close

Вход

Забыли?

вход по аккаунту

?

Why and how to activate parkin for neurodegeneration therapy

код для вставки
Nilotinib and Bosutinib in Parkinson’s
Models
Charbel Moussa, MD, PhD
Assistant Professor
Laboratory for Dementia and Parkinsonism
Department of Neuroscience
Georgetown University Medical Center
Outline
•
•
•
•
WHY
Parkin and a-Synuclein in neurodegeneration (PD )
Parkin-beclin-1 interaction and autophagic a-Synuclein clearance
Parkin is an excellent therapeutic target in PD
• HOW
• Parkin activation via tyrosine kinase inhibition leads to autophagic
a-Synuclein clearance.
• Conclusions
• Nilotinib and Bosutinib are therapeutic candidates for Parkinsonism
Parkinson’s disease (PD) is predominantly sporadic, but some disease-causing
mutations suggest a genetic component in the pathogenesis of this disorder
Parkinson's disease
susceptibility loci
Locia
Genetic variants (penetrance)
Full
Partialb
A53T, A30P, E46K
REP1, rs2736990,
duplication,
rs11931074
triplication
SNCA (PARK1/4)
Chromosome
4q21
LRRK2 (PARK8)
12p12
R1441C/G/H, I2020T,
Y1699C, G2019S
R1628P, G2385R,
rs1994090
GBA
MAPT
1q21
17q21
—
—
N370S, L444P, others
H1 haplotype,
rs393152
BST1
PARK16c
4p15
1q32
—
—
rs4538475
rs947211, rs823128
Autosomal dominant mutations in aSNCA cause late onset (age-related>65
years) PD
Familial and sporadic PD are both characterized by death of dopaminergic neurons in the Substantia
Nigra pars compacta (SN) AND cytosolic a-Synuclein inclusions known as Lewy bodies (LBs)
Mutations in the autosomal recessive gene Parkin are the most common causes (50%)
of familial autosomal recessive early onset (Juvenile 14+ years old) PD
 Genea
 parkin (PARK2)
 6q25
 Chromosome
 Protein function
 E3 ubiquitin protein ligase
 Clinical phenotypeb
 AR-JP, often indistinguishable 
from PD, but also with
dystonia, reflex changes
Mitochondrial serine-threonine  AR-JP, similar to parkin

kinase
Unknown, possible role in
 AR-JP, similar to parkin

stress response
Lysosomal cation transporter  Kufor-Rakeb disease (AR),

ATPase
parkinsonism and dementia
E3 ubiquitin protein ligase
 Pallido-pyramidal syndrome

(AR), dystonia and
parkinsonism
Phospholipase A2
 NBIA-2 (AR), dystonia,

parkinsonism, and dementia
Pantothenate kinase
 NBIA-1 (AR), dystonia,

parkinsonism, and dementia
Copper transporter ATPase
 Wilson's disease (AR),

parkinsonism, liver failure,
neuropsychiatric symptoms
Progranulin, growth factor
 Frontotemporal dementia (AD), 
dementia with or without
parkinsonism
Unknown, enriched in Golgi
 Spinocerebellar ataxia (AD),

apparatus
with or without parkinsonism
GTP cyclohydrolase I, dopamine Dopa-responsive dystonia (AD), 
biosynthesis
occasionally phenocopied by
parkin
 Pathology
Nigrostriatal degeneration, no
inclusions
 PINK1 (PARK6)
 1p36

