Bogomoletz Institute of Physiology, Kiev

,
Ukraine
nana@biph.kiev.ua

Molecular Mechanisms
of Pain
part II

Dr. NANA VOITENKO
AACIMP
KIEV - 2011
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Part A:
Classification of receptors to glutamate.
Structure and molecular organization of AMPA receptors.
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.


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Part D:
- Protein kinase Ca as a molecular target for perspective treatment of persistent
pain.
- Antisense oligonucleotides – a new strategy of pain treatment

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GLUTAMATE RECEPTORS
Ionotropic GluRs

NMDA
NR1

AMPA

NR2A-D

Metabotropic GluRs

Kainate
GluR 5,6,7 KA 1,2

Group I Group II
mGlu1 mGlu5

Group III
mGlu4

mGlu6

mGlu7

mGlu8

mGlu2 mGlu3

NR3A
GluR 1-4

Mediating effects include:

Modulatory effects on:

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

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

Drug News Perspect 2003, 16(8): 513

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Its name is derived from its ability to be activated by the artificial glutamate analog AMPA. 4 . The receptor was discovered by Tage Honore and colleagues at the School of Pharmacy in Copenhagen.  AMPARs are trafficked from the dendrite into the synapse and incorporated through some series of signaling cascades increasing efficacy of synaptic transmission. AMPARs are found in many parts of the brain and are the most commonly found receptor in the nervous system.AMPA receptors  is a subtype of ionotropic transmembrane receptor for glutamate that mediates fast synaptic transmission in the central nervous system. and published in 1982 in the Journal of Neurochemistry.

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 5 .

Ca2+-permeable and -impermeable AMPARs 6 .

. Neuron.Important future of AMPA receptors: they are not static. 2009 7 . exocytosis and endocytosis at the postsynaptic membrane lateral diffusion of glutamate receptors in the plasma membrane Petrini et al.

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

AMPA receptors at dorsal horn synapse 9 .

but not saline injection into a hindpaw (n = 10/time point). 10 .Determination of Sensory Abnormalities after Complete Freund’s Adjuvant (CFA)-induced Peripheral Inflammation Increased mechanical hypersensitivity on the ipsilateral side after CFA.

Lumbar Spinal Cord with Dorsal Root Region 11 .

CNQX and GYKI 53655 blocked kainate-induced cobalt loading of DH neurons. B. CFA (but not saline) increased cobalt uptake loading in DH on ipsilateral. Immunostaining with neuronal marker NeuN. Right . but not contralateral. side. C.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. whereas AP5 had no effect. 12 Voitenko group.Identification of Ca2+-permeable AMPARs in DH Neurons by Kainate-induced Cobalt Uptake Loading A. unpublished data .

Park et al.13 Pain. Mol. 2008 .Inflammation Does Not Change the Expression of Total GluR1 and GluR2 in Dorsal Horn 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)..

Copyright ©2009 Society for Neuroscience 14 Park. Voitenko.-S.29:3206-19 . statistics of the densitometric analysis (relative to the naive animals . et al..0 h). Neurosci. J. and 3 d after saline or CFA. bottom. Actin was used as a control. 1 d. 2009. The amount of sample loaded for the total (T) was 10% of that for the biotinylated surface (S). Surface expression of GluR2 and NR1 in DH neurons at 1 d after CFA or saline. C.Dorsal Horn GluR2 Internalization during Inflammation GluR2 expression in 150 k-g spin fraction (A) and plasma membrane fraction (B) from ipsilateral DH at 2 h. N. J. Top. Representative Western blots.

N. Post. Scale bars. Copyright ©2009 Society for Neuroscience 15 2009. Pre. J. Voitenko.-S.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. Neurosci.. 100 nm. postsynaptic structure. et al. Presynaptic terminal. J.29:3206-19 Park. .

