Enhancing 7-dehydrocholesterol suppresses brain ferroptosis and tissue injury after neonatal hypoxia-ischemia.

Neonatal hypoxic-ischemic brain injury (HIBI) results in part from excess reactive oxygen species and iron-dependent lipid peroxidation (i.e. ferroptosis). The vitamin D precursor 7-dehydrocholesterol (7-DHC) may inhibit iron-dependent lipid peroxidation. Primary neurons underwent oxygen and glucose deprivation (OGD) injury and treatment with 7-DHC-elevating medications such as cariprazine (CAR) or vehicle. Postnatal day 9 mice underwent sham surgery or carotid artery ligation and hypoxia and received intraperitoneal CAR. In neurons, CAR administration resulted in significantly increased cell survival compared to vehicle controls, whether administered 48 h prior to or 30 min after OGD, and was associated with increased 7-DHC. In the mouse model, malondialdehyde and infarct area significantly increased after HIBI in the vehicle group, which were attenuated by post-treatment with CAR and were negatively correlated with tissue 7-DHC concentrations. Elevating 7-DHC concentrations with CAR was associated with improved cellular and tissue viability after hypoxic-ischemic injury, suggesting a novel therapeutic avenue.

Effects of Neonatal Hypoxic-Ischemic Injury on Brain Sterol Synthesis and Metabolism.

BACKGROUND: Neonatal hypoxic-ischemic brain injury (HIBI) results from disruptions to blood supply and oxygen in the perinatal brain. The goal of this study was to measure brain sterol metabolites and plasma oxysterols after injury in a neonatal HIBI mouse model to assess for potential therapeutic targets in the brain biochemistry as well as potential circulating diagnostic biomarkers. METHODS: Postnatal day 9 CD1-IGS mouse pups were randomized to HIBI induced by carotid artery ligation followed by 30 minutes at 8% oxygen or to sham surgery and normoxia. Brain tissue was collected for sterol analysis by liquid chromatography with tandem mass spectrometry (LC-MS/MS). Plasma was collected for oxysterol analysis by LC-MS/MS. RESULTS: There were minimal changes in brain sterol concentrations in the first 72 hours after HIBI. In severely injured brains, there was a significant increase in desmosterol, 7-DHC, 8-DHC, and cholesterol 24 hours after injury in the ipsilateral tissue. Lanosterol, 24-dehydrolathosterol, and 14-dehydrozymostenol decreased in plasma 24 hours after injury. Severe neonatal HIBI was associated with increased cholesterol and sterol precursors in the cortex at 24 hours after injury. CONCLUSIONS: Differences in plasma oxysterols were seen at 24 hours but were not present at 30 minutes after injury, suggesting that these sterol intermediates would be of little value as early diagnostic biomarkers.

Chronic Aripiprazole and Trazodone Polypharmacy Effects on Systemic and Brain Cholesterol Biosynthesis.

The concurrent use of several medications is a common practice in the treatment of complex psychiatric conditions. One such commonly used combination is aripiprazole (ARI), an antipsychotic, and trazodone (TRZ), an antidepressant. In addition to their effects on dopamine and serotonin systems, both of these compounds are inhibitors of the 7-dehydrocholesterol reductase (DHCR7) enzyme. To evaluate the systemic and nervous system distribution of ARI and TRZ and their effects on cholesterol biosynthesis, adult mice were treated with both ARI and TRZ for 21 days. The parent drugs, their metabolites, and sterols were analyzed in the brain and various organs of mice using LC-MS/MS. The analyses revealed that ARI, TRZ, and their metabolites were readily detectable in the brain and organs, leading to changes in the sterol profile. The levels of medications, their metabolites, and sterols differed across tissues with notable sex differences. Female mice showed higher turnover of ARI and more cholesterol clearance in the brain, with several post-lanosterol intermediates significantly altered. In addition to interfering with sterol biosynthesis, ARI and TRZ exposure led to decreased ionized calcium-binding adaptor molecule 1 (IBA1) and increased DHCR7 protein expression in the cortex. Changes in sterol profile have been also identified in the spleen, liver, and serum, underscoring the systemic effect of ARI and TRZ on sterol biosynthesis. Long-term use of concurrent ARI and TRZ warrants further studies to fully evaluate the lasting consequences of altered sterol biosynthesis on the whole body.

