Subcellular segregation of kainate receptors and temporal lobe epilepsy

Transcription

1 Subcellular segregation of kainate receptors and temporal lobe epilepsy Christophe Mulle Interdisciplinary Institute for Neuroscience CNRS University of Bordeaux

2 Kainate receptors: a family of ionotropic glutamate receptors Glutamate receptor ion channels Five genes GluK1 to GluK5 Heterotetrameric receptors CA3 DG from Contractor, Mulle, Swanson, TINS 2011

3 Kainate receptors in the hippocampus Carta et al, EJN 2014

4 TLE: recurrent synapses in DG operate via aberrant KARs Epilepsy 100 µm oml PP AMPA-R Epilepsy iml gl rmf rmf KA-R G rmf MF CA3 h Valérie Crépel, INSERM, Marseille EPSP KA Epsztein et al., 2005, 2010 Artinian et al., 2011 Pinheiro et al., 2013

5 Recurrent Mf synapses on DG cells operate via KARs in TLE: implication for epilepsy? Control AMPA-R EPSP AMPA Role of aberrant KARs in Chronic Epilepsy Epileptic KA-R? EPSP KA

6 Reduced Number of Ictal Discharges in GluK2 KO Mice and in Presence of KAR Blockers

7 Aberrant postsynaptic KARs essential contributor to temporal lobe epilepsy Chronic models of temporal lobe epilepsy Peret et al - Cell Reports 2014

8 Subcellular segregation of KARs in control conditions and TLE

9 Mechanism for (recruitment and) stabilization of synaptic KARs: role of N-Cadherins CTD Δ 14 destabilization of KARs at MF-CA3 synapse Alteration of N-cadherins reduction of KAR-EPSCs 14 last aa interaction N-Cadherin/βcatenin complexes N-Cadherin/β-catenin complexes play a crucial role in stabilization of KARs Fièvre, Carta et al Nature Communications, 2016

10 Mechanisms for recruitment of aberrant postsynaptic KARs? Cadherin 9 labeling

11 What next? a translational approach Fact: >30% of TLE patients are intractable to pharmacological treatments they undergo surgical removal of the temporal lobe. Reduce the expression or activity of pathological KARs at ectopic synapses: a gene therapy approach, develop micrornas to invalidate KARs under the control of a DG specific promoter. develop GluK2/GluK5 antagonists prevent or disrupt the stabilization of ectopic KARs an interacting peptide approach. Disrupt Cadherin/KAR interactions (FFRE/FRC project). Collaboration with Valérie Crépel, INSERM Marseille, Support from AST Aquitaine and INSERM transfer

12 What next? a translational approach Tests on resected hippocampus from patients with TLE organotypic cultures collab Valérie Crépel, NSERM Fabrice Bartholoméi, Didier Scavarda, Service de Neurophysiologie Clinique - La Timone, APHM Eugène et al, J. Neurosci Meth, 2014

13 Funding ANR, FRC, FFRE Mario Carta Adam Gorlewicz Sabine Fièvre Collaboration Valérie Crépel, INSERM Marseille

14 Subcellular segregation of KARs at Mf-CA3 synapses GluK2 GluK2 -/- GluK5 -/- GluK5 st radiatum st lucidum st pyramidale Ruiz et al, J Neurosci 2005

15 Mechanism for recruitment and stabilization of synaptic KARs: Cadherin 9 could explain synapse specifity CA3 DG

16 CA3 synaptic circuits operation and plasticity role in memory Rebola, Carta and Mulle, Nature Rev Neuro, 2017

17 Functional mapping of GluRs: focal uncaging of glutamate CA3 pyramidal cell / GFP Distinct behaviour of AMPAR and KAR: strict subcellular segregation of KARs

18 CA3 pyramidal cells: Anatomy and structure 3D reconstruction MFB A/C PP 1µm 1µm 3D reconstruction 10µm 10µm CA3 MF 50µm myfp, injected in vivo at P2 and collected at P24. Confocal microscopy, 100x. thorny excrescences

19 Mossy fiber synapses: short term plasticity dynamics frequency facilitation paired pulse facilitation MPFA: quantal analysis Lanore et al, J Neuroscience 2012

20 Mf-CA3 short term plasticity and spike transfer -80 mv Feedforward inhibition 0 mv -30 mv -60 mv current-clamp minimal stimulation, 20Hz Sachidhanandam et al, J Neurosci 2009 Torborg et al, J Neuroscience 2011

