Synapse Complexity Swartz Foundation Banbury Meeting, CSHL 15 th April 2009 Seth Grant Genes to Cognition program Wellcome Trust Sanger Institute, Cambridge UK

MASC

PSD MASC Post-Synaptic Proteome MASCMAGUK Associated Signaling Complex PSDPost Synaptic Density PSPPost Synaptic Proteome (total set of postsynaptic proteins) Presynaptic terminal Postsynaptic terminal

NR2 PSD-95 fyn NMDA receptor Genetic evidence for postsynaptic complexes Grant, et al, Science. 258, Migaud et al, Nature, 396; Sprengel et al, Cell 92, Learning impairments Plasticity impairments MAGUK proteins

PSD95 NR    Husi et al. Nature Neuroscience, 3 (7), Husi & Grant. J. Neurochem, 77, Collins et al, J. Neurochem Fernandez et al, submitted Proteomic characterisation of complexes 2.5 Mda 77 proteins (2000) 186 (2005) 158(2009) 118 core (2009)

Migaud et al, Nature, 396; ; Husi et al, Nature Neuroscience, 2000 synaptic strength gene expression mRNA turnover Protein turnover Behaviours Cognition & plasticity NRC / MASC 2-3 MDa 186 proteins 47 genes in human disease 48 synaptic plasticity 42 rodent behaviour

Complexity PSD MASC PSD proteins MASC proteins MASC occupies ~10 % of the PSP - a core subset - same classes of proteins - ideal model of PSP - multiple isolation methods available

Grant. (2006) Biochemical Society Transactions. 34, Post Synaptic Density 1124 ER:microsomes491 Splicesome311 NRC/MASC186 Nucleolus147 Peroxisomes181 Mitochondria179 Phagosomes140 Golgi81 Choroplasts81 Lysosomes27 Exosomes21

Is there some logic to this complexity ?

Pocklington, et al, Molecular Systems Biol Molecular Networks: modular protein interaction networks

upstream downstream Upstream and Downstream modules A useful tool for handling complexity

Human cortex PSD: 1462 protein network Alex Bayes, Mark Collins, Louie Van De Lagemaat, Ian Whittle, Jyoti Choudhary

The origin and evolution of synapses and the brain Emes et al, Nature Neuroscience 11, 799 (2008)

6 myr 75 myr 600 myr 1000 myr Tree of life

6 myr 75 myr 600 myr 1000 myr chordates metazoans eukaryotes Origin of the brain

The first neurons and first brains

protosynapse combinations expansion Emes et al, Nature Neuroscience 11, 799 (2008)

570 genes: 186 NRC/MASC; 570 PSD Number of orthologues compared to mouse Comparative genomics

Synapse orthologues in single cell eukaryotes control response to environment Yeast behaviours: -Environmental responses -- stress -- nutrients -- pH 143 PSD proteins

Origin of the brain synapse first modelneuron first model

6 myr 75 myr 600 myr 1000 myr chordates metazoans eukaryotes Origin of the brain protosynapse

Unicellular eukaryotes Invertebrates Vertebrates Evolutionary elaboration and expansion of the protosynapse

upstream downstream

upstream downstream Yyeast Iinvertebrate Vvertebrates Method: Proportion of each functional class with earliest identifiable orthologue Origins of functional classes

Evolution of ‘learning molecules’ neurotransmitter receptors second messengers protein synthesis GO terms Yyeast Wworm Ffly Zzebrafish Cchicken Mmouse Hhuman

DLG NRC / MASC NR1 NR2 Increased combinations of complexes in vertebrates from expansion in paralogues Invertebrates Vertebrates Proteins NR111 NR214 DLG14 Complexes 116 Theoretically possible MASC combinations in mammals >10 30

Protosynapse ‘first synapse’ Synapse evolution

Protosynapse ‘first synapse’ first neurons Synapse evolution

Protosynapse ‘first synapse’ first neurons Synapse evolution

Brain size vs Synapse complexity

6 myr 75 myr 600 myr 1000 myr complex synapses big brains Big synapse proteomes evolved before big brains

How are complex synapses used in complex brains ? Examine synapse proteome composition in different in classes of neurons in mouse Chris Anderson, Cathy Vickers, Andrew Pocklington

anatomical expression level profiling >150 MASC/PSD proteins in 22 regions of mouse brain variation in levels & overlap Measured: mRNA in situ microarray protein immunohistochem western

Combinations of synapse proteome define brain regions, neuron types

Expression barcode MASC anatomical variation phylogeny recent ancient upstream downstream

Expression barcode MASC anatomical variation phylogeny recent ancient upstream downstream Protosynapse has most conserved & uniform expression pattern

Evolutionary expansions gave combinations used to generate anatomical diversity Combinations

Signaling networks in the PSP a complex combinatorial signalling machine

NMDA receptor activation with a synaptic plasticity protocol how many substrates change? Marcelo Coba, Andrew Pocklington, Mark Collins, Jyoti Choudhary (Science Signalling 2009) NMDA stimulation (long term depression, LTD) > 200 phosphorylation sites. > 120 proteins > 200 phosphorylation sites. > 120 proteins PSD

9 of 21 kinases tested NMDA receptor activation with a synaptic plasticity protocol how many kinases change? Marcelo Coba

Combinatorial outputs 10 phosphorylation sites on 4 proteins Stimulus: No change Increase phos Decrease phos Marcelo Coba

Combinatorial outputs 10 phosphorylation sites on 4 proteins Stimulus: No change Increase phos Decrease phos

Combinatorial outputs 10 phosphorylation sites on 4 proteins Stimulus: No change Increase phos Decrease phos

Combinatorial outputs 10 phosphorylation sites on 4 proteins Stimulus: No change Increase phos Decrease phos

sitesstates GluR138 (2 3 ) 10 proteins (2 100 ) PSP Combinatorial outputs = states Are any two synapses the same? Can a synapse ever be in the same state twice? What are the physiological constraints?

kinases substrates Postsynaptic kinase-substrate network - mapping 743 phosphorylation events Marcelo Coba, Andrew Pocklington

Building the network defining minimal units

How many substrates for a kinase? 20.5 (  8.3) protein substrates phosphorylated by each kinase k1 P PP kinase divergence

How many sites were phosphorylated by more than one kinase? 65% (129) sites phosphorylated by multiple kinases kinase convergence k1k2 kn P redundance / robustness multiple upstream receptors

Hubs – highly connected Sites Proteins

coincidence detectors

NMDA receptor mGluR receptor Dopamine receptor Combinations of sites Functional orchestration

Synapse evolution and expansion model of synapse diversity and behaviour

Acknowledgements Proteomics Marcelo Coba Alex Bayes Bilal Malik Mark Collins Jyoti Choudhary Expression Christopher Anderson Cathy Vickers Informatics Andrew Pocklington J. Douglas Armstrong Mike Croning Richard Emes Support Wellcome Trust Medical Research Council European Union