SRC UPREGULATION OF NMDA RECEPTORS:A COMMON LINK IN PAIN AND

SCHIZOPHRENIAMICHAEL SALTER MD PHD FRSC

U TORONTO CENTRE FOR THE STUDY OF PAIN

Mg2+

Cytoplasmic

2134

Glu

Cytoplasmic

K+

Na+

Ca2+

N

C

Scaffolding/Signalling Proteins

YP

YP

YP

YP

YP

Phosphorylation by Src kinase

21 3 4

Gly

N

C

K+

Na+

Ca2+

0.01

0.001

2 min0.1

0.01

2 min0.1

kinase activator

phosphatase

0.001

NMDA

phosphatase inhibitor

kinase inhibitor

Src upregulation of NMDA receptors Src upregulation of NMDA receptors in pathologic pain statesin pathologic pain states

Suppression of Src upregulation of Suppression of Src upregulation of NMDA receptors by neuregulin 1 – NMDA receptors by neuregulin 1 – ErbB4 signalingErbB4 signaling

Src upregulation of NMDA receptors Src upregulation of NMDA receptors in pathologic pain statesin pathologic pain states

Suppression of Src upregulation of Suppression of Src upregulation of NMDA receptors by neuregulin 1 – NMDA receptors by neuregulin 1 – ErbB4 signalingErbB4 signaling

NMDA receptor complex

ND2

SrcGingrich et al, PNAS 101: 6237-6242, 2004

NMDA receptor complex

SrcSrc 40-49

ND2

Tat Src40-49

Spinal cord

L5DRG

Sham

Nerve injury

0 1 2 3 40

5

10

15

20

50%

Th

resh

old (

g)

Time after surgery (weeks)

Experimental model of chronic constriction injury of a sensory nerve

Experimental models of chronic injury of a peripheral nerve

PNI-induced behaviours are reversed by Src40-49Tat

PNI-induced behaviours are reversed by Src40-49Tat

Src40-49Tat i.v. suppresses Src-ND2 association

PNI-induced increase in GluN2B tyrosine phosphorylation is reversed by Src40-49Tat

PNI-induced behaviours are suppressed Src-/- mice

Effect of Src40-49Tat is occluded in Src null mice

Liu et al. Nature Medicine 14:1325-1332, 2008

Src upregulation of NMDA receptors Src upregulation of NMDA receptors in pathologic pain statesin pathologic pain states

Suppression of Src upregulation of Suppression of Src upregulation of NMDA receptors by neuregulin 1 – NMDA receptors by neuregulin 1 – ErbB4 signalingErbB4 signaling

a

b

30 ms

10 mV

sSrc(40-58)

a

b Src (40-58)

Slope (

V/S

)

A

Slope (%

)

100

200

300a b

8

4

1

Ba

b

Anti-Src1

IgG

a

b

Slope (%

)

100

200

300a b

8

4

1

Slope (

V/S

)

Time (min)

0 20 40 60

fEP

SP

Slope (%

)

100

200

300

Time (min)

0 20 40 60

fEP

SP

slope (%)

100

200

300

Src (40-58)

sSrc(40-58)

Anti-Src1

IgG

2 mV2 m

V15 ms

15 ms

a

b

a

b

a b

a

b

a

b

a b

10 mV

Blocking Src Prevents LTP Induction in CA1

30 ms

Lu et al, Science 279: 1363, 199840 58

KPASADGHRGPSAAFVPPA

CAKb

STEP

NMDAR

Glu

AMPAR KAIR

Basal

Mg2+

Tetanus /Theta burst

Input

CAKb

Glu

Na+

P

CAKbP

Glu

Potentiated

Ca2+

ND2

SrcND2

SrcND2

Src

PTPαCsk

+--

GPCR, EphB and other signaling

P

AMPAR KAIR

Induction

Salter & Pitcher, 2011

ErbB Family of Receptors

Trends Cardiovasc Med 2:80-86, 2003

ErbB4 interacts with PSD-95 at excitatory synapses

Huang et al, Neuron 26:443-455, 2000

Ma et al, J. Neurosci. 23:3164 –3175, 2003

ErbB4 localizes in the PSD in spines

NRG1: 8p12-p21

ERBB4: 2p33.3-q34

Mei & Xiong Nature Rev Neuroci 9:437–452, 2008

b c

a

Time (min)

fEP

SP

slo

pe (

%)

0

25

50

75

100

125

APV

NRG

NBQX

Neuregulin

fEP

SP

slo

pe (

%)

fEP

SP

slo

pe (

%)

0 20 40 60 80

100

150

200

250

a b c

a b c

20 40 60 80Interval (ms)

0 250 500

Rat

io (

P2/

P1)

1.0

1.5

2.0

Time (min)

0 20 40 60 80

100

150

200

250

Control

Neuregulin

Time (min)

Huang et al, Neuron 26:443-455, 2000

Pitcher et al, NeuroReport 19:139-143, 2008

Pitcher et al, NeuroReport 19:139-143, 2008

Loss of function of ErbB4 enhances tbLTP in the hippocampal CA1

NRG1b prevents but does not reverse theta burst stimulation (TBS)-induced LTP in the hippocampal CA1

