Cholesterol Turnover Research Articles

Thyroid hormone receptor beta (THRB) dependent regulation of diurnal hepatic lipid metabolism in adult male mice

Thyroid hormones (THs) are critical regulators of systemic energy metabolism and homeostasis. In the liver, high TH action protects against steatosis by enhancing cholesterol and triglyceride turnover, with thyroid hormone receptor beta (THRB) signaling playing a pivotal role. This study probed the potential interaction between THRB action and another critical regulator of liver energy metabolism, the circadian clock. Liver transcriptome analysis of THRB deficient (THRBKO) mice under normal chow conditions revealed a modest impact of THRB deletion. Temporal transcriptome and lipidome profiling uncovered significant alterations in diurnal metabolic rhythms attributable to THRB deficiency pointing to a pro-steatotic state with elevated levels of cholesterol, tri- and diacylglycerides, and fatty acids. These findings were confirmed by THRB agonization in hepatocytes under steatosis-promoting conditions in vitro. Integration of transcriptome profiles from THRBKO mice and mice with induced high or low TH action identified a subset of TH responsive but THRB insensitive genes implicated in immune processes. In summary, our study reveals a complex time-of-day dependent interaction of different TH-related signals in the regulation of liver physiology indicating an opportunity for chronopharmacological approaches to TH/THRB manipulation in fatty liver diseases.

Bile Acid Signaling in Metabolic and Inflammatory Diseases and Drug Development.

Bile acids are the end products of cholesterol catabolism. Hepatic bile acid synthesis accounts for a major fraction of daily cholesterol turnover in humans. Biliary secretion of bile acids generates bile flow and facilitates biliary secretion of lipids, endogenous metabolites, and xenobiotics. In intestine, bile acids facilitate the digestion and absorption of dietary lipids and fat-soluble vitamins. Through activation of nuclear receptors and G protein-coupled receptors and interaction with gut microbiome, bile acids critically regulate host metabolism and innate and adaptive immunity and are involved in the pathogenesis of cholestasis, metabolic dysfunction-associated steatotic liver disease, alcohol-associated liver disease, type-2 diabetes, and inflammatory bowel diseases. Bile acids and their derivatives have been developed as potential therapeutic agents for treating chronic metabolic and inflammatory liver diseases and gastrointestinal disorders. SIGNIFICANCE STATEMENT: Bile acids facilitate biliary cholesterol solubilization and dietary lipid absorption, regulate host metabolism and immunity, and modulate gut microbiome. Targeting bile acid metabolism and signaling holds promise for treating metabolic and inflammatory diseases.

Unbiased insights into the multiplicity of the CYP46A1 brain effects in 5XFAD mice treated with low dose-efavirenz

Cytochrome P450 46A1 (CYP46A1) is the CNS-specific cholesterol 24-hydroxylase that controls cholesterol elimination and turnover in the brain. In mouse models, pharmacologic CYP46A1 activation with low-dose efavirenz or by gene therapy mitigates the manifestations of various brain disorders, neurologic, and nonneurologic, by affecting numerous, apparently unlinked biological processes. Accordingly, CYP46A1 is emerging as a promising therapeutic target; however, the mechanisms underlying the multiplicity of the brain CYP46A1 activity effects are currently not understood. We proposed the chain reaction hypothesis, according to which CYP46A1 is important for the three primary (unifying) processes in the brain (sterol flux through the plasma membranes, acetyl-CoA, and isoprenoid production), which in turn affect a variety of secondary processes. We already identified several processes secondary to changes in sterol flux and herein undertook a multiomics approach to compare the brain proteome, acetylproteome, and metabolome of 5XFAD mice (an Alzheimer’s disease model), control and treated with low-dose efavirenz. We found that the latter had increased production of phospholipids from the corresponding lysophospholipids and a globally increased protein acetylation (including histone acetylation). Apparently, these effects were secondary to increased acetyl-CoA production. Signaling of small GTPases due to their altered abundance or abundance of their regulators could be affected as well, potentially via isoprenoid biosynthesis. In addition, the omics data related differentially abundant molecules to other biological processes either reported previously or new. Thus, we obtained unbiased mechanistic insights and identified potential players mediating the multiplicity of the CYP46A1 brain effects and further detailed our chain reaction hypothesis.

