立命館大学審査博士論文 神経系における細胞骨格関連 - R-Cube

 Novel functions of cytoskeletal protein ezrin in nervous system
2015
3
March, 2015
Doctoral Program In Advanced Life Sciences
Graduate School of Life Sciences
Ritsumeikan University
MATSUMOTO Yosuke
Supervisor
Professor ASANO Shinji
1
7
14
14
19
21
21
RhoA/ROCK
RhoA
23
25
26
27
28
29
33
1
Dotti et al., 1988; Arimura and
Kaibuchi, 2007
1-1
º
1
º
2
3
4/5
1
1
Rho
GTPase
RhoA Rac1 Cdc42
1-2 Govek et
al., 2005
Dent and Getler, 2003; Gupton and Gertler, 2010
Rho
GDP
GTP
Rho
Rac1
Cdc42
p21-activated kinase PAK
Hayashi et al., 2007
Rho kinase
RhoA
ROCK
Redmond and Ghosh, 2001
II
myosin light chain 2
MLC2
ROCK
Kollins et al., 2009
Rho
Schmidt and Hall, 2002; Tcherkezian and Lamarche-Vane,
2007
Rho guanine nucleotide exchange factor
GDP
Rho
GTP
1-3A
GTPase-activating protein RhoGAP
GDP
RhoGEF
Rho
GTPase
Rho
nucleotide dissociation inhibitor RhoGDI
GTP
1-3B
Rho
Rho
GDP
1-3C
2
Rho
Rho guanine
Ezrin/Radixin/Moesin
º
Tsukita and Yonemura, 1999
1-4
ERM
N
four-point-one, ezrin, radixin, moesin FERM
C
II
nf2
ERM
FERM
85%
º
61%
84%
ERM
ERM
º
ERM
N
C
phosphatidylinositol 4,5-bisphosphate PIP2
1-5
3T3
lysophosphatidic acid LPA
567
º
ROCK
C
564
º
C
3
558
311-583
564
FERM
Matsui et al., 1998
RhoA/ROCK
C
RhoA
N
FERM
Takahashi et al., 1997
RhoA
GDP
RhoGDI
RhoGDI
RhoA
Vil2-/-
Casaletto et al., 2011
LLC-PK1
Speck et al., 2003
RhoA
RhoA
RhoA/ROCK
RhoA
ERM
13H9
ERM
ERM
Goslin et al., 1989; Everett and Nichol, 1990
º
Paglini et al., 1998
º
4
Vil2kd/kd
1
Tamura et al., 2005
Vil2+/+
Vil2kd/kd
Vil2kd/kd
Vil2kd/kd
Vil2+/+
Vil2+/+
RhoA
Vil2kd/kd
3
RhoA/ROCK
MLC2
Rac1
ROCK
Cdc42
Vil2+/+
II
Vil2kd/kd
Vil2kd/kd
8
Vil2+/+
V
V
RhoA/ROCK/MLC2
5
2
ERM
lrrk2
repeat kinase 2 LRRK2
leucine-rich
Jaleel et al., 2007: Parisiadou et al., 2009
6-hydroxydopamine 6-OHDA
6-OHDA
iPS
6
Vil2kd/kd
Tamura et al., 2005
Vil2kd/kd
C57BL/6J Jcl
ICR
12
BKC
Vil2kd/kd
2
3
1-6 Tamura et al., 2005
5%
10%
Tamura et al., 2005
Hatano et al., 2013
3 mm
95ºC
10
Hatano et al., 2014
50 mM
10
1 M Tris-HCl
12,000 rpm
pH 8.0
PCR
KOD FX TOYOBO
PCR
º
7
1
PCR
380 bp
290 bp
PCR
Vil2+/+: Vil2+/kd: Vil2kd/kd = 1:2:1
15.5
HBSS
0.25% trypsin/EDTA
Invitrogen
20
Viesselmann et al., 2011
10% FBS
Invitrogen
24
60 mm
B27 Invitrogen
37.5 mM NaCl
Neurobasal
GlutaMAX Invitrogen
0.3% glucose
Invitrogen
5%
blebbistatin
Opti-MEM
15
0.1 mg/ml poly-D-lysine
CO2
37ºC
40 µM Y-27632
37ºC
Wako
50 µM
Wako
10
mM
pH 7.4
4%
4%
15%
º
2
º
100 mM
20 µm
0.3% TritonX-100
8
100 mM
4 C
2
IgG
4
4 C
Vector
IgG
2
Vector
VECTASTIN ABC
0.02% DAB
0.0045%
º
Vector
0.3%
(
50 mM Tris-HCl
)
pH 7.6
º
FD Rapid
GolgiStain Kit