21 Properties of CA3-CA3 (A/C) synapses paired-pulse facilitation PP A/C long-term potentiation associative pairing Mf Silva et al, Nature Communications 2016

22 Mossy fiber synapses: short term plasticity and spike transfer current-clamp recording Mf-EPSPs 20 Hz burst stimulation 20 Hz 0 mv -30 mv -60 mv GluK2 -/- wild-type GluK2 -/- spikes s Sachidhanadam et al, J Neuroscience s

23 Kainate receptors in the hippocampus Carta et al, EJN 2014

24 Postsynaptic KARs at Mf-CA3 synapses mv CA1 Mf-EPSP -62 wt vs GluK2 -/- Sch. -64 CA3 fm pp DG wt -66 vs GluK3-/ s Sachidhanandam et al J Neurosci 2009

25 Postsynaptic KARs at Mf-CA3 synapses and synaptic integration KAR-EPSCs UPB 310 Mf-EPSP (AMPAR+ KAR) GYKI µM Postsynaptic KARs shape the EPSP waveform important for excitability Pinheiro et al, Cerebral Cortex 2013

26 Recombinant KARs: GluK2 vs GluK2/GluK5 GluK2 GluK2/GluK5 UBP 310 an antagonist with selectivity GluK2/GluK5 but not GluK2 Barberis et al, J. Neuroscience 2008 Pinheiro et al, Cer Cortex, 2013

27 Functional diversity of excitatory synapses Cellular mechanisms and functional consequences Glu Glu Structure/spine architecture Position along dendrite Glu Receptor equipment Presynaptic properties Types and extent of synaptic plasticity

28 Single-unit recordings from CA3 cells in freely moving mice wild-type GluK2 -/- collab Y. Cho

29 KARs and Temporal Lobe Epilepsy (TLE) A chronic Disease % of Adult Epilepsy Pharmaco-resistant Associated with Comorbidity Distresses: - Social - Emotional - Attention - Memory - Language

30 An unexpected link between KARs and Alzheimer disease? (on-going work) Pei Zhang Gaël Barthet

31 Early synaptic deficits with chronic elevation of Aß in APP/PS1 mice Amyloid deposits in DG - CA3 Region Silvia Viana da Silva APP/PS1 transgenic mice: Overexpression of APP swedish mutation + PS1 with deletion of exon 9 accumulation of Ab peptides acceleration of plaque deposits Immunolabeling by Adam Gorlewicz

32 Properties of CA3-CA3 (A/C) synapses in APP/PS1 mice paired-pulse facilitation PP A/C long-term potentiation associative pairing Mf Silva et al, Nat Comm 2016

33 No plasticity impairment at hippocampal Mf-CA3 synapses APP/PS1 at 6 months paired pulse facilitation PP A/C CA3 Mf TRAIN: 3 x 10s apart

34 Decreased KAR-EPSCs at Mf-CA3 synapses in APP/PS1 mice Wt APP/PS1 *Barthet, *Zhang et al, unpublished

35 Proteolysis of N-Cadherin Loss or reduction of g-secretase cleavage leads to increase in N-Cadh CTF1 Barthet et al, FASEB J 201

36 Molecular determinants: role of N-Cadherins Overexpression of dominant negative form of N-Cadh

37 Reduced KAR-EPSCs in PS-KO CA3 pyramidal cells AAV-cre in CA3 Ps1fl/fl mice AMPAR-EPSCs control PS KO KAR-EPSCs *** PS is involved in subcellular targeting of KARs Possible involvement of N-Cdh cleavage by g-secretase.

38 Synaptic plasticity in CA3 pyramidal cells moderate short-term plasticity NMDA dependent LTP of AMPA EPSCs Perf. path A/C Stratum lucidum Mossy fiber Robust short-term plasticity presynaptic LTP, not dependent on NMDARs LTP of NMDA EPSCs Metaplastic switch to prime for LTP of AMPA EPSCs CA3 Pyramidal cell

39 KARs and Temporal Lobe Epilepsy (TLE) A chronic Disease % of Adult Epilepsy Pharmaco-resistant Timm's staining control Associated with Comorbidity Distresses - Social - Emotional - Attention - Memory - Language rmf chronic epilepsy Recurrent mossy fibers in the dentate gyrus in TLE

