the role of shank3/prosap2 in regulating the function of

C. Sala- IN-CNR

The role of Shank3/ProSAP2 in regulating the function of brain

synapses

Carlo SalaCNR Istituto di Neuroscienze

MilanoCatania, 2006

1

C. Sala- IN-CNR

Or brain is a massive network of electrically active cells, neurons, that communicate with each other via specialized cell junctions: the synapses

Two major types of synapses exist in our brain: excitatory and inhibitory synapses

Excitation and inhibition in our brain are mediated mainly by the neurotransmitters glutamate and -amino butyric acid (GABA), respectively.

Remarkably, neurons take exquisite care in outfitting each synapse type with characteristic structural and neurochemical features

The drastic reduction in the number or in the function of synapses is observed in the brain of patients suffering from various mental retardations including

down syndrome, fragile X syndrome, ecc…

4

from Kristen Harris and colleagues

Type 1 excitatorysynapses

Type 2 inhibitorysynapses

1.25 m 1.25 m

GABA

neurexin

SV2A

VAMP

Syntaxin

-catenin

Rab3

CASK SNAP25

Synapsin

VGCC

Bassoon

Piccolo

ERCRim

Veli

N-cadherin

NLG-2

-catenin

GABAR GABAR

Piccolo

Bassoon

? GephyrinDystroglycan

neurexin

DystrophinGABARAP

MA

P1B

microtubulesmicrotubules

GABAergic synapse5

from Kristen Harris and colleagues

Type 1 excitatorysynapses

Type 2 inhibitorysynapses

1.25 m1.25 m

Glutamate

SAP97

N-cadherin

mGluR

N-catenin

PP1Spin/Neu

drebrin

Kal-7

NMDAREpHR

OPH

F-actin

NLG

-PIX

Abp1

GKAP

Cort

SynGAP

Shank

PICK1

PKC

AMPAR

GRIP/ABP

SynCAM

IRSp53

Shank

Hom

er

IRSp53

neurexinephrin

SPAR

nNOSliprin

CamKII CamKII

CamKII PSD-95

TARP

integrins

SV2A

VAMP

Syntaxin

-catenin

Rab3

CASK CASKSNAP25

Synapsin

VGCC

Bassoon

Piccolo

ERCRim

Veli

Piccolo

Bassoon

talin-actinin

SER IP3R

profilin

F-actinF-actin

SER

TARP

PSD-95

6

PSD95 VGAT merge

Synapses in hippocampal neuron

excitatory inhibitory

7

Brain and neurons need a correct balance between excitatory and inhibitory synapses to work well

Normal cognitive functions

8

Cognitive disfunctions

Possibly incorrect balance between excitatory (E) and inhibitory (I) synapses

9

Where is Shank in the synapses?

C. Sala- IN-CNR

10

Glutamate

SAP97

N-cadherin

mGluR

N-catenin

PP1Spin/Neu

drebrin

Kal-7

NMDAREpHR

OPH

F-actin

NLG

-PIX

Abp1

GKAP

Cort

SynGAP

Shank

PICK1

PKC

AMPAR

GRIP/ABP

SynCAM

IRSp53

Shank

Hom

er

IRSp53

neurexinephrin

SPAR

nNOSliprin

CamKII CamKII

CamKII PSD-95

TARP

integrins

SV2A

VAMP

Syntaxin

-catenin

Rab3

CASK CASKSNAP25

Synapsin

VGCC

Bassoon

Piccolo

ERCRim

Veli

Piccolo

Bassoon

talin-actinin

SER IP3R

profilin

F-actinF-actin

SER

TARP

PSD-95

11

Shank

GKAP

mGluR

Homer

Shank: a higher order scaffold in the PSD

NMDAR

PSD-95

NR

2A\B

12


NMDAR

PSD-95

NR

2A\B

Shank

GKAP

IP3R

SER

Homer

13

Shank1 regulates dendritic spine morphology and synaptic function

Hayashi, M. K., Tang, C., Verpelli, C., Narayanan, R., Stearns, M. H., Xu, R. M., Li, H., Sala, C., and Hayashi, Y. (2009). The postsynaptic density proteins Homer and Shank form a polymeric network structure. Cell 137, 159- 171.

Hung, A. Y., Futai, K., Sala, C., Valtschanoff, J. G., Ryu, J., Woodworth, M. A., Kidd, F. L., Sung, C. C., Miyakawa, T., Bear, M. F., et al. (2008). Smaller dendritic spines, weaker synaptic transmission, but enhanced spatial learning in mice lacking Shank1. J Neurosci 28, 1697-1708.

Sala C, Roussignol G, Meldolesi J, Fagni L (2005) Key role of the PSD scaffold proteins, Shank and Homer, in the functional architecture of Ca2+ homeostasis at dendritic spines in hippocampal neurons. J Neurosci 25:4587-92

Roussignol G, Ango F, Romorini S, Tu JC, Sala C, Worley PF, Bockaert J, Fagni L (2005) Shank expression is sufficient to induce functional dendritic spine synapses in aspiny neurons. J Neurosci 25:3560-3570.

Romorini S, Piccoli G, Jiang M, Grossano P, Tonna N, Passafaro M, Zhang M, Sala C (2004) Multimerizationand interaction with PSD-95/GKAP regulate the association of Shank with synapses.J Neurosci 24:9391-404.

