VEGF modulates NMDA receptor activity in cerebellar granule cells prior to synapse formation

Although blood vessels and nerves are functionally different, anatomical parallels between both systems can be clearly identified, as illustrated by Andreas Vesalius centuries ago. Both systems display comparable patterning in ramifying and hierarchically ordered networks interconnecting nearly all domains of the body. More recently, several molecules were identified to affect both neural and vascular functions, and they are therefore termed angioneurins. The phenomenon of common principles in the wiring of both systems is denoted as the neurovascular link. The vascular endothelial growth factor (VEGF) is a striking example of this cross-talk between nerves and vessels. Indeed, VEGF is a key factor in blood vessel development and has also important, yet incompletely characterized roles in neuronal wiring. Interestingly, VEGF is expressed in the cerebellum, when granule neurons are still migrating and nerve connections are being formed.NMDA type glutamate receptors (NMDARs) are best known for their role in synaptogenesis and synaptic plasticity. Much less is known about their developmental role before neurons form synapses. Prompted by findings that VEGF and NMDARs regulate similar neurological processes, we hypothesized that VEGF receptor-2 (Flk1) activation might regulate NMDAR function. Because both NMDARs (NR1 and NR2B subunits) and Flk1 are expressed by migrating granule cells (GCs) and regulate GC migration, this study aimed to explore a possible link between VEGF and NMDARs in GCs before synaptogenesis, when there are no excitatory glutamatergic synapticinputs on GCs.We identified VEGF as a modulator of NMDAR function before synapse formation and documented a unique link between a receptor for a classic neurotransmitter (NMDAR) and a receptor for an angiogenic factor and neuronal guidance cue (Flk1), both of which are needed for proper GC migration in the developing cerebellum. A combination of electrophysiological, biochemical, calcium (Ca2+) imaging and other cell-biological studies in organotypic slices, primary GCs and cells in culture revealed that VEGF enhances NMDAR-mediated currents and Ca2+ influx in immature GCs before synapse formation. The VEGF receptor Flk1 forms a complex with the NMDAR subunits NR1 and NR2B. In response to VEGF, the number of Flk1/NR2B co-clusters on the cell surface increases. Stimulation of Flk1 by VEGF activates Src-family kinases, which increases tyrosine phosphorylation of NR2B. Inhibition of Src-family kinases abolishes the VEGF-dependent NR2B phosphorylation and amplification of NMDAR-mediated currents and Ca2+ influx in GCs.In conclusion, this study identifies for the first time VEGF as a modulator of NMDARs before synapse formation and highlights a link between an activity independent neurovascular guidance cue (VEGF) and an activity-regulated neurotransmitter receptor (NMDAR). While it is becoming increasingly evident that blood vessels co-opted axon guidance signals to navigate, there is still little evidence that molecules involved in angiogenesis also guide migrating neurons. Moreover, less is known about the mechanisms via which these molecules act. The results obtained during this thesis shed new light on how an activity-independent signal (VEGF) regulates a receptor for a classic neurotransmitter and provide a unique insight into the molecular basis of brain wiring.General Introduction 1 Literature Overview 3 1 The molecular biology of VEGF and its receptors 3 1.1 The VEGF-family 3 1.1.1 VEGF: gene organization 3 1.1.2 Regulation of VEGF expression 4 1.1.3 VEGF protein sequence and structure 5 1.2 VEGF receptors 6 1.2.1 VEGFR tyrosine kinases 6 1.2.2 Neuropilin (co-)receptors 11 2 The neurovascular link: a new role for VEGF 13 2.1 The neurovascular link in health and disease 13 2.2 VEGF: a key angiogenic growth factor 14 2.3 VEGF functions in the nervous system 15 2.3.1 VEGF signaling converges vessel and neuronal wiring during development 16 2.3.2 Role of VEGF in the (adult) nervous system 19 2.3.3 VEGF: role in neurological diseases? 22 3 Development of the cerebellum 25 3.1 Cerebellar granule cell migration as model system of neuronal migration 25 3.2 Leaving the nest: GC precursor migration from the rhombic lip to the upper external GC layer (EGL) 27 3.3 GC precursor proliferation and differentiation in the EGL 29 3.4 Layer specific migration of GCs in the early postnatal cerebellum 31 3.4.1 The EGL 31 3.4.2 The EGL-ML interface; switching from tangential to radial migration 32 3.4.3 The ML 32 3.4.4 The PCL 35 3.4.5 The IGL 35 3.5 Intrinsic program of in vitro migrating GCs 35 3.6 Potential modulators and regulators of GC migration 36 3.7 The adult cerebellar circuitry 41 4 Calcium signaling in neuronal motility: the role of NMDARs 43 4.1 Calcium (Ca2+) homeostasis and signaling 43 4.2 Neuronal Ca2+ channels at the plasma membrane 44 4.2.1 Voltage dependent Ca2+ channels (VDCCs) 46 4.2.2 NMDARs: members of the ionotropic superfamily 47 4.3 Concluding remark 70 Rationale and Objectives 71 Experimental Procedures 73 5 Materials and Methods 73 5.1 Animals 73 5.2 Cell culture experiments 73 5.2.1 Ex vivo cerebellar electroporation 73 5.2.2 Culture and treatment of organotypic cerebellar slices 73 5.2.3 Cerebellar GC and glia purification and cultivation 74 5.2.4 Chemotaxis assay 75 5.2.5 Transfection of HEK-293 cells 75 5.2.6 Transfection of mouse embryonic fibroblasts (MEFs) 76 5.3 Electrophysiology and Ca2+ imaging 77 5.3.1 Electrophysiological recordings 77 5.3.2 Ca2+ imaging 78 5.4 Histology 79 5.5 Protein analysis 79 5.6 Microscopic analysis 81 5.6.1 Analysis of Flk1 and NR2B co-clusters 81 5.7 Statistical analysis 82 Results 83 6 VEGF regulates NMDAR activity in cerebellar GCs 83 6.1 Effect of VEGF on NMDAR-mediated Ca2+ influx and currents in GCs 83 6.1.1 VEGF enhances NMDAR-mediated Ca2+ influx in vitro 83 6.1.2 VEGF enhances NMDAR-mediated currents ex vivo 88 6.2 Interaction between Flk1 and NMDARs: role of Src-family-kinases (SFKs)? 91 6.2.1 Flk1 interacts with NMDARs 91 6.2.2 Flk1 activation by VEGF enhances NR2B phosphorylation via SFKs 94 6.2.3 VEGF enhances NMDAR-mediated Ca2+ influx and currents via SFKs 98 6.3 VEGF promotes GC migration via NMDAR activation 101 Discussion 105 7 VEGF modulates NMDAR activity in GCs through SFKs prior to synapse formation 105 7.1 Identification of a novel link between NMDAR and Flk1 in cerebellar GCs 105 7.2 VEGF promotes GC migration via NMDAR activation 107 7.3 Future perspectives 108 7.3.1 Is VEGF able to modulate NMDAR activity at the synapse? 108 7.3.2 VEGF/NMDAR link in health and disease 110 7.3.3 VEGF: modulator of other Ca2+ channels in cerebellar GCs as well? 111 7.4 Conclusion 113 References 115 Curriculum Vitae 139 Summary 145nrpages: 156status: publishe