Regulation of cholesterol metabolism: New players for an old physiological process.

Identified more than two centuries ago, cholesterol plays a pivotal role in human physiology. Since cholesterol metabolism is a physiologically significant process, it is not surprising that its alterations are associated with several pathologies. The discovery of new molecular targets or compounds able to modulate this sophisticated metabolism has been capturing the attention of research groups worldwide since many years. Endogenous and exogenous compounds are known to regulate cellular cholesterol synthesis and uptake, or reduce cholesterol absorption at the intestinal level, thereby regulating cholesterol homeostasis. However, there is a great need of new modulators and diverse new pathways have been uncovered. Here, after illustrating cholesterol metabolism and its well-known regulators, some new players of this important physiological process are also described.

CRISPR/Cas9 and piggyBac Transposon-Based Conversion of a Pathogenic Biallelic TBCD Variant in a Patient-Derived iPSC Line Allows Correction of PEBAT-Related Endophenotypes.

Induced pluripotent stem cells (iPSCs) have been established as a reliable in vitro disease model system and represent a particularly informative tool when animal models are not available or do not recapitulate the human pathophenotype. The recognized limit in using this technology is linked to some degree of variability in the behavior of the individual patient-derived clones. The development of CRISPR/Cas9-based gene editing solves this drawback by obtaining isogenic iPSCs in which the genetic lesion is corrected, allowing a straightforward comparison with the parental patient-derived iPSC lines. Here, we report the generation of a footprint-free isogenic cell line of patient-derived TBCD-mutated iPSCs edited using the CRISPR/Cas9 and piggyBac technologies. The corrected iPSC line had no genetic footprint after the removal of the selection cassette and maintained its "stemness". The correction of the disease-causing TBCD missense substitution restored proper protein levels of the chaperone and mitotic spindle organization, as well as reduced cellular death, which were used as read-outs of the TBCD KO-related endophenotype. The generated line represents an informative in vitro model to understand the impact of pathogenic TBCD mutations on nervous system development and physiology.

Long-Lasting Impact of Sugar Intake on Neurotrophins and Neurotransmitters from Adolescence to Young Adulthood in Rat Frontal Cortex.

The detrimental impact of fructose, a widely used sweetener in industrial foods, was previously evidenced on various brain regions. Although adolescents are among the highest consumers of sweet foods, whether brain alterations induced by the sugar intake during this age persist until young adulthood or are rescued returning to a healthy diet remains largely unexplored. To shed light on this issue, just weaned rats were fed with a fructose-rich or control diet for 3 weeks. At the end of the treatment, fructose-fed rats underwent a control diet for a further 3 weeks until young adulthood phase and compared with animals that received from the beginning the healthy control diet. We focused on the consequences induced by the sugar on the main neurotrophins and neurotransmitters in the frontal cortex, as its maturation continues until late adolescence, thus being the last brain region to achieve a full maturity. We observed that fructose intake induces inflammation and oxidative stress, alteration of mitochondrial function, and changes of brain-derived neurotrophic factor (BDNF) and neurotrophin receptors, synaptic proteins, acetylcholine, dopamine, and glutamate levels, as well as increased formation of the glycation end-products Nε-carboxymethyllysine (CML) and Nε-carboxyethyllysine (CEL). Importantly, many of these alterations (BDNF, CML, CEL, acetylcholinesterase activity, dysregulation of neurotransmitters levels) persisted after switching to the control diet, thus pointing out to the adolescence as a critical phase, in which extreme attention should be devoted to limit an excessive consumption of sweet foods that can affect brain physiology also in the long term.

Mechanism Underlying Naringenin Hypocholesterolemic Effects: Involvement of Estrogen Receptor α Subtype.

Naringenin (Nar) is one of major citrus flavonoids predominantly found in grapefruit and orange. In vivo studies have demonstrated Nar potential as a normolipidemic agent capable to reduce circulating cholesterol in hypercholesterolemic rabbits, rats, and patients, suggesting a new role for this molecule in cardiovascular disease prevention. Although Nar cholesterol-lowering effects are known, the underlying mechanisms have not yet been elucidated. Interestingly, Nar binds to the estrogen receptors (ERs), modulating both transcriptional and membrane-initiating signals. Although estrogen and ERs are deeply involved in lipid metabolism, no data are available regarding a putative role of these nuclear receptors as mediators of the hypocholesterolemic effect exerted by Nar. Thus, the aim of this work was to study the involvement of ERs in Nar-induced modulation of cholesterol metabolism. Results obtained in HepG2 cell line demonstrate that Nar can modulate the molecular network of cholesterol homeostasis. However, these effects were only partially dependent on the activity of estrogen receptor α. As a whole, our data highlight new molecular mechanisms by which Nar influences cholesterol metabolism, opening a new scenery about dietary impact on human health.

