1 2013 Vol: 19(11):1524-1528. DOI: 10.1038/nm.3356

Chloride extrusion enhancers as novel therapeutics for neurological diseases

Dysfunction of the potassium-chloride cotransporter KCC2 has been linked to many neurological diseases, including pain, anxiety and epilepsy. Now, Yves De Koninck and his colleagues report that they have developed a novel small-molecule compound that is orally bioavailable and can activate KCC2 and reduce chronic pain in rats.

Mentions
Figures
Figure 1: Screening for, and improvement of KCC2-dependent intracellular Cl− lowering compounds. (a) Expression of KCC2 in NG108 cells stably transfected with Clomeleon (NG108-cl). Top, immunoblots of KCC2 in HEK293-cl cells (negative control), hippocampal neurons (positive control) and NG108-cl cells (total lysates versus after KCC2 immunoprecipitation; IP). Asterisk indicates a nonspecific band not observed after IP. Bottom, immunoblot of NKCC1 in total lysates of all cell types. Input 1 and 2: β-actin used as loading control. The top band in Input 1 corresponds to IgGs serving as control for the amount of antibodies used for IP. MW, molecular weight. (b) Relationship between [Cl−] and the ratio of Clomeleon YFP to CFP fluorescence. [Cl−]i was clamped to [Cl−]e by membrane permeabilization using 0.15% Triton X-100 (means ± s.d.; n = 4 assays). Inset, effect of pH on fluorescence ratio (open circles: with 0.15% Triton X-100, closed squares: without Triton X-100; means ± s.d.; n = 4 assays). (c) Chemical structures of the CL-058 analogs tested in d. (d) Concentration-response curves of the CL-058 hit compound and selected analogs. In c and d, [Cl−]i was measured after a 5-h exposure to compounds. (e) Concentration-response curves of CLP257 in NG108-cl and HEK293-cl cells (means ± s.e.m.; n = 4 assays). Inset shows [Cl−]i response of NG108-cl cells to 1.25 μM CLP257 over time (means ± s.e.m.; n = 4 assays). (f) Effect of CLP257 pretreatment on 45-min Rb+ influx assays in CCC-expressing X. laevis oocytes (means ± s.e.m.; n = 12–20 oocytes. Kruskall-Wallis H = 41; ***P < 0.001). (g) Effect of the KCC2 antagonist VU0240551 on response to CLP257. Shown are [Cl−]i in NG108-cl cells after 5-h exposure to CLP257 500 nM + DMSO vehicle or VU0240551 (means ± s.e.m.; n = 4 assays). Figure 2: CLP257 restores Cl− transport in adult spinal cord slices with impaired KCC2 function. (a) Diagram illustrating inversion of KCC2 transport upon raising [K+]e. (b) Color-coded lifetime image of lamina II cells loaded with the Cl− indicator MQAE in slices treated with BNDF or BDNF with 15 mM [K+]e; lower lifetime values correspond to high [Cl−]. Scale bars, 50 μm. (c) Time-lapse recording of Cl− accumulation in the cell bodies of neurons upon extracellular application of 15 mM KCl. Measurements taken every 10 s. Scale bars: vertical, 50 ps; horizontal, 20 s. Insets show examples of photon-distribution histograms fitted to extract the lifetimes shown in b (arrows), during the control period (top) and after Cl− equilibration in 15 mM KCl (bottom). Scale bars: vertical, 40 photons; horizontal, 2 ns. (d) Effects of CLP257 on the efficacy of Cl− transport in lamina II cells. Slope of the change in fluorescence 2 h after addition of CLP257 (100 μM) to a slice pretreated with BDNF for 2 h. (e) Comparison of the rate of Cl− changes measured in SDH neurons in spinal cord slices treated with BDNF or taken from PNI rats, before and 2 h after addition of CLP257 (means ± s.e.m.; n = 4–12, H: 32; *P < 0.05; ***P < 0.001). Ctrl represents the rate of Cl− transport measured in naive animals. (f,g) Effect of CLP257 (25 μM, >1 h) on EGABA in SDH neurons in slices from rats after PNI (f) or BDNF (g) treatment. Left, responses to 30-ms GABA puffs (black arrowheads) in lamina II neurons in the presence of a Cl− load (30 mM in the recording pipette). Right, I-V relationships from representative cells. (h) Pooled EGABA of neurons from PNI rats under control conditions (n = 11 cells) or treated with CLP257 (n = 8 cells; Mann-Whitney U = 9, **P < 0.01) and from BDNF-treated slices with (n = 8 cells) or without CLP257 (n = 7 cells; U: 7, *P < 0.05). (i) Quantification of GABAA responses (normalized to baseline) in neurons after 5 min of CLP257 incubation (25 μM, n = 7 cells, Wilcoxon W = −4, P > 0.05; 100 μM, n = 5 cells, W: −5, P > 0.05) using 120 mM CsCl–filled pipettes and in the presence of the KCC2 antagonist VU0240551. Inset shows representative responses of SDH neurons to GABA puffs before and after application of CLP257 (100 μM). Figure 3: CLP257 increases plasmalemmal KCC2 protein in BDNF-treated adult rat spinal cord slices. (a) Two cell surface biotinylation and total immunoblots (Exp. 1 and Exp. 2) of KCC2 monomers and dimers after BDNF + 100 μM CLP257 treatment. Input 1: total biotinylated cell surface proteins used as loading control. Input 2: overexposed film of a β-actin immunoblot showing no signal. Input 3: total β-actin protein used as loading control. (b) Quantification of the ratio of surface to total KCC2 monomer and dimer expression in response to treatment of slices with BDNF + 100 μM CLP257 versus control BDNF + DMSO; each spinal cord served as its own control (means ± s.e.m.; n = 16 pairs from 16 rats; Wmonomers: 9; Wdimers: 26; *P < 0.05; ***P < 0.001). Figure 4: In vivo assessment of the efficacy and pharmacokinetics of CLP257 and its prodrug CLP290. (a) Input/output relationship between the field electrophysiological response recorded in the superficial layers of the spinal dorsal horn and the strength of mechanical stimuli applied to the receptive field (footpad) after local spinal administration of vehicle (saline) or CLP257 in normal animals. Inset: representative example of field responses upon local spinal administration of saline, CLP257 or tetrodotoxin (TTX). (means ± s.e.m., n = 7 rats). (b) Same analysis as in a but in PNI rats (means ± s.e.m.; W: 21, *P < 0.05). (c) Effect of CLP257 on the normalized maximal field response in control animals (Ctrl, n = 7 rats) and animals with peripheral nerve injury (PNI, n = 6 rats, W: 28, *P < 0.05). (d) Black bars: mean (± s.e.m.) maximal field responses to mechanical stimulation of the receptive field in control (Ctrl, n = 7 rats) and PNI rats (PNI, n = 6 rats, U: 4, *P < 0.05). Red bars: effect of CLP257 application in control (n = 7 rats) and PNI rats (n = 6 rats, W: 27, *P < 0.05). (e) Effect of CLP257 on paw withdrawal threshold in PNI rats. CLP257 was dissolved in 20% 2-hydroxypropyl-β-cyclodextrin (HPCD) and administered i.p. (means ± s.e.m.; n = 8–10; *P < 0.05, **P < 0.01; ***P < 0.001). BW, body weight. (f) Pharmacokinetic profile of CLP257 and the carbamate prodrug CLP290 in rats after intravenous (i.v.), i.p. or oral (p.o.) administration (means ± s.e.m.; n = 3 animals per time point). Inset: structure of CLP290. (g) Analgesic effect of CLP290 administered p.o. in a PNI rat (means ± s.e.m.; n = 7–32; *P < 0.05). (h) Effect of CLP290 and pregabalin (at equipotent analgesic dose) on motor performance in rats as measured by time spent on an accelerating rotorod (means ± s.e.m.; n = 4–12; U: 6; *P < 0.05).
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References
  1. Arion, D.; Lewis, D.A. Altered expression of regulators of the cortical chloride transporters NKCC1 and KCC2 in schizophrenia Arch. Gen. Psychiatry 68, 21-31 (2011) .
    • . . . Although no CNS disorders have been associated with KCC2 mutations, loss of activity of this transporter has emerged as a key mechanism underlying several neurological and psychiatric disorders, including epilepsy, motor spasticity, stress, anxiety, schizophrenia, morphine-induced hyperalgesia and chronic pain1, 2, 3, 4, 5, 6, 7, 8, 9 . . .
    • . . . As evidence is mounting that KCC2 dysfunction and Cl− homeostasis are central to many CNS disorders1, 2, 3, 4, 5, 6, 7, 8, we believe that Cl− extrusion enhancers such as CLP257 and CLP290 represent an important new method of treatment for a wide range of neurological and psychiatric indications. . . .
  2. Boulenguez, P. Down-regulation of the potassium-chloride cotransporter KCC2 contributes to spasticity after spinal cord injury Nat. Med. 16, 302-307 (2010) .
    • . . . Although no CNS disorders have been associated with KCC2 mutations, loss of activity of this transporter has emerged as a key mechanism underlying several neurological and psychiatric disorders, including epilepsy, motor spasticity, stress, anxiety, schizophrenia, morphine-induced hyperalgesia and chronic pain1, 2, 3, 4, 5, 6, 7, 8, 9 . . .