CYP46A1-mediated cholesterol turnover induces sex-specific changes in cognition and counteracts memory loss in ovariectomized mice.

The brain-specific enzyme CYP46A1 controls cholesterol turnover by converting cholesterol into 24S-hydroxycholesterol (24OH). Dysregulation of brain cholesterol turnover and reduced CYP46A1 levels are observed in Alzheimer's disease (AD). In this study, we report that CYP46A1 overexpression in aged female mice leads to enhanced estrogen signaling in the hippocampus and improved cognitive functions. In contrast, age-matched CYP46A1 overexpressing males show anxiety-like behavior, worsened memory, and elevated levels of 5α-dihydrotestosterone in the hippocampus. We report that, in neurons, 24OH contributes to these divergent effects by activating sex hormone signaling, including estrogen receptors. CYP46A1 overexpression in female mice protects from memory impairments induced by ovariectomy while having no effects in gonadectomized males. Last, we measured cerebrospinal fluid levels of 24OH in a clinical cohort of patients with AD and found that 24OH negatively correlates with neurodegeneration markers only in women. We suggest that CYP46A1 activation is a valuable pharmacological target for enhancing estrogen signaling in women at risk of developing neurodegenerative diseases.

Tuning of liver circadian transcriptome rhythms by thyroid hormone state in male mice

Thyroid hormones (THs) are important regulators of systemic energy metabolism. In the liver, they stimulate lipid and cholesterol turnover and increase systemic energy bioavailability. It is still unknown how the TH state interacts with the circadian clock, another important regulator of energy metabolism. We addressed this question using a mouse model of hypothyroidism and performed circadian analyses. Low TH levels decreased locomotor activity, food intake, and body temperature mostly in the active phase. Concurrently, liver transcriptome profiling showed only subtle effects compared to elevated TH conditions. Comparative circadian transcriptome profiling revealed alterations in mesor, amplitude, and phase of transcript levels in the livers of low-TH mice. Genes associated with cholesterol uptake, biosynthesis, and bile acid secretion showed reduced mesor. Increased and decreased cholesterol levels in the serum and liver were identified, respectively. Combining data from low- and high-TH conditions allowed the identification of 516 genes with mesor changes as molecular markers of the liver TH state. We explored these genes and created an expression panel that assesses liver TH state in a time-of-day dependent manner. Our findings suggest that the liver has a low TH action under physiological conditions. Circadian profiling reveals genes as potential markers of liver TH state.

Alterations of the chemical profile of cholesterol in cancer tissue as traced with ToF-SIMS.

Cancer has become one of the leading causes of death, with approximately ten million people worldwide dying from cancer each year. In most cases, cancer spreads to remote organs and develops a resistance to therapy. To reduce the deadly impact of cancer, novel targets for markers for early detection are necessary. Given the notable influence of rapid chemical turnover on isotope effects, the heightened turnover rate of cholesterol in cancer offers a promising way for investigation. Time-of-Flight Secondary Ion Mass Spectrometry (ToF-SIMS) offers a valuable tool of tracking cholesterol dynamics. Consequently, we employed ToF-SIMS to assess cholesterol alterations, aiming to uncover potential diagnostic vulnerabilities stemming from heightened cholesterol synthesis. Our study explored the chemical profile of cholesterol influenced by cancer cell metabolism using mammary glands from mice, both with and without cancer. Results revealed a significant increase in the fractional abundance of fragment cholesterol peaks (C27H45+) in cancerous tissues, indicating dysregulated cholesterol metabolism within cancer cells. This suggests potential structural weaknesses or incomplete synthesis. Further investigation into carbon isotope incorporation suggests that the isotopic patterns might be due to the integration of heavier carbon isotopes, although these patterns could be affected by other isotopic influences. Nevertheless, understanding isotope effect of cholesterol profiles have the potential to advance our understanding of cancer biology and improve diagnostic approaches.