Fat Checking: Emerging Role of Lipids in Metabolism and Disease.

Lipids are hydrophobic molecules involved in a plethora of biological functions; for example, they are employed for the storage of energy, serve as essential constituents of cell membranes and participate in the assembly of bilayer configuration [...].

NGF Modulates Cholesterol Metabolism and Stimulates ApoE Secretion in Glial Cells Conferring Neuroprotection against Oxidative Stress.

Cholesterol plays a crucial role in the brain, where its metabolism is particularly regulated by astrocytic activity. Indeed, adult neurons suppress their own cholesterol biosynthesis and import this sterol through ApoE-rich particles secreted from astrocytes. Recent evidence suggests that nerve growth factor (NGF) may exert neurotrophic activity by influencing cell metabolism. Nevertheless, the effect of NGF on glial cholesterol homeostasis has still not been elucidated. Thus, the aim of this project is to assess whether NGF could influence cholesterol metabolism in glial cells. To reach this objective, the U373 astrocyte-derived cell line was used as an experimental model. Immunoblot and ELISA analysis showed that proteins and enzymes belonging to the cholesterol metabolism network were increased upon NGF treatment in glial cells. Furthermore, NGF significantly increased ApoE secretion and the amount of extracellular cholesterol in the culture medium. Co-culture and U373-conditioned medium experiments demonstrated that NGF treatment efficiently counteracted rotenone-mediated cytotoxicity in N1E-115 neuronal cells. Conversely, neuroprotection mediated by NGF treatment was suppressed when N1E-115 were co-cultured with ApoE-silenced U373 cells. Taken together, these data suggest that NGF controls cholesterol homeostasis in glial cells. More importantly, NGF exerts neuroprotection against oxidative stress, which is likely associated with the induction of glial ApoE secretion.

Brain Cholesterol Biosynthetic Pathway Is Altered in a Preclinical Model of Fragile X Syndrome.

Fragile X Syndrome (FXS) is the most frequent form of inherited X-linked pathology, associated with an intellectual and developmental disability, and currently considered the first monogenic cause of autism spectrum disorder (ASD). Low levels of total cholesterol reported in the serum of FXS patients, and evidence that FMRP targets a subset of mRNAs encoding proteins of lipid synthesis and transport suggests that the cholesterol metabolism impairments could be involved in FXS. Thus, the aim of the presented work was to investigate the modulations of the cholesterol biosynthetic pathway and its end-products in a recently developed Fmr1-Δexon 8 rat model of FXS. Here, we show that this experimental model mimics what is found in FXS patients, exhibiting a lower serum cholesterol content, accompanied by a reduction in food intake and body weight compared to WT animals. Moreover, alterations of proteins committed to cholesterol synthesis and uptake have been observed in the amygdala, prefrontal cortex and nucleus accumbens. Interestingly, the end-products show a brain region-dependent modulation in Fmr1-Δexon 8 rats. Overall, our results demonstrate that the cholesterol biosynthetic pathway is altered in some brain regions of this preclinical model of FXS. This finding has relevance for future studies to delve deeper into the involvement of this metabolic process in FXS, and thus its possible role as a therapeutic target.

Perinatal supplementation with omega-3 fatty acids corrects the aberrant social and cognitive traits observed in a genetic model of autism based on FMR1 deletion in rats.

Background and objective: Autism spectrum disorder (ASD) is a complex neurodevelopmental disorder for which no treatments exist. Fragile X syndrome (FXS) is the most common form of inherited mental retardation and the most frequent monogenic cause of ASD. Given the lack of pharmacological treatments for ASD, increasing interest is devoted to non-pharmacological approaches, including dietary interventions. Omega-3 polyunsaturated fatty acids (PUFAs) are critical for neurobehavioraldevelopment. This study had two aims: 1. To validatethe recently developed Fmr1-Δexon 8 rat model of FXS; 2. To assess the impact of omega-3 PUFAs dietary supplementation during pregnancy and lactation on the altered behavior displayed by Fmr1-Δexon 8 rats.Methods: Female Fmr1-Δexon 8 and wild-type Sprague-Dawley rats were fed with either an omega-3 PUFAs enriched diet or with an isocaloric control diet during pregnancy and lactation. Behavioral experiments were carried out on the infant (Postnatal days (PNDs) 9 and 13), juvenile (PND 35) and adult (PND 90) male offspring.Results: Fmr1-Δexon 8 pups showed hypolocomotion, reduced ultrasonic vocalizations (USVs) emission and impaired social discrimination compared to wild-type controls. Juvenile and adult Fmr1-Δexon 8 rats showed deficits in the social and cognitive domains, that were counteracted by perinatal omega-3 PUFAs supplementation.Conclusion: Our results support the validity of the Fmr1-Δexon 8 rat model to mimic key autistic-like features and support an important role of omega-3 PUFAs during of neurodevelopment. Although the mechanisms underlying the beneficial effects of omega-3 PUFAs supplementation in ASD needs to be clarified, this dietary intervention holds promise to mitigate core and comorbid autistic features.

