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The Role of Wnt Signalling in Excitatory Hippocampal Synapse Formation and Function

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The Role of Wnt Signalling in Excitatory Hippocampal Synapse Formation and Function The role of Wnt signalling in excitatory hippocampal synapse formation and function Derek Anane UCL PhD Thesis Supervisors: Prof. Patricia C. Salinas Dr. Alasdair Gibb 1 I, Derek Anane confirm that the work presented in this thesis is my own. Where information has been derived from other sources, I confirm that this has been indicated in the thesis. 2 Abstract The formation of a functional neural network is dependent on the correct assembly of cell-cell contacts. Apposition of pre and post synaptic terminals is a highly regulated process culminating in the formation of the functional junction points called synapses. Whilst much is understood of the processes involved in bringing axon and target together less is understood of the mechanisms controlling synapse assembly and maintenance. Wnts are highly glycosylated secretary proteins which have been demonstrated to be involved at several stages of the developing nervous system. Through a range of signalling pathways Wnts are able to produce cellular effects including embryonic patterning, fate and movement. Much recent research has been focused on the role of Wnts in synapse formation and function. In this thesis I present data from hippocampal cultures showing Wnt7a regulation of excitatory synapses. Exposure of developing hippocampal neurons to Wnt7a results in an increase in the density of surface GluA1, GluA2 and GluN1 puncta on dendritic spines. Wnt7a also regulates the co-localisation of postsynaptic glutamate receptor puncta with presynaptic sites labelled with vesicular glutamate transporter protein (vGlut). Interestingly the Wnt7a mediated increase in excitatory synapse formation is no longer present on neurons from mature cultures. At the main postsynaptic site of excitatory synaptic transmission I identified Wnt7a and Dvl mediated maturation of glutamatergic receptor localisation. Both exogenous Wnt7a and overexpression of Dvl in 14DIV hippocampal cultures caused an increase in the size and number of receptor puncta located on spines and the proportion of spines containing GluA1, GluA2 and GluN1 puncta. In my thesis I also present in vivo functional data, using animals null for both Wnt7a and Dvl expression. These animals demonstrate defects in evoked post synaptic currents and paired pulse ratio at the CA3-CA1 hippocampal synapse. In conclusion this report demonstrates the crucial role of Wnt7a – Dvl signalling in the regulation of excitatory synapse formation in the hippocampus. Furthermore amongst the myriad ways in which Wnt7a-Dvl signalling affects development of the CNS, Wnt7a acts directly at postsynaptic sites to increase synaptic strength. 3 Thesis Contents Title Page…………………………………………………………………………………...…1 Abstract…………………………………………………………………………………...…..3 Thesis Contents……………………………………………………………………………….4 List of Abbreviations………………………………………………………………................8 Chapter 1: Introduction…………………………………………………………………….11 1.1 Synapses: Presynaptic terminus……………………………………………………….13 1.2 Synapses: Postsynaptic terminal……………………………………………………….17 1.2.1 GABAergic postsynaptic specialisation……………………………………………17 1.2.2 Excitatory postsynaptic specialisation: Dendritic spines…………………………21 1.2.3 Metabotropic glutamate receptors…………………………………………………26 1.2.4 Kainate receptors…………………………………………………………………...26 1.2.5 AMPA receptors…………………………………………………………………….27 1.2.6 NMDA receptors…………………………………………………………………….29 1.2.7 Developmental regulation of glutamate receptor expression…………………….31 1.3 Synaptogenesis…………………………………………………………………………..35 1.3.1 Axon extension and target recognition…………………………………………….35 1.3.2 Synapse maturation…………………………………………………………………36 1.4 Synaptogenic Molecules………………………………………………………………...37 1.4.1 Trans-synaptic molecules…………………………………………………………..38 1.4.2 Secreted molecules…………………………………………………………………..40 1.5 Wnts……………………………………………………………………………………...43 1.5.1 Wnts: expression and secretion…………………………………………………….43 1.5.2 Wnt signalling pathways……………………………………………………………45 4 1.6 Wnts and Synapse formation…………………………………………………………49 1.6.1 Dendritic and axonal development………………………………………………...50 1.6.2 Wnts as synaptic organisers………………………………………………………51 1.6.3 Wnts at stable synapses……………………………………………………………..53 1.7 Project aims…………………………………………………………………………… 56 Chapter 2: Materials and methods………………………………………………………...57 2.1 Hippocampal Cell Culture……………………………………………………………...57 2.2 Hippocampal cell calcium phosphate transfection……………………………………57 2.3 Immunofluorescence……………………………………………………………………58 2.4 Image acquisition and analysis of synaptic puncta…………………………………...58 2.5 Dendritic spine-puncta colocalisation analysis………………………………………..59 2.6 Mouse breeding and genotyping……………………………………………………….59 2.7 Acute slice preparation…………………………………………………………………60 2.8 Recording of postsynaptic currents……………………………………………………60 2.9 Analysis of miniature currents…………………………………………………………61 2.10 Analysis of paired currents……………………………………………………………62 2.11 Statistical analysis……………………………………………………………………..63 2.12 Solutions………………………………………………………………………………..63 2.13 Antibodies……………………………………………………………………………...67 Chapter 3: Wnt7a signalling promotes the formation of functional glutamatergic synapses in the Hippocampus………………………………………………………………69 3.1 Introduction……………………………………………………………………………..69 5 3.2 Results…...