2 CFA -0.8 -1. J.29:3206-19 . B.. The evoked EPSCs were blocked by GYKI 52466.2 M em brane potential (m V ) -80 -60 -40 ** 0. N.0 Copyright ©2009 Society for Neuroscience Normalized current Saline CFA Normalized EPSC amplitudes 1. Voitenko.4 0. Examples of the EPSCs at 70 mV and 40 mV holding potentials.8 0. et al. but not by SYM 2081.4 -0.2 CFA 1 0.0 -20 0 20 Saline 1. J.4 50 pA 10 ms 16 Park. Neurosci. I–V curves at 1 d postsaline or CFA.AMPAR-mediated Evoked EPSCs at Synapses between Primary Afferents and SG Neurons A GYKI 52466 B SYM 2081 50 pA 10 ms C 0. 40 Saline -0.2 0 0 2 4 6 8 10 12 14 16 Time (min) A. C. 2009.6 -0. Increased sensitivity of EPSC amplitude to PhTx433 after 1 d CFA.4 PhTX 433 0.-S.6 0.

NR1 (5 nm gold. J.NMDA Receptors and PKC couple to AMPAR complex PICK1 coimmunoprecipitates with GluR2 and PKC (A).-S. 100 nm. while stargazin . E. PSD-95 coimmunoprecipitates with NR2B and stargazin (C).with PSD-95 and GluR2 (D). while GluR2 with PICK1 and PKC.29:3206-19 . J. Copyright ©2009 Society for Neuroscience 17 Park.. 2009. arrowheads) and GluR2 (15 nm gold) colocalize at DH synapses. Neurosci. Scale bar. et al. Voitenko. N.

Proposed Mechanism for GluR2 Internalization from Synapses 18 .

Conclusion Persistent peripheral inflammation induces GluR2 internalization from synapses via NMDA Receptor-triggered PKC activation in DH neurons 19 .

Trafficking of Ca2+-permeable AMPARs during inflammatory pain.Part C: Involvement of spinal extrasynaptic AMPARs in the maintaining of persistent pain. 20 .

Combined Electrophysiology and Calcium Imaging in Substantia Gelatinosa Neuron Transmitted light image of Substantia Gelatinosa in transverse DH slice (left). 21 . SG neuron loaded with 200 mM of fura-2 (right).

abolished current and [Ca 2+]i transients. NBQX and GYKI. D. 22 Voitenko group. Simultaneous recording of AMPA-induced current (lower row) and [Ca2+]i transients (upper row) in soma and dendrites of SG neurons. unpublished data .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 Kopach et. Statistics of current amplitudes (left) and [Ca2+]i transients (right) in different groups of SG neurons. AMPARs antagonist. A. Pain. 2011. al. B-C.

E. D. al. 2011. . Pain.Inflammation Potentiates AMPARs-mediated Current and [Ca2+]i Transients in “Tonic” but not in “Transient” SG Neurons A. B-C. AMPA-induced current (lower row) and [Ca2+]i transients (upper row) in “tonic” neurons 24h after saline or CFA. A relationship of the [Ca2+]i transient amplitudes and a normalized value of integrated current in the timeframe of AMPA application. D E 23 Kopach et. Statistics of current amplitudes (left) and [Ca 2+]i transients (right) in SG neurons. A scatter dot plot of currents in SG neurons.

CFA-induced inward rectification was completely reversed by IEM. IEM was applied during steady-state current level. Right. A. B-C. 24 . 2011.Inflammation-induced Increase of the Number of Extrasynaptic Ca2+-permeable AMPARs A. Dotted lines are exponential fitting of current. Note. Kopach et. Statistics of IEM inhibition of current amplitude. AMPA-induced currents after 5 min preapplication of IEM at 24 h post-CFA or saline. Scatter dot plot illustrated a spread of rectification index (I+30mV/I-50mV) and statistics in “tonic” neurons from 1 d salineand CFA-treated rats. Pain. I-V relationship of AMPARsmediated currents in “tonic” neurons at 1 d post-saline and CFA. Left. al. B.

The Altered Level of GluR1 and GluR2 in the Plasma Membrane Fraction & Cytosol after CFA Kopach et. 2008 . al. Pain. 2011. Pain.25 Mol.. Park et al.

