epHero - a tandem-fluorescent probe to track the fate of apoptotic cells during efferocytosis.

The efficient removal of apoptotic cells via efferocytosis is critical for maintaining optimal tissue function. This involves the binding and engulfment of apoptotic cells by phagocytes and the subsequent maturation of the phagosome, culminating in lysosomal fusion and cargo destruction. However, current approaches to measure efferocytosis rely on labelling apoptotic targets with fluorescent dyes, which do not sufficiently distinguish between changes to the engulfment and acidification of apoptotic material. To address this limitation, we have developed a genetically coded ratiometric probe epHero which when expressed in the cytoplasm of target cells, bypasses the need for additional labelling steps. We demonstrate that epHero is a pH-sensitive reporter for efferocytosis and can be used to simultaneously track changes to apoptotic cell uptake and acidification, both in vitro and in mice. As proof-of-principle, we modify extracellular nutrition to show how epHero can distinguish between changes to cargo engulfment and acidification. Thus, tracking efferocytosis with epHero is a simple, cost-effective improvement on conventional techniques.

Basal autophagic flux measured in blood correlates positively with age in adults at increased risk of type 2 diabetes.

Preclinical data show that autophagy delays age-related disease. It has been postulated that age-related disease is-at least in part-caused by an age-related decline in autophagy. However, autophagic flux has never been measured in humans across a spectrum of aging in a physiologically relevant context. To address this critical gap in knowledge, the objective of this cross-sectional observational study was to measure basal autophagic flux in whole blood taken from people at elevated risk of developing type 2 diabetes and correlate it with chronological age. During this study, 119 people were recruited and five people were excluded during sample analysis such that 114 people were included in the final analysis. Basal autophagic flux measured in blood and correlations with parameters such as age, body weight, fat mass, AUSDRISK score, blood pressure, glycated hemoglobin HbA1c, blood glucose and insulin, blood lipids, high-sensitivity C-reactive protein, plasma protein carbonylation, and plasma ß-hexosaminidase activity were analysed. Despite general consensus in the literature that autophagy decreases with age, we found that basal autophagic flux increased with age in this human cohort. This is the first study to report measurement of basal autophagic flux in a human cohort and its correlation with age. This increase in basal autophagy could represent a stress response to age-related damage. These data are significant not only for their novelty but also because they will inform future clinical studies and show that measurement of basal autophagic flux in a human cohort is feasible.

Intermittent fasting plus early time-restricted eating versus calorie restriction and standard care in adults at risk of type 2 diabetes: a randomized controlled trial.

Intermittent fasting appears an equivalent alternative to calorie restriction (CR) to improve health in humans. However, few trials have considered applying meal timing during the 'fasting' day, which may be a limitation. We developed a novel intermittent fasting plus early time-restricted eating (iTRE) approach. Adults (N = 209, 58 ± 10 years, 34.8 ± 4.7 kg m-2) at increased risk of developing type 2 diabetes were randomized to one of three groups (2:2:1): iTRE (30% energy requirements between 0800 and 1200 hours and followed by a 20-h fasting period on three nonconsecutive days per week, and ad libitum eating on other days); CR (70% of energy requirements daily, without time prescription); or standard care (weight loss booklet). This open-label, parallel group, three-arm randomized controlled trial provided nutritional support to participants in the iTRE and CR arms for 6 months, with an additional 12-month follow-up. The primary outcome was change in glucose area under the curve in response to a mixed-meal tolerance test at month 6 in iTRE versus CR. Glucose tolerance was improved to a greater extent in iTRE compared with CR (-10.10 (95% confidence interval -14.08, -6.11) versus -3.57 (95% confidence interval -7.72, 0.57) mg dl-1 min-1; P = 0.03) at month 6, but these differences were lost at month 18. Adverse events were transient and generally mild. Reports of fatigue were higher in iTRE versus CR and standard care, whereas reports of constipation and headache were higher in iTRE and CR versus standard care. In conclusion, incorporating advice for meal timing with prolonged fasting led to greater improvements in postprandial glucose metabolism in adults at increased risk of developing type 2 diabetes. ClinicalTrials.gov identifier NCT03689608 .

Modifying Dietary Protein Impacts mTOR Signaling and Brain Deposition of Amyloid ß in a Knock-In Mouse Model of Alzheimer Disease.

