Elsa M. Janle

AMP-activated protein kinase signaling activation by resveratrol modulates amyloid-β peptide metabolism.

Alzheimer disease is an age-related neurodegenerative disorder characterized by amyloid-β (Aβ) peptide deposition into cerebral amyloid plaques. The natural polyphenol resveratrol promotes anti-aging pathways via the activation of several metabolic sensors, including the AMP-activated protein kinase (AMPK). Resveratrol also lowers Aβ levels in cell lines; however, the underlying mechanism responsible for this effect is largely unknown. Moreover, the bioavailability of resveratrol in the brain remains uncertain. Here we show that AMPK signaling controls Aβ metabolism and mediates the anti-amyloidogenic effect of resveratrol in non-neuronal and neuronal cells, including in mouse primary neurons. Resveratrol increased cytosolic calcium levels and promoted AMPK activation by the calcium/calmodulin-dependent protein kinase kinase-β. Direct pharmacological and genetic activation of AMPK lowered extracellular Aβ accumulation, whereas AMPK inhibition reduced the effect of resveratrol on Aβ levels. Furthermore, resveratrol inhibited the AMPK target mTOR (mammalian target of rapamycin) to trigger autophagy and lysosomal degradation of Aβ. Finally, orally administered resveratrol in mice was detected in the brain where it activated AMPK and reduced cerebral Aβ levels and deposition in the cortex. These data suggest that resveratrol and pharmacological activation of AMPK have therapeutic potential against Alzheimer disease.

Brain-Targeted Proanthocyanidin Metabolites for Alzheimer's Disease Treatment

While polyphenolic compounds have many health benefits, the potential development of polyphenols for the prevention/treatment of neurological disorders is largely hindered by their complexity as well as by limited knowledge regarding their bioavailability, metabolism, and bioactivity, especially in the brain. We recently demonstrated that dietary supplementation with a specific grape-derived polyphenolic preparation (GP) significantly improves cognitive function in a mouse model of Alzheimers disease (AD). GP is comprised of the proanthocyanidin (PAC) catechin and epicatechin in monomeric (Mo), oligomeric, and polymeric forms. In this study, we report that following oral administration of the independent GP forms, only Mo is able to improve cognitive function and only Mo metabolites can selectively reach and accumulate in the brain at a concentration of ∼400 nm. Most importantly, we report for the first time that a biosynthetic epicatechin metabolite, 3′-O-methyl-epicatechin-5-O-β-glucuronide (3′-O-Me-EC-Gluc), one of the PAC metabolites identified in the brain following Mo treatment, promotes basal synaptic transmission and long-term potentiation at physiologically relevant concentrations in hippocampus slices through mechanisms associated with cAMP response element binding protein (CREB) signaling. Our studies suggest that select brain-targeted PAC metabolites benefit cognition by improving synaptic plasticity in the brain, and provide impetus to develop 3′-O-Me-EC-Gluc and other brain-targeted PAC metabolites to promote learning and memory in AD and other forms of dementia.

Identification of brain-targeted bioactive dietary quercetin-3-O-glucuronide as a novel intervention for Alzheimer’s disease

