The post-synaptic density of human postmortem brain tissues: an experimental study paradigm for neuropsychiatric illnesses.
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.
Hahn
Chang-Gyu
CG
Department of Psychiatry, University of Pennsylvania, Philadelphia, PA, USA. hahnc@mail.med.upenn.edu
Banerjee
Anamika
A
Macdonald
Matthew L
ML
Cho
Dan-Sung
DS
Kamins
Joshua
J
Nie
Zhiping
Z
Borgmann-Winter
Karin E
KE
Grosser
Tilo
T
Pizarro
Angel
A
Ciccimaro
Eugene
E
Arnold
Steven E
SE
Wang
Hoau-Yan
HY
Blair
Ian A
IA
D001923:Brain Chemistry; D002102:Cadaver; D006801:Humans; D046912:Multiprotein Complexes / Q000032:analysis / Q000302:isolation & purification* / Q000648:ultrastructure; D009419:Nerve Tissue Proteins / Q000032:analysis / Q000302:isolation & purification* / Q000648:ultrastructure; D009422:Nervous System Diseases / Q000503:physiopathology; D010455:Peptides / Q000032:analysis; D013347:Subcellular Fractions / Q000737:chemistry* / Q000648:ultrastructure; D013570:Synaptic Membranes / Q000737:chemistry* / Q000648:ultrastructure; D013574:Synaptosomes / Q000737:chemistry / Q000648:ultrastructure
D046912:Multiprotein Complexes; D009419:Nerve Tissue Proteins; D010455:Peptides; C033652:postsynaptic density proteins