Thesis discussion andrea robotti
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REGULATION OF NEURAL DEVELOPMENT AND REGENERATION POTENTIAL BY PLASMA MEMBRANE
GANGLIOSIDES
Andrea Robotti
• So far it has been shown that :
1) PMGS is localized asymmetrically in non-polarized early stage neurons. This determines a specific neurite as the future axon, by actin cytoskeleton remodeling via the inhibition of RhoA pathway in a PI3K-Rac1-dependent manner.
2) inhibition of PMGS lowers membrane GM1 levels and retards axonal growth
3) PMGS over-expression enhances GM1 levels and notably accelerates axonal growth
Rodriguez J.A. et al., J Neurosci, 2001. 21(21): p. 8387-95 Da Silva J.S. et al., Nat. Neurosc., 2005 .8 : p. 606-15
• Plasma Membrane Ganglioside Sialidase (PMGS / Neu3) is the unique enzyme that specifically hydrolyzes oligosialogangliosides to produce GM1 in the plasma membrane.
Hasegawa, T., et al., J Biol Chem, 2000. 275(19): p. 14778. Miyagi, T., et al., J Biol Chem, 1999. 274(8): p. 5004-11. Wada, T., et al., Biochem Biophys Res Commun, 1999. 261(1): p. 21-7.
• The modulation of the PMGS activity is able to modify the endogenous levels of plasma membrane GM1 in neurons, as shown in previous works from our lab.
CNS does not regenerate spontaneously after injury.
1. Reduced capacity of injured neurons to induce axonal re-growth.
Loss of function
More than 70% of axons from PMGS over-expressing neurons regenerate after transection in vitro while, less than 25% of control axons, do so.
Control
OE PMGS
CNS does not regenerate spontaneously after injury.
1. Reduced capacity of injured neurons to induce axonal re-growth.
Loss of function
2. Formation of a non-permissive barrier for axonal progression.
3. Presence of specific inhibitory factors as:
Myelin-associated inhibitors: - MAG - Nogo - OMgp - Sem3A
Extra-cellular Matrix associated Glial Scar components:
- CSPG
Are axons of PMGS over-
expressing neurons able to
grow across surfaces covered
with either myelin
or its derived factors?
Is PMGS a tool for axon regeneration
in the presence of an inhibitory
environment ?
Crude Myelin
OE
PM
GS
-HA
Co
ntr
ol
Axons of PMGS over-expressing neurons can grow across myelin-covered surfaces
Mature Oligodendrocytes
Co
ntr
ol
OE
PM
GS
-HA
Cont r ol Over exp. PM GS
** **
**
*
0
140
20
40
60
80
100
120
Untreated + Mag-Fc + Sem3A
Axo
n l
en
gth
(µ
m)
OE-PMGS
+ Mag-Fc + Sem3AUntreated
Tub
HA
Axons of PMGS over-expressing neurons can grow in the presence of myelin-derived inhibitors (Mag and Sem3A)
Which is the mechanism underlying this effect?
PMGS over-expression enhances axonal growth in
the presence of myelin inhibitors.
MYELIN-ASSOCIATED INHIBITORS
MAGNogo
Nogo 66
NiG
OMgpOligo
TrkA
Neuron
PMGS
GM1
RhoA
RhoA
GDP
GTP
Rac1 Rac1GDP GTP
PI3K
GROWTH ENHANCEMENTAND REGENERATION
GROWTH INHIBITION
?GT1b
NgR p75
Ca2+
?
GD1a
Glial Scar
P
EGFR
NGF stimulation differentiates PC12 to a “neuron like” phenotype and this differentiation is delayed
by contact with myelin extracts.
We generated a PC12 clone stably expressing PMGS-HA to study its effect in axon growth
inhibition.
These cells express p75, NgR, TrkA and EGFR, signaling proteins target of our study.
The PC12 model
PMGS-HA PC12 are able to differentiate in presence of myelin
PC12 on PLL PC12 on Myelin
PMGS-HA PC12 on PLL PMGS-HA PC12 on Myelin
PC12 WT or
PMGS-HA
In DMEM
NO FCS/HS
+ NGF
100 ng/ml
48 hrs
PC12 on PLL PC12 on Myelin
PMGS-HA PC12 on PLL PMGS-HA PC12 on Myelin
PC12 WT or
PMGS-HA
In DMEM
NO FCS/HS
+ NGF
100 ng/ml
PMGS-HA PC12 are able to differentiate in presence of myelin
72 hrs
To study the mechanism by which PMGS overcomes inhibition, we have focused on :
1. Changes on membrane composition :
- gangliosides
- other lipids
2. Receptor re-organization in membrane domains :
- binding of inhibitors
- receptor localization in/out raft domains
PMGS induces the content of two different forms of GM1
Fractions
2
3
4
5
6
7
8
9
GT1b GD1a/bW
T P
C12
CLO
NE
#2
WT
PC
12 o
n M
yelin
CLO
NE
#2
on M
yelin
WT
PC
12C
LON
E #
2W
T P
C12
on
Mye
lin
CLO
NE
#2
on M
yelin
PMGS induces the reduction of GT1b and the enhancement of GM1 in raft fractions
GM1a
CLO
NE
#2
on M
yelin
WT
PC
12 o
n M
yelin
WT
PC
12C
LON
E #
2
CLO
NE
#2
+ M
ag-F
c
CLO
NE
#2
+ M
ag-F
c
CLO
NE
#2
+ M
ag-F
c
Raft
WT PC12 PMGS-HA PC12
Cer
Diacylglycerol
0
20
40
60
80
100
WT PMGS-HA
Qu
anti
ty in
a.u
.