Retinol and Pro-Vitamin A Carotenoid Nutritional Status during Pregnancy Is Associated with Newborn Hearing Screen Results.

The prenatal period is critical for auditory development; thus, prenatal influences on auditory development may significantly impact long-term hearing ability. While previous studies identified a protective effect of carotenoids on adult hearing, the impact of these nutrients on hearing outcomes in neonates is not well understood. The purpose of this study is to investigate the relationship between maternal and umbilical cord plasma retinol and carotenoid concentrations and abnormal newborn hearing screen (NHS) results. Mother-infant dyads (n = 546) were enrolled at delivery. Plasma samples were analyzed using HPLC and LC-MS/MS. NHS results were obtained from medical records. Statistical analysis utilized Mann-Whitney U tests and logistic regression models, with p ≤ 0.05 considered statistically significant. Abnormal NHS results were observed in 8.5% of infants. Higher median cord retinol (187.4 vs. 162.2 µg/L, p = 0.01), maternal trans-ß-carotene (206.1 vs. 149.4 µg/L, p = 0.02), maternal cis-ß-carotene (15.9 vs. 11.2 µg/L, p = 0.02), and cord trans-ß-carotene (15.5 vs. 8.0 µg/L, p = 0.04) were associated with abnormal NHS. Significant associations between natural log-transformed retinol and ß-carotene concentrations and abnormal NHS results remained after adjustment for smoking status, maternal age, and corrected gestational age. Further studies should investigate if congenital metabolic deficiencies, pesticide contamination of carotenoid-rich foods, maternal hypothyroidism, or other variables mediate this relationship.

Effects of Psychotropic Medication on Somatic Sterol Biosynthesis of Adult Mice.

Polypharmacy is commonly used to treat psychiatric disorders. These combinations often include drugs with sterol biosynthesis inhibiting side effects, including the antipsychotic aripiprazole (ARI), and antidepressant trazodone (TRZ). As the effects of psychotropic medications are poorly understood across the various tissue types to date, we investigated the effects of ARI, TRZ, and ARI + TRZ polypharmacy on the post-lanosterol biosynthesis in three cell lines (Neuro2a, HepG2, and human dermal fibroblasts) and seven peripheral tissues of an adult mouse model. We found that both ARI and TRZ strongly interfere with the function of 7-dehydrocholesterol reductase enzyme (DHCR7) and lead to robust elevation in 7-dehydrocholesterol levels (7-DHC) and reduction in desmosterol (DES) across all cell lines and somatic tissues. ARI + TRZ co-administration resulted in summative or synergistic effects across the utilized in vitro and in vivo models. These findings suggest that at least some of the side effects of ARI and TRZ are not receptor mediated but arise from inhibiting DHCR7 enzyme activity. We propose that interference with sterol biosynthesis, particularly in the case of simultaneous utilization of medications with such side effects, can potentially interfere with functioning or development of multiple organ systems, warranting further investigation.

Individual and simultaneous treatment with antipsychotic aripiprazole and antidepressant trazodone inhibit sterol biosynthesis in the adult brain.

Polypharmacy, or the simultaneous use of multiple drugs to treat a single patient, is a common practice in psychiatry. Unfortunately, data on the health effects of commonly used combinations of medications are very limited. In this study, we therefore investigated the effects and interactions between two commonly prescribed psychotropic medications with sterol inhibiting side effects, trazodone (TRZ), an antidepressant, and aripiprazole (ARI), an antipsychotic. In vitro cell culture experiments revealed that both medications alone disrupted neuronal and astroglial sterol biosynthesis in dose-dependent manners. Furthermore, when ARI and TRZ were combined, exposure resulted in an additive 7-dehydrocholesterol (7-DHC) increase, as well as desmosterol (DES) and cholesterol decreases in both cell types. In adult mice, at baseline, we found that the three investigated sterols showed significant differences in distribution across the eight assessed brain regions. Furthermore, experimental mice treated with ARI or TRZ, or a combination of both medications for 8 days, showed strong sterol disruption across all brain regions. We show ARI or TRZ alone elevated 7-DHC and decreased DES levels in all brain regions, but with regional differences. However, the combined utilization of these two medications for 8 days did not lead to additive changes in sterol disturbances. Based on the complex roles of 7-DHC derived oxysterols, we conclude that individual and potentially simultaneous use of medications with sterol biosynthesis-inhibiting properties might have undesired side effects on the adult brain, with as yet unknown long-term consequences on mental or physical health.

