A potential defense mechanism against amyloid deposition in cerebellum

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 25

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 26

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 27

Senile plaques

(Amyloid β)

Neurofibrillary

Tangles (tau)

Fig. 1 Neuropathological hallmarks of AD. Bielshosky’s silver staining visualizes

extracellular amyloid plaques composed of amyloid-β and neurofibrillary tangles of

hyperphosphorylated tau. Modified from ( Blennow K et al., 2006) [1].

 28

A Production

A polymers & deposition

Potential effective druggable

targets for disease-modifying

therapy

Tau NFT formations

Neuronal dysfunction &

loss

AD

Fig. 2 The amyloid cascade hypothesis. The series of key pathogenic incidents leading to

AD by the amyloid cascade hypothesis. Accumulating Aβ turns into Aβ oligomerization

and gradually deposits as the forms of fibrils and senile plaques. Furthermore, Aβ

aggregation alters the kinase/phosphatase activity that leads to the Tau protein

hyperphosphorylated, which causes the formation of NFT; and eventual synaptic and

neuronal dysfunction and AD. (Modified from San X et al., 2015) ) [1].

 29

sAPP

APP

-Secretase

(BACE 1)

A

A

A

Extracellular space

AICD

AICD

Cytosol

C99

AICD

-Secretase

Membrane

APP = Amyloid precursor protein

AICD = APP intracellular domain

Fig. 3 Generation of Aβ. In amyloidogenic pathway, APP is first cleaved byβsecretase (BACE-1) to generate C99, the direct substrate of γ-secretase. γ-Secretase

cleaves C99 in the middle of its transmembrane domain to generate extracellular Aβ

and APP intracellular domain (AICD). (Modified from Lichtenthaler et al., 2011)

[12].

 30

40

42

48 49

…VGGVVIATVIVITLVML…

A42 product line

42

…VGGVVIA

A40 product line

40

…VGGVV

AICD49-99

49

LVML…

50

AICD50-99

VML…

Fig. 4 Stepwise tripeptide release from C99 by γ-secretase. Cleavage at the 48th

site of C99 liberates AICD49-99 and triggers the Aβ42 product line by successively

releasing the tripeptides VIT and TVI. Cleavage at the 49th site leads to AICD50-99

production and the Aβ40 product line by releasing ITL, VIV, and IAT. Modified

from Funamoto S et al., 2020 [11].

 31

CTX

CBL

82E1

Anti-A42

Fig. 5 Aβ deposition in brain. (a) In human, Aβ plaques (blue) develop first in

the neocortex at early stage. In severe cases of AD, Aβ plaques found in CBL.

Modified from Godert M et all., 2015 [13]. (b) Immunofluorescent doublestaining with 82E1 and anti-Aβ42 antibodies showed less Aβ deposition in CBL

than CTX in both 12-month tg2576 mice. Scale bar indicates 1 mm.

 32

Fig. 6 Less Aβ burden in CBL of APPNL-G-F . APPNL-G-F is an AD model mice in

which aggregation prone Aβ42 is produced predominantly in whole brain.

Importantly, the DAB immunohistochemistry staining with anti-Aβ (6E10) showed

less Aβ deposit in CBL of 3-month old APPNL-G-F mice than CTX that is similar with

human brain. Scale bar indicate (1mm). ( Shahnur A et al., 2021) [20].

 33

CBL

CTX

CBL

CTX

CBL

CTX

CBL

CBL

CTX

CTX

CBL

CTX

CBL

CTX

CBL

CTX

CBL

CTX

CBL

CBL

CTX

CTX

CBL

CBL

CTX

CBL

13 M

13 week

7 week

3 week

CTX

CBL

CTX

kDa

10

CTX

1 week

Aβ

82E1

kDa

APP

100

6E10

3500

Aβ level (AU)

3000

2500

CTX

2000

CBL

1500

1000

500

1 Week

3 Week

7 Week

13 Week

6M

Fig. 7 Aβ in CTX and CBL. (a) TS soluble and insoluble fractions from CTX and CBL

in APPNL-G-F mice were subjected to immunoprecipitation with 4G8 antibody and

western blotting with 82E1 antibody for Aβ detection. (b) No difference on Aβ levels

between CTX and CBL in 1- and 3-week mice, however the level Aβ in CTX of 6-month

old mice was 4.5 times higher than that in CBL. APP expression levels were consistent

in CTX and CBL in all mice tested. Data represent mean ± SD. n = 3 mice per group. * p

< 0.05 (paired t-test). ( Shahnur A et al., 2021) [20].

