@University of Electro-Communications
September 9, 2016

Michisuke Yuzaki

Department of Physiology

Visualization of active circuits by f-MRI

Hersher, Nature Med, Mar 13, 2012

“Functional networks” visualized by rs-fMRI
Yu et al, Schizophrenic Res, 2013

normalschizophrenia

Functional/morphological changes @ synapses
Neuralactivities
Increase

Long-term
memoryfunctional changes:(LTP・LTD)= # of postsynaptic
AMPA receptors
nerve
terminal

LTD (long-term depression)

glutamate

post-synaptic
currents
LTP (long-term potentiation）

AMPA
receptors

spine

Functional/morphological changes @ synapses
Neuralactivities
Increase

Long-term
memoryLonger-term
memoryfunctional changes:(LTP・LTD)= # of postsynaptic
AMPA receptors

morphologicalchanges
new gene
expression

nerve
terminal

LTD (long-term depression)

glutamate
AMPA
receptors

LTP (long-term potentiation）

spine

Menu

 Cbln1 as a prototype of C1q family
 C1q-like 1 (C1qL1)—Yet another synapse organizer C1q family in the hippocampus

Synapse organizers―key molecules inneuropsychiatric disorders
Ménage à Trois
Secreted type
Cell-adhesive typePreNL

NMDARPostglypican

LARPTPLRRTM1/2

TSP

LRRTM4

NGL-3
TrkC
IL1RAPL1
Slitrks

astrocytes

NRX

FGF22/23
Wnt-7a2-1
Ca channel

Sandwich-type

NRXCbln
C1qL

GluD1/2
GluK2/4

C1q family

C1q proteins: secreted signaling molecules

globularC1q domain

trimermultimerComplement C1q →recognize foreign objects → removal

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

Cbln1 rescues ataxia in Cbln1 KO in vivo
Cbln1 KO(adult)

30 d after

beforeCbln1single inj.
2 d after

Ito-Ishida et al, J Neurosci, 2008

Cbln1 induces rapid & transient synaptogeneis in vivo

control (KO)maturegranule cell axons

2d laterin vivo

35 d later

free spines+Cbln1denditic spines(Purkinje cells)

Cbln1 is necessary forsynapse maintenance!!

Nrx-Cbln1-GluD2: a sandwich-type synapse organizer
Granule cell axon (parallel fiber)

SVNrx
Cbln1
GluD2NPresynaptic action
Mishina’s group Cell, 2010
Matsuda & Yuzaki EJN, 2011

WhatBidirectional
is GluD2? !
Postsynaptic action

Purkinje cell
dendriteshankhomer

PSD93

Matsuda et al Science 2010
Yuzaki Curr Opin Neurobiol, 2010

Orphan receptor GluD2Cbln1
N
N-terminal
domain (NTD)• Belongs to iGluR family
Cloned by Seeburg & Mishina’s groups ’93• Orphan receptor• No currents by Glu analogs

Ligand-binding
domain (LBD)

intracellular

Not orphan
anymore!

Purkinje cell
dendrites

D-Ser: yet another GluD2 ligand to regulate LTD
Granule cell axon (parallel fiber)

Nrx
Cbln1

NGluD2
D-Ser

Bergmannglia
Kakegawa et al
Nat Neurosci 2011

AMPAR
endocytosis (LTD)& Motor learningSummary

Cbln1: a new synaptic organizer• Released from cerebellar granule cells→binds to Nrx(PF) & GluD2(PCs)• Necessary for development throughout adulthood• At PF-PC synapses• Forms and maintains presynaptic boutons• Regulates LTD via D-Ser from glia
• Activity-dep.release via TeNT-insensitive vesicles

Menu

 Cbln1 as a prototype of C1q family C1q-like 1 (C1qL1)—Yet another synapse organizer C1q family in the hippocampus

The C1q family (32 members)
C1qtnf9 (CTRP9)
Adipoq (ACRP30, adiponectin)
Col8a1 (Collagen VIII1)

….
….

C1qC
C1qB
C1qA
Mmrn2 (Emilin4)
Mmrn1 (Emilin3)
Emilin2 (FOAP-10)
Emilin1 (gp115)….
Cbln subfamily

C1qL subfamily

C1qdc1 (EEG-1L, caprin2)
Cbln4
Cbln2
Cbln1
Cbln3
C1qL4
C1qL1 (CRF, C1QRF)
C1qL3
C1qL2

Synapseformation??

