Cholesterol sensing and metabolic adaptation in tissue immunity.

Cholesterol metabolites, particularly oxidized forms known as oxysterols, play crucial roles in modulating immune and metabolic processes across various tissues. Concentrations of local cholesterol and its metabolites influence tissue-specific immune responses by shaping the metabolic and spatial organization of immune cells in barrier organs like the small intestine (SI) and lungs. We explore recent molecular and cellular evidence supporting the metabolic adaptation of innate and adaptive immune cells in the SI and lung, driven by cholesterol and cholesterol metabolites. Further research should unravel the detailed molecular mechanisms and spatiotemporal adaptations involving cholesterol metabolites in distinct mucosal tissues in homeostasis or infection. We posit that pharmacological interventions targeting the generation or sensing of cholesterol metabolites might be leveraged to enhance long-term immune protection in mucosal tissues or prevent autoinflammatory states.

Modulation of Brain Cholesterol Metabolism through CYP46A1 Overexpression for Rett Syndrome.

Rett syndrome (RTT) is a rare neurodevelopmental disorder caused by mutation in the X-linked gene methyl-CpG-binding protein 2 (Mecp2), a ubiquitously expressed transcriptional regulator. RTT results in mental retardation and developmental regression that affects approximately 1 in 10,000 females. Currently, there is no curative treatment for RTT. Thus, it is crucial to develop new therapeutic approaches for children suffering from RTT. Several studies suggested that RTT is linked with defects in cholesterol homeostasis, but for the first time, therapeutic evaluation is carried out by modulating this pathway. Moreover, AAV-based CYP46A1 overexpression, the enzyme involved in cholesterol pathway, has been demonstrated to be efficient in several neurodegenerative diseases. Based on these data, we strongly believe that CYP46A1 could be a relevant therapeutic target for RTT. Herein, we evaluated the effects of intravenous AAVPHP.eB-hCYP46A1-HA delivery in male and female Mecp2-deficient mice. The applied AAVPHP.eB-hCYP46A1 transduced essential neurons of the central nervous system (CNS). CYP46A1 overexpression alleviates behavioral alterations in both male and female Mecp2 knockout mice and extends the lifespan in Mecp2-deficient males. Several parameters related to cholesterol pathway are improved and correction of mitochondrial activity is demonstrated in treated mice, which highlighted the clear therapeutic benefit of CYP46A1 through the neuroprotection effect. IV delivery of AAVPHP.eB-CYP46A1 is perfectly well tolerated with no inflammation observed in the CNS of the treated mice. Altogether, our results strongly suggest that CYP46A1 is a relevant target and overexpression could alleviate the phenotype of Rett patients.

Simvastatin prevents BMP-2 driven cell migration and invasion by suppressing oncogenic DNMT1 expression in breast cancer cells.

Both epigenetic and genetic changes in the cancer genome act simultaneously to promote tumor development and metastasis. Aberrant DNA methylation, a prime epigenetic event, is often observed in various cancer types. The elevated DNA methyltransferase 1 (DNMT1) enzyme creates DNA hypermethylation at CpG islands to drive oncogenic potential. This study emphasized to decipher the molecular mechanism of endogenous regulation of DNMT1 expression for finding upstream signaling molecules. Cancer database analyses found an upregulated DNMT1 expression in most cancer types including breast cancer. Overexpression of DNMT1 showed an increased cell migration, invasion, and stemness potential whereas 5-azacytidine (DNMT1 inhibitor) and siRNA mediated knockdown of DNMT1 exhibited inhibition of such cancer activities in breast cancer MDA-MB-231 and MCF-7 cells. Infact, cancer database analyses further found a positive correlation of DNMT1 transcript with both cholesterol pathway regulatory genes and BMP signaling molecules. Experimental observations documented that the cholesterol-lowering drug, simvastatin decreased DNMT1 transcript as well as protein, whereas BMP-2 treatment increased DNMT1 expression in breast cancer cells. In addition, expression of various key cholesterol regulatory genes was found to be upregulated in response to BMP-2 treatment. Moreover, simvastatin inhibited BMP-2 induced DNMT1 expression in breast cancer cells. Thus, this study for the first time reveals that both BMP-2 signaling and cholesterol pathways could regulate endogenous DNMT1 expression in cancer cells.