 34

CTX

CBL

CTX

CBL

CTX

CBL

CTX

CBL

13 week

CTX

CTX

CBL

CTX

7 week

CBL

CTX

CBL

kDa

CTX

CBL

3 week

CBL

10

Aβ

82E1

kDa

10

Aβ

82E1

CTX

CTX

CBL

CBL

35

400

30

350

300

25

***

Aβ level (AU)

Aβ level (AU)

***

20

15

10

250

200

100

50

3 Week

7 Week

13 Week

**

150

3 Week

7 Week

13 Week

Fig. 8 Soluble and insoluble Aβ in CTX and CBL. (a) TS soluble and (b) TS insoluble

fractions from CTX and CBL in APPNL-G-F mice were subjected to immunoprecipitation

with 4G8 antibody and western blotting with 82E1 antibody for Aβ detection. (c) Less

difference on Aβ levels between CTX and CBL in 3- , 7- and 13-week mice in TS

soluble fraction. (d) Difference on Aβ levels between CTX and CBL in 3- , 7- and 13week mice in TS insoluble fraction was enhanced. Data represent mean ± SD. n = 3 mice

per group. * p < 0.05. ** p < 0.01. *** P< 0.001 (paired t-test).

 35

1.2

p = 0.108

CTX

Aβ level (AU)

1.0

0.8

0.6

0.4

0.2

CBL

0.0

CTX

CBL

Fig. 9 ISF A level in CTX and CBL. (a) Representative image of probe insertion

sites in CTX and CBL for microdialysis. (b) ISF A level between CTX and CBL in

4-month old APPNL-G-F mice. No Significant difference was observed between them.

Data represent mean ± SD. n = 4 mice per group. p = 0.108 (paired t-test). Courtesy

of Dr. M. Nakano. ( Shahnur A et al., 2021) [20].

 36

HF-A42

Iba-1

Merge

CBL

CTX

HF-A42+ Iba-1+ cells/mm2

35

P=0.337

30

25

20

15

10

CTX

CBL

Fig. 10 Microglial engulfment of A. (a) HiLyte™ Fluor 555 labeled Aβ1-42 (HF-Aβ42)

was injected into CTX and CBL of 8-week mice and observed its localization with Iba-1

positive cells after 72 hours of the injection. Arrows indicate positions of HF-A in Iba-1

positive microglia cells. Scale bar indicates 20 μm. (b) Density of HF-Aβ42 and Iba-1

positive cells in CTX and CBL. Data represent mean ± SD. n = 4 mice per group. p = 0.33

(paired t-test). ( Shahnur A et al., 2021) [20].

 37

0h

2h

24 h

72 h

CBL

CTX

***

0.35

0.30

300

CTX

CBL

CTX

CBL

250

***

200

0.25

% 0f 0 h

HF-A42+ area per mm2

0.40

0.20

0.15

0.10

**

0.05

0.00

150

***

***

100

50

24

Time after injection (h)

72

24

72

Time after injection (h)

Fig. 11 HF-Aβ42 diffusion in brain tissues at low concentration. HF-Aβ42 was

stereotaxically injected into the brain parenchyma. (a) Representative coronal sections of

C57BL6 mouse brains injected with HF-Aβ42 at 0.5mg/ml conc. (b) Quantification of HFAβ42 positive areas in CBL and CTX at the time indicated. HF-Aβ42 positive areas in CBL

expanded around six-times than that in CTX right after injection (0 h). Importantly, the HFAβ42 positive area decreased sharply after 24 hours. In contrast, HF-Aβ42 positive areas in

CTX tended to be constant up to 72 h. (c) Normalization with 0 h indicate that diffusion rate

is higher in CBL than CTX after 24 h. Scale bar indicates 2 mm. Data represent mean ± SD.

n = 35 mice per group. ** p < 0.01. *** P< 0.001 (unpaired t-test). ( Shahnur A et al.,

2021) [20].