Neonatal Hypoxic-Ischemic Brain Injury Alters Brain Acylcarnitine Levels in a Mouse Model.

Hypoxic-ischemic brain injury (HIBI) leads to depletion of ATP, mitochondrial dysfunction, and enhanced oxidant formation. Measurement of acylcarnitines may provide insight into mitochondrial dysfunction. Plasma acylcarnitine levels are altered in neonates after an HIBI, but individual acylcarnitine levels in the brain have not been evaluated. Additionally, it is unknown if plasma acylcarnitines reflect brain acylcarnitine changes. In this study, postnatal day 9 CD1 mouse pups were randomized to HIBI induced by carotid artery ligation, followed by 30 min at 8% oxygen, or to sham surgery and normoxia, with subgroups for tissue collection at 30 min, 24 h, or 72 h after injury (12 animals/group). Plasma, liver, muscle, and brain (dissected into the cortex, cerebellum, and striatum/thalamus) tissues were collected for acylcarnitine analysis by LC-MS. At 30 min after HIBI, acylcarnitine levels were significantly increased, but the differences resolved by 24 h. Palmitoylcarnitine was increased in the cortex, muscle, and plasma, and stearoylcarnitine in the cortex, striatum/thalamus, and cerebellum. Other acylcarnitines were elevated only in the muscle and plasma. In conclusion, although plasma acylcarnitine results in this study mimic those seen previously in humans, our data suggest that the plasma acylcarnitine profile was more reflective of muscle changes than brain changes. Acylcarnitine metabolism may be a target for therapeutic intervention after neonatal HIBI, though the lack of change after 30 min suggests a limited therapeutic window.

Ubiquitous Aberration in Cholesterol Metabolism across Pancreatic Ductal Adenocarcinoma.

Pancreatic cancer (PC) is characterized by metabolic deregulations that often manifest as deviations in metabolite levels and aberrations in their corresponding metabolic genes across the clinical specimens and preclinical PC models. Cholesterol is one of the critical metabolites supporting PC, synthesized or acquired by PC cells. Nevertheless, the significance of the de novo cholesterol synthesis pathway has been controversial in PC, indicating the need to reassess this pathway in PC. We utilized preclinical models and clinical specimens of PC patients and cell lines and utilized mass spectrometry-based sterol analysis. Further, we also performed in silico analysis to corroborate the significance of de novo cholesterol synthesis pathway in PC. Our results demonstrated alteration in free sterol levels, including free cholesterol, across in vitro, in vivo, and clinical specimens of PC. Especially, our sterol analyses established consistent alterations in free cholesterol across the different PC models. Overall, this study demonstrates the significance and consistency in deviation of cholesterol synthesis pathway in PC while showing the aberrations in sterol metabolite intermediates and the related genes using preclinical models, in silico platforms, and the clinical specimens.

Sterol Biosynthesis Inhibition in Pregnant Women Taking Prescription Medications.

Sterol biosynthesis is a critical homeostatic mechanism of the body. Sterol biosynthesis begins during early embryonic life and continues throughout life. Many commonly used medications, prescribed >200 million times in the United States annually, have a sterol biosynthesis inhibition side effect. Using our high-throughput LC-MS/MS method, we assessed the levels of post-lanosterol sterol intermediates (lanosterol, desmosterol, and 7-dehydrocholesterol (7-DHC)) and cholesterol in 1312 deidentified serum samples from pregnant women. 302 samples showing elevated 7-DHC were analyzed for the presence of 14 medications known to inhibit the 7-dehydrocholesterol reductase enzyme (DHCR7) and increase 7-DHC. Of the 302 samples showing 7-DHC elevation, 43 had detectable levels of prescription medications with a DHCR7-inhibiting side effect. Taking more than one 7-DHC-elevating medication in specific combinations (polypharmacy) might exacerbate the effect on 7-DHC levels in pregnant women, suggesting a potentially additive or synergistic effect. As 7-DHC and 7-DHC-derived oxysterols are toxic, and as DHCR7-inhibiting medications are considered teratogens, our findings raise potential concerns regarding the use of prescription medication with a DHCR7-inhibiting side effect during pregnancy. The use of prescription medications during pregnancy is sometimes unavoidable, but choosing a medication without a DHCR7-inhibiting side effect might lead to a heathier pregnancy and prevent putatively adverse outcomes for the developing offspring.