Recapsoma®: A Novel Mixture Based on Bergamot, Ipomoea Batatas, Policosanol Extracts and Liposomal Berberine for the Treatment of Hypercholesterolemia.

Cardiovascular disease (CVD) is considered one of the major causes of mortality worldwide. Epidemiological studies have shown that regular consumption of phenols is inversely associated with cardiovascular disease, and the use of nutraceuticals and functional foods can provide protective, preventive, and possibly curative effects in CVD. A novel mixture of different natural substances named Recapsoma® (bergamot, liposomal berberine, Ipomoea batatas, oleuropein, polycosanols, and vitamin E) has been produced, and its anti-dyslipidaemic efficacy has been tested, specifically studying the in vitro effects on the mechanisms of action underlying cholesterol synthesis, triglycerides, and LDL-cholesterol oxidation. The work has demonstrated the ability of this herbal extract mixture to inhibit the action of PCSK, ACAT, PAP, and HMGR and to increase the LDL receptor (LDLR), underlying the synergistic effect of the mixture over the single components. Such results suggest that the Recapsoma® mixture could be used as a tool for controlling hypercholesterolemia, and an alternative to statins, especially for those patients with metabolic syndrome.

Statins affect cancer cell plasticity with distinct consequences for tumor progression and metastasis.

Statins are among the most commonly prescribed drugs, and around every fourth person above the age of 40 is on statin medication. Therefore, it is of utmost clinical importance to understand the effect of statins on cancer cell plasticity and its consequences to not only patients with cancer but also patients who are on statins. Here, we find that statins induce a partial epithelial-to-mesenchymal transition (EMT) phenotype in cancer cells of solid tumors. Using a comprehensive STRING network analysis of transcriptome, proteome, and phosphoproteome data combined with multiple mechanistic in vitro and functional in vivo analyses, we demonstrate that statins reduce cellular plasticity by enforcing a mesenchymal-like cell state that increases metastatic seeding ability on one side but reduces the formation of (secondary) tumors on the other due to heterogeneous treatment responses. Taken together, we provide a thorough mechanistic overview of the consequences of statin use for each step of cancer development, progression, and metastasis.

Monocyte mitochondrial dysfunction, inflammaging, and inflammatory pyroptosis in major depression.

BACKGROUND: The macrophage theory of depression states that macrophages play an important role in Major Depressive Disorder (MDD). METHODS: MDD patients (N = 140) and healthy controls (N = 120) participated in a cross-sectional study investigating the expression of apoptosis/growth and lipid/cholesterol pathway genes (BAX, BCL10, EGR1, EGR2, HB-EGF, NR1H3, ABCA1, ABCG1, MVK, CD163, HMOX1) in monocytes (macrophage/microglia precursors). Gene expressions were correlated to a set of previously determined and reported inflammation-regulating genes and analyzed with respect to various clinical parameters. RESULTS: MDD monocytes showed an overexpression of the apoptosis/growth/cholesterol and the TNF genes forming an inter-correlating gene cluster (cluster 3) separate from the previously described inflammation-related gene clusters (containing IL1 and IL6). While upregulation of monocyte gene cluster 3 was a hallmark of monocytes of all MDD patients, upregulation of the inflammation-related clusters was confirmed to be found only in the monocytes of patients with childhood adversity. The latter group also showed a downregulation of the cholesterol metabolism gene MVK, which is known to play an important role in trained immunity and proneness to inflammation. CONCLUSIONS: The upregulation of cluster 3 genes in monocytes of all MDD patients suggests a premature aging of the cells, i.e. mitochondrial apoptotic dysfunction and TNF "inflammaging", as a general feature of MDD. The overexpression of the IL-1/IL-6 containing inflammation clusters and the downregulation of MVK in monocytes of patients with childhood adversity indicates a shift in this condition to a more severe inflammation form (pyroptosis) of the cells, additional to the signs of premature aging and inflammaging.

Cyclovirobuxine D inhibits dengue virus replication by impeding the complete autophagy in a cholesterol-dependent manner.