Epidemiological and preclinical studies indicate that polyphenol intake from moderate consumption of red wines may lower the relative risk for developing Alzheimers disease (AD) dementia. There is limited information regarding the specific biological activities and cellular and molecular mechanisms by which wine polyphenolic components might modulate AD. We assessed accumulations of polyphenols in the rat brain following oral dosage with a Cabernet Sauvignon red wine and tested brain‐targeted polyphenols for potential beneficial AD disease‐modifying activities. We identified accumulations of select polyphenolic metabolites in the brain. We demonstrated that, in comparison to vehicle‐control treatment, one of the brain‐targeted polyphenol metabolites, quercetin‐3‐O‐glucuronide, significantly reduced the generation of β‐amyloid (Aβ) peptides by primary neuron cultures generated from the Tg2576 AD mouse model. Another brain‐targeted metabolite, malvidin‐3‐O‐glucoside, had no detectable effect on Aβ generation. Moreover, in an in vitro analysis using the photo‐induced cross‐linking of unmodified proteins (PICUP) technique, we found that quercetin‐3‐O‐glucuronide is also capable of interfering with the initial protein‐protein interaction of Aβ1–40 and Aβ1–42 that is necessary for the formation of neurotoxic oligomeric Aβ species. Lastly, we found that quercetin‐3‐O‐glucuronide treatment, compared to vehicle‐control treatment, significantly improved AD‐type deficits in hippocampal formation basal synaptic transmission and long‐term potentiation, possibly through mechanisms involving the activation of the c‐Jun N‐terminal kinases and the mitogen‐activated protein kinase signaling pathways. Brain‐targeted quercetin‐3‐O‐glucuronide may simultaneously modulate multiple independent AD disease‐modifying mechanisms and, as such, may contribute to the benefits of dietary supplementation with red wines as an effective intervention for AD.—Ho, L., Ferruzzi, M. G., Janle, E. M., Wang, J., Gong, B., Chen, T.‐Y., Lobo, J., Cooper, B., Wu, Q. L., Talcott, S. T., Percival, S. S., Simon, J. E., Pasinetti, G. M. Identification of brain‐targeted bioactive dietary quercetin‐3‐O‐glucuronide as a novel intervention for Alzheimers disease. FASEB J. 27, 769–781 (2013). www.fasebj.org

Role of intestinal microbiota in the generation of polyphenol-derived phenolic acid mediated attenuation of Alzheimer's disease β-amyloid oligomerization.

SCOPE Grape seed polyphenol extract (GSPE) is receiving increasing attention for its potential preventative and therapeutic roles in Alzheimers disease (AD) and other age-related neurodegenerative disorders. The intestinal microbiota is known to actively convert many dietary polyphenols, including GSPE, to phenolic acids. There is limited information on the bioavailability and bioactivity of GSPE-derived phenolic acid in the brain. METHODS AND RESULTS We orally administered GSPE to rats and investigated the bioavailability of 12 phenolic acids known to be generated by microbiota metabolism of anthocyanidins. GSPE treatment significantly increased the content of two of the phenolic acids in the brain: 3-hydroxybenzoic acid and 3-(3´-hydroxyphenyl)propionic acid, resulting in the brain accumulations of the two phenolic acids at micromolar concentrations. We also provided evidence that 3-hydroxybenzoic acid and 3-(3´-hydroxyphenyl)propionic acid potently interfere with the assembly of β-amyloid peptides into neurotoxic β-amyloid aggregates that play key roles in AD pathogenesis. CONCLUSION Our observation suggests important contribution of the intestinal microbiota to the protective activities of GSPE (as well as other polyphenol preparations) in AD. Outcomes from our studies support future preclinical and clinical investigations exploring the potential contributions of the intestinal microbiota in protecting against the onset/progression of AD and other neurodegenerative conditions.

Pharmacokinetics and Tissue Distribution of 14C-Labeled Grape Polyphenols in the Periphery and the Central Nervous System Following Oral Administration

Grape polyphenols confer potential health benefits, including prevention of neurodegenerative diseases. To determine the absorption and tissue distribution of the complex grape polyphenol mixture, (14)C-labeled polyphenols were biosynthesized by grape cell suspension cultures, during co-incubation with radioisotopically labeled sucrose, and fractionated into polyphenolic subfractions. The pharmacokinetics and distribution of grape polyphenols into blood, brain, and peripheral interstitial fluid were determined by tracking the (14)C label. The blood peak (14)C concentration of the fractions ranged from 15 minutes to 4 hours. Absorption and tissue distribution varied greatly between fractions. Concentrations in interstitial fluid were lower than in blood. The amount of residual label in the brain at 24 hours ranged from 0.1% to 1.7% of the dose, depending on the fraction. (14)C label found in the brain tissue and brain microdialysate indicated that grape polyphenols or their metabolites are able to cross the blood-brain barrier. Using (14)C-labeled plant polyphenols it is possible to track the compounds or their metabolic products into any tissue and determine distribution patterns in spite of low concentrations. A central question regarding the potential role of dietary polyphenolics in neurodegenerative research is whether they are bioavailable in the brain. Our observations indicate that some grape-derived polyphenolics do reach the brain, which suggests their potential value for applications in neurodegenerative disorders.

