BEHAVIORAL AND SIGNALING EFFECTS OF BERRY FRUIT - BARBARA SHUKITT-HALE Neuroscience Laboratory
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BEHAVIORAL AND SIGNALING
EFFECTS OF BERRY FRUIT
BARBARA SHUKITT-HALE
Neuroscience Laboratory
USDA Human Nutrition Research Center on
Aging, Tufts University
Boston, Massachusetts
WhatIsAging? • Agingresultsfromacomplexsetofprocessesthat rendersthebodylessabletoactivateprotective mechanismstocounteractstressors thatcandamagethe bodyleadingtoaprogressivedysregulation with advancingage. • Intheneuroscienceworld,agingischaracterizedby progressivelossesinneuronalfunction accompaniedby behavioraldeclines(decreasesinmotorand cognitiveperformance) inbothhumansand animals.
TheFourStagesOfLife 1)YoubelieveinSantaClaus. 2)Youdon'tbelieveinSantaClaus. 3)YouareSantaClaus. 4)YoulooklikeSantaClaus.
Rotarod Test
RatAgeStudy
110 a* Rotarod
88
LatencytoFall(s)
66
44 b b
22
b
b
0
6 12 15 18 22
Age(months)
*Meanswithdifferentlettersaresignificantlydifferentfromeachother(P
Typicalagerelatedlossofspatialmemoryseen
inoldratsinMorrisWaterMazeparadigm
Oldrat YoungRat
ReversalTest:
(1)representsoriginalplatformescapepositionontrainingdays.
(2)representsreversedplatformescapepositiononcurrenttest day.
MorrisWaterMaze
RatAgeStudy
Trial1 Trial2
85
LatencytoPlatform(s)
68
*
51
34
17
0
6 12 15 18 22
Age(months)
*Statistically significant (P
OxidativeStressAnd
InflammationInBrainAging
• Atrestthebrainutilizes20%ofthebody’soxygen.
• Markerstobothinflammationandoxidativestress
increaseasafunctionofageandthebrainbecomes
moresensitivetothem.
• Whencoupledwithgeneticchangesthebrain
becomesmorevulnerabletodiseasessuchas
Alzheimerdisease.
•Wemustfindstrategiestoimprovebehavior, possiblybychangingtheneuronalenvironment byalteringoxidativestressandinflammatory components. • Researchinourlabandothershasshownthat thebehavioraldeficitsseeninagingcanbe retardedorevenreversedbythepolyphenolicsin berryfruits,possiblybyincreasingantioxidant and/orantiinflammatorylevels.
BEHAVIORAL STUDIES
Procedures • Inallofoursupplementationstudiesinaging,the ratswere19monthsofageandsupplementedfor 8weeks,typicallyat2%ofthediet. • Intheoriginalstudy,anAIN93dietwasused,and inallothersanNIH31dietwasused. • Inthepurplegrapejuiceandplumjuicestudies theanimalsdrankthejuice.Forallothers,the fruit,vegetable,ornutextractwasaddedintothe diet.
SummaryOfFindings • Nutritionalinterventionscanforestallagerelated deficitsinlearningandmemory,butdidnotaffect motorbehavior. • Nutritionalinterventionscanreversedeficitsinlearning andmemoryanddeclinesinmotorbehavioral performance. • Thebeneficialeffectsofberryfruitonbehavioral performancewereseenevenwhensuperimposedon analreadywellfortified,healthydiet.
ReversalsofBrainAgingMarkersafter
PhytochemicalSupplementation
Dietary intervention improved behavior
– Morris water maze performance
– Motor behavioral performance (rod walking, accelerod)
– Motor behavioral learning
– Exploration of a novel environment
Dietary intervention improved neuronal function
– Striatal calcium sequestration
– Cerebellar noradrenergic sensitivity
– Muscarinic receptor sensitivity
Localizationofanthocyanins inthecortex
&hippocampuswascorrelatedwithrat
testperformance.Rotarod
60
LATENCYTOFALL(sec)
50
40
30
20
10
0
CONTROL BLUEBERRY STRAWBERRY
DIETRotarod Performance ShukittHaleetal.Nutr.Neurosci.2009;12:135140.