The Diagnostic Use of the Plasma Quantification of 24S-Hydroxycholesterol and Other Oxysterols in Neurodegenerative Disease.

Cholesterol regulates fluidity and structure of cellular membranes. The brain is involved in signal transduction, synaptogenesis, and membrane trafficking. An impairment of its metabolism was observed in different neurodegenerative diseases, such as Multiple Sclerosis, Alzheimer, and Huntington diseases. Because of the blood-brain barrier, cholesterol cannot be uptaken from the circulation and all the cholesterol is locally synthetized. The excess cholesterol in neurons is converted into 24S-hydroxycholesterol (24OHC) by the cholesterol 24-hydroxylase (CYP46A1). The plasmatic concentration of 24OHC results in the balance between cerebral production and liver elimination. It is related to the number of metabolically active neurons in the brain. Several factors that affect the brain cholesterol turnover and the liver elimination of oxysterols, the genetic background, nutrition, and lifestyle habits were found to significantly affect plasma levels of 24OHC. Reduced levels of 24OHC were found related to the loss of metabolically active cells and the degree of brain atrophy. The dysfunction of the blood-brain barrier, inflammation, and increased cholesterol turnover might overlap with this progressive reduction giving temporary increased levels of 24OHC.The study of plasma 24OHC is likely to offer an insight into brain cholesterol turnover with a limited diagnostic power.

Blocking cholesterol formation and turnover improves cellular and mitochondria function in murine heart microvascular endothelial cells and cardiomyocytes.

Background: Statins and proprotein convertase subtilisin/kexin type 9 inhibitors (PCSK9i) are cornerstones of therapy to prevent cardiovascular disease, acting by lowering lipid concentrations and only partially identified pleiotropic effects. This study aimed to analyze impacts of atorvastatin and synthetic peptide PCSK9i on bioenergetics and function of microvascular endothelial cells and cardiomyocytes. Methods: Mitochondrial function and abundance as well as intracellular nucleotides, membrane potential, cytoskeleton structure, and cell proliferation rate were evaluated in mouse heart microvascular endothelial cells (H5V) and cardiomyocytes (HL-1) under normal and hypoxia-mimicking conditions (CoCl2 exposure). Results: In normal conditions PCSK9i, unlike atorvastatin, enhanced mitochondrial respiratory parameters, increased nucleotide levels, prevented actin cytoskeleton disturbances and stimulated endothelial cell proliferation. Under hypoxia-mimicking conditions both atorvastatin and PCSK9i improved the mitochondrial respiration and membrane potential in both cell types. Conclusion: This study demonstrated that both treatments benefited the endothelial cell and cardiomyocyte bioenergetics, but the effects of PCSK9i were superior.

ABCD1 Transporter Deficiency Results in Altered Cholesterol Homeostasis.

X-linked adrenoleukodystrophy (X-ALD), the most common peroxisomal disorder, is caused by mutations in the peroxisomal transporter ABCD1, resulting in the accumulation of very long-chain fatty acids (VLCFA). Strongly affected cell types, such as oligodendrocytes, adrenocortical cells and macrophages, exhibit high cholesterol turnover. Here, we investigated how ABCD1 deficiency affects cholesterol metabolism in human X-ALD patient-derived fibroblasts and CNS tissues of Abcd1-deficient mice. Lipidome analyses revealed increased levels of cholesterol esters (CE), containing both saturated VLCFA and mono/polyunsaturated (V)LCFA. The elevated CE(26:0) and CE(26:1) levels remained unchanged in LXR agonist-treated Abcd1 KO mice despite reduced total C26:0. Under high-cholesterol loading, gene expression of SOAT1, converting cholesterol to CE and lipid droplet formation were increased in human X-ALD fibroblasts versus healthy control fibroblasts. However, the expression of NCEH1, catalysing CE hydrolysis and the cholesterol transporter ABCA1 and cholesterol efflux were also upregulated. Elevated Soat1 and Abca1 expression and lipid droplet content were confirmed in the spinal cord of X-ALD mice, where expression of the CNS cholesterol transporter Apoe was also elevated. The extent of peroxisome-lipid droplet co-localisation appeared low and was not impaired by ABCD1-deficiency in cholesterol-loaded primary fibroblasts. Finally, addressing steroidogenesis, progesterone-induced cortisol release was amplified in X-ALD fibroblasts. These results link VLCFA to cholesterol homeostasis and justify further consideration of therapeutic approaches towards reducing VLCFA and cholesterol levels in X-ALD.