Unknown
 DJ-1 (PARK7)
 1p36

 ATP13A2 (PARK9)
 22q13

 FBXO7 (PARK15)
 22q12

 PLA2G6 (PARK14)
 22q13

 PANK2
 20p13

 ATP7B
 13q14

 GRN
 17q21

 ATXN2 (SCA2)
 12q24

 GCH1 (DYT5)
 14q22

Unknown
Unknown
Unknown
Neuroaxonal dystrophy, iron
accumulation
Neuroaxonal dystrophy, iron
accumulation
Basal ganglia copper
accumulation and degeneration
Ubiquitin/TDP-43 inclusions
Polyglutamine-repeat nuclear
inclusions
No nigral degeneration, no
inclusions
IMPORTANT, sporadic PD pathology includes cytosolic a-Synuclein inclusions or Lewy
bodies (LBs), while loss of parkin function (via mutations) is not usually associated with
cytosolic LBs
Possible quality control process in the cell
1- Ubiquitin-proteasome system?
2- Autophagy-lysosome system?
• a-Synuclein impairs autophagy (quality control mechanism) and leads to
autophagosome accumulation, resulting in neuronal death
• Parkin is a cytosolic E3 ubiquitin ligase that targets specific substrates for
proteasomal and autophagic degradation
• α-Synuclein may be among several parkin substrates in the human brain
• Could parkin-mediated autophagic clearance of a-Synuclein explain SN neuron
death, despite lack of LBs formation and genetic relationship between parkin and
a-Synuclein?
Possible mechanisms of Parkin activation and amyloid
clearance
Normal conditions
Inactive
Parkin
Aging/Pathology
Inactive
Parkin
Ubiquitin
Ubiquitination
Parkin
active
De-ubiquitination?
Parkin
unstable
degradation
Clearance
Amino acids
Decreased solubility/
inactivity
Interacts
Parkin
Beclin-1
Maturation
Fusion
Amyloids
Phagophore-AV10
Autophagosome-AV20
Lysosome: degradation
Proteasome
Proteasome
Recycling
Substrate
ubiquitination
Ubiquitin
Insoluble parkin is associated with loss of tyrosine hydroxylase (TH+) neurons in sporadic PD.
Caudate
Ctl # 1683
PRK8+DAPI
18 mm
Caudate
PD/AD #
2215
PRK8+DAPI
18 mm
H
PRK8+GFAP
PRK8+GFA
P
PRK8+GFAP
30 mm
40
TH+DAB
25 mm
mm
30 mm
TH+DAB
25 mm
Midbrain/SN
SN- PD
Control
# 1855
PRK8+DAPI
18 mm
PRK8+GFAP
30 mm
25 mm
TH+DAB
Midbrain/SN
PD # 2315
PRK8+DAPI
18 mm
30 mm
PRK8+GFAP
25 mm
TH+DAB
Midbrain/SN
PD/Dementia #
2243
AB5112+DAPI
18 mm
AB5112+GFAP
30 mm
25 mm
TH+DAB
Thus, despite the lack of genetic link
between parkin and a-Synuclein, the
role of parkin is supported in both
familial and sporadic PD.
Subcellular fractionation to isolate autophagic vacuoles (AVs)
LC3
LC3
LC3
LC3
20-10%
24-20%
Metrazimade
10% -Lys
Human parkin ELISA in autophagic
vacuoles form human samples
(% control)
Phagophore-AV10
Debris