    • . . . As evidence is mounting that KCC2 dysfunction and Cl− homeostasis are central to many CNS disorders1, 2, 3, 4, 5, 6, 7, 8, we believe that Cl− extrusion enhancers such as CLP257 and CLP290 represent an important new method of treatment for a wide range of neurological and psychiatric indications. . . .
  3. Coull, J.A. Trans-synaptic shift in anion gradient in spinal lamina I neurons as a mechanism of neuropathic pain Nature 424, 938-942 (2003) .
    • . . . Although no CNS disorders have been associated with KCC2 mutations, loss of activity of this transporter has emerged as a key mechanism underlying several neurological and psychiatric disorders, including epilepsy, motor spasticity, stress, anxiety, schizophrenia, morphine-induced hyperalgesia and chronic pain1, 2, 3, 4, 5, 6, 7, 8, 9 . . .
    • . . . We measured the effect of CLP257 on Cl− transport in spinal slices obtained from rats with peripheral nerve injury (PNI; as an experimental model of neuropathic pain)20 and control slices pretreated with brain-derived neurotrophic factor (BDNF), which mediates downregulation of KCC2 activity21, 22 in models of pain hypersensitivity3, 9, 23, 24, 25, 26, 27 . . .
    • . . . We tested whether CLP257 modulates Cl− extrusion capacity using voltage-clamp recordings from superficial dorsal horn (SDH) neurons in spinal cord slices treated with BDNF and isolated from rats with PNI3 . . .
    • . . . We tested the antinociceptive efficacy of CLP257 in PNI rats3, 20, 28, 29 (Fig. 4a–d) . . .
    • . . . Expression of KCC2 is limited to the central nervous system, unlike the case for other CCCs that are expressed in several tissues3, 12, 32, 33, 34, such as the kidneys and blood vessels, where ionic homeostasis is important . . .
    • . . . This constrained expression makes KCC2 a prime target for the control of neuronal [Cl−]i and should limit adverse side effects in other organs (for example, it is not expressed in choroid plexus, inner ear or sensory fibers3, 12, 32, 33) . . .
    • . . . As evidence is mounting that KCC2 dysfunction and Cl− homeostasis are central to many CNS disorders1, 2, 3, 4, 5, 6, 7, 8, we believe that Cl− extrusion enhancers such as CLP257 and CLP290 represent an important new method of treatment for a wide range of neurological and psychiatric indications. . . .
  4. Hewitt, S.A.; Wamsteeker, J.I.; Kurz, E.U.; Bains, J.S. Altered chloride homeostasis removes synaptic inhibitory constraint of the stress axis Nat. Neurosci. 12, 438-443 (2009) .
    • . . . Although no CNS disorders have been associated with KCC2 mutations, loss of activity of this transporter has emerged as a key mechanism underlying several neurological and psychiatric disorders, including epilepsy, motor spasticity, stress, anxiety, schizophrenia, morphine-induced hyperalgesia and chronic pain1, 2, 3, 4, 5, 6, 7, 8, 9 . . .
    • . . . As evidence is mounting that KCC2 dysfunction and Cl− homeostasis are central to many CNS disorders1, 2, 3, 4, 5, 6, 7, 8, we believe that Cl− extrusion enhancers such as CLP257 and CLP290 represent an important new method of treatment for a wide range of neurological and psychiatric indications. . . .
  5. Huberfeld, G. Perturbed chloride homeostasis and GABAergic signaling in human temporal lobe epilepsy J. Neurosci. 27, 9866-9873 (2007) .
    • . . . Although no CNS disorders have been associated with KCC2 mutations, loss of activity of this transporter has emerged as a key mechanism underlying several neurological and psychiatric disorders, including epilepsy, motor spasticity, stress, anxiety, schizophrenia, morphine-induced hyperalgesia and chronic pain1, 2, 3, 4, 5, 6, 7, 8, 9 . . .
    • . . . As evidence is mounting that KCC2 dysfunction and Cl− homeostasis are central to many CNS disorders1, 2, 3, 4, 5, 6, 7, 8, we believe that Cl− extrusion enhancers such as CLP257 and CLP290 represent an important new method of treatment for a wide range of neurological and psychiatric indications. . . .
  6. Hyde, T.M. Expression of GABA signaling molecules KCC2, NKCC1, and GAD1 in cortical development and schizophrenia J. Neurosci. 31, 11088-11095 (2011) .
    • . . . Although no CNS disorders have been associated with KCC2 mutations, loss of activity of this transporter has emerged as a key mechanism underlying several neurological and psychiatric disorders, including epilepsy, motor spasticity, stress, anxiety, schizophrenia, morphine-induced hyperalgesia and chronic pain1, 2, 3, 4, 5, 6, 7, 8, 9 . . .
