The Post-Synaptic Density of Human Postmortem Brain Tissues: An Experimental Study Paradigm for Neuropsychiatric Illnesses

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.

Postmortem Brain: An Underutilized Substrate for Studying Severe Mental Illness

“…Using synaptosomes as a starting point, highly enriched PSDs were isolated using density gradient purification, detergent extraction, and pHbased differential extraction. The authors concluded that a PSD fraction can be isolated from human postmortem brain tissues with a reasonable degree of integrity (Hahn et al, 2009). Another group has used high-speed centrifugation, detergents, and sucrose gradients to isolate a 'detergentresistant membrane fraction' that they refer to as membrane microdomains (Behan et al, 2009;Parkin et al, 1999).…”

“…The PSD is perhaps the most well-characterized microdomain in the CNS, and several groups have developed cutting edge approaches to interrogate this complex structure in postmortem brain (Cheng et al, 2010;Fernandez et al, 2009;Hahn et al, 2009;MacDonald et al, 2012). Identification and characterization of novel microdomains is currently feasible using standard biochemical techniques.…”

Chronic Cerebral Hypoperfusion Induced Synaptic Proteome Changes in the rat Cerebral Cortex

“…Synaptosomes were prepared by the method of Hahn et al with minor modifications (Hahn et al, 2009). Briefly, the cerebral cortical tissue was homogenized using a Dounce type glass homogenizer (Small Clearance, Kontes Glass Co.) with 40 strokes in 1 ml of homogenization buffer (320 mM sucrose, 0.1 mM CaCl 2 , 1 mM MgCl 2 ) supplemented with protease and phosphatase inhibitor cocktails (Sigma-Aldrich, St.Louis, MO, USA).…”

Common Proteomic Changes in the Hippocampus in Schizophrenia and Bipolar Disorder and Particular Evidence for Involvement of Cornu Ammonis Regions 2 and 3

“…129 Thus, our findings implicating cytoskeletal function and CME may be related. Future work will need to consider the functional aspects of CME within cellular domains such as within the postsynaptic density 130 where CME changes would directly affect NMDA recycling.…”