LAMP
Autophagosome-AV20
Control
Parkinson
200
180
160
140
*
*
120
100
80
AV-10
AV-20
Lysosome
LAMP
Lysosome
Human a-Synuclein ELISA in autophagic
vacuoles form human samples
(% control)
Debris
Control
Parkinson
200
*
180
160
140
*
120
100
80
AV-10
AV-20
Lysosome
50K
D-
Parkin
(Soluble)
42K
D-
b-actin
Case #
50KD-
50KD-
42KD-
Control
Parkinson
705 417 2052 487
2140 2074 1877 1955
100
*
50
*
50
0
Control
Phospho-parkin
(ser 378)
(insoluble)
Actin
(insoluble)
Control
Parkinson
Parkin
Phospho-parkin
300
*
200
*
100
0
Control
Soluble parkin
Phospho-parkin
Insoluble parkin
100
50
0
Co
100
0
Parkin
(insoluble)
150
Human parkin EILISA
(% control)
705 417 2052 487 2140 2074 1877 1955
Parkin level by densitometry (AU)
Case #
Parkinson
150
Parkin
150
Insoluble parkin levels relative
to actin (% control)
Control
Soluble protein levels relative
to actin (% control)
Parkin soubility is decreased in sporadic PD
ol
ol
ol
on
on
on
ntr
ntr
ntr
ins
ins
ins
o
o
k
k
k
r
r
r
C
C
Pa
Pa
Pa
Parkinson
Parkinson
120-
Total-Abl
120-
T412-Abl
42-
Actin
300
*
*
Total-Abl
100
120-
T412-Abl
42-
*
300
*
200
*
100
Lv-Abl
Lv-LacZ
Lv-Abl
Lv-LacZ
Lv-Abl
Abl/actin
120-
Total-Abl
120-
T412-Abl
62-
Syn
17-
0
Lv-LacZ
Lv-Syn
42-
Actin
Parkinson
Actin
Protein level/ actin (%control)
T412 Abl/Abl
Lv-Syn
120-
Lv-LacZ
Protein level/actin (%control)
Abl/actin
Lv-LacZ
200
Control
400
Syn
17-
0
Syn/actin
Lv-Syn
p-Abl/Abl
Lv-LacZ
Abl/actin
400
Lv-Syn
Parkinson
Densitometry (% control)
control
Lv-LacZ
Abl is a tyrosine kinase that is increased/activated in sporadic human PD brains
T412 Abl/Abl
300
Syn/actin
* *
200
*
100
0
Lv-LacZ
Lv-Abl
Tyrosine kinase inhibitors (TKIs) induce autophagy and are FDA-approved and welltolerated treatments for chronic myelogenous leukemia (CML).
Syn+Nilo
Syn+DMSO
Syn+Nilo
Syn+DMSO
Syn+Nilo
Syn+DMSO
Tyrosine kinase inhibitors (TKIs), Nilotinib and Bosutinib penetrate the brain at sufficient
concentrations, induce autophagy and clear monomeric and aggregated a-Synuclein
62-
Nilotinib
Bosutinib
Imatinib
DMSO
400
Syn
3917-
Human
Syn
17-
1917-
LC3-I
LC3-II
1917-
LC3-1
LC3-II
50-
Beclin-1
50-
Beclin-1
55-
Atg-12
42-
Actin
200
55-
Atg-12
42-
Actin
0
4hr
6hr
8hr
10hr
12hr
Syn/actin
300
*
#
LC3II/LC3I
#
*
200
#
100
**
*
0
Control
LC3II/actin
A53T+DMSO A53T+Nilo
Beclin-1/actin
Atg12/actin
200
Densitometry (% control)
2 hr
Densitometry (% control)
Concentration (nM)
600
*
150
*
Syn/actin
LC3II/actin
LC3II/LC3I
Beclin-1/actin
100
Atg12/actin
*
50
**
0
Lv-Syn+DMSO
Lv-Syn+Nilo