Phytosterols reverse antiretroviral-induced hearing loss, with potential implications for cochlear aging.

Cholesterol contributes to neuronal membrane integrity, supports membrane protein clustering and function, and facilitates proper signal transduction. Extensive evidence has shown that cholesterol imbalances in the central nervous system occur in aging and in the development of neurodegenerative diseases. In this work, we characterize cholesterol homeostasis in the inner ear of young and aged mice as a new unexplored possibility for the prevention and treatment of hearing loss. Our results show that cholesterol levels in the inner ear are reduced during aging, an effect that is associated with an increased expression of the cholesterol 24-hydroxylase (CYP46A1), the main enzyme responsible for cholesterol turnover in the brain. In addition, we show that pharmacological activation of CYP46A1 with the antiretroviral drug efavirenz reduces the cholesterol content in outer hair cells (OHCs), leading to a decrease in prestin immunolabeling and resulting in an increase in the distortion product otoacoustic emissions (DPOAEs) thresholds. Moreover, dietary supplementation with phytosterols, plant sterols with structure and function similar to cholesterol, was able to rescue the effect of efavirenz administration on the auditory function. Altogether, our findings point towards the importance of cholesterol homeostasis in the inner ear as an innovative therapeutic strategy in preventing and/or delaying hearing loss.

Quantitative characterizations of the cholesterol-related pathways in the retina and brain of hamsters

The retina and brain are separated from the systemic circulation by the anatomical barriers, which are permeable (the outer blood-retinal barrier) and impermeable (the blood-brain and inner blood-retina barriers) to cholesterol. Herein we investigated whether whole-body cholesterol maintenance affects cholesterol homeostasis in the retina and brain. We used hamsters, whose whole-body cholesterol handling is more similar to those in humans than in mice, and conducted separate administrations of deuterated water and deuterated cholesterol. We assessed the quantitative significance of the retinal and brain pathways of cholesterol input and compared the results with those from our previous studies in mice. The utility of the measurements in the plasma of deuterated 24-hydroxycholesterol, the major cholesterol elimination product from the brain, was investigated as well. We established that despite a sevenfold higher serum LDL to HDL ratio and other cholesterol-related differences, in situ biosynthesis remained the major source of cholesterol for hamster retina, although its quantitative significance was reduced to 53% as compared to 72%–78% in the mouse retina. In the brain, the principal pathway of cholesterol input was also the same, in situ biosynthesis, accounting for 94% of the total brain cholesterol input (96% in mice); the interspecies differences pertained to the absolute rates of the total cholesterol input and turnover. We documented the correlations between deuterium enrichments of the brain 24-hydroxycholesterol, brain cholesterol, and plasma 24-hydroxycholesterol, which suggested that deuterium enrichment of plasma 24-hydroxycholesteol could be an in vivo marker of cholesterol elimination and turnover in the brain.