BACKGROUND: Alzheimer disease (AD) is a neurodegenerative condition defined by the build-up of amyloid plaques in the brain and intraneuronal tangles of the protein tau. Autophagy is a cellular cleaning process involved in the degradation of proteins, including proteins directly responsible for amyloid plaques, but its activity is compromised in AD. The mechanistic target of rapamycin complex (mTORC) 1 inhibits autophagy when activated by amino acids. OBJECTIVES: We hypothesized that reducing amino acid intake by decreasing dietary protein could promote autophagy, which in turn could prevent amyloid plaque deposition in AD mice. METHODS: Homozygote (2-mo-old) and heterozygote (4-mo-old) amyloid precursor protein NL-G-F mice, a model of brain amyloid deposition, were used in this study to test this hypothesis. Male and female mice were fed with isocaloric low-protein, control, or high-protein diets for 4 mo and killed for analysis. Locomotor performance was measured using the inverted screen test, and body composition was measured using EchoMRI. Samples were analyzed using western blotting, enzyme-linked immunosorbent assay, mass spectrometry, and immunohistochemical staining. RESULTS: mTORC1 activity in the cerebral cortex was inversely covaried with protein consumption in both homozygote and heterozygote mice. Low-protein diet improved metabolic parameters and restored locomotor performance only in male homozygous mice. Dietary protein adjustment did not affect amyloid deposition in homozygous mice. However, in the heterozygous amyloid precursor protein NL-G-F mice, amyloid plaque was lower in male mice consuming the low protein compared with that in mice fed with the control diet. CONCLUSIONS: This study showed that reducing protein intake reduces mTORC1 activity and may prevent amyloid accumulation, at least in male mice. Moreover, dietary protein is a tool that can be used to change mTORC1 activity and amyloid deposition in the mouse brain, and the murine brain's response to dietary protein is sex specific.

The Break-Fast study protocol: a single arm pre-post study to measure the effect of a protein-rich breakfast on autophagic flux in fasting healthy individuals.

BACKGROUND: Autophagy is a cellular process that cleanses cells and is particularly important during ageing. Autophagy has been extensively studied in vitro and in animal models and is known to be sensitive to nutrition. However, human data are limited because autophagic flux (autophagic degradative activity) has been challenging to measure in humans. This protocol paper describes the Break-Fast study, in which autophagic flux will be measured using a recently developed blood test, before and after ingestion of whey protein. This aims to determine whether an acute nutritional intervention can change autophagy in humans. METHODS: A minimum of forty healthy participants (both male and female) aged 20-50 years, BMI 18.5-29.9 kg/m2 will be recruited into this single arm pre-post study. Participants will visit the clinic after an overnight fast for a first blood collection after which they will consume a whey protein-rich drink. A second blood collection will be performed 60 minutes after consumption of the drink. The primary outcome is the change in autophagic flux at 60 minutes post drink. Secondary outcomes include changes in blood glucose, autophagy-related proteins and mRNA, plasma hormones (e.g. insulin, C-peptide, adiponectin, GLP-1, GIP, ghrelin), cytokines, amino acids and lipids, protein synthesis, and correlation between molecular cell damage and autophagic flux. DISCUSSION: This study will provide information about whether autophagy responds to nutrients in humans, and if nutritional strategies could be used to treat or prevent autophagy-related diseases such as Alzheimer's disease or cancer. TRIAL REGISTRATION: Australian New Zealand Clinical Trials Registry (ANZCTR), anzctr.org.au ACTRN12621001029886. Registered on 5 August 2021.

Biological Sensing of Nitric Oxide in Macrophages and Atherosclerosis Using a Ruthenium-Based Sensor.

