Spatial and Temporal Protein Modules Signatures Associated with Alzheimer Disease in 3xTg-AD Mice Are Restored by Early Ubiquinol Supplementation.

Despite its robust proteopathic nature, the spatiotemporal signature of disrupted protein modules in sporadic Alzheimer's disease (AD) brains remains poorly understood. This considered oxidative stress contributes to AD progression and early intervention with coenzyme Q10 or its reduced form, ubiquinol, delays the progression of the disease. Using MALDI-MSI and functional bioinformatic analysis, we have developed a protocol to express how deregulated protein modules arise from hippocampus and cortex in the AD mice model 3xTG-AD in an age-dependent manner. This strategy allowed us to identify which modules can be efficiently restored to a non-pathological condition by early intervention with ubiquinol. Indeed, an early deregulation of proteostasis-related protein modules, oxidative stress and metabolism has been observed in the hippocampus of 6-month mice (early AD) and the mirrored in cortical regions of 12-month mice (middle/late AD). This observation has been validated by IHC using mouse and human brain sections, suggesting that these protein modules are also affected in humans. The emergence of disrupted protein modules with AD signature can be prevented by early dietary intervention with ubiquinol in the 3xTG-AD mice model.

Assessment of genotoxicity induced by subchronic exposure to graphene in HaCaT human skin cell line.

The applications of graphene-based materials (GBMs) and their processing involve prolonged contact with cellular barriers such as human skin. Even though the potential cytotoxicity of graphene has been studied in recent years, the impact of long-term graphene exposure has rarely been explored. We tested in the HaCaT epithelial cells, in vitro, the effect of subchronic treatments with sublethal doses of four different, well-characterized GBMs, two commercial graphene oxides (GO) and two few-layer graphenes (FLG). Cells were exposed weekly to low doses of the GBMs for 14 days, 30 days, 3 months, and 6 months. GBMs-cells uptake was assessed by confocal microscopy. Cell death and cell cycle were determined by fluorescence microscopy and cytometry. DNA damage was measured by comet assay and γ-H2AX staining, followed by the determination of p-p53 and p-ATR by immunolabeling. Subchronic exposure to different GBMs at noncytotoxic doses has potential genotoxic effects on HaCaT epithelial cells that can be recovered depending on the GBM and exposure time. Specifically, GO-induced genotoxicity can be detected after 14 and 30 days from treatment. At this time, FLG appears less genotoxic than GO, and cells can recover more quickly when genotoxic pressure disappears after some days of removal of the GBM. Long-term exposure, 3 and 6 months, to different GBMs induces permanent, nonreversible, genotoxic damage comparable to the exerted by arsenite. This should be considered for the production and future applications of GBMs in scenarios where low concentrations of the material interact chronically with epithelial barriers.

CoQ10 reduces glioblastoma growth and infiltration through proteome remodeling and inhibition of angiogenesis and inflammation.

PURPOSE: Most monotherapies available against glioblastoma multiforme (GBM) target individual hallmarks of this aggressive brain tumor with minimal success. In this article, we propose a therapeutic strategy using coenzyme Q10 (CoQ10) as a pleiotropic factor that crosses the blood-brain barrier and accumulates in cell membranes acting as an antioxidant, and in mitochondrial membranes as a regulator of cell bioenergetics and gene expression. METHODS: Xenografts of U251 cells in nu/nu mice were used to assay tumor growth, hypoxia, angiogenesis, and inflammation. An orthotopic model was used to explore microglial infiltration, tumor growth, and invasion into the brain parenchyma. Cell proliferation, migration, invasion, proteome remodeling, and secretome were assayed in vitro. Conditioned media were used to assay angiogenesis, monocyte chemoattraction, and differentiation into macrophages in vitro. RESULTS: CoQ10 treatment decreased tumor volume in xenografts and orthotopic models, although its effect on tumor cell proliferation was not direct. Tumors from mice treated with CoQ10 were less hypoxic and vascularized, having less infiltration from inflammatory cells. Treatment-induced downregulation of HIF-1α and NF-kB led to a complete remodeling of the tumor cells proteome and secretome, impacting angiogenesis, monocyte infiltration, and their differentiation into macrophages. Besides, tumor cell migration and invasion were drastically restricted by mechanisms involving modulation of the actin cytoskeleton and downregulation of matrix metalloproteases (MMPs). CONCLUSIONS: CoQ10 has a pleiotropic effect on GBM growth, targeting several hallmarks simultaneously. Thus, its integration into current treatments of this fatal disease should be considered.

