a carboxylated zn-phthalocyanine inhibits the fibril formation of alzheimer’s amyloid β peptide...
TRANSCRIPT
A carboxylated Zn-phthalocyanine inhibits the fibril formation of Alzheimer’s amyloid β peptide
Atsushi Nagai
Dept. Laboratory Medicine
Shimane University Faculty of Medicine
Background and Purpose
3
Alzheimer’s Disease
Alzheimer’s disease (AD) is a common dementia disease of the elderly
Histopathological features of AD1. Degenerative and dystrophic neurons2. Reactive glial cells3. Extracellular Aβ peptide deposition4. Intracellular neurofibrillary tangles
Genetic and animal studies demonstrated the vital role of Aβ peptide in AD pathology
4
Current Management of Alzheimer’s Disease
• Diagnosis– Mainly depends on clinical assessment– Neuro-imaging is valuable, but either
expensive or nonspecific– Definitive diagnosis can be done by autopsy
examination of patient’s brain.• Treatment
– Currently no disease modifying therapy is available
5
Near-infrared Imaging
• Diagnostic tools– Near-infrared imaging is a technique to
detect target molecules with near-infrared probe.
Mol. Pharmaceutics 2013, 10, 3946−3958
• Example– NIR fluorophore conjugated folic
acid is used as a probe to visualize folate receptor
– Phthalocyanine-LHRH is used to visualize tumor in vivo
6
Phthalocyanine
• Water insoluble or tends to aggregate in water, resulting quenching of fluorescence properties
• Water soluble phthalocyanine substituted with sodium carboxylate group was prepared.
• Phthalocyanine is a large macrocyclic compound having optical properties in near-infrared region
UV-vis Spectra of ZnPc(COONa)8 in Buffer Solutions and MeOH at R.T.
Wavelength / nm
ε /
105
M-1c
m-1
673673
pH9.2
pH6.9
MeOH
642pH4.0
yield 46%
Syntheses and Properties of ZnPc Complex
Perspective
• Digagnostic strategy• Can phthalocyanine bind to A be detected
with NIRS?
• Therapeutic strategy• Potential of phthalocyanine for inhibition of
amyloidogenesis
Aim of this study
To investigate the interaction of
phthalocyanine with Aβ peptide
during fibril formation process
Methods and Results
1. Inhibition of Aβ fibril formation
2. Destabilization of Aβ fibril
3. Binding to Aβ
4. Secondary structure and hydrophobicity
5. Inhibition of oligomer formation
6. Cell toxicity
Phthalocyanines:
Phthalocyanines used in the study
(A) (B)
(C) (D)
ZnPc(COONa)8
(water soluble)
ZnPc(COONa)16
(water soluble)
ZnPc(OOC5H11)8
(water insoluble)
PdPc dimer(water insoluble)
0
40
80
0.1 1 5 10 20 µM
ThT
fluor
esce
nce
(% c
ontr
ol)
† † ††
0
40
80
120
0.1 1 5 10 20 µM
ThT
fluor
esce
nce
(% c
ontr
ol)
‡ ‡✴
ThT
fluor
esce
nce
(% c
ontr
ol)
0.1 1 5 10 20 µM
✴
‡
✴
0
100
200
300
400
0.1 1 5 10 20 µM
ThT
fluor
esce
nce
(% c
ontr
ol)
0
40
80
120
† †
ZnPc(COONa)8: ZnPc(COONa)16:
ZnPc(OOC6H13)8: PdPc dimer:
Effects of phthalocyanines on Aβ1-40 fibril formation
A1-40 (50 M)
*48 h incubation
Effects of phthalocyanines on Aβ1-42 fibril formation
