21st US-Japan Cellular and Gene Therapy Conference

Neurodegenerative Diseases: Biology, Cellular and Gene Therapy
FDA White Oak Campus, Building 31, Great Room, 1503 A
Silver Spring, Maryland 20993
Thursday, March 1, 2018
9:00 am – 5:00 pm

Aging and Anti-Aging ofCognitive Functions

Tatsuhiro Hisatsune
Graduate School of Frontier Sciences
The University of Tokyo, Japan

2

開発の背景（認知症を例として）

Rise in the Number of Dementia Patients

Japan
2014

World

4.6 million

46 million

ThirtyYears

SevenYears
2025

2015

7.0 million(Estimation)

https://www.asahi.com/articles/ASK4R7J67K4RUBQU00S.html2050

ThreeTimes

132 million(Estimation)

http://www.worldalzreport2015.org/

http://www.who.int/mental_health/neurology/dementia/dementia_thematicbrief_epidemiology.pdf

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The Course of Healthy Aging, Mild Cognitive Impairment(MCI), and Alzheimer’s Disease (AD)http://www.nia.nih.gov/Alzheimers/Publications/ADProgress2004_2005/

3/30

Cognitive function of elderly people (≥ 65 years-old)
CDR: 0*

CDR: 0.5

CN: Cognitive Normal

MCI: Mild Cognitive Impairment

10%/y

CDR: 1
Dementia

Reversion
Cognitive function (DSM-5)*
Learning & memory

MCI due to AD

AD

Slight Decline

Language

NormalExecutive function

NormalSocial cognition

NormalComplex attention

NormalPerceptual-motor

NormalDefinite
Declinehttp://www.alz.org/jp• CDR: Clinical Dementia Rating• DSM-5: Diagnostic and Statistical Manual of Mental Disorders-5th version

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Neuroinflammation: as a target of regenerative medication for MCI
NeurovasculardamageSenile Plaques

Aging

Toxic Aβ peptide

Neuroinflammation
Defect in Neurovasculardrainage of Aβ peptide

Control of
Neuroinflammation

Glial Activation(Astrocytes, Microglia)
Inflammatory
Cytokines

Normal Neurovascularfunction

Normal Neurogenesis

Tau Phosphorylation

Alteration in neural network

Decline inCognitive Functions

Reversion
Cognitive normal

Memory decline

Dementia

Regenerative medication for the prevention of the onset of Dementia

5/30Strategies to control neuroinflammation
Many trials with:• Nonsteroidal Anti-Inflammatory Drugs (NSAIDs)
NSAIDs, such as ibuprofen and indomethacin
COX-2 inhibitors, such as rofecoxib and celecoxib• To control inflammatory cytokines
Antagonists, Specific blockers
Antibodies toward inflammatory cytokines• Anti-inflammatory small molecules
Anti-inflammatory drugs, such as ChE inhibitors
Anti-inflammatory natural products
Ref “Can Alzheimer’s Disease Be Prevented?” NIH Publication No. 09-5503, April 20096/30

Age-related declines in Cognitive Functions in Animal and Human
19952000

2005

Mouse

NINDS

Stroke monkey

2015

2020

Aging study

Adult Hippocampal Neurogenesis

NIH

2010

Alzheimer’s Disease Model Mouse

Stroke model

MonkeyAdult
Neurogenesis

StrokeAging study
Mild Cognitive Impairment
Alzheimer’s Disease

Sato et al. (2009)

Neuroinflammation<Glial Activation>

Human
My research history

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Alzheimer’s Disease (AD) Model Mouse【B6C3-Tg(APPswe,PSEN1dE9)85Dbo/J】

Wild TypeAD Transgenic

100μm

Senile Plaques (18 month olds: Red, Anti-Aβ staining)

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Control of memory decline and neuroinflammation
by Choline-esterase inhibitor (ChEI)

SalineChEI

Memory performance

Water Maze (Probe test)

AD model mice (App/Psen tg +HFD)

ChEI has:no effect onplaque formation

suppressive effectonneuroinflammation

**

WtTg
Control**

Saline ChEI
AD modelSenile Plaques

Iba1 GFAP

Wild typeMatsuda et al. J Alzheimer`s Disease (2017)

App/Psen tg

SalineChEI

AD model mice (App/Psen tg +HFD)

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ChEI Recovers Adult Neurogenesis in AD model mouse
Wt-ND

Tg-ND

AD-Vehicle

AD-Rivastigmine (ChEI)

GFP

50 µm

5 µm
immature

**

**

**

immature

**

**

**

mature**p<0.01****

matureMatsuda et al. J Alzheimer`s Disease (2017)

totalControl

AD (Tg-HFD)