Macrophage-derived nitric oxide (NO) plays a critical role in atherosclerosis and presents as a potential biomarker. We assessed the uptake, distribution, and NO detection capacity of an irreversible, ruthenium-based, fluorescent NO sensor (Ru-NO) in macrophages, plasma, and atherosclerotic plaques. In vitro, incubation of Ru-NO with human THP1 monocytes and THP1-PMA macrophages caused robust uptake, detected by Ru-NO fluorescence using mass-cytometry, confocal microscopy, and flow cytometry. THP1-PMA macrophages had higher Ru-NO uptake (+13%, p < 0.05) than THP1 monocytes with increased Ru-NO fluorescence following lipopolysaccharide stimulation (+14%, p < 0.05). In mice, intraperitoneal infusion of Ru-NO found Ru-NO uptake was greater in peritoneal CD11b+F4/80+ macrophages (+61%, p < 0.01) than CD11b+F4/80− monocytes. Infusion of Ru-NO into Apoe−/− mice fed high-cholesterol diet (HCD) revealed Ru-NO fluorescence co-localised with atherosclerotic plaque macrophages. When Ru-NO was added ex vivo to aortic cell suspensions from Apoe−/− mice, macrophage-specific uptake of Ru-NO was demonstrated. Ru-NO was added ex vivo to tail-vein blood samples collected monthly from Apoe−/− mice on HCD or chow. The plasma Ru-NO fluorescence signal was higher in HCD than chow-fed mice after 12 weeks (37.9%, p < 0.05). Finally, Ru-NO was added to plasma from patients (N = 50) following clinically-indicated angiograms. There was lower Ru-NO fluorescence from plasma from patients with myocardial infarction (−30.7%, p < 0.01) than those with stable coronary atherosclerosis. In conclusion, Ru-NO is internalised by macrophages in vitro, ex vivo, and in vivo, can be detected in atherosclerotic plaques, and generates measurable changes in fluorescence in murine and human plasma. Ru-NO displays promising utility as a sensor of atherosclerosis.

Intermittent fasting activates markers of autophagy in mouse liver, but not muscle from mouse or humans.

OBJECTIVES: Intermittent fasting (IF) activates autophagy in cardiac muscle and pancreatic islets. We examined the effect of IF on markers of autophagy in liver and skeletal muscle in mice and in humans. METHODS: Ten-wk-old C57 BL/6 J male mice were ad libitum (AL) fed a high-fat diet (HFD) or chow diet for 8 wk, before randomization to AL or IF (24-h fast, 3 non-consecutive days per week) for 8 wk (8-16 per group). Tissue was collected in the fed or 22-h fasted state. Fifty women (51 ± 2 y, 31.8 ± 4.3 kg/m2) were randomly assigned to one of two IF protocols (24-hfast, 3 non-consecutive days per week) and fed at 70% (IF70) or 100% (IF100) of energy requirements for 8 wk. Vastus lateralis muscle was collected at 0800 after 12- and 24-h fasts. Microtubule-associated protein light chain 1 (Map1 lc3 b), Beclin1 (Becn1), Sequestosome 1 (Sqstm1/p62), and Lysosomal associated membrane protein 2 (Lamp2) were assessed by quantitative polymerase chain reaction and LC3, BECLIN1 and LAMP1 protein content by immunoblotting. RESULTS: Fasting increased hepatic LC3 I protein and Map1 lc3 b mRNA levels in IF mice fed chow or HFD. LAMP1 protein and Beclin1 mRNA levels in liver were also increased by fasting, but only in chow-fed mice. IF did not activate markers of autophagy in mouse muscle. In humans, a 24-h fast increased SQSTM1. BECLIN1, SQSTM1 and LAMP2 mRNA levels were decreased in IF70 after a 12-h overnight fast . CONCLUSION: Markers of autophagy in liver, but not in muscle, were elevated in response to IF in mice. In humans, autophagy markers in muscle were reduced, likely in response to weight loss.

Lysosomal gene Hexb displays haploinsufficiency in a knock-in mouse model of Alzheimer's disease.

Lysosomal network abnormalities are an increasingly recognised feature of Alzheimer's disease (AD), which appear early and are progressive in nature. Sandhoff disease and Tay-Sachs disease (neurological lysosomal storage diseases caused by mutations in genes that code for critical subunits of ß-hexosaminidase) result in accumulation of amyloid-ß (Aß) and related proteolytic fragments in the brain. However, experiments that determine whether mutations in genes that code for ß-hexosaminidase are risk factors for AD are currently lacking. To determine the relationship between ß-hexosaminidase and AD, we investigated whether a heterozygous deletion of Hexb, the gene that encodes the beta subunit of ß-hexosaminidase, modifies the behavioural phenotype and appearance of disease lesions in App NL-G-F/NL-G-F (App KI/KI ) mice. App KI/KI and Hexb +/- mice were crossed and evaluated in a behavioural test battery. Neuropathological hallmarks of AD and ganglioside levels in the brain were also examined. Heterozygosity of Hexb in App KI/KI mice reduced learning flexibility during the Reversal Phase of the Morris water maze. Contrary to expectation, heterozygosity of Hexb caused a small but significant decrease in amyloid beta deposition and an increase in the microglial marker IBA1 that was region- and age-specific. Hexb heterozygosity caused detectable changes in the brain and in the behaviour of an AD model mouse, consistent with previous reports that described a biochemical relationship between HEXB and AD. This study reveals that the lysosomal enzyme gene Hexb is not haplosufficient in the mouse AD brain.

