( A ) Subunit membrane topology. The three transmembrane domains and the hydrophobic pore-lining region are indicated by grey and white rounded rectangles respectively. The S1 and S2 domains form the ligand-binding domain. ( B ) Tetrameric assembly of a functional iGluR showing the hydrophobic regions that co-operate to form the ion channel pore.
Molecular Mechanisms of Pain. Part 2
Bogomoletz Institute of Physiology, Kiev, Ukraine [email_address] Dr. NANA VOITENKO AACIMP KIEV - 2011 Molecular Mechanisms of Pain part II
<ul><li>Part B: </li></ul><ul><li>Spinal AMPARs-mediated synaptic transmission during inflammation. </li></ul><ul><li>Molecular mechanism of GluR2 internalization from synapses. </li></ul><ul><li>Part C: </li></ul><ul><li>Involvement of spinal extrasynaptic AMPARs in the maintaining of persistent pain. </li></ul><ul><li>Trafficking of Ca 2+ -permeable AMPARs during inflammatory pain. </li></ul><ul><li>Part D : </li></ul><ul><li>- Protein kinase Ca as a molecular target for perspective treatment of persistent pain . </li></ul><ul><li>- Antisense oligonucleotides – a new strategy of pain treatment </li></ul><ul><li>Part A: </li></ul><ul><li>Classification of receptors to glutamate. </li></ul><ul><li>Structure and molecular organization of AMPA receptors. </li></ul>
GLUTAMATE RECEPTORS <ul><li>Mediating effects include : </li></ul><ul><li>Membrane depolarization </li></ul><ul><li>Ca 2+ and Na + influx </li></ul><ul><li>K + efflux </li></ul><ul><li>Free radical formation </li></ul><ul><li>Modulatory effects on : </li></ul><ul><li>iGluR </li></ul><ul><li>K + and Ca 2+ channels </li></ul><ul><li>Neurotransmitter release </li></ul>Ionotropic GluRs Metabotropic GluRs NMDA NR1 NR3A NR2A-D AMPA GluR 1-4 Kainate GluR 5 ,6, 7 KA 1,2 Group I mGlu 1 mGlu 5 Group II mGlu 2 mGlu 3 Group III mGlu 4 mGlu 6 mGlu 8 mGlu 7 Drug News Perspect 2003, 16(8): 513
AMPA receptors <ul><li>is a sub type 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. </li></ul><ul><li>AMPARs are trafficked from the dendrite into the synapse and incorporated through some series of signaling cascades increasing efficacy of synaptic transmission . </li></ul>
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
<ul><li>exocytosis and endocytosis at the postsynaptic membrane </li></ul><ul><li>lateral diffusion of glutamate receptors in the plasma membrane </li></ul>Petrini et al., Neuron. 2009 Important future of AMPA receptors: they are not static.
Part B: Spinal AMPARs-mediated synaptic transmission during inflammation. Molecular mechanism of GluR2 internalization from synapses.
Determination of Sensory Abnormalities after C omplete Freund’s A djuvant (CFA)-induced Peripheral Inflammation Increased mechanical hypersensitivity on the ipsilateral side after CFA, but not saline injection into a hindpaw (n = 10/time point) .
Identification of Ca 2+ -permeable AMPARs in DH Neurons by 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
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 Glu R 1 protein (A) and Glu R 2 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).
Proposed Mechanism for GluR2 Internalization from Synapses
Conclusion Persistent peripheral inflammation induces GluR2 internalization from synap ses via NMDA Receptor- t riggered PKC a ctivation in D H n eurons
Part C: Involvement of spinal extrasynaptic AMPARs in the maintaining of persistent pain. Trafficking of Ca 2+ -permeable AMPARs during inflammatory pain.
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).