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.

An altered peripheral IL6 response in major depressive disorder.

Major depressive disorder (MDD) is one of the most prevalent major psychiatric disorders with a lifetime prevalence of 17%. Recent evidence suggests MDD is not only a brain dysfunction, but a systemic disease affecting the whole body. Central and peripheral inflammatory changes seem to be a centerpiece of MDD pathology: a subset of patients show elevated blood cytokine and chemokine levels that partially normalize with symptom improvement over the course of anti-depressant treatment. As this inflammatory process in MDD is poorly understood, we hypothesized that the peripheral tissues of MDD patients will respond differently to inflammatory stimuli, resulting in an aberrant transcriptional response to elevated pro-inflammatory cytokines. To test this, we used MDD patient- and control-derived dermal fibroblast cultures to investigate their response to an acute treatment with IL6, IL1ß, TNFα, or vehicle. Following RNA isolation and subsequent cDNA synthesis, quantitative PCR was used to determine the relative expression level of several families of inflammation-responsive genes. Our results showed comparable expression of the tested genes between MDD patients and controls at baseline. In contrast, MDD patient fibroblasts had a diminished transcriptional response to IL6 in all the gene sets tested (oxidative stress response, mitochondrial function, and lipid metabolism). We also found a significant increase in baseline and IL6 stimulated transcript levels of the IL6 receptor gene. This IL6 receptor transcript increase in MDD fibroblasts was accompanied by an IL6 stimulated increase in induction of SOCS3, which dampens IL6 receptor signaling. Altogether our results demonstrate that there is an altered transcriptional response to IL6 in MDD, which may represent one of the molecular mechanisms contributing to disease pathophysiology. Ultimately we hope that these studies will lead to validation of novel MDD drug targets focused on normalizing the altered IL6 response in patients.

A highly sensitive method for analysis of 7-dehydrocholesterol for the study of Smith-Lemli-Opitz syndrome.

We describe a highly sensitive method for the detection of 7-dehydrocholesterol (7-DHC), the biosynthetic precursor of cholesterol, based on its reactivity with 4-phenyl-1,2,4-triazoline-3,5-dione (PTAD) in a Diels-Alder cycloaddition reaction. Samples of biological tissues and fluids with added deuterium-labeled internal standards were derivatized with PTAD and analyzed by LC-MS. This protocol permits fast processing of samples, short chromatography times, and high sensitivity. We applied this method to the analysis of cells, blood, and tissues from several sources, including human plasma. Another innovative aspect of this study is that it provides a reliable and highly reproducible measurement of 7-DHC in 7-dehydrocholesterol reductase (Dhcr7)-HET mouse (a model for Smith-Lemli-Opitz syndrome) samples, showing regional differences in the brain tissue. We found that the levels of 7-DHC are consistently higher in Dhcr7-HET mice than in controls, with the spinal cord and peripheral nerve showing the biggest differences. In addition to 7-DHC, sensitive analysis of desmosterol in tissues and blood was also accomplished with this PTAD method by assaying adducts formed from the PTAD "ene" reaction. The method reported here may provide a highly sensitive and high throughput way to identify at-risk populations having errors in cholesterol biosynthesis.

Probing lipid-protein adduction with alkynyl surrogates: application to Smith-Lemli-Opitz syndrome.

