Thesis discussion andrea robotti

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.


Mye

lin

Mye

lin

PMGS increases the amount of Ceramides and Diacylglycerol

PC

SM

PLL Myelin PLL Myelin


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


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


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!!!

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Thesis discussion andrea robotti