols in herpes simplex virus entry and infectivity.

Herpes simplex virus 1 (HSV-1) required cholesterol or desmosterol for virion-induced membrane fusion. HSV successfully entered DHCR24(-/-) cells, which lack a desmosterol-to-cholesterol conversion enzyme, indicating that entry can occur independently of cholesterol. Depletion of desmosterol from these cells resulted in diminished HSV-1 entry, suggesting a general sterol requirement for HSV-1 entry and that desmosterol can operate in virus entry. Cholesterol functioned more effectively than desmosterol, suggesting that the hydrocarbon tail of cholesterol influences viral entry.

Desmosterol in brain is elevated because DHCR24 needs REST for Robust Expression but REST is poorly expressed.

Cholesterol synthesis in the fetal brain is inhibited because activity of DHCR24 (24-dehydrocholesterol reductase) is insufficient, causing concentrations of the precursor desmosterol to increase temporarily to 15-25% of total sterols at birth. We demonstrate that failure of DHCR24 to be adequately upregulated during periods of elevated cholesterol synthesis in the brain results from the presence in its promoter of the repressor element 1 (RE1) nucleotide sequence that binds the RE1-silencing transcription factor (REST) and that REST, generally reduced in neural tissues, uncharacteristically but not without precedent, enhances DHCR24 transcription. DHCR24 and REST mRNA levels are reduced 3- to 4-fold in fetal mouse brain compared to liver (p < 0.001). Chromatin immunoprecipitation assays suggested that REST binds to the human DHCR24 promoter in the vicinity of the predicted human RE1 sequence. Luminescent emission from a human DHCR24 promoter construct with a mutated RE1 sequence was reduced 2-fold compared to output from a reporter with wild-type RE1 (p < 0.005). Silencing REST in HeLa cells resulted in significant reductions of DHCR24 mRNA (2-fold) and DHCR24 protein (4-fold). As expected, relative concentrations of Δ(24)-cholesterol precursor sterols increased 3- to 4-fold, reflecting the inhibition of DHCR24 enzyme activity. In contrast, mRNA levels of DHCR7 (sterol 7-dehydrocholesterol reductase), a gene essential for cholesterol synthesis lacking an RE1 sequence, and concentrations of HMGR (3-hydroxy-3-methyl-glutaryl-CoA reductase) enzyme protein were both unaffected. Surprisingly, a dominant negative fragment of REST consisting of just the DNA binding domain (about 20% of the protein) and full-length REST enhanced DHCR24 expression equally well. Furthermore, RE1 and the sterol response element (SRE), the respective binding sites for REST and the SRE binding protein (SREBP), are contiguous. These observations led us to hypothesize that REST acts because it is bound in close proximity to SREBP, thus amplifying its ability to upregulate DHCR24. It is likely that modulation of DHCR24 expression by REST persisted in the mammalian genome either because it does no harm or because suppressing metabolically active DHCR24 while providing abundant quantities of the multifunctional sterol desmosterol during neural development proved useful.