Influence of Chocolate Matrix Composition on Cocoa Flavan-3-ol Bioaccessibility In Vitro and Bioavailability in Humans

Conflicting data exist regarding the influence of chocolate matrices on the bioavailability of epicatechin (EC) from cocoa. The objective of this study was to assess the bioavailability of EC from matrices varying in macronutrient composition and physical form. EC bioavailability was assessed from chocolate confections [reference dark chocolate (CDK), high sucrose (CHS), high milk protein (CMP)] and cocoa beverages [sucrose milk protein (BSMP), non-nutritive sweetener milk protein (BNMP)], in humans and in vitro. Six subjects consumed each product in a randomized crossover design, with serum EC concentrations monitored over 6 h post consumption. Areas under the serum concentration-time curve (AUC) were similar among chocolate matrices. However, AUCs were significantly increased for BSMP and BNMP (132 and 143 nM h) versus CMP (101 nM h). Peak serum concentrations (C(MAX)) were also increased for BSMP and BNMP (43 and 42 nM) compared to CDK and CMP (32 and 25 nM). Mean T(MAX) values were lower, although not statistically different, for beverages (0.9-1.1 h) versus confections (1.8-2.3 h), reflecting distinct shapes of the pharmacokinetic curves for beverages and confections. In vitro bioaccessibility and Caco-2 accumulation did not differ between treatments. These data suggest that bioavailability of cocoa flavan-3-ols is likely similar from typical commercial cocoa based foods and beverages, but that the physical form and sucrose content may influence T(MAX) and C(MAX).

Targeting multiple pathogenic mechanisms with polyphenols for the treatment of Alzheimer's disease-experimental approach and therapeutic implications

Alzheimers disease (AD) is the most prevalent neurodegenerative disease of aging and currently has no cure. Its onset and progression are influenced by multiple factors. There is growing consensus that successful treatment will rely on simultaneously targeting multiple pathological features of AD. Polyphenol compounds have many proven health benefits. In this study, we tested the hypothesis that combining three polyphenolic preparations (grape seed extract, resveratrol, and Concord grape juice extract), with different polyphenolic compositions and partially redundant bioactivities, may simultaneously and synergistically mitigate amyloid-β (Aβ) mediated neuropathology and cognitive impairments in a mouse model of AD. We found that administration of the polyphenols in combination did not alter the profile of bioactive polyphenol metabolites in the brain. We also found that combination treatment resulted in better protection against cognitive impairments compared to individual treatments, in J20 AD mice. Electrophysiological examination showed that acute treatment with select brain penetrating polyphenol metabolites, derived from these polyphenols, improved oligomeric Aβ (oAβ)-induced long term potentiation (LTP) deficits in hippocampal slices. Moreover, we found greatly reduced total amyloid content in the brain following combination treatment. Our studies provided experimental evidence that application of polyphenols targeting multiple disease-mechanisms may yield a greater likelihood of therapeutic efficacy.

Differential carbonylation of proteins as a function of in vivo oxidative stress.