    • . . . As evidence is mounting that KCC2 dysfunction and Cl− homeostasis are central to many CNS disorders1, 2, 3, 4, 5, 6, 7, 8, we believe that Cl− extrusion enhancers such as CLP257 and CLP290 represent an important new method of treatment for a wide range of neurological and psychiatric indications. . . .
  7. Price, T.J.; Cervero, F.; De Koninck, Y. Role of cation-chloride-cotransporters (CCC) in pain and hyperalgesia Curr. Top. Med. Chem. 5, 547-555 (2005) .
    • . . . Although no CNS disorders have been associated with KCC2 mutations, loss of activity of this transporter has emerged as a key mechanism underlying several neurological and psychiatric disorders, including epilepsy, motor spasticity, stress, anxiety, schizophrenia, morphine-induced hyperalgesia and chronic pain1, 2, 3, 4, 5, 6, 7, 8, 9 . . .
    • . . . As evidence is mounting that KCC2 dysfunction and Cl− homeostasis are central to many CNS disorders1, 2, 3, 4, 5, 6, 7, 8, we believe that Cl− extrusion enhancers such as CLP257 and CLP290 represent an important new method of treatment for a wide range of neurological and psychiatric indications. . . .
  8. Tornberg, J.; Voikar, V.; Savilahti, H.; Rauvala, H.; Airaksinen, M.S. Behavioural phenotypes of hypomorphic KCC2-deficient mice Eur. J. Neurosci. 21, 1327-1337 (2005) .
    • . . . Although no CNS disorders have been associated with KCC2 mutations, loss of activity of this transporter has emerged as a key mechanism underlying several neurological and psychiatric disorders, including epilepsy, motor spasticity, stress, anxiety, schizophrenia, morphine-induced hyperalgesia and chronic pain1, 2, 3, 4, 5, 6, 7, 8, 9 . . .
    • . . . As evidence is mounting that KCC2 dysfunction and Cl− homeostasis are central to many CNS disorders1, 2, 3, 4, 5, 6, 7, 8, we believe that Cl− extrusion enhancers such as CLP257 and CLP290 represent an important new method of treatment for a wide range of neurological and psychiatric indications. . . .
  9. Ferrini, F. Morphine hyperalgesia gated through microglia-mediated disruption of neuronal Cl− homeostasis Nat. Neurosci. 16, 183-192 (2013) .
    • . . . Although no CNS disorders have been associated with KCC2 mutations, loss of activity of this transporter has emerged as a key mechanism underlying several neurological and psychiatric disorders, including epilepsy, motor spasticity, stress, anxiety, schizophrenia, morphine-induced hyperalgesia and chronic pain1, 2, 3, 4, 5, 6, 7, 8, 9 . . .
    • . . . We measured the effect of CLP257 on Cl− transport in spinal slices obtained from rats with peripheral nerve injury (PNI; as an experimental model of neuropathic pain)20 and control slices pretreated with brain-derived neurotrophic factor (BDNF), which mediates downregulation of KCC2 activity21, 22 in models of pain hypersensitivity3, 9, 23, 24, 25, 26, 27 . . .
    • . . . A Cl− load was imposed through the recording pipette to measure Cl− extrusion capacity via the reversal potential for GABAA currents (EGABA)9 . . .
    • . . . Fluorescence lifetime imaging of MQAE was conducted as previously described9, 10 . . .
    • . . . Whole-cell clamp recordings were performed in parasagittal slices of lumbar spinal cord from rats as described previously9, 42, 43 . . .
    • . . . For whole-cell experiments involving measurements of EGABA under Cl− load, borosilicate patch pipettes had a resistance of 4 to 5 MΩ when filled with a potassium methylsulfate-based intrapipette solution9, 43 . . .
    • . . . All recordings were performed at physiological temperature (30-34 °C) in standard ACSF9, 42 supplemented with 10 mM 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX), 40 mM D-(−)-2-amino-5-phosphonovaleric acid (AP5) and 1 mM TTX . . .
  10. Doyon, N. Efficacy of synaptic inhibition depends on multiple, dynamically interacting mechanisms implicated in chloride homeostasis PLoS Comput. Biol. 7, e1002149 (2011) .
    • . . . Recent reports indicate that enhancing KCC2 activity may be the favored therapeutic strategy to restore inhibition and normal function in pathological conditions involving impaired Cl− transport10, 11, 12 . . .
    • . . . We assessed the functioning of KCC2 by quantitatively measuring K+-driven Cl− influx via fluorescence lifetime measurements of the Cl− probe MQAE10 (Fig. 2a–c) . . .
    • . . . Extracellular MQAE was washed out for 10 min in the presence of 1 μM tetrodotoxin, 10 μM CNQX, 40 μM AP5, 1 μM strychnine and 10 μM gabazine to minimize KCC2-independent Cl− transport . . .
  11. De Koninck, Y. Altered chloride homeostasis in neurological disorders: a new target Curr. Opin. Pharmacol. 7, 93-99 (2007) .
    • . . . Recent reports indicate that enhancing KCC2 activity may be the favored therapeutic strategy to restore inhibition and normal function in pathological conditions involving impaired Cl− transport10, 11, 12 . . .
  12. Kahle, K.T. Roles of the cation-chloride cotransporters in neurological disease Nat. Clin. Pract. Neurol. 4, 490-503 (2008) .
    • . . . Recent reports indicate that enhancing KCC2 activity may be the favored therapeutic strategy to restore inhibition and normal function in pathological conditions involving impaired Cl− transport10, 11, 12 . . .
    • . . . Expression of KCC2 is limited to the central nervous system, unlike the case for other CCCs that are expressed in several tissues3, 12, 32, 33, 34, such as the kidneys and blood vessels, where ionic homeostasis is important . . .
    • . . . In fact, unlike other KCCs (KCC1, KCC3 and/or KCC4), KCC2 is not involved in volume regulation and is not typically found in cells where these KCCs are expressed12. . . .
  13. Kuner, T.; Augustine, G.J. A genetically encoded ratiometric indicator for chloride: capturing chloride transients in cultured hippocampal neurons Neuron 27, 447-459 (2000) .
    • . . . As a model, we chose the NG-108 cell line, which expresses low levels of KCC2 protein (Fig. 1a), and into which we stably transfected the Cl−-sensitive indicator Clomeleon13, yielding NG108-cl cells . . .
  14. Garbarini, N.; Delpire, E. The RCC1 domain of protein associated with Myc (PAM) interacts with and regulates KCC2 Cell Physiol. Biochem. 22, 31-44 (2008) .
    • . . . We calibrated and validated the fluorometric assay by placing NG108-cl and KCC2-lacking HEK293-cl cells14, 15, 16 in isosmolar extracellular medium containing increasing Cl− concentration in permeabilized cells to clamp [Cl−]i at different levels . . .
  15. Inoue, K.; Yamada, J.; Ueno, S.; Fukuda, A. Brain-type creatine kinase activates neuron-specific K+-Cl− co-transporter KCC2 J. Neurochem. 96, 598-608 (2006) .
    • . . . We calibrated and validated the fluorometric assay by placing NG108-cl and KCC2-lacking HEK293-cl cells14, 15, 16 in isosmolar extracellular medium containing increasing Cl− concentration in permeabilized cells to clamp [Cl−]i at different levels . . .
  16. Simard, C.F. Homooligomeric and heterooligomeric associations between K+-Cl− cotransporter isoforms and between K+-Cl− and Na+-K+-Cl− cotransporters J. Biol. Chem. 282, 18083-18093 (2007) .
    • . . . We calibrated and validated the fluorometric assay by placing NG108-cl and KCC2-lacking HEK293-cl cells14, 15, 16 in isosmolar extracellular medium containing increasing Cl− concentration in permeabilized cells to clamp [Cl−]i at different levels . . .
    • . . . We further selected hits for lack of effects on [Cl−]i in HEK293-cl cells, which do not express KCC2 but do express NKCC1, KCC1, KCC3 and KCC4 (refs. 16,18) (Fig. 1a) . . .
    • . . . Oocytes were then incubated for another 45 min in a regular flux medium containing either CLP257 or DMSO, bathed several times in a wash solution and lysed in pure nitric acid16 . . .
  17. Lipinski, C.A.; Lombardo, F.; Dominy, B.W.; Feeney, P.J. Experimental and computational approaches to estimate solubility and permeability in drug discovery and development settings Adv. Drug Deliv. Rev. 46, 3-26 (2001) .
    • . . . We then carried out high-throughput screening of a library of 92,500 drug-like17 compounds to identify small molecules able to reduce [Cl−]i and confirmed a total of 78 as positive hits . . .
    • . . . Further selection based on lack of cytotoxic effects, ease of synthesis and drug-like properties17 identified the hit compound CL-058 for further characterization and optimization (Fig. 1c,d). . . .
    • . . . We synthesized more than 300 unique analogs of CL-058 to improve potency and drug-like properties17 . . .
  18. Hiki, K. Cloning, characterization, and chromosomal location of a novel human K+-Cl− cotransporter J. Biol. Chem. 274, 10661-10667 (1999) .
