Michisuke Yuzaki

Department of Physiology

@University of Electro-CommunicationsSeptember 9, 2016

Visualization of active circuits by f-MRI

Hersher, Nature Med, Mar 13, 2012

Yu et al, Schizophrenic Res, 2013

normal schizophrenia

“Functional networks” visualized by rs-fMRI

IncreaseIncrease

Neural activities

glutamate

nerve terminal

spine

AMPAreceptors

LTP (long-term potentiation）

post-synaptic currents

Functional/morphological changes @ synapses

LTD (long-term depression)

Long-term memory

= # of postsynaptic AMPA receptors

functional changes:(LTP・LTD)

IncreaseIncrease

Neural activities

glutamate

nerve terminal

spine

AMPAreceptors

LTP (long-term potentiation）

LTD (long-term depression)

Long-term memory

= # of postsynaptic AMPA receptors

functional changes:(LTP・LTD)

AMPAreceptors

morphological changes

new gene expression

Longer-term memory

Functional/morphological changes @ synapses

Menu

Cbln1 as a prototype of C1q family

C1q family in the hippocampus

C1q-like 1 (C1qL1)—Yet another synapse organizer

2-1Ca channel

Secreted type

astro

cyte

s

TSP FGF22/23Wnt-7a

Sandwich-type

CblnC1qL

GluK2/4

NRX

Synapse organizers―key molecules in neuropsychiatric disorders

GluD1/2

NRX

LRR-TM1/2NL

Pre

Post

LAR-PTP

NGL-3TrkCIL1RAPL1Slitrks

LRR-TM4

Cell-adhesive type

NMDAR

glypican

C1q family

Ménage à Trois

trimer

globular C1q domain

multimer

Complement C1q →recognize foreign objects → removal

C1q proteins: secreted signaling molecules

32 members → Some C1q proteins regulate CNS synapses! Cbln1-4・・・Naｔ Neurosci ‘05; Science ‘10, ‘16 C1qL1-4・・・Neuron ’15, ‘16 C1q （complement）・・・Stevens et al, Cell ‘07

Cbln1single inj.

2 d after

Ito-Ishida et al, J Neurosci, 2008

Cbln1 KO(adult)

30 d after

before

Cbln1 rescues ataxia in Cbln1 KO in vivo

control (KO)

free spines

35 d later

denditic spines(Purkinje cells)

granule cell axons

+Cbln1

2d later

Cbln1 induces rapid & transient synaptogeneis in vivo

Cbln1 is necessary for synapse maintenance!!

mature in vivo

N

Postsynaptic action

homer

shank

PSD93

Nrx-Cbln1-GluD2: a sandwich-type synapse organizer

Matsuda et al Science 2010Yuzaki Curr Opin Neurobiol, 2010

Cbln1

Granule cell axon (parallel fiber)

Nrx Presynaptic action

Mishina’s group Cell, 2010Matsuda & Yuzaki EJN, 2011

GluD2

SV

Purkinje cell dendrite

What is GluD2?What is GluD2?Bidirectional !

•Belongs to iGluR familyN Cloned by Seeburg & Mishina’s groups ’93

•Orphan receptor• No currents by Glu analogs

Ligand-binding domain (LBD)

N-terminal domain (NTD)

Purkinje cell dendrites

Orphan receptor GluD2Cbln1

Not orphan anymore!

intracellular

N

D-Ser: yet another GluD2 ligand to regulate LTD

Cbln1

Granule cell axon (parallel fiber)

Nrx

GluD2

D-SerD-Ser

Bergmann glia

Kakegawa et alNat Neurosci 2011

AMPAR endocytosis (LTD) & Motor learning

Cbln1: a new synaptic organizer

•Released from cerebellar granule cells→binds to Nrx(PF) & GluD2(PCs)

Summary

•Activity-dep.release via TeNT-insensitive vesicles

•At PF-PC synapses•Forms and maintains presynaptic boutons•Regulates LTD via D-Ser from glia

•Necessary for development throughout adulthood

Menu

Cbln1 as a prototype of C1q family

C1q family in the hippocampus

C1q-like 1 (C1qL1)—Yet another synapse organizer

C1qdc1 (EEG-1L, caprin2)Cbln4

Cbln2Cbln1

Cbln3C1qL4C1qL1 (CRF, C1QRF)

C1qL3C1qL2

C1qBC1qC

C1qA

Mmrn2 (Emilin4)Mmrn1 (Emilin3)

Emilin2 (FOAP-10)Emilin1 (gp115)

C1qtnf9 (CTRP9)Adipoq (ACRP30, adiponectin)

Col8a1 (Collagen VIII1)

Cbln subfamily

….

….

….

C1qL subfamily

The C1q family (32 members)

Synapse formation

??