AMPARs-mediated Current and [Ca 2+ ] i Transients in Soma & Dendrites of SG Neurons Kopach et. al. Pain, 2011 . SG neurons were identified according to their pattern of action potential firing A, Simultaneous recording of AMPA-induced current (lower row) and [Ca 2+ ] i transients (upper row) in soma and dendrites of SG neurons. B-C, AMPARs antagonist, NBQX and GYKI, abolished current and [Ca 2+ ] i transients. D, Statistics of current amplitudes (left) and [Ca 2+ ] i transients (right) in different groups of SG neurons. Voitenko group, unpublished data
Inflammation Potentiates AMPARs-mediated Current and [Ca 2+ ] i Transients in “Tonic” but not in “Transient” SG Neurons A, AMPA-induced current (lower row) and [Ca 2+ ] 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 [Ca 2+ ] i transients (right) in SG neurons. E, A relationship of the [Ca 2+ ] i transient amplitudes and a normalized value of i ntegrated current in the timeframe of AMPA application. Kopach et. al. Pain, 2011 . D E
Inflammation-induced Increase of the Number of Extrasynaptic Ca 2+ -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. Kopach et. al. Pain, 2011 .
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 .
Peripheral Inflammation Induces GluA1 Membrane Insertion at Extrasynaptic Sites of SG Neurons <ul><li>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. </li></ul><ul><li>B. Expression of GluA1 in synaptosomal fraction from DH. </li></ul><ul><li>The number of GluA1- and GluA2-labeled immunogold particles at synapses, extrasynaptic membranes, and cytoplasm of SG neurons 1 d post- CFA or saline. </li></ul><ul><li>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. </li></ul>Kopach et. al. Pain, 2011 . B
Proposed Model for AMPA Receptors Recycling At synapses (green), 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). Newly exocytosed receptors exhibit high mobility and accumulate at synapses.
Conclusion Increased functional expression of extrasynaptic Ca 2+ -permeable AMPARs contributes to the maintaining of persistent inflammation
Part D: Role of Protein Kinase C subtype in AMPARs-mediated Pain Plasticity Antisense oligonucleotides – a new strategy of pain treatment
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.
Antisense oligonucleotides: scheme of action cmbi.bjmu.edu.cn
Bourinet et al., THE EMBO JOURNAL (2005) 24 , 315 - 324 Localization of AS ODN in lumbar spinal cord
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. S tatistics of paw withdrawal latency value in different groups of rats.
Paw withdrawal latency and RI values in 4 days of As ODN treatment. S - saline-injected; C – CFA-injected Voitenko group, under preparation CFA _________________ 0 2 4 6 8 10 12 14 S C S C S C PWL (s) ________________ i.t. Saline ________________ ________________ i.t. AS-ODN i.t. MS-ODN # * * Rectification index Saline Naive AS-ODN MS-ODN * #
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 CFA-induced Rectification of Synaptic Currents CFA-induced Potentiation of Extrasynaptic AMPARs current and [Ca 2+ ] i transients in “tonic” SG neurons
Inflammation INCREASED EXCITABILITY IMPAIRED SYNAPTIC EFFICACY CENTRAL SENSITIZATION PAIN Ca 2+ influx through Synaptic AMPARs Increased synaptic GluR2 internalization Ca 2+ influx through extrasynaptic AMPARs Increased GluR1 insertion into extrasynaptic sites PKC
<ul><li>Further investigations of molecular mechanisms of pain </li></ul><ul><li>Improvement of AS ODN design </li></ul><ul><li>Increasing of bioactivity of AS ODN </li></ul><ul><li>Improvement of target delivery of genetic materials </li></ul>Future aims
<ul><li>Grants: </li></ul><ul><li>NIH (NS058886, NS057343) and the JHU Blaustein Pain Research Fund (Y.X.T); </li></ul><ul><li>JDRF 1-2004-30 and INTAS 8061 (N.V.); the Intramural Research Program of NIDCD (R.S.P.); </li></ul><ul><li>NIH NS036715 and Howard Hughes Medical Institute (R.L.H). </li></ul>Olga Kopach Andrij Sotnik Viacheslav Viatchenko-Karpinski Pavel 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 Park Xiaowei Guan Ji-Tian Xu Jordan P. Steinberg Kogo Takamiya Richard L. Huganir Acknowledgements: