Abstract
Recent molecular genetics studies have suggested various trans-synaptic processes for pathophysiologic mechanisms of neuropsychiatric illnesses. Examination of pre- and post-synaptic scaffolds in the brains of patients would greatly aid further investigation, yet such an approach in human postmortem tissue has yet to be tested. We have examined three methods using density gradient based purification of synaptosomes followed by detergent extraction (Method 1) and the pH based differential extraction of synaptic membranes (Methods 2 and 3). All three methods separated fractions from human postmortem brains that were highly enriched in typical PSD proteins, almost to the exclusion of pre-synaptic proteins. We examined these fractions using electron microscopy (EM) and verified the integrity of the synaptic membrane and PSD fractions derived from human postmortem brain tissues. We analyzed protein composition of the PSD fractions using two dimensional liquid chromatography tandem mass spectrometry (2D LC-MS/MS) and observed known PSD proteins by mass spectrometry. Immunoprecipitation and immunoblot studies revealed that expected protein-protein interactions and certain posttranscriptional modulations were maintained in PSD fractions. Our results demonstrate that PSD fractions can be isolated from human postmortem brain tissues with a reasonable degree of integrity. This approach may foster novel postmortem brain research paradigms in which the stoichiometry and protein composition of specific microdomains are examined.
Highlights
Recent studies in neuropsychiatric illnesses have implicated various trans-synaptic mechanisms [1,2,3,4,5,6,7], for which protein compositions in sub-cellular microdomains are crucial [8,9,10,11,12]
The soluble fractions obtained after the pH 6 and pH 8 Triton X-100 extractions were designated the synaptic vesicular fraction (SV) and the presynaptic membrane fraction (PPF), respectively
The postsynaptic density (PSD) contains molecular machineries for N-methyl-D-aspartic acid (NMDA), amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor (AMPA) and mGlu receptors, all of which have been implicated in the pathophysiology of various neuropsychiatric illnesses [40,41,42,43]
Summary
Recent studies in neuropsychiatric illnesses have implicated various trans-synaptic mechanisms [1,2,3,4,5,6,7], for which protein compositions in sub-cellular microdomains are crucial [8,9,10,11,12]. Others, have recently applied similar approaches to human postmortem brains [18,19], but the feasibility and validity have not been fully elucidated. We evaluate a research paradigm to isolate biochemical fractions enriched in the PSD of human postmortem brains, which can be used as a platform to profile the stoichiometry of specific receptor complexes, when combined with a proteomics approach
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