Protein fingerprints of cultured CA3-CA1 hippocampal neurons: comparative analysis of the distribution of synaptosomal and cytosolic proteins.
BACKGROUND
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. 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.


RESULTS
To 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.


CONCLUSIONS
Because 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.
Corti
Valeria
V
Proteome Biochemistry, San Raffaele Scientific Institute, Milan, Italy. corti.valeria@hsr.it
Sanchez-Ruiz
Yovan
Y
Piccoli
Giovanni
G
Bergamaschi
Andrea
A
Cannistraci
Carlo V
CV
Pattini
Linda
L
Cerutti
Sergio
S
Bachi
Angela
A
Alessio
Massimo
M
Malgaroli
Antonio
A
D000818:Animals; D015153:Blotting, Western; D002478:Cells, Cultured; D003600:Cytosol / Q000378:metabolism*; D015180:Electrophoresis, Gel, Two-Dimensional; D006624:Hippocampus / Q000378:metabolism*; D009474:Neurons / Q000378:metabolism*; D010449:Peptide Mapping / Q000379:methods*; D040901:Proteomics / Q000379:methods; D051381:Rats; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D013574:Synaptosomes / Q000378:metabolism*