 38

0h

2h

24 h

72 h

CBL

CTX

0.8

0.6

**

**

CTX

CBL

200

0.5

150

0.4

% 0f 0 h

HF-A42+ area per mm2

0.7

250

CTX

CBL

0.3

0.2

100

50

0.1

0.0

24

Time after injection (h)

72

24

72

Time after injection (h)

Fig. 12 HF-Aβ42 diffusion in brain tissues at high concentration. HF-Aβ42 was

stereotaxically injected into the brain parenchyma. (a) Representative coronal sections of

C57BL6 mouse brains injected with HF-Aβ42 at 2mg/ml conc. (b) Quantification of HFAβ42 positive areas in CBL and CTX at the time indicated. HF-Aβ42 positive areas in CBL

expanded around four-times than that in CTX right after injection (0 h). Importantly, the

HF-Aβ42 positive area decreased sharply after 72 hours. In contrast, HF-Aβ42 positive

areas in CTX was tended to be constant up to 72 hours. (f) Normalization with 0-h indicate

that diffusion rate is higher in CBL than CTX after 72 h Scale bar indicates 2 mm. Data

represent mean ± SD. n = 710 mice per group. * p < 0.05.** p < 0.01. (unpaired t-test).

 39

HF-555

-DAEFRHDSGYEVHHQKLVFFAEDVGSNKGAIIGLMVGGVVIA

Anti-A antibody (4G8)

CTX

HF- Aβ42

Merge

72 h

0h

Anti-A

antibody (4G8)

CBL

HF- Aβ42

Merge

72 h

0h

Anti-A

antibody (4G8)

Fig. 13 Immunological detection of HF-Aβ42 in brain tissues. (a) Hilyte Fluor-555

labeled Aβ42 sequence that can recognize anti-Aβ (4G8) at 17-24 amino acid. (b)

Immunostaining demonstrated 4G8 (green), HF-Aβ42 (red) and Merge (yellow) staining.

Top panel for CTX and bottom panel for CBL. Scale bar indicates 500 μm.

 40

0h

24 h

72 h

CBL

CTX

2h

CTX

CBL

0.25

0.20

0.15

0.10

% 0f 0 h

FAM-ScA42+ area per mm2

0.05

0.00

24

72

Time after injection (h)

CTX

CBL

500

450

400

350

300

250

200

150

100

50

24

72

Time after injection (h)

Fig. 14 FAM-scAβ42 diffusion in brain tissues. FAM-scAβ42 was stereotaxically

injected into the brain parenchyma. (a) Representative coronal sections of C57BL6 mouse

brains injected with FAM-scAβ42. (b) Quantification of FAM-scAβ42 positive areas at the

time indicated. FAM-scAβ42 positive areas in CBL was 2.5-times larger than that in CTX

at right after injection (0 h). FAM-scAβ42 positive areas reached to the maximum level at

2 hours both in CTX and CBL. Importantly, we detected no significant difference in the

FAM-scAβ42 positive area between CTX and CBL after 2 hours of injection. (c)

Normalization with 0-h indicate that diffusion rate is simlar in CTX and CBL untill 72 h.

Scale bar indicates 2 mm. Data represent mean ± SD. n=56 mice per group. *, p < 0.05

(unpaired t-test). ( Shahnur A et al., 2021) [20].