Shenlian extract protected ox-LDL-loaded macrophages against ER stress by promoting LAL-LXRα mediated cholesterol flux

Ethnopharmacological relevanceShenlian (SL) extract is consisted of extracts from Salvia miltiorrhiza Bunge and Andrographis paniculata (Burm.f.) Nees, two herbs commonly used in Chinese clinical formula to treat atherosclerosis by removing blood stasis and clearing away heat. Pharmacologically, the anti-atherosclerotic effects of these two herbs are related to unresolved inflammation and the macrophage anergy or apoptosis in lesions led by the lipid flux blockage and ER stress. However, the deeper understanding of SL extract in protecting macrophage in plaques remains unknown. Aim of the studyThis study aimed to investigate the underlying mechanism of SL extract in protecting ER-stressed macrophages from apoptosis in atherosclerosis. MethodsThe ApoE−/− atherosclerotic mice model and ox-LDL loaded macrophages model were established to assess the effect of SL extract on ER stress in vivo and in vitro. Key markers related to ER stress in plaque were determined by immunohistochemical staining. Proteins involved in apoptosis and ER stress in macrophages loaded by ox-LDL were assessed by Western blot. ER morphology was observed by electron microscope. Lipid flux was temporally and quantitatively depicted by Oil red staining. The LAL and LXRα were blocked by lalistat and Gsk 2033 respectively to investigate whether SL extract protected the function of macrophages by the activation of LAL-LXRα axis. ResultsOur study reported that, in ApoE−/− atherosclerotic mice, SL extract effectively relieved ER stress of carotid artery plaque. In lipid-overloaded macrophage models, SL extract significantly alleviated ER stress by promoting cholesterol degradation and efflux, which finally prevented apoptosis of foam cells induced by ox-LDL. Blockage of ER stress by 4-Phenylbutyric acid (4-PBA), an inhibitor of Endoplasmic Reticulum (ER) stress, largely attenuated the protective effects of SL extract on macrophage. By utilizing the selective antagonists against both LAL and LXRα, this study further revealed that the beneficial effects of SL extract in macrophages was dependent on the proper functionalization of LAL-LXRα axis. ConclusionsBy highlighting the therapeutic significance of macrophage protection in resolving atherosclerosis inflammation, our study pharmacologically provided convincing mechanistic evidence of SL extract in the activation LAL-LXRα axis and revealed its promising potential in the promotion of cholesterol turnover and prevention of ER stress induced apoptosis in lipid-loaded macrophages.

The human microglial surveillant phenotype is preserved by de novo neurosteroidogenesis through the control of cholesterol homeostasis: Crucial role of 18 kDa Translocator Protein

Neurodegenerative disease-associated microglia commonly exhibit harmful cholesterol accumulation that impairs their ability to resolve the neuroinflammatory response, contributing to disease onset and progression. Neurosteroids, whose levels have been often found significantly altered in brain diseases, are the most potent endogenous anti-inflammatory molecules exerting beneficial effects on activities of brain cells, including microglia. For the first time, the impact of neurosteroidogenesis on cholesterol homeostasis for the immune surveillance phenotype maintenance was investigated in a human microglia in vitro model. To enhance and inhibit neurosteroidogenesis, pharmacological stimulation and knock-down of 18 kDa Translocator Protein (TSPO), which is involved in the neurosteroidogenesis rate-limiting step, were used as experimental approaches, respectively. The obtained results point to an essential autocrine control of neurosteroidogenesis in orchestrating cholesterol trafficking in human microglia. TSPO pharmacological stimulation ensured cholesterol turnover by strengthening cholesterol efflux systems and preserving healthy immune surveillant phenotype. Conversely, TSPO knock-down induced an impairment of the controlled interplay among cholesterol synthesis, efflux, and metabolism mechanisms, leading to an excessive cholesterol accumulation and acquisition of a chronically activated dysfunctional phenotype. In this model, the exogenous neurosteroid administration restored proper the cholesterol clearance. The TSPO ability in promoting native neurosteroidogenesis opens the way to restore cholesterol homeostasis, and thus to maintain microglia proper functionality for the treatment of neuroinflammation-related brain diseases.

Emerging Roles of Gut Microbial Modulation of Bile Acid Composition in the Etiology of Cardiovascular Diseases.