FD NeuroTechnologies
250 µm
º
º
Axioplan II, Carl Zeiss
º
95%
º
50%
1
15
1
2
2
70%
2
70%
0.5%
1
95%
2
50%
100%
Axioplan II, Carl Zeiss
9
1
1
1
4%
4%
10
º
0.1% Triton X-100
BSA
4ºC
10
1%
30
4ºC
2
fluorescein isothiocyanate FITC
Jackson ImmunoResearch
Alexa Fluor 633
45
IgG
IgG
Vector
rhodamine phalloidin Invitrogen
FV-1000D FV-10i Olympus
Hirai et al., 2011
10 µm
1
3
Shelly et al., 2007
ImageJ
40 µm
2
40 µm
2
neuronal class III β-tubulin
NeuronJ
º
protease inhibitors Nacalai Tesque
5 mM Tris-HCl, pH 7.4, 250 mM sucrose
10
lysis buffer
20
4ºC
17,500×g
protease inhibitors
phosphatase inhibitors Nacalai Tesque
Cell BioLabs
RIPA buffer 25 mM Tris-HCl pH 7.6, 150
mM NaCl, 1% Nonidet P-40, 1% sodium deoxycholate, 0.1% SDS
10
50 mM Tris-HCl, pH 6.8, 2%
SDS, 2% 2-mercaptoethanol, 20% glycerol, 0.01% BPB
SDS-PAGE
5
PVDF
2
HRP
95ºC
5%
60
Millipore
4ºC
14,000×g
SDS
IgG
10
IgG
4ºC
Millipore
1
HRP
Immobilon Western
Chemiluminescent HRP Substrate Millipore
LAS-3000 Fujifilm
ImageJ
Rho
lysis buffer 25 mM HEPES, pH 7.5, 150
mM NaCl, 1% Nonidet P-40, 10 mM MgCl2, 2% glycerol
14,000×g
4ºC
10
GST-rhotekin-RBD Cytoskeleton
11
GST-PAK-PBD
Cell BioLabs
RhoA
3
1
Rac1 Cdc42
lysis buffer
SDS
SDS-PAGE
4ºC
5
PVDF
95ºC
RhoA Rac1
Cdc42
6
1 cm
2.7 mm
bregma
Uhobby
0.8 mm
0.02%
6-OHDA
2 µl
0.7 mm
10 µg
5
6-OHDA
2
6
6-OHDA
18
5
3
100 cm
10 cm
º
º
2 cm
24 C
15
º
12
15
º
1
10
º
3
I
II
III
IV
4
I
I
1 cm
º
10 cm
º
4
60
SMART
º
Bio Research Center
º
10
º
1
º
2
7
60
º
º
I
%
º
2
HPLC
EDTA 50 mg/l
3,4-dihydroxybenzylamine hydrobromide
0.1 N
dihydroxyphenylacetic acid
15
DOPAC
13
4ºC 15,000×g
Vil2+/+
Vil2+/+
2-1
1
Vil2kd/kd
3
Vil2kd/kd
1
Vil2kd/kd
2-2A
Vil2+/+
80 kDa
2-2B
Vil2kd/kd
Vil2+/+
10 µg
Vil2kd/kd
Vil2kd/kd
5%
14
1 µg
0.5 µg
Vil2kd/kd
neuronal class III β-tubulin
rhodamine phalloidin
2-3A,B
Vil2+/+
48
Vil2kd/kd
2-3C
Vil2+/+: 8.8 ± 1.4%
Vil2kd/kd
1
Vil2kd/kd: 17.3 ± 2.3%
Vil2+/+: 55.8 ± 4.2%
3
Vil2kd/kd: 43.1 ± 4.1%
3
Vil2kd/kd
Vil2+/+: 2.7 ± 0.2 Vil2kd/kd: 1.5 ± 0.2
2-3D
2-3E,F
96
Vil2+/+
2-4A,B
Vil2kd/kd
48
Vil2+/+: 4.2 ± 0.6
Vil2kd/kd: 1.4 ± 0.2
2-4C
2-4D,E
Vil2kd/kd
RhoA
GTPase
RhoA Rac1 Cdc42
Govek et al., 2005; Negishi and Katoh, 2002
Vil2kd/kd
RhoA
Rac1
RhoA
15
Cdc42
GTP
Vil2kd/kd
RhoA
Vil2+/+
RhoA
2-5A,D
Vil2+/+
Rac1
3
2-5B,E
Cdc42
2-5C,F
Vil2kd/kd
RhoA
Vil2kd/kd
RhoA
MLC2
2-6A,B
MLC2
ROCK
º
Vil2+/+
2-6C,D
Vil2kd/kd
Y-27632
2-7A,B
MLC2
ERM
2-7C-E
MLC2
Vil2kd/kd
ROCK
ROCK
ROCK
º
II
Vil2kd/kd
RhoA
RhoA
ROCK
Y-27632
Bito et
al., 2000; Ishizaki et al., 2000; Da Silva et al., 2003; Peris et al., 2012
ROCK
Vil2kd/kd
16
Y-27632
2-8A-D
Vil2kd/kd
DMSO
2-8E
Vil2+/+
Vil2+/+
Vil2kd/kd
Y-27632