RASPBERRY STUDY
INCLINED SCREEN
500
LATENCY TO FALL (sec)
b
400
300 a
200
100
0
CONTROL RASPBERRY
DIETROD WALKING
12
b
LATENCY TO FALL (sec)
9 a,b
a
6
3
0
0% 10% 50%
GRAPE JUICE CONCENTRATION
ShukittHaleetal.Nutr.2006;22:295302.GRAPE JUICE STUDY - MWM
TRIAL 1 vs TRIAL 2, DAYS 1-4
LATENCY TO PLATFORM (sec)
60
TRIAL 1
TRIAL 2
48
36
*
24
12
0
0% 10% 50%
GRAPE JUICE CONCENTRATION
ShukittHaleetal.Nutr.2006;22:295302.MORRISWATERMAZE
TRIAL1vs TRIAL2,DAYS34
60
TRIAL1 TRIAL2
LATENCYTOPLATFORM(sec)
48
*
36
*
24
12
0
CONTROL BLUEBERRY STRAWBERRY
DIET
*=differentthanTrial1It’snotjustfruitsandvegetables
thataregoodforyourbrain
WALNUTS • Polyphenols
– twiceasmuchasalmonds,
peanuts,hazlenuts1
– syringic acid,juglone,ellagic
acid,proanthocyanidins2
• PUFAs,Melatonin,folate,
gammatocopherol3
(
1Macfarlane,B.J.1988AmJClin Nutr;47:270274
2Colaric M.2005.JAgricFoodChem;53:63906396
3USDANationalNutrientDatabase,2005WalnutStudies
TRIAL1vs TRIAL2,DAYS3 4
65
LATENCYTOPLATFORM(sec)
TRIAL1 TRIAL2 #
52
39
* *
26 *
13
0
CONTROL 2%WALNUT 6%WALNUT 9%WALNUT
Willisetal.Brit.J. Nutr.2009;101:114044.Neurological
Benefit
Memory Motor
Blackberries X X
Black currant X
Blueberries X X
Concord grape juice X X
Cranberries X X
Plum Juice X
Raspberries X
Spinach X
Strawberries X X
Walnuts X X
Coffee X XMECHANISMS
PossibleMechanismsintheBeneficial
EffectsofthePolyphenolics
• Functionalantioxidant/antiinflammatoryeffects
–Decreasedsensitivitytooxidativestress
–Decreasedsensitivitytoneurotoxinsandinflammatoryagents
• Directeffectsonthebrain
–Increasedcalciumclearance
–Membraneeffects
–Alterationsinsignaling
• Decreasedoxidativestress/inflammatorysignaling
• Increasedprotectivesignaling
• Signalinginlearningandmemory
–Increasedneurogenesis
–Increasedarborization
–Increasedautophagy (naturalhousekeeping)A.ProSurvival B.Anti OS/Inflammatory
BFS ROS
BFS
IGF1
ERK
PKCY RAS
AKT DirectROS
PKA Scavenging
GSK3E pp38MAPK
NFKB
-
CREB MITOCHONDRIA
ARC Inactivation
Elk1 ofBAD
+ ROS
ROS
ROS
P53 JNK
Caspases
TNF,II1E,
Bcl 2 COX2,iNOS
CourtesyofJamesJoseph,PhD.Microglial Cells Slightlyactivated Veryactivated Microglial cellsarethebrain’simmunecellsand becomeactivatedinresponseinflammatorychallenges. Thesecellsareinthehippocampusofagedrats.
ActivatedMicrogliainthe
CA3Hippocampal Region
Control 6%Walnut 9%Walnut
Immunohistochemistry forOx6(brown)demonstratesthenumberof
activatedmicrogliaintheCA3regionhippocampusofagedanimals.ActivatedMicroglial intheDentate
Control 6%Walnut 9%Walnut
Dentate
Activatedmicrogliaarealsofoundinthedentategyrus;
animalsthatconsumedawalnutsupplementeddietappear
tohavefewerOx6positivecellsinthedentate.WalnutsDecreasetheNumberofActivated
MicrogliaintheAgedHippocampus
10
Control
9 *
Ox6positivecellspersection
8 6%Walnut
7 9%Walnut
*
6
5
**
4
3 **
2
1
0
Dentate CA3 CA1 Hippocampus
QuantificationofOx6positivemicrogliarevealsthatdietarywalnutsupplementation is
associatedwithsignificantlyfeweractivatedmicrogliainthedentate,CA3,andthe
hippocampusasawhole.(mean± SEM;n=5control,7for6%walnut,and6for9%walnut;
*pKainic AcidDietStudy
MorrisWaterMazeDay3Probe
50 Ringer’s Kainic acid b
b
LatencytoCrossthePlatform
40
30 a,b
a
20
a
10 a
0
Control Blueberry Piroxicam
Diet
a=KAdifferentthanmatcheddietRinger’sgroup;b=differentthancontrolRinger’sgroup;
ShukittHaleetal.Nutr.Neurosci. 2008;11:17282.TheEffectsofKainic Acid
onOX6Activation
8 Ringer’s Kainic acid
a,b
a,b
6
OX6Activation
a,b,c
4
2
0
Control Blueberry Piroxicam
Diet
a=KAdifferentthanmatcheddietRinger’sgroup;b=differentthancontrolRinger’sgroup;
c=differentthancontrolKAgroup.InflammatoryandProtectiveSignalsin
HippocampusinControl,BBorPXMTreated
AnimalsGivenKAorVehicle
14
Hipp.RelativemRNAExpression
12
10
8
6
4
2
0
Control KA BB BB+KA PX PX+KA
ShukittHaleetal.Nutr.Neurosci. 2008;11:17282.1.40
IGF1
1.12 c
DensityUnits
0.84 b
0.56 a
0.28
0.00
CONTROL BLUEBERRY STRAWBERRY
Meansnotsharingacommonletteraresignificantly
DIET
differentfromeachother(pErrorsInTheRadialArmWaterMaze
40
CONTROL
Mean#ofMemoryErrors
32
BLUEBERRY
pCorrelationsBetweenNeurogenesis,IGF1,and
RAWMDay4PerformanceInOldControl
FedandBlueberrySupplementedRats
• Totalerrors(anywrongarmentrance)andneurogenesis
r=0.677,pTherefore,polyphenolicscanhavedirecteffects
onthebrain,i.e.,notmediatedthroughoxidative
stressorinflammatorypathways,bydirectly
increasingsignalingandneurogenesis.
