Molecular Nutrition Group School of Chemistry Food and

Molecular Nutrition Group School of Chemistry, Food and Pharmacy Flavonoids and Brain Health: Multiple effects underpinned by common mechanisms Dr. Jeremy P E Spencer © University of Reading 2008 www. reading. ac. uk

Ageing and Incidence of Neurodegenerative Diseases Age (years) Prevalence of PD (%) 35 -59 0. 2 0. 07 60 -69 0. 3 0. 18 70 -79 Life Expectancy is increasing Prevalence of AD (%) 3. 2 1. 72 80 -89 10. 8 6. 2 Increased health care costs and general demand on the NHS. • Reduced quality of life for the elderly population.

Diet and brain function Human Studies Molecular Mechanisms? Animal Investigations Flavonoids as neuroprotective agents Flavonoids, in particular flavanols, influence neural activity, measured with f. MRI ¯ Francis et al (2006) J. Cardiovasc. Pharmacol. ¯ Fisher et al (2006) J. Cardiovasc. Pharmacol. ¯ Kuriyama et al (2006) AJCN Flavonoid extracts from fruit and vegetables have been reported to attenuate cognitive decline and neuronal dysfunction in animal models and humans. ¯ Joseph et al. (1998, 1999) J. Neurosci. ¯ Unno et al (2004) Exp. Gerontol. ¯ Haque et al (2006) J. Nutr. ¯ Williams et al (2008) Free Radic. Biol. Med.

Flavonoids: source Fruit and vegetables: (All classes) Tea: (Flavanols) Citrus: (Flavanone) Red wine: (Flavanol, Flavonols) Cocoa: (Flavanols and procyanidins) Berries: (Anthocyanins)

Flavonoids: structure R 1 R 2 HO O R 3 R 1 OH OH OH HO Flavanol O O R 2 O R 4 R 5 + Isoflavone R 2 OH OH R 1 Anthocyanin R 1 R 2 HO O R 3 O HO OH OH O Flavonol OH O Flavanone R 3

Plant-derived flavonoids and brain function

Effects of a Blueberry-rich diet on Spatial Working Memory Correct Choices (All trials) 8 Number correct (out of 8) * * 9 weeks 12 weeks 6 4 2 0 Baseline 3 weeks Young Williams et al: FRBM, 2008 6 weeks Old Blueberry

Effects of flavonoid supplementation on spatial memory in older animals

Human intervention Cognitive tests Blood sample Day 1 Cognitive tests Test Drink Blood sample Day 2 Urine collected 09. 00 - 13. 00 hours Urine collected 13. 00 - 17. 00 hours Urine collected 17. 00 - 09. 00 hours the next day Cognitive tests Placebo Blood sample Day 3 Urine collected 09. 00 - 13. 00 hours Urine collected 13. 00 - 17. 00 hours Urine collected 17. 00 - 09. 00 hours the next day

Cognitive Tests • Memory: Working memory: Serial Sevens task Explicit and implicit long-term memory: Immediate cued word recall and word-stem completion, respectively Spatial memory: Brooks grid, computerised 3 D maze Visual memory: Face Recognition Test • Executive Function: Computerised Stroop Test, Go-No. Go task • Mood: Visual Analogue Scales. • Motor skill: Static balance and Dynamic balance

Improvements in Human Executive Function (Attention) Go-No. Go task: measures Executive function/attention Subjects : 18 -30 yrs Subjects: 60 -75 yrs 60 56 56 * 52 48 Flavonoid 44 Placebo 40 Mean no. of correctly detected targets 60 Flavonoid Placebo 52 * 48 44 40 36 36 Pre-drink 1 h post 5 h post Sustained ability to correctly detect target stimuli following flavonoid supplementation compared to the placebo (* p < 0. 05; n=14).

How are these effects mediated?

Proposed mechanisms underlying the neuroprotective effects of flavonoids Antioxidant effects Modulation of Neuronal and glial signalling Modulation of Receptor Function Influences on gene expression Modulation of Membrane Fluidity Inhibitors of Neuroinflammation

Biotransformation of flavonoids Oral Ingestion of flavonoid Neurons glia Oligomers cleaved Stomach Oligomeric Flavonoids cells Blood-brain barrier Monomeric units jejunum O-methylated A-ring glucuronides O-methylated glucuronides Small Intestine O-methylated ileum Portal vein Further metabolism Sulphates glucuronides Liver aglycone glucuronides Colon Kidney Flavonoid Phenolic acids Gut microflora Renal excretion of glucuronides Urine Spencer , et al: Antiox Redox Signal, 2001; J Nutr, 2003; Biochem J, 2003; ABB, 2004; Brit. J. Nutr. 2008