DMSO
Y-27632
DMSO
2-8E
Vil2+/+
Y-27632
Vil2kd/kd
Y-27632-treated Vil2+/+: 4.0 ± 0.3, Y-27632-treated Vil2kd/kd: 3.4 ± 0.3
Y-27632
2-8F,G
Vil2kd/kd
ROCK
II
blebbistatin
Vil2+/+
Y-27632
2-8E
2-9A,B
Vil2kd/kd
blebbistatin
DMSO
blebbistatin
2-9C
Vil2+/+
DMSO
blebbistatin
Vil2kd/kd
blebbistatin-treated Vil2+/+: 7.7 ± 0.8, blebbistatin -treated Vil2kd/kd: 8.4 ± 1.0
2-9C
ROCK
2-9D,E
RhoA/ROCK/MLC2
2-10
17
Vil2kd/kd
Vil2kd/kd
Vil2kd/kd
2-11
Vil2kd/kd
2-12A
Vil2kd/kd
Vil2+/+: 368.0 ± 16.1 µm, Vil2kd/kd:
2-12B
299.3 ± 4.5 µm
2-12C
Vil2kd/kd
V
neuronal class III β-tubulin
2-13A
microtubule associated proteins 2 MAP2
2-13B
2-14A,B
6
2/3
Larkum et al., 1999
5
1
1
2/3
5
Laramée et al., 2013
Vil2kd/kd
V
18
2-14C
Vil2+/+: 212.4 ± 8.5 µm, Vil2kd/kd: 159.9 ± 11.7 µm
2-14D
6-OHDA
Vil2+/+: 4.9 ± 0.3, Vil2kd/kd: 4.0 ± 0.2
6
%
º
6-OHDA
3-1A
º
50%
6-OHDA
3-1B
6-OHDA
18
6
6-OHDA
º
%
2
3-2A
6
º
40%
6-OHDA
3-2B
6-OHDA
TH
L-DOPA
19
L-DOPA
DOPA
6-OHDA
TH
6-OHDA
TH
3-3A
DOPAC
HPLC
6-OHDA
DOPAC
3-3B,C
6-OHDA
º
3-4
6-OHDA
2
º
20
6-OHDA
RhoA/ROCK/MLC2
RhoA
ROCK
1C11
Da Silva et al., 2003
II
RhoA
Dent et al., 2007; Kollins
et al., 2009
RhoA
II
RhoA
MLC2
II
Vil2+/+
Amano et al., 1998; Krey et al., 2013
RhoA/ROCK
MLC2
II
2-10A
Vil2kd/kd
blebbistatin
ROCK
RhoA/ROCK/MLC2
2-10B
II
2-8,9
Y-27632
Vil2kd/kd
blebbistatin
Y-27632
2-9D,E
II
21
Vil2+/+
Vil2kd/kd
V
in
vitro
in vivo
ERM
Goslin et al., 1989; Antoine-Bertrand et al., 2011; Marsick et al., 2012
º
Gonzalez-Agosti and Solomon, 1996
º
microscale chromophore-assisted
laser inactivation micro-CALI
º
al., 1999
30%
º
Castelo et
º
Paglini et al., 1998
º
ERM
deleted in colorectal carcinoma DCC
netrin-1
Netrin-1
ERM
ERM
Antoine-Bertrand et al., 2011
N1E-115
DCC
DCC
C
siRNA
22
55%
netrin-1
netrin-1
º
Vil2kd/kd
5%
º
3
Everett and Nichol, 1990;
Paglini et al., 1998
2-1
RhoA
GTPase
RhoA
Rac1
Cdc42
Govek et al.,
2005
RhoA/ROCK/MLC2
23
Speck
LLC-PK1
Speck et al., 2003
RhoA
RhoA
Vil2−/−
Casaletto
MLC2
Casaletto et
al., 2011
RhoA
Vil2kd/kd
RhoA/ROCK/MLC2
Schmieder
MDCK
RhoGDI
podocalyxin
RhoA
Schmieder et al., 2004
RhoA
1
Rac1
Cdc42
PAK
Hayashi et al., 2007; Redmond
and Ghosh, 2001
FERM
Rac1
Schulz et al., 2010
RhoA
GTPase
24
RhoA/ROCK
RhoA
ROCK
Matsui et al., 1998;
Jeon et al., 2002
RhoA/ROCK
4-1A
ROCK
Vil2+/+
Y-27632
º
2-7A,C-E
Vil2kd/kd
RhoA
º
2-6C,D
ERM
ROCK
RhoA/ROCK
4-1B
ERM
3-kinase PI3 kinase
phosphoinositide
protein kinase C PKC
LRRK2
Gallo,
2008; Parisiadou et al., 2009; Kim et al., 2010
LRRK2
G2019S
lrrk2
2019
Jaleel et al., 2007
LRRK2
G2019S
º
ERM
Parisiadou et al., 2009
25
ERM
ERM
3-4
ERM
º
ERM
Kashimoto et al., 2013
Vil2kd/kd
26
1.