Lipid modifications aid in regulating (and misregulating) protein function and localization. However, efficient methods to screen for a lipid's ability to modify proteins are not readily available. We present a strategy to identify protein-reactive lipids and apply it to a neurodevelopmental disorder, Smith-Lemli-Opitz syndrome (SLOS). Alkynyl surrogates were synthesized for polyunsaturated fatty acids, phospholipids, cholesterol, 7-dehydrocholesterol (7-DHC), and a 7-DHC-derived oxysterol. To probe for protein-reactive lipids, we used click chemistry to biotinylate the alkynyl tag and detected the lipid-adducted proteins with streptavidin Western blotting. In Neuro2a cells, the trend in amount of protein adduction followed known rates of lipid peroxidation (7-DHC >> arachidonic acid > linoleic acid >> cholesterol), with alkynyl-7-DHC producing the most adduction among alkynyl lipids. 7-DHC reductase-deficient cells, which cannot properly metabolize 7-DHC, exhibited significantly more alkynyl-7-DHC-protein adduction than control cells. Model studies demonstrated that a 7-DHC peroxidation product covalently modifies proteins. We hypothesize that 7-DHC generates electrophiles that can modify the proteome, contributing to SLOS's complex pathology. These probes and methods would allow for analysis of lipid-modified proteomes in SLOS and other disorders exhibiting 7-DHC accumulation. More broadly, the alkynyl lipid library would facilitate exploration of lipid peroxidation's role in specific biological processes in numerous diseases.

Oxidative stress and glutathione response in tissue cultures from persons with major depression.

There is evidence that major depressive disorder (MDD) is associated with increased peripheral markers of oxidative stress. To explore oxidation and antioxidant response in MDD, we assayed human dermal fibroblast cultures derived from skin biopsies of age-, race-, and sex-matched individuals in depressed and normal control groups (n = 16 each group), cultured in glucose and galactose conditions, for relative protein carbonylation (a measure of oxidative stress), glutathione reductase (GR) expression, and total glutathione concentration. In control-group fibroblasts, galactose induced a significant increase from the glucose condition in both protein carbonylation and GR. The cells from the MDD group showed total protein carbonylation and GR expression in the glucose condition that was significantly higher than control cells in glucose and equivalent to controls in galactose. There was a small decrease in protein carbonylation in MDD cells from glucose to galactose and no significant change in GR. There was no difference in total glutathione among any of the groups. Increased protein carbonylation and GR expression, cellular responses to oxidative stress induced by galactose in control fibroblasts, are present in fibroblasts derived from MDD patients and are not explainable by reduced GR or total glutathione in the depressed patients. These studies support the role of oxidative stress in the pathophysiology of MDD. Further confirmation of these findings could lead to the development of novel antioxidant approaches for the treatment of depression.

An oxysterol biomarker for 7-dehydrocholesterol oxidation in cell/mouse models for Smith-Lemli-Opitz syndrome.

The level of 7-dehydrocholesterol (7-DHC) is elevated in tissues and fluids of Smith-Lemli-Opitz syndrome (SLOS) patients due to defective 7-DHC reductase. Although over a dozen oxysterols have been identified from 7-DHC free radical oxidation in solution, oxysterol profiles in SLOS cells and tissues have never been studied. We report here the identification and complete characterization of a novel oxysterol, 3ß,5α-dihydroxycholest-7-en-6-one (DHCEO), as a biomarker for 7-DHC oxidation in fibroblasts from SLOS patients and brain tissue from a SLOS mouse model. Deuterated (d7)-standards of 7-DHC and DHCEO were synthesized from d7-cholesterol. The presence of DHCEO in SLOS samples was supported by chemical derivatization in the presence of d7-DHCEO standard followed by HPLC-MS or GC-MS analysis. Quantification of cholesterol, 7-DHC, and DHCEO was carried out by isotope dilution MS with the d7-standards. The level of DHCEO was high and correlated well with the level of 7-DHC in all samples examined (R = 0.9851). Based on our in vitro studies in two different cell lines, the mechanism of formation of DHCEO that involves 5α,6α-epoxycholest-7-en-3ß-ol, a primary free radical oxidation product of 7-DHC, and 7-cholesten-3ß,5α,6ß-triol is proposed. In a preliminary test, a pyrimidinol antioxidant was found to effectively suppress the formation of DHCEO in SLOS fibroblasts.

