Skip to main content
SpringerLink
Log in

Gephyrin Cleavage in In Vitro Brain Ischemia Decreases GABAA Receptor Clustering and Contributes to Neuronal Death

  • Published:
Molecular Neurobiology Aims and scope Submit manuscript

Abstract

GABA (γ-aminobutyric acid) is the major inhibitory neurotransmitter in the central nervous system, and changes in GABAergic neurotransmission modulate the activity of neuronal networks. Gephyrin is a scaffold protein responsible for the traffic and synaptic anchoring of GABAA receptors (GABAAR); therefore, changes in gephyrin expression and oligomerization may affect the activity of GABAergic synapses. In this work, we investigated the changes in gephyrin protein levels during brain ischemia and in excitotoxic conditions, which may affect synaptic clustering of GABAAR. We found that gephyrin is cleaved by calpains following excitotoxic stimulation of hippocampal neurons with glutamate, as well as after intrahippocampal injection of kainate, giving rise to a stable cleavage product. Gephyrin cleavage was also observed in cultured hippocampal neurons subjected to transient oxygen-glucose deprivation (OGD), an in vitro model of brain ischemia, and after transient middle cerebral artery occlusion (MCAO) in mice, a model of focal brain ischemia. Furthermore, a truncated form of gephyrin decreased the synaptic clustering of the protein, reduced the synaptic pool of GABAAR containing γ2 subunits and upregulated OGD-induced cell death in hippocampal cultures. Our results show that excitotoxicity and brain ischemia downregulate full-length gephyrin with a concomitant generation of truncated products, which affect synaptic clustering of GABAAR and cell death.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Fig. 1
Fig. 2
Fig. 3
Fig. 4
Fig. 5
Fig. 6
Fig. 7

Similar content being viewed by others

References

  1. Szydlowska K, Tymianski M (2010) Calcium, ischemia and excitotoxicity. Cell Calcium 47(2):122–129. doi:10.1016/j.ceca.2010.01.003

    Article  CAS  PubMed  Google Scholar 

  2. Lai TW, Zhang S, Wang YT (2014) Excitotoxicity, and stroke: identifying novel targets for neuroprotection. Prog Neurobiol 115:157–188. doi:10.1016/j.pneurobio.2013.11.006

    Article  CAS  PubMed  Google Scholar 

  3. Schwartz-Bloom RD, Sah R (2001) gamma-Aminobutyric acidA neurotransmission and cerebral ischemia. J Neurochem 77(2):353–371

    Article  CAS  PubMed  Google Scholar 

  4. Wu C, Sun D (2014) GABA receptors in brain development, function, and injury. Metab Brain Dis 30(2):367–379. doi:10.1007/s11011-014-9560-1

    Article  PubMed  PubMed Central  Google Scholar 

  5. Mele M, Ribeiro L, Inacio AR, Wieloch T, Duarte CB (2014) GABA(A) receptor dephosphorylation followed by internalization is coupled to neuronal death in in vitro ischemia. Neurobiol Dis 65:220–232. doi:10.1016/j.nbd.2014.01.019

    Article  CAS  PubMed  Google Scholar 

  6. Bedet C, Bruusgaard JC, Vergo S, Groth-Pedersen L, Eimer S, Triller A, Vannier C (2006) Regulation of gephyrin assembly and glycine receptor synaptic stability. J Biol Chem 281(40):30046–30056. doi:10.1074/jbc.M602155200

    Article  CAS  PubMed  Google Scholar 

  7. Yu W, Jiang M, Miralles CP, Li RW, Chen G, de Blas AL (2007) Gephyrin clustering is required for the stability of GABAergic synapses. Mol Cell Neurosci 36(4):484–500. doi:10.1016/j.mcn.2007.08.008

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  8. Tyagarajan SK, Fritschy JM (2014) Gephyrin: a master regulator of neuronal function? Nat Rev Neurosci 15(3):141–156. doi:10.1038/nrn3670

