Enrichment of presynaptic and postsynaptic markers by size-based gating analysis of synaptosome preparations from rat and human cortex (original) (raw)
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Panel of synaptic protein ELISAs for evaluating neurological phenotype
Experimental Brain Research, 2010
The purpose of this study was to develop ELISAs for key neural proteins, three synaptic and one glial, that exist in different intracellular compartments, which would be used as a measure of synaptic phenotype. These assays would be valuable to neurologically phenotype transgenic mouse models of human disease and also human disease itself using minimal amounts of post-mortem tissue. We showed that supernatant from crude brain tissue homogenates extracted in RIPA buffer containing 0.1% SDS bind to synaptophysin, synaptosome-associated protein of 25 kDa (SNAP-25), postsynaptic density-95 (PSD-95), and glial fibrillary acidic protein (GFAP) antibody pairs with high affinity and selectivity. Overall, RIPA + 0.1% SDS were more efficient than RIPA + 2% SDS or a buffer containing only 1% Triton-X-100. Diluting the brain extracts resulted in dose-dependent binding to the antibody pairs for each neural protein, with EC50s that varied from 8.6 µg protein for PSD-95 to 0.23 µg for GFAP. The assays were used to measure synaptic marker protein levels at various times during mouse development and GFAP in a model of disease accompanied by neuroinflammation. Comparison of ELISAs with Western blots by measuring marker levels in brain extract from developing mice showed a greater relative difference in values derived from ELISA. These ELISAs should be valuable to phenotype the synapse in neurological disease and their rodent models.
Journal of Biological Chemistry, 2003
The postsynaptic density contains multiple protein complexes that together relay the presynaptic neurotransmitter input to the activation of the postsynaptic neuron. In the present study we took two independent proteome approaches for the characterization of the protein complement of the postsynaptic density, namely 1) two-dimensional gel electrophoresis separation of proteins in conjunction with mass spectrometry to identify the tryptic peptides of the protein spots and 2) isolation of the trypsin-digested sample that was labeled with isotope-coded affinity tag, followed by liquid chromatography-tandem mass spectrometry for the partial separation and identification of the peptides, respectively. Functional grouping of the identified proteins indicates that the postsynaptic density is a structurally and functionally complex organelle that may be involved in a broad range of synaptic activities. These proteins include the receptors and ion channels for glutamate neurotransmission, proteins for maintenance and modulation of synaptic architecture, sorting and trafficking of membrane proteins, generation of anaerobic energy, scaffolding and signaling, local protein synthesis, and correct protein folding and breakdown of synaptic proteins. Together, these results imply that the postsynaptic density may have the ability to function (semi-) autonomously and may direct various cellular functions in order to integrate synaptic physiology.
Proteomic comparison of different synaptosome preparation procedures
Amino Acids
Synaptosomes are frequently used research objects in neurobiology studies focusing on synaptic transmission as they mimic several aspects of the physiological synaptic functions. They contain the whole apparatus for neurotransmission, the presynaptic nerve ending with synaptic vesicles, synaptic mitochondria and often a segment of the postsynaptic membrane along with the postsynaptic density is attached to its outer surface. As being artificial functional organelles, synaptosomes are viable for several hours, retain their activity, membrane potential, and capable to store, release, and reuptake neurotransmitters. Synaptosomes are ideal subjects for proteomic analysis. The recently available separation and protein detection techniques can cope with the reduced complexity of the organelle and enable the simultaneous qualitative and quantitative analysis of thousands of proteins shaping the structural and functional characteristics of the synapse. Synaptosomes are formed during the hom...
Molecular Brain, 2014
Background: Synapses are fundamental components of brain circuits and are disrupted in over 100 neurological and psychiatric diseases. The synapse proteome is physically organized into multiprotein complexes and polygenic mutations converge on postsynaptic complexes in schizophrenia, autism and intellectual disability. Directly characterising human synapses and their multiprotein complexes from post-mortem tissue is essential to understanding disease mechanisms. However, multiprotein complexes have not been directly isolated from human synapses and the feasibility of their isolation from post-mortem tissue is unknown. Results: Here we establish a screening assay and criteria to identify post-mortem brain samples containing well-preserved synapse proteomes, revealing that neocortex samples are best preserved. We also develop a rapid method for the isolation of synapse proteomes from human brain, allowing large numbers of post-mortem samples to be processed in a short time frame. We perform the first purification and proteomic mass spectrometry analysis of MAGUK Associated Signalling Complexes (MASC) from neurosurgical and post-mortem tissue and find genetic evidence for their involvement in over seventy human brain diseases. Conclusions: We have demonstrated that synaptic proteome integrity can be rapidly assessed from human post-mortem brain samples prior to its analysis with sophisticated proteomic methods. We have also shown that proteomics of synapse multiprotein complexes from well preserved post-mortem tissue is possible, obtaining structures highly similar to those isolated from biopsy tissue. Finally we have shown that MASC from human synapses are involved with over seventy brain disorders. These findings should have wide application in understanding the synaptic basis of psychiatric and other mental disorders.
