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© University of Reading 2008 www.reading.ac.uk
Molecular Nutrition Group
School of Chemistry, Food and Pharmacy
10 April 2023
Flavonoids and Brain Health: Multiple effects underpinned by common mechanisms
Dr. Jeremy P E Spencer
10.880-89
3.270-79
0.360-69
0.235-59
Prevalenceof AD (%)
Age (years)
6.2
1.72
0.18
0.07
Prevalenceof PD (%)
1900 1920 1940 1960 1980 2000 2020
40
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: source
Fruit 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
OH
R1
R2
O
O
HO
OH
OH
R1
R2
R3
OHO
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
corr
ect
(out
of
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
of C
orre
ct C
hoic
es (o
ut o
f 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)
Mean n
o.
of
corr
ect
ly
dete
cted t
arg
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
Flavonoid
Placebo
*
Go-NoGo task: measures Executive function/attention
Pre-drink 1h post 5h post
Flavonoid
Placebo
*
36
40
44
48
52
56
60
Pre-drink 1h post 5h postM
ean n
o.
of
corr
ect
ly
dete
cted t
arg
ets
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 flavonoids
Oral 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 glucuronides
O-methylatedglucuronides
O-methylated
Sulphates
Furthermetabolism
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
OCH3
OH
OHO
OH
OH
Epicatechin-7-sulphate
OH
OH
OO
OH
OH
SO
O
O
Epicatechin-7-b-D-glucuronide OH
OH
OOH
OH
COOH
OH
OO
OH
OH
Epicatechin OH
OH
OHO
OH
OH0.1-3
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
mo
l/g
bra
in t
issu
e
0
0.04
0.08
0.12
0.16
0.2
Pelargonidin
nm
ol/g
bra
in t
issu
e
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
ed
ucti
on
***
***
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 Apoptosis
CREBc-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
Ban
d in
ten
sit
y
***
0
0.4
0.8
1.2
1.6
Ban
d in
ten
sit
y
0
0.4
0.8
1.2
1.6
Ban
d in
ten
sit
y
0
0.4
0.8
1.2
1.6
Ban
d in
ten
sit
y
basal
54
H2O2
3’MEC
46
active JNK
total JNK
Flavonoid: 0.3 MOS: Peroxide: 50 M
H2O2H2O2
EC
basal H2O2
3’MECH2O2
H2O2
ECSpencer 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
44
42pERK1/2
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 B
an
d I
nte
nsit
y
pERK2
pERK1
***
***
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 nM
UO126
EC 300 nM
LY294002
EC 300 nM
LY294003
UO126
Rela
tive b
an
d in
ten
sit
y
pCREB (Ser-133)
MEK inhibitor
PI3K inhibitor
O
OH
OH
OH
HO
OH
O
O
N
O
LY294002
O
O
OH
H2N
PD98059
Epicatechin
PI3 KinaseInhibitorMEK
Inhibitor O
OH
OH
OH
OH
HO
O
Quercetin
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-9
Caspase-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
Activation
Inhibition 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
Acquisition
Post-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 CREB
pCREB1 (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 0.001
Williams 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 B
Pro-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 44
pERK 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 NR4A2
0.0
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
‘GlutamateRelease’
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
ERK
PI3K
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 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
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