Ubiquinol Supplementation Improves Gender-Dependent Cerebral Vasoreactivity and Ameliorates Chronic Inflammation and Endothelial Dysfunction in Patients with Mild Cognitive Impairment.

Ubiquinol can protect endothelial cells from multiple mechanisms that cause endothelial damage and vascular dysfunction, thus contributing to dementia. A total of 69 participants diagnosed with mild cognitive impairment (MCI) received either 200 mg/day ubiquinol (Ub) or placebo for 1 year. Cognitive assessment of patients was performed at baseline and after 1 year of follow-up. Patients' cerebral vasoreactivity was examined using transcranial Doppler sonography, and levels of Ub and lipopolysaccharide (LPS) in plasma samples were quantified. Cell viability and necrotic cell death were determined using the microvascular endothelial cell line bEnd3. Coenzyme Q10 (CoQ) levels increased in patients supplemented for 1 year with ubiquinol versus baseline and the placebo group, although higher levels were observed in male patients. The higher cCoQ concentration in male patients improved cerebral vasoreactivity CRV and reduced inflammation, although the effect of Ub supplementation on neurological improvement was negligible in this study. Furthermore, plasma from Ub-supplemented patients improved the viability of endothelial cells, although only in T2DM and hypertensive patients. This suggests that ubiquinol supplementation could be recommended to reach a concentration of 5 µg/mL in plasma in MCI patients as a complement to conventional treatment.

A comprehensive systematic review of CSF proteins and peptides that define Alzheimer's disease.

BACKGROUND: During the last two decades, over 100 proteomics studies have identified a variety of potential biomarkers in CSF of Alzheimer's (AD) patients. Although several reviews have proposed specific biomarkers, to date, the statistical relevance of these proteins has not been investigated and no peptidomic analyses have been generated on the basis of specific up- or down- regulation. Herein, we perform an analysis of all unbiased explorative proteomics studies of CSF biomarkers in AD to critically evaluate whether proteins and peptides identified in each study are consistent in distribution; direction change; and significance, which would strengthen their potential use in studies of AD pathology and progression. METHODS: We generated a database containing all CSF proteins whose levels are known to be significantly altered in human AD from 47 independent, validated, proteomics studies. Using this database, which contains 2022 AD and 2562 control human samples, we examined whether each protein is consistently present on the basis of reliable statistical studies; and if so, whether it is over- or under-represented in AD. Additionally, we performed a direct analysis of available mass spectrometric data of these proteins to generate an AD CSF peptide database with 3221 peptides for further analysis. RESULTS: Of the 162 proteins that were identified in 2 or more studies, we investigated their enrichment or depletion in AD CSF. This allowed us to identify 23 proteins which were increased and 50 proteins which were decreased in AD, some of which have never been revealed as consistent AD biomarkers (i.e. SPRC or MUC18). Regarding the analysis of the tryptic peptide database, we identified 87 peptides corresponding to 13 proteins as the most highly consistently altered peptides in AD. Analysis of tryptic peptide fingerprinting revealed specific peptides encoded by CH3L1, VGF, SCG2, PCSK1N, FBLN3 and APOC2 with the highest probability of detection in AD. CONCLUSIONS: Our study reveals a panel of 27 proteins and 21 peptides highly altered in AD with consistent statistical significance; this panel constitutes a potent tool for the classification and diagnosis of AD.

Postprandial endotoxemia may influence the development of type 2 diabetes mellitus: From the CORDIOPREV study.

BACKGROUND & AIMS: Insulin resistance (IR) and impaired beta-cell function are key determinants of type 2 diabetes mellitus (T2DM). Intestinal absorption of bacterial components activates the toll-like receptors inducing inflammation, and this in turn IR. We evaluated the role of endotoxemia in promoting inflammation-induced insulin resistance (IR) in the development of T2DM, and its usefulness as predictive biomarker. METHODS: We included in this study 462 patients from the CORDIOPREV study without T2DM at baseline. Of these, 107 patients developed T2DM according to the American Diabetes Association (ADA) diagnosis criteria after a median follow-up of 60 months (Incident-DIAB group), whereas 355 patients did not developed it during this period of time (Non-DIAB group). RESULTS: We observed a postprandial increase in lipopolysaccharides (LPS) levels in the Incident-DIAB at baseline (P < 0.001), whereas LPS levels were not modified in the Non-DIAB. Disease-free survival curves based on the LPS postprandial fold change improved T2DM Risk Assessment as compared with the previously described FINDRISC score (hazard ratio of 2.076, 95% CI 1.149-3.750 vs. 1.384, 95% CI 0.740-2.589). Moreover, disease-free survival curves combining the LPS postprandial fold change and FINDRISC score together showed a hazard ratio of 3.835 (95% CI 1.323-11.114), linked to high values of both parameters. CONCLUSION: Our results suggest that a high postprandial endotoxemia precedes the development of T2DM. Our results also showed the potential use of LPS plasma levels as a biomarker predictor of T2DM development. CLINICAL TRIALS.GOV. IDENTIFIER: NCT00924937.

