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Treatment during a vulnerable developmental period rescues a genetic epilepsy

Nature Medicine volume 21, pages 1436–1444 (2015)Cite this article

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Abstract

The nervous system is vulnerable to perturbations during specific developmental periods. Insults during such susceptible time windows can have long-term consequences, including the development of neurological diseases such as epilepsy. Here we report that a pharmacological intervention timed during a vulnerable neonatal period of cortical development prevents pathology in a genetic epilepsy model. By using mice with dysfunctional Kv7 voltage-gated K+ channels, which are mutated in human neonatal epilepsy syndromes, we demonstrate the safety and efficacy of the sodium-potassium-chloride cotransporter NKCC1 antagonist bumetanide, which was administered during the first two postnatal weeks. In Kv7 current–deficient mice, which normally display epilepsy, hyperactivity and stereotypies as adults, transient bumetanide treatment normalized neonatal in vivo cortical network and hippocampal neuronal activity, prevented structural damage in the hippocampus and restored wild-type adult behavioral phenotypes. Furthermore, bumetanide treatment did not adversely affect control mice. These results suggest that in individuals with disease susceptibility, timing prophylactically safe interventions to specific windows during development may prevent or arrest disease progression.

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Figure 1: Altered hippocampal excitability and structure induced by Kv7 channelopathy.
Figure 2: Abnormal spontaneous in vivo network activity in neonatal mutants.
Figure 3: Bumetanide treatment does not affect neurocognitive development of healthy mice.
Figure 4: Timed bumetanide treatment normalizes in vivo unit and network activity.
Figure 5: Prophylactic bumetanide treatment during a vulnerable window protects against behavioral hyperactivity.
Figure 6: Bumetanide treatment restores hippocampal structure and reduces neuroinflammation and abnormal ECoG events.

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References

  1. Kwan, P., Schachter, S. & Brodie, M. Drug-resistant epilepsy. N. Engl. J. Med. 365, 919–926 (2011).

    CAS  PubMed  Google Scholar 

  2. Ben-Ari, Y. & Holmes, G.L. Effects of seizures on developmental processes in the immature brain. Lancet 5, 1055–1063 (2006).

    PubMed  Google Scholar 

  3. Turrigiano, G.G. & Nelson, S.B. Homeostatic plasticity in the developing nervous system. Nat. Rev. Neurosci. 5, 97–107 (2004).

    CAS  PubMed  Google Scholar 

  4. Lewis, D.A. & Levitt, P. Schizophrenia as a disorder of neurodevelopment. Annu. Rev. Neurosci. 25, 409–432 (2002).

    CAS  PubMed  Google Scholar 

  5. Allin, M. et al. Personality in young adults who are born preterm. Pediatrics 117, 309–316 (2006).

    PubMed  Google Scholar 

  6. Hensch, T.K. Critical period regulation. Annu. Rev. Neurosci. 27, 549–579 (2004).

    CAS  PubMed  Google Scholar 

  7. Katagiri, H., Fagiolini, M. & Hensch, T.K. Optimization of somatic inhibition at critical period onset in mouse visual cortex. Neuron 53, 805–812 (2007).

    CAS  PubMed  Google Scholar 

  8. Levelt, C.N. & Hübener, M. Critical-period plasticity in the visual cortex. Annu. Rev. Neurosci. 35, 309–330 (2012).

    CAS  PubMed  Google Scholar 

  9. Peters, H.C., Hu, H., Pongs, O., Storm, J.F. & Isbrandt, D. Conditional transgenic suppression of M channels in mouse brain reveals functions in neuronal excitability, resonance and behavior. Nat. Neurosci. 8, 51–60 (2005).

    CAS  PubMed  Google Scholar 

  10. Brown, D.A. & Passmore, G.M. Neural KCNQ (Kv7) channels. Br. J. Pharmacol. 156, 1185–1195 (2009).

    CAS  PubMed  PubMed Central  Google Scholar 

  11. Wang, H.S. et al. KCNQ2 and KCNQ3 potassium channel subunits: molecular correlates of the M channel. Science 282, 1890–1893 (1998).

    CAS  PubMed  Google Scholar 

  12. Cooper, E.C., Harrington, E., Jan, Y.N. & Jan, L.Y. M channel KCNQ2 subunits are localized to key sites for control of neuronal network oscillations and synchronization in mouse brain. J. Neurosci. 21, 9529–9540 (2001).

    CAS  PubMed  PubMed Central  Google Scholar 

  13. Devaux, J.J., Kleopa, K.A., Cooper, E.C. & Scherer, S.S. KCNQ2 is a nodal K+ channel. J. Neurosci. 24, 1236–1244 (2004).

