© University of Reading 2008 www.reading.ac.uk

Molecular Nutrition GroupSchool of Chemistry, Food and Pharmacy

4 May 2023

Flavonoids and Brain Health: Multiple effects underpinned by common mechanismsDr. Jeremy P E Spencer

10.880-89

3.270-790.360-69

0.235-59

Prevalenceof AD (%)

Age (years)

6.2

1.72

0.18

0.07

Prevalenceof PD (%)

1900 1920 1940 1960 1980 2000 202040

45

50

55

60

65

70

75

80

85

Life

Exp

ecta

ncy

at B

irth

Year

Men Women

Increased health care costs and general demand on the NHS.

• Reduced quality of life for the elderly population.

Ageing and Incidence of Neurodegenerative Diseases

Life Expectancyis increasing

Diet and brain function

Molecular Mechanisms?

Flavonoids asneuroprotective agents

Animal Investigations

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.

Human Studies

Flavonoids, in particular flavanols, influenceneural activity, measured with fMRI

Francis et al (2006) J. Cardiovasc. Pharmacol. Fisher et al (2006) J. Cardiovasc. Pharmacol. Kuriyama et al (2006) AJCN

Flavonoids: sourceFruit and vegetables:(All classes)

Red wine:(Flavanol, Flavonols)

Cocoa:(Flavanols and procyanidins)

Tea:(Flavanols)

Berries:(Anthocyanins)

Citrus:(Flavanone)

Flavonoids: structure

Flavanol

Flavonol

Isoflavone

Flavanone

Anthocyanin

R2

HO O

R1

R3

OHOH

O

O

R1

R2 R4

R3

R5

O+

OH

HO

OH

OHR1

R2

O

O

HO

OHOH

R1

R2

R3OHO

OH O

R1

R2

R3

Plant-derived flavonoids and brain function

Correct Choices (All trials)

0

2

4

6

8

Baseline 3 weeks 6 weeks 9 weeks 12 weeks

Num

ber c

orre

ct (o

ut o

f 8)

Young Old

Effects of a Blueberry-rich diet on Spatial Working Memory

Blueberry

* * * *

Williams et al: FRBM, 2008

2.00

3.00

4.00

5.00

6.00

7.00

8.00

0 10 20 30 40 50

Time on Diet (days)

Num

ber o

f Cor

rect

Cho

ices

(out

of

8)

Placebo

Blueberry

Anthocyanins

Flavanols

Effects of flavonoid supplementation on spatial memory in older animals

Human intervention

Cognitivetests

Test Drink

Placebo

Day 1

Day 2

Day 3

Blood sample

Blood sample Blood sample Blood sample

Blood sample Blood sample

Urine collected 09.00 - 13.00 hoursUrine collected 13.00 - 17.00 hoursUrine collected 17.00 - 09.00 hours the next day

Urine collected 09.00 - 13.00 hoursUrine collected 13.00 - 17.00 hoursUrine collected 17.00 - 09.00 hours the next day

Blood sample

Cognitivetests

Cognitivetests

Day 1

Day 2

Day 3

Cognitivetests

Cognitivetests

Cognitivetests

Cognitivetests

Cognitivetests

• 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

Cognitive Tests

Improvements in Human Executive Function (Attention)

Mea

n no

. of c

orre

ctly

d

etec

ted

targ

ets

Sustained ability to correctly detect target stimuli following flavonoid supplementation compared to the placebo (* p < 0.05; n=14).

Subjects : 18-30 yrs Subjects: 60-75 yrs

36

40

44

48

52

56

60

FlavonoidPlacebo

*

Go-NoGo task: measures Executive function/attention

Pre-drink 1h post 5h post

FlavonoidPlacebo

*

36

40

44

48

52

56

60

Pre-drink 1h post 5h postM

ean

no. o

f cor

rect

ly

det

ecte

d ta

rget

s

How are these effects mediated?

Proposed mechanisms underlying the neuroprotective effects of flavonoids

Antioxidanteffects

Modulation ofReceptor Function

Modulation ofNeuronal and glial

signalling

Modulation ofMembrane Fluidity

Influences on gene expression

Inhibitors of Neuroinflammation

Biotransformation of flavonoidsOral Ingestionof flavonoid

Monomeric

units

OligomericFlavonoids

Stomach

Small Intestine

jejunum

ileum

Colon

Liver

Kidney

Urine

Portalvein

Renal excretionof glucuronides

Oligomerscleaved

cells

Blood-brainbarrier

Neuronsglia

glucuronides

A-ring glucuronides

aglycone

O-methylated glucuronidesO-methylated

glucuronides

O-methylated

Sulphates

Furthermetabolism

Phenolic acidsGut microflora

Flavonoid

Spencer , et al: Antiox Redox Signal, 2001; J Nutr, 2003; Biochem J, 2003; ABB, 2004; Brit. J. Nutr. 2008