The mTOR-lysosome axis at the centre of ageing.

Age-related diseases represent some of the largest unmet clinical needs of our time. While treatment of specific disease-related signs has had some success (for example, the effect of statin drugs on slowing progression of atherosclerosis), slowing biological ageing itself represents a target that could significantly increase health span and reduce the prevalence of multiple age-related diseases. Mechanistic target of rapamycin complex 1 (mTORC1) is known to control fundamental processes in ageing: inhibiting this signalling complex slows biological ageing, reduces age-related disease pathology and increases lifespan in model organisms. How mTORC1 inhibition achieves this is still subject to ongoing research. However, one mechanism by which mTORC1 inhibition is thought to slow ageing is by activating the autophagy-lysosome pathway. In this review, we examine the special bidirectional relationship between mTORC1 and the lysosome. In cells, mTORC1 is located on lysosomes. From this advantageous position, it directly controls the autophagy-lysosome pathway. However, the lysosome also controls mTORC1 activity in numerous ways, creating a special two-way relationship. We then explore specific examples of how inhibition of mTORC1 and activation of the autophagy-lysosome pathway slow the molecular hallmarks of ageing. This body of literature demonstrates that the autophagy-lysosome pathway represents an excellent target for treatments that seek to slow biological ageing and increase health span in humans.

Human autophagy measurement: an underappreciated barrier to translation.

Preclinical research shows that autophagy is a modifiable process that holds promise for preventing human age-related disease. However, this knowledge has not been clinically translated. Here, we discuss recent developments in the ability to measure human autophagy, and why it is a critical step for translation.

The composition of the gut microbiota following early-life antibiotic exposure affects host health and longevity in later life.

Studies investigating whether there is a causative link between the gut microbiota and lifespan have largely been restricted to invertebrates or to mice with a reduced lifespan because of a genetic deficiency. We investigate the effect of early-life antibiotic exposure on otherwise healthy, normal chow-fed, wild-type mice, monitoring these mice for more than 700 days in comparison with untreated control mice. We demonstrate the emergence of two different low-diversity community types, post-antibiotic microbiota (PAM) I and PAM II, following antibiotic exposure. PAM II but not PAM I mice have impaired immunity, increased insulin resistance, and evidence of increased inflammaging in later life as well as a reduced lifespan. Our data suggest that differences in the composition of the gut microbiota following antibiotic exposure differentially affect host health and longevity in later life.

PICALM regulates cathepsin D processing and lysosomal function.

Degradation and clearance of cellular waste in the autophagic and endo-lysosomal systems is important for normal physiology and prevention of common late-onset diseases such as Alzheimer's disease (AD). Phosphatidylinostol-binding clathrin assembly protein (PICALM) is a robust AD risk factor gene and encodes an endosomal protein clathrin-binding cytosolic protein, reduction of which is known to exacerbate tauopathy. Although PICALM is known to regulate initiation of autophagy, its role in maturation of lysosomal enzymes required for proteolysis has not been studied. We sought to determine the importance of PICALM for cellular degradative function by disrupting exon 1 of PICALM using CRISPR/Cas9 in HeLa cells. PICALM disruption increased numbers of early endosomes. Proteomic analysis of endosome-enriched samples showed that disrupting exon 1 of PICALM increased the abundance of lysosomal enzymes in these organelles, and western blotting revealed disruption to processing and maturation of the lysosomal protease, cathepsin D, and a deficit in autophagy. This study shows PICALM is important for the correct maturation of lysosomal enzymes and efficient proteolytic function in the lysosome.

Inhibiting mTOR activity using AZD2014 increases autophagy in the mouse cerebral cortex.