    Article  CAS  PubMed  Google Scholar 

  9. Smith KR, Muir J, Rao Y, Browarski M, Gruenig MC, Sheehan DF, Haucke V, Kittler JT (2012) Stabilization of GABAA receptors at endocytic zones is mediated by an AP2 binding motif within the GABAA receptor beta3 subunit. J Neurosci 32(7):2485–2498. doi:10.1523/JNEUROSCI.1622-11.2011

    Article  CAS  PubMed  Google Scholar 

  10. Prior P, Schmitt B, Grenningloh G, Pribilla I, Multhaup G, Beyreuther K, Maulet Y, Werner P et al (1992) Primary structure and alternative splice variants of gephyrin, a putative glycine receptor-tubulin linker protein. Neuron 8(6):1161–1170

    Article  CAS  PubMed  Google Scholar 

  11. Saiyed T, Paarmann I, Schmitt B, Haeger S, Sola M, Schmalzing G, Weissenhorn W, Betz H (2007) Molecular basis of gephyrin clustering at inhibitory synapses: role of G- and E-domain interactions. J Biol Chem 282(8):5625–5632. doi:10.1074/jbc.M610290200

    Article  CAS  PubMed  Google Scholar 

  12. Jacob TC, Moss SJ, Jurd R (2008) GABA(A) receptor trafficking and its role in the dynamic modulation of neuronal inhibition. Nat Rev Neurosci 9(5):331–343. doi:10.1038/nrn2370

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  13. Tretter V, Jacob TC, Mukherjee J, Fritschy JM, Pangalos MN, Moss SJ (2008) The clustering of GABAA receptor subtypes at inhibitory synapses is facilitated via the direct binding of receptor alpha 2 subunits to gephyrin. J Neurosci 28(6):1356–1365. doi:10.1523/JNEUROSCI.5050-07.2008

    Article  CAS  PubMed  Google Scholar 

  14. Tretter V, Kerschner B, Milenkovic I, Ramsden SL, Ramerstorfer J, Saiepour L, Maric HM, Moss SJ et al (2011) Molecular basis of the gamma-aminobutyric acid A receptor alpha3 subunit interaction with the clustering protein gephyrin. J Biol Chem 286(43):37702–37711. doi:10.1074/jbc.M111.291336

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  15. Mukherjee J, Kretschmannova K, Gouzer G, Maric HM, Ramsden S, Tretter V, Harvey K, Davies PA et al (2011) The residence time of GABAARs at inhibitory synapses is determined by direct binding of the receptor alpha1 subunit to gephyrin. J Neurosci 31(41):14677–14687. doi:10.1523/JNEUROSCI.2001-11.2011

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  16. Kowalczyk S, Winkelmann A, Smolinsky B, Forstera B, Neundorf I, Schwarz G, Meier JC (2013) Direct binding of GABAA receptor beta2 and beta3 subunits to gephyrin. Eur J Neurosci 37(4):544–554. doi:10.1111/ejn.12078

    Article  PubMed  Google Scholar 

  17. Maric HM, Mukherjee J, Tretter V, Moss SJ, Schindelin H (2011) Gephyrin-mediated gamma-aminobutyric acid type A and glycine receptor clustering relies on a common binding site. J Biol Chem 286(49):42105–42114. doi:10.1074/jbc.M111.303412

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  18. Sassoe-Pognetto M, Panzanelli P, Sieghart W, Fritschy JM (2000) Colocalization of multiple GABAA receptor subtypes with gephyrin at postsynaptic sites. J Comp Neurol 420(4):481–498

    Article  CAS  PubMed  Google Scholar 

  19. Tyagarajan SK, Ghosh H, Yevenes GE, Nikonenko I, Ebeling C, Schwerdel C, Sidler C, Zeilhofer HU et al (2011) Regulation of GABAergic synapse formation and plasticity by GSK3beta-dependent phosphorylation of gephyrin. Proc Natl Acad Sci U S A 108(1):379–384. doi:10.1073/pnas.1011824108

