ACTIVATION OF THE UNFOLDED
PROTEIN RESPONSE IN GAUCHER
DISEASE
Gali Maor and Mia Horowitz
EWGGD, Paris June 27-30, 2012
Gaucher Disease
Due to the defective activity of
lysosomal glucocerebrosidase (GCase)
in Gaucher disease there is:
★Accumulation of substrate
★ERAD of mutant GCase variants
EP Gaucher
ERAD of mutant GCase variants
ERAD has been shown for the following genotypes
(mutations):
N370S/N370S, N370S/L444P, N370S/V394, N370S/recTl,
N370S/V394L, R120W/R120W, L444P/R120W,
G202R/G202R+M362I, L444P/P415R, G202R/G202R,
K157Q/D140H+E326K, R463C/?, R131C/R131C,
L444P/L444P, L444P/R415P, D409H/D409H
Every ER resident or passenger protein undergoes ERQC
and, if needed, ERAD!!!
Quantitation of ERAD
TOTAL AMOUNT
(% OF NORMAL)
A
AMOUNT IN
LYSOSOMES (%
OF A)
B
N370S/N370S
47.3
79.8
37.7
N370S/N370S
70.6
89.8
63.4
N370S/N370S
49.8
84.9
42.2
N370S/V394L
45.2
27.2
12.2
R463C/?
31.9
63.9
20.4
TYPE 3
14.8
34.5
4.9
TYPE2
15.5
4.1
0.6
P415R/L444P
20.3
1.9
0.4
WT
100
89.8
89.8
ERAD
GENOYYPE
AXB
There is a correlation between the severity of the disease and
the ERAD level
ERAD of mutant GCase variants
ERAD of mutant GCase variants may lead to
pathogenesis
The Unfolded Protein Response
1
3
Douglas M. Cyr & Daniel
N. Hebert, Embo
Reports April 2012
2
1
(CHOP)
Test UPR by :
1.Changes in mRNA and protein levels of Bip, CHOP
2.Phosphorylation of eIF2α
3.Splicing of Xbp1 mRNA
Gaucher Disease
CUPR exists in GD derived cells
CUPR also exists in carriers of GD mutations
CUPR upregulates GBA transcription
Relative mRNA quantity
UPR in GD patients and carriers:
Bip and CHOP mRNA levels
12
CHOP
10
BiP
** P <
0.01
**
*
8
** *
**
**
**
* P < 0.05
*
*
6
4
* *
**
3
4
*
*
2
0
1
2
5
6
Patient Number
7
8
9
Patient number
Genotype
Disease type
Relative Chop
mRNA
Relative BiP
mRNA
1
WT
-
1
1
2
N370S/N370S
1
5.5±2.6*
6.2±3.1**
3
L444P/R120W
2
2.8±1.4*
4.2±0.6*
4
G202R/G202R+M362I
2
3.3±1.6*
3±1.7*
5
L444P/L415W
2
8.9±2.1**
6±2*
6
G202R/G202R
2
8.7±3.1**
8.5±4*
7
K157Q/D140H+E326K
1 (severe)
4.3±2.19 *
3.6±1.48 *
8
K157Q/D140H+E326K
1
9.2±2.94 **
7.2±2.87**
9
N370S/N370S
1 (severe)
4.1
3.4 ±1.82*
BiP
Relative protein
quantity
UPR in GD patients:
Bip and CHOP proteins
4
3,5
3
2,5
2
1,5
1
0,5
0
*
*
** P < 0.01
β-tubulin
* P < 0.05
Relative protein
quantity
Genotype
12
10
8
6
4
2
0
**
*
**
*
CHOP
β-tubulin
Gnotype
Bip and CHOP mRNA and protein levels are significantly
elevated in GD fibroblasts
UPR in GD patients: Xbp1 splicing
Forward primer
Reverse primer
No splicing
Splicing
Forward primer
Reverse primer
Forward primer
Reverse primer
Spliced xbp1
mRNA quantity
1,2
1
**
*
0,8
0,6
**
0,4
0,2
0
GAPDH
Level of Xbp1 splicing is significantly
elevated in GD fibroblasts
Genotype
*
P-EIF2α
EIF2α
Relative protein quantity
UPR in GD patients:
Phosphorylation of eIF2α
5
4,5
4
3,5
3
2,5
2
1,5
1
0,5
0
**
**
Genotype
* P < 0.01
** P < 0.05
Levels of phosphorylated eIF2α protein are
significantly elevated in GD fibroblasts
Unfolded Protein Response in GD
Relative mRNA quantity (%)
Relative GCase activity
(%)
6
120
100
80
60
40
20
0
0
1
CBE treatment (days)
10
5
*
* P < 0.05
CHOP
*
BiP
4
3
2
1
0
WT
WT+CBE
WT+Tg
Patient
Disease type
Relative Chop
mRNA
Relative BiP mRNA
WT
-
1
1
WT+CBE treatment
-
0.82±0.24
0.86±0.41
WT+Tg treatment
-
5.04±0.42*
3.9±1.28*
Substrate accumulation does not lead to UPR
UPR in GD mice (Grabowski):
Bip and CHOP proteins
A
4,5
Relative mRNA quantity
4
3,5
* P < 0.05
CHOP
BiP
Genotype
Relative Chop
mRNA
Relative BiP
mRNA
WT
1
1
2
D409H/D409H
0.155±0.04
0.69±0.08
1,5
D409V/D409V
*2.7±0.5
*2.02±0.4
KO
0.7±0.06
1.08±0.3
WT+Tg
*3.81
*2.91
*
3
*
2,5
1
0,5
0
WT
D409H/D409H D409V/D409V
KO
Thapsigargin
Genotype
Bip and CHOP mRNA levels are significantly
elevated only in D409V homozygous mice!!!