RhoA/ROCK/MLC2
2.
ERM
Vil2kd/kd
in vitro
in vivo
1
27
Vil2kd/kd
HPLC
1.
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Ezrin mediates neuritogenesis via down-regulation of RhoA activity in cultured cortical
neurons.
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2.
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6-hydroxydopamine.
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28
1.
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2.
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3.
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4.
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in
mammalian
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5.
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6.
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7.
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8.
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12.
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17.
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18.
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21.
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22.
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30
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31.
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32.
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31
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37.
Schmidt A, Hall A. Guanine nucleotide exchange factors for Rho GTPases: turning on
the switch. Genes Dev. 2002 Jul 1;16(13):1587-609.
38.
Schmieder S, Nagai M, Orlando RA, Takeda T, Farquhar MG. Podocalyxin activates RhoA
and induces actin reorganization through NHERF1 and Ezrin in MDCK cells. J Am Soc
Nephrol. 2004 Sep;15(9):2289-98.
39.
Schulz A, Geissler KJ, Kumar S, Leichsenring G, Morrison H, Baader SL. Merlin
inhibits neurite outgrowth in the CNS. J Neurosci. 2010 Jul 28;30(30):10177-86.
40.
Shelly M, Cancedda L, Heilshorn S, Sumbre G, Poo MM. LKB1/STRAD promotes
axon initiation during neuronal polarization. Cell. 2007 May 4;129(3):565-77.
41.
Speck O, Hughes SC, Noren NK, Kulikauskas RM, Fehon RG. Moesin functions
antagonistically to the Rho pathway to maintain epithelial integrity. Nature. 2003 Jan
2;421(6918):83-7.
42.
Takahashi K, Sasaki T, Mammoto A, Takaishi K, Kameyama T, Tsukita S, Takai Y.
Direct interaction of the Rho GDP dissociation inhibitor with ezrin/radixin/moesin
initiates the activation of the Rho small G protein. J Biol Chem. 1997 Sep
12;272(37):23371-5.
43.
Tamura A, Kikuchi S, Hata M, Katsuno T, Matsui T, Hayashi H, Suzuki Y, Noda T,
Tsukita
S, Tsukita
S. Achlorhydria
by
ezrin
knockdown: defects in
the
formation/expansion of apical canaliculi in gastric parietal cells. J Cell Biol. 2005 Apr
11;169(1):21-8.
44.
Tcherkezian J, Lamarche-Vane N. Current knowledge of the large RhoGAP family of
proteins. Biol Cell. 2007 Feb;99(2):67-86.
45.
Tsukita
S,
Yonemura
S.
Cortical
actin
organization:
lessons
from
ERM
(ezrin/radixin/moesin) proteins. J Biol Chem. 1999 Dec 3;274(49):34507-10.
46. Viesselmann C, Ballweg J, Lumbard D, Dent EW. Nucleofection and primary culture of
embryonic mouse hippocampal and cortical neurons. J Vis Exp. 2011 Jan 24;(47). pii:
2373.
32
Primer
GenoA1
EK29
En2A
1
Sequence
5’-CATGGTGCCACACAGGACTC-3’
5’-GTGTGGCACTCTGCCTTCAAG-3’
5’-AGCGGATCTCAAACTCTCCTC-3’
Primer sequences used in genotyping.
Antigen
Ezrin
Source
Cell Signaling Technology
Species (Clone)
Rabbit
Ezrin
Acris
Mouse (3C12)
Radixin
Moesin
pan-ERM
phospho-ERM
GAPDH
β-actin
α-tubulin
Class III β-tubulin
Class III β-tubulin
MAP2
MLC2
Phospho-MLC2
RhoA
Rac1
Cdc42
TH
Gift from Dr. Tsukita
Gift from Dr. Tsukita
Cell Signaling Technology
Cell Signaling Technology
Sigma
Cell Signaling Technology
Abcam
Covance
Sigma
Chemicon
Cell Signaling Technology
Cell Signaling Technology
Cytoekeleton
Cell BioLabs
Cytoekeleton
Sigma
Rat (R21)
Mouse (2287)
Rabbit
Rabbit
Rabbit
Rabbit
Mouse
Rabbit
Mouse (2G10)
Mouse (HM-2)
Rabbit
Rabbit
Mouse
Mouse
Mouse
Mouse
2
Antibodies used in this study.
