MLECULAR NUTRITIN GRUP Established 2004 Dietary Flavonoids: Modulators of Brain Ageing? Jeremy P E Spencer School of Food Biosciences University of Reading, UK
The Sensitivity of the Brain to xidative Stress High oxygen consumption Rich in polyunsaturated fatty acids. High levels of transition metals Lower antioxidant defences Presence of oxidisable Parkinson s Disease Age-Related Decline xidative Stress Alzheimer s Disease Ischaemia/ Reperfusion neurotransmitters
Why is there interest in diet? Increasing life expectancy Increasing number of patients with neurodegenerative diseases 5 portions a day! To develop therapeutic or dietary interventions to delay the onset or counteract the progression of neurodegenerative diseases
Flavonoids: source Tea: (Flavanols) Fruit and vegetables: (All classes) Citrus: (Flavanone) Red wine: (Flavanol, Flavonols) Cocoa: (Flavanols and procyanidins) Berries: (Anthocyanins)
Flavonoids: structure R 1 R 2 R 1 H Flavanol R 3 H + R 1 R 2 R 2 R 4 R 5 Isoflavone R 3 R 1 Anthocyanin R 1 R 2 R 2 H R 3 H R 3 Flavonol Flavanone
Ageing and Incidence of Neurodegenerative Diseases 85 80 Men Women Age (years) Prevalence of AD (%) Prevalence of PD (%) Life Expectancy at Birth 75 70 65 60 55 50 Life Expectancy is increasing 35-59 60-69 70-79 80-89 0.2 0.3 3.2 10.8 0.07 0.18 1.72 6.2 45 40 1900 1920 1940 1960 1980 2000 2020 Year Increased health care costs and general demand on the NHS. Reduced quality of life for the elderly population.
Evidence for Flavonoid Action? Epidemiological Studies Molecular Mechanisms? Supplementation studies in humans and animals Flavonoids as beneficial compounds against degenerative diseases. Flavonoids may reduce the risk of death from coronary heart disease and cancer Hertog et al. (1993) Lancet. Flavonoid extracts from fruit and vegetables have been reported to attenuate cognitive decline and neuronal dysfunction in animal models and humans. Joseph et al. (1999) J. Neuroscience.
How Might They Work at the Cellular Level?
Flavonoids as Antioxidants Classical Antioxidant Property: Quercetin B-ring catechol group Un-saturation in the C-ring H A C B Carbonyl group Presence of transition metal ion binding functions Powerful scavengers of RS and RNS: Fenton systems: Fe 2+ Cu + and peroxide N - and HCl Metal chelators
Biotransformation of flavonoids in the body ral Ingestion of flavonoid * 5 * Neurons glia Stomach ligomeric Flavonoids Small Intestine jejunum ileum ligomers cleaved Monomeric units 1 A-ring glucuronides -methylated glucuronides -methylated aglycone Portal vein cells Further metabolism -methylated Blood-brain barrier Sulphates glucuronides Liver 4 2 3 Colon glucuronides Kidney Flavonoid Phenolic acids Gut microflora Urine Renal excretion of glucuronides
Metabolism Reduces Antioxidant Potential Increasing level in the circulation H CH 3 3 --Methylepicatechin Reduction in Antioxidant potential C - Epicatechin 7--β-Dglucuronide H Epicatechin
Do Flavonoids Access the Brain?
1.2 1 2h Conjugated Free Pelargonidin nmol/g tissue 0.8 0.6 0.4 0.2 0 Brain Lungs Heart Liver Spleen Kidney Plasma nmol/g or ml 1 0.9 0.8 0.7 0.6 0.5 0.4 0.3 0.2 0.1 0 18h p-hba Conjugated Free Brain Lungs Heart Liver Spleen Kidney Plasma
Levels of antioxidants in brain µmol/g brain tissue 3 2.5 2 1.5 1 0.5 0 0.2 0.16 0.12 0.08 0.04 nmol/g brain tissue Total GSH Vitamin E Vitamin C Pelargonidin 0
Possible Mechanisms of Bioactivity xidative Stress Biomarkers of damage i.e. 8--Guan. Lipid ox. Mitochondrial dysfunction MAPK Signaling Mitochondrial Swelling Cytochrome c release Respiratory complexes Apoptosis Necrosis MAP kinase JNK, c-jun, AP-1 Neurodegeneration Age-Related Decline Alzheimer s Disease Parkinson s Disease Ischaemia/ Reperfusion
Signal Signal Small G-protein Plasma membrane MAPKKKs/ MAPKK MAPK PI3-kinase Ca(II) Pro-survival pathway p38 JNK ERK Akt/PKB ERK: Pro-survival JNK and p38: Pro-death Tau PLA 2 Cytosolic Targets Nuclear Targets CREB c-jun Neuronal Death/Survival
Effects of Flavonoids on Neurons
Morphological Changes Control x. Stress EC + x. Stress MeEC + x. Stress
Flavonoid modulation of Death Signalling Epicatechin/3 MEC attenuate oxldl-induced JNK activation (EC/3 MEC 10 µm, oxldl 12.5 µg/ml) basal active JNK oxldl oxldl EC oxldl 3 MEC 54 46 Band intensity 1.6 1.2 0.8 0.4 *** *** total JNK 0 Control oxldl oxldl/ec oxldl/3 MEC