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Imidazole-di-peptides
Anti-inflammatory natural products from vertebrate muscle

Anserine

Carnosine
Chicken (100g):

Anserine (0.6g)
Carnosine (0.2g)

Ref. Boldyrev et al, Physiol. Rev. (2013)

Salmon (100g):

Anserine (0.5g)

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Carnosine protects neurovascular damage andmemory decline in High Fat Diet-AD model mouse

Sigma C9625

AD model mouse- Carnosine

Senile Plaques

Memory performance test
Contextual Fear Conditioning

Memory performance

Imidazoledipeptide
Carnosine(β-alanyl-histidine)

Control-－－Carnosine

Control AD model mouse

Neurovascular damage
CA3 area

RAGE/CD31－Control－Carnosine

Alzheimer’s Disease (AD) model mouse

Herculano et al. J Alzheimer`s Disease 33: 983-997 (2013)

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WT+Ans

CH3－Purification(>93 %) from Salmon

GFAPADDAPI

AD + Ans

Glial Inflammation

(C)

AD

Kaneko et al. Scientific Reports (2017)

AD+Ans

Merge

4540

Time in Target Quadrant (%)

WT(B)

*3530
2520151050

WTWT+AnsAD AD+Ans(D)

IL-1β in Hippocampus (pg/mg)(A)

Inflammatory cytokine

Imidazoledipeptide
Anserine (Ans)(β-alanyl-methyl histidine)

Memory performance

Anserine protects neuroinflammation andmemory decline in 18 months old AD model mouse

2.5

*

21.5
10.5
0

AD AD+Ans
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Anserine recovers degeneration of brain capillary pericytes
Endothelialcell

Endothelial Cell

(A) WT
Pericyte100%

Pericyte
PDGFR-β(B) AD
28%
Merge
(C) AD+Anserine
60%
Pericyte staining
with anti-PDGFR-β

Kaneko et al. Scientific Reports (2017)14/30

Aging and Anti-Aging of Cognitive Functions in Mouse
1) Alzheimer’s Disease model mouse showed declines incognitive function and glial neuroinflammation.
2) The control of neuroinflammation in AD model mouse leaded
to the improvement of cognitive functions.
3) Cognitive declines in Stroke model mouse were also preventedin (less glial neuroinflammation) P2Y1R knock-out mouse.
Memory performance
Freezing time (%)

ShamStroke (MCAO)

Iba1
WT

P2Y1R KO

Chin et al. J Neuroinflammation (2013)

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Aging and Anti-Aging of Cognitive Functions- Translation from Animal Models to Human clinical trials Species

Behavior
Tests

Brain
Examinations

Mouse

HumanMorris
Water Maze

Wechsler
Memory Scale

Contextual
Fear Conditioning

Clinical Dementia
Rating (CDR)

Brain Tissue
Staining

Brain MRIs
VBM, fMRI
ASL, DTI
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Imidazoledipeptide ameliorates memory decline in elderly people
Elderly People(60-80 years old)

Random Grouping(Active:Placebo=1:1)

Statistics
Test ScheduleTwo-wayrepeated ANOVA(pre and post tests)

Active6 monthImidazoledipeptide

BL: Base Line, FU: Follow Up

6 monthPlacebo

Active vs Placebo
VBM analysis (p < 0.005)

Wechsler Memory ScaleLogical Memory (delayed recall)(Test food from
Nipponham)

Preservation of
prefrontal cortex

FUBL

Carnosine 0.25 g/dAnserine 0.75 g/d

Active GroupBL

Preservation of
episodic verbal memory
P < 0.05*

Placebo GroupFU
Decrease in Inflammatory
cytokine, IL-8, in plasma
P < 0.01*

BLActive(n=42)

Placebo(n=42)

BL: Base Line, FU: Follow UpFU

ActiveBLFUPlacebo

Hisatsune et al. J Alzheimer Dis (2016)17/30

Alteration in network activity (fMRI study)
Resting-state fMRI analysis
PCC: Posterior
Cingulate Cortex

Functional connectivity between
the hippocampus and the PCC

Hippocampus
Spearman’s r = 0.446, P = 0.043

Change (FU-BL) in memory score

Rokicki et al. Frontier Aging Neuroscience (2015)
In collaboration with NCNP, IBIC (Dr. Matsuda)

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Arterial Spin Labeling (ASL):to evaluate brain blood flow
LabeledNo label