Despite advances in preventive measures and treatment options, cardiovascular disease (CVD) remains the number one cause of death globally. Recent research has challenged the traditional risk factor profile and highlights the potential contribution of non-traditional factors in CVD, such as the gut microbiota and its metabolites. Disturbances in the gut microbiota have been repeatedly associated with CVD, including atherosclerosis and hypertension. Mechanistic studies support a causal role of microbiota-derived metabolites in disease development, such as short-chain fatty acids, trimethylamine-N-oxide, and bile acids, with the latter being elaborately discussed in this review. Bile acids represent a class of cholesterol derivatives that is essential for intestinal absorption of lipids and fat-soluble vitamins, plays an important role in cholesterol turnover and, as more recently discovered, acts as a group of signaling molecules that exerts hormonal functions throughout the body. Studies have shown mediating roles of bile acids in the control of lipid metabolism, immunity, and heart function. Consequently, a picture has emerged of bile acids acting as integrators and modulators of cardiometabolic pathways, highlighting their potential as therapeutic targets in CVD. In this review, we provide an overview of alterations in the gut microbiota and bile acid metabolism found in CVD patients, describe the molecular mechanisms through which bile acids may modulate CVD risk, and discuss potential bile-acid-based treatment strategies in relation to CVD.

24-hydroxycholesterol replacement rate measured in blood is a non-invasive biomarker of brain demyelination and remyelination in cuprizone-treated mice

In mice, dietary cuprizone causes brain demyelination with subsequent spontaneous remyelination upon return to normal chow. Heavy water (2H2O) labeling with mass spectrometric analysis can be used to measure brain de novo synthesis of several myelin components including cholesterol, phospholipids, galactocereboside (GalC) and myelin-associated proteins. 24-hydroxycholesterol (24-OHC), the major metabolite of brain cholesterol, is detected in blood and is believed to be specifically derived from CNS cholesterol metabolism. We assessed changes in syntheses of myelin components in brain and of blood sterols during cuprizone-induced experimental demyelination and remyelination, with and without thyroid hormone (T3) treatment.Mice were fed cuprizone for 4 weeks, then returned to control diet and treated with either placebo or T3 (0.005 mg/day). 2H2O was administered for the last 14 days of cuprizone diet, and for either 6, 12 or 19 days of treatment during recovery from cuprizone, after which blood and corpus callosum (CC) samples were collected (n = 5/time point/treatment). 2H incorporation into cholesterol and 24-OHC in blood and CC, and incorporation into phospholipid (PL)-palmitate, GalC, myelin basic protein (MBP) and 2′,3′-cyclic-nucleotide 3′-phosphodiesterase (CNPase) in CC were measured.Cuprizone significantly (p < 0.05) decreased syntheses of cholesterol, 24-OHC, GalC, MBP, CNPase and PL-palmitate in the CC and these effects were all reversed during recovery. T3 treatment significantly (p < 0.05) increased syntheses of cholesterol, 24-OHC and palmitate compared to placebo. 24-OHC and cholesterol turnover rates in brain and blood were nearly identical and 24-OHC rates in blood paralleled rates in CC, indicating that blood 24-OHC derives primarily from the brain and reflects oligodendrocyte function.In summary, changes in synthesis of several lipid and protein components in brain during cuprizone-induced demyelination and remyelination are measurable through stable isotope labeling. Blood 24-OHC turnover rates closely reflect flux rates of brain cholesterol in response to cuprizone and T3, which alter oligodendrocyte function. Labeling of blood 24-OHC has potential as a non-invasive marker of brain de novo cholesterol synthesis and breakdown rates in demyelinating conditions.

Identification of Side Chain Oxidized Sterols as Novel Liver X Receptor Agonists with Therapeutic Potential in the Treatment of Cardiovascular and Neurodegenerative Diseases.

