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Bogomoletz Institute of Physiology, Kiev, Ukraine

nana@biph.kiev.ua

Dr. NANA VOITENKODr. NANA VOITENKOAACIMPAACIMP

KIEV - 2011KIEV - 2011

Molecular Mechanisms of Pain

part II

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Part B:- Spinal AMPARs-mediated synaptic transmission during inflammation. - Molecular mechanism of GluR2 internalization from synapses.

Part C:- Involvement of spinal extrasynaptic AMPARs in the maintaining of persistent

pain.- Trafficking of Ca2+-permeable AMPARs during inflammatory pain.

Part D: - Protein kinase Ca as a molecular target for perspective treatment of persistent

pain. - Antisense oligonucleotides – a new strategy of pain treatment

Part A: - Classification of receptors to glutamate.- Structure and molecular organization of AMPA receptors.

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GLUTAMATE RECEPTORS

Ionotropic GluRs Metabotropic GluRs

NMDA

NR1

NR3A

NR2A-D

AMPA

GluR 1-4

Kainate

GluR 5,6,7 KA 1,2

Group I

mGlu1 mGlu5

Group II

mGlu2 mGlu3

Group III

mGlu4 mGlu6

mGlu8mGlu7

Drug News Perspect 2003, 16(8): 513

Mediating effects include:

- Membrane depolarization- Ca2+ and Na+ influx- K+ efflux- Free radical formation

Modulatory effects on:

- iGluR- K+ and Ca2+ channels - Neurotransmitter release

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AMPA receptors is a subtype of ionotropic transmembrane receptor for

glutamate that mediates fast synaptic transmission in the central nervous system. Its name is derived from its ability to be activated by the artificial glutamate analog AMPA. The receptor was discovered by Tage Honore and colleagues at the School of Pharmacy in Copenhagen, and published in 1982 in the Journal of Neurochemistry. AMPARs are found in many parts of the brain and are the most commonly found receptor in the nervous system.

AMPARs are trafficked from the dendrite into the synapse and incorporated through some series of signaling cascades increasing efficacy of synaptic transmission.

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Molecular Organization of AMPARs : Homomeric & Heteromeric Receptors

GluR2 subunit determines functional properties of GluR-channel

MRC Centre for Synaptic Plasticity Nature 454(7200):118-121, 2008

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Ca2+-permeable and -impermeable AMPARs

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exocytosis and endocytosis at the postsynaptic membranelateral diffusion of glutamate receptors in the plasma membrane

Petrini et al., Neuron. 2009

Important future of AMPA receptors: they are not static.

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Part B:

Spinal AMPARs-mediated synaptic transmission during

inflammation.

Molecular mechanism of GluR2 internalization from

synapses.

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AMPA receptors at dorsal horn synapse

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Determination of Sensory AbnormalitiesDetermination of Sensory Abnormalities after after

CComplete Freund’s omplete Freund’s AAdjuvantdjuvant (CFA)-induced (CFA)-induced

Peripheral InflammationPeripheral Inflammation

Increased mechanical hypersensitivity on the ipsilateral side after CFA, but not saline injection into a hindpaw (n = 10/time point).

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0 2 h 4 h 6 h 1 d 2 d 3 d 4 d 5 d 6 d 7 d

PW

L (

s)

Control

CFA

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Lumbar Spinal Cord with Dorsal Root Region

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Identification of CaIdentification of Ca2+2+-permeable AMPARs in DH Neurons -permeable AMPARs in DH Neurons by Kainate-induced Cobalt Uptake Loadingby Kainate-induced Cobalt Uptake Loading

A, CNQX and GYKI 53655 blocked kainate-induced cobalt loading of DH neurons, whereas AP5 had no effect. B, Immunostaining with neuronal marker NeuN. C, CFA (but not saline) increased cobalt uptake loading in DH on ipsilateral, but not contralateral, side. Right - statistics of the number of cobalt-positive neurons in laminae I-II and laminae III-VII 1 d post-saline and 1 d post-CFA.

Voitenko group, unpublished data

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Inflammation Does Not Change the Expression of Total GluR1 and GluR2 in Dorsal Horn

Park et al., Mol. Pain, 2008

Top: representative Western blots showing GluR1 protein (A) and GluR2 protein (B) in total soluble fraction from the ipsilateral and contralateral dorsal horns of naïve rats (n = 4/time point) and the rats at 2 and 24 h after saline (S) or CFA injection (C).

Bottom: statistics of the densitometric analysis expressed relative to the control (β-actin).

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Dorsal Horn GluR2 Internalization during Inflammation

Copyright ©2009 Society for Neuroscience Park, J.-S., Voitenko, N. et al. J. Neurosci. 2009;29:3206-19

GluR2 expression in 150 k-g spin fraction (A) and plasma membrane fraction (B) from ipsilateral DH at 2 h, 1 d, and 3 d after saline or CFA. Top, Representative Western blots; bottom, statistics of the densitometric analysis (relative to the naive animals - 0 h). C, Surface expression of GluR2 and NR1 in DH neurons at 1 d after CFA or saline. The amount of sample loaded for the total (T) was 10% of that for the biotinylated surface (S). Actin was used as a control.

