Identification of synaptic vesicle, pre- and postsynaptic proteins in human cerebrospinal fluid using liquid-phase isoelectric focusing

Synaptic pathology is central in the pathogenesis of several psychiatric di sorders, for example in Alzheimer's disease (AD) and schizophrenia. Quantif ication of specific synaptic proteins has proved to be a useful method to e stimate synapitc density in the brain. Using this approach, several synapti c proteins have been demonstrated to be altered In both AD and schizophreni a. until recently, the analysts of synaptic pathology has been limited to p ostmortem tissue. In living subjects, these synaptic proteins may be studie d through analysis of cerebrospinal fluid (CSF). In an earlier study perfor med by us, one synaptic vesicle specific protein, synaptotagmin, was detect ed in CSF for the first time using a procedure based on affinity chromatogr aphy, reversed-phase chromatography, sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and chemiluminescence immunoblotting. Howeve r, other synaptic proteins were not detectable with this procedure. Therefo re, we have developed a procedure including precipitation of CSF proteins w ith trichloroacetic acid, followed by liquid-phase isoelectric focusing usi ng the Rotofor Cell, and finally analysis of Rotofor fractions by Western b lotting for identification of synaptic proteins in CSF. Five synaptic prote ins, rab3a, synaptotagmin, growth-associated protein (GAP-43), synaptosomal -associated protein (SNAP-25) and neurogranin, have been demonstrated in CS F using this method. The major advantage of liquid-phase isoelectric focusi ng (IEF) using the Rotofor cell is that it provides synaptic proteins from CSF in sufficient quantities for identification. This method may also be su itable for identification of other types of trace amounts of brain-specific proteins in CSF. These results demonstrate that several synaptic proteins can be identified and measured in CSF to study synaptic function and pathol ogy in degenerative disorders.