Top.5 1 0. . Pain. A. bottom. Expression of GluA1 in synaptosomal fraction from DH.5 0 Syn Extra Cyto Syn Extra Cyto GluR1 GluR2 Ratio (CFA/saline) Number of immunogold labelings Peripheral Inflammation Induces GluA1 Membrane Insertion at Extrasynaptic Sites of SG Neurons 3 2 1 0 Syn Extra Cyto GluR1 Syn Extra Cyto GluR2 A.5 Saline CFA B 2 1. Ratios of the number of GluA1. densitometric analysis. actin was used as a control. B. The number of GluA1. Surface expression of GluA1 in DH neuron 1 d post-CFA or saline. and cytoplasm of SG neurons 1 d post. extrasynaptic membranes. al. B.and GluA2-labeled particles at extrasynaptic membranes. The level of sample loaded for the total (T) was 10% of that for the biotinylated surface (S).and GluA2-labeled immunogold particles at synapses. synapses and cytoplasm of SG neurons in inflamed rats to saline-treated.2. 26 Kopach et.CFA or saline. Representative Western blot. 2011.

there are mobile and immobile pools of AMPARs. Mobile receptors leaving synapses can be trapped at EZs (red) either for transient stabilization or for endocytosis (red arrow) and recycling (blue arrow).Proposed Model for AMPA Receptors Recycling At synapses (green). Newly exocytosed receptors exhibit high mobility and accumulate at synapses. 27 .

Conclusion Increased functional expression of extrasynaptic Ca2+-permeable AMPARs contributes to the maintaining of persistent inflammation 28 .

Part D: Role of Protein Kinase C  subtype in AMPARs-mediated Pain Plasticity Antisense oligonucleotides – a new strategy of pain treatment 29 .

30 .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. If binding takes places this DNA/RNA hybrid can be degraded by the enzyme RNase H. Antisense DNA can be used to target a specific. complementary (coding or non-coding) RNA.

Antisense oligonucleotides: scheme of action 31 cmbi.bjmu.edu.cn .

324 . THE EMBO JOURNAL (2005) 24.. 315 .Localization of AS ODN in lumbar spinal cord 32 Bourinet et al.

Statistics of paw withdrawal latency value in different groups of rats. 33 Voitenko group. unpublished data .In Vivo Gene Silencing of PKCα Attenuates Inflammation-induced Hyperalgesia 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.

12 PWL (s) 10 # 8 6 4 * * Rectification index 14 * # 2 0 S C ________________ S C S C ________________ _____________ ___i.t. 34 under preparation . MS-ODN i. Saline Naive Saline AS-ODN MS-ODN _________________ CFA S .Paw withdrawal latency and RI values in 4 days of As ODN treatment.saline-injected. AS-ODN i.t. C – CFA-injected Voitenko group.t.

2.In Vivo Gene Silencing of PKCα Reverses: CFA-induced Rectification of Synaptic Currents CFA-induced Potentiation of Extrasynaptic AMPARs current and [Ca2+]i transients in “tonic” SG neurons Dendrite Soma 0. unpublished data 60 s . F340/F 380 AMPA 5 M AS-ODN I-V curve of evoked AMPA-mediated EPSCs in AS-ODN-treated and MS-ODN-treated inflammatory animals 50 pA MS-ODN 35 Voitenko group.

Inflammation Increased GluR1 insertion into extrasynaptic sites Increased synaptic GluR2 internalization PKC Ca2+ influx through extrasynaptic AMPARs INCREASED EXCITABILITY Ca2+ influx through Synaptic AMPARs IMPAIRED SYNAPTIC EFFICACY CENTRAL SENSITIZATION PAIN 36 .

Future aims •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 37 .

Steinberg Kogo Takamiya Richard L.).H). Huganir Grants: NIH (NS058886. Maryland. NIH NS036715 and Howard Hughes Medical Institute (R.P.S.Acknowledgements: Bogomoletz Institute of Physiology.V. 38 . NS057343) and the JHU Blaustein Pain Research Fund (Y. JDRF 1-2004-30 and INTAS 8061 (N.). USA Yuan-Xiang Tao Ronald S. Petralia Jang-Su Park Xiaowei Guan Ji-Tian Xu Jordan P. National Academy of Sciences of Ukraine Olga Kopach Andrij Sotnik Viacheslav Viatchenko-Karpinski Pavel Belan Johns Hopkins University School of Medicine.X. Baltimore. the Intramural Research Program of NIDCD (R.T).L.

THANKS! 39 Vadim Holovanov Artist: .