    • . . . We further selected hits for lack of effects on [Cl−]i in HEK293-cl cells, which do not express KCC2 but do express NKCC1, KCC1, KCC3 and KCC4 (refs. 16,18) (Fig. 1a) . . .
  19. Delpire, E. Small-molecule screen identifies inhibitors of the neuronal K-Cl cotransporter KCC2 Proc. Natl. Acad. Sci. USA 106, 5383-5388 (2009) .
    • . . . We also observed functional, dose-dependent antagonism between CLP257 and the recently characterized KCC2 antagonist VU0240551 (ref. 19) (Fig. 1g) . . .
  20. Mosconi, T.; Kruger, L. Fixed-diameter polyethylene cuffs applied to the rat sciatic nerve induce a painful neuropathy: ultrastructural morphometric analysis of axonal alterations Pain 64, 37-57 (1996) .
    • . . . We measured the effect of CLP257 on Cl− transport in spinal slices obtained from rats with peripheral nerve injury (PNI; as an experimental model of neuropathic pain)20 and control slices pretreated with brain-derived neurotrophic factor (BDNF), which mediates downregulation of KCC2 activity21, 22 in models of pain hypersensitivity3, 9, 23, 24, 25, 26, 27 . . .
    • . . . We tested the antinociceptive efficacy of CLP257 in PNI rats3, 20, 28, 29 (Fig. 4a–d) . . .
    • . . . Under isoflurane anesthesia (induction at 4%, maintenance at 2%), a polyethylene cuff (PNI)20, 28 or loose chromic gut ligatures (CCI)29 were implanted around the left sciatic nerve of 190- to 200-g male Sprague-Dawley rats . . .
  21. Rivera, C. BDNF-induced TrkB activation down-regulates the K+-Cl− cotransporter KCC2 and impairs neuronal Cl− extrusion J. Cell Biol. 159, 747-752 (2002) .
    • . . . We measured the effect of CLP257 on Cl− transport in spinal slices obtained from rats with peripheral nerve injury (PNI; as an experimental model of neuropathic pain)20 and control slices pretreated with brain-derived neurotrophic factor (BDNF), which mediates downregulation of KCC2 activity21, 22 in models of pain hypersensitivity3, 9, 23, 24, 25, 26, 27 . . .
  22. Rivera, C. Mechanism of activity-dependent downregulation of the neuron-specific K-Cl cotransporter KCC2 J. Neurosci. 24, 4683-4691 (2004) .
    • . . . We measured the effect of CLP257 on Cl− transport in spinal slices obtained from rats with peripheral nerve injury (PNI; as an experimental model of neuropathic pain)20 and control slices pretreated with brain-derived neurotrophic factor (BDNF), which mediates downregulation of KCC2 activity21, 22 in models of pain hypersensitivity3, 9, 23, 24, 25, 26, 27 . . .
  23. Coull, J.A. BDNF from microglia causes the shift in neuronal anion gradient underlying neuropathic pain Nature 438, 1017-1021 (2005) .
    • . . . We measured the effect of CLP257 on Cl− transport in spinal slices obtained from rats with peripheral nerve injury (PNI; as an experimental model of neuropathic pain)20 and control slices pretreated with brain-derived neurotrophic factor (BDNF), which mediates downregulation of KCC2 activity21, 22 in models of pain hypersensitivity3, 9, 23, 24, 25, 26, 27 . . .
    • . . . Thus, CLP257 can restore Cl− extrusion capacity in neurons with reduced KCC2 activity23 . . .
  24. Zhang, W.; Liu, L.Y.; Xu, T.L. Reduced potassium-chloride co-transporter expression in spinal cord dorsal horn neurons contributes to inflammatory pain hypersensitivity in rats Neuroscience 152, 502-510 (2008) .
    • . . . We measured the effect of CLP257 on Cl− transport in spinal slices obtained from rats with peripheral nerve injury (PNI; as an experimental model of neuropathic pain)20 and control slices pretreated with brain-derived neurotrophic factor (BDNF), which mediates downregulation of KCC2 activity21, 22 in models of pain hypersensitivity3, 9, 23, 24, 25, 26, 27 . . .
  25. Lu, Y.; Zheng, J.; Xiong, L.; Zimmermann, M.; Yang, J. Spinal cord injury-induced attenuation of GABAergic inhibition in spinal dorsal horn circuits is associated with down-regulation of the chloride transporter KCC2 in rat J. Physiol. (Lond.) 586, 5701-5715 (2008) .
