Flavonoids and Brain Health: Multiple effects underpinned by common mechanisms Dr. Jeremy P E Spencer 28 March 2017

Ageing and Incidence of Neurodegenerative Diseases Age (years) Prevalence of AD (%) Prevalence of PD (%) 35-59 0.2 0.07 60-69 0.3 0.18 70-79 3.2 1.72 Life Expectancy is increasing 80-89 10.8 6.2 Increased health care costs and general demand on the NHS. Reduced quality of life for the elderly population.

Animal Investigations neuroprotective agents Diet and brain function Human Studies Animal Investigations Molecular Mechanisms? Flavonoids as neuroprotective agents Flavonoids, in particular flavanols, influence neural activity, measured with fMRI 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 HO O R1 R3 OH R1 O R3 R1 O R2 R4 OH R5 HO O + Isoflavone Flavanol R2 OH OH Anthocyanin R1 R1 R2 R2 HO O HO O R3 R3 OH OH O OH O Flavonol Flavanone

Plant-derived flavonoids and brain function

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

Effects of flavonoid supplementation on spatial memory in older animals

Human intervention Day 1 Day 1 Day 2 Day 2 Day 3 Day 3 Cognitive tests tests Blood sample Day 1 Day 1 Cognitive Cognitive Cognitive tests tests tests Test Drink Blood sample Blood sample Blood sample Day 2 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 Cognitive Cognitive tests tests tests Placebo Blood sample Blood sample Blood sample Day 3 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 3D maze Visual memory: Face Recognition Test Executive Function: Computerised Stroop Test, Go-NoGo task Mood: Visual Analogue Scales. Motor skill: Static balance and Dynamic balance

Improvements in Human Executive Function (Attention) Go-NoGo task: measures Executive function/attention Subjects : 18-30 yrs Subjects: 60-75 yrs 60 60 * Flavonoid 56 56 Placebo 52 52 Mean no. of correctly detected targets * Mean no. of correctly detected targets 48 48 44 Flavonoid 44 Placebo 40 40 36 36 Pre-drink 1h post 5h post Pre-drink 1h post 5h 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 Monomeric units Oligomeric Flavonoids Stomach Small Intestine jejunum ileum Colon Liver Kidney Urine Portal vein Renal excretion of glucuronides Oligomers cleaved cells Blood-brain barrier Neurons glia glucuronides A-ring glucuronides aglycone O-methylated Sulphates Further metabolism Phenolic acids Gut microflora Flavonoid Spencer , et al: Antiox Redox Signal, 2001; J Nutr, 2003; Biochem J, 2003; ABB, 2004; Brit. J. Nutr. 2008

3’-O-methyl-epicatechin Major Flavonoid Metabolites Epicatechin 3’-O-methyl-epicatechin 0.1-3 0.9-25 0.1-8 0.9-5 Plasma concentration mM Epicatechin-7-b-D- glucuronide 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 Ox. Stress (steady-state peroxide) Control % MTT reduction *** 20 40 60 80 100 120 Control Ox. Stress EC Me-EC EC-Gluc Vehicle EC + Ox. Stress Me-EC + Ox. Stress Spencer et al: Biochem J, 2001; FRBM, 2004

Increases in cognition performance and memory Neurotransmitter Receptor PKC PI 3-kinase Tyrosine kinase MAP kinase cascades JNK p38 ERK1/2 Akt/PKB Neuronal survival and Plasticity Neuronal Apoptosis CREB c-jun 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 *** 0.4 0.8 1.2 1.6 Band intensity basal 54 H2O2 3’MEC 46 active JNK total JNK Flavonoid: 0.3 mM OS: Peroxide: 50 mM EC Spencer et al: Biochem J, 2001; FRBM, 2004; Schroeter et al: Biochem J, 2001

Flavonoid Activation of Pro-survival Signalling vehicle 0.1 0.3 1.0 mM 44 42 pERK1/2 Total ERK ( 15 min; 310 K; n=4) 0.0 0.2 0.4 0.6 0.8 1.2 EC 0.1 EC 0.3 EC 1 EC 3 EC 10 Relative Band Intensity pERK2 pERK1 ***