Major Flavonoid Metabolites Epicatechin 3’-O-methyl-epicatechin 0. 1 -3 0. 9 -5 Plasma concentration m. M Epicatechin-7 -b-Dglucuronide 0. 9 -25 0. 1 -8 Epicatechin-7 -sulphate

Brain Uptake of Flavonoids Abd el Mohsen et al: FRBM, 2002; Free Radic Res. , 2004 ; Br J Nutr. , 2006

Inhibition of Neuronal Injury by Flavonoids Control Ox. Stress (steady-state peroxide) 120 *** % MTT reduction 100 *** 80 60 40 EC + Ox. Stress Me-EC + Ox. Stress 20 0 Control Vehicle EC Me-EC Ox. Stress Spencer et al: Biochem J, 2001; FRBM, 2004 EC-Gluc

Neurotransmitter Receptor Tyrosine kinase PKC PI 3 -kinase MAP kinase cascades JNK p 38 ERK 1/2 Akt/PKB c-jun CREB Neuronal Apoptosis Neuronal survival and Plasticity Brain Ageing Increases in cognition performance and memory Williams et al: FRBM, 2004 Spencer et al: Genes & Nutr, 2007; Brit J Nutr, 2008; Chem Soc Rev, 2009

Flavonoid Attenuation of Death Signalling active JNK basal Flavonoid: 0. 3 m. M OS: Peroxide: 50 m. M H 2 O 2 EC 54 46 1. 6 *** total JNK Band intensity 1. 2 0. 8 *** 0. 4 0 basal Spencer et al: Biochem J, 2001; FRBM, 2004; Schroeter et al: Biochem J, 2001 H 2 O 2 3’MEC H 2 O 2 3’MEC

Flavonoid Activation of Pro-survival Signalling vehicle 1. 2 Relative Band Intensity 1. 0 0. 1 0. 3 1. 0 44 p. ERK 2 m. M p. ERK 1/2 42 p. ERK 1 Total ERK *** 0. 8 (15 min; 310 K; n=4) 0. 6 0. 4 0. 2 0. 0 vehicle EC 0. 1 EC 0. 3 EC 1 EC 3 EC 10 m. M

Flavonoids mediate CREB Activation Epicatechin [m. M] basal 0. 1 0. 3 10 p. CREB (Ser-133) total CREB Relative band intensity 1. 6 Epicatechin: 300 n. M; 15 min; O 126: 10 m. M; LY 294002: 35 m. M p. CREB (Ser-133) 1. 2 MEK inhibitor 0. 8 PI 3 K inhibitor 0. 4 0 basal EC 300 n. M UO 126 EC 300 n. M LY 294002 LY 294003 UO 126

Similarity between flavonoids and kinase inhibitors PI 3 Kinase Inhibitor MEK Inhibitor Quercetin PD 98059 LY 294002 Epicatechin

Flavonoid interactions with neuronal and glial signalling Activation ROS/RNS Inhibition Scavenging by Flavonoids ASK 1 Microglia/Astrocyte Activation by Flavonoids JNK 1/2 i. NOS Cys. DA DHBT-1 NO • Akt PI 3 K ERK 1/2 MEK 1/2 BAD Bcl-x. L Inhibition by Flavonoids STAT-1 Caspase-9 TNF-a p 38 Caspase-3 CREB CD 23 TNF-a IFN IL-1 b Neuronal Apoptosis Neuron Vafeiadou et al: EMID Drug Targets, 2007; ABB, 2009 Vauzour et al: J Neurochem, 2007; Genes & Nutr, 2008; ABB, 2008

Flavonoid-Induced Signalling in Cancer Prevention Lee et al (2006) FRBM 40, 323 -334 Lee et al: FRBM, 2006 Nguyen et al: FRBM, 2006 Vauzour et al: ABB, 2007

Interaction of Flavonoids with the brains architecture of memory

The Sensory Input to the Hippocampus Rendeiro et al: Genes & Nutr, 2009 26

Newly acquired Sensory information Post-translational modification of proteins Acquisition Hippocampus Short-term memory Memory Loss Rapid Retrieval Consolidation De Novo protein synthesis Training/ Practice Memory Recall Storage Slower Retrieval Long-term memory Cortex Spencer et al: Proceed Nutr Soc, 2006; Chem Soc Rev, 2009

Control of Memory at the Molecular Level ERK 1/2/5 Ca. MK II/IV PKA PKC PKB/Akt CREB m. TOR Neurotrophins i. e. BDNF Arc/Arg 3. 1 B-actin Synapse re-modelling e. NOS NO Translation Efficiency Synaptic plasticity Angiogenesis Neurogenesis Memory and Learning Spencer et al: Chem Soc Rev, 2009