    CAS  PubMed  PubMed Central  Google Scholar 

  14. Biervert, C. et al. A potassium channel mutation in neonatal human epilepsy. Science 279, 403–406 (1998).

    CAS  PubMed  Google Scholar 

  15. Singh, N.A. et al. A novel potassium channel gene, KCNQ2, is mutated in an inherited epilepsy of newborns. Nat. Genet. 18, 25–29 (1998).

    CAS  PubMed  Google Scholar 

  16. Charlier, C. et al. A pore mutation in a novel KQT-like potassium channel gene in an idiopathic epilepsy family. Nat. Genet. 18, 53–55 (1998).

    CAS  PubMed  Google Scholar 

  17. Neubauer, B.A. et al. KCNQ2 and KCNQ3 mutations contribute to different idiopathic epilepsy syndromes. Neurology 71, 177–183 (2008).

    CAS  PubMed  Google Scholar 

  18. Weckhuysen, S. et al. KCNQ2 encephalopathy: emerging phenotype of a neonatal epileptic encephalopathy. Ann. Neurol. 71, 15–25 (2012).

    CAS  PubMed  Google Scholar 

  19. Orhan, G. et al. Dominant-negative effects of KCNQ2 mutations are associated with epileptic encephalopathy. Ann. Neurol. 75, 382–394 (2014).

    CAS  PubMed  Google Scholar 

  20. Booth, D. & Evans, D.J. Anticonvulsants for neonates with seizures. Cochrane Database Syst. Rev. CD004218 (2004).

  21. Evans, D.J., Levene, M.I. & Tsakmakis, M. Anticonvulsants for preventing mortality and morbidity in full term newborns with perinatal asphyxia. Cochrane Database Syst. Rev. CD001240 (2007).

  22. Koyama, R. et al. GABAergic excitation after febrile seizures induces ectopic granule cells and adult epilepsy. Nat. Med. 18, 1271–1278 (2012).

    CAS  PubMed  Google Scholar 

  23. Dzhala, V.I. et al. NKCC1 transporter facilitates seizures in the developing brain. Nat. Med. 11, 1205–1213 (2005).

    CAS  PubMed  Google Scholar 

  24. Dzhala, V.I., Brumback, A.C. & Staley, K.J. Bumetanide enhances phenobarbital efficacy in a neonatal seizure model. Ann. Neurol. 63, 222–235 (2008).

    CAS  PubMed  Google Scholar 

  25. Wang, D.D. & Kriegstein, R. GABA regulates excitatory synapse formation in the neocortex via NMDA receptor activation. J. Neurosci. 28, 5547–5558 (2008).

    CAS  PubMed  PubMed Central  Google Scholar 

  26. Kilb, W., Sinning, A. & Luhmann, H.J. Model-specific effects of bumetanide on epileptiform activity in the in vitro–intact hippocampus of the newborn mouse. Neuropharmacology 53, 524–533 (2007).

    CAS  PubMed  Google Scholar 

  27. Margineanu, D.G. & Klitgaard, H. Differential effects of cation-chloride cotransport-blocking diuretics in a rat hippocampal slice model of epilepsy. Epilepsy Res. 69, 93–99 (2006).

    CAS  PubMed  Google Scholar 

  28. Liu, Y., Shangguan, Y., Barks, J.D.E. & Silverstein, F.S. Bumetanide augments the neuroprotective efficacy of phenobarbital plus hypothermia in a neonatal hypoxia-ischemia model. Pediatr. Res. 71, 559–565 (2012).

    CAS  PubMed  PubMed Central  Google Scholar 

  29. Kahle, K.T., Barnett, S.M., Sassower, K.C. & Staley, K.J. Decreased seizure activity in a human neonate treated with bumetanide, an inhibitor of the Na+-K+-2Cl− cotransporter NKCC1. J. Child Neurol. 24, 572–576 (2009).

    PubMed  Google Scholar 

  30. Pressler, R.M. et al. Bumetanide for the treatment of seizures in newborn babies with hypoxic ischaemic encephalopathy (NEMO): an open-label, dose-finding and feasibility phase 1/2 trial. Lancet Neurol. 14, 469–477 (2015).

    CAS  PubMed  Google Scholar 

  31. Khazipov, R. et al. Early motor activity drives spindle bursts in the developing somatosensory cortex. Nature 432, 758–761 (2004).