Nilotinib clears human a-Synuclein in transgenic and gene transfer models
A
A
Tg-A53T+DMSO
B
Lv-LacZ+Nilo
B
Lv-Syn+DMSO
VTA
SN
A53T+DMSO
DF
D
0.4 mm
Substantia nigra
Animal 1
0.2nm
Substantia nigra
H
I
18 mm
Human Syn+DAB
E
F
TH+DAB
G
St
Brainstem
r
A53T+DMSO
E
A53T+Nilo
Animal 1 1
C
TH+DAB
F
A53T+Nilo
65mm
Animal 3
Animal 2
TH+DAB
Human Syn+DAB
G
J
K
L
25 mm
Ctx
A53T+DMSO
H
25mm
A53T+Nilo
Animal 3 4
Animal 3
TH+DAB+Nissl
M
N
O
25 mm
Human Syn+DAB
Lv-Syn+Nilo
65mm
Human Syn+DAB
Adapted from www.sciencedirect.com
C
A53T+DMSO
Tg-A53T+Nilo
Hip-CA3
10mm
Nissl
EM studies show that Nilotinib clears autophagic vacuoles in SN of PD models
Lv-Syn+Nilo
Lv-Syn+DMSO
A
B
Animal 1
Animal 1
nucleus
AV
Cytosol
AV
SN
C
D
nucleus
Animal 2
Animal 2
AV
AV
AV
Cytosol
SN
E
F
Animal 3
Animal 3
AV
AV
AV
SN
Nilotinib and Bosutinib decrease brain and blood a-Synuclein levels
Control
Control
*
600
*
400
200
0
1000
800
*
600
*
400
200
0
l
Ct
SO
3T
A5
M
+D
3
A5
s
Bo
T+
(5
)
/kg
g
m
3T
A5
o
+B
)
/kg
g
1m
s(
Human a -Synuclein (ng/ml) in
the blood of A53T-Tg mice
A53T + Nil (1mg/kg)
A53T + DMSO
2000
A53T + Nil (5mg/kg)
*
1500
A53T + Nil (1mg/kg)
*
1000
Human a-Synuclein (ng/ml) in
the blood of A53T-Tg mice
A53T + Nil (5mg/kg)
800
Human a-Synuclein (ng/ml) in
the brain of A53T-Tg mice
Human a-Synuclein (ng/ml) in
the brain of A53T-Tg mice
A53T + DMSO
500
0
2000
1500
*
1000
*
500
0
l
Ct
g)
g)
g/k
g/k
m
m
5
(1
3
s(
os
A5
Bo
B
+
+
3T
3T
A5
A5
S
DM
T+
O
Summary -1
Parkin solubility is decreased in sporadic PD, suggesting that loss of parkin
function is due to changes in protein stability independent of disease-causing
mutations
Insoluble parkin is associated with loss of tyrosine hydroxylase (TH+) neurons
in sporadic PD
Insoluble parkin is associated with loss of TH+ neurons and decreased interaction with the key
autophagy enzyme Beclin-1 in sporadic PD.
A
Midbrain Ctl
Case # 2201
B
C
35 mm
75 mm
TH+DAB
Parkin+GFAP+DAPI
D
Midbrain PD
Case # 2315
35 mm
E
PLA: Parkin+beclin-1+DAPI
F
35 mm
75 mm
Parkin+GFAP+DAPI
G
Midbrain Ctl
Case # 1252
Parkin+DAPI
J
Parkin+DAPI
Midbrain PD
Case # 2312
TH+DAB
35 mm
PLA: Parkin+beclin-1+DAPI
H
I
Beclin-1+DAPI
Beclin-1+parkin
K
L
Beclin-1+DAPI
Beclin-1+parkin
35 mm
35 mm
Parkin is a hybrid HECT (Homologous to E6-AP C Terminal) and RING (Really Interesting
New Gene) E3 ubiquitin ligase that is endogenously inactive
LC3
Debris
LC3
Phagophore-AV10
LC3
LAMP
LC3
Autophagosome-AV20
LAMP
Lysosome
98
62
50
39
45
IgG Input
Pa
rk
+
Pa DM
rk SO
+D
+
M Ub
SO
E +K
Pa 1-E 0
rk
2
+N -E