    • . . . We measured the effect of CLP257 on Cl− transport in spinal slices obtained from rats with peripheral nerve injury (PNI; as an experimental model of neuropathic pain)20 and control slices pretreated with brain-derived neurotrophic factor (BDNF), which mediates downregulation of KCC2 activity21, 22 in models of pain hypersensitivity3, 9, 23, 24, 25, 26, 27 . . .
  26. Cramer, S.W. The role of cation-dependent chloride transporters in neuropathic pain following spinal cord injury Mol. Pain 4, 36 (2008) .
    • . . . We measured the effect of CLP257 on Cl− transport in spinal slices obtained from rats with peripheral nerve injury (PNI; as an experimental model of neuropathic pain)20 and control slices pretreated with brain-derived neurotrophic factor (BDNF), which mediates downregulation of KCC2 activity21, 22 in models of pain hypersensitivity3, 9, 23, 24, 25, 26, 27 . . .
  27. Jolivalt, C.G.; Lee, C.A.; Ramos, K.M.; Calcutt, N.A. Allodynia and hyperalgesia in diabetic rats are mediated by GABA and depletion of spinal potassium-chloride co-transporters Pain 140, 48-57 (2008) .
    • . . . We measured the effect of CLP257 on Cl− transport in spinal slices obtained from rats with peripheral nerve injury (PNI; as an experimental model of neuropathic pain)20 and control slices pretreated with brain-derived neurotrophic factor (BDNF), which mediates downregulation of KCC2 activity21, 22 in models of pain hypersensitivity3, 9, 23, 24, 25, 26, 27 . . .
  28. Pitcher, G.M.; Ritchie, J.; Henry, J.L. Nerve constriction in the rat: model of neuropathic, surgical and central pain Pain 83, 37-46 (1999) .
    • . . . We tested the antinociceptive efficacy of CLP257 in PNI rats3, 20, 28, 29 (Fig. 4a–d) . . .
    • . . . Under isoflurane anesthesia (induction at 4%, maintenance at 2%), a polyethylene cuff (PNI)20, 28 or loose chromic gut ligatures (CCI)29 were implanted around the left sciatic nerve of 190- to 200-g male Sprague-Dawley rats . . .
  29. Bennett, G.J.; Xie, Y.K. A peripheral mononeuropathy in rat that produces disorders of pain sensation like those seen in man Pain 33, 87-107 (1988) .
    • . . . We tested the antinociceptive efficacy of CLP257 in PNI rats3, 20, 28, 29 (Fig. 4a–d) . . .
    • . . . Under isoflurane anesthesia (induction at 4%, maintenance at 2%), a polyethylene cuff (PNI)20, 28 or loose chromic gut ligatures (CCI)29 were implanted around the left sciatic nerve of 190- to 200-g male Sprague-Dawley rats . . .
  30. Gilron, I. Gabapentin and pregabalin for chronic neuropathic and early postsurgical pain: current evidence and future directions Curr. Opin. Anaesthesiol. 20, 456-472 (2007) .
    • . . . Pregabalin can cause sedation and dizziness, greatly affecting motor performance30 . . .
  31. Bos, R. Activation of 5–HT2A receptors upregulates the function of the neuronal K-Cl cotransporter KCC2 Proc. Natl. Acad. Sci. USA 110, 348-353 (2013) .
    • . . . This work validates KCC2 as a druggable target and Cl− modulation as a new mechanism of action for the development of therapeutics (also see ref. 31) . . .
  32. Kanaka, C. The differential expression patterns of messenger RNAs encoding K-Cl cotransporters (KCC1,2) and Na-K-2Cl cotransporter (NKCC1) in the rat nervous system Neuroscience 104, 933-946 (2001) .
    • . . . Expression of KCC2 is limited to the central nervous system, unlike the case for other CCCs that are expressed in several tissues3, 12, 32, 33, 34, such as the kidneys and blood vessels, where ionic homeostasis is important . . .
  33. Yang, K.; Huang, Z.W.; Huang, J.; Zhang, X.J.; Xiao, B.K. Expression of the neuron-specific potassium chloride cotransporter KCC2 in adult rat cochlear Neurosci. Lett. 441, 205-209 (2008) .
    • . . . Expression of KCC2 is limited to the central nervous system, unlike the case for other CCCs that are expressed in several tissues3, 12, 32, 33, 34, such as the kidneys and blood vessels, where ionic homeostasis is important . . .
  34. Mercado, A.; Mount, D.B.; Gamba, G. Electroneutral cation-chloride cotransporters in the central nervous system Neurochem. Res. 29, 17-25 (2004) .
    • . . . Expression of KCC2 is limited to the central nervous system, unlike the case for other CCCs that are expressed in several tissues3, 12, 32, 33, 34, such as the kidneys and blood vessels, where ionic homeostasis is important . . .