    Article  CAS  PubMed  Google Scholar 

  20. Tyagarajan SK, Ghosh H, Yevenes GE, Imanishi SY, Zeilhofer HU, Gerrits B, Fritschy JM (2013) Extracellular signal-regulated kinase and glycogen synthase kinase 3beta regulate gephyrin postsynaptic aggregation and GABAergic synaptic function in a calpain-dependent mechanism. J Biol Chem 288(14):9634–9647. doi:10.1074/jbc.M112.442616

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  21. Bevers MB, Neumar RW (2008) Mechanistic role of calpains in postischemic neurodegeneration. J Cereb Blood Flow Metab 28(4):655–673. doi:10.1038/sj.jcbfm.9600595

    Article  CAS  PubMed  Google Scholar 

  22. Vosler PS, Brennan CS, Chen J (2008) Calpain-mediated signaling mechanisms in neuronal injury and neurodegeneration. Mol Neurobiol 38(1):78–100. doi:10.1007/s12035-008-8036-x

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  23. Gomes JR, Lobo AC, Melo CV, Inacio AR, Takano J, Iwata N, Saido TC, de Almeida LP et al (2011) Cleavage of the vesicular GABA transporter under excitotoxic conditions is followed by accumulation of the truncated transporter in nonsynaptic sites. J Neurosci 31(12):4622–4635. doi:10.1523/JNEUROSCI.3541-10.2011

    Article  CAS  PubMed  Google Scholar 

  24. Baliova M, Knab A, Franekova V, Jursky F (2009) Modification of the cytosolic regions of GABA transporter GAT1 by calpain. Neurochem Int 55(5):288–294. doi:10.1016/j.neuint.2009.03.012

    Article  CAS  PubMed  Google Scholar 

  25. Almeida RD, Manadas BJ, Melo CV, Gomes JR, Mendes CS, Graos MM, Carvalho RF, Carvalho AP et al (2005) Neuroprotection by BDNF against glutamate-induced apoptotic cell death is mediated by ERK and PI3-kinase pathways. Cell Death Differ 12(10):1329–1343. doi:10.1038/sj.cdd.4401662

    Article  CAS  PubMed  Google Scholar 

  26. Caldeira MV, Curcio M, Leal G, Salazar IL, Mele M, Santos AR, Melo CV, Pereira P et al (2013) Excitotoxic stimulation downregulates the ubiquitin-proteasome system through activation of NMDA receptors in cultured hippocampal neurons. Biochim Biophys Acta 1832(1):263–274. doi:10.1016/j.bbadis.2012.10.009

    Article  CAS  PubMed  Google Scholar 

  27. Hardingham GE, Fukunaga Y, Bading H (2002) Extrasynaptic NMDARs oppose synaptic NMDARs by triggering CREB shut-off and cell death pathways. Nat Neurosci 5(5):405–414. doi:10.1038/nn835

    CAS  PubMed  Google Scholar 

  28. Nygren J, Wieloch T (2005) Enriched environment enhances recovery of motor function after focal ischemia in mice, and downregulates the transcription factor NGFI-A. J Cereb Blood Flow Metab 25(12):1625–1633. doi:10.1038/sj.jcbfm.9600157

    Article  CAS  PubMed  Google Scholar 

  29. Lardi-Studler B, Smolinsky B, Petitjean CM, Koenig F, Sidler C, Meier JC, Fritschy JM, Schwarz G (2007) Vertebrate-specific sequences in the gephyrin E-domain regulate cytosolic aggregation and postsynaptic clustering. J Cell Sci 120(Pt 8):1371–1382. doi:10.1242/jcs.003905

    Article  CAS  PubMed  Google Scholar 

  30. Konermann S, Brigham MD, Trevino AE, Hsu PD, Heidenreich M, Cong L, Platt RJ, Scott DA et al (2013) Optical control of mammalian endogenous transcription and epigenetic states. Nature 500(7463):472–476. doi:10.1038/nature12466