33
Dilution (Application)
1:1000 (WB)
1:100 (IF)
1:1000 (WB)
1:100 (IHC)
1:1000 (WB)
1:1000 (WB)
1:1000 (WB)
1:1000 (WB)
1:5000 (WB)
1:5000 (WB)
1:100 (IF)
1:100 (IF)
1:5000 (IHC)
1:3000 (IHC)
1:100 (WB)
1:100 (WB)
1:500 (WB)
1:500 (WB)
1:500 (WB)
1:10000 (IHC)
Stage 1
Stage 2
Stage 3
Stage 4/5
Dendrites
Filopodia
Spines
Lamellipodia
Neurites
Axon
Growth cone
1-1
Schematic representation of morphological changes in cultured neurons.
↓
↓
↓
RhoA
Rac1
Cdc42
↓
↓
↓
ROCK
PAK
PAK
↓
↓
↓
↓
↓
↓
↓
↓
↓
↓
Retraction
1-2
Elongation
Rho signaling pathways in neuronal morphogenesis.
34
A RhoGEF
Rho
GDP
→→→→→
Rho
GEF
↓
↓
↓
Rho activity:
Increased
GTP
Effector
B RhoGAP
Rho
GDP
←←←←←
Rho
GAP
↓
↓
↓
Rho activity:
Decreased
GTP
Effector
C RhoGDI
Rho
GDP
→→→→→
↓
↓
↓
GDI
Rho activity:
Decreased
1-3
Rho
Effector
Regulation of Rho GTPase activities.
35
GTP
N
C
Four-point-one, ezrin,
radixin, moesin (FERM)
domain
Ezrin
Actin
binding
domain
T567
Phosphorylation
Radixin
85%
586
T564
583
Moesin
84%
T558
577
Merlin
61%
591
1-4
Structures of ezrin, radixin, moesin and merlin.
36
Membrane proteins
L1-CAM
DCC receptor
etc…
→→→→→→→→
Phosphorylation
P-Thr
Ezrin
Radixin
Moesin
F-actin
Inactive
Active
“Closed form”
“Opened form”
1-5
Activation of ERM proteins.
1-6
Targeting strategy of mouse ezrin gene (gene symbol Vil2).
Tamura et al., J Cell Biol 2005.
37
Stage 2
Stage 3
Merge
α-tubulin
Phalloidin
Ezrin
Stage 1
2-1
Distribution of ezrin was observed in wild-type cultured cortical neurons at the
stages 1, 2 and 3 by immunofluorescence.
Neurons at the stages 1, 2 and 3 were stained with an anti-ezrin antibody, rhodamine
phalloidin, and an anti-α-tubulin antibody, respectively. In the bottom lane, neurons were
triple stained with an anti-ezrin antibody (green), rhodamine phalloidin (red) and an
anti-α-tubulin antibody (blue). Scale bars, 50 µm.
38
A
+/+
kd/kd
0.2 µg
+/+
kd/kd
0.5 µg
+/+
kd/kd
1 µg
+/+
kd/kd
10 µg
+/+
B
kd/kd
Ezrin
80 kDa
2-2
Detection of ezrin in the Vil2+/+ and Vil2kd/kd neurons.
A, Immunofluorescence of the Vil2+/+ and Vil2kd/kd neurons at stage 1 using an anti-ezrin
antibody. Scale bar, 50 µm. B, Western blotting of cell extracts (10 µg, 1 µg, 0.5 µg or 0.2
µg) from the Vil2+/+ and Vil2kd/kd neurons (2 DIV) with an anti-ezrin antibody.
39
βIII-tubulin/Phalloidin
+/+
kd/kd
A
2-3
B
Neuritogenesis is impaired by ezrin knockdown.
A, B, The Vil2+/+ (A) and Vil2kd/kd (B) neurons were fixed at 2 DIV and stained with an
anti-neuronal class III β-tubulin antibody (green) and rhodamine phalloidin (red). Scale bars,
50 µm.
40
C
D
120%
Stage: ■ 1 ■ 2 ■ 3
3
Number of neurites
Neurons in stage (%)
100%
*
80%
60%
40%
20%
0%
**
+/+
kd/kd
F
200
Length of axon (µm)
25
Length of neurites (µm)
1
0
+/+ kd/kd
E
20
15
10
5
0
2-3
***
2
+/+
150
100
50
0
kd/kd
+/+
kd/kd
Continued.
C, Stacked bar graph showing stage progression in the Vil2+/+ (n = 153) and Vil2kd/kd (n = 162)
neurons. Stages of cells were defined by the length of the longest neurite as reported
previously. D-F, Quantitation of number (D) and length (E) of neurites, and length of axon
(F) in the Vil2+/+ (gray columns, n = 50) and Vil2kd/kd (green columns, n = 50) neurons. Three
independent experiments were performed. *p<0.05, **p<0.01, ***p<0.001, Student's t test.
Data represent mean ± SE.
