The role of Shank3/ProSAP2 in regulating the function of brain synapses. Carlo Sala CNR Istituto di Neuroscienze Milano. C.

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1 1 The role of Shank3/ProSAP2 in regulating the function of brain synapses Carlo Sala CNR Istituto di Neuroscienze Milano Catania, 2006 C. Sala- IN-CNR

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4 4 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 C. Sala- IN-CNR

5 Type 1 excitatory synapses Bassoon Piccolo ERC GABAergic synapse Rim Rab3 GABA Type 2 inhibitory synapses SV2A Synapsin Piccolo Bassoon 5 -catenin Veli CASK VAMP SNAP25 Syntaxin N-cadherin GABAR neurexin VGCC GABAR neurexin NLG-2 Dystroglycan -catenin MAP1B GABARAP? Gephyrin Dystrophin 1.25 m 1.25 m microtubules microtubules from Kristen Harris and colleagues

6 Piccolo Type 1 excitatory Bassoon ERC synapsesrim Rab3 Glutamate Piccolo Type 2 inhibitory Bassoon Synapsin synapses SV2A 6 -catenin Veli CASK VAMP SNAP25 CASK talin Syntaxin N-cadherin AMPAR TARP ephrin neurexin EpHR NLG TARP VGCC NMDAR SynCAM integrins mglur N-catenin PICK1 PKC Kal-7 drebrin Spin/Neu GRIP/ABP liprin PP1 SynGAP CamKII PSD-95 nnos SPAR 1.25 m profilin SAP97 GKAP -PIX Abp1 Cort Shank CamKII CamKII PSD-95 IRSp53 Shank IRSp53 OPH -actinin Homer 1.25 m F-actin F-actin SER IP3R F-actin from Kristen Harris and colleagues

7 Synapses in hippocampal neuron 7 PSD95 VGAT merge excitatory inhibitory

8 Brain and neurons need a correct balance between excitatory and inhibitory synapses to work well 8 Normal cognitive functions

9 Possibly incorrect balance between excitatory (E) and inhibitory (I) synapses 9 Cognitive disfunctions

10 10 Where is Shank in the synapses? C. Sala- IN-CNR

11 Glutamate SAP97 N-cadherin mglur N-catenin PP1 Spin/Neu drebrin Kal-7 NMDAR EpHR OPH F-actin NLG -PIX Abp1 GKAP Cort SynGAP Shank PICK1 PKC AMPAR GRIP/ABP SynCAM IRSp53 Shank Homer IRSp53 neurexin ephrin SPAR nnos liprin CamKII CamKII CamKII PSD-95 TARP integrins SV2A VAMP Syntaxin -catenin Rab3 CASK CASK SNAP25 Synapsin VGCC Bassoon Piccolo ERC Rim Veli Piccolo Bassoon talin -actinin SER IP3R profilin F-actin F-actin SER TARP PSD-95 11

12 Shank: a higher order scaffold in the PSD 12 PSD-95 NMDAR NR2A\B mglur Shank GKAP Homer

13 Shank: a higher order scaffold in the PSD 13 PSD-95 NMDAR NR2A\B GKAP Shank IP3R Homer SER

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

15 DIV 11 DIV 17 GFP GFP+Shank1B 15 GFP GFP+ Shank1B 0.15 s 50 pa FM4-64 Puncta / μm 2 Frequency (Hz) DIV11 DIV * * ** DIV11 DIV17 Sala et al. Neuron (2001)

16 - dendritic spine density is reduced 16 from Hung and Sheng

17 17 In order to study the functions of Shank3 we knocked down Shank3 expression in neurons

18 18 SHANK3 gene is located on the chromosome 22

19 SHANK3 gene is transcribed in mrna and translated in protein 19

20 We can block Shank3 protein production by degrading Shank3 mrna with the sirna sirns for Shank3 sirna control Shank3 PSD N. of clusters 10 m sirna control sirns for Shank3 20

21 Shank3 sirna can be transfer to neurons using lentivirus 21 We can block Shank3 expression in neurons for studying Shank3 function

22 What happen to dendritic spines and synapses in neurons knocked down for Shank3? 22

23 Dendritic spines morphology in hippocampal neurons knocked down for Shank3: dendritic spines are reduced in dimendion and number 23 sirna control GFP DsRed merge sirna for Shank3 GFP DsRED merge 3 p < sirna control sirna for Shank3 2,1 p < ,2 p < 0.02 Spines/10 m 2,5 2 1,5 1 0,5 ** m 2 1,9 1,8 1,7 m 1,15 1,1 ** * 1,05 0 Number 1,6 Length 1 Width

24 Shank: a higher order scaffold in the PSD 24 PSD-95 NMDAR NR2A\B mglur Shank GKAP IP3R Homer Homer SER

25 not infected sishank3 PanShank Shank3 Shank1 Actin PSD-95 Homer Syn eef2 Abi1 IRSp53 MAPK mglur5 GluR2/3 NR1 sishank3 not infected 1,4 1,2 1 0,8 0,6 0,4 0,2 0 1,4 1,2 1 0,8 0,6 0,4 0, Expression of synaptic proteins in hippocampal neurons knocked down for Shank3: mglur5 is reduced * * PanShank Normalized intensity Shank3 Shank1 Normalized intensity actin PSD/95 Homer Syn eef2 Abi1 IRSp53 MAPK mglur5 GluR2/3 NR1 sirna control sirna for Shank3

26 mglur5 phopsphorilation of ERK1/2 is inhibited in neurons knocked down for Shank3 26 sirna control sirna for Shank3 NT DHPG 30 NMDA KCl NT DHPG 30 NMDA KCl perk1/2 Actin DHPH perk 1/2 level sirna control sirns for Shank3

27 In presence of Shank3 27 NMDAR mglur5 Shank3 Dendritic spines maturation AAAA AAAA mglur dependent ERK1/2 activation Synthesis of proteins that regulate dendritic spines

28 In absence of Shank3 28 NMDAR mglur5 Shank3 Dendritic spines maturation AAAA AAAA mglur dependent ERK1/2 activation Synthesis of proteins that regulate dendritic spines

29 In absence of Shank3 the ratio excitatory/inhibitory synapses might be altered 29 In presence of Shank3 In absence of Shank3

30 Possible pharmacological rescue 30 NMDAR restore Shank3 sinthesys mglur5 Dendritic spines maturation AAAA AAAA restore ERK1/2 activity mglur dependent ERK1/2 activation restore Synthesis the of synthesis proteins of proteins that regulatethat regulates dendritic spines

31 31 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

32 From the pluripotent cells derives our organs 32

33 We can take a unipotent cell (skin fibroblast) and transform the cell into a pluripotent cell 33

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35 We can take a human unipotent cell (skin fibroblast) and transform the cell into a pluripotent cell 35

36 What we can do with the ips cells 36 Genetic reprogramming

37 Chiara Verpelli Antonella Gianfelice Valentina Cea Hanako Tsushima Francesca Rossi Monica Patti Alice Zanchi CNR Institute of Neuroscience Milano, Italy Albert Y. Hung Morgan Sheng MIT Cambridge MA Maria Passafaro DTI/CNR Milano Clara Bonaglia Robero Giorda La Nostra Famiglia, Bosisio Vania Broccoli Univ. San Raffaele 37