Blood flow

RFpulseRF pulse
Label0s

Image
2.35s

time

Image
No label

0s

2.35s

time
19/30

Effect of ACS on the preservation of brain blood flowat the frontal areasp < 0.05(*)

ActivePlacebo

Ding et al. Aging and Disease (2017)

Brain Areas ofBlood flow preservation
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Table 3

ApoE４ as a risk gene for ADJapanese(2313)
E3/E371.8 %

E4/E3： 18.6 %E4/E4： 1.9 %Lindsay et al., JAMA 278(16):1349-1356 (1997)

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Acceleration AD in ApoE4 carrier
AD free (% of total)

10080
60
40
0 ApoE4
20

1 ApoE4
2 ApoE460

Corder et al. Science (1993)

68 70

7480

Age at onset

90

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Volume 9, Number 3; xxx-xx, June 2018
http://dx.doi.org/10.14336/AD.2017.0809

Original Article

Anserine/Carnosine Supplementation Preserves Blood Flow in
the Prefrontal Brain of Elderly People Carrying APOE e4
Qiong Ding1, Kitora Tanigawa1, Jun Kaneko1, Mamoru Totsuka2, Yoshinori Katakura3,
Etsuko Imabayashi4, Hiroshi Matsuda4, Tatsuhiro Hisatsune1*
Department of Integrated Biosciences, Graduate School of Frontier Sciences, The University of Tokyo,
Department of Applied Biochemistry, Graduate School of Agriculture and Life Sciences, The University of Tokyo,
3Graduate School of Systems Life Sciences, Kyushu University, Higashi-ku, Fukuoka, Japan
4Integrative Brain Imaging Center (IBIC), National Center of Neurology and Psychiatry, Tokyo, Japan
12

APOE4+ group

APOE4− group

E4/E4

E4/E3

E4/E2

E3/E3

E3/E2

E2/E2Active

1702300

Placebo2203300

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Effect of ACS on the preservation of the prefrontal tract
in an ApoE4 double carrier
ApoE4/E4
Pre-DTI

Post-DTI
1 year

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Randomized Controlled Trial to assess the effect ofAnserine/Carnosine on the prevention of Alzheimer’s Disease
Participants: Mild Cognitive Impairment (MCI)
1VisitRecruit

Informed Consent○

20 week
Baseline

344 week

8 week

512 week

6○

○

Post
Follow-up observation◎Intervention Period
Medical Examination○Anserine/Carnosine○△ (1 g) / day△

CDR(Clinical Dementia Rating)○○Blood Test○○

Genotyping of ApoE○

MMSE, WMS-LM2
ADAS-cog, GDS-S-J

Under UMIN# R000023455
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Interventional Clinical Study (individuals with MCI)
StableReversion

MCI:

CN: Cognitive Normal
20~30%/y

CDR: 0Recruit MCIConversion

Mild Cognitive Impairment

Post 12w(Follow-up)

Dementia
CDR: 1, 2, 3

CDR: 0.5

54

Active (Total)
Pre 0w(Baseline)

10%/y

27

Active (ApoE4+)

27
Withdrawn

Withdrawn

2225
Active256419

Stable4
Reversion

Placebo

Imidazoledipeptide(Anserine/Carnosine)
0.75g/0.25g per day

Reversion

MCI

Reversion

MCI
StableStableMasuoka et al. Presentation at Neuroscience 2017, Masuoka et al. (in revision)

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CLINICALDEMENTIA RATING(CDR):

00.5

1

2

3

Impairment
None0Memory

Orientation
Judgment &Problem
Solving
Community
Affairs

Home andHobbies

Questionable
0.5

Mild1Moderate2

No memory loss orSlight inconsistentforgetfulness

Consistent slightforgetfulness;
Partialrecollection of events;"benign" forgetfulness

Moderate memory loss;
more marked for recentevents;
defect interfereswith everyday activities

Severe memory loss;
Only highly learned
material retained;
new material
rapidly lost

Fully oriented

Fully oriented except forModerate difficulty with

Severe difficulty with time Oriented to person only
70’s Malerelationships;
usually (ApoE4/E3) BL, FU
disoriented to time, often
to placetime relationships;
slightdifficultywith time (Excerpt):
Memory Questions
for
Informant