  35. Nightingale, S. The neuropathic pain market Nat. Rev. Drug Discov. 11, 101-102 (2012) .
    • . . . Drug development was previously limited by the poor understanding of the pathophysiology of neuropathic pain35, as reflected by the classes of currently approved drugs such as anticonvulsants36, antidepressants37 and opioid narcotics38 . . .
  36. Vargas-Espinosa, M.L.; Sanmarti-Garcia, G.; Vazquez-Delgado, E.; Gay-Escoda, C. Antiepileptic drugs for the treatment of neuropathic pain: a systematic review Med. Oral Patol. Oral Cir. Bucal 17, e786-e793 (2012) .
    • . . . Drug development was previously limited by the poor understanding of the pathophysiology of neuropathic pain35, as reflected by the classes of currently approved drugs such as anticonvulsants36, antidepressants37 and opioid narcotics38 . . .
  37. Dharmshaktu, P.; Tayal, V.; Kalra, B.S. Efficacy of antidepressants as analgesics: a review J. Clin. Pharmacol. 52, 6-17 (2012) .
    • . . . Drug development was previously limited by the poor understanding of the pathophysiology of neuropathic pain35, as reflected by the classes of currently approved drugs such as anticonvulsants36, antidepressants37 and opioid narcotics38 . . .
  38. Smith, H.S. Opioids and neuropathic pain Pain Physician 15, ES93-ES110 (2012) .
    • . . . Drug development was previously limited by the poor understanding of the pathophysiology of neuropathic pain35, as reflected by the classes of currently approved drugs such as anticonvulsants36, antidepressants37 and opioid narcotics38 . . .
  39. Hudmon, A. A mechanism for Ca2+/calmodulin-dependent protein kinase II clustering at synaptic and nonsynaptic sites based on self-association J. Neurosci. 25, 6971-6983 (2005) .
    • . . . Rat hippocampal cultures were prepared as described previously39 . . .
  40. Chorin, E. Upregulation of KCC2 activity by zinc-mediated neurotransmission via the mZnR/GPR39 receptor J. Neurosci. 31, 12916-12926 (2011) .
    • . . . After a control period of 50 s, perfusion solution was switched to ACSF containing 15 mM KCl (osmolarity adjusted using mannitol) to reverse KCC2-mediated Cl− transport40 . . .
  41. Digman, M.A.; Caiolfa, V.R.; Zamai, M.; Gratton, E. The phasor approach to fluorescence lifetime imaging analysis Biophys. J. 94, L14-L16 (2008) .
    • . . . Briefly, based on the work of Digman et al.41, we converted the photon timing histograms of each acquired lifetime image to phasor plots . . .
  42. Bonin, R.P. Pharmacological enhancement of δ-subunit–containing GABAA receptors that generate a tonic inhibitory conductance in spinal neurons attenuates acute nociception in mice Pain 152, 1317-1326 (2011) .
    • . . . Whole-cell clamp recordings were performed in parasagittal slices of lumbar spinal cord from rats as described previously9, 42, 43 . . .
    • . . . To study the effects of CLP257 on GABAA receptor function, a CsCl-based intrapipette solution42, 44 was used and the ACSF was supplemented with the KCC2 inhibitor VU0240551 (10 μM; Tocris Cookson, Ellisville, MO) . . .
  43. Cordero-Erausquin, M.; Coull, J.A.; Boudreau, D.; Rolland, M.; De Koninck, Y. Differential maturation of GABA action and anion reversal potential in spinal lamina I neurons: impact of chloride extrusion capacity J. Neurosci. 25, 9613-9623 (2005) .
    • . . . Whole-cell clamp recordings were performed in parasagittal slices of lumbar spinal cord from rats as described previously9, 42, 43 . . .
  44. Labrakakis, C.; Lorenzo, L.E.; Bories, C.; Ribeiro-da-Silva, A.; De Koninck, Y. Inhibitory coupling between inhibitory interneurons in the spinal cord dorsal horn Mol. Pain 5, 24 (2009) .
    • . . . To study the effects of CLP257 on GABAA receptor function, a CsCl-based intrapipette solution42, 44 was used and the ACSF was supplemented with the KCC2 inhibitor VU0240551 (10 μM; Tocris Cookson, Ellisville, MO) . . .
  45. Chaplan, S.R.; Bach, F.W.; Pogrel, J.W.; Chung, J.M.; Yaksh, T.L. Quantitative assessment of tactile allodynia in the rat paw J. Neurosci. Methods 53, 55-63 (1994) .
    • . . . Mechanical allodynia was quantified by assessing the paw withdrawal threshold using von Frey filaments (Stoelting, USA) using the up and down method45 . . .
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