Interplay Between Mitochondrial Oxidative Disorders and Proteostasis in Alzheimer's Disease.

Although the basis of Alzheimer's disease (AD) etiology remains unknown, oxidative stress (OS) has been recognized as a prodromal factor associated to its progression. OS refers to an imbalance between oxidant and antioxidant systems, which usually consist in an overproduction of reactive oxygen species (ROS) and reactive nitrogen species (RNS) which overwhelms the intrinsic antioxidant defenses. Due to this increased production of ROS and RNS, several biological functions such as glucose metabolism or synaptic activity are impaired. In AD, growing evidence links the ROS-mediated damages with molecular targets including mitochondrial dynamics and function, protein quality control system, and autophagic pathways, affecting the proteostasis balance. In this scenario, OS should be considered as not only a major feature in the pathophysiology of AD but also a potential target to combat the progression of the disease. In this review, we will discuss the role of OS in mitochondrial dysfunction, protein quality control systems, and autophagy associated to AD and suggest innovative therapeutic strategies based on a better understanding of the role of OS and proteostasis.

Influence of gender and menopausal status on gut microbiota.

OBJECTIVES: We explore the differences in the gut microbiota associated with gender and hormonal status. STUDY DESIGN: We included 76 individuals in this study: 17 pre-menopausal women, 19 men matched by age, as a control group for the pre-menopausal women, 20 post-menopausal women and 20 men matched by age as a control group for the post-menopausal women; all 4 groups were also matched by body mass index (BMI) and nutritional background. MAIN MEASUREMENTS: We analyzed the differences in the gut microbiota, endotoxemia, intestinal incretins, pro-inflammatory cytokines, and plasma levels of energy homeostasis regulatory hormones between pre- and post-menopausal women and compared them with their respective male control groups. RESULTS: We found a higher Firmicutes/Bacteroidetes ratio, a higher relative abundance of Lachnospira and Roseburia, and higher GLP-1 plasma levels in pre-menopausal women than in post-menopausal women, who had similar levels to men. In contrast, we observed a lower relative abundance of the Prevotella, Parabacteroides and Bilophila genera, and IL-6 and MCP-1 plasma levels in pre-menopausal women than in post-menopausal women, who had similar levels to the men. We also found higher GiP and leptin plasma levels in women than in men, irrespective of the menopausal status of the women. In addition, adiponectin levels were higher in pre-menopausal women than in their corresponding age-matched male control group. CONCLUSIONS: Our results suggest that the differences in the composition of gut microbiota between genders and between women of different hormonal status may be related to the sexual dimorphism observed in the incidence of metabolic diseases and their co-morbidities.

Endotoxemia is modulated by quantity and quality of dietary fat in older adults.

BACKGROUND: Aging is an important determinant of the rate of atherosclerosis development, mainly through low-grade inflammation. Diet, and particularly its fat content, modulates the inflammatory response in fasting and postprandial states. OBJECTIVE: We aimed to study the effects of dietary fat on endotoxemia in healthy older adults. MATERIALS AND METHODS: Twenty healthy older adults were randomized to three diets, lasting three-weeks each, using a crossover design: 1. A Mediterranean diet enriched in MUFA with virgin olive oil. 2. An SFA-rich diet. 3. A low-fat high-carbohydrate diet enriched in n-3 PUFA (α-linolenic acid of plant origin) (CHO-PUFA diet). At the end of each period, after a 12-h fast, the subjects received a meal with a composition similar to the dietary period just completed. We determined the fasting and the postprandial plasma levels of lipopolysaccharide (LPS) and LPS-binding protein (LBP). RESULTS: In the fasting state, we observed lower LPS plasma levels after the consumption of the CHO-PUFA diet (P=0.046) in comparison with the consumption of the Med and SFA-rich diets. In the postprandial measurements, we observed a statistically significant increase in plasma levels of LPS (P=0.044) and a decrease in LBP (P=0.003) after the intake of the CHO-PUFA meal, whereas no postprandial changes were observed after the ingestion of the Med and SFA-rich meals. CONCLUSION: Our results, together with those obtained in a previous study, support the concept that the consumption of the Med Diet, in contrast to a low-fat PUFA diet, constitutes a more suitable dietary lifestyle for preventing the development of atherosclerosis in a population at risk, such as older adults.