Long-lasting impact of perinatal dietary supplementation of omega 3 fatty acids on mevalonate pathway: potential role on neuron trophism in male offspring hippocampal formation.

Objective: We were aimed at evaluating the long-term impact of perinatal an omega-3 fatty acid-enriched diet on the mevalonate/cholesterol pathway in the brain of male offspring.Methods: Female rats were fed with standard or omega-3 fatty acid-enriched diet during pregnancy and lactation. Liver, brain and plasma were collected from infant, adolescent and adult male offspring for subsequent biochemical and morphological analyses.Results: The omega-3 enriched diet induced region-dependent changes of the 3-hydroxy 3-methylglutaryl Coenzyme A reductase in the brain and affected notably RhoA/CREB signaling and the nerve growth factor content in the hippocampus. Our data reveal a long-lasting impact of perinatal omega-3 fatty acid supplementation on hippocampal nerve growth factor levels mediated by reduced 3-hydroxy 3-methylglutaryl Coenzyme A reductase activation state and enhanced CREB signaling.Discussion: These data underline the importance of the perinatal omega-3 enriched diet for adult brain function and reveal a new pathway important for nerve growth factor regulation.

The extra-nuclear interactome of the estrogen receptors: implications for physiological functions.

Over the last decades, a great body of evidence has defined a novel view of the cellular mechanism of action of the steroid hormone 17ß-estradiol (E2) through its estrogen receptors (i.e., ERα and ERß). It is now clear that the E2-activated ERs work both as transcription factors and extra-nuclear plasma membrane-localized receptors. The activation of a plethora of signal transduction cascades follows the E2-dependent engagement of plasma membrane-localized ERs and is required for the coordination of gene expression, which ultimately controls the occurrence of the pleiotropic effects of E2. The definition of the molecular mechanisms by which the ERs locate at the cell surface (i.e., palmitoylation and protein association) determined the quest for understanding the specificity of the extra-nuclear E2 signaling. The use of mice models lacking the plasma membrane ERα localization unveiled that the extra-nuclear E2 signaling is operational in vivo but tissue-specific. However, the underlying molecular details for such ERs signaling diversity in the perspective of the E2 physiological functions in the different cellular contexts are still not understood. Therefore, to gain insights into the tissue specificity of the extra-nuclear E2 signaling to physiological functions, here we reviewed the known ERs extra-nuclear interactors and tried to extrapolate from available databases the ERα and ERß extra-nuclear interactomes. Based on literature data, it is possible to conclude that by specifically binding to extra-nuclear localized proteins in different sub-cellular compartments, the ERs fine-tune their molecular activities. Moreover, we report that the context-dependent diversity of the ERs-mediated extra-nuclear E2 actions can be ascribed to the great flexibility of the physical structures of ERs and the spatial-temporal organization of the logistics of the cells (i.e., the endocytic compartments). Finally, we provide lists of proteins belonging to the potential ERα and ERß extra-nuclear interactomes and propose that the systematic experimental definition of the ERs extra-nuclear interactomes in different tissues represents the next step for the research in the ERs field. Such characterization will be fundamental for the identification of novel druggable targets for the innovative treatment of ERs-related diseases.

Glial contribution to cyclodextrin-mediated reversal of cholesterol accumulation in murine NPC1-deficient neurons in vivo.

Niemann-Pick type C disease is a rare and fatal lysosomal storage disorder presenting severe neurovisceral symptoms. Disease-causing mutations in genes encoding either NPC1 or NPC2 protein provoke accumulation of cholesterol and other lipids in specific structures of the endosomal-lysosomal system and degeneration of specific cells, notably neurons in the central nervous system (CNS). 2-hydroxypropyl-beta-cyclodextrin (CD) emerged as potential therapeutic approach based on animal studies and clinical data, but the mechanism of action in neurons has remained unclear. To address this topic in vivo, we took advantage of the retina as highly accessible part of the CNS and intravitreal injections as mode of drug administration. Coupling CD to gold nanoparticles allowed us to trace its intracellular location. We report that CD enters the endosomal-lysosomal system of neurons in vivo and enables the release of lipid-laden lamellar inclusions, which are then removed from the extracellular space by specific types of glial cells. Our data suggest that CD induces a concerted action of neurons and glial cells to restore lipid homeostasis in the central nervous system.