41
A
B
+/+
kd/kd
C
5
4
3
1
0
2-4
**
2
+/+
kd/kd
E
25
Length of axon (µm)
Number of neurites
6
Length of neurites (µm)
D
20
15
10
5
0
+/+
kd/kd
250
200
150
100
50
0
+/+
kd/kd
Morphological abnormalities in Vil2kd/kd neurons in 4 DIV.
A, B, The Vil2+/+ (A) and Vil2kd/kd (B) neurons were fixed at 4 DIV and stained with an
anti-neuronal class III β-tubulin antibody. Scale bars, 50 µm. C-E, Quantitation of number (C)
and length (D) of neurites, and length of axon (E) in the Vil2+/+ (white columns, n = 5) and
Vil2kd/kd (black columns, n = 5) neurons. Three independent experiments were performed.
**p<0.01, Student's t test. Data represent mean ± SE.
42
A
+/+
kd/kd
B
+/+ kd/kd
Active
Rac1
Active
Cdc42
Total
RhoA
Total
Rac1
Total
Cdc42
E
3
2
1
+/+
kd/kd
1.5
Active/Total Cdc42 ratio
*
4
0
F
1.5
Active/Total Rac1 ratio
5
Active/Total RhoA ratio
C
Active
RhoA
D
2-5
+/+ kd/kd
1
0.5
0
+/+
kd/kd
1
0.5
0
+/+
kd/kd
Increased RhoA activity in the Vil2kd/kd neurons.
A-C, The amounts of active and total RhoA (A), Rac1 (B) and Cdc42 (C) from cell lysates of
the Vil2+/+ and Vil2kd/kd neurons (2 DIV). Representative patterns were presented. D-F, The
ratios of active RhoA (D), Rac1 (E) and Cdc42 (F) to total amount of proteins were compared
between the Vil2+/+ (white columns) and Vil2kd/kd (black columns) neurons. Each experiment
was performed in triplicate. *p<0.05 , Student's t test. Data represent mean ± SE.
43
A
+/+ kd/kd
C
pERM
pEzrin
pRadixin
pMoesin
MLC2
panERM
Ezrin
Radixin
Moesin
GAPDH
GAPDH
D
1.5
**
3
2
1
0
+/+
Phospho/Total ratio
4
Phospho/Total ratio
kd/kd
pMLC2
Ser19
B
2-6
+/+
1
0.5
0
kd/kd
Ezrin
Radixin Moesin
Up-regulation of MLC2 phosphorylation.
A, Western blotting of the Vil2+/+ and Vil2kd/kd neurons (2 DIV) using an antibody recognizing
phospho-MLC2 (Ser19, top), MLC2 (middle) and GAPDH (bottom), respectively.
Representative blotting patterns were shown. 8 µg of cell lysate was applied onto each lane. B,
The ratio of phosphorylated MLC2 to total MLC2 in the lysate of the Vil2+/+ (white column)
and Vil2kd/kd (black column) neurons was shown. C, Western blotting of the Vil2+/+ and
Vil2kd/kd neurons (2 DIV) using an antibody recognizing phospho-ERM (top), pan-ERM
(middle) and GAPDH (bottom), respectively. Representative blotting patterns were shown. 8
µg of cell lysate was applied onto each lane. D, The ratios of phosphorylated ezrin, radixin
and moesin to each total protein in the lysate of the Vil2+/+ (white columns) and Vil2kd/kd
(black columns) neurons were shown. Each experiment was performed in triplicate.
**p<0.01 , Student's t test. Data represent mean ± SE.
44
kd/kd
+/+
Y-27632
-
-
+
+
pMLC2
Ser19
pERM
GAPDH
C
1.4
D
0.5
+/+
*
0.8
0.6
*
0.4
0.2
kd/kd
+/+
E
1.6
pMoesin/GAPDH ratio
pRadixin/GAPDH ratio
1
2-7
1
1.6
1.5
0
1.2
0
2
pEzrin/GAPDH ratio
B
pMLC2/GAPDH ratio
A
1.4
1.2
1
0.8
0.6
0.4
0.2
0
kd/kd
+/+
1.4
1.2
1
0.8
0.6
0.4
0.2
0
+/+
kd/kd
Phosphorylation was affected by Y-27632 in the MLC2, not in the ERM
proteins.
A, Western blotting of the DMSO- or Y-27632-treated Vil2+/+ and Vil2kd/kd neurons (2 DIV)
using an antibody recognizing phospho-MLC2 (Ser19, top), phospho-ERM (middle) and
GAPDH (bottom), respectively. Representative blotting patterns were shown. 8 µg of cell
lysate was applied onto each lane. B-E, The ratios of phosphorylated MLC2, ezrin, radixin
and moesin to GAPDH in the lysate of the DMSO-treated (white columns) and
Y-27632-treated (black columns) Vil2+/+ and Vil2kd/kd neurons were shown. Each experiment
was performed in triplicate. *p<0.05, Student's t test. Data represent mean ± SE.