    CAS  PubMed  Google Scholar 

  32. Ben-Ari, Y. Developing networks play a similar melody. Trends Neurosci. 24, 353–360 (2001).

    CAS  PubMed  Google Scholar 

  33. Hanganu, I.L., Ben-Ari, Y. & Khazipov, R. Retinal waves trigger spindle bursts in the neonatal rat visual cortex. J. Neurosci. 26, 6728–6736 (2006).

    CAS  PubMed  PubMed Central  Google Scholar 

  34. Weber, Y.G. et al. Immunohistochemical analysis of KCNQ2 potassium channels in adult and developing mouse brain. Brain Res. 1077, 1–6 (2006).

    CAS  PubMed  Google Scholar 

  35. Safiulina, V.F., Zacchi, P., Taglialatela, M., Yaari, Y. & Cherubini, E. Low expression of Kv7-M channels facilitates intrinsic and network bursting in the developing rat hippocampus. J. Physiol. (Lond.) 586, 5437–5453 (2008).

    CAS  Google Scholar 

  36. Bullitt, E. Expression of c-fos–like protein as a marker for neuronal activity following noxious stimulation in the rat. J. Comp. Neurol. 296, 517–530 (1990).

    CAS  PubMed  Google Scholar 

  37. Streit, W.J. & Kreutzberg, G.W. Lectin binding by resting and reactive microglia. J. Neurocytol. 16, 249–260 (1987).

    CAS  PubMed  Google Scholar 

  38. Khalilov, I. et al. A novel in vitro preparation: the intact hippocampal formation. Neuron 19, 743–749 (1997).

    CAS  PubMed  Google Scholar 

  39. Ben-Ari, Y., Cherubini, E., Corradetti, R. & Gaiarsa, J.-L.L. Giant synaptic potentials in immature rat CA3 hippocampal neurons. J. Physiol. (Lond.) 416, 303–325 (1989).

    CAS  Google Scholar 

  40. Leinekugel, X. et al. Correlated bursts of activity in the neonatal hippocampus in vivo. Science 296, 2049–2052 (2002).

    CAS  PubMed  Google Scholar 

  41. Seelke, A.M.H. & Blumberg, M.S. Developmental appearance and disappearance of cortical events and oscillations in infant rats. Brain Res. 1324, 34–42 (2010).

    CAS  PubMed  PubMed Central  Google Scholar 

  42. Vanhatalo, S. & Kaila, K. Development of neonatal EEG activity: from phenomenology to physiology. Semin. Fetal Neonatal Med. 11, 471–478 (2006).

    PubMed  Google Scholar 

  43. Ben-Ari, Y. Excitatory actions of GABA during development: the nature of the nurture. Nat. Rev. Neurosci. 3, 728–739 (2002).

    CAS  PubMed  Google Scholar 

  44. Wang, D.D. & Kriegstein, A.R. Blocking early GABA depolarization with bumetanide results in permanent alterations in cortical circuits and sensorimotor gating deficits. Cereb. Cortex 21, 574–587 (2011).

    PubMed  Google Scholar 

  45. Talos, D.M. et al. Antiepileptic effects of levetiracetam in a rodent neonatal seizure model. Pediatr. Res. 73, 24–30 (2013).

    CAS  PubMed  Google Scholar 

  46. Noebels, J.L. & Sidman, R.L. Inherited epilepsy: spike-wave and focal motor seizures in the mutant mouse tottering. Science 204, 1334–1336 (1979).

    CAS  PubMed  Google Scholar 

  47. Ryan, L.J. & Sharpless, S.K. Genetically determined spontaneous and brief spindle episodes in mice. Exp. Neurol. 66, 493–508 (1979).

    CAS  PubMed  Google Scholar 

  48. Vanhatalo, S., Hellström-Westas, L. & de Vries, L.S. Bumetanide for neonatal seizures: based on evidence or enthusiasm? Epilepsia 50, 1292–1293 (2009).

    PubMed  Google Scholar 

  49. Chabwine, J.N. & Vanden Eijnden, S. A claim for caution in the use of promising bumetanide to treat neonatal seizures. J. Child Neurol. 26, 657–658 (2011).

    PubMed  Google Scholar 

  50. Töpfer, M. et al. Consequences of inhibition of bumetanide metabolism in rodents on brain penetration and effects of bumetanide in chronic models of epilepsy. Eur. J. Neurosci. 39, 673–687 (2014).

    PubMed  Google Scholar 

  51. Löscher, W., Puskarjov, M. & Kaila, K. Cation-chloride cotransporters NKCC1 and KCC2 as potential targets for novel antiepileptic and antiepileptogenic treatments. Neuropharmacology 69, 62–74 (2013).