    CAS  PubMed  PubMed Central  Google Scholar 

  31. Parsons MP, Raymond LA (2014) Extrasynaptic NMDA receptor involvement in central nervous system disorders. Neuron 82(2):279–293. doi:10.1016/j.neuron.2014.03.030

    Article  CAS  PubMed  Google Scholar 

  32. Liu Z, Cao J, Gao X, Ma Q, Ren J, Xue Y (2011) GPS-CCD: a novel computational program for the prediction of calpain cleavage sites. PLoS One 6(4), e19001. doi:10.1371/journal.pone.0019001

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  33. Kawasaki BT, Hoffman KB, Yamamoto RS, Bahr BA (1997) Variants of the receptor/channel clustering molecule gephyrin in brain: distinct distribution patterns, developmental profiles, and proteolytic cleavage by calpain. J Neurosci Res 49(3):381–388

    Article  CAS  PubMed  Google Scholar 

  34. Takano J, Tomioka M, Tsubuki S, Higuchi M, Iwata N, Itohara S, Maki M, Saido TC (2005) Calpain mediates excitotoxic DNA fragmentation via mitochondrial pathways in adult brains: evidence from calpastatin mutant mice. J Biol Chem 280(16):16175–16184. doi:10.1074/jbc.M414552200

    Article  CAS  PubMed  Google Scholar 

  35. Higuchi M, Tomioka M, Takano J, Shirotani K, Iwata N, Masumoto H, Maki M, Itohara S et al (2005) Distinct mechanistic roles of calpain and caspase activation in neurodegeneration as revealed in mice overexpressing their specific inhibitors. J Biol Chem 280(15):15229–15237. doi:10.1074/jbc.M500939200

    Article  CAS  PubMed  Google Scholar 

  36. Farrant M, Nusser Z (2005) Variations on an inhibitory theme: phasic and tonic activation of GABAA receptors. Nat Rev Neurosci 6(3):215–229. doi:10.1038/nrn1625

    Article  CAS  PubMed  Google Scholar 

  37. Sola M, Kneussel M, Heck IS, Betz H, Weissenhorn W (2001) X-ray crystal structure of the trimeric N-terminal domain of gephyrin. J Biol Chem 276(27):25294–25301. doi:10.1074/jbc.M101923200

    Article  CAS  PubMed  Google Scholar 

  38. Sola M, Bavro VN, Timmins J, Franz T, Ricard-Blum S, Schoehn G, Ruigrok RW, Paarmann I et al (2004) Structural basis of dynamic glycine receptor clustering by gephyrin. EMBO J 23(13):2510–2519. doi:10.1038/sj.emboj.7600256

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  39. Kim EY, Schrader N, Smolinsky B, Bedet C, Vannier C, Schwarz G, Schindelin H (2006) Deciphering the structural framework of glycine receptor anchoring by gephyrin. EMBO J 25(6):1385–1395. doi:10.1038/sj.emboj.7601029

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  40. Hanus C, Ehrensperger MV, Triller A (2006) Activity-dependent movements of postsynaptic scaffolds at inhibitory synapses. J Neurosci 26(17):4586–4595. doi:10.1523/JNEUROSCI.5123-05.2006

    Article  CAS  PubMed  Google Scholar 

  41. Specht CG, Izeddin I, Rodriguez PC, El Beheiry M, Rostaing P, Darzacq X, Dahan M, Triller A (2013) Quantitative nanoscopy of inhibitory synapses: counting gephyrin molecules and receptor binding sites. Neuron 79(2):308–321. doi:10.1016/j.neuron.2013.05.013

    Article  CAS  PubMed  Google Scholar 

  42. Dobie FA, Craig AM (2011) Inhibitory synapse dynamics: coordinated presynaptic and postsynaptic mobility and the major contribution of recycled vesicles to new synapse formation. J Neurosci 31(29):10481–10493. doi:10.1523/JNEUROSCI.6023-10.2011