3’-O-methyl-epicatechinOCH3

OH

OHO

OHOH

Epicatechin-7-sulphateOH

OH

OO

OHOH

SOO

O

Epicatechin-7-b-D-glucuronide OH

OH

OOH

OH

COOH

OH

OO

OHOH

Epicatechin OHOH

OHO

OHOH 0.1-3

0.9-25 0.1-8

0.9-5

Plasmaconcentration

M

Major Flavonoid Metabolites

Brain Uptake of Flavonoids

0

0.5

1

1.5

2

2.5

3

TotalGSH

VitaminE

VitaminC

mol

/g b

rain

tiss

ue

0

0.04

0.08

0.12

0.16

0.2

Pelargonidin

nmol

/g b

rain

tiss

ue

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

Inhibition of Neuronal Injury by Flavonoids

ControlOx. Stress (steady-state peroxide)

EC + Ox. Stress Me-EC + Ox. Stress

% M

TT r

educ

tion

***

***

0

20

40

60

80

100

120

Control

Ox. Stress

EC Me-EC EC-GlucVehicle

Spencer et al: Biochem J, 2001; FRBM, 2004

Neurotransmitter Receptor

PKC PI 3-kinaseTyrosine kinase

MAP kinase cascades

JNK p38 ERK1/2 Akt/PKB

Neuronal survival and PlasticityNeuronal ApoptosisCREBc-jun

Brain Ageing Increases in cognition performance and memory

Williams et al: FRBM, 2004Spencer et al: Genes & Nutr, 2007; Brit J Nutr, 2008; Chem Soc Rev, 2009

Flavonoid Attenuation of Death Signalling

***

0

0.4

0.8

1.2

1.6

Band

inte

nsit

y

***

0

0.4

0.8

1.2

1.6

Band

inte

nsit

y

0

0.4

0.8

1.2

1.6

Band

inte

nsit

y

0

0.4

0.8

1.2

1.6

Band

inte

nsit

y

basal

54

H2O23’MEC

46

active JNK

total JNK

Flavonoid: 0.3 MOS: Peroxide: 50 M

H2O2H2O2EC

basal H2O23’MEC

H2O2H2O2EC

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 M

4442

pERK1/2

Total ERK

(15 min; 310 K; n=4)

0.0

0.2

0.4

0.6

0.8

1.0

1.2

vehicle EC 0.1 MEC 0.3 EC 1 EC 3 EC 10

Rela

tive

Ban

d In

tens

ity

pERK2pERK1

***

***

Flavonoids mediate CREB Activation

pCREB (Ser-133)

total CREB

basal 0.1 0.3 1 3 10

Epicatechin [M]

Epicatechin: 300 nM; 15 min;O126: 10 M; LY294002: 35 M

0

0.4

0.8

1.2

1.6

basal EC 300 nM EC 300 nMUO126

EC 300 nMLY294002

EC 300 nMLY294003

UO126

Rela

tive

ban

d in

tens

ity pCREB (Ser-133)

MEK inhibitor

PI3K inhibitor

O

OH

OHOH

HO

OH

O

O

NO

LY294002

O

O

OHH2N

PD98059

Epicatechin

PI3 KinaseInhibitorMEK

Inhibitor O

OHOH

OHOH

HO

OQuercetin

Similarity between flavonoids and kinase inhibitors

Flavonoid interactions with neuronal and glial signalling

IFNIL-1

TNF-CD23

p38

STAT-1

iNOS NO •

TNF-Caspase-8

Caspase-9Caspase-3

NeuronalApoptosis

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 byFlavonoids

ActivationInhibition CysDA

Vafeiadou et al: EMID Drug Targets, 2007; ABB, 2009Vauzour et al: J Neurochem, 2007; Genes & Nutr, 2008; ABB, 2008