0
40
80
0.1 1 5 10 20ZnPc(COONa)8: µM
ThT
fluor
esce
nce
(% c
ontr
ol)
††
††
0.1 1 5 10 20
ThT
fluor
esce
nce
(% c
ontr
ol)
ZnPc(COONa)16: µM0
40
80
120
✴
✴
0.1 1 5 10 20
ThT
fluor
esce
nce
(% c
ontr
ol)
µMZnPc(OOC6H13)8:0
100
200
300
400
0
40
80
120
ThT
fluor
esce
nce
(% c
ontr
ol)
0.1 1 5 10 20PdPc dimer: µM
A1-42 (12.5 M)
*24 h incubation
Aβ fibril formation kineticsTh
T flu
ores
cenc
e
0 20 40 60 800
20
40
60
h
h0 20 40 60 800
4
8
12
16
ThT
fluor
esce
nce
+ ZnPc(COONa)8
A1-40 (50 M)
A1-40
0 20 40 60 800
10
20
ThT
fluor
esce
nce
0
2
4
6
0 20 40 60 80
ThT
fluor
esce
nce
h
h
+ ZnPc(COONa)8
A1-42 (12.5 M)
A1-42
Aβ fibril morphology
+ ZnPc(COONa)8
A1-40 A1-42
A1-40
Bar=200 nm
+ ZnPc(COONa)8A1-42
0
10
20
30
0 0.1 1 10 50 M
ThT
fluor
esce
nce
✴
†
‡
†
0
20
40
0 0.1 1 10 50
ThT
fluor
esce
nce
M
†
††
‡
0 0.1 1 50 10 M 0 0.1 1 50 10 M
28
17
10
KDaKDa
Aβ fibril destabilization
28
17
10
A1-40 fibril (50 M) + ZnPc(COONa)8 A1-42 fibril (25 M) + ZnPc(COONa)8
buffer
Aβ1-42
Aβ1-42 fibril
Aβ1-42
0 1 5 10
ZnPc(COONa)8 (μM)
+ -
++
Aβ antibody
Binding of phthalocyanine to Aβ1-42 peptide or fibril
Incubation (24 h)
ZnPc(COONa)8+ Immunoprecipitation with Aβ Ab
buffer
Aβ1-42
Aβ1-42 fibril
Aβ1-42
NIR scan
0
1
2
3
4
5
6
7
ANS
fluor
esce
nce
400 450 500 550 600 nm
random
turn sheet
α helix
ZnPc(COONa)8
0%
20%
40%
60%
80%
100%
- + - +0 h 2 h
Secondary structure and hydrophobicity of Aβ peptide or fibril
CD spectroscopyCD spectroscopy ANS fluorescence assay ANS fluorescence assay
emission spectra
A1-40 A1-42
Molecular species of Aβ1-42 during fibril formation
+ ZnPc(COONa)8
0 h 2 h 4 h 6 h 8 h 16h 24 h
ZnPc(COONa)8: - - - - - -+ + + + + +0 h 1 h 2 h 4 h 8 h 24 h
352817
10
KDa
delayed oligomer formation
Coomassie blue staining (SDS PAGE)
Coomassie blue staining (SDS PAGE)
Dot blot immunoassay with oligomer specific AbDot blot immunoassay
with oligomer specific Ab
A1-42
A1-42
A1-42 (50 μM)
0 0.1 1 5 10
MTT
- % c
ontr
ol
ZnPc(COONa)8
µM
MTT
- % c
ontr
ol
0 0.1 1 5 10 µM
✳ ✳
40
60
80
100
120
40
60
80
100
120
Cell viability assay of neuronal cell
ZnPc(COONa)8
ZnPc(COONa)8 protects A1 neuronal cells from Aβ-induced toxicity
A1-42
A1-42 (5 M)
22
Summary of the results
• Water soluble ZnPc(COONa)8
– has near-infrared properties
– binds to Aβ peptide
– inhibits its oligomer and subsequent fibril formation process
– destabilizes preformed Aβ fibrils
– decreases β-sheet conformation and hydrophobicity
– possesses neuroprotective feature against Aβ peptide
23
Conclusion
Phthalocyanine could be used as a safe diagnostic
probe as well as a therapeutic tool for AD.
24
Acknowledgements
• Shimane University• Faculty of Medicine,
• Dept. Laboratory Medicine• Shatera Tabassum• Abdullah Md. Sheikh• Shozo Yano
• Interdisciplinary Faculty of Science and Engineering, • Dept. Material Science• Takahisa Ikeue• Makoto Handa
Laboratory Medicine
• Funded by JSPS KAKENHI Grant Number 24310102