Autophagy is a catabolic process that collects and degrades damaged or unwanted cellular materials such as protein aggregates. Defective brain autophagy has been linked to diseases such as Alzheimer's disease. Autophagy is regulated by the protein kinase mTOR (mechanistic target of rapamycin). Although already demonstrated in vitro, it remains contentious whether inhibiting mTOR can enhance autophagy in the brain. To address this, mice were intraperitoneally injected with the mTOR inhibitor AZD2014 for seven days. mTOR complex 1 (mTORC1) activity was decreased in liver and brain. Autophagic activity was increased by AZD2014 in both organs, as measured by immunoblotting for LC3 (microtubule-associated proteins-1A/1B light chain 3B) and measurement of autophagic flux in the cerebral cortex of transgenic mice expressing the EGFP-mRFP-LC3B transgene. mTOR activity was shown to correlate with changes in LC3. Thus, we show it is possible to promote autophagy in the brain using AZD2014, which will be valuable in tackling conditions associated with defective autophagy, especially neurodegeneration.

Measurement of autophagic flux in humans: an optimized method for blood samples.

Autophagic flux is a critical cellular process that is vastly under-appreciated in terms of its importance to human health. Preclinical studies have demonstrated that reductions in autophagic flux cause cancer and exacerbate chronic diseases, including heart disease and the pathological hallmarks of dementia. Autophagic flux can be increased by targeting nutrition-related biochemical signaling. To date, translation of this knowledge has been hampered because there has been no way to directly measure autophagic flux in humans. In this study we detail a method whereby human macroautophagic/autophagic flux can be directly measured from human blood samples. We show that whole blood samples can be treated with the lysosomal inhibitor chloroquine, and peripheral blood mononuclear cells isolated from these samples could be used to measure autophagic machinery protein LC3B-II. Blocking of autophagic flux in cells while still in whole blood represents an important advance because it preserves genetic, nutritional, and signaling parameters inherent to the individual. We show this method was reproducible and defined LC3B-II as the best protein to measure autophagic flux in these cells. Finally, we show that this method is relevant to assess intra-individual variation induced by an intervention by manipulating nutrition signaling with an ex vivo treatment of whole blood that comprised leucine and insulin. Significantly, this method will enable the identification of factors that alter autophagic flux in humans, and better aid their translation in the clinic. With further research, it could also be used as a novel biomarker for risk of age-related chronic disease.Abbreviations: AMPK: AMP-activated protein kinase; ACTB: actin beta; ATG5: autophagy related 5; BAF: bafilomycin A1; CQ: chloroquine; DMSO: dimethyl sulfoxide; DPBS: Dulbecco's phosphate-buffered saline; EDTA: ethylenediaminetetraacetic acid; KO: knockout; MAP1LC3A/LC3A: microtubule associated protein 1 light chain 3 alpha; MAP1LC3B/LC3B: microtubule associated protein 1 light chain 3 beta; MAP1LC3C/LC3C: microtubule associated protein 1 light chain 3 gamma; MTOR: mechanistic target of rapamycin kinase; NBR1: NBR1 autophagy cargo receptor; PBMCs: peripheral blood mononuclear cells; PMNs: polymorphonuclear cells; RPMI: Roswell Park Memorial Institute; SQSTM1: sequestosome 1; TBST: Tris-buffered saline containing 0.1% (v:v) Tween 20; TEM: transmission electron microscopy.

Comparison of chloroquine-like molecules for lysosomal inhibition and measurement of autophagic flux in the brain.

Measurement of autophagic flux in vivo is critical to understand how autophagy can be used to combat disease. Neurodegenerative diseases have a special relationship with autophagy, which makes measurement of autophagy in the brain a significant research priority. Currently, measurement of autophagic flux is possible through use of transgenic constructs, or application of a lysosomal inhibitor such as chloroquine. Unfortunately, chloroquine is not useful for measuring autophagic flux in the brain and the use of transgenic animals necessitates cross-breeding of transgenic strains and maintenance of lines, which is costly. To find a drug that could block lysosomal function in the brain for the measurement of autophagic flux, we selected compounds from the literature that appeared to have similar properties to chloroquine and tested their ability to inhibit autophagic flux in cell culture and in mice. These chemicals included chloroquine, quinacrine, mefloquine, promazine and trifluoperazine. As expected, chloroquine blocked lysosomal degradation of the autophagic protein LC3B-II in cell culture. Quinacrine also inhibited autophagic flux in cell culture. Other compounds tested were not effective. When injected into mice, chloroquine caused accumulation of LC3B-II in heart tissue, and quinacrine was effective at blocking LC3B-II degradation in male, but not female skeletal muscle. None of the compounds tested were useful for measuring autophagic flux in the brain. During this study we also noted that the vehicle DMSO powerfully up-regulated LC3B-II abundance in tissues. This study shows that chloroquine and quinacrine can both be used to measure autophagic flux in cells, and in some peripheral tissues. However, measurement of flux in the brain using lysosomal inhibitors remains an unresolved research challenge.

