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 AMOUNT IN
ERAD (% OF NORMAL) LYSOSOMES (% AXB
A OF A)
GENOYYPE 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

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

Douglas M. Cyr & Daniel

N. Hebert, Embo
2 Reports April 2012
3

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

UPR in GD patients and carriers:
Bip and CHOP mRNA levels
12 CHOP ** P < ** * **
**
0.01

Relative mRNA quantity

10 BiP **
** * P < 0.05
8
* *
*
6 *
*
* * **
4

0
1 2 3 4 5 6 7 8 9
Patient Number

Patient number Genotype Disease type Relative Chop Relative BiP

mRNA 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*
 UPR in GD patients:
Bip and CHOP proteins
4

Relative protein
3.5 *
3 *

quantity
2.5
2
1.5
1
0.5
0
BiP
** P < 0.01
β-tubulin
* P < 0.05
Genotype

12

Relative protein
10 **
*

quantity
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

Splicing No splicing

Forward primer Reverse primer Forward primer Reverse primer

1.2
**

mRNA quantity
1 *
*
0.8
0.6 **
0.4
Spliced xbp1 0.2
0

GAPDH

Level of Xbp1 splicing is significantly

elevated in GD fibroblasts
Genotype
 UPR in GD patients:
Phosphorylation of eIF2α

5
**

Relative protein quantity

4.5
4 **
3.5
3
2.5
2
1.5
1
0.5
P-EIF2α 0

EIF2α

* P < 0.01
Genotype
** P < 0.05

Levels of phosphorylated eIF2α protein are

significantly elevated in GD fibroblasts
 Unfolded Protein Response in GD

6
*

Relative mRNA quantity (%)

CHOP * P < 0.05 *
120 5
Relative GCase activity

100 BiP
4
80
60 3
(%)

40
20 2
0 1
0 1 10
CBE treatment (days) 0
WT WT+CBE WT+Tg

Patient Disease type Relative Chop Relative BiP mRNA

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

4 CHOP
* P < 0.05

3.5 BiP Genotype Relative Chop Relative BiP

*
Relative mRNA quantity

mRNA mRNA
3

2.5 * 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

1 KO 0.7±0.06 1.08±0.3
0.5 WT+Tg *3.81 *2.91

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

Splicing No splicing

Forward Reverse Forward Reverse

primer primer primer primer

3.5
Relative mRNA quantity

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

9 4
*

Relative spliced Xbp1 mRNA

8 CHOP **P < 0.01
3.5 **
Relative mRNA quantity

7 BiP 3 * P < 0.05 **

6
2.5

quantity
5
2
4
1.5
3
1
2
0.5
1
0
0
Normal WT/84GG WT/N370S
Normal WT/N370S WT/84GG
Genotype
Genotype

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
9 **

Relative GCase mRNA expression

* P < 0.05
8 ** *
7 ** *
6 *
*
5
4
3
*
2
1
0

Genotype

Genotype Disease type Relative mRNA

WT -
quantity
1
There are
N370S/N370S 1 *4.4±1.2 elevated levels of
L444P/R120W
R131C/R131C
2
2
3.3±1.7
3±1.1
GBA mRNA in GD
L444P/L444P 3 5.1±1.3* derived and
carrier derived
N370S/WT - *5.7±0.8
WT+CBE - 0.99±0.43
84GG/WT - 1.7±0.1*
fibroblasts
N370S/WT - 5.3±2.3*
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
+1

-350 AP-1 PEA CAAT

GBA

Vector
delivery
to living
cells
 Luciferase in HEK293 cells
1.6
No Thapsigargin * P < 0.01 *
1.4
Thapsigargin
1.2

Luminscence
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