Dengue virus (DENV) is the most common mosquito-borne flavivirus, and it affects millions of people globally every year. Currently, there are no approved drugs for the treatment of dengue infection. By screening a natural product library, we identified a novel compound, cyclovirobuxine D (Cvb D), that displays anti-DENV activity. Cvb D inhibits DENV replication in vitro in a dose-dependent manner and protects suckling mice against lethal DENV infection. Mechanistically, Cvb D regulates the expression of genes related to the cellular cholesterol pathway. As a result, Cvb D increases cellular cholesterol synthesis and accumulation, activates mTOR, and inhibits viral-dependent autophagy. Cvb D does not suppress autophagy initiation but impedes the nuclear translocation of the lysosome transcription factor TFEB. In addition, Cvb D restricts the replication of other positive-strand RNA viruses such as Zika virus and Coxsackievirus B3. We speculate that Cvb D could be a broad-spectrum antiviral drug candidate for use against positive-strand RNA viruses that require autophagy for optimal replication.

New Conjugated Compound T5 Epidioxy-Sterol-ANB Inhibits the Growth of Mycobacterium tuberculosis Affecting the Cholesterol and Folate Pathways.

The upsurge and persistence of drug resistant strains of Mycobacterium tuberculosis (Mtb) is an important limitant to the battery of drugs available for the elimination of tuberculosis (TB). To avoid future scarcity of antibiotics against Mtb, it is important to discover new effective anti-mycobacterial agents. In this study, we present data from a series of experiments to determine in vitro and in vivo anti-mycobacterial activity of a library of epidioxy-sterol analogs. We test 15 compounds for their ability to reduce the viability of Mtb. We found that one compound called T5 epidioxy-sterol-ANB display significant potency against Mtb in vitro specifically inside macrophages but without effectivity in axenic cultures. A viability assay confirms that this T5 compound is less toxic for macrophages in vitro as compared to the current Mtb drug Rifampicin at higher concentrations. We use a transcriptomic analysis of Mtb inside macrophages after T5 epidioxy-sterol-ANB treatment, and we found a significant down-regulation of enzymes involved in the cholesterol and folic acid pathways. In vivo, significant differences were found in the lungs and spleen CFUs of Mtb infected mice treated with the T5 epidioxy-sterol-ANB as compared with the untreated control group, which provides additional evidence of the effectivity of the T5 compound. Altogether these results confirm the potential of this T5 epidioxy-sterol-ANB compound against Mtb.

Intrarenal arteriosclerosis and telomere attrition associate with dysregulation of the cholesterol pathway.

BACKGROUND: Recently, we demonstrated that arteriosclerosis in the smaller intrarenal arteries is associated with shorter telomere length, independently of history of cardiovascular events and calendar age. This suggests that intrarenal arteriosclerosis reflects replicative senescence, although the underlying molecular alterations remain unclear. RESULTS: Shorter intrarenal telomere length associated significantly with the presence of renal arteriosclerosis (T/S ratio 0.91±0.15 vs. 1.20±0.23 with vs. without arteriosclerosis, p=0.007, test cohort; T/S ratio 0.98 ±0.26 vs. 1.03 ±0.18 with vs. without arteriosclerosis, p=0.02, validation cohort). The presence versus absence of intrarenal arteriosclerosis was associated with differential expression of 1472 transcripts. Pathway analysis revealed enrichment of molecules involved in the superpathway of cholesterol biosynthesis as the most significant. The differential expression of these genes was confirmed in the independent validation cohort. Furthermore, the specific mRNA expression of the molecules in the superpathway of cholesterol biosynthesis associated significantly with intrarenal telomere length, and with history of cardiovascular events. INTERPRETATION: Our study illustrates that the superpathway of cholesterol biosynthesis interacts with the previously published association between shorter telomere length and arteriosclerosis. METHODS: This study included a test cohort of 40 consecutive kidney donors (calendar age 48.0 ± 15), with biopsies obtained prior to transplantation. Intrarenal leucocyte telomere length content was assessed using quantitative RT-PCR. Whole genome microarray mRNA expression analysis was performed using Affymetrix Gene 2.0 ST arrays. We investigated the associations between mRNA gene expression, telomere length as marker of replicative senescence, and intrarenal arteriosclerosis (Banff "cv" score = vascular fibrous intimal thickening = intimal hyperplasia) using adjusted multiple regression models. For biological interpretation and pathway overrepresentation analysis, we used Ingenuity Pathway Analysis. The significant pathways and genes were validated in an independent validation cohort of 173 kidney biopsies obtained prior to transplantation.