45
βIII-tubulin/Phalloidin
+/+ + DMSO
+/+ + Y-27632
B
A
kd/kd + DMSO
kd/kd + Y-27632
C
2-8
D
Y-27632 rescues neuritogenesis.
A-D, The Vil2+/+ and Vil2kd/kd neurons treated with DMSO (A,C) or 40 µM Y-27632 (24 h,
B,D) were fixed at 2 DIV and stained with an anti-neuronal class III β-tubulin antibody
(green) and rhodamine phalloidin (red). Scale bars, 50 µm.
46
E
F
40
**
***
4
3
###
2
1
0
Length of neurites (µm)
Number of neurites
5
***
*
20
10
0
kd/kd
+/+
30
+/+
kd/kd
G
Length of axon (µm)
250
**
150
100
50
0
2-8
*
200
+/+
kd/kd
Continued.
E-G, The number (E) and length (F) of neurites, and length of axon (G) were quantified in the
Vil2+/+ and Vil2kd/kd neurons treated with DMSO (white columns, n = 30) or 40 µM Y-27632
(black columns, n = 30). Three independent experiments were performed. *p<0.05, **p<0.01,
***p<0.001 (DMSO-treated vs. Y-27632-treated), ###p<0.001 (DMSO-treated Vil2+/+ vs.
DMSO-treated Vil2kd/kd), Student's t test. Data represent mean ± SE.
47
+/+ + Blebbistatin
kd/kd + Blebbistatin
A
2-9
B
Blebbistatin rescues neuritogenesis.
A, B, The Vil2+/+ and Vil2kd/kd neurons treated with 50 µM blebbistatin (24 h) were fixed at 2
DIV and stained with an anti-neuronal class III β-tubulin antibody. Scale bar, 50 µm.
48
C
10
***
***
9
Number of neurites
8
7
6
5
4
3
1
0
D
#
2
+/+
kd/kd
E
35
180
2-9
160
25
Length of axon (µm)
Length of neurites (µm)
30
20
15
10
5
0
200
140
120
100
80
60
40
20
0
+/+
kd/kd
+/+
kd/kd
Continued.
C-E, The number (C) and length (D) of neurites, and length of axon (E) were quantified in the
Vil2+/+ and Vil2kd/kd neurons treated with DMSO (white columns, n = 10) or 50 µM
blebbistatin (black columns, n = 10). Three independent experiments were performed.
***p<0.001 (DMSO-treated vs. blebbistatin-treated), #p<0.05 (DMSO-treated Vil2+/+ vs.
DMSO-treated Vil2kd/kd), Student's t test. Data represent mean ± SE.
49
A Vil2+/+ neurons
B Vil2kd/kd neurons
Activate
Ezrin
RhoA
Ezrin
RhoA
Inactivation
2-10
ROCK
ROCK
MLC2
MLC2
Neuritogenesis
Impairment of
Neuritogenesis
Schematic representation of the relationship between ezrin and RhoA/Rho
kinase/MLC2 pathway in neuritogenesis.
50
A
B
Vil2+/+
Vil2kd/kd
Cx
Hip
Am
Hy
2-11
Ezrin is not detected in Vil2kd/kd mice brain.
Immunohistochemical analyses of ezrin expression in adult (8 week-old) wild-type (A) and
Vil2kd/kd (B) mouse brains using an anti-ezrin antibody (clone, 3C12). Scale bars, 1 mm. Cx,
cerebral cortex; Hip, hippocampus; Am, amygdala; Hy, hypothalamus.
51
A
Vil2+/+
Vil2kd/kd
C
B
140
450
120
350
***
300
250
200
150
100
Number of neurons (%)
Length of layers (µm)
400
80
60
40
20
50
0
0
+/+
2-12
100
+/+
kd/kd
kd/kd
Structure of cerebral cortex in the adult Vil2+/+ and Vil2kd/kd mice.
A, Nissl staining in the cerebral cortex of Vil2+/+ and Vil2kd/kd mice. The length of cortical
layers (B), and the number of Nissl-positive neurons (C) in Vil2+/+ and Vil2kd/kd mouse brains.
Three independent experiments were performed. ***p < 0.001, Student’s t test. Data
represent mean ± SE. Scale bar, 100 µm.
52
A
βIII-tubulin
Vil2+/+
Vil2kd/kd
a
b
a
2-13
b
Impairment of neuronal morphology in Vil2kd/kd cerebral cortex.
Immunohistochemistry of Vil2+/+ and Vil2kd/kd cerebral cortices using antibodies against class
III β-tubulin (A) and MAP2 (B). High magnification images in Vil2+/+ (a) and Vil2kd/kd (b)
were shown. Scale bars, 50 µm.