Pitcher et al, Nature Medicine 17:470-478, 2011

NRG1b has no effect on basal NMDAR EPSCs in CA1

Nor

mal

ized

NM

DA

EP

SC

A

mpl

itud

e (%

)Neuregulin Prevents Endogenous Src Activation-Dependent

Potentiation of Schaffer Collateral-Evoked NMDA Excitatory Postsynaptic Currents

NRG1b prevents endogenous Src activation-induced potentiation of NMDAR EPSCs in CA1

ErbB4 mediates NRG1b suppression of Srcpotentiation of NMDAR EPSCs in CA1

NRG1b prevents endogenous Src potentiation of NMDAR EPSCs in Prefrontal Cortex

NRG1b–ErbB4 signaling prevents TBS-induced GluN2B phosphorylation in hippocampal CA1

NRG1b does not affect LTP in Src-/- slices

EPQ(pY)EEIPIA enhancement of synaptic transmission is lost inSrc-/- slices

NRG1b signaling reduces depolarization of CA1 neurons during the period of TBS

NRG1b signaling reduces depolarization of CA1 neurons during the period of TBS

NRG1b signaling reduces depolarization of CA1 neurons during the period of TBS

Control

NRG1b

NMDAR

Glu

Mg2+AMPAR KAIR

ND2

Basal SynapticTransmission

AMPAR KAIR

Src

Glu

ND2

EnhancedSynaptic Plasticity

Src

ErbB4

ErbB4

NRG1

X

Glu

ND2

PhysiologicalSynaptic Plasticity

Src

ErbB4

NRG1

+

-

Theta BurstStimulation

Glu

ND2Src

ErbB4

SuppressedSynaptic Plasticity

NRG1

+-

Ca2+

Ca2+

Salter & Pitcher, FEBS Letters, 2011

Graham Pitcher

Declan AliRand AskalanDavid NgJon DavidowJeff GingrichYueqiao HuangLorraine KaliaGary KeilSimon BeggsKen PelkeyXuejun LiuXian-Min Yu Yutian Wang

Janice Hicks Sandra Singhroy David Wong

U of TEvelyn LambeNathalie GoodfellowJohn MacDonald

Tufts UnivKathleen Yee

UNCRichard Weinberg

U. Alabama / Georgia Health Sci. U.Lin MeiRan-Sook WooYang Z. Huang

Mt. Sinai HospitalJohn RoderYou-Ming Lu

Declan AliRand AskalanSimon BeggsJon DavidowJeff GingrichYueqiao HuangLorraine KaliaGary KeilChun KwanKen PelkeyXuejun LiuYi NongGraham Pitcher Tuan TrangXian-Min Yu Yutian WangWenbo Zhang Janice Hicks Sandra Singhroy David Wong

Mt. Sinai HospitalJohn RoderYou-Ming Lu

Yale UniversityPaul LombrosoTri-Hung Nguyen

U of T / RobartsJohn MacDonaldWei-Yang LuZhigang Xiong

Sapporo Medical Univ.Terukatsu SasakiHiroshi Aoto

U of T / UBCYutian WangWilliam JuReza Ahmadian

N I HRobert WentholdRonald Petralia

U of TEvelyn LambeNathalie Goodfellow

Tufts UnivKathleen Yee

UNCRichard Weinberg

U. Alabama / Georgia Health Sci. U.Lin MeiRan-Sook WooYang Z. Huang

PNI-induced increase in GluN2B tyrosine phosphorylation is suppressed in Src-/- mice

Liu et al. Nature Medicine 14:1325-1332, 2008

Mg2+

Cytoplasmic

2134

Glu

Cytoplasmic

N

C

Scaffolding/Signalling Proteins

YP

YP

YP

YP

YP

Phosphorylation by Src kinase

21 3 4

Gly

N

C

K+

Na+

Ca2+

PNI-induced cold sensitization reversed by Src40-49Tat

Schizophrenia Genetic Susceptibility(Neuregulin1 (NRG1) & ErbB4)

NMDAR HypofunctionPCP

Ketamine

Hippocampus&

Theta Wave (EEG)

Human ErbB4 (side)

Human ErbB4 (top)

b c

a

Time (min)

fEP

SP

slo

pe

(%

)

0

25

50

75

100

125

APV

NRG

NBQX

Neuregulin

fEP

SP

slo

pe

(%

)fE

PS

P s

lop

e (

%)

0 20 40 60 80

100

150

200

250

a b c

a b c

20 40 60 80Interval (ms)

0 250 500

Ra

tio (

P2

/P1

)

1.0

1.5

2.0

Time (min)

0 20 40 60 80

100

150

200

250

Control

Neuregulin

Time (min)

Effect of NRG-1b on NR2 Tyrosine Phosphorylation at Schaffer Collateral CA1 Synapses

pY

NR2

pY

NR2

Hahn, C-G., Nat. Med. 17:425-427, 2011

Src hypofunction induced by enhanced NRG1-ErbB4 signalingin schizophrenia