Synaptic and extrasynaptic NMDA receptors in hippocampal neurons [Elektronische Ressource] : regulation of nuclear shape and cell fate / presented by Malte Wittmann

Dissertation

submitted to the
Combined Faculties for the Natural Sciences and for Mathematics
of the Ruperto-Carola University of Heidelberg, Germany
for the degree of
Doctor of Natural Sciences





















presented by


Diplom-Biologe Malte Wittmann

born in: Braunschweig

Oral examination: ................................................










Title


Synaptic and extrasynaptic NMDA receptors in
hippocampal neurons: regulation of nuclear shape
and cell fate

























Referees: Prof. Dr. Hilmar Bading

Prof. Dr. Christoph Schuster Acknowledgements



I would like to thank my referee Professor Hilmar Bading for the opportunity to prepare my
PhD thesis in his laboratory. I am very grateful for the many ideas and discussions we had and
his supervision throughout the projects.

I would further like to thank my second referee Professor Christoph Schuster for his good
advice and the discussions we had.

Many thanks to Gillian Queisser for his wonderful 3D reconstructions and mathematical
calculations.

Many thanks to Andrea Helwig for the sharp electron microscopic images.

Further I would like to thank C. Peter Bengtson and Simon Wiegert for carrying out the
electrophysiological recordings as well as Frank Hofmann for recordings on Multi Electrode
Arrays.

Many thanks to Ruth Jelinek for general support in the lab and assistance during biochemical
experiments.

I would also like to thank Matthias Klugmann for the brain sections as well as the lively
discussions and technical advice.

Thanks to all other members of the Bading laboratory for close interactions, support and the
friendly atmosphere created.


Special thanks to the Boehringer Ingelheim Fonds (B.I.F.) for the generous financial support
granted for the thesis Table of contents 1
1. Summary ...............................................................................................4
2. Zusammenfassung................................................................................5
3. Introduction ..........................................................................................7
3.1. Nuclear lamina..................................................................................................7
3.1.1. Lamins and apoptosis...............................................................................8
3.1.2. Nuclear morphology.................................................................................8
3.1.3. Lamins in genetic deseases....................................................................... 9
3.2. The Involvement of NMDA Receptors in Neuron Death .............................. 10
3.2.1. NMDA receptor overview...................................................................... 11
3.2.2. The Prevalence of NR2B Subunits in Extrasynaptic NMDA Receptors 12
3.2.3. NR2B-containing Receptors and Neuronal Death.................................. 13
3.2.4. Extrasynaptic NMDA receptor activation leads to death ....................... 14
3.2.5. Signaling cascades regulating survival and death .................................. 14
3.2.6. CREB: A Calcium Regulated Transcription Factor ............................... 15
3.2.7. MAP kinases and cell death.................................................................... 17
3.2.8. The “Source Specificity” vs “Calcium Load” Hypotheses .................... 17
3.2.9. Mitochondria..........................................................................................18
3.2.10. PSD-95 and the Coupling of NMDA Receptors to Mitochondria
and nNOS Production............................................................................. 19
3.2.11. Conantokins............................................................................................21

4. Results..................................................................................................22
4.1. Nuclear infoldings..........................................................................................22
4.1.1. Localizations by fluorescence microscopy.............................................
4.1.2. Electron microscopy...............................................................................23
4.1.3. 3D reconstructions of nuclear infoldings................................................ 26
4.1.4. Destabilization of nuclear infoldings by activation of
extrasynaptic NMDA receptors.............................................................. 30
4.1.5. Induction of nuclear infoldings by stimulation of synaptic
NMDA receptors .................................................................................... 33
4.1.6. Live imaging of the induction of infoldings........................................... 34
4.1.7. Intracellular signaling cascades involved in the generation of
nuclear infoldings ................................................................................... 35
4.1.8. Decay of nuclear infoldings.................................................................... 40
4.1.9. Functional assays: bicuculline-induced calcium spikes ......................... 42
4.1.10. Functional assays – Patch clamp recordings .......................................... 44
4.1.11. Functional assays – the immediate early gene cfos................................ 45
4.1.12. Nuclear pore complexes ......................................................................... 46
4.2. Excitotoxicity.................................................................................................48
4.2.1. Stimulation of extrasynaptic NMDA receptors induces necrotic
cell death 48
4.2.2. Stimulation of synaptic NMDA receptors enhances cell survival.......... 51
4.2.3. Extrasynaptic NMDA receptors and MAP kinases ................................ 54
4.2.4. CREB shut-off........................................................................................55
4.2.5. Specific blockade of NR2B reduces excitotoxicity 58
4.2.6. Patch clamp analysis of conG effects..................................................... 61
4.2.7. Development of a NR2B antagonist specific for extrasynaptic
NMDA receptors .................................................................................... 62 Table of contents 2
4.2.7.1. Modification of conG ..................................................................... 62
4.2.7.2. Conantokin-G protects against oxygen-glucose deprivation.......... 65
4.2.7.3. Colocalization of conG-BSA and synaptic markers....................... 66
4.2.7.4. Multi Electrode Array (MEA) recordings ...................................... 67
4.2.7.5. Coupling of conG-BSA to gold beads............................................ 71
4.2.7.6. Patch-clamp analysis of the effects of conG-gold beads................ 74
4.2.7.7. Coupling of conG to latex beads .................................................... 74
4.2.7.8. Coupling of conG to beads via a myc tag....................................... 75
4.2.7.9. Coupling of conG-biotin to streptavidin beads............................... 78
4.2.7.10. Patch-clamping of conG-biotin coupled to beads .......................... 83

5. Discussion ............................................................................................84
5.1. Nuclear shape.................................................................................................84
5.1.1. Nuclear infoldings..................................................................................
5.1.2. Destabilization of nuclear infoldings...................................................... 86
5.1.3. Synaptic NMDA receptors induce infoldings in nuclear membranes .... 86
5.1.4. Signaling cascades regulating infolding of nuclear membranes ............ 87
5.1.5. Stability of nuclear infoldings ................................................................ 88
5.1.6. Differences in synaptic activity in infolded nuclei................................. 89
5.1.7. Enhanced upregulation of immediate early genes.................................. 90
5.1.8. The density of nuclear pore complexes stays constant........................... 90
5.2. Cell fate..........................................................................................................92
5.2.1. Extrasynaptic NMDA receptors mediate excitotoxicity.........................
5.2.2. tediate shut-off pathways.................. 93
5.2.3. Potency of Conantokin-G ....................................................................... 94
5.2.4. Modifications of Conantokin-G ............................................................. 95
5.2.5. Coupling of conG to beads ....................................................