Lee et al (2006)FRBM 40, 323-334

Flavonoid-Induced Signalling in Cancer Prevention

Lee et al: FRBM, 2006 Nguyen et al: FRBM, 2006Vauzour et al: ABB, 2007

Interaction of Flavonoids with the brains architecture

of memory

26

The Sensory Input to the Hippocampus

Rendeiro et al: Genes & Nutr, 2009

Newly acquiredSensory

information

Short-termmemory

Consolidation

Long-termmemory

MemoryRecall

Slower Retrieval

Rapid Retrieval

Training/Practice

MemoryLoss

AcquisitionPost-translationalmodification ofproteins

De Novo protein synthesis

Hippocampus

Cortex

Storage

Spencer et al: Proceed Nutr Soc, 2006; Chem Soc Rev, 2009

ERK1/2/5

CREB

CaMK II/IV PKA PKB/AktPKC

Neurotrophinsi.e. BDNF

eNOSNO

Angiogenesis

mTOR

Arc/Arg3.1B-actin

Synaptic plasticity

Memory and Learning

Neurogenesis

Synapse re-modelling

TranslationEfficiency

Control of Memory at the Molecular Level

Spencer et al: Chem Soc Rev, 2009

Changes in Hippocampal CREBpCREB1 (Ser 133) CREB1

Y Y O BO B

Hippocampus

CortexpCREB1 (Ser 133) CREB1

0

1

2

3

4

Hippocampus Cortex

a

***

Relative BandIntensity

pCREB/CREB

Aged

Young

Aged + BB

a = p 0.001

*** = p 0.001Williams et al: FRBM, 2008

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

Hippocampal changes in pro- and mature BDNF

Y Y O BO BPro-BDNF

BDNF

GAPDH

***

a

Y O B

RelativeBand

Intensity

0.0

0.5

1.0

1.5 ***

b

Pro-BDNF

Mature DDNFa/b = p 0.001

*** = p 0.001

Changes in Hippocampal ERK1/2

pERK 44pERK 42

Y Y O BO B

ERK2

pERK 44 pERK 42

a

***

b

**

RelativeBand

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

ERKPKC

CREB

PKA CaMK

Hippocampal changes in Akt

pAkt (Ser 473)

Akt

PKA C

CaMKIV (Thr 196)CaMKIV

Y Y O BO B

pAkt (Ser 473)

CaMKIV(Thr 196)

PKA (Thr 197)

***

b

RelativeBand

Intensity

0.0

0.5

1.0

1.5

2.0

a a

Aged

Young

Aged + BB

a/b = p 0.001

*** = p 0.001

PI3K

TrkB

Akt

BDNF

Enhancement of Hippocampal Protein Synthesis

ERK

PI3K

TrkB

mTORHomer2Arc/Arg3.1

Akt

BDNF

mTOR(Ser 2448)

0.0

0.2

0.4

0.6

0.8

1.0

1.2***

a

RelativeBand

Intensity

Phospho-mTOR/Total mTOR

mTOR(Ser 2481)

Arc/Arg3.1 NR4A20.0

0.5

1.0

1.5

2.0

2.5

BandIntensity

***

a

Aged

Young

Aged + BB

ERKPKC

CREB

PI3KBDNF

TrkB

mTORHomer2

Postsynaptic

NMDA-R

PKA CaMK

Arc/Arg3.1

Akt

BDNF

‘Enhancementof Protein Synthesis’

Presynaptic‘Glutamate

Release’

AMPA-R

(1) (2)

(3)

Proposed mechanism of action I

Spencer et al: Chem Soc Rev, 2009

Postsynaptic

Presynaptic

TrkB

NMDA-RAMPA-R

‘Increased Synaptic

Receptor Density’

‘Dendritic spinal growth’

- mushroom spines

Synapse following LTP

ERKPI3K

mTORHomer2

Arc/Arg3.1Akt

‘GlutamateRelease’

‘F-actinExpansion’ Cofilin

‘SustainedActivation of Arc, mTOR’

Proposed mechanism of action II

Interactions with the architecture of memory and cognition

New nerve cell growth (neurogenesis)

Increased neuronal communication(synaptic plasticity)

Plant Bioactives

Cell Signalling and Gene Expression

Vascular EffectsNeuronal Morphology

37

Flavonoids improve Peripheral and Cerebral Blood Flow

Nitric oxide-dependentVasodilatation

Acute changes in brain blood flow

Modulation of vascular signalling and factors linked

with neurogenesis

Acute improvements in vascular responsiveness

Heiss et al. JAMA, 2003

Neurogenesis?

38

Neurogenesis

39

The Future:Brain Imaging, morphology and Networks

40

Summary

• Flavonoid-rich diets are capable of reversing age-related declines in spatial working memory.

• The effects of dietary flavonoids/metabolites areseemingly 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

AcknowledgementsDr. Manal Abd El MohsenDr. Giulia CoronaDr. Ana Rodriquez-MateosDr. Maria-Jose Oruna-ConchaDr. Katerina VafeiadouDr. David Vauzour

Prof. Judi EllisDr. Laurie ButlerDr. Claire Williams

Vanessa CollinsGeorgina DoddEva HernandezPauline HowSusie JenningsSara NeshatdoustCatarina RendeiroCaroline SaundersSetarah TabatabaeeXenofon Tzounis

43