40 Recurrent MF onto DG Cells Operate via Aberrant KARs Epilepsy 100 µm oml PP AMPA-R Epilepsy iml gl rmf rmf KA-R G rmf MF CA3 h work of Valérie Crépel, INSERM, Marseille EPSP KA Epsztein et al., 2005, 2010 Artinian et al., 2011 Pinheiro et al., 2013

41 Organotypic hippocampal slices retain synaptic segregation of KARs P5-7 DIV 7-14 culture on insert CA3 CA1 DG Mf stimulation A/C stimulation WT GluK2 -/- WT LY LY LY Ctrl 60 pa 50 ms Ctrl 100 pa 50 ms Ctrl 100 pa 50 ms

42 Aberrant postsynaptic KARs are essential contributor to temporal lobe epilepsy Chronic models of temporal lobe epilepsy Mechanisms allowing recruitment of postsynaptic KARs should be better understood Peret et al - Cell Reports 2014

43 Quantitative reexpression of GluK2: kinetics Normalized traces

44 Subcellular distribution of SEP GluK2a in CA3 PCs Live staining for the SEP moiety using anti-gfp nanobody-atto647 (red) coll Ingrid Chamma, Olivier Thoumine

45 Molecular determinants: role of N-Cadherins Overexpression of dominant negative form of N-Cadh

46 High affinity Kainate binding in the human hippocampus control Temporal lobe epilepsy The autoradiography carried out with [ 3 H]kainate High Low Control Epileptic Represa et al Brines et al

47 In vitro model of Temporal Lobe Epilepsy: Hippocampal Organotypic Slices organotypic slice culture treated with pilocarpine Synaptoporin staining EPSC KA EPSC AMPA Mossy fiber sprouting

48 Inter-ictal spikes in vivo in mouse model of TLE In vivo recording from pilocarpine-treated mice Local Field Potential

49 The slow kinetics of EPSCs are Important Determinants of Epileptiform Activity

50 Quantitative reexpression of GluK2: focal Glu uncaging GluK2 -/- slices, CA3 Pyramidal cells Single cell electroporation GluK2a + GFP

51 Quantitative reexpression of GluK2: kinetics Normalized traces WT GluK2 -/- Aberrant/ectopic KARs upon overexpression of GluK2 are homomeric GluK2 KARs

52 Synaptic KARs with GluK2a re-expression

53 Part I - Interim conclusions Distinct behaviour of AMPARs and KARs: strict subcellular segregation of KARs no extrasynaptic receptors Single cell electroporation necessary for quantitative replacement approach for study of receptor trafficking Synaptic KARs are heteromers containing GluK2 and GluK5 ectopic KARs following overexpression are likely homomers of GluK2. Synaptic KARs are readily rescued in amplitude and decay following GluK2 reexpression at Mf- but not A/C synapses. Data suggest limited amount of synaptic slots for KARs limited amount of GluK5 for heteromer formation strong transcriptional or post-transcriptional regulation of GluK2 What are molecular determinants of this subcellular segregation? Fièvre, Carta et al Nat Communications, 2016

54 Molecular determinants: Rexpression of mutated GluK2a HEK 293 cells Jaskolski et al J Neurosci 2004

55 Specific role of N-Cadherins for KARs: focal Glu uncaging focal Glu uncaging

56

57 Mechanism for recruitment and stabilization of synaptic KARs Transssynaptic Recruitment by C1ql2/3 Stabilization by Cadherins limited number of slots Regulated amount of GluK2 protein sumoylation, park2 interaction

58 Sprouting of recurrent mossy fibers in dentate gyrus in TLE Non epileptic Epileptic Rodents Human Timm/CV Golgi/CaBP rmf rmf rmf Tauck and Nadler, 1985 Represa et al., 1987 Hester and Danzer, 2013 Gabriel et al. 2004

59 Epileptiform discharges are strongly reduced in GluK2 -/- mice and in presence of KAR antagonists organotypic slice culture

60 Animal Model of Temporal Lobe Epilepsy Status epilepticus Latent period (several weeks) Chronic phase 1 w 2 w 3 w PILOCARPINE (ip)

61 Molecular determinants: Rexpression of mutated GluK2a HEK 293 cells Jaskolski et al J Neurosci 2004 KAR-EPSCs Mf stimulation

62 Molecular determinants: role of N-Cadherins Targeted expression of Cre in N-Cadh floxed mice single cell electroporation in organotypic slices