    Article  CAS  PubMed  Google Scholar 

  43. Alldred MJ, Mulder-Rosi J, Lingenfelter SE, Chen G, Luscher B (2005) Distinct gamma2 subunit domains mediate clustering and synaptic function of postsynaptic GABAA receptors and gephyrin. J Neurosci 25(3):594–603. doi:10.1523/JNEUROSCI.4011-04.2005

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  44. Dejanovic B, Schwarz G (2014) Neuronal nitric oxide synthase-dependent S-nitrosylation of gephyrin regulates gephyrin clustering at GABAergic synapses. J Neurosci 34(23):7763–7768. doi:10.1523/JNEUROSCI.0531-14.2014

    Article  CAS  PubMed  Google Scholar 

  45. Samdani AF, Dawson TM, Dawson VL (1997) Nitric oxide synthase in models of focal ischemia. Stroke 28(6):1283–1288

    Article  CAS  PubMed  Google Scholar 

  46. Muir J, Arancibia-Carcamo IL, MacAskill AF, Smith KR, Griffin LD, Kittler JT (2010) NMDA receptors regulate GABAA receptor lateral mobility and clustering at inhibitory synapses through serine 327 on the gamma2 subunit. Proc Natl Acad Sci U S A 107(38):16679–16684. doi:10.1073/pnas.1000589107

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  47. Mtchedlishvili Z, Kapur J (2006) High-affinity, slowly desensitizing GABAA receptors mediate tonic inhibition in hippocampal dentate granule cells. Mol Pharmacol 69(2):564–575. doi:10.1124/mol.105.016683

    Article  CAS  PubMed  Google Scholar 

  48. Bannai H, Levi S, Schweizer C, Inoue T, Launey T, Racine V, Sibarita JB, Mikoshiba K et al (2009) Activity-dependent tuning of inhibitory neurotransmission based on GABAAR diffusion dynamics. Neuron 62(5):670–682. doi:10.1016/j.neuron.2009.04.023

    Article  CAS  PubMed  Google Scholar 

  49. Santos MS, Park CK, Foss SM, Li H, Voglmaier SM (2013) Sorting of the vesicular GABA transporter to functional vesicle pools by an atypical dileucine-like motif. J Neurosci 33(26):10634–10646. doi:10.1523/JNEUROSCI.0329-13.2013

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  50. Baptista MS, Melo CV, Armelao M, Herrmann D, Pimentel DO, Leal G, Caldeira MV, Bahr BA et al (2010) Role of the proteasome in excitotoxicity-induced cleavage of glutamic acid decarboxylase in cultured hippocampal neurons. PLoS One 5(4), e10139. doi:10.1371/journal.pone.0010139

    Article  PubMed  PubMed Central  Google Scholar 

  51. Wei J, Wu JY (2008) Post-translational regulation of L-glutamic acid decarboxylase in the brain. Neurochem Res 33(8):1459–1465. doi:10.1007/s11064-008-9600-5

    Article  CAS  PubMed  Google Scholar 

  52. Gonzalez MI, Cruz Del Angel Y, Brooks-Kayal A (2013) Down-regulation of gephyrin and GABAA receptor subunits during epileptogenesis in the CA1 region of hippocampus. Epilepsia 54(4):616–624. doi:10.1111/epi.12063

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  53. Forstera B, Belaidi AA, Juttner R, Bernert C, Tsokos M, Lehmann TN, Horn P, Dehnicke C et al (2010) Irregular RNA splicing curtails postsynaptic gephyrin in the cornu ammonis of patients with epilepsy. Brain 133(Pt 12):3778–3794. doi:10.1093/brain/awq298

    Article  PubMed  Google Scholar 

  54. Fang M, Shen L, Yin H, Pan YM, Wang L, Chen D, Xi ZQ, Xiao Z et al (2011) Downregulation of gephyrin in temporal lobe epilepsy neurons in humans and a rat model. Synapse 65(10):1006–1014. doi:10.1002/syn.20928

    Article  CAS  PubMed  Google Scholar 

Download references

Acknowledgments

This work was supported by FCT, FEDER, and COMPETE (PEst-C/SAU/LA0001/2013-2014; PTDC/NEU-NMC/0198/2012). We thank Professor Günter Schwarz (Institute of Biochemistry, University of Cologne, Germany) for the kind gift of the EGFP-geph.FL and EGFP-geph.T plasmids. We are also thankful to Elisabete Carvalho for the assistance in the preparation of cultured hippocampal neurons.

