Hahn Chang-Gyu, Banerjee Anamika, Macdonald Matthew L, Cho Dan-Sung, Kamins Joshua, Nie Zhiping, Borgmann-Winter Karin E, Grosser Tilo, Pizarro Angel, Ciccimaro Eugene, Arnold Steven E, Wang Hoau-Yan, Blair Ian A
Department of Psychiatry, University of Pennsylvania, Philadelphia, PA, USA.
PLoS One. 2009;4(4):e5251. doi: 10.1371/journal.pone.0005251. Epub 2009 Apr 16.
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.
近期的分子遗传学研究提出了神经精神疾病病理生理机制的各种跨突触过程。检查患者大脑中突触前和突触后的支架将极大地有助于进一步研究，但这种在人类尸检组织中的方法尚未得到验证。我们研究了三种方法，即基于密度梯度纯化突触体，然后用去污剂提取（方法1），以及基于pH值的突触膜差异提取（方法2和3）。所有这三种方法都从人类尸检大脑中分离出了富含典型突触后致密部（PSD）蛋白的组分，几乎排除了突触前蛋白。我们使用电子显微镜（EM）检查了这些组分，并验证了源自人类尸检脑组织的突触膜和PSD组分的完整性。我们使用二维液相色谱串联质谱（2D LC-MS/MS）分析了PSD组分的蛋白质组成，并通过质谱观察到了已知的PSD蛋白。免疫沉淀和免疫印迹研究表明，PSD组分中维持了预期的蛋白质-蛋白质相互作用和某些转录后调节。我们的结果表明，可以从人类尸检脑组织中分离出具有合理完整性的PSD组分。这种方法可能会促进新的尸检脑研究范式，即研究特定微结构域的化学计量和蛋白质组成。