    PubMed  Google Scholar 

  52. Puskarjov, M., Kahle, K.T., Ruusuvuori, E. & Kaila, K. Pharmacotherapeutic targeting of cation-chloride cotransporters in neonatal seizures. Epilepsia 55, 806–818 (2014).

    CAS  PubMed  PubMed Central  Google Scholar 

  53. Minlebaev, M., Ben-Ari, Y.Y. & Khazipov, R. Network mechanisms of spindle-burst oscillations in the neonatal rat barrel cortex in vivo. J. Neurophysiol. 97, 692–700 (2007).

    CAS  PubMed  Google Scholar 

  54. Rheims, S. et al. Layer-specific generation and propagation of seizures in slices of developing neocortex: role of excitatory GABAergic synapses. J. Neurophysiol. 100, 620–628 (2008).

    PubMed  Google Scholar 

  55. Tyzio, R. et al. Oxytocin-mediated GABA inhibition during delivery attenuates autism pathogenesis in rodent offspring. Science 343, 675–679 (2014).

    CAS  PubMed  Google Scholar 

  56. Deidda, G. et al. Reversing excitatory GABAAR signaling restores synaptic plasticity and memory in a mouse model of Down syndrome. Nat. Med. 21, 318–326 (2015).

    CAS  PubMed  Google Scholar 

  57. Dzhala, V.I., Saponjian, Y., De Konnick, Y. & Staley, K. Modulation of NKCC1 and KCC2 cotransporters for control of catastrophic drug-resistant seizures. (Program no. 521.05, 2014 Neuroscience Meeting Planner) (Society for Neuroscience, Washington, DC, 2014).

  58. Huberfeld, G., Wittner, L. & Clemenceau, S. Perturbed chloride homeostasis and GABAergic signaling in human temporal lobe epilepsy. J. Neurosci. 27, 9866–9873 (2007).

    CAS  PubMed  PubMed Central  Google Scholar 

  59. Miles, R., Blaesse, P., Huberfeld, G., Wittner, L. & Kaila, K. in Jasper's Basic Mechanisms of the Epilepsies 4th edn. (eds. Noebels, J.L., Avoli, M., Rogawski, M.A., Olsen, R.W. & Delgado-Escueta, A.V.) 1–13 (National Center for Biotechnology Information, Bethesda, MD, USA) (2012).

  60. Tremblay, P., Meiner, Z., Galou, M. & Heinrich, C. Doxycycline control of prion protein transgene expression modulates prion disease in mice. Proc. Natl. Acad. Sci. USA 95, 12580–12585 (1998).

    CAS  PubMed  Google Scholar 

  61. Abràmoff, M.D., Magelhães, P.J. & Ram, S.J. Image processing with ImageJ. Biophotonics Int. 11, 36–42 (2004).

    Google Scholar 

  62. Adelsberger, H., Garaschuk, O. & Konnerth, A. Cortical calcium waves in resting newborn mice. Nat. Neurosci. 8, 988–990 (2005).

    CAS  PubMed  Google Scholar 

  63. Paxinos, G., Hallyday, G., Watson, C., Koutcherov, Y. & Wang, H. Atlas of the Developing Mouse Brain at E17.5, P0 and P6. (Academic Press, 2007).

  64. Hazan, L., Zugaro, M. & Buzsáki, G. Klusters, NeuroScope, NDManager: a free software suite for neurophysiological data processing and visualization. J. Neurosci. Methods 155, 207–216 (2006).

    PubMed  Google Scholar 

  65. Rossant, C. et al. Spike sorting for large, dense electrode arrays. bioRxiv http://biorxiv.org/content/biorxiv/early/2015/02/16/015198.full.pdf (2015).

  66. Kadir, S.N., Goodman, D.F.M. & Harris, K.D. High-dimensional cluster analysis with the masked EM algorithm. Neural Comput. 26, 2379–2394 (2014).

    PubMed  PubMed Central  Google Scholar 

  67. Heyser, C.J. Assessment of developmental milestones in rodents. Curr. Protoc. Neurosci. 25, 8.18 (2003).

    Google Scholar 

  68. Freitag, S., Schachner, M. & Morellini, F. Behavioral alterations in mice deficient for the extracellular matrix glycoprotein tenascin-R. Behav. Brain Res. 145, 189–207 (2003).

    CAS  PubMed  Google Scholar 

  69. Morellini, F. & Schachner, M. Enhanced novelty-induced activity, reduced anxiety, delayed resynchronization to daylight reversal and weaker muscle strength in tenascin-C–deficient mice. Eur. J. Neurosci. 23, 1255–1268 (2006).

    PubMed  Google Scholar 

  70. Sawallisch, C. et al. The insulin receptor substrate of 53 kDa (IRSp53) limits hippocampal synaptic plasticity. J. Biol. Chem. 284, 9225–9236 (2009).