Flavonoids mediate CREB Activation pCREB (Ser-133) total CREB basal 0.1 0.3 1 3 10 Epicatechin [mM] Epicatechin: 300 nM; 15 min; O126: 10 mM; LY294002: 35 mM 1.6 pCREB (Ser-133) 1.2 MEK inhibitor Relative band intensity 0.8 PI3K inhibitor 0.4 basal EC 300 nM EC 300 nM EC 300 nM EC 300 nM UO126 LY294002 LY294003 UO126

Similarity between flavonoids and kinase inhibitors Quercetin PI3 Kinase Inhibitor MEK Inhibitor PD98059 LY294002 Epicatechin

Flavonoid interactions with neuronal and glial signalling IFN IL-1b TNF-a CD23 p38 STAT-1 iNOS NO • Caspase-8 Caspase-9 Caspase-3 Neuronal Apoptosis ROS/RNS Microglia/Astrocyte Neuron ASK1 JNK1/2 BAD Bcl-xL DHBT-1 ERK1/2 Akt MEK1/2 PI3K CREB Activation by Flavonoids Scavenging by Flavonoids Inhibition by Activation Inhibition CysDA 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

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

Control of Memory at the Molecular Level ERK1/2/5 CREB CaMK II/IV PKA PKB/Akt PKC Neurotrophins i.e. BDNF eNOS NO Angiogenesis mTOR Arc/Arg3.1 B-actin Synaptic plasticity Memory and Learning Neurogenesis Synapse re-modelling Translation Efficiency Spencer et al: Chem Soc Rev, 2009

Changes in Hippocampal CREB Y Y O O B B pCREB1 (Ser 133) Hippocampus CREB1 pCREB1 (Ser 133) *** Cortex CREB1 4 Relative Band Intensity pCREB/ CREB 3 Young 2 a Aged Aged + BB 1 a = p  0.001 *** = p  0.001 Hippocampus Cortex Williams et al: FRBM, 2008

Hippocampal changes in pro- and mature BDNF Y Y O O B B Pro-BDNF 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 Mature DDNF Y O B

Changes in Hippocampal ERK1/2 pERK 44 pERK 42 Y O B ERK2 a *** b ** Relative Band Intensity pERK/ERK 0.0 0.5 1.0 1.5 2.0 Aged Young Aged + BB a/b = p  0.001 *** = p  0.001 ** = p  0.01 CREB PKC PKA CaMK ERK

Hippocampal changes in Akt pAkt (Ser 473) Akt PKA C CaMKIV (Thr 196) CaMKIV Y O B pAkt (Ser 473) (Thr 196) PKA (Thr 197) *** b Relative Band Intensity 0.0 0.5 1.0 1.5 2.0 a Aged Young Aged + BB a/b = p  0.001 *** = p  0.001 Hippocampal changes in Akt PI3K TrkB Akt BDNF

Enhancement of Hippocampal Protein Synthesis mTOR (Ser 2448) 0.0 0.2 0.4 0.6 0.8 1.0 1.2 *** a Relative Band Intensity Phospho-mTOR/ Total mTOR (Ser 2481) Arc/Arg3.1 NR4A2 0.5 1.5 2.0 2.5 Aged Young Aged + BB BDNF TrkB PI3K Akt ERK mTOR Arc/Arg3.1 Homer2

Proposed mechanism of action I ERK PKC CREB PI3K BDNF TrkB mTOR Homer2 Postsynaptic NMDA-R PKA CaMK Arc/Arg3.1 Akt ‘Enhancement of Protein Synthesis’ Presynaptic ‘Glutamate Release’ AMPA-R (1) (2) (3) Spencer et al: Chem Soc Rev, 2009

Proposed mechanism of action II Synapse following LTP ‘Glutamate Release’ Presynaptic ‘Increased Synaptic Receptor Density’ AMPA-R NMDA-R TrkB Postsynaptic PI3K ERK ‘F-actin Expansion’ Akt Cofilin Arc/Arg3.1 ‘Sustained Activation of Arc, mTOR’ mTOR Homer2 ‘Dendritic spinal growth’ - mushroom spines

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

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

Neurogenesis? Neurogenesis

The Future: Brain Imaging, morphology and Networks

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 age-associated 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