Liver X receptor-α activation enhances cholesterol secretion in lactating mammary epithelium.

Liver X receptors (LXRs) are ligand-dependent transcription factors activated by cholesterol metabolites. These receptors induce a suite of target genes required for de novo synthesis of triglycerides and cholesterol transport in many tissues. Two different isoforms, LXRα and LXRß, have been well characterized in liver, adipocytes, macrophages, and intestinal epithelium among others, but their contribution to cholesterol and fatty acid efflux in the lactating mammary epithelium is poorly understood. We hypothesize that LXR regulates lipogenesis during milk fat production in lactation. Global mRNA analysis of mouse mammary epithelial cells (MECs) revealed multiple LXR/RXR targets upregulated sharply early in lactation compared with midpregnancy. LXRα is the primary isoform, and its protein levels increase throughout lactation in MECs. The LXR agonist GW3965 markedly induced several genes involved in cholesterol transport and lipogenesis and enhanced cytoplasmic lipid droplet accumulation in the HC11 MEC cell line. Importantly, in vivo pharmacological activation of LXR increased the milk cholesterol percentage and induced sterol regulatory element-binding protein 1c (Srebp1c) and ATP-binding cassette transporter a7 (Abca7) expression in MECs. Cumulatively, our findings identify LXRα as an important regulator of cholesterol incorporation into the milk through key nodes of de novo lipogenesis, suggesting a potential therapeutic target in women with difficulty initiating lactation.

Biallelic pathogenic variants in the lanosterol synthase gene LSS involved in the cholesterol biosynthesis cause alopecia with intellectual disability, a rare recessive neuroectodermal syndrome.

PURPOSE: Lanosterol synthase (LSS) gene was initially described in families with extensive congenital cataracts. Recently, a study has highlighted LSS associated with hypotrichosis simplex. We expanded the phenotypic spectrum of LSS to a recessive neuroectodermal syndrome formerly named alopecia with mental retardation (APMR) syndrome. It is a rare autosomal recessive condition characterized by hypotrichosis and intellectual disability (ID) or developmental delay (DD), frequently associated with early-onset epilepsy and other dermatological features. METHODS: Through a multicenter international collaborative study, we identified LSS pathogenic variants in APMR individuals either by exome sequencing or LSS Sanger sequencing. Splicing defects were assessed by transcript analysis and minigene assay. RESULTS: We reported ten APMR individuals from six unrelated families with biallelic variants in LSS. We additionally identified one affected individual with a single rare variant in LSS and an allelic imbalance suggesting a second event. Among the identified variants, two were truncating, seven were missense, and two were splicing variants. Quantification of cholesterol and its precursors did not reveal noticeable imbalance. CONCLUSION: In the cholesterol biosynthesis pathway, lanosterol synthase leads to the cyclization of (S)-2,3-oxidosqualene into lanosterol. Our data suggest LSS as a major gene causing a rare recessive neuroectodermal syndrome.

Neuronal Dysfunction Associated with Cholesterol Deregulation.

Cholesterol metabolism is crucial for cells and, in particular, its biosynthesis in the central nervous system occurs in situ, and its deregulation involves morphological changes that cause functional variations and trigger programmed cell death. The pathogenesis of rare diseases, such as Mevalonate Kinase Deficiency or Smith⁻Lemli⁻Opitz Syndrome, arises due to enzymatic defects in the cholesterol metabolic pathways, resulting in a shortage of downstream products. The most severe clinical manifestations of these diseases appear as neurological defects. Expanding the knowledge of this biological mechanism will be useful for identifying potential targets and preventing neuronal damage. Several studies have demonstrated that deregulation of the cholesterol pathway induces mitochondrial dysfunction as the result of respiratory chain damage. We set out to determine whether mitochondrial damage may be prevented by using protective mitochondria-targeted compounds, such as MitoQ, in a neuronal cell line treated with a statin to induce a biochemical block of the cholesterol pathway. Evidence from the literature suggests that mitochondria play a crucial role in the apoptotic mechanism secondary to blocking the cholesterol pathway. Our study shows that MitoQ, administered as a preventive agent, could counteract the cell damage induced by statins in the early stages, but its protective role fades over time.

Melittin inhibits tumor growth and decreases resistance to gemcitabine by downregulating cholesterol pathway gene CLU in pancreatic ductal adenocarcinoma.