Polyphenols From Grape and Blueberry Improve Episodic Memory in Healthy Elderly with Lower Level of Memory Performance: A Bicentric Double-Blind, Randomized, Placebo-Controlled Clinical Study.

Polyphenols are promising nutritional bioactives exhibiting beneficial effect on age-related cognitive decline. This study evaluated the effect of a polyphenol-rich extract from grape and blueberry (PEGB) on memory of healthy elderly subjects (60-70 years-old). A bicentric, randomized, double-blind, placebo-controlled trial was conducted with 215 volunteers receiving 600 mg/day of PEGB (containing 258 mg flavonoids) or a placebo for 6 months. The primary outcome was the CANTAB Paired Associate Learning (PAL), a visuospatial learning and episodic memory test. Secondary outcomes included verbal episodic and recognition memory (VRM) and working memory (SSP). There was no significant effect of PEGB on the PAL on the whole cohort. Yet, PEGB supplementation improved VRM-free recall. Stratifying the cohort in quartiles based on PAL at baseline revealed a subgroup with advanced cognitive decline (decliners) who responded positively to the PEGB. In this group, PEGB consumption was also associated with a better VRM-delayed recognition. In addition to a lower polyphenol consumption, the urine metabolomic profile of decliners revealed that they excreted more metabolites. Urinary concentrations of specific flavan-3-ols metabolites were associated, at the end of the intervention, with the memory improvements. Our study demonstrates that PEGB improves age-related episodic memory decline in individuals with the highest cognitive impairments.

Polyphenol-rich extract from grape and blueberry attenuates cognitive decline and improves neuronal function in aged mice.

Ageing is characterised by memory deficits, associated with brain plasticity impairment. Polyphenols from berries, such as flavan-3-ols, anthocyanins, and resveratrol, have been suggested to modulate synaptic plasticity and cognitive processes. In the present study we assessed the preventive effect of a polyphenol-rich extract from grape and blueberry (PEGB), with high concentrations of flavonoids, on age-related cognitive decline in mice. Adult and aged (6 weeks and 16 months) mice were fed a PEGB-enriched diet for 14 weeks. Learning and memory were assessed using the novel object recognition and Morris water maze tasks. Brain polyphenol content was evaluated with ultra-high-performance LC-MS/MS. Hippocampal neurotrophin expression was measured using quantitative real-time PCR. Finally, the effect of PEGB on adult hippocampal neurogenesis was assessed by immunochemistry, counting the number of cells expressing doublecortin and the proportion of cells with dendritic prolongations. The combination of grape and blueberry polyphenols prevented age-induced learning and memory deficits. Moreover, it increased hippocampal nerve growth factor (Ngf) mRNA expression. Aged supplemented mice displayed a greater proportion of newly generated neurons with prolongations than control age-matched mice. Some of the polyphenols included in the extract were detected in the brain in the native form or as metabolites. Aged supplemented mice also displayed a better survival rate. These data suggest that PEGB may prevent age-induced cognitive decline. Possible mechanisms of action include a modulation of brain plasticity. Post-treatment detection of phenolic compounds in the brain suggests that polyphenols may act directly at the central level, while they can make an impact on mouse survival through a potential systemic effect.

Pattern of polyphenol intake and the long-term risk of dementia in older persons.