………………………………………………………………………………72 3.2.1 Wnt7a promotes the formation of excitatory pre and postsynaptic sites………72 3.2.2 The synaptogenic effect of Wnt7a is related to neuronal maturity…77 3.2.3 Wnt7a signalling affects the formation of functional synapses in both mixed hippocampal cultures and at the MF-CA3 synapse………………………………………83 3.3 Discussion………………………………………………………………………………..86 3.3.1 Wnt7a regulates the formation of synapses both pre- and post-synaptically…86 3.3.2 Exposure of mature neurons to exogenous Wnt7a does not affect the localisation of pre- or post-synaptic proteins……………………………………………………88 3.3.3 Exposure of neuronal cultures to Wnt7a increases the amplitude and frequency of excitatory miniature synaptic currents without affecting inhibitory currents. ……..90 Chapter 4: Wnt signalling regulates the localisation of glutamatergic receptors on dendritic spines by acting directly at the postsynaptic terminal………………………92 4.1 Introduction……………………………………………………………………………92 4.2 Results…………………………………………………………………………………..94 4.2.1 Exogenous Wnt7a increases accumulation of glutamate receptors and presynaptic inputs onto the dendritic spines of developing neurons…………………….94 4.2.2 Exogenous Wnt7a does not affect accumulation of synaptic proteins onto the dendritic spines of mature neurons……………………………………………100 4.2.3 Postsynaptic Wnt activation increases accumulation of glutamate receptors and presynaptic inputs onto the dendritic spines of developing neurons………….106 4.2.4 Postsynaptic Wnt activation does not affect accumulation of synaptic proteins onto the dendritic spines of mature neurons…………………………………………….114 4.3 Discussion…………………………………………………………………………120 4.3.1 The role of Wnt7a signalling on NMDAR to synaptic sites………..120 4.3.2 Wnt7a signalling regulates increases in spine maturity through glutamate receptor complementation………………………………………………………………121 4.3.3 Postsynaptic expression of Wnt signalling activates effects both post and presynaptically……………………………………………………………………………..122 6 Chapter 5: Wnt 7a regulates the number and strength of functional synaptic sites…124 5.1 Introduction……………………………………………………………………………124 5.2 Results………………………………………………………………………………….126 5.2.1 Wnt7a gain of function enhances evoked postsynaptic current amplitude and decreases synaptic failure……………………………………...………………………….126 5.2.2 Wnt7a/Dvl1 loss of function at the MF-CA3 synapse decreases evoked postsynaptic current amplitude and increases the frequency of synaptic failures……………………………………………………………………………………...127 5.2.3 Wnt7a gain of function decreases paired pulse ratios in hippocampal cultures……………………………………………………………………………………..130 5.2.4 Wnt7a/Dvl1 loss of function at MF-CA3 synapses results in a decreased Paired pulse ratio…………………………………………………………………………………..132 5.3 Discussion………………………………………………………………………………134 Chapter 6: Discussion……………………………………………………………………..137 6.1 Summary of Results…………………………………………………………………...137 6.2 The role of Wnt7a signalling in pre and postsynaptic excitatory synapse formation… ………………………………………………………………………………………………139 6.3 Wnt7a signalling strongly regulates synaptic transmission in the CA3 region of the hippocampus and the implications for memory and learning………………………….141 6.4 The role of Wnt7a signalling in neurodegenerative and neurological disorders …144 Acknowledgements………………………………………………………………………...147 References………………………………………………………………………………….148 7 List of Abbreviations AChR – Acetyl choline receptor AMPA - α-amino-3-hydroxyl-5-methyl-4-isoxazole-propionic acid AMPA-R – AMPA receptor BDNF – Brain-derived neurotrophic factor BSA – Bovine serum albumin CaMKII – Ca 2+ /calmodulin-dependent kinase 2 CaMKK-– Ca 2+ /calmodulin-dependent kinase kinase CK1 α - Casein kinase 1 α CNS – Central nervous system CRD – Cysteine-rich domain Daam1- Dishevelled associated activator of morphogenesis 1 DFz2 – Drosophila Frizzled 2 DIV – Days in vitro Dkk1 – Dikkopf-1 Dvl1 – Dishevelled-1 EPSC – Excitatory postsynaptic current ER – Endoplasmic reticulum Evi- Eveness interrupted FGF – Fibroblast growth factor FGF-R – FGF receptor Fz – Frizzled 8 GABA - γ-aminobutyric acid GC – Granule cell dGRIP- Drosophila glutamate receptor interacting protein GSK3β – Glycogen synthase kinase 3β GTPase- Guanosine Triphosphate hydroxase HBSS – Hank’s balanced salt solution HSPG – Heperan sulfate proteoglycan
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