1. REGULATION OF NEURAL
DEVELOPMENT AND REGENERATION
POTENTIAL BY PLASMA MEMBRANE
GANGLIOSIDES
Andrea Robotti

2. • So far it has been shown that :
• 1)Plasma Membrane Ganglioside Sialidase (PMGS / Neu3)
PMGS is localized asymmetrically in non-polarized early stage neurons.
is the unique enzyme that specifically hydrolyzes cytoskeleton
This determines a specific neurite as the future axon, by actin
remodeling via the inhibition of RhoA pathway in a PI3K-Rac1-dependent
oligosialogangliosides to produce GM1 in the plasma
manner.
membrane.
2) inhibition of PMGS lowers membrane GM1 levels and retards axonal growth
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.
3) PMGS over-expression enhances GM1 levels261(1): p. 21-7. accelerates axonal
Wada, T., et al., Biochem Biophys Res Commun, 1999. and notably
growth
• 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.
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

3. CNS does not regenerate spontaneously after injury.
Loss of function
1. Reduced capacity of injured neurons to induce axonal re-growth.
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

4. CNS does not regenerate spontaneously after injury.
Loss of function
1. Reduced capacity of injured neurons to induce axonal re-growth.
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

5. Are axons of PMGS over-
Is PMGS a tool for axon regeneration
expressing neurons able to
in the presence of an inhibitory
grow across surfaces covered
environment ?
with either myelin
or its derived factors?

6. Axons of PMGS over-expressing neurons can grow across
myelin-covered surfaces
Crude Myelin Mature Oligodendrocytes
Control
Control
OE PMGS-HA
OE PMGS-HA

7. Axons of PMGS over-expressing neurons can grow in the presence of
myelin-derived inhibitors (Mag and Sem3A)
140
C ont r ol Ov er ex p. P M GS *
120
Tub
*
*
100
Axon length (µm)
80
60
40 ** **
20
HA
0
Untreated + Mag-Fc + Sem3A
Untreated + Mag-Fc + Sem3A
OE-PMGS

8. PMGS over-expression enhances axonal growth in
the presence of myelin inhibitors.
Which is the mechanism underlying this effect?

9. MY ELIN-ASSOCIATED INHIBITORS
Nogo Glial
MAG Scar
Oligo OMgp
Nogo 66
NiG
EGFR ? PMGS TrkA
NgR p75
GD1a
? GM1
GT1b
GTP
Neuron
RhoA P
Ca2+
PI3K
GDP
RhoA
GROWTH ENHANCEMENT
GROWTH INHIBITION
AND REGENERATION
GDP GTP
Rac1 Rac1

10. The PC12 model
NGF stimulation differentiates PC12 to a “neuron
like” phenotype and this differentiation is delayed
by contact with myelin extracts.
These cells express p75, NgR, TrkA and EGFR,
signaling proteins target of our study.
We generated a PC12 clone stably expressing
PMGS-HA to study its effect in axon growth
inhibition.

11. PMGS-HA PC12 are able to differentiate in presence of myelin
PC12 on PLL PC12 on Myelin
48 hrs
PC12 WT or
PMGS-HA
In DMEM
NO FCS/HS
+ NGF
100 ng/ml
PMGS-HA PC12 on PLL PMGS-HA PC12 on Myelin

12. PMGS-HA PC12 are able to differentiate in presence of myelin
PC12 on PLL PC12 on Myelin
72 hrs
PC12 WT or
PMGS-HA
In DMEM
NO FCS/HS
+ NGF
100 ng/ml
PMGS-HA PC12 on PLL PMGS-HA PC12 on Myelin

13. 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

14. PMGS induces the content of two different forms of GM1

15. Raft
9
8
7
6
5
4
3
2
WT
PC
Fractions
CL 12
ON CLO
E# NE
WT 2+ #
PC Ma 2
12 g- F
CL on c
ON My
GT1b
E# elin
2o
nM
yel
in
WT
PC
CL CL 1
ON ON 2
E
#2 E
WT + M #2
PC ag-
12 Fc
CL on
GD1a/b
ON My
E# elin
2o
nM
yel
in
WT
P
CL C L C 12
ON
E # ON E
WT 2+ #2
enhancement of GM1 in raft fractions
PC Ma
12 g- F
CL on c
ON My
GM1a
E# elin
2o
PMGS induces the reduction of GT1b and the
nM
yel
in

16. PMGS increases the amount of Ceramides and Diacylglycerol
Ceramide WT PC12 PMGS-HA PC12
WT PC12 PMGS-HA PC12
1,0
0,8
Cer
0,6
Quantity in a.u.
0,4
rl
rl
n
n
Cer
GF
GF
Ct
Ct
eli
eli
My
My
+N
+N
0,2
0,0
C NGF M
Diacylglycerol
100
Quantity in a.u. 80
60
40
20
A
G
WT
-H
0
DA
GS
WT PMGS-HA
PM