OBJECTIVE: To investigate the optimal combination of dietary polyphenols associated with the long-term risk of dementia in a large prospective French cohort of older persons, the Three-City (3C) Study. METHODS: We included 1,329 older adults without dementia from the 3C study with assessment of intake of 26 polyphenol subclasses who were followed up for 12 years for dementia. Using partial least squares for Cox models, we identified a pattern of polyphenol intake associated with dementia risk. RESULTS: The pattern combined several flavonoids (dihydroflavonols, anthocyanins, isoflavonoids, flavanones), stilbenes (including resveratrol), lignans, and other subclasses (hydroxybenzaldehydes, naphthoquinones, furanocoumarins). Compared with participants in the lower quintile of pattern score, those in the higher quintile had a 50% lower risk of dementia (95% confidence interval 20%-68%, p for trend <0.01) in multivariate models. CONCLUSIONS: In this French cohort, a polyphenol pattern provided by a diet containing specific plant products (nuts, citrus, berries, leafy vegetables, soy, cereals, olive oil) accompanied by red wine and tea was associated with lower dementia risk.

A mixed grape and blueberry extract is safe for dogs to consume.

BACKGROUND: Grape and blueberry extracts are known to protect against age-related cognitive decline. However, beneficial effects achieved by mixing grape and blueberry extracts have yet to be evaluated in dogs, or their bioavailability assessed. Of concern to us were cases of acute renal failure in dogs, after their ingestion of grapes or raisins. The European Pet Food Industry Federation (2013) considers only the grape or raisin itself to be potentially dangerous; grape-seed extracts per-se, are not considered to be a threat. Our aim was therefore to evaluate the renal and hepatic safety, and measure plasma derivatives of a polyphenol-rich extract from grape and blueberry (PEGB; from the Neurophenols Consortium) in dogs. Polyphenol expression was analyzed by UHPLC-MS/MS over 8 hours, for dogs given PEGB at 4 mg/kg. Safety was evaluated using four groups of 6 dogs. These groups received capsules containing no PEGB (control), or PEGB at 4, 20, or 40 mg/kg BW/d, for 24 weeks. Blood and urine samples were taken the week prior to study commencement, then at the end of the 24-wk study period. Routine markers of renal and liver damage, including creatinine (Creat), blood urea nitrogen, albumin, minerals, alkaline phosphatase (ALP), and alanine transaminase (ALT) were measured. Biomarkers for early renal damage were also evaluated in plasma (cystatin C (CysC), and neutrophil gelatinase-associated lipocalin (NGAL)), and urine (CysC, clusterin (Clu), and NGAL). Ratios of urinary biomarkers to Creat were calculated, and compared with acceptable maximal values obtained for healthy dogs, as reported in the literature. RESULTS: While several PEGB-specific polyphenols and metabolites were detected in dog plasma, at the end of the PEGB consumption period, our biomarker analyses presented no evidence of either renal or liver damage (Creat, BUN, ionogram, albumin and ALT, ALP). Similarly, no indication of early renal damage could be detected. Plasma CysC, urinary CysC/Creat, Clu/Creat, and NGAL/Creat ratios were all beneath reported benchmarked maximums, with no evidence of PEGB toxicity. CONCLUSIONS: Long-term consumption of a pet specific blend of a polyphenol-rich extract from grape and blueberry (PEGB; from the Neurophenols Consortium), was not associated with renal or hepatic injury, and can therefore be considered safe.

Dietary Polyphenol Supplementation Prevents Alterations of Spatial Navigation in Middle-Aged Mice.

Spatial learning and memory deficits associated with hippocampal synaptic plasticity impairments are commonly observed during aging. Besides, the beneficial role of dietary polyphenols has been suggested as potential functional food candidates to prevent this memory decline. Indeed, polyphenols could potentiate the signaling pathways of synaptic plasticity underlying learning and memory. In this study, spatial learning deficits of middle-aged mice were first highlighted and characterized according to their navigation patterns in the Morris water maze task. An eight-week polyphenol-enriched diet, containing a polyphenol-rich extract from grape and blueberry (PEGB; from the Neurophenols Consortium) with high contents of flavonoids, stilbenes and phenolic acids, was then successful in reversing these age-induced effects. The use of spatial strategies was indeed delayed with aging whereas a polyphenol supplementation could promote the occurrence of spatial strategies. These behavioral results were associated with neurobiological changes: while the expression of hippocampal calmodulin kinase II (CaMKII) mRNA levels was reduced in middle-aged animals, the polyphenol-enriched diet could rescue them. Besides, an increased expression of nerve growth neurotrophic factor (NGF) mRNA levels was also observed in supplemented adult and middle-aged mice. Thus these data suggest that supplementation with polyphenols could be an efficient nutritional way to prevent age-induced cognitive decline.