Sala C, Futai K, Yamamoto K, Worley PF, Hayashi Y, Sheng M (2003) Inhibition of dendritic spine morphogenesis and synaptic transmission by activity-inducible protein Homer1a. J Neurosci 23:6327-6337.

Sala C, Piech V, Wilson NR, Passafaro M, Liu G, Sheng M (2001) Regulation of dendritic spine morphology and synaptic function by Shank and Homer. Neuron 31:115-130.

14

0.15 s

50 pA

GFP

GFP+Shank1B

DIV 11 DIV 17

FM4-

64 P

unct

a/ μ

m2

DIV11 DIV170.00

0.04

0.08

0.12

0.16 *

**

Freq

uenc

y (H

z)

DIV11 DIV170

2

4

6

*

GFPGFP+Shank1B

Sala et al. Neuron (2001)

15

- dendritic spine density is reduced

from Hung and Sheng

16

In order to study the functions of Shank3we knocked down Shank3 expression

in neurons

17

SHANK3 gene is located on the chromosome 2218

SHANK3 gene is transcribed in mRNA and translated in protein

19

Shank3

PSD-95

siRNS for Shank3 siRNA control

0

2

4

6

8

10

12

N. o

f clu

ster

s 10

m

siRNS for Shank3

siRNA control

We can block Shank3 protein production by degrading Shank3 mRNA with the siRNA

20

Shank3 siRNA can be transfer to neurons using lentivirus

We can block Shank3 expression in neurons for studying Shank3 function

21

What happen to dendritic spines and synapses in neurons knocked down for Shank3?

22

siRNAcontrol

siRNA forShank3

Number Width0

0,5

1

1,5

2

2,5

3

1,6

1,7

1,8

1,9

2

2,1

1

1,05

1,1

1,15

1,2

Spi

nes/

10m

m m

p < 0.003p < 0.003 p < 0.02

**** *

Length

GFP DsRED merge

GFP DsRed merge

Dendritic spines morphology in hippocampal neurons knocked down for Shank3:dendritic spines are reduced in dimendion and number

siRNA controlsiRNA for Shank3

23

Shank

GKAP

mGluR

Homer


NMDAR

PSD-95

NR

2A\B

IP3R

SER

Homer

24

MAPK

Syn

Abi1

IRSp53

eEF2

GluR2/3

mGluR5

NR1

Actin

PSD-95

PanShank

Shank1

Shank3

Homer

not infecte

d

siShank3

not infecte

d

siShank3

0

0,2

0,4

0,6

0,8

1

1,2

1,4

Pan

Sha

nk

Sha

nk3

Sha

nk1

0

0,2

0,4

0,6

0,8

1

1,2

1,4

actin

PS

D/9

5

Hom

er

Syn

eEF2

Abi

1

IRS

p53

MAP

K

mG

luR

5

Glu

R2/

3

NR

1Nor

mal

ized

inte

nsity

Nor

mal

ized

inte

nsity

Expression of synaptic proteins in hippocampal neurons

knocked down for Shank3:mGluR5 is reduced

**

siRNA controlsiRNA for Shank3

25

siRNA for Shank3

pERK1/2

Actin

mGluR5 phopsphorilation of ERK1/2 is inhibited in neuronsknocked down for Shank3

DHPG 30’NT KClNMDA DHPG 30’NT KClNMDA

siRNA control

DH

PH p

ERK

1/2

leve

l

0

2

4

6

8

siRNS for Shank3

siRNA control

26

NM

DA

RAAAA

mGluR5

Dendritic spinesmaturation

mGluR dependent ERK1/2 activation

AAAA

Shank3

Synthesis of proteins that regulatedendritic spines

In presence of Shank3 27

NM

DA

RAAAA

mGluR5



AAAA

Shank3


In absence of Shank3 28

In absence of Shank3In presence of Shank3

In absence of Shank3 the ratio excitatory/inhibitory synapses might be altered

29

NM

DA

RAAAA

mGluR5



AAAA

Shank3


Possible pharmacological rescue

restore ERK1/2activity

restore the synthesisof proteins that regulatesdendritic spines

restore Shank3sinthesys

30

We plan to test the pharmacological rescue in two models:

-using rat neuronal cultures knocked down for Shank3-using mouse knock out for Shank3

-using induced pluripotent stem cells (iPS cells) derived from patients fibroblasts

31

From the pluripotent cells derives our organs32

We can take a unipotent cell (skin fibroblast) and transform the cell into a pluripotent cell

33

We can take a human unipotent cell (skin fibroblast) and transform the cell into a pluripotent cell

35

What we can do with the iPS cells

Geneticreprogramming

36

Chiara Verpelli

Monica Patti

Francesca Rossi

Hanako Tsushima

CNR Institute of NeuroscienceMilano, Italy

Antonella Gianfelice

Valentina Cea

Alice Zanchi

Clara BonagliaRobero GiordaLa Nostra Famiglia, Bosisio

Maria PassafaroDTI/CNR Milano

Morgan ShengMIT Cambridge MA

Albert Y. Hung

Univ. San RaffaeleVania Broccoli

37

the role of shank3/prosap2 in regulating the function of

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