Changes in Hippocampal CREB Y Y O O B B p. CREB 1 (Ser 133) Hippocampus *** CREB 1 p. CREB 1 (Ser 133) Cortex CREB 1 4 Relative Band Intensity 3 p. CREB/ CREB Young 2 a Aged + BB 1 a = p 0. 001 0 Williams et al: FRBM, 2008 Hippocampus Cortex *** = p 0. 001

Hippocampal changes in pro- and mature BDNF Y Y O O B B Pro-BDNF GAPDH Pro-neurotrophin precursors also mediate biological functions *** • Polymorphism that replaces valine for methionine at position 66 of the pro domain, is associated with memory defects and abnormal hippocampal function in humans *** 1. 5 Relative Band Intensity 1. 0 0. 5 a b Pro-BDNF a/b = p 0. 001 *** = p 0. 001 0. 0 Y O B Mature DDNF

Changes in Hippocampal ERK 1/2 CREB PKC PKA Ca. MK Y Y O O B B p. ERK 44 ERK p. ERK 42 ERK 2 *** 2. 0 Relative Band Intensity 1. 5 Young Aged p. ERK/ERK ** 1. 0 0. 5 Aged + BB b a/b = p 0. 001 a *** = p 0. 001 ** = p 0. 01 0. 0 p. ERK 44 p. ERK 42

Hippocampal changes in Akt Y Y O O B B Ca. MKIV (Thr 196) Ca. MKIV p. Akt (Ser 473) Akt *** PKA C 2. 0 Relative Band Intensity 1. 5 Young BDNF Trk. B Aged + BB 1. 0 PI 3 K b Akt 0. 5 a a 0. 0 a/b = p 0. 001 Ca. MKIV (Thr 196) p. Akt (Ser 473) PKA (Thr 197) *** = p 0. 001

Enhancement of Hippocampal Protein Synthesis 1. 2 *** Relative Band Intensity Phospho-m. TOR/ Total m. TOR 1. 0 0. 8 0. 6 0. 4 a 0. 2 BDNF Trk. B 0. 0 m. TOR (Ser 2448) m. TOR (Ser 2481) *** PI 3 K 2. 5 Akt ERK Arc/Arg 3. 1 Band Intensity Young 2. 0 Aged + BB 1. 5 m. TOR Homer 2 1. 0 a 0. 5 0. 0 Arc/Arg 3. 1 NR 4 A 2

Proposed mechanism of action I ‘Glutamate Release’ (1) Presynaptic (2) BDNF AMPA-R NMDA-R Trk. B Postsynaptic BDNF PI 3 K CREB (3) Akt PKC PKA Ca. MK ERK Arc/Arg 3. 1 Spencer et al: Chem Soc Rev, 2009 m. TOR Homer 2 ‘Enhancement of Protein Synthesis’

Proposed mechanism of action II Synapse following LTP ‘Glutamate Release’ Presynaptic ‘Increased Synaptic Receptor Density’ AMPA-R NMDA-R Trk. B Postsynaptic PI 3 K ‘F-actin Expansion’ ‘Dendritic spinal growth’ - mushroom spines ERK Cofilin Arc/Arg 3. 1 ‘Sustained Activation of Arc, m. TOR’ Akt m. TOR Homer 2

Interactions with the architecture of memory and cognition Plant Bioactives Cell Signalling and Gene Expression Neuronal Morphology Increased neuronal communication (synaptic plasticity) Vascular Effects New nerve cell growth (neurogenesis)

Flavonoids improve Peripheral and Cerebral Blood Flow Acute improvements in vascular responsiveness Nitric oxide-dependent Vasodilatation Acute changes in brain blood flow Modulation of vascular signalling and factors linked with neurogenesis Heiss et al. JAMA, 2003 37

Neurogenesis? Neurogenesis 38

The Future: Brain Imaging, morphology and Networks 39

40

Summary • Flavonoid-rich diets are capable of reversing age-related declines in spatial working memory. • 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 flavonoids on the reversal of the ageassociated cognitive decline might be mediated through modifications of CREB and CREB-dependant gene expression

Acknowledgements Dr. Manal Abd El Mohsen Dr. Giulia Corona Dr. Ana Rodriquez-Mateos Dr. Maria-Jose Oruna-Concha Dr. Katerina Vafeiadou Dr. David Vauzour Prof. Judi Ellis Dr. Laurie Butler Dr. Claire Williams Vanessa Collins Georgina Dodd Eva Hernandez Pauline How Susie Jennings Sara Neshatdoust Catarina Rendeiro Caroline Saunders Setarah Tabatabaee Xenofon Tzounis

43