 41

0h

2h

72 h

CBL

CTX

24 h

0.50

0.45

0.40

0.35

0.30

0.25

0.20

0.15

0.10

0.05

0.00

CTX

CBL

CTX

CBL

450

**

400

350

**

% 0f 0 h

HF-A40+

area per

mm2

300

250

200

150

100

50

24

Time after injection (h)

72

24

72

Time after injection (h)

Fig. 15 HF-Aβ40 diffusion in brain tissues. HF-Aβ40 was stereotaxically injected

into the brain parenchyma. (a) Representative coronal sections of C57BL6 mouse

brains injected with HF-Aβ40 at 2mg/ml concentration. (b) Quantification of HF-Aβ42

positive areas in CBL and CTX at the time indicated. HF-Aβ40 positive areas in CBL

expanded around four-times than that in CTX right after injection (0 h). HF-Aβ40

positive areas reached to the maximum level at 24 hours both in CTX and CBL. (c)

Normalization with 0-h indicate that diffusion rate is higher in CBL than CTX after 72

h. Scale bar indicates 2 mm. Data represent means ± SD, n= 4 5 per group, * P <

0.05, ** P < 0.01 (unpaired t-test).

 42

HF-555

-DAEFRHDSGYEVHHQKLVFFAEDVGSNKGAIIGLMVGGVV

Anti-A antibody (4G8)

CTX

HF- Aβ42

Merge

72 h

0h

Anti-A

antibody (4G8)

CBL

HF- Aβ42

Merge

72 h

0h

Anti-A

antibody (4G8)

Fig. 16 Immunological detection of HF-Aβ40 in brain tissues. (a) Hilyte Fluor-555

labeled Aβ40 sequence that can recognize anti-Aβ (4G8) at 17-24 amino acid. (b)

Immunostaining demonstrated 4G8 (green), HF-Aβ40 (red) and Merge (yellow) staining.

Top panel for CTX and bottom panel for CBL. Scale bar indicates 500 μm.

 43

0h

2h

72 h

CBL

CTX

24 h

CTX

CBL

**

CTX

CBL

250

0.80

200

0.70

0.60

% 0f 0 h

Alexa-OVA+ area per mm2

0.90

0.50

0.40

0.30

0.20

150

100

50

0.10

0.00

24

Time after injection (h)

72

24

72

Time after injection (h)

Fig. 17 Alexa-OV diffusion in brain tissues. Alexa-OV was stereotaxically injected into the

brain parenchyma. (a) Representative coronal sections of C57BL6 mouse brains injected

with Alexa-OV at 2mg/ml concentration. (b) Quantification of Alexa-OV positive areas at

the time indicated. Alexa-OV in CBL was 6-times larger than that in CTX right after

injection (0 h). Importantly, Alexa-OV positive areas in CTX and CBL decreased similarly

after 2 hours of injection. (c) Normalization with 0-h indicate that diffusion rate is higher in

CBL than CTX after 72 h. Scale bar indicates 2 mm. Data represent mean ± SD. n = 35

mice per group. *, p < 0.05. **, p < 0.01 (unpaired t-test). ( Shahnur A et al., 2021) [20].

 44

CTX

LYVE-1

HF-A42

CBL

Merge

LYVE-1

HF-A42

Merge

0h

2h

24 h

72 h

HF-A42+ area fraction (%)

14

12

CTX

CBL

10

**

24

72

Time after injection (h)

Fig. 18 Drainage of HF-Aβ42 from brain tissues into DcLNs. (a) Detection of brain-injected

HF-Aβ42 in Dclns. Dclns were removed in each tome point after HF-Aβ42 injection in CTX (left

panel) and CBL (right panel). We detected robust HF-Aβ42 signals in DcLNs in 2 and 24 hours

after CBL injection. HF-Aβ42 (red), LYVE-1 (green), and Merge (yellow). Scale bar indicates

500 μm. (b) Quantification for HF-Aβ42 positive area fraction in DcLns sections. HF-Aβ42

positive area fraction in DcLNs of CBL injection reached at the maximum level at 2 hours and

decreased over time, while that of CTX was faint. Data represent mean ± SD. n = 710 mice per

group. *, p < 0.05. **, p < 0.01 (unpaired t-test). ( Shahnur A et al., 2021) [20].

