Synapse Complexity

Swartz FoundationBanbury Meeting, CSHL

15th April 2009

Seth Grant

Genes to Cognition programwww.genes2cognition.org

Wellcome Trust Sanger Institute, Cambridge UK

MASC

PSDMASC

Post-Synaptic Proteome

MASC MAGUK Associated Signaling ComplexPSD Post Synaptic Density------------------------------------------------------------------------------------------PSP Post Synaptic Proteome (total set of postsynaptic proteins)

Presynaptic terminal

Postsynaptic terminal

NR

2

PSD-95fyn

NMDA receptor

Genetic evidence for postsynaptic complexes

Grant, et al, Science. 258, 1903-10. 1992Migaud et al, Nature, 396; 433-439. 1998Sprengel et al, Cell 92, 279-89. 1998

Learning impairmentsPlasticity impairments

MAGUK proteins

PSD95

NR

Husi et al. Nature Neuroscience, 3 (7), 661-669. 2000.Husi & Grant. J. Neurochem, 77, 281-291. 2001Collins et al, J. Neurochem. 2005Fernandez 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; 433-439. 1998; Husi et al, Nature Neuroscience, 2000

synaptic strength gene expressionmRNA turnoverProtein turnover

BehavioursCognition & plasticity

NRC / MASC

2-3 MDa186 proteins

47 genes in human disease48 synaptic plasticity42 rodent behaviour

Complexity

PSDMASC

PSD 700 -1500 proteins

MASC 77 - 185 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, 59-63. 2006

Post Synaptic Density 1124ER:microsomes 491Splicesome 311NRC/MASC 186Nucleolus 147Peroxisomes 181Mitochondria 179Phagosomes 140Golgi 81Choroplasts 81Lysosomes 27Exosomes 21

Is there some logic to this complexity ?

Pocklington, et al, Molecular Systems Biol 2006.

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

Unicellulareukaryotes

Invertebrates Vertebrates

Evolutionary elaboration and expansion of the protosynapse

upstream

downstream

upstream

downstream

Y yeastI invertebrateV vertebrates

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

Y yeastW wormF flyZ zebrafishC chickenM mouseH human

DLG

NRC / MASC

NR

1NR

2

Increased combinations of complexes in vertebratesfrom expansion in paralogues

Invertebrates Vertebrates

Proteins

NR1 1 1NR2 1 4DLG 1 4

Complexes1 16

Theoretically possible MASC combinations in mammals >1030

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 s

ynapse

s

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

0 4 4 3 1

1 4 3 2 3

2 3 2 2 1

anatomicalvariation

phylogeny

recent

ancient

upstream

downstream

Expression barcode

MASC

0 4 4 3 1

1 4 3 2 3

2 3 2 2 1

anatomicalvariation

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

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

sites states

GluR1 3 8 (23)

10 proteins 100 1030 (2100)

PSP 104 1060

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

Number of kinases with n substrates

0

1

2

3

4

5

6

7

8

9

0-5 5-10 10-15 15-20 20-25 25-30 30-35 35-40

n substrate

nu

mb

er o

f ki

nas

es

How many substrates for a kinase?

20.5 ( 8.3) protein substrates phosphorylated by each kinase

k1

PPPkinasedivergence

Number of sites with n kinases

0

10

20

30

40

50

60

70

80

1 2 3 4 5 6 8 9 10 12

n kinase

num

ber

of

site

s

How many sites were phosphorylated by more than one kinase?

65% (129) sites phosphorylated by multiple kinases

kinaseconvergence

k1 k2 kn

P

• redundance / robustness• multiple upstream receptors

Hubs – highly connected

SitesProteins

coincidence detectors

NMDA receptor

mGluR receptor

Dopaminereceptor

Combinations of sites

Functional orchestration

EPMBEHAVIOUR

OF NOE FC RR

Synapse evolution and expansion model of synapse diversity and behaviour

-20 0 20 40 60

50

100

150

200

250

AcknowledgementsProteomicsMarcelo CobaAlex Bayes

Bilal Malik

Mark CollinsJyoti Choudhary

ExpressionChristopher AndersonCathy Vickers

InformaticsAndrew PocklingtonJ. Douglas Armstrong

Mike CroningRichard Emes

Support

Wellcome TrustMedical Research CouncilEuropean Union

www.genes2cognition.orgwww.g2conline.org