Diapositive 1

1
APOLIPOPROTEIN D
ERIC RASSART SONIA DO CARMO, LAURENCE TERRISSE
2
HISTORIC
1972
Isolation
McConathy and Alaupovic
1986
Cloning of the human gene
Drayna et al.
1990
In situ localization in rabbit
Provost et al.
1990
ApoD is GCDFP-24
Balbin et al.
1998
High levels in Alzheimer and other
neuropathologies
Terrisse et al.
3
FUNCTIONS AND LIGANDS
4
AMINO ACID SEQUENCE
5
TISSUE DISTRIBUTION OF mRNA
6
In situ HYBRIDIZATION IN RABBIT
Steroid hormone production sites
Seminiferous tubules
Testis
Adrenals
7
In situ HYBRIDIZATION IN RABBIT
Spinal cord
Brain
W
A
G
W
G
Cerebellum
Cerebellum
8
REGULATION OF ApoD EXPRESSION
9
MODULATION OF EXPRESSION
10
PRIMARY CULTURES
s
0
4
7
10
14
21
21
s
0
4
7
10
14
Apo D
C57/Bl
Balb/c
Apo E
?-actin
12 -
12-
8 -
8-
4-
4-
s
0
4
7
10
14
21
s
0
4
7
10
14
21
days
days
11
IMMORTALIZED CELL LINES
12
(No Transcript)
13
PROGRESSIVE DELETIONS
14
SRE AND APP ELEMENTS
- serum / serum
pSV2-luc
15
FACTORS BINDING SRE REGION
SRE2
- - - - - -
- - - - - -
- - - - -
- - - - -
- - - - -
- - - - -
- - - - -
- - - - -
- - - - -
- - - - -
- - - - -
- - - - - -
- - - - - -
- - - - -
SRE1
SRE1
mSRE1
SRE c-fos
UNR
SRE2
1
4
5
6
7
8
9
10
11
12
13
14
2
3
A -
B -
C -
D -
16
EPITOPE MAPPINGandBINDING STUDIES
17
CARTOGRAPHY OF ApoD
?-Galactosidase-ApoD
a.a 128-169
a.a 128-169
ApoD-pEX
a.a 99-169
ApoD-pEX
a.a 99-169
a.a 7-99
a.a 7-78
a.a 7-99
a.a 7-78
MW (kDa)
pEX
pEX
200
97
69
D1
D2
D1, D3, D5, D7 et D8


b gal-ApoD 99-169
D1, D2 et D4
b gal - ApoD 7-99

D1, D3, D5, D6, D7, D8 et D9
D1, D2 et D4
b gal - ApoD 128-169
b gal - ApoD 7-78
RV SmaI
HincII BamHI Sau3AI
SmaI BamHI
119 352
417 501
629 bp
18
INHIBITION OF ApoD-PREGNENOLONE BINDING
Binding ()
140
120
100
80
60
40
20
0
apoD
D1
D2
D4
D5
D6
D7
D8
D9
caro
Antibody
19
ApoD in the CENTRAL NERVOUS SYSTEM
20
LIPOPROTEINS IN ALZHEIMER
1,2
ug/mL
1
0,8
ApoD
ApoE
0,6
0,4
0,2
0
ctrl
alz
op
n11 n41 n29
21
ApoD CONCENTRATION
20
1,2
18
1
16
14
0,8
ApoD (ug/ml)
12
ALZ
ALZ
ApoD/ total proteins
0,6
10
CT
CT
8
0,4
6
4
0,2
2
0
0
LCR
Serum
LCR
Serum
22
RAT HIPPOCAMPUS AFTER ECL
Expression
ApoD ipsi
ApoE ipsi
ApoD contra
ApoE contra
Days post-lesion
23
RAT HIPPOCAMPUS AFTER ECL
ApoD protein
Days post-lesion
24
ApoD IN ApoE -/- MICE
K5
K1
K2
K3
K4
C1
C2
C3
C4
C5
Apo D
Frontal cortex
Cerebellum
25
CONCLUSIONS
ApoD is a multi-ligand, multi-function protein.
It is present in many species evolutive
importance?
It is expressed differentially in several tissues.
Its expression is associated with growth arrest.
SREs and APP regulatory elements are the major
determinants of promoter activity during growth
arrest. /
26
CONCLUSIONS
High levels of protein are present in Alzheimer
disease and several other neuropathologies.
A possible role as an inflammatory
protein? presence of APRE in the
promoter increased in several nervous system
injuries neuropathologies in human nerve
crush, ECL, TBI, Kainic acid
27
FUTURE PERSPECTIVES
28
FACTORS BINDING SRE REGION
SRE2
- - - - - -
- - - - - -
- - - - -
- - - - -
- - - - -
- - - - -
- - - - -
- - - - -
- - - - -
- - - - -
- - - - -
- - - - - -
- - - - - -
- - - - -
SRE1
SRE1
mSRE1
SRE c-fos
UNR
SRE2
1
4
5
6
7
8
9
10
11
12
13
14
2
3
A -
B -
C -
D -
29
LIGAND BINDING OF MUTANTS
293-T WT F36Y V56A L61I Q69N E73D A83V

L85I V87A A96V Y105F Y108F I 117L
F121Y F125Y
Mammalian expression vector pCDNA3.1
30
TRANSGENICS
Thy1/ApoD
NSE/ApoD
NF-L/ApoD
31
KNOCK-OUT
Normal
Recombinant
x
ApoD -/-
32
ACKNOWLEDGEMENTS
Pierre Provost Diane Séguin Laurence
Terrisse Karine Marcoux Sonia Do Carmo Jacinthe
Sirois Christian Trahan David Fournier
Judes Poirier, Mc Gill University Ross Milne,
Ottawa Heart Institute Gérard Siest, Université
de Nancy
Canadian Institutes for Health Research