Prenatal Exposure to BPA: The Effects on Hepatic Lipid Metabolism in Male and Female Rat Fetuses.

Bisphenol A (BPA) is an organic chemical compound widely used for manufacturing plastics. BPA exposure originates principally from the diet, but it can also originate from dermal contact. In over 90% of individuals, including pregnant women, BPA is detectable in several body fluids. The effects of this exposure on the fetus are under active investigation in several research laboratories. The aim of our work was to study the impact of prenatal exposure to BPA in the liver of rat fetuses from a sex-dependent point of view. We particularly investigated the effects of prenatal BPA exposure on hepatic lipids because of their crucial role, not only for the liver, but also for the whole-body functions. Our results demonstrate that the liver of rat fetuses, in utero exposed to a very low dose of BPA (2.5 µg/kg/day), displays significant modulations with regard to proteins involved in cholesterol and fatty acid biosynthesis and trafficking. Moreover, an impact on inflammatory process has been observed. All these effects are dependent on sex, being observable only in female rat fetuses. In conclusion, this work demonstrates that maternal exposure to BPA compromises hepatic lipid metabolism in female offspring, and it also reveals the perspective impact of BPA on human health at doses currently considered safe.

Neurotrophins as Key Regulators of Cell Metabolism: Implications for Cholesterol Homeostasis.

Neurotrophins constitute a family of growth factors initially characterized as predominant mediators of nervous system development, neuronal survival, regeneration and plasticity. Their biological activity is promoted by the binding of two different types of receptors, leading to the generation of multiple and variegated signaling cascades in the target cells. Increasing evidence indicates that neurotrophins are also emerging as crucial regulators of metabolic processes in both neuronal and non-neuronal cells. In this context, it has been reported that neurotrophins affect redox balance, autophagy, glucose homeostasis and energy expenditure. Additionally, the trophic support provided by these secreted factors may involve the regulation of cholesterol metabolism. In this review, we examine the neurotrophins' signaling pathways and their effects on metabolism by critically discussing the most up-to-date information. In particular, we gather experimental evidence demonstrating the impact of these growth factors on cholesterol metabolism.

Effects of Late-Life Caloric Restriction on Age-Related Alterations in the Rat Cortex and Hippocampus.

BACKGROUND: A major problem of aging is the disruption of metabolic homeostasis. This is particularly relevant in the brain where it provokes neurodegeneration. Caloric restriction is a physiologic intervention known to delay the deleterious consequences of aging in several species ranging from yeast to mammals. To date, most studies on experimental models have started this dietary intervention from weaning, which is very difficult to be translated to human beings. Here, we study the effects of a more realistic dietary regimen in rats, starting at an advanced age and lasting for six months. METHODS: we analyzed in the cortex and hippocampus, the proteins involved in the energetic balance of the cells, cholesterol metabolism, oxidative stress response, inflammation, synaptic impairment, and brain trophism. RESULTS: our results suggest that caloric restriction in late life can revert only some age-related changes studied here.

Understanding and Treating Niemann-Pick Type C Disease: Models Matter.

Biomedical research aims to understand the molecular mechanisms causing human diseases and to develop curative therapies. So far, these goals have been achieved for a small fraction of diseases, limiting factors being the availability, validity, and use of experimental models. Niemann-Pick type C (NPC) is a prime example for a disease that lacks a curative therapy despite substantial breakthroughs. This rare, fatal, and autosomal-recessive disorder is caused by defects in NPC1 or NPC2. These ubiquitously expressed proteins help cholesterol exit from the endosomal-lysosomal system. The dysfunction of either causes an aberrant accumulation of lipids with patients presenting a large range of disease onset, neurovisceral symptoms, and life span. Here, we note general aspects of experimental models, we describe the line-up used for NPC-related research and therapy development, and we provide an outlook on future topics.

ProNGF/p75NTR Axis Drives Fiber Type Specification by Inducing the Fast-Glycolytic Phenotype in Mouse Skeletal Muscle Cells.