 45

HF- 555

- DAEFRHDSGYEVHHQKLVFFAEDVGSNKGAIIGLMVGGVVIA

Anti-A 42antibody

Anti-A 42

antibody

HF- Aβ42

Merge

2h

Fig. 19 Immunological detection of HF-Aβ42 in DcLNs. (a) Hilyte Fluor-555 labeled

Aβ42 sequence that can recognize anti-Aβ42 antibody. (b) Immunostaining demonstrated

anti-Aβ42 (green), HF-Aβ42 (red) and Merge (yellow). Scale bar indicates 200 μm.

 46

CTX

LYVE-1

Alexa-OV

CBL

Merge

LYVE-1

Alexa-OV

Merge

0h

2h

24 h

72 h

Alexa-OVA+ area fraction(%)

25

20

**

CTX

CBL

15

**

10

24

Time after injection (h)

72

Fig. 20 Drainage of Alexa-OV from brain tissues into DcLNs. (a) Detection of brain-injected AlexaOV in Dclns. Dclns were removed in each tome point after Alexa-OV injection in CTX (left panel) and

CBL (right panel). We detected robust Alexa-OV signals in DcLNs in 2 hours after CTX injection and

24 hours after CBL injection. Alexa-OV(red), LYVE-1 (green), and Merge (yellow). Scale bar indicates

500 μm. (b) Quantification for Alexa-OV positive area fraction in DcLns sections. Alexa-OV positive

area fraction in DcLNs of CTX injection reached at the maximum level at 2 hours while that of CBL

maximum at 24 hours and decreased over time respectively. Data represent mean ± SD. n = 35 mice

per group. *, p < 0.05. **, p < 0.01 (unpaired t-test).

 47

anti-LYVE-1

2 mo

2 mo

9 mo

9 mo

14 mo

14 mo

anti- Aβ42

2nd ab

Fig. 21 Detection of endogenous Aβ42 in DcLNs. (a) Immunostaining of APPNL-F mouse

brain sections with anti-Aβ antibody (82E1). Age-dependent Aβ deposition was observed in

CTX. No Aβ deposition in CBL was detected even in 14-month old mice. Scale bar

indicates 1 mm. (b) Dclns were collected and analyzed by immunohistochemistry with

anti-lymphatic vessel endothelial hyaluronan receptor-1 (LYVE-1) antibody and anti-Aβ42

antibody in DcLNs. We detected anti-Aβ42 antibody-dependent signals in DcLNs.

Treatment of DcLNs specimens with only secondary antibody showed no signal. Scale bar

indicates 500 μm. ( Shahnur A et al., 2021) [20].

 48

Acknowledgement

First of all, I would like to express my deepest sense of gratitude to my

supervisor, Dr. Satoru Funamoto, Laboratory of Neuropathology, Graduate School

of Life and Medical Sciences, Doshisha University, for his constant inspiration,

scholastic guidance, immense encouragement, valuable suggestion, timely and

solitary instruction, cordial behaviors, constructive criticism and providing all

facilities for successful completion of the research work.

I would like to convey my eternal gratitude to Dr. Tomohiro Miyasaka and

Dr. Nobuto Kakuda, Laboratory of Neuropathology, Graduate School of Life and

Medical Sciences, Doshisha University, for their constructive advice, experimental

support and active help.

My sincere thanks to Dr. Moniruzzaman Mohammad for sharing experience

and knowledge during the time of study as well as all other members of Laboratory

of Neuropathology for their kind cooperation in every step of my research.

During of my PhD, first time I get a taste of my fatherhood with a boy Shah

Muhammad Samir. I would like to acknowledge a super lady and she is my wife

Shahanaz Parvin. Mrs. Parveen has been extremely supportive of me throughout

this entire process and has made countless sacrifices to ensure that I can

complete this PhD.

& 49

...