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

2. 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.

3. 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.

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

5. Flavonoids: structureHO 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

6. Plant-derived flavonoids and
brain function

7. 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

8. Effects of flavonoid supplementation on spatial memory in older animals

9. 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 hours
Urine collected hours
Urine collected hours the next day
Cognitive
Cognitive
Cognitive
tests
tests
tests
Placebo
Blood sample
Blood sample
Blood sample
Day 3
Day 3
Urine collected hours
Urine collected hours
Urine collected hours the next day

10. 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

11. Improvements in Human Executive Function (Attention)
Go-NoGo task: measures Executive function/attention
Subjects : yrs
Subjects: 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).

12. How are these effects mediated?

13. 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

14. 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

15. 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

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

17. 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

18. 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

19. 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

20. 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
***

21. 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

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

23. 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

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

25. Interaction of Flavonoids with the brains architecture of memory

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

27. 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

28. 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

29. Changes in Hippocampal CREBY 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

30. Hippocampal changes in pro- and mature BDNFY 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

31. 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

32. 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

33. 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

34. 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

35. 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

36. 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)

37. 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

38. Neurogenesis?
Neurogenesis

39. The Future:
Brain Imaging, morphology and Networks

41. 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

42. 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