Cbln1 and C1qL1 in two different inputs to PC
Climbing fiber (CF)

Parallel fiber (PF)C1qL1

GluGlu

Cbln1

AMPAR

GluD2

AMPAR

Proximal dendrites

cerebellum

Purkinje GranulecellcellInferiorolive

PC
GC

Inf. olive

Distal dendrites

C1qL1 localizes at CF synaptic clefts (vGluT2+)

C1qL1
vGluT2 (CF)Severe reduction of CF synapses in C1qL1 KO
WT (P39)

C1qL-1 KO (P39)vGluT2 (climbing fiber terminals)

What are receptors
for C1qL1?

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

CFKakegawa et al
Neuron 2015

Brain Angiogenesis Inhibitor (Bai) 3: adhesion GPCRs

・ Highly expressed in brain
・ Pull-down assay identified C1ql1-3 as ligands
Bolliger et al, PNAS 2010

C1qL1

Bai3

N・ Linked to psychiatric disorders
Lanoue et al, Mol. Psychiat 2013

CCell-adhesion GPCRs

Bai3 is located at CF synapses

vGluT2 (CF)Bai3
Calbindin• Retracted• Decreased #Formation of CF synapses during developmentｰP3
Innervation bymultiple CFs

P3ｰP7

P7ｰP11

P12ｰ

Functional selection Translocation ofof a “winner CF”
the winner CF
strengthen
weakenactivity

Singlewinner CF
dep. on P/Q Ca2+ channel

Normal in C1qL1 KO
Kano & Watanabe’s model

Elimination ofloser CFs

Abnormal CF synapse formation in C1qL1 KO
P3ｰP7ｰP3
Innervation bymultiple CFs

P7ｰP11

P12ｰ

Functional selectionof a “winner CF”

C1qL1 determines
winner CF?

activity
dep. on P/Q Ca2+ channel

Normal in C1qL1 KO
Kano & Watanabe’s modelNo single
winner!

Incomplete
elimination!

Abnormal in C1qL1KO

Like Shiva – "the Destroyer" and "the Creator"ｰP3
Innervation bymultiple CFs

P3ｰP7

P7ｰP11

P7ｰ

Functional selectionof “winner CF”

in a shorter time

activity

Elimination ofloser CFs

C1qL1-Bai3 accelerates
Can C1qL1 function-Maturation of a strong CF
in maturebrain?
-Eliminationof weakCFs

C1qL1-Bai3 restores normal CF synapses in adultWeak (loser) CFs
Single winner CF strengthened"the Destroyer" and "the Creator"

SummaryDual roles of C1qL1 in synapse maturation

・ During development
・Strengthening of a single winner CF
・Weakening of a looser CFs
・ Even after maturation
・ Strengthening of strong CF・ Elimination of weak CF・ Bai3 serves as a receptor

Summary

Cbln1 & C1ql1: new synaptic organizers

Climbing fiber (CF)C1qL1

Parallel fiber (PF)

Glu

Bai3

AMPAR
Glu

Cbln1
GluD2

AMPAR

Proximal dendrites@CF synapses

Purkinje GranulecellcellInferior
olive

Distal dendrites@PF synapses• Forms and maintains PF・ Strengthen strong CFHowaboutoutside
・ Eliminate weak CFs

cerebellum?

Menu Cbln1 as a prototype of C1q family
 C1q-like 1 (C1qL1)—Yet another synapse organizer C1q family in the hippocampus

Expression of C1qL subfamily in adult brain (ISH)
C1qL1

C1qL3

C1qL2

C1qL4

Iijima et al, Eur J Neurosci 2010
C1qL1 in inf. olive
C1qL2 & 3 in the dentate gyrus

C1qL2 and C1qL3 localize at MF-CA3 synapses
null

WT
IHC: C1qｌ2
CA1
CA2
DG

CA3C1qｌ3

C1qL2/3 do not regulate MF–CA3 synapse formation
WT

C1ql2/3-null

VGluT10.4VGluT1/calb

Relative frequency

Lucidum

0.3

WTC1ql2/3-null

0.2
0.10

Size of VGluT1 puncta (m2)

C1qL2/3 determine synaptic localization of GluK2/3
IHC:

C1qｌ2

WTnull

IHC: GluK2/3

C1qｌ2/3-null

WT

CA1

GluK2-null

GluK2/3 CA2
MAP2

CA2
DG

RaCA3

Lu
CA3

C1qｌ3

GluK2/3

WT

WTnullnull

GluK2/3

No synaptic
GluK2/3!
CA3

IB: GluK2/3

C1qL2/3 specifically bind to GluK2 and GluK4+ C1ql2-HAor C1ql3-HA

ATD
LBD***TMD293 cells

CTD

40iGluR
20***

C1ql2-HA
C1ql3-HA

What happens to******
Neto1 in C1qL2/3KO?

0KAR co-subunit

C1qL2/3 as a master regulator of KARs
WT

C1qL2-null

Neto1

WT
GluK4GluK4

GluK5GluK5

Functions ofKARs in KO?