UPR in GD mice: Xbp1 splicing
A
Forward
primer
Reverse
primer
No splicing
Splicing
Forward
primer
Relative mRNA quantity
Reverse
primer
Forward
primer
Reverse
primer
3,5
3
*
* P < 0.05
2,5
*
2
1,5
1
0,5
0
Genotype
Level of Xbp1 splicing is significantly elevated in D409V
homozygous mouse fibroblasts
Gaucher Disease
CUPR exists in GD derived cells
CUPR also exists in carriers of GD mutations
CUPR upregulates GBA transcription
Gaucher Disease
CUPR exists in GD derived cells
CUPR also exists in carriers of GD mutations
CUPR upregulates GBA transcription
Gaucher Disease
CUPR exists in GD derived cells
CUPR also exists in carriers of GD mutations
CUPR upregulates GBA transcription
Unfolded Protein Response in
GD carriers
8
CHOP
7
BiP
*
6
5
4
3
2
1
4
Relative spliced Xbp1 mRNA
quantity
Relative mRNA quantity
9
3,5
3
**P < 0.01
**
* P < 0.05
**
2,5
2
1,5
1
0,5
0
0
Normal
WT/N370S
Genotype
WT/84GG
Normal
WT/84GG
Genotype
WT/N370S
There is UPR in GD carriers even without a
detected protein
Gaucher Disease
CUPR exists in GD derived cells
CUPR also exists in carriers of GD mutations
CUPR upregulates GBA transcription
GBA mRNA levels in GD fibroblasts
**
There are elevated levels of
GBA mRNA in GD derived
fibroblasts
Does it result from response
to UPR??????
GBA mRNA levels in GD fibroblasts
**
Relative GCase mRNA expression
9
8
7
6
* P < 0.05
**
**
*
*
*
*
5
4
3
*
2
1
0
Genotype
Genotype
Disease type
Relative mRNA
quantity
WT
-
1
N370S/N370S
1
*4.4±1.2
L444P/R120W
2
3.3±1.7
R131C/R131C
2
3±1.1
L444P/L444P
3
5.1±1.3*
N370S/WT
-
*5.7±0.8
WT+CBE
-
0.99±0.43
84GG/WT
-
1.7±0.1*
N370S/WT
-
5.3±2.3*
There are
elevated levels of
GBA mRNA in GD
derived and
carrier derived
fibroblasts
GBA mRNA levels in GD fibroblasts
**
In UPR there are genes that are upregulated
by the transcription factor CHOP.
Does the GBA gene promoter have a UPR
Responsive element?
GBA mRNA levels in GD fibroblasts
CHOP
-350
AP-1
PEA
+1
CAAT
GBA
Vector
delivery
to living
cells
Luciferase in HEK293 cells
1,6
No Thapsigargin
* P < 0.01
*
1,4
Thapsigargin
Luminscence
1,2
1
0,8
0,6
0,4
0,2
0
CAAT mut
Normal
Upon UPR induction (with thapsigargin) normal (but
not CAAT mutated) GBA promoter activity elevates.
GBA promoter binds CHOP
Gaucher Disease
Since UPR in general may lead to cell death,
UPR in GD may lead to death of cells as well.
Dopaminergic cells (Parkinson disease)?!
Conclusions
There is UPR in GD patients
There is UPR in one animal models (out of few we have tested)
There is UPR in GD carriers including the 84GG carriers
There is upregulation of the GBA gene in patients in response
to UPR, through CHOP binding
Even without ERAD there is UPR (ER stress) that may lead
to death of cells for example in the case of 84GG carriers
and PD
ERAD of mutant GCase variants
Gaucher disease
(mutant GCase)
Parkinson disease
ERAD of mutant GCase variants may lead to
pathogenesis
Thanks
Collaborator:
Dr. M. Filocamo
Gali Maor
Thank you for your attention