The gluN2B subunit of NMDARs and its role in glutamatergic synapses : towards the characterization of the proteome of postsynaptic densities from wild-type and GluN2B-null cultured neurons

摘要

N-methyl-D-aspartate receptors (NMDARs) are unique regulators ofglutamatergic synaptic transmission in the central nervous system (CNS). Theseionotropic glutamate receptors play a crucial role in shaping the strength of synapticconnections, which underlies mechanisms of learning and memory formation. TheGluN2B subunit of NMDARs is crucial for the organization of postsynapticmacromolecular complexes involved in glutamatergic transmission, and previous resultsfrom our group have shown that in the absence of GluN2B there is dramatic reductionof synaptic NMDARs in hippocampal neurons, despite the presence of other GluN2subunits. To determine the composition of the molecular complexes organized byGluN2B we proposed to evaluate biochemically the postsynaptic densities (PSDs)isolated from cultured cortical neurons of wild-type and GluN2B (-/-) mice, since thePSDs are organized postsynaptic structures specialized for postsynaptic signalling andplasticity, and is at PSDs that receptors, associated signaling molecules andcytoskeleton elements are assembled through scaffold proteins.The study of the PSD is a valuable approach to understand which moleculardeterminants govern glutamatergic synaptic transmission. PSDs can be purifiedbiochemically from brain tissue through several centrifugation steps, and are thereforeamenable to characterization. However, since GluN2B (-/-) mice are not viable, in thepresent work we developed and validated a protocol for the purification of PSDs fromcultured cortical neurons isolated from GluN2B (-/-) embryos and wild-type littermates,and maintained in culture for 15 days, to guarantee synapse maturation. This procedureallows isolating PSDs from 38M cultured neurons and yields, in average, the amount ofprotein required for biochemical assays (10 to 20μg), namely mass spectrometryanalysis. The isolated fractions were fully characterized biochemically, and acomparison between the crude homogenate, synaptosomal and PSD fractions suggeststhat our method is reliable and efficient in yielding final pure fractions.We found that PSDs isolated from GluN2B (-/-) cultured cortical neurons exhibita reduction of approximately 50% in protein content, when compared to PSDs isolatedfrom wild-type cultured cortical neurons, suggesting that GluN2B indeed is anindispensable element for the maintenance of postsynaptic macromolecular complexes.Quantitative analysis of the protein content of the isolated PSDs, by iTRAQ-labeling inconjunction with tandem mass liquid chromatography and mass spectrometry, isunderway, in collaboration with Dr. Ka Wan Li neuroproteomics group. Thecharacterization of the full proteome of GluN2B (-/-) cultured cortical neuron PSDs willprovide new insights regarding the role of GluN2B at the synapse.