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Postsynaptic Immunogold Labeling for GluR2 in Superficial DH Synapses

Immunogold labeling for GluR2 (arrowheads) both the synapse and adjacent cytoplasmic structures 1 d after saline and only in cytoplasm 1 d after CFA. Pre, Presynaptic terminal; Post, postsynaptic structure. Scale bars, 100 nm.

Copyright ©2009 Society for Neuroscience Park, J.-S., Voitenko, N. et al. J. Neurosci. 2009;29:3206-19

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A, The evoked EPSCs were blocked by GYKI 52466, but not by SYM 2081.B, Increased sensitivity of EPSC amplitude to PhTx-433 after 1 d CFA. C, I–V curves at 1 d post-saline or CFA. Examples of the EPSCs at 70 mV and 40 mV holding potentials.

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PhTX 433N

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M em brane potential (m V) **

GYKI 52466 SYM 2081

10 ms

50 pA

Saline CFA

10 ms

50 pA

A B

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AMPAR-mediated Evoked EPSCs at Synapses

between Primary Afferents and SG Neurons

Copyright ©2009 Society for Neuroscience Park, J.-S., Voitenko, N. et al. J. Neurosci. 2009;29:3206-19

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NMDA Receptors and PKC couple to AMPAR complex

PICK1 coimmunoprecipitates with GluR2 and PKC (A), while GluR2 - with PICK1 and PKC. PSD-95 coimmunoprecipitates with NR2B and stargazin (C), while stargazin - with PSD-95 and GluR2 (D).E, NR1 (5 nm gold, arrowheads) and GluR2 (15 nm gold) colocalize at DH synapses. Scale bar, 100 nm.

Copyright ©2009 Society for Neuroscience Park, J.-S., Voitenko, N. et al. J. Neurosci. 2009;29:3206-19

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Proposed Mechanism for

GluR2 Internalization from Synapses

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Conclusion

Persistent peripheral inflammation induces GluR2 internalization from synapses via NMDA Receptor-triggered PKC activation in DH neurons

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Part C:

Involvement of spinal extrasynaptic AMPARs in the

maintaining of persistent pain.

Trafficking of Ca2+-permeable AMPARs during inflammatory pain.

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Combined Electrophysiology and Calcium Imaging

in Substantia Gelatinosa Neuron

Transmitted light image of Substantia Gelatinosa in transverse DH slice (left); SG neuron loaded with 200 mM of fura-2 (right).

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AMPARs-mediated Current and [Ca2+]i Transients

in Soma & Dendrites of SG Neurons SG neurons were identified according to their pattern of action potential firing

A, Simultaneous recording of AMPA-induced current (lower row) and [Ca2+]i transients (upper row) in soma and dendrites of SG neurons. B-C, AMPARs antagonist, NBQX and GYKI, abolished current and [Ca2+]i transients.D, Statistics of current amplitudes (left) and [Ca2+]i transients (right) in different groups of SG neurons.

Voitenko group, unpublished dataKopach et. al. Pain, 2011.

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Inflammation Potentiates AMPARs-mediated Current and [Ca2+]i Transients in “Tonic”

but not in “Transient” SG Neurons

A, AMPA-induced current (lower row) and [Ca2+]i transients (upper row) in “tonic” neurons 24h after saline or CFA. B-C, A scatter dot plot of currents in SG neurons.D, Statistics of current amplitudes (left) and [Ca2+]i transients (right) in SG neurons. E, A relationship of the [Ca2+]i transient amplitudes and a normalized value of integrated current in the timeframe of AMPA application.

D E

Kopach et. al. Pain, 2011.

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Inflammation-induced Increase of the Number of Extrasynaptic Ca2+-permeable AMPARs

A. Left, AMPA-induced currents after 5 min pre-application of IEM at 24 h post-CFA or saline. Right, IEM was applied during steady-state current level. Dotted lines are exponential fitting of current. B, Statistics of IEM inhibition of current amplitude.

A, I-V relationship of AMPARs-mediated currents in “tonic” neurons at 1 d post-saline and CFA. Note, CFA-induced inward rectification was completely reversed by IEM. B-C, Scatter dot plot illustrated a spread of rectification index (I+30mV/I-50mV) and statistics in “tonic” neurons from 1 d saline- and CFA-treated rats.

AA

Kopach et. al. Pain, 2011.

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The Altered Level of GluR1 and GluR2 in the Plasma Membrane Fraction & Cytosol after CFA

Park et al., Mol. Pain, 2008 Kopach et. al. Pain, 2011.

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Peripheral Inflammation Induces GluA1 Membrane Insertion at Extrasynaptic Sites

of SG Neurons

A. Surface expression of GluA1 in DH neuron 1 d post-CFA or saline. Top, Representative Western blot; bottom, densitometric analysis. The level of sample loaded for the total (T) was 10% of that for the biotinylated surface (S). -actin was used as a control.