Compliance with Ethical Standards

ᅟ

Conflict of Interest

The authors declare that they have no conflict of interest.

Welfare of Animals

Experiments were performed according to the European Union Directive 86/609/EEC and the legislation Portaria n. 1005/92, issued by the Portuguese Government for the protection of animals used for experimental and other scientific purposes. The procedures were approved by the CNC-FMUC Ethical Committee for Animal Research (ORBEA 19–201325022013) and by DGAV (Reference: 421/2013).

Author information

Author notes

    Authors and Affiliations

    1. CNC-Center for Neuroscience and Cell Biology, University of Coimbra, 3004-504, Coimbra, Portugal

      João T. Costa, Miranda Mele, Márcio S. Baptista, João R. Gomes, Rui J. Nobre, Luís Pereira de Almeida & Carlos B. Duarte

    2. Institute for Interdisciplinary Research, University of Coimbra (IIIUC), Coimbra, 3030-789, Portugal

      Miranda Mele & Rui J. Nobre

    3. Laboratory for Experimental Brain Research, Division of Neurosurgery, Department of Clinical Sciences, Lund University, BMC A13, S-22184, Lund, Sweden

      Karsten Ruscher & Tadeusz Wieloch

    4. Faculty of Pharmacy, University of Coimbra, Coimbra, Portugal

      Luís Pereira de Almeida

    5. Department of Life Sciences, University of Coimbra, 3004-517, Coimbra, Portugal

      Carlos B. Duarte

    6. Center for Neuroscience and Cell Biology, Faculty of Medicine, Polo I, Rua Larga, University of Coimbra, 3004-504, Coimbra, Portugal

      Carlos B. Duarte

    Authors
    1. João T. Costa
      View author publications

      You can also search for this author in PubMed Google Scholar

    2. Miranda Mele
      View author publications

      You can also search for this author in PubMed Google Scholar

    3. Márcio S. Baptista
      View author publications

      You can also search for this author in PubMed Google Scholar

    4. João R. Gomes
      View author publications

      You can also search for this author in PubMed Google Scholar

    5. Karsten Ruscher
      View author publications

      You can also search for this author in PubMed Google Scholar

    6. Rui J. Nobre
      View author publications

      You can also search for this author in PubMed Google Scholar

    7. Luís Pereira de Almeida
      View author publications

      You can also search for this author in PubMed Google Scholar

    8. Tadeusz Wieloch
      View author publications

      You can also search for this author in PubMed Google Scholar

    9. Carlos B. Duarte
      View author publications

      You can also search for this author in PubMed Google Scholar

    Corresponding author

    Correspondence to Carlos B. Duarte.

    Additional information

    João T. Costa and Miranda Mele contributed equally to this work.

    Rights and permissions

    Reprints and permissions

    About this article

    Check for updates. Verify currency and authenticity via CrossMark

    Cite this article

    Costa, J.T., Mele, M., Baptista, M.S. et al. Gephyrin Cleavage in In Vitro Brain Ischemia Decreases GABAA Receptor Clustering and Contributes to Neuronal Death. Mol Neurobiol 53, 3513–3527 (2016). https://doi.org/10.1007/s12035-015-9283-2

    Download citation

    • Received:

    • Accepted:

    • Published:

    • Issue Date:

    • DOI: https://doi.org/10.1007/s12035-015-9283-2

    Keywords

    Search

    Navigation