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The post-synaptic density of human postmortem brain tissues: an experimental study paradigm for neuropsychiatric illnesses.

Hahn CG, Banerjee A, Macdonald ML, Cho DS, Kamins J, Nie Z, Borgmann-Winter KE, Grosser T, Pizarro A, Ciccimaro E, Arnold SE, Wang HY, Blair IA - PLoS ONE (2009)

Bottom Line: 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).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.

View Article: PubMed Central - PubMed

Affiliation: Department of Psychiatry, University of Pennsylvania, Philadelphia, PA, USA. hahnc@mail.med.upenn.edu

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.

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Related in: MedlinePlus

Electron micrographs of osmicated insoluble pellets obtained by Method 3).(A, B) Thin sections of SPM pellets show surprisingly intact synaptosomes and intact synaptic vesicles (A, B) as well as filamentous crossbridges (see arrows in B). (C) Synaptic membrane fractions extracted by Triton-X 100 at pH 6.0 show paired electron dense profiles representing synaptic junctions (see arrows in C). (D) PSD fraction obtained as an insoluble phase after synaptic membranes were extracted with Triton-X 100 at pH 8.0. Note that presynaptic specialization is removed and that the PSD is thinner than shown in A and B.
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pone-0005251-g003: Electron micrographs of osmicated insoluble pellets obtained by Method 3).(A, B) Thin sections of SPM pellets show surprisingly intact synaptosomes and intact synaptic vesicles (A, B) as well as filamentous crossbridges (see arrows in B). (C) Synaptic membrane fractions extracted by Triton-X 100 at pH 6.0 show paired electron dense profiles representing synaptic junctions (see arrows in C). (D) PSD fraction obtained as an insoluble phase after synaptic membranes were extracted with Triton-X 100 at pH 8.0. Note that presynaptic specialization is removed and that the PSD is thinner than shown in A and B.

Mentions: The integrity of isolated fractions was tested using EM. Figure 3A and B show a thin section electron micrograph of SPM and demonstrate strikingly intact synaptsomes that contain synaptic vesicles and filamentous connections to other synaptosomes. Figure 3C represents the soluble fraction of pH 6 Triton X-100 extraction and shows paired pieces of electron dense membranes. The more electron dense membrane is thought to be postsynaptic. Figure 3D shows that the PSD fraction is largely devoid of the fine substructures of synaptic junctions.


The post-synaptic density of human postmortem brain tissues: an experimental study paradigm for neuropsychiatric illnesses.

Hahn CG, Banerjee A, Macdonald ML, Cho DS, Kamins J, Nie Z, Borgmann-Winter KE, Grosser T, Pizarro A, Ciccimaro E, Arnold SE, Wang HY, Blair IA - PLoS ONE (2009)

Electron micrographs of osmicated insoluble pellets obtained by Method 3).(A, B) Thin sections of SPM pellets show surprisingly intact synaptosomes and intact synaptic vesicles (A, B) as well as filamentous crossbridges (see arrows in B). (C) Synaptic membrane fractions extracted by Triton-X 100 at pH 6.0 show paired electron dense profiles representing synaptic junctions (see arrows in C). (D) PSD fraction obtained as an insoluble phase after synaptic membranes were extracted with Triton-X 100 at pH 8.0. Note that presynaptic specialization is removed and that the PSD is thinner than shown in A and B.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2666803&req=5

pone-0005251-g003: Electron micrographs of osmicated insoluble pellets obtained by Method 3).(A, B) Thin sections of SPM pellets show surprisingly intact synaptosomes and intact synaptic vesicles (A, B) as well as filamentous crossbridges (see arrows in B). (C) Synaptic membrane fractions extracted by Triton-X 100 at pH 6.0 show paired electron dense profiles representing synaptic junctions (see arrows in C). (D) PSD fraction obtained as an insoluble phase after synaptic membranes were extracted with Triton-X 100 at pH 8.0. Note that presynaptic specialization is removed and that the PSD is thinner than shown in A and B.
Mentions: The integrity of isolated fractions was tested using EM. Figure 3A and B show a thin section electron micrograph of SPM and demonstrate strikingly intact synaptsomes that contain synaptic vesicles and filamentous connections to other synaptosomes. Figure 3C represents the soluble fraction of pH 6 Triton X-100 extraction and shows paired pieces of electron dense membranes. The more electron dense membrane is thought to be postsynaptic. Figure 3D shows that the PSD fraction is largely devoid of the fine substructures of synaptic junctions.

Bottom Line: 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).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.

View Article: PubMed Central - PubMed

Affiliation: Department of Psychiatry, University of Pennsylvania, Philadelphia, PA, USA. hahnc@mail.med.upenn.edu

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.

Show MeSH
Related in: MedlinePlus