The nuclear receptors-liver X receptors (LXR α and β) are potential therapeutic targets in cardiovascular and neurodegenerative diseases because of their key role in the regulation of lipid homeostasis and inflammatory processes. Specific oxy(phyto)sterols differentially modulate the transcriptional activity of LXRs providing opportunities to develop compounds with improved therapeutic characteristics. We isolated oxyphytosterols from Sargassum fusiforme and synthesized sidechain oxidized sterol derivatives. Five 24-oxidized sterols demonstrated a high potency for LXRα/β activation in luciferase reporter assays and induction of LXR-target genes APOE, ABCA1 and ABCG1 involved in cellular cholesterol turnover in cultured cells: methyl 3β-hydroxychol-5-en-24-oate (S1), methyl (3β)-3-aldehydeoxychol-5-en-24-oate (S2), 24-ketocholesterol (S6), (3β,22E)-3-hydroxycholesta-5,22-dien-24-one (N10) and fucosterol-24,28 epoxide (N12). These compounds induced SREBF1 but not SREBP1c-mediated lipogenic genes such as SCD1, ACACA and FASN in HepG2 cells or astrocytoma cells. Moreover, S2 and S6 enhanced cholesterol efflux from HepG2 cells. All five oxysterols induced production of the endogenous LXR agonists 24(S)-hydroxycholesterol by upregulating the CYP46A1, encoding the enzyme converting cholesterol into 24(S)-hydroxycholesterol; S1 and S6 may also act via the upregulation of desmosterol production. Thus, we identified five novel LXR-activating 24-oxidized sterols with a potential for therapeutic applications in neurodegenerative and cardiovascular diseases.

CYP46A1 activation by low-dose efavirenz enhances brain cholesterol metabolism in subjects with early Alzheimer’s disease

BackgroundEfavirenz is an anti-HIV drug, and cytochrome P450 46A1 (CYP46A1) is a CNS-specific enzyme that metabolizes cholesterol to 24-hydroxycholesterol (24HC). We have previously shown that allosteric CYP46A1 activation by low-dose efavirenz in a transgenic mouse model of Alzheimer’s disease (AD) enhanced both cholesterol elimination and turnover in the brain and improved animal performance in memory tests. Here, we sought to determine whether CYP46A1 could be similarly activated by a low-dose efavirenz in human subjects. MethodsThis pilot study enrolled 5 subjects with early AD. Participants were randomized to placebo (n = 1) or two daily efavirenz doses (50 mg and 200 mg, n = 2 for each) for 20 weeks and evaluated for safety and CYP46A1 target engagement (plasma 24HC levels). A longitudinal mixed model was used to ascertain the statistical significance of target engagement. We also measured 24HC in CSF and conducted a unique stable isotope labeling kinetics (SILK) study with deuterated water to directly measure CYP46A1 activity changes in the brain.ResultsIn subjects receiving efavirenz, there was a statistically significant within-group increase (P ≤ 0.001) in the levels of plasma 24HC from baseline. The levels of 24HC in the CSF of subjects on the 200-mg dose of efavirenz were also increased. Target engagement was further supported by the labeling kinetics of 24HC by deuterated water in the SILK study. There were no serious adverse effects in any subjects.ConclusionsOur findings suggest efavirenz target engagement in human subjects with early AD. This supports the pursuit of a larger trial for further determination and confirmation of the efavirenz dose that exerts maximal enzyme activation, as well as evaluation of this drug’s effects on AD biomarkers and clinical symptomatology.Trial registrationClinicalTrials.gov, NCT03706885.

Alzheimer’s-Associated Upregulation of Mitochondria-Associated ER Membranes After Traumatic Brain Injury