Biological activities of 7-dehydrocholesterol-derived oxysterols: implications for Smith-Lemli-Opitz syndrome.

Smith-Lemli-Opitz syndrome (SLOS) is a metabolic and developmental disorder caused by mutations in the gene encoding the enzyme 7-dehydrocholesterol reductase (Dhcr7). This reductase catalyzes the last step in cholesterol biosynthesis, and levels of 7-dehydrocholesterol (7-DHC), the substrate for this enzyme, are elevated in SLOS patients as a result of this defect. Our group has previously shown that 7-DHC is extremely prone to free radical autoxidation, and we identified about a dozen different oxysterols formed from oxidation of 7-DHC. We report here that 7-DHC-derived oxysterols reduce cell viability in a dose- and time-dependent manner, some of the compounds showing activity at sub-micromolar concentrations. The reduction of cell survival is caused by a combination of reduced proliferation and induced differentiation of the Neuro2a cells. The complex 7-DHC oxysterol mixture added to control Neuro2a cells also triggers the gene expression changes that were previously identified in Dhcr7-deficient Neuro2a cells. Based on the identification of overlapping gene expression changes in Dhcr7-deficient and 7-DHC oxysterol-treated Neuro2a cells, we hypothesize that some of the pathophysiological findings in the mouse SLOS model and SLOS patients might be due to accumulated 7-DHC oxysterols.

p75 neurotrophin receptor-mediated apoptosis in sympathetic neurons involves a biphasic activation of JNK and up-regulation of tumor necrosis factor-alpha-converting enzyme/ADAM17.

During the development of the sympathetic nervous system, the p75 neurotrophin receptor (p75NTR) has a dual function: promoting survival together with TrkA in response to NGF, but inducing cell death upon binding pro or mature brain-derived neurotrophic factor (BDNF). Apoptotic signaling through p75NTR requires activation of the stress kinase, JNK. However, the receptor also undergoes regulated proteolysis, first by a metalloprotease, and then by gamma-secretase, in response to pro-apoptotic ligands and this is necessary for receptor mediated neuronal death (Kenchappa, R. S., Zampieri, N., Chao, M. V., Barker, P. A., Teng, H. K., Hempstead, B. L., and Carter, B. D. (2006) Neuron 50, 219-232). Hence, the relationship between JNK activation and receptor proteolysis remains to be defined. Here, we report that JNK3 activation is necessary for p75NTR cleavage; however, following release of the intracellular domain, there is a secondary activation of JNK3 that is cleavage dependent. Receptor proteolysis and apoptosis were prevented in sympathetic neurons from jnk3(-/-) mice, while activation of JNK by ectopic expression of MEKK1 induced p75NTR cleavage and cell death. Proteolysis of the receptor was not detected until 6 h after BDNF treatment, suggesting that JNK3 promotes cleavage through a transcriptional mechanism. In support of this hypothesis, BDNF up-regulated tumor necrosis factor-alpha-converting enzyme (TACE)/ADAM17 mRNA and protein in wild-type, but not jnk3(-/-) sympathetic neurons. Down-regulation of TACE by RNA interference blocked BDNF-induced p75NTR cleavage and apoptosis, indicating that this metalloprotease is responsible for the initial processing of the receptor. Together, these results demonstrate that p75NTR-mediated activation of JNK3 is required for up-regulation of TACE, which promotes receptor proteolysis, leading to prolonged activation of JNK3 and subsequent apoptosis in sympathetic neurons.

Oxysterols from free radical chain oxidation of 7-dehydrocholesterol: product and mechanistic studies.