53
B
MAP2
Vil2+/+
Vil2kd/kd
a
b
a
2-13
b
Continued.
Immunohistochemistry of Vil2+/+ and Vil2kd/kd cerebral cortices using antibodies against class
III β-tubulin (A) and MAP2 (B). High magnification images in Vil2+/+ (a) and Vil2kd/kd (b)
were shown. Scale bars, 50 µm.
54
A
B
Vil2+/+
Vil2kd/kd
Vil2+/+
Vil2kd/kd
C
D
150
100
50
0
*
4
3
2
1
0
+/+
2-14
6
5
**
200
Number of
basal dendrites
Length of
apical dendrites (µm)
250
kd/kd
+/+
kd/kd
Ezrin knockdown causes abnormal dendritic outgrowth.
Golgi staining of Vil2+/+ and Vil2kd/kd cerebral cortices. Representative images of apical
dendrites (A) and basal dendrites (shown by triangles, B) of layer V pyramidal neurons were
shown. C, Quantification of length of apical dendrites (C) and number of basal dendrites (D)
in the Vil2+/+ and Vil2kd/kd layer V pyramidal neurons. *p < 0.05, **p < 0.01, Student’s t test.
Data represent mean ± SE. Scale bar, 5 µm.
55
A
6-OHDA (6 w)
Vehicle (6 w)
1 day
7 day
1 day
7 day
B
***
I
3-1
6-OHDA (6 w)
II
Time in target quadrant (%)
Time in target quadrant (%)
Vehicle (6 w)
50
45
40
35
30
25
20
15
10
5
0
III
IV
50
45
40
35
30
25
20
15
10
5
0
I
II
III
IV
Intrastriatal Injection of 6-OHDA Impairs Long-Term Spatial Memory at 6
weeks.
A, Representative traces of the swimming paths of vehicle- and 6-OHDA-microinjected mice
at the 1 and 7 d of acquisition trials. 6-OHDA-microinjected mice had significantly impaired
water maze performance that was associated with (B) a preference of the target quadrant
(quadrant I). ***p < 0.001, Student’s t test. Data represent mean ± SE.
56
A
6-OHDA (18 w)
Vehicle (18 w)
1 day
7 day
1 day
7 day
B
Vehicle (18 w)
40
6-OHDA ( 18 w)
*
Time in target quadrant (%)
Time in target quadrant (%)
45
35
30
25
20
15
10
5
0
I
3-2
II
III
IV
50
45
40
35
30
25
20
15
10
5
0
I
II
III
IV
Intrastriatal Injection of 6-OHDA Impairs Long-Term Spatial Memory at 18
weeks.
A, Representative traces of the swimming paths of vehicle- and a 6-OHDA-microinjected
mouse at the 1 and 7 d of acquisition trials. 6-OHDA-microinjected mice had significantly
impaired water maze performance that was associated with (B) a preference of the target
quadrant (quadrant I). *p < 0.05, Student’s t test. Data represent mean ± SE.
57
A
Vehicle
3-3
6-OHDA
Striatum
Striatum
Substantia nigra
Substantia nigra
Immunohistochemical and neurochemical analyses in the nigrostriatal DA
system.
A, Representative photomicrographs of striatum and substantia nigra of vehicle- or
6-OHDA-microinjected mice at 6 weeks after the microinjection. These slices were
immunostained by an antibody against TH. Scale bars, 1 mm (in striatum), 200 µm (in
substantia nigra).
58
C
30
6
25
5
DOPAC levels (pmol/mg tissue)
DA levels (pmol/mg tissue)
B
20
15
10
*
5
0
3-3
4
3
2
1
0
Vehicle 6-OHDA
*
Vehicle 6-OHDA
Continued.
B, C, Striatal dopamine (DA) and DOPAC contents after intrastriatal injection of 6-OHDA.
Amounts of DA (B) and DOPAC (C) in the striatum from treated mice were measured using
an HPLC-ECD system. *p<0.05, Student's t test. Data represent mean ± SE.
59
B
Radixin
*
250
% of vehicle
Ezrin
300
6-OHDA
Vehicle
A
200
150
100
Moesin
GAPDH
3-4
50
0
Ezrin
Radixin
Moesin
Level of ERM proteins in the hippocampus.
A, Western blotting for ezrin, radixin, moesin and GAPDH (control protein) presented protein
bands of 82, 80, 75 and 37 kDa, respectively. B, Quantitative results were obtained by
measuring the optical density of each band using computerized image analysis. *p<0.05,
Student's t test. Data represent mean ± SE.
60
A
B
RhoA
LRRK2
PI3K
ROCK
PKC
?
Ezrin
Ezrin
Radixin
Moesin
RhoA
Phosphorylation
ROCK
Inhibition
4-1
Ezrin acts as negative regulator for RhoA in up-stream of RhoA/ROCK.
61