Consumption of Two Healthy Dietary Patterns Restored Microbiota Dysbiosis in Obese Patients with Metabolic Dysfunction.

SCOPE: The consumption of two healthy diets (Mediterranean (MED) and low-fat (LF) diets) may restore the gut microbiome dysbiosis in obese patients depending on the degree of metabolic dysfunction. METHODS AND RESULTS: The differences in bacterial community at baseline and after 2 years of dietary intervention of 106 subjects from the CORDIOPREV study were analyzed, 33 of whom were obese patients with severe metabolic disease (5 criteria for metabolic syndrome) (MetS-OB), 32 obese patients without metabolic dysfunction (2 or less criteria for metabolic syndrome) (NonMetS-OB) and 41 non-obese subjects (NonMetS-NonOB). Our study showed a marked dysbiosis in people with severe metabolic disease (Met-OB), compared with obese people without MetS (NonMetS-OB) and non-obese people (NonMetS-NonOB). This disbiotic pattern was reversed by consumption of both MED (35% of calories as fat (22% MUFA fat, 6% PUFA fat and <10% saturated fat) or LF (<30% total fat (<10% saturated fat, 12%-14% MUFA fat and 6-8% PUFA fat) diets, whereas no significant microbiota changes were observed in NonMetS-NonOB and NonMetS-OB groups. CONCLUSION: Our results suggest that the chronic intake of two healthy dietary patterns partially restores the gut microbiome dysbiosis in obese patients with coronary heart disease, depending on the degree of metabolic dysfunction.

Effect of Dietary Lipids on Endotoxemia Influences Postprandial Inflammatory Response.

Metabolic syndrome (MetS) results in postprandial metabolic alterations that predisposes one to a state of chronic low-grade inflammation and increased oxidative stress. We aimed to assess the effect of the consumption of the quantity and quality of dietary fat on fasting and postprandial plasma lipopolysaccharides (LPS). A subgroup of 75 subjects with metabolic syndrome was randomized to receive 1 of 4 diets: HSFA, rich in saturated fat; HMUFA, rich in monounsaturated fat; LFHCC n-3, low-fat, rich in complex carbohydrate diet supplemented with n-3 polyunsaturated fatty acids; LFHCC low-fat, rich in complex carbohydrate diet supplemented with placebo, for 12 weeks each. We administered a fat challenge reflecting the fatty acid composition of the diets at postintervention. We determined the plasma lipoproteins and glucose and gene expression in peripheral blood mononuclear cells (PBMC) and adipose tissue. LPS and LPS binding protein (LBP) plasma levels were determined by ELISA, at fasting and postprandial (4 h after a fat challenge) states. We observed a postprandial increase in LPS levels after the intake of the HSFA meal, whereas we did not find any postprandial changes after the intake of the other three diets. Moreover, we found a positive relationship between the LPS plasma levels and the gene expression of IkBa and MIF1 in PBMC. No statistically significant differences in the LBP plasma levels at fasting or postprandial states were observed. Our results suggest that the consumption of HSFA diet increases the intestinal absorption of LPS which, in turn, increases postprandial endotoxemia levels and the postprandial inflammatory response.

The gut microbial community in metabolic syndrome patients is modified by diet.

Intestinal microbiota changes may be involved in the development of metabolic syndrome (MetS), which is a multicomponent disorder frequently associated with obesity. The aim of this study was to test the effect of consuming two healthy diets: a Mediterranean diet and a low-fat high-carbohydrate diet, for 2years in the gut microbiota of MetS patients and those in the control group. We analyzed the differences in the bacterial community structure between the groups after 2years of dietary intervention (Mediterranean or low-fat diet) through quantitative polymerase chain reaction using primers, targeting specific bacterial taxa. We observed, at basal time, that the abundance of Bacteroides, Eubacterium and Lactobacillus genera is lower in the control group than in MetS patients, while Bacteroides fragilis group, Parabacteroides distasonis, Bacteroides thetaiotaomicron, Faecalibacterium prausnitzii, Fusobacterium nucleatum, Bifidobacterium longum, Bifidobacterium adolescentis, Ruminococcus flavefaciens subgroup and Eubacterium rectale are depleted in MetS patients (all P values <.05). Additionally, we found that long-term consumption of Mediterranean diet partially restores the population of P. distasonis, B. thetaiotaomicron, F. prausnitzii, B. adolescentis and B. longum in MetS patients (all P values <.05). Our results suggest that the Mediterranean diet could be a useful tool to restore potentially beneficial members of the gut microbiota, although the stability of these changes over time still remains to be assessed.