    CAS  PubMed  PubMed Central  Google Scholar 

  71. Fellini, L., Schachner, M. & Morellini, F. Adult but not aged C57BL/6 male mice are capable of using geometry for orientation. Learn. Mem. 13, 473–481 (2006).

    PubMed  PubMed Central  Google Scholar 

  72. Fellini, L. & Morellini, F. Geometric information is required for allothetic navigation in mice. Behav. Brain Res. 222, 380–384 (2011).

    PubMed  Google Scholar 

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Acknowledgements

We thank K. Sauter and S. Schillemeit for excellent technical support, H. Voss for expert animal caretaking, I. Hermans-Borgmeyer and C.H. Peters for in situ hybridization, M. Bindszus, R. Schubert and J. Szeremeta for help with genotyping and A. Isbrandt for comments on the manuscript. The study was supported by the German Federal Ministry of Education and Research (NGFNplus/EMINet project 01GS0831; I.L.H.-O.: 01GQ0809; D.I. and A.N.), the Deutsche Forschungsgemeinschaft (DFG; IS63/3-1/2 and IS63/4-1 (D.I.); SFB936, B3 (F.M. and D.I.); SPP1665 IS63/5-1 (D.I.); and Ha4466/3-1 (I.L.H.-O.)), the Human Frontier Science Program (D.I.), Werner-Otto-Stiftung (Hamburg, Germany) (D.I.), Hamburg macht Kinder gesund e.V. (Hamburg, Germany) (D.I.), L'Agence Nationale de la Recherche (ANR, ANR-13-NEUC-005-01 MOTION; C.B.), and INSERM (C.B.).

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  1. Stephan Lawrence Marguet and Vu Thao Quyen Le-Schulte: These authors contributed equally to this work.

Authors and Affiliations

  1. Experimental Neurophysiology, German Center for Neurodegenerative Diseases (DZNE), Bonn, Germany

    Stephan Lawrence Marguet, Andrea Merseburg, Igor Jakovcevski & Dirk Isbrandt

  2. Institute for Molecular and Behavioral Neuroscience, University of Cologne, Cologne, Germany

    Stephan Lawrence Marguet, Andrea Merseburg, Igor Jakovcevski & Dirk Isbrandt

  3. Experimental Neuropediatrics, University Medical Center Hamburg-Eppendorf, Hamburg, Germany

    Stephan Lawrence Marguet, Vu Thao Quyen Le-Schulte, Andrea Merseburg, Axel Neu, Ronny Eichler, Fabio Morellini & Dirk Isbrandt

  4. Aix Marseille Université, Institut National de la Santé et de la Recherche Médicale (INSERM), Institut de Neurosciences des Systèmes (INS), Unité Mixte de Recherche (UMR) S1106, Marseille, France

    Anton Ivanov & Christophe Bernard

  5. Developmental Neurophysiology, Center for Experimental Medicine, University Medical Center Hamburg-Eppendorf, Hamburg, Germany

    Ileana Livia Hanganu-Opatz

  6. Behavioral Biology Unit, Center for Molecular Neurobiology, University Medical Center Hamburg-Eppendorf, Hamburg, Germany

    Fabio Morellini

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Contributions

S.L.M. and V.T.Q.L.-S. contributed equally to the manuscript. D.I. conceived the study. D.I., S.L.M., C.B. and V.T.Q.L.-S. wrote the manuscript. V.T.Q.L.-S., I.L.H.-O. and D.I. established experimental conditions for in vivo electrophysiological experiments and performed the initial data analysis. S.L.M., V.T.Q.L.-S. and D.I. performed—and S.L.M., V.T.Q.L.-S., A.M. and D.I. analyzed—all in vivo electrophysiological experiments. S.L.M. wrote Matlab routines for data analysis and graphical representation. A.N., A.I. and C.B. designed, performed and analyzed in vitro electrophysiological experiments. V.T.Q.L.-S. and I.J. performed immunohistochemical experiments. V.T.Q.L.-S. performed immunoblotting experiments. V.T.Q.L.-S., A.M., R.E. and F.M. acquired and analyzed behavioral data. F.M., D.I. and S.L.M. performed statistical analysis. All coauthors contributed to the revision of the manuscript.

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Correspondence to Dirk Isbrandt.

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Marguet, S., Le-Schulte, V., Merseburg, A. et al. Treatment during a vulnerable developmental period rescues a genetic epilepsy. Nat Med 21, 1436–1444 (2015). https://doi.org/10.1038/nm.3987

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