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