Pa
P
il 3
rk
+N ark o+
ilo +N Ub
+U ilo
b +K
Pa +M 0
rk G1
+B 3
Pa
o 2
rk Pa
+B rk s+
os +N Ub
+U ilo
b+ + K
M 0
G
13
2
Par+Ub+MG132
Par+Ub+Bos+MG132
Par+Ub+Bos
Par+Ub+MG132
53
T
+B
os
+N
ilo
SO
50
0
Parkin self-ubiquitination
(% control)
A
53
T
+D
M
*
Par+Ub+Nilo
A
53
T
T
150
Par+Ub+Nilo+MG132
A
W
Parkin (ng/ml) in A53T mice
Nilotinib and Bosutinib increase parkin activity
*
100
188
IP: Ub
WB: parkin
28
Actin
500
400
300
*
*
*
*
200
100
0
TKIs reverse the failure of parkin-Beclin-1 interaction in transgenic models of a-Synuclein
A
SN Wild type mice
Parkin+Beclin-1+DAPI
D
SN Tg-A53T+Nilo
Parkin+Beclin-1+DAPI
G
G
45 mm
45 mm
SN Tg-A53T+DMSO
Parkin+Ub+DAPI
45 mm
B
SN Parkin-/- mice
45 mm
Parkin+Beclin-1+DAPI
E
SN Tg-A53T+Bos
Parkin+Beclin-1+DAPI
H
45 mm
SN Tg-A53T+Nilo
Parkin+Ub+DAPI
45 mm
C
SN Tg-A53T+DMSO
Parkin+Beclin-1+DAPI
FF
SN Wild type mice
Parkin+Ub+DAPI
I
45 mm
45 mm
SN Tg-A53T+Bos
Parkin+Ub+DAPI
45 mm
Nilotinib and Bosutinib fail to clear a-Synuclein in parkin-/- mice
Lv-LacZ+DMSO
B
Lv-LacZ+Bos
C
Lv-Syn+DMSO
D
Lv-Syn+Bos
C57BL/6
A
hSyn+DAB
35 mm
35 mm
F
SN
35 mm
H
G
C57BL/6
E
35 mm
TH+DAB
Lv-LacZ+DMSO
J
Lv-Syn+DMSO
18 mm
K
Lv-Syn+Bos
SN
18 mm
Lv-Syn+Nilo
L
Park-/-
I
18 mm
18 mm
hSyn+DAB
Lv-LacZ+DMSO
N
Lv-Syn+DMSO
35 mm
O
Lv-Syn+Bos
SN
P
35 mm
Lv-Syn+Nilo
Park-/-
M
35 mm
35 mm
TH+DAB
R
Lv-LacZ+DMSO
400 mm
S
Lv-Syn+DMSO
Lv-Syn+Bos
SN
T
400 mm
Lv-Syn+Nilo
Park-/-
Q
400 mm
400 mm
TH+DAB
30 mm
30 mm
30 mm
SN
30 mm
Lv
-S
Human a-Syn (ng/ml) in autophagic
yn
+D
Lv M compartments of Lv-Syn injected SN
-S S
y O
Lv n+ (W
-S Ni
T
yn lo )
(
+B W
Lv
os T)
-S
yn
(W
+D
T)
Lv M
-S S
y O
Lv n+ (P
-S Ni ar
yn lo k -/+B (P )
os ark -/
(P )ar
k -/Lv
)
-L
ac
Z+
D
M
S
O
TKIs facilitate autophagic a-Synuclein clearance in wild type but not parkin-/- mice
*
200
*
AV10
AV20
Lysosome
*
*
* *
* *
150
100
50
Brain human a-Synuclein (ng/ml)
0
300
200
100
* *
0
O ilo os T) T) T) -/- /-) /-) /-)
MSZ+N Z+BO (Wo (Ws (WPark ark- ark- arkD
P P P
+ c
c
il
o
cZ La La DMSn+N n+B
O ( ilo ( os (
S
La
N
M + +B
n+ Sy Sy
+D Syn Syn
Sy
n
Sy
Interacts
Parkin
Beclin-1
Maturation
Fusion
Amyloids
Phagophore-AV10
Autophagosome-AV20
Lysosome: degradation
Blood human a -Synuclein (ng/ml)
250
50
40
30
20
10
* *
0
O ilo T) T) T) T) -/- /-) /-) /-)
MSZ+N s (WO (Wo (Ws (WPark ark- ark- arkD
P P P
+ c
o
il
o
cZ La n+B DMSn+N n+B
O ( ilo ( os (
S
La
N
+
y
y
y
M + +B
S yn S S
+D Syn Syn
S
n
Sy
Dopamine
Dopamine
HVA
600
*
*
400
*
*
** *
200
*
*
*
**
0
Rotarod performance (% control)
il
s
s
O
O
//-)
/-)
/-)