Despite its undisputable role in the homeostatic regulation of the nervous system, the nerve growth factor (NGF) also governs the relevant cellular processes in other tissues and organs. In this study, we aimed at assessing the expression and the putative involvement of NGF signaling in skeletal muscle physiology. To reach this objective, we employed satellite cell-derived myoblasts as an in vitro culture model. In vivo experiments were performed on Tibialis anterior from wild-type mice and an mdx mouse model of Duchenne muscular dystrophy. Targets of interest were mainly assessed by means of morphological, Western blot and qRT-PCR analysis. The results show that proNGF is involved in myogenic differentiation. Importantly, the proNGF/p75NTR pathway orchestrates a slow-to-fast fiber type transition by counteracting the expression of slow myosin heavy chain and that of oxidative markers. Concurrently, proNGF/p75NTR activation facilitates the induction of fast myosin heavy chain and of fast/glycolytic markers. Furthermore, we also provided evidence that the oxidative metabolism is impaired in mdx mice, and that these alterations are paralleled by a prominent buildup of proNGF and p75NTR. These findings underline that the proNGF/p75NTR pathway may play a crucial role in fiber type determination and suggest its prospective modulation as an innovative therapeutic approach to counteract muscle disorders.

Facts about Fats: New Insights into the Role of Lipids in Metabolism, Disease and Therapy.

Although initially regarded as a passive system to store energy, lipids are now considered to play crucial, structural and functional roles in almost all the biological processes involved in the regulation of physiological and pathological conditions [...].

Impact of Sex and Age on the Mevalonate Pathway in the Brain: A Focus on Effects Induced by Maternal Exposure to Exogenous Compounds.

The mevalonate pathway produces cholesterol and other compounds crucial for numerous cellular processes. It is well known that age and sex modulate this pathway in the liver. Recently, similar effects were also noted in different brain areas, suggesting that alterations of the mevalonate pathway are at the root of marked sex-specific disparities in some neurodevelopmental disorders related to disturbed cholesterol homeostasis. Here, we show how the mevalonate pathway is modulated in a sex-, age- and region-specific manner, and how maternal exposure to exogenous compounds can disturb the regulation of this pathway in the brain, possibly inducing functional alterations.

A Short-Term Western Diet Impairs Cholesterol Homeostasis and Key Players of Beta Amyloid Metabolism in Brain of Middle Aged Rats.

SCOPE: Cholesterol homeostasis is crucial for brain functioning. Unhealthy nutrition can influence cerebral physiology, but the effect of western diets on brain cholesterol homeostasis, particularly at middle age, is unknown. Given the link between brain cholesterol alteration and beta amyloid production, the aim is to evaluate whether a diet rich in fat and fructose affects the protein network implicated in cholesterol synthesis and shuttling between glial cells and neurons, as well as crucial markers of beta amyloid metabolism. METHODS AND RESULTS: Middle aged rats are fed a high fat-high fructose (HFF) or a control diet for 4 weeks. Inflammatory markers and cholesterol levels significantly increase in hippocampus of HFF rats. A higher activation of 3-hydroxy 3-methylglutaryl coenzyme-A reductase, coupled with lower levels of apolipoprotein E, LXR-beta, and lipoproteins receptors is measured in hippocampus from HFF rats. The alteration of critical players of cholesterol homeostasis is associated with increased level of amyloid precursor protein, presenilin 1, and nicastrin, and decreased level of insulin degrading enzyme. CONCLUSIONS: Overall these data show that a western diet is associated with perturbation of cholesterol homeostasis in middle aged rats, mostly in hippocampus. This might trigger molecular events involved in the onset of neurodegenerative diseases.

Maternal Dietary Exposure to Low-Dose Bisphenol A Affects Metabolic and Signaling Pathways in the Brain of Rat Fetuses.

Bisphenol A (BPA) is a synthetic compound widely used for the production of polycarbonate plasticware and epoxy resins. BPA exposure is widespread and more than 90% of individuals have detectable amounts of the molecule in their body fluids, which originates primarily from diet. Here, we investigated whether prenatal exposure to BPA affects the mevalonate (MVA) pathway in rat brain fetuses, and whether potential effects are sex-dependent. The MVA pathway is important for brain development and function. Our results demonstrate that the fetal brain, exposed in utero to a very low dose of BPA (2.5 µg/kg/day), displayed altered MVA pathway activation, increased protein prenylation, and a decreased level of pro-BDNF. Interestingly, the BPA-induced effects on estrogen receptor α were sex-dependent. In conclusion, this work demonstrates intergenerational effects of BPA on the brain at very low doses. Our results reveal new targets for BPA-induced interference and underline the impacts of BPA on health.