DA
G
WT
PM
GS
-HA
Ctr
l
Ctr
l
+ N
GF
+ N
GFCer
Ceramide
0,0
0,2
0,4
0,6
0,8
1,0
C NGF M
Qua
ntity
in a
.u.
WT PC12 PMGS-HA PC12
Mye
lin
Mye
lin
PMGS increases the amount of Ceramides and Diacylglycerol
PC
SM
PLL Myelin PLL Myelin
WT PC12 PMGS-HA PC12
Phosphatidyl-Choline
0
20
40
60
80
100
WT WTM PMGS-HA PMGS-HA M
Qua
ntity
in a
.u.
PC
SM
Sphingomyelin
0
20
40
60
80
100
WT WT M PMGS-HA PMGS-HA M
Qua
ntity
in a
.u.
PMGS increases the amount of two main raft lipids: Sphingomyelin and Phosphatidyl-Choline
Cholesterol is increased in PMGS-HA PC12
WT = 100 %
#2 = +8,93%
Chol = +10,31%
WT + 2.0 MCD = -7,56%
WT + 2.5 MCD = -7,22%2,50
2,70
2,90
3,10
3,30
3,50
wt clone chol 2 MCD 2.5 MCDu
g o
f ch
ole
ster
ol
/ u
g o
f p
rote
in
3,0
3,2
3,4
3,6
WT PMGS-HA NeuAc2en
ug
of
ch
ole
ste
rol
/ u
g o
f p
rote
in
MYELIN-ASSOCIATED INHIBITORS
MAGNogo
Nogo 66
NiG
OMgpOligo
TrkA
Neuron
PMGS
GM1
RhoA
RhoA
GDP
GTP
Rac1 Rac1GDP GTP
PI3K
GROWTH ENHANCEMENTAND REGENERATION
GROWTH INHIBITION
?GT1b
NgR p75
Ca2+
?
GD1a
Glial Scar
P
EGFR
2
3
4
5
6
7
8
9
Fractions
WT
PC
12C
LON
E #
2C
LON
E #
2+C
hol
WT+
2mM
βM
CD
WT
PC
12C
LON
E #
2C
LON
E #
2+C
hol
WT+
2mM
βM
CD
p75Binding
Mag-Fc
WT
PC
12C
LON
E #
2C
LON
E #
2+C
hol
WT+
2mM
βM
CD
GT1b
Mag binding is reduced in PMGS-HA PC12
Raft
2 3 4 5 6 7 8 9 2 3 4 5 6 7 8 9 Fractions
Flotillin I
NgR
Myelin p75
WT PC12 PMGS-HA PC12
2 3 4 5 6 7 8 9 2 3 4 5 6 7 8 9 Fractions
PMGS
NgR
p75
Raft Raft
Raft Raft
PLL
p75 and NgR flotation is altered in PMGS-HA PC12
2 3 4 5 6 7 8 9
p75Myelin
PLL
NgR
2 3 4 5 6 7 8 9
Myelin
p75
NgR
p75
NgR
p75
NgRPLL
Fractions
WT PC12 PMGS-HA PC12Raft Raft
CSPG binding is reduced in PMGS-HA PC12
2 3 4 5 6 7 8 9 2 3 4 5 6 7 8 9 Fractions
EGFR
pEGFR
EGFR
pEGFRMyelin
Raft
WT PC12 PMGS-HA PC12
Conclusions1.- The over-expression of PMGS in rat hippocampal neurons enhances axonal growth in the presence of myelin-producing oligodendrocytes, myelin extracts, myelin-derived growth inhibitors (Mag, Sem3A) and CSPG.
2.- A PC12 cell line stably expressing active PMGS-HA has been produced. These cells grow long axon-like neurites after NGF stimulation, even in the presence of inhibitory myelin extracts.
3.- The lipidic composition of PMGS-HA PC12 membrane is altered: a) PMGS-HA-PC12 show a higher content of PMGS product GM1, and a reduced content of its substrata GD1a and GT1b. b) PMGS-HA-PC12 present a modest increase in the levels of other lipids as ceramide, sphingomyelin, phosphatidyl-choline and diacylglycerol. c) Membrane cholesterol is moderately increased in PMGS-HA-PC12. This effect is rescued by PMGS activity inhibitors.
4.- The binding of the inhibitory molecules Mag-Fc and CSPG to PMGS-HA-PC12 raft membrane fractions is reduced.
5.- The content of Mag receptors (GT1b and p75) and of CSPG effector (pEGFR) in membrane fractions is rearranged.
6.- Both p75 and NgR localization in rafts is reduced in PMGS-HA PC12 and the effect is more evident in the presence of Myelin.
We propose that the effect of PMGS activity overcomes axon growth inhibition by changing the membrane composition. This reduces the binding of myelin inhibitors and disorganizes the interactions of their receptors, disturbing intracellular inhibitory signaling and allowing axon extension.
I would like to thank……
Josè Abad-Rodriguez Carlos G. Dotti
Ferdinando Di Cunto
Bianca, Etienne, Lola, Fabian, Eva, Jorge, Vanessa, Sven, Froylan, Federica, Simona, Laura, Giulia, Flavio, Cristian, Iva,
Villy, Mauricio, Aga, Enrico, Gaia, Paola, Marta, Federico, Ylenia, Serena, Enzo, Stefania
For giving me the chance to live such an exciting experience across different labs and countries and to do
science in such a great way!!!