/-)
MSilo/Bo DMS yn+Nn+Bo ark- ark- ark- ark- arkD
P
P
P
P
P
+
y
S
+
O ( SO ( ilo ( os (
cZ cZ+ N -Syn Lv- Lv-S
S
a
N
B
L
M
M
+
+
Lv
+D +D yn yn
Lv Lv-La
cZ -Syn Lv-S Lv-S
a
-L Lv
Lv
L
Dopamine and HVA % control
Dopamine and HVA levels (% control)
TKIs control dopamine levels and improve motor performance in a parkin-dependent manner
HVA
400
200
100
*
50
0
o
o
SO
SO
Nil
Nil
DM
DM
n+
Z+
+
+
y
c
Z
-S
yn
-La
ac
-S
Lv
Lv
v-L
Lv
*
*
*
100
0
M
+D
WT
150
*
300
SO
O
ilo
os
ilo
os
MS
+N
+B
+N
+B
D
T
T
T
T
+
3
3
W
W
3T
A5
A5
A5
Possible mechanisms of Parkin activation and amyloid
clearance
Normal conditions
Inactive
Parkin
TKIs
Parkin
active
Aging/Pathology
Inactive
Parkin
Ubiquitin
Ubiquitination
Ubiquitin
De-ubiquitination?
DUBs as quality control
degradation
Clearance
Amino acids
Decreased solubility/
inactivity
Interacts
Parkin
Beclin-1
Maturation
Fusion
Amyloids
Phagophore-AV10
Autophagosome-AV20
Lysosome: degradation
Proteasome
Substrate
ubiquitination
Proteasome
Recycling
Parkin
unstable
Loss of quality control:
DUBs de-ubiquitinate
Parkin, reducing its
proteasomal recycling
Summary - 2
• Parkin is inactivated/insoluble in sporadic late onset PD
• Parkin responds to a-Synuclein stress and forms complexes with Beclin-1 to
facilitate autophagic clearance
• TKIs activate parkin via ubiquitination and control dopamine levels and aSynuclein clearance
• Lack of LBs in parkin-linked early onset PD may be due to loss of aSynuclein into the blood due to lack of sequestration by parkin, while LBs in
late onset sporadic PD may reflect age-dependent failure of parkin activity
(insolubility) to clear protein over time
• TKIs are great therapeutic candidates for PD and other related disorders
An ON/OFF strategy that exploits post-mitotic (non-dividing) neuronal biology
to induce clearance of intraneuronal debris,
without causing the self-cannibalization seen in rapidly dividing tumor cells
CLINICAL TRIALS !!
Disease onset
Nilotinib
Bosutinib
1/100s of
clinical dose
AUTOPHAGY ON
AUTOPHAGY ON
Nilotinib Bosutinib
4-8hrs 6-12hrs
Time in years
AUTOPHAGY OFF
Nilotinib Bosutinib
4-8hrs 6-12hrs
Time in days
Parkin
Beclin-1
Parkin
Debris accumulation:
Tau, b-amyloid
a-Synuclein, TDP-43,
Damaged organelles
Neurodegeneration
Autophagic clearance
Re-accumulation
Beclin-1
Halting
neurodegeneration
Funding:
NIH-NIA AG30378
Georgetown University
Merck & Co
THANK YOU!
Документ
Категория
Без категории
Просмотров
6
Размер файла
3 176 Кб
Теги
1/--страниц
Пожаловаться на содержимое документа