Flavonoid Activation of Pro-survival Signalling (15 min; 310 K; n=4) 1.2 perk2 44 vehicle 0.1 0.3 1.0 µm perk1/2 Relative Band Intensity 1.0 0.8 0.6 0.4 0.2 perk1 *** *** 42 Total ERK 0.0 vehicle EC 0.1 EC 0.3 EC 1 EC 3 EC 10 µm
Flavonoids mediate CREB Activation Epicatechin [µm] basal 0.1 0.3 1 3 10 pcreb (Ser-133) total CREB Relative band intensity 1.6 1.2 0.8 0.4 0 pcreb (Ser-133) basal (epicatechin 300 nm; 15 min; U126 10 µm, LY294002 35 µm) MEK inhibitor EC 300 nm EC 300 nm U126 PI3K inhibitor EC 300 nm LY294002 EC 300 nm LY294003 U126
Possible route to EC-induced CREB phosphorylation U126 ERK EC PI3K LY294002 CREB Neuronal Survival
Similarity between flavonoids and kinase inhibitors MEK Inhibitor H PI3 Kinase Inhibitor H 2 N Quercetin N PD98059 LY294002 H Epicatechin
Effects of a blueberry-rich diet on age-associated decline in cognitive function in rats
Study design Animal groups Young (5-6 month) Control diet ld (22-24 month) Control diet + 2% freeze-dried blueberries ld (22-24 month) Control diet
40 35 30 25 20 15 10 5 0 Results Daily Food Intake Intake (g) 1 4 7 10 13 16 19 22 25 28 31 34 37 40 43 46 49 52 55 58 61 64 67 70 73 76 79 YUNG LD SUPP
Weights 650 600 550 Weight (g) 500 450 400 350 300 0 1 2 3 4 5 6 7 8 9 10 11 12 Week YUNG LD SUPP
Measurement of spatial learning and memory T-maze alternation task Baseline and 3, 6, 9, 12 weeks Animals have been trained for 5 weeks to find food placed in opposite arms.
Test phase I Supplement Diet animal allowed to eat one treat Training phase (5 weeks) mins Spatial learning Alternate phase II Number of correct choices + Time to chose measured Test phase II
Performance on T-maze alteration task Correct Choices (All trials) 8 * * * * Number correct (out of 8) 6 4 2 0 Baseline 3 weeks 6 weeks 9 weeks 12 weeks Young ld Blueberry
Time Taken to Choice (All trials) 140 120 Time (seconds) 100 80 60 40 *** *** *** *** 20 0 Baseline 3 weeks 6 weeks 9 weeks 12 weeks Young ld Supplement
ngoing work: Investigate the mechanism underlying the beneficial effect of blueberries on cognitive decline At the end of feeding period Sacrifice of animals Hippocampal tissue CoronaL slices Total/active CREB AKT,ERK, PKA BDNF DNA Microarray Gene expression Immunohistochemistry p-creb in hippocampal CA1 Lipofuscin
CREB in the Brain Transcription factor CREB is expressed in numerous tissues but plays a large regulatory role in the nervous system. CREB is believed to play a key role in promoting neuronal survival, proliferation, neurite outgrowth and neuronal differentiation in certain neuronal populations. Additionally, CREB signaling is involved in learning and memory in several organisms.
CREB, BDNF and Memory
Hippocampal changes in CREB C C ld ld B B Control ld Blueberry 380,000 360,000 340,000 320,000 300,000 * * 280,000 260,000 * 240,000 220,000 200,000 CREB 1 CREB 2
Hippocampal changes in pro-bdnf probdnf C C ld ld B B Relative Band Density 1.5 1.2 0.9 *** All neurotrophins are synthesized as preproneurotrophin precursors Proneurotrophin precursors also mediate biological functions 0.6 0.3 0.0 a Young ld ld + BB Polymorphism that replaces valine for methionine at position 66 of the pro domain, is associated with memory defects and abnormal hippocampal function in humans
Hippocampal changes in ERK 450,000 400,000 350,000 Control Young Control ld ld Blueberry 300,000 250,000 200,000 p-erk 1 p-erk 2 400,000 Control Young Control ld ld Blueberry 350,000 300,000 Total ERK 1 Total ERK 2
Hippocampal changes in Akt 300000 Control ld Blueberry 250000 200000 150000 100000 50000 0 pakt Total AKT
PI3K Normal Learning Akt ERK + + CREB + Ca(II)/Calmodulin Neuronal Survival BDNF PKA Anthocyanins Memory/ LTP
Summary Ageing and neurodegeneration are linked with oxidative processes in the brain. The effects of dietary flavonoids/metabolites are seemingly independent of their antioxidant potential. Flavonoids appear to induce cellular effects via specific interactions within cell signalling cascades such as the MAP kinase pathway. The beneficial effects of flavonoid-rich foods on the reversal of the age-associated cognitive decline might be mediated through modifications of CREB and CREB-dependant gene expression cascades
Dr. David Vauzour Dr. Katerina Vafeiadou Dr Manal Abd El Mohsen Acknowledgements Eva Hernandez Susie Pollard Xenofon Tzounis Vanessa Clarke Pauline How BBSRC and MRC