Severe memory loss;only fragments remainoriented for place atexamination; may have
Yes
No
1. Does he/she have a problem with his/hermemoryor thinking?
geographicdisorientationelsewhere
UnableSolves everyday problems Slight impairment inModerate difficulty in
Severely impaired in
Yes to makeNo
1a. If yes, is this asolving
consistentproblem (ashandlingopposedto inconsistent)?
judgments or solve
problems,problems,handling problems,& handles business &problems
similarities, ands imilarities, andsimilarities, andfinancial affairs well;differences; socialdifferences; social
judgment good in relation differences
RarelyUsually
Can he/she recall recent events?
Sometimes
judgment usually
judgment usuallyimpaired
to past2.
performance
maintained
Independentfunctionat remember
Slight impairment
theseUnable
to function
No pretenseof independentSometimes
function outside homeRarely
Usually
3. Canhe/shea short inlistof items(shopping)?
independently at theseusual level in job,
activities
Appears well enough toAppears too ill to be
activities although may
shopping, volunteer andbetaken
tofunctions
taken
Yes to functions
Nostillbeengaged
insome;social4.
groupsHas there been some decline in memory during the past year? outside a family homeoutside a family homeappears normal to casualinspection
relationships

Only interferedsimple choresLife at 5.
home,Ishobbies,
Life at impaired
home, hobbies,
but definite
his/her memory
to such aMild
degreethat it would havepreserved; very restrictedof function
at pre-retirement
and intellectual
and intellectualinterests
withinterests
his/her activities
of dailylife impairmenta few years
ago (orinterests, poorly
well maintainedslightly impairedactivities)? (collateralsources opinion)home; more difficult
chores abandoned; more maintained
andUsually
6. Does he/she completely forget a majorcomplicatedevent (e.g.,hobbiestrip, party,interests abandoned

family wedding) within a few weeks of the event?

Usually in
Requires assistancedressing, hygiene,keeping of personal
Usually
8. Does he/she completely forget important information of the distanteffects
Fully capable of self-care

Personal Care

Severe3

Needs prompting

7. Does he/she forget pertinent details of the major event?

past (e.g., birthdate, wedding date, place of employment)?

http://alzheimer.wustl.edu/adrc2/Images/CDRWorksheet.PDF

No significant function inYes
homeNoSometimes

R Rarely

Sometimes
Requiresmuch help with
Rarelypersonal care; frequent
S incontinence
RaSometimes

Rarely27/30

Reports regarding reversion from MCI
Effect of reversion from mild cognitive impairment (MCI)
on cumulative incidence of dementia

Figure 2

Cumulative incidence of dementia in subjects whodeveloped MCI and did not revert to CN, subjects whodeveloped MCI and reverted to CN, and subjects whowere CN at baseline

Roberts et al. Neurology (2014)

Figure 2 Effect of reversion from mild cognitiveimpairment (MCI) on cumulative incidence ofdementia

Aerts et al. Neurology (2017)28/30

Summary (Human Clinical Studies)
1) Regenerative Medicine to reduce the risk of Dementia can befeasible in Individuals with Mild Cognitive Impairment (MCI).
MCI is a reversible transition stage between cognitive normal(CN) and Alzheimer’s Disease (AD). Reversion, from MCI toCN, reduces the risk of the onset of AD, to about 50%.
2) Neuroinflammation as a downstream cellular responses is atarget to develop regenerative medicine for Dementia.
3) Anserine/Carnosine, natural products from animal meat(imidazoledipeptides), suppressed neuroinflammation-related
neurovascular dysfunction and cognitive declines in elders.
4) In MCI Individuals carrying ApoE4, a major AD risk gene,
Anserine/Carnosine supplementation induced reversion andmay reduce the risk of AD onset. Large scale clinical study toverify its effect is definitely awaited.
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Collaborators and Acknowledgements
National Center of Neurology and PsychiatryIntegrative Brain Imaging Center (IBIC)
Etsuko Imabayashi and Hiroshi Matsuda
Kyusyu University
Graduate School of System Life Sciences
Yoshinori Katakura
The University of Tokyo
Graduate School of Agricultural and Life Sciences
Hideo Satsu, Mamoru Totsuka

Novartis Pharma K.K. Japan
Yasushi Kajii, Kazuhiro Toriyama
Tokai Bussan Co. Ltd., Tokyo
Nobuya Yanai, Kenichiro Sato
R&D Center, Nippon Meat Packers,
Inc. (Nipponham Group)., Tsukuba
Takashi Matsumoto, Mikako SatoThe University of Tokyo, GSFS,Jun Kaneko, Bruno Herculano, Akiko Enya, Kota Enomoto, Takeshi Matsuda,Qiong Ding, Kitora Tanigawa, Marie Hori, Risa Fujinaga, Nobutaka Masuoka
Supported by:
The Ministry of Agriculture,
Forestry and Fisheries (MAFF)
MEXT/JSPS KAKENHI Grant
Number JP25115004
JST/CREST, Sentan Program

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