B. Expression of GluA1 in synaptosomal fraction from DH.

GluA1

-actin

AT S T S

Saline CFA

Per

cent

age

surf

ace

expr

esse

d (S

/T)

Saline CFA Saline CFA0

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60

80

GluA1

N-cadherin

Saline CFAB

020406080

100

Rel

ativ

e le

vel

*

GluA1

-actin

AT S T S

Saline CFA

Per

cent

age

surf

ace

expr

esse

d (S

/T)

Saline CFA Saline CFA0

20

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60

80

GluA1

N-cadherin

Saline CFAB

020406080

100

Rel

ativ

e le

vel

*

A. The number of GluA1- and GluA2-labeled immunogold particles at synapses, extrasynaptic membranes, and cytoplasm of SG neurons 1 d post- CFA or saline.

B. Ratios of the number of GluA1- and GluA2-labeled particles at extrasynaptic membranes, synapses and cytoplasm of SG neurons in inflamed rats to saline-treated.

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Syn Extra Cyto Syn

GluR2

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GluR1

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io(C

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Syn Extra Cyto Syn Extra Cyto

GluR1

Syn Extra Cyto

GluR1

Num

bero

fim

mun

ogol

dla

belin

gs

GluR2

B

Kopach et. al. Pain, 2011.

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Proposed Model for AMPA Receptors Recycling

At synapses (green), there are mobile and immobile pools of AMPARs. Mobile receptorsleaving synapses can be trapped at EZs (red) either for transient stabilization or for

endocytosis (red arrow) and recycling (blue arrow). Newly exocytosed receptors exhibit high mobility and accumulate at synapses.

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Conclusion Increased functional expression of extrasynaptic Ca2+-permeable AMPARs contributes to the maintaining of

persistent inflammation

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Part D:

Role of Protein Kinase C subtype in

AMPARs-mediated Pain Plasticity

Antisense oligonucleotides – a new strategy of pain treatment

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AS ODN – Antisense oligonucleotides

Antisense oligonucleotides are single strands of DNA or RNA that are complementary to a chosen sequence. In the case of antisense RNA they prevent protein translation of

certain messenger RNA strands by binding to them. Antisense DNA can be used to target a specific, complementary (coding or non-coding) RNA. If binding takes places

this DNA/RNA hybrid can be degraded by the enzyme RNase H.

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Antisense oligonucleotides: scheme of action

cmbi.bjmu.edu.cn

32Bourinet et al., THE EMBO JOURNAL (2005) 24, 315 - 324

Localization of AS ODN in lumbar spinal cord

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In Vivo Gene Silencing of PKCα Attenuates Inflammation-induced Hyperalgesia

Voitenko group, unpublished data

Effect of anti-sense (AS-) and miss-sense (MS) oligonucleotydes for PKCα on CFA-induced hyperalgesia. Time course of hyperalgesia development following CFA injection, in MS-ODN-treated and AS-ODN-treated rats.Statistics of paw withdrawal latency value in different groups of rats.

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PWL

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Sham-operated AS-ODNMS-ODN CFA+saline

Before surgery

After surgery CFA

inf lammation

ODN administration

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*,#

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Paw withdrawal latency and RI values in 4 days of As ODN treatment.

S - saline-injected; C – CFA-injected

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Voitenko group, under preparation

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In Vivo Gene Silencing of PKCα Reverses:

Voitenko group, unpublished data

I-V curve of evoked AMPA-mediated EPSCs in AS-ODN-treated and MS-ODN-treated inflammatory animals

AMPA 5 Mm

0.2, DF /F340 380

60 s

50 pA

MS-ODN

Dendrite

Soma

AS-ODN

CFA-induced Rectification of Synaptic Currents

CFA-induced Potentiation of Extrasynaptic AMPARs current and [Ca2+]i transients in “tonic” SG neurons

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Inflammation

INCREASED EXCITABILITY IMPAIRED SYNAPTIC EFFICACY

CENTRAL SENSITIZATION

PAIN

Ca2+ influx through Synaptic AMPARs

Increased synaptic GluR2 internalization

Ca2+ influx through

extrasynapticAMPARs

Increased GluR1 insertion

into extrasynaptic sites

PKC

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•Further investigations of molecular mechanisms of pain•Improvement of AS ODN design •Increasing of bioactivity of AS ODN •Improvement of target delivery of genetic materials

Future aims

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Olga KopachAndrij SotnikViacheslav Viatchenko-KarpinskiPavel Belan

Bogomoletz Institute of Physiology,National Academy of Sciences of Ukraine

Johns Hopkins University School of Medicine, Baltimore, Maryland, USA

Yuan-Xiang Tao Ronald S. Petralia Jang-Su ParkXiaowei GuanJi-Tian XuJordan P. SteinbergKogo Takamiya Richard L. Huganir

Acknowledgements:

Grants: NIH (NS058886, NS057343) and the JHU Blaustein Pain Research Fund (Y.X.T); JDRF 1-2004-30 and INTAS 8061 (N.V.); the Intramural Research Program of NIDCD (R.S.P.); NIH NS036715 and Howard Hughes Medical Institute (R.L.H).

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THANKS!

Artist: Vadim Holovanov