Dietary Flavonoids: Modulators of Brain Ageing?
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1 MLECULAR NUTRITIN GRUP Established 2004 Dietary Flavonoids: Modulators of Brain Ageing? Jeremy P E Spencer School of Food Biosciences University of Reading, UK
2 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
3 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
4 Flavonoids: source Tea: (Flavanols) Fruit and vegetables: (All classes) Citrus: (Flavanone) Red wine: (Flavanol, Flavonols) Cocoa: (Flavanols and procyanidins) Berries: (Anthocyanins)
5 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
6 Ageing and Incidence of Neurodegenerative Diseases Men Women Age (years) Prevalence of AD (%) Prevalence of PD (%) Life Expectancy at Birth Life Expectancy is increasing Year Increased health care costs and general demand on the NHS. Reduced quality of life for the elderly population.
7 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.
8 How Might They Work at the Cellular Level?
9 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
10 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 Colon glucuronides Kidney Flavonoid Phenolic acids Gut microflora Urine Renal excretion of glucuronides
11 Metabolism Reduces Antioxidant Potential Increasing level in the circulation H CH Methylepicatechin Reduction in Antioxidant potential C - Epicatechin 7--β-Dglucuronide H Epicatechin
12 Do Flavonoids Access the Brain?
13 h Conjugated Free Pelargonidin nmol/g tissue Brain Lungs Heart Liver Spleen Kidney Plasma nmol/g or ml h p-hba Conjugated Free Brain Lungs Heart Liver Spleen Kidney Plasma
14 Levels of antioxidants in brain µmol/g brain tissue nmol/g brain tissue Total GSH Vitamin E Vitamin C Pelargonidin 0
15 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
16 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
17 Effects of Flavonoids on Neurons
18 Morphological Changes Control x. Stress EC + x. Stress MeEC + x. Stress
19 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 Band intensity *** *** total JNK 0 Control oxldl oxldl/ec oxldl/3 MEC
20 Flavonoid Activation of Pro-survival Signalling (15 min; 310 K; n=4) 1.2 perk2 44 vehicle µm perk1/2 Relative Band Intensity perk1 *** *** 42 Total ERK 0.0 vehicle EC 0.1 EC 0.3 EC 1 EC 3 EC 10 µm
21 Flavonoids mediate CREB Activation Epicatechin [µm] basal pcreb (Ser-133) total CREB Relative band intensity pcreb (Ser-133) basal (epicatechin 300 nm; 15 min; U µm, LY µm) MEK inhibitor EC 300 nm EC 300 nm U126 PI3K inhibitor EC 300 nm LY EC 300 nm LY U126
22 Possible route to EC-induced CREB phosphorylation U126 ERK EC PI3K LY CREB Neuronal Survival
23 Similarity between flavonoids and kinase inhibitors MEK Inhibitor H PI3 Kinase Inhibitor H 2 N Quercetin N PD98059 LY H Epicatechin
24 Effects of a blueberry-rich diet on age-associated decline in cognitive function in rats
25 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
26 Results Daily Food Intake Intake (g) YUNG LD SUPP
27 Weights Weight (g) Week YUNG LD SUPP
28 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.
29 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
30 Performance on T-maze alteration task Correct Choices (All trials) 8 * * * * Number correct (out of 8) Baseline 3 weeks 6 weeks 9 weeks 12 weeks Young ld Blueberry
31 Time Taken to Choice (All trials) Time (seconds) *** *** *** *** 20 0 Baseline 3 weeks 6 weeks 9 weeks 12 weeks Young ld Supplement
32 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
33 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.
34 CREB, BDNF and Memory
35 Hippocampal changes in CREB C C ld ld B B Control ld Blueberry 380, , , , ,000 * * 280, ,000 * 240, , ,000 CREB 1 CREB 2
36 Hippocampal changes in pro-bdnf probdnf C C ld ld B B Relative Band Density *** All neurotrophins are synthesized as preproneurotrophin precursors Proneurotrophin precursors also mediate biological functions 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
37 Hippocampal changes in ERK 450, , ,000 Control Young Control ld ld Blueberry 300, , ,000 p-erk 1 p-erk 2 400,000 Control Young Control ld ld Blueberry 350, ,000 Total ERK 1 Total ERK 2
38 Hippocampal changes in Akt Control ld Blueberry pakt Total AKT
39 PI3K Normal Learning Akt ERK + + CREB + Ca(II)/Calmodulin Neuronal Survival BDNF PKA Anthocyanins Memory/ LTP
40
41 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
42 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
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