Granule cell

Purkinje cell

Inferior olive

Cbln1

Parallel fiber (PF)

AMPAR

Glu

Distal dendrites

GluD2

C1qL1

GluAMPAR

Proximal dendrites

Climbing fiber (CF)

cerebellum

Inf. olive

PC

GC

Cbln1 and C1qL1 in two different inputs to PC

C1qL1 localizes at CF synaptic clefts (vGluT2+)

vGluT2 (CF)C1qL1

Severe reduction of CF synapses in C1qL1 KO

WT (P39) C1qL-1 KO (P39)

vGluT2 (climbing fiber terminals)

Kakegawa et alNeuron 2015

• decreased # of CF synapses• retracted CF territory• multiple CFs innervate single PC

CF

CFCF

What are receptors for C1qL1?

What are receptors for C1qL1?

Brain Angiogenesis Inhibitor (Bai) 3: adhesion GPCRs

Bolliger et al, PNAS 2010

・ Highly expressed in brain

・ Pull-down assay identified C1ql1-3 as ligands

・ Linked to psychiatric disordersLanoue et al, Mol. Psychiat 2013

Bai3 C

N

Cell-adhesion GPCRs

C1qL1

Bai3 is located at CF synapses

Bai3vGluT2 (CF)

Calbindin

vGluT2 (CF)

• Retracted• Decreased #

Formation of CF synapses during development

ｰP3Innervation by multiple CFs

Functional selection of a “winner CF”

P3ｰP7

activity

P7ｰP11Translocation of the winner CF

Single winner CF

Normal in C1qL1 KOdep. on P/Q Ca2+ channel

Kano & Watanabe’s model

Elimination of loser CFs

P12ｰ

strengthen

weaken

Abnormal CF synapse formation in C1qL1 KO

ｰP3Innervation by multiple CFs

Functional selection of a “winner CF”

P3ｰP7

activity

Normal in C1qL1 KOdep. on P/Q Ca2+ channel

Kano & Watanabe’s modelAbnormal in C1qL1KO

P7ｰP11

No single winner!

P12ｰ

Incomplete elimination!

winner CF?C1qL1 determines

winner CF?

Like Shiva – "the Destroyer" and "the Creator"

ｰP3Innervation by multiple CFs

Functional selection of “winner CF”

P3ｰP7

activity

P7ｰP11 P7ｰ

Elimination of loser CFs

C1qL1-Bai3 accelerates-Maturation of a strong CF-Elimination of weak CFs

in a shorter time

Can C1qL1 function Can C1qL1 function in mature brain?

C1qL1-Bai3 restores normal CF synapses in adult

Single winner CF strengthened

Weak (loser) CFs

"the Destroyer" and "the Creator"

Dual roles of C1qL1 in synapse maturation

・ Even after maturation・ Strengthening of strong CF・ Elimination of weak CF

・ Bai3 serves as a receptor

・ During development・Strengthening of a single winner CF・Weakening of a looser CFs

Summary

Granule cell

Purkinje cell

Inferior olive

Parallel fiber (PF)

AMPAR

Glu

Distal dendrites

GluAMPAR

Proximal dendrites

Climbing fiber (CF)

Cbln1GluD2

C1qL1

Summary Cbln1 & C1ql1: new synaptic organizers

Bai3

• Forms and maintains PF ・ Strengthen strong CF・ Eliminate weak CFs

@CF synapses @PF synapses

How about outside How about outside cerebellum?

Menu

Cbln1 as a prototype of C1q family

C1q family in the hippocampus

C1q-like 1 (C1qL1)—Yet another synapse organizer

Expression of C1qL subfamily in adult brain (ISH)

C1qL2 & 3 in the dentate gyrus

Iijima et al, Eur J Neurosci 2010C1qL1 in inf. olive

C1qL1

C1qL2

C1qL3

C1qL4

C1qｌ2CA1

CA2

CA3DG

IHC:

C1qｌ3

WT null

C1qL2 and C1qL3 localize at MF-CA3 synapses

C1qL2/3 do not regulate MF–CA3 synapse formation

WT C1ql2/3-null

VGluT1/calb

VGluT1

Rel

ativ

e fre

quen

cy

0

0.1

0.2

0.3

0.4

Size of VGluT1 puncta (m2)

WTC1ql2/3-null

Lucidum

C1qL2/3 determine synaptic localization of GluK2/3

GluK2/3

C1qｌ2CA1

CA2

CA3DG

IHC:

C1qｌ3

WT null

null

null

WT

WT

C1qｌ2/3-null GluK2-nullWTGluK2/3MAP2

GluK2/3

CA2

CA3

RaLu

IHC: GluK2/3

No synaptic No synaptic GluK2/3!

IB: GluK2/3CA3

C1qL2/3 specifically bind to GluK2 and GluK4

ATD

LBD

TMD

CTD

iGluR

+ C1ql2-HAor C1ql3-HA

293 cells

20

40

0

***

***

******

C1ql2-HAC1ql3-HA

KAR co-subunit

Neto1 in C1qL2/3KO?What happens to

Neto1 in C1qL2/3KO?