Additionally,berryfruitsmayprotectneuronsby
activatingproteinkinase signalingcascades.Inadditiontoneutralizingfreeradicals,berries
maypreventthedeleteriouseffectsof
inflammationandoxidativestressbyblockingthe
stresssignalswhosegenerationresultsin
downstreamproductionofcytokines.CELL STUDIES
Effects of Walnuts on LPS-Activated BV2
Microglia: NITRIC OXIDE RELEASE
NITRITE
Walnut Oil
60
50
*
μM Nitrite
40
30
*
20 *
*
10
0
C LPS 0.00001 0.0001 0.001 0.01 0.1 1
Walnut extract (mg/ml)Effects of Walnuts on LPS-Activated BV2
Microglia: iNOS EXPRESSION
iNOS
Walnut Oil
120000
96000
O.D. Units
* *
72000 *
48000
*
24000
0
C LPS 0.01 0.1 1
Walnut Extract (mg/ml)Effects of Walnuts on LPS-Activated BV2
Microglia: TNF EXPRESSION
TNF
Walnut Oil
60
50
* *
pg/mg TNFD
40 * *
30
20
10
0
Control LPS 0.01 0.1 1
Walnut Extract (mg/ml)Effects of Walnuts on LPS-Activated
Hippocampal Neurons: ROS ACTIVATION
ROS
PERCENTAGE OF VIABLE CELLS
Control Walnut Oil LPS
1600 d
d
1280
960 bd
bd
640
b
320
0
Control WO .1 WO .2 WO .5 WO 1
Control LPSp-MAPK
Control Aß DA
1250
a,b
Average Fluorescence Intensity
a
1000
a
750
b
500
250
0
Cont BB
From:J.A.Josephetal./JournalofAlzheimers Disease2007,11(4),433446.PKC Gamma
Control Aß DA
a
Average Fluorescence Intensity
1300 a
1040
780 b
b
520
260
0
Cont BB
From:J.A.Josephetal./JournalofAlzheimers Disease2007,11(4),433446.p- PKC
Control Aß DA
a,b
Average Fluorescence Intensity
1450
b
1160
a a
870
a
580
290
0
Cont BB
From:J.A.Josephetal./JournalofAlzheimers Disease2007,11(4),433446.p-p38
Control Aß DA
a
Average Fluorescence Intensity 800
640
a
480
a,b
320
b
160
0
Cont BBNFB
Control Aß DA
a a
Average Fluorescence Intensity
750
600 a
a
450
b
300
150
0
Cont BB
From:J.A.Josephetal./JournalofAlzheimersDisease2007,11(4),433446.p-JNK
Control Aß DA
1000
Average Fluorescence Intensity
a
800
600 a a,b
400
b
200
0
Cont BB
From:J.A.Josephetal./JournalofAlzheimersDisease2007,11(4),433446.p-CREB
Control Aß DA
700
a
Average Fluorescence Intensity
560
a
420 b
b
280 a
140
0
Cont BB
From:J.A.Josephetal./JournalofAlzheimersDisease2007,11(4),433446.From:G.J.Breweretal./JournalofNutritionalBiochemistry21(2010)991–998
From:G.J.Breweretal./JournalofNutritionalBiochemistry21(2010)991–998
Conclusions • Eatingberries,grapesand walnutsmayhelppreventor reverseagerelateddeclinesin cognitionandbrain functioning. • Thismaybeduetothe activityofpolyphenolsand othercomponentssuch asomegafattyacids. • Thesecomponentsmayactto inhibitorreduceinflammation andoxidativestressand/or enhanceprotective mechanisms. • Polyphenolicscanhavedirect effectsonthebrainbydirectly increasingneuronalsignaling cascadesandneurogenesis.
Therefore,itappearsthatthe
significanteffectsofpolyphenolics
onmotorandcognitivebehavioraredue
toamultiplicityofdirectandindirect
actions,theformerinvolvingeffects
onneuronalcommunicationandthe
latterinvolvingantioxidantand
antiinflammatoryactivity.In loving memory of Dr. Jim Joseph
NeuroscienceLab NeuroscienceLab(past)
B.ShukittHale,Ph.D. J.A.Joseph,Ph.D.
D.Bielinski,Ph.D G.Casadesus,Ph.D.
D.Fisher K.Youdim,Ph.D.
A.N.Carey,Ph.D. F.Lau,Ph.D.
S.M.Poulose,Ph.D. L.Willis,Ph.D.
S.Gomes A.Martin,MD,Ph.D.
M.G.Miller C.AndresLacueva,Ph.D.
I.CantutiCastelvetri,Ph.D.
R.Galli Ph.D.
A.Neuman,K.Luskin
L.Simon,H.Stellwagen
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