Synaptic vesicle life cycle and synaptic turnover
Journal of Physiology-Paris, 1993
Cholinergic synaptic vesicles contain a mixture of soluble low molecular mass constituents. Besides acetylcholine these include Ca 2+, ATP, GTP, small amounts of ADP and AMP, and also the diadenosine polyphosphates AP4A and ApsA. In synaptic vesicles isolated from the electric ray these diadenosine polyphosphates occur in mmol concentrations and might represent a novel cotransmitter. The membrane proteins of cholinergic synaptic vesicles presumably are identical to those in other types of electron-lucent synaptic vesicles. A presumptive exception are the transmitter-specific carriers. The life cycle of the synaptic vesicle in intact neurons and in situ was investigated by analysis of all cytoplasmic membrane compartments that share membrane integral proteins with synaptic vesicles. The results suggest that the synaptic vesicle membrane compartment might originate from the trans-Golgi network and, after cycles of exo-and endocytosis in the nerve terminal, might fuse into an endosomal membrane compartment early on retrograde transport. Tracer experiments using membrane proteins and soluble contents suggest that the synaptic vesicle membrane compartment does not intermix with the presynaptic plasma membrane on repeated cycles of exo-and endocytosis if low frequency stimulation is applied. A cDNA has been isolated from the electric ray electric lobe that codes for o-rab3, a small GTP-binding protein highly homologous to mammalian rab3. While abundant in the nerve terminals of the electric organ and at the neuromuscular junction this protein occurs only in limited subpopulations of nerve terminals in electric ray brain. Immunocytochemical analysis using the colloidal gold technique and a monospecific antibody against o-rab3 suggests that the GTP-binding protein remains attached to recycling synaptic vesicles. No evidence was found for a major contribution of an intraterminal endosomal sorting compartment involved in synaptic vesicle recycling.
Quantification of synapse formation and maintenance in vivo in the absence of synaptic release
Neuroscience, 2004
Outgrowing axons in the developing nervous system secrete neurotransmitters and neuromodulatory substances, which is considered to stimulate synaptogenesis. However, some synapses develop independent of presynaptic secretion. To investigate the role of secretion in synapse formation and maintenance in vivo, we quantified synapses and their morphology in the neocortical marginal zone of munc18-1 deficient mice which lack both evoked and spontaneous secretion [Science 287 (2000) 864]. Histochemical analyses at embryonic day 18 (E18) showed that the overall organization of the neocortex and the number of cells were similar in mutants and controls. Western blot analysis revealed equal concentrations of pre- and post-synaptic marker proteins in mutants and controls and immunocytochemical analyses indicated that these markers were targeted to the neuropil of the synaptic layer in the mutant neocortex. Electron microscopy revealed that at E16 immature synapses had formed both in mutants and controls. These synapses had a similar synapse diameter, active zone length and contained similar amounts of synaptic vesicles, which were immuno-positive for two synaptic vesicle markers. However, these synapses were three times less abundant in the mutant. Two days later, E18, synapses in the controls had more total and docked vesicles, but not in the mutant. Furthermore, synapses were now five times less abundant in the mutant. In both mutant and controls, synapse-like structures were observed with irregular shaped vesicles on both sides of the synaptic cleft. These 'multivesicular structures' were immuno-positive for synaptic vesicle markers and were four times more abundant in the mutant. We conclude that in the absence of presynaptic secretion immature synapses with a normal morphology form, but fewer in number. These secretion-deficient synapses might fail to mature and instead give rise to multivesicular structures. These two observations suggest that secretion of neurotransmitters and neuromodulatory substances is required for synapse maintenance, not for synaptogenesis. Multivesicular structures may develop out of unstable synapses.
We have estimated the densities of synapses per unit volume in the whole mouse brain. To do this, we combined Spinning Disc confocal Microscopy (SDM) that acquires images of synaptic proteins labeled with fluorophores in large brain areas, and FIB-SEM, a three-dimensional electron microscopy technique that provides a resolution beyond that of the confocal microscope, but in relatively small areas. The postsynaptic scaffold proteins PSD95 and SAP102 were genetically labeled to visualize excitatory synapses with SDM. We calculated the densities of synaptic puncta (with SDM) and the actual number of synapses (with FIB-SEM) in the CA1 region of the hippocampus. We then calculated the quantitative relationship between the densities of fluorescent puncta and the number of synapses. Finally, we applied this conversion factor to other regions of the brain where densities of puncta were available. We observed three different groups of brain regions, one with the highest densities of synapses...
Single-Synapse Analysis of a Diverse Synapse Population: Proteomic Imaging Methods and Markers
Neuron, 2010
A lack of methods for measuring the protein compositions of individual synapses in situ has so far hindered the exploration and exploitation of synapse molecular diversity. Here, we describe the use of array tomography, a new high-resolution proteomic imaging method, to determine the composition of glutamate and GABA synapses in somatosensory cortex of Line-H-YFP Thy-1 transgenic mice. We find that virtually all synapses are recognized by antibodies to the presynaptic phosphoprotein synapsin I, while antibodies to 16 other synaptic proteins discriminate among 4 subtypes of glutamatergic synapses and GABAergic synapses. Cell-specific YFP expression in the YFP-H mouse line allows synapses to be assigned to specific presynaptic and postsynaptic partners and reveals that a subpopulation of spines on layer 5 pyramidal cells receives both VGluT1-subtype glutamatergic and GABAergic synaptic inputs. These results establish a means for the high-throughput acquisition of proteomic data from individual cortical synapses in situ.