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2014 IBC Summer Internship Program

Alzheimer’s Disease treatment with Chinese herbal extracts in APP/PSEN1 transgenic mice

Supervisor: Rita P. Y. Chen Ph. D.

Institute of Biological Chemistry, Academia Sinica

Presented by: Po-Ting, HoAug. 29, 2014

2 Mo/HuAPPswe HuPSEN1dE9

Increase total Aβ

Aβ aggregation

plaques

Dementia

Neur

on d

amag

e

Aβ deposit

s

Morris Water Maze

ELISA

Behavior test

Aβ quantification

INTR

OD

UCT

ION

locationIHC/ Thioflavin S stain

Vorhees, C. V. 2006. Nature protocolsZhang, C. (2009) Harvard Medical School.

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3rd month

Genotyping Treatments Sacrifice

10th month

Treatments started

Experimental Timeline

My Internship

Period

Morris Water Maze

4

11thGenotyping Treatments Sacrifice

Experimental Timeline

Morris Water MazeTreatments started

3rd month 10th month

5 Chinese Herbal Extracts Treatment

Extract A

Extract B

APP

WT

• Dosage: 250 mg/kg a day by gavage

Mat

eria

l

6

11thGenotyping Treatments Sacrifice

Experimental Timeline

Treatments started

3rd month

Morris Water Maze

10th month

7Morris Water Maze

• Behavior testing: to study spatial learning and memory.

N

E

S

W

100 cm

35 cm

Water + milk

Hidden platform

Visual cues

Mat

eria

l

Vorhees, C. V. 2006. Nature protocols.

Q3

8 Spatial learning test Probe test

Day 1 Day 2 Day 3 Day 5Day 4 Day 6

4 trials a day

swim for 90 sec.

guide for 30 sec.

Grab them to the

platform for 30 sec.

Stay on the platform for

30 sec.

Remove platform

1 trial

Swim for 60 sec.

yes

no

no yes Latency time

Duration and cross number of

Q3Met

hods

Vorhees, C. V. 2006. Nature protocols.

9Re

sult

sSpatial Learning trials

D1 D2 D3 D4 D50.0

20.0

40.0

60.0

80.0

100.0897 994 APP WT

Lat

ency

(sec

)Extract A Extract B

N=9 N=9 N=4 N=10

10Re

sult

sProbe trial

897 994 APP WT0.0

2.0

4.0

6.0

8.0

10.0

Q3 (Cross number)

Cro

ss n

umbe

r (b

outs

)

897 994 APP WT0.0

5.0

10.0

15.0

20.0

25.0

30.0

Q3 (Duration, sec)T

ime

(sec

)

Extract AExtract A

Extract BExtract B

*

* P <0.05 compared with APP mice.

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11thGenotyping Treatments Sacrifice

Experimental Timeline

3rd month

Treatments started

10th month

Morris Water Maze

12 Sacrifice

http://www.zviratka-verunky.estranky.cz/fotoalbum/zvirata/mysi/illustrated_brown_mouse.jpg.-.htmlHatziioannouk T. 2012. Nature Reviews Microbiology 10, 852-867http://www.biologycorner.com/worksheets/rat_head.html

Serum

HeartLungsLiverKidneysspleen

HippocampusCerebral cortexCerebellumBrain StemOlfactory lobes

ELISA

Toxicity test

Met

hods

Brain tissue IHC/ ThS stain

13• The results of probe trial suggested that the treatments with Chinese herbal

extracts A and B might have potential to improve memory deficits.

• Important factors that do matter:

mice should find the platform by memory instead of sights.

a) The proportion of milk and water b) The depth of water

Conc

lusi

onCo

nclu

sion

14

I would like to thank Professor Chen for her expert advice and encouragement throughout this difficult project, as well as Ramus for his brilliance in the lab.

Ackn

owle

dgm

ents

15

Questions?

Zhang, C. (2009) Harvard Medical School.

(APP Intracellular Domain)

B6C3-Tg (APPswe/PSEN1dE9) Double Transgenic Mice

Mo/HuAPPswe

• Swedish mutations amyloid precursor protein

• Elevate the amount of Aβ

• Exon-9-deleted mutant human presenilin 1

• Increase the level of Aβ40 and Aβ42 in the brain

• Both genes are directed to CNS neurons

The Jackson Labortory

Mutant Mouse Regional Resource Centers supported by NIH

Purpose: Make sure there are the presences of transgenes, APP and PSEN, construct in transgenic mice.

Mr. PC 1047 1048 1051 1052 1074

APP377 bps

PSEN608 bps

Genotyping

E

N

S

W

Morris Water Maze

Q2 Q1

Q3 Q4

Treatment NLearning trials

D1 latency (s) D2 latency (s) D3 latency (s) D4 latency (s) D5 latency (s)

Extract A 9 (9M) 76.4 ± 5.3 35.7 ± 9.5 31.7 ± 5.8 49.8 ± 7.8 37.0 ± 9.4

Extract B 9 (9F) 72.9 ± 6.1 48.2 ± 6.6 32.1 ± 4.7 41.8 ± 4.6 34.6 ± 4.5

APP 4 (1M, 3F) 80.8 ± 6.8 55.9 ± 7.7 53.9 ± 7.8 33.7 ± 4.4 59.5 ± 9.3

WT 10 (6M, 4F) 63.0 ± 6.9 41.1 ± 5.8 29.1 ± 5.4 21.2 ± 3.5 29.7 ± 5.0

Results of learning trials

Results of Probe trial

Treatment N

Probe trial: Quadrant 3

Cross number Duration (sec)

Extract A 9 (9M) 5.7 ± 1.4* 20.1 ± 3.7

Extract B 9 (9F) 4.1 ± 0.7 14.8 ± 4.0

APP 4 (1M, 3F) 2.0 ± 1.2 8.7 ± 5.1

WT 10 (6M, 4F) 5.1 ± 1.5 15.1 ± 5.4