17. PMGS increases the amount of two main raft lipids:
Sphingomyelin and Phosphatidyl-Choline
WT PC12 PMGS-HA PC12
PC
SM
SM
PC
PLL Myelin PLL Myelin
Phosphatidyl-Choline Sphingomyelin
100 100
80 Quantity in a.u. 80
60
Quantity in a.u.
60
40 40
20 20
0 0
WT WTM PMGS-HA PMGS-HA M WT WT M PMGS-HA PMGS-HA M

18. Cholesterol is increased in PMGS-HA PC12
ug of cholesterol / ug of protein
3,50
WT = 100 % 3,30
#2 = +8,93%
3,10
Chol = +10,31%
2,90
WT + 2.0 MCD = -7,56%
2,70
WT + 2.5 MCD = -7,22%
2,50
wt clone chol 2 MCD 2.5 MCD
3,6
ug of cholesterol / ug of protein
3,4
3,2
3,0
WT PMGS-HA NeuAc2en

19. MY ELIN-ASSOCIATED INHIBITORS
Nogo Glial
MAG Scar
Oligo OMgp
Nogo 66
NiG
EGFR ? PMGS TrkA
NgR p75
GD1a
? GM1
GT1b
GTP
Neuron
RhoA P
Ca2+
PI3K
GDP
RhoA
GROWTH ENHANCEMENT
GROWTH INHIBITION
AND REGENERATION
GDP GTP
Rac1 Rac1

20. Raft
9
8
7
6
5
4
3
2
Fractions
WT
PC
12
CL
CL O NE
ON #2
E #2+
WT Ch
+2m ol
Binding
Mβ
MC
D
WT
PC
12
CL
CL ON
ON E#
E 2
#2+
p75
WT Ch
+2 m ol
Mag-Fc
Mβ
MC
D
WT
PC
12
CL
CL ON
ON E#
E 2
#2+
WT Ch
GT1b
+2 m ol
Mβ
MC
D
Mag binding is reduced in PMGS-HA PC12

21. p75 and NgR flotation is altered in PMGS-HA PC12
Raft Raft
2 3 4 5 6 7 8 9 Fractions 2 3 4 5 6 7 8 9
NgR
Raft Raft
PLL WT PC12 p75 PMGS-HA PC12
NgR NgR
PMGS
PLL PLL
p75 Raft p75 Raft
NgR 2 3 4 5 6 7 8 9 Fractions
NgR 2 3 4 5 6 7 8 9
Myelin Myelin
p75 NgR p75
2 3 4 5 6 7 8 9 2 3 4 5 6 7 8 9 Fractions
Myelin p75
Flotillin I
WT PC12 PMGS-HA PC12

22. CSPG binding is reduced in PMGS-HA PC12
Raft
WT PC12 PMGS-HA PC12
2 3 4 5 6 7 8 9 Fractions 2 3 4 5 6 7 8 9
pEGFR pEGFR
Myelin
EGFR EGFR

23. Conclusions
4.- The binding of the inhibitory molecules Mag-Fc and CSPG
1.- ThePMGS-HA-PC12 raft membrane fractionsneurons enhances axonal growth in
to over-expression of PMGS in rat hippocampal is reduced.
the presence of myelin-producing oligodendrocytes, myelin extracts, myelin-derived growth
inhibitors (Mag, Sem3A) and CSPG.
5.- The content of Mag receptors (GT1b and p75) and of CSPG effector
2.-(pEGFR) in line stably expressing active PMGS-HA has been produced. These cells
A PC12 cell membrane fractions is rearranged.
grow long axon-like neurites after NGF stimulation, even in the presence of inhibitory myelin
extracts.
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.
3.- The lipidic composition of PMGS-HA PC12 membrane is altered:
a) PMGS-HA-PC12 show a higher content of PMGS product GM1, and
We propose that thesubstrata GD1a and GT1b. overcomes axon
a reduced content of its effect of PMGS activity
growth inhibition by changing the membrane composition. This
b) PMGS-HA-PC12 present a modest increase in the levels of other
reducesceramide, sphingomyelin, phosphatidyl-choline and
lipids as the binding of myelin inhibitors and disorganizes the
diacylglycerol.
interactions of their receptors, disturbing intracellular inhibitory
c) Membrane cholesterol is moderately increased in PMGS-HA-PC12.
signaling and allowing axon extension.
This effect is rescued by PMGS activity inhibitors.

24. 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!!!