Melittin is a Chinese traditional medicine for treating chronic inflammation, immunological diseases and cancers, however, the efficacy of melittin and its mechanism for treating pancreatic ductal adenocarcinoma (PDAC) are still unknown. Here we investigated the anti-cancer activity of melittin and its regulated mechanism(s) in the PDAC models. Melittin was found to suppress tumor growth by promoting cell apoptosis and cell-cycle arrest. Interestingly, the microarray analyses demonstrated that melittin significantly regulated cholesterol biosynthesis pathway during treatment. For instance, the cholesterol pathway gene clusterin (CLU) was highly downregulated by melittin which also enhanced gemcitabine sensitivity in PDAC cells by inhibiting CLU expression. In contrast, overexpression of CLU significantly diminished melittin mediated tumor suppression and gemcitabine sensitization, suggesting that CLU is the target of melittin. Furthermore, in the xenograft mouse model, the combination therapy of melittin and gemcitabine is more efficacious for inhibiting PDAC tumor growth than either single regimen. Taken together, our study has indicated that melittin is capable of suppressing tumor growth and promoting gemcitabine sensitivity in PDAC by downregulating cholesterol pathway.

Inhibition of the 3-hydroxy-3-methyl-glutaryl-CoA reductase induces orofacial defects in zebrafish.

BACKGROUND: Orofacial clefts (OFCs) are common birth defects, which include a range of disorders with a complex etiology affecting formation of craniofacial structures. Some forms of syndromic OFCs are produced by defects in the cholesterol pathway. The principal enzyme of the cholesterol pathway is the 3-hydroxy-3-methyl-glutaryl-CoA reductase (HMGCR). Our aim is to study whether defects of HMGCR function would produce orofacial malformation similar to those found in disorders of cholesterol synthesis. METHODS: We used zebrafish hmgcrb mutants and HMGCR inhibition assay using atorvastatin during early and late stages of orofacial morphogenesis in zebrafish. To describe craniofacial phenotypes, we stained cartilage and bone and performed in situ hybridization using known craniofacial markers. Also, we visualized neural crest cell migration in a transgenic fish. RESULTS: Our results showed that mutants displayed loss of cartilage and diminished orofacial outgrowth, and in some cases palatal cleft. Late treatments with statin show a similar phenotype. Affected-siblings displayed a moderate phenotype, whereas early-treated embryos had a minor cleft. We found reduced expression of the downstream component of Sonic Hedgehog-signaling gli1 in ventral brain, oral ectoderm, and pharyngeal endoderm in mutants and in late atorvastatin-treated embryos. CONCLUSION: Our results suggest that HMGCR loss-of-function primarily affects postmigratory cranial neural crest cells through abnormal Sonic Hedgehog signaling, probably induced by reduction in metabolites of the cholesterol pathway. Malformation severity correlates with the grade of HMGCR inhibition, developmental stage of its disruption, and probably with availability of maternal lipids. Together, our results might help to understand the spectrum of orofacial phenotypes found in cholesterol synthesis disorders. Birth Defects Research (Part A) 106:814-830, 2016. © 2016 Wiley Periodicals, Inc.

Geranylgeraniol and Neurological Impairment: Involvement of Apoptosis and Mitochondrial Morphology.

Deregulation of the cholesterol pathway is an anomaly observed in human diseases, many of which have in common neurological involvement and unknown pathogenesis. In this study we have used Mevalonate Kinase Deficiency (MKD) as a disease-model in order to investigate the link between the deregulation of the mevalonate pathway and the consequent neurodegeneration. The blocking of the mevalonate pathway in a neuronal cell line (Daoy), using statins or mevalonate, induced an increase in the expression of the inflammasome gene (NLRP3) and programmed cell death related to mitochondrial dysfunction. The morphology of the mitochondria changed, clearly showing the damage induced by oxidative stress and the decreased membrane potential associated with the alterations of the mitochondrial function. The co-administration of geranylgeraniol (GGOH) reduced the inflammatory marker and the damage of the mitochondria, maintaining its shape and components. Our data allow us to speculate about the mechanism by which isoprenoids are able to rescue the inflammatory marker in neuronal cells, independently from the block of the mevalonate pathway, and about the fact that cell death is mitochondria-related.