C1qL3-null C1qL2/3-null

C1qｌ2/3-null GluK2-null

C1qL2 & C1qL3
GluK2, GluK4
KARs(Neto1,GluK5…)

Pathological significance
15in epilepsy?

MF

WT+ GYKI

Stim

DG

WTC1qL2/3-null

100 pA
50 ms

30 ms

+ GYKI52466 (AMPAR blocker)

GluK2-null

10* *5C1ql2/3-null

GYKI / ContRec.

CA3

GYKI-resistant EPSC (%)

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

0240 pA* *100 ms
1Reduced EPSC summation0Less seizure-like activities in C1ql2/3 KO
Field RecordingrMF

glMF

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

MF sprouting w/opostsynaptic KARs!!

DGWT

GluK2-null

C1ql2/3-null

400 V
400 msParoxysmal Recurrent
 postsynaptic KAR in rMFDischargeBursts

Summary

C1ql2/3 recruits KARs at MF–CA3 synapses

Presynapse (MF terminal)

Nrx3(S5)
C1ql2/3
GluK2/4
L-Glu

Current integration

recruit

CA3 neurons (wt)
DGCs (chronic epilepsy)
Matsuda et al Neuron, 2016

Menu

 Cbln1 as a prototype of C1q family
 C1q-like 1 (C1qL1)—Yet another synapse organizer C1q family in the hippocampus
 How is C1q family regulated trans-synaptically?

Cbln1 and C1qL2/3 bind to specific Nrx isoforms
NrxNrxNrx

Pre (PF terminal)Pre (MF terminal)

Nrx1-3(AS4)

Nrx3(AS5)

Cbln1

C1ql2/3

GluD2

GluK2/4
D-Ser

L-Glu

Post
Purkinje cellMatsuda et al, Eur J Neurosci 2011

Post
CA3 pyramidal cellMatsuda et al, Neuron 2016

Cbln1 and C1qL2/3 bind to specific Nrx isoforms• Symmetry mismatch: how is hexameic Cbln1 recognized?
 Bind to Nrx & GluD2
Pre
Nrx1-3(S4)

?

Cbln1
GluD2
D-Ser

monomer

 No binding!

hexamer (dimer of trimer)?

tetramer (dimer of dimer)

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

1 Nrx binds to 1 Cbln1 hexamer
Negative –stain EM class average

Cbln1

Cbln1

Nrx+

Nrx

Isothermal Titration
Calorimetry

10 nm

KD = 44 nM
N = 0.895

Cbln1+

KD = NA

1 Nrx
1 Cbln1

Cbln1 trimer binds to GluD2 ATD monomer
Cbln1(hexamer)

Cbln1(trimer)

GluD2-ATD(dimer)GluD2-ATD(monomer)

KD = 125 nM

F76DHigh M range“Avidity” enhances Cbln1-GluD2 bindingD-Ser signaling requires Cbln1 binding to GluD2PreStoichiometry

2 monomerNrx1-3(S4):Cbln1?

D-Ser

2 hexamer
1 tetramer

:

GluD2

Amino-terminal
domain (ATD)
Ligand-binding
domain (LBD)

GluD2GW
Post

GluD2WT• Allosteric interaction?
ATD-LBD glycan wedge (GluD2GW)
No Cbln1 binding (GluD2Cbln1)
Monomeric ATD (GluD2mono)

D-Ser-dependent

LTD was impaired!
Elegheert et al Science 2016

Summary

Structural paradigm for transsynaptic
Nrx-Cbln1-GluD2 signaling

Parallel fiber(Granule cell axons)

Presynapse
Avidityenhanced binding formation
Parallel fiberclustering

Nrx
GluD2
NTD
LBD

Ito-Ishida,Neuron, 2012

AMPAR
Cbln1

Purkinje cell

D-Ser

Purkinje cell
dendriteElegheert, Science, 2016

Allostericinteraction
LTDregulation

Summary

C1q family: Synaptic Ménage á Trois
cerebellum

PF-PC
synapseshippocampus

CF-PC
synapses

Nrx1-3
(S4)

?

Cbln1

C1qL1

GluD2

Cblns
C1qLs

Bai3

PP–CA2/DG
synapses

MF–CA3synapses

?

Nrx3
(S5)

Cbln1/4

C1qL2/3

GluD1

GluK2/4• Synapse formation/maturation• Synapse elimination• Regulation of postsynaptic iGluRsAcknowledement: collaborators…

Dr. Masahiko Watanabe
Hokkaido Univ.

Dr. Kenji Sakimura
Niigata Univ.

Dr. Radu Aricescu
Univ Oxford, UKDr. Wataru Kakegawa
Dr. Keiko Matsuda