C1qL2/3 as a master regulator of KARs

WT C1qL2/3-nullC1qL3-nullC1qL2-null

GluK5

C1qｌ2/3-null GluK2-nullWTGluK4GluK4

GluK5

Neto1

C1qL2 & C1qL3

GluK2, GluK4

KARs in KO?Functions of KARs in KO?

KARs(Neto1,GluK5…)

Slow KAR-dep. EPSC is missing in C1qL2/3 KO

CA3 MF

DGStim

Rec.

0

5

10

15

GYK

I-res

ista

nt E

PSC

(%)

0

2

1

GYK

I/

Con

t

GluK2-nullC1qL2/3-nullWT+ GYKI

+ GYKI52466 (AMPAR blocker)

* *

* *

50 ms

100 pA

30 ms

WT C1ql2/3-null

40 pA

100 ms

Reduced EPSC summation

Pathological significance Pathological significance in epilepsy?

Less seizure-like activities in C1ql2/3 KO

rMF

DG

Field Recording

MF

gl

GluK2-null C1ql2/3-null

P9 Slice culture + Pilocarpine 5 div for 2 d Record at 9-12 div

WT

400 ms400 V

Paroxysmal Discharge

Recurrent Bursts

MF sprouting w/o postsynaptic KARs!!

postsynaptic KAR in rMF

C1ql2/3 recruits KARs at MF–CA3 synapses

Current integration

C1ql2/3

Nrx3(S5)

Presynapse (MF terminal)

GluK2/4L-Glu

recruit

Matsuda et al Neuron, 2016

CA3 neurons (wt)DGCs (chronic epilepsy)

Summary

Menu

Cbln1 as a prototype of C1q family

C1q family in the hippocampus

C1q-like 1 (C1qL1)—Yet another synapse organizer

How is C1q family regulated trans-synaptically?

Cbln1 and C1qL2/3 bind to specific Nrx isoforms

C1ql2/3

Nrx3(AS5)

Pre (MF terminal)

GluK2/4L-Glu

PostCA3 pyramidal cell

Nrx

Nrx

Matsuda et al, Neuron 2016

Nrx

Cbln1

Nrx1-3(AS4)

Pre (PF terminal)

GluD2D-Ser

Post Purkinje cell

Matsuda et al, Eur J Neurosci 2011

Cbln1 and C1qL2/3 bind to specific Nrx isoforms

Cbln1

Nrx1-3(S4)

Pre

GluD2D-Ser

Post

hexamer (dimer of trimer)

monomer

tetramer (dimer of dimer)

• Symmetry mismatch: how is hexameic Cbln1 recognized?

Bind to Nrx & GluD2

No binding!

• Allosteric interaction? Cbln1 binding vs. D-Ser biidng?

?

?

1 Nrx binds to 1 Cbln1 hexamer

Nrx

+

Cbln1

Isothermal Titration Calorimetry

Negative –stain EM class average

1 Cbln1

10 nm

+KD = NA

KD = 44 nMN = 0.895

1 Nrx

Cbln1

Nrx Cbln1

Cbln1 trimer binds to GluD2 ATD monomer

GluD2-ATD(dimer)

High M rangeKD = 125 nM

Cbln1(hexamer)

Cbln1(trimer)

GluD2-ATD(monomer)

F76D

“Avidity” enhances Cbln1-GluD2 binding

D-Ser signaling requires Cbln1 binding to GluD2

Cbln1

Nrx1-3(S4)

Pre

GluD2

Post

Ligand-binding domain (LBD)

Amino-terminal domain (ATD)

D-Ser?

• Allosteric interaction?ATD-LBD glycan wedge (GluD2GW)No Cbln1 binding (GluD2Cbln1)Monomeric ATD (GluD2mono)

D-Ser-dependent LTD was impaired!

2 monomer

2 hexamer

1 tetramer

::

Stoichiometry

GluD2GW

GluD2WT

Elegheert et al Science 2016

Presynapseformation

Ito-Ishida, Neuron, 2012

clustering

GluD2NTDLBD

Nrx

D-Ser

LTD regulation

AMPAR

Elegheert, Science, 2016

Purkinje celldendrite

Parallel fiber(Granule cell axons)

Purkinje cell

Parallel fiber

Cbln1

SummaryStructural paradigm for transsynaptic

Nrx-Cbln1-GluD2 signaling

Allosteric interaction

Avidity-enhanced binding

PF-PC synapses

cerebellum

GluD2

Cbln1

• Synapse formation/maturation• Synapse elimination• Regulation of postsynaptic iGluRs

CblnsC1qLs

C1q family: Synaptic Ménage á Trois

Nrx1-3(S4)

C1qL1

CF-PC synapses

Bai3

?

PP–CA2/DG synapses

hippocampus

Cbln1/4

GluD1

?C1qL2/3

MF–CA3 synapses

GluK2/4

Nrx3(S5)

Summary

Dr. Masahiko WatanabeHokkaido Univ.

Dr. Kenji SakimuraNiigata Univ.

Dr. Radu AricescuUniv Oxford, UK

Acknowledement: collaborators…

Dr. Wataru Kakegawa

Dr. Keiko Matsuda