Traumatic brain injury (TBI) can lead to neurodegenerative diseases such as Alzheimer’s disease (AD) through mechanisms that remain incompletely characterized. Similar to AD, TBI models present with cellular metabolic alterations and modulated cleavage of amyloid precursor protein (APP). Specifically, AD and TBI tissues display increases in amyloid-β as well as its precursor, the APP C-terminal fragment of 99 a.a. (C99). Our recent data in cell models of AD indicate that C99, due to its affinity for cholesterol, induces the formation of transient lipid raft domains in the ER known as mitochondria-associated endoplasmic reticulum (ER) membranes (“MAM” domains). The formation of these domains recruits and activates specific lipid metabolic enzymes that regulate cellular cholesterol trafficking and sphingolipid turnover. Increased C99 levels in AD cell models promote MAM formation and significantly modulate cellular lipid homeostasis. Here, these phenotypes were recapitulated in the controlled cortical impact (CCI) model of TBI in adult mice. Specifically, the injured cortex and hippocampus displayed significant increases in C99 and MAM activity, as measured by phospholipid synthesis, sphingomyelinase activity and cholesterol turnover. In addition, our cell type-specific lipidomics analyses revealed significant changes in microglial lipid composition that are consistent with the observed alterations in MAM-resident enzymes. Altogether, we propose that alterations in the regulation of MAM and relevant lipid metabolic pathways could contribute to the epidemiological connection between TBI and AD.Graphical

Cholesterol-24-hydroxylase (CYP46) in the old brain: Analysis of positive populations and factors triggering its expression in astrocytes.

Cholesterol-24-hydroxylase (CYP46), a member of the cytochrome P450 superfamily of enzymes, is selectively expressed in the brain and is mainly responsible for cholesterol turnover in the central nervous system. Although increased cyp46A1 gene expression has been linked to cognitive alterations in aging and observed in neurodegenerative diseases and after traumatic brain injury, a detailed characterization of the brain regions and cell types in which CYP46 is expressed in old individuals has not been performed. Using immunohistochemistry and immunofluorescence, we investigated the specific regions and cell populations in the brain, in which cyp46A1 is expressed in 24-month-old mice. We found that CYP46 is localized in the same neuronal populations in young and old brains, mainly in the hippocampus, in cortical layers, and in Purkinje neurons of the cerebellum. No increase in CYP46 levels was found in astrocytes in old mice brains, in primary astrocyte-neuron cocultures aged in vitro, or in primary cultures of senescent astrocytes. However, interleukin-6 treatment strongly induced cyp46A1 expression in reactive astrocytes characterized by high GFAP levels but had no effect in nonactivated astrocytes. Our data suggest that cholesterol-24-hydroxylase expression is triggered in reactive astrocytes in response to proinflammatory signals, probably as part of a response mechanism to injury.

Effects of SLCO1B1 Genetic Variant on Metabolite Profile in Participants on Simvastatin Treatment.

Organic-anion-transporting polypeptide 1B1 (OATP1B1), encoded by the solute carrier organic anion transporter family member 1B1 gene (SLCO1B1), is highly expressed in the liver and transports several endogenous metabolites into the liver, including statins. Previous studies have not investigated the association of SLCO1B1 rs4149056 variant with the risk of type 2 diabetes (T2D) or determined the metabolite signature of the C allele of SLCO1B1 rs4149056 (SLCO1B1 rs4149056-C allele) in a large randomly selected population. SLCO1B1 rs4149056-C inhibits OATP1B1 transporter and is associated with increased levels of blood simvastatin concentrations. Our study is to first to show that SLCO1B1 rs4149056 variant is not significantly associated with the risk of T2D, suggesting that simvastatin has a direct effect on the risk of T2D. Additionally, we investigated the effects of SLCO1B1 rs4149056-C on plasma metabolite concentrations in 1373 participants on simvastatin treatment and in 1368 age- and body-mass index (BMI)-matched participants without any statin treatment. We found 31 novel metabolites significantly associated with SLCO1B1 rs4149056-C in the participants on simvastatin treatment and in the participants without statin treatment. Simvastatin decreased concentrations of dicarboxylic acids, such as docosadioate and dodecanedioate, that may increase beta- and peroxisomal oxidation and increased the turnover of cholesterol into bile acids, resulting in a decrease in steroidogenesis due to limited availability of cholesterol for steroid synthesis. Our findings suggest that simvastatin exerts its effects on the lowering of low-density lipoprotein (LDL) cholesterol concentrations through several distinct pathways in the carriers of SLCO1B1 rs4149056-C, including dicarboxylic acids, bile acids, steroids, and glycerophospholipids.