Studies on the plasticity of glutamate receptors and its role for neuronal calcium homeostasis

Abstract: Calcium is the most important and versatile signaling molecule in the brain. Neurons are equipped with a variety of tools to utilize this simple ion in diverse ways. From controlling the millisecond fast neurotransmitter release, to reshaping neuronal structure and function. A brief pulse of glutamate, released from a presynaptic neuron, activates the NMDA receptor and mGluR5 which triggers an increase in the cytosolic calcium concentration in the postsynaptic neuron. The NMDA receptor and mGluR5 are important mediators of synaptic plasticity - the ability of synapses to change their strength in response to changed patterns of neuronal activity. Because of the fundamental role calcium signaling has for numerous processes in the brain it is also highly sensitive to disturbances. A sustained high level of calcium can even cause neuronal death. We have in this work studied the plasticity of neuronal glutamate receptors and how novel accessory proteins can modulate their calcium mobilization. In study I, we identified Norbin as a novel interacting partner to mGluR5. Norbin facilitated the cell surface expression of mGluR5 and positively modulated mGluR5 mediated calcium signaling. Studies in Norbin transgenic animals confirmed and demonstrated the relevance of this interaction. Mice lacking Norbin displayed decreased cell surface expression of mGluR5, impaired synaptic plasticity, and a behavioral phenotype that corresponds to hypofunctional mGluR5 activity. In study II, using super-resolution imaging, we investigated the spatial relationship between Norbin, postsynaptic density protein 95 (PSD-95), mGluR5, and actin in dendritic spines. We found Norbin to have a high degree of colocalization with actin and a much lower degree with PSD-95. We also found that Norbin, as expected, have a high degree of colocalization with mGluR5 and the mGluR5-interacting protein Homer. Co-immunoprecipitation experiments confirmed our imaging results. In study III, we identified p11 as another novel partner to mGluR5. Similar to Norbin, p11 promoted mGluR5 plasma membrane expression and modulated mGluR5 cell signaling. The known antidepressant effect of the mGluR5 specific antagonist MPEP was abolished in p11 knockout mice with a specific deletion of p11 in parvalbumin positive GABAergic interneurons. In study IV, we investigated the spatial and functional relationship between the NMDA receptor and Na+,K+-ATPase in cultured hippocampal neurons. We found that sub-saturating concentrations of ouabain, a Na+,K+-ATPase ligand, attenuated NMDA receptor mediated calcium influx. Experiments performed in HEK293 cells indicated that the effects of ouabain was mediated by the neuronspecific Na+,K+-ATPase α3 isoform. Using single-molecule super-resolution microscopy dSTORM, we found a close spatial proximity between the NMDA receptor and the Na+,K+-ATPase in postsynaptic membranes.