Abstract
BackgroundAll studies aimed at understanding complex molecular changes occurring at synapses face the problem of how a complete view of the synaptic proteome and of its changes can be efficiently met. This is highly desirable when synaptic plasticity processes are analyzed since the structure and the biochemistry of neurons and synapses get completely reshaped. Because most molecular studies of synapses are nowadays mainly or at least in part based on protein extracts from neuronal cultures, this is not a feasible option: these simplified versions of the brain tissue on one hand provide an homogeneous pure population of neurons but on the other yield only tiny amounts of proteins, many orders of magnitude smaller than conventional brain tissue. As a way to overcome this limitation and to find a simple way to screen for protein changes at cultured synapses, we have produced and characterized two dimensional electrophoresis (2DE) maps of the synaptic proteome of CA3-CA1 hippocampal neurons in culture.ResultsTo obtain 2D maps, hippocampal cultures were mass produced and after synaptic maturation, proteins were extracted following subfractionation procedures and separated by 2D gel electrophoresis. Similar maps were obtained for the crude cytosol of cultured neurons and for synaptosomes purified from CA3-CA1 hippocampal tissue. To efficiently compare these different maps some clearly identifiable reference points were molecularly identified by mass spectrometry and immunolabeling methods. This information was used to run a differential analysis and establish homologies and dissimilarities in these 2D protein profiles.ConclusionBecause reproducible fingerprints of cultured synapses were clearly obtained, we believe that our mapping effort could represent a simple tool to screen for protein expression and/or protein localization changes in CA3-CA1 hippocampal neurons following plasticity.
Highlights
All studies aimed at understanding complex molecular changes occurring at synapses face the problem of how a complete view of the synaptic proteome and of its changes can be efficiently met
To evaluate the extent of enrichment in synaptic proteins and the degree of contamination with glia and myelin proteins, subcellular fractions were analyzed by standard electron microscopy (EM) and by western blotting
The general appearance of these 2D maps, the molecular weight (MW) and pI distributions, the number and the relative intensity of individual protein spots were all reproducible in different experiments
Summary
All studies aimed at understanding complex molecular changes occurring at synapses face the problem of how a complete view of the synaptic proteome and of its changes can be efficiently met. Synapses are complex structures that regulate neuronal communication and mediate virtually all functions of the nervous system They are highly polarised structures because of the asymmetric distribution of cytosolic and membrane proteins, such as ion channels and signaling molecules. In the postsynaptic compartment membrane clusters of neurotransmitter receptors are highly enriched and in excitatory terminals these are embedded in a characteristic electron-dense structure called the postsynaptic density In this area neurotrasmitter receptors are tightly associated directly or indirectly with other proteins subserving as scaffolding elements, regulators of signalling and of exo-endocytosis (see [2] for review)
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