This study reports for the first time qualitative and quantitative differences in carbonylated proteins shed into blood as a function of increasing levels of OS. Carbonylated proteins in freshly drawn blood from pairs of diabetic and lean rats were derivatized with biotin hydrazide, dialyzed, and enriched with avidin affinity chromatography. Proteins thus selected were used in several ways. Differences between control and diabetic subjects in relative concentration of proteins was achieved by differential labeling of tryptic digests with iTRAQ reagents followed by reversed phase chromatography (RPC) and tandem mass spectrometry (MS/MS). Identification and characterization of OS induced post-translational modification sites in contrast was achieved by fractionation of affinity selected proteins before proteolysis and RPC-MS/MS. Relative quantification of peptides bearing oxidative modifications was achieved for the first time by selective reaction monitoring (SRM). Approximately 1.7% of the proteins in Zucker diabetic rat plasma were selected by the avidin affinity column as compared to 0.98% in lean animal plasma. Among the 35 proteins identified and quantified, Apo AII, clusterin, hemopexin precursor, and potassium voltage-gated channel subfamily H member 7 showed the most dramatic changes in concentration. Seventeen carbonylation sites were identified and quantified, 11 of which changed more than 2-fold in oxidation state. Three types of carbonylation were identified at these sites: direct oxidative cleavage from reactive oxygen species, glycation and addition of advanced glycation end products, and addition of lipid peroxidation products. Direct oxidation was the dominant form of carbonylation observed while hemoglobin and murinoglobulin 1 homologue were the most heavily oxidized proteins.

Complementary Approaches To Gauge the Bioavailability and Distribution of Ingested Berry Polyphenolics

Two different strategies for investigating the likely fate, after ingestion, of natural, bioactive berry constituents (anthocyanins and other non-nutritive flavonoids) are compared. A model of the human gastrointestinal tract (TIM-1) that mimicked the biological environment from the point of swallowing and ingestion through the duodenum, jejunum, and ileum (but not the colon) was used to monitor the stability and bioaccessibility of anthocyanins from both maqui berry and wild blueberry. TIM-1 revealed that most anthocyanins were bioaccessible between the second and third hours after intake. Alternatively, biolabeled anthocyanins and other flavonoids generated in vitro from berry and grape cell cultures were administered to in vivo (rodent) models, allowing measurement and tracking of the absorption and transport of berry constituents and clearance through the urinary tract and colon. The advantages and limitations of the alternative strategies are considered.

Chocolate matrix factors modulate the pharmacokinetic behavior of cocoa flavan-3-ol phase II metabolites following oral consumption by Sprague-Dawley rats.

The impact of carbohydrates and milk on the bioavailability of catechin (C) and epicatechin (EC) from chocolate has been previously studied. However, little data exist regarding potential modulation of the phase II metabolism by these chocolate matrix factors. The objectives of this study were to assess the impact of matrix composition on qualitative and quantitative profiles of circulating catechins and their metabolites following administration of commercially relevant chocolate confections. Sprague-Dawley rats were administered 1.5 g of a confection (reference dark, high sucrose, or milk chocolate) by intragastric gavage, and plasma samples were collected over 8 h. High-performance liquid chromatography-mass spectrometry analysis was performed to quantify C, EC, and their metabolites. The predominant metabolites were O-glucuronides (two metabolites) and O-Me-O-glucuronides (three metabolites). Plasma concentrations of metabolites were generally the highest for high sucrose treatment and lowest for milk treatment, while the reference dark treatment generally resulted in intermediate concentrations. The O-Me-(+/-)-C/EC-O-beta-glucuronide (peak 4) was significantly higher for the high sucrose treatment (2325 nM h) versus the milk treatment (1300 nM h). Additionally, C(MAX) values for (+/-)-C/EC-O-beta-glucuronide (peak 3) and two O-Me-(+/-)-C/EC-O-beta-glucuronides (peaks 4 and 6) were significantly higher for the high sucrose treatment (4012, 518, and 2518 nM, respectively) versus the milk treatment (2590, 240, and 1670 nM, respectively). Milk and sucrose appear to modulate both metabolism and plasma pharmacokinetics and, to a lesser extent, the overall bioavailability of catechins from chocolate confections.