Free radical chain oxidation of highly oxidizable 7-dehydrocholesterol (7-DHC), initiated by 2,2'-azobis(4-methoxy-2,4-dimethylvaleronitrile), was carried out at 37 degrees C in benzene for 24 h. Fifteen oxysterols derived from 7-DHC were isolated and characterized with 1D and 2D NMR spectroscopy and mass spectrometry. A mechanism that involves abstraction of hydrogen atoms at C-9 and/or C-14 is proposed to account for the formation of all of the oxysterols and the reaction progress profile. In either the H-9 or H-14 mechanism, a pentadienyl radical intermediate is formed after abstraction of H-9 or H-14 by a peroxyl radical. This step is followed by the well-precedented transformations observed in peroxidation reactions of polyunsaturated fatty acids such as oxygen addition, peroxyl radical 5-exo cyclization, and S(H)i carbon radical attack on the peroxide bond. The mechanism for peroxidation of 7-DHC also accounts for the formation of numerous oxysterol natural products isolated from fungal species, marine sponges, and cactaceous species. In a cell viability test, the oxysterol mixture from 7-DHC peroxidation was found to be cytotoxic to Neuro2a neuroblastoma cells in the micromolar concentration range. We propose that the high reactivity of 7-DHC and the oxysterols generated from its peroxidation may play important roles in the pathogenesis of Smith-Lemli-Opitz syndrome, X-linked dominant chondrodysplasia punctata, and cerebrotendinous xanthomatosis, all of these being metabolic disorders characterized by an elevated level of 7-DHC.

NRIF is a regulator of neuronal cholesterol biosynthesis genes.

Cholesterol is a critical component of neuronal membranes, required for normal signal transduction. We showed previously that adult hippocampal neurons co-express high levels of cholesterogenic enzymes, and that their expression is under the control of the p75 neurotrophin receptor (p75NTR). Most of the cellular effects of p75NTR are mediated via interacting proteins, including neurotrophin receptor interacting factor (NRIF). In this study, we tested the hypothesis that p75NTR-dependent regulation of cholesterol and lipid biosynthesis genes is mediated by NRIF. We found that in vitro down regulation of NRIF expression decreased the mRNA for two main cholesterogenic enzymes, 3-hydroxy-3-methylglutaryl-coenzyme A reductase (Hmgcr; EC 2.3.3.10) and 7-dehydrocholesterol reductase (Dhcr7; EC 1.3.1.21). Further analyses revealed that NRIF-dependent and Dhcr7-dependent transcriptional changes show a high degree of overlap, and that NRIF reduction resulted in reduced expression of sterol-sensing domain protein SCAP, followed by a decrease in mRNA levels of SRE-motif containing genes (HMGCR, FASN, SREBP2, S1P, and SQS1). Finally, a reduction in cholesterol biosynthesis-related gene expression was also observed in hippocampal tissue of mice with NRIF deletion. Our combined in vitro and in vivo studies suggest that hippocampal neuronal cholesterol biosynthesis is regulated through the p75NTR interacting factor NRIF.

Protein kinase A-induced phosphorylation of the p65 subunit of nuclear factor-kappaB promotes Schwann cell differentiation into a myelinating phenotype.

Axon-Schwann cell interactions are critical for myelin formation during peripheral nerve development and regeneration. Axonal contact promotes Schwann cell precursors to differentiate into a myelinating phenotype, and cAMP-elevating agents can mimic this; however, the mechanisms underlying this differentiation are poorly understood. We demonstrated previously that the transcription factor nuclear factor-kappaB (NF-kappaB) is required for myelin formation by Schwann cells (Nickols et al., 2003), although how it is activated during this process remained to be determined. Here, we report that culturing Schwann cells with sensory neurons results in the activation of cAMP-dependent protein kinase (PKA), and this kinase phosphorylates the p65 subunit of NF-kappaB at S276. The phosphorylation was also induced in cultured Schwann cells by treatment with forskolin, dibutyryl-cAMP, or by overexpression of a catalytic subunit of PKA, and this increased the transcriptional activity of NF-kappaB. In developing perinatal rat sciatic nerve, the kinetics of p65 phosphorylation at S276 paralleled that of PKA and NF-kappaB activation. To elucidate the role of p65 phosphorylation in myelin formation, we overexpressed an S276A mutant of p65 in cultured Schwann cells, which blocked PKA-mediated transcriptional activation of NF-kappaB. When the Schwann cells expressing the mutant were cocultured with sensory neurons, there was a 45% reduction in the number of myelinated fibers relative to controls, demonstrating a requirement for p65 phosphorylation by PKA during myelin formation.