-
Cell Nov 2023Neurons build synaptic contacts using different protein combinations that define the specificity, function, and plasticity potential of synapses; however, the diversity...
Neurons build synaptic contacts using different protein combinations that define the specificity, function, and plasticity potential of synapses; however, the diversity of synaptic proteomes remains largely unexplored. We prepared synaptosomes from 7 different transgenic mouse lines with fluorescently labeled presynaptic terminals. Combining microdissection of 5 different brain regions with fluorescent-activated synaptosome sorting (FASS), we isolated and analyzed the proteomes of 18 different synapse types. We discovered ∼1,800 unique synapse-type-enriched proteins and allocated thousands of proteins to different types of synapses (https://syndive.org/). We identify shared synaptic protein modules and highlight the proteomic hotspots for synapse specialization. We reveal unique and common features of the striatal dopaminergic proteome and discover the proteome signatures that relate to the functional properties of different interneuron classes. This study provides a molecular systems-biology analysis of synapses and a framework to integrate proteomic information for synapse subtypes of interest with cellular or circuit-level experiments.
Topics: Animals; Mice; Brain; Mice, Transgenic; Proteome; Proteomics; Synapses; Synaptosomes
PubMed: 37918396
DOI: 10.1016/j.cell.2023.09.028 -
Sub-cellular Biochemistry 2007Our knowledge of the complex synaptic proteome and its relationship to physiological or pathological conditions is rapidly expanding. This has been greatly accelerated... (Review)
Review
Our knowledge of the complex synaptic proteome and its relationship to physiological or pathological conditions is rapidly expanding. This has been greatly accelerated by the application of various evolving proteomic techniques, enabling more efficient protein resolution, more accurate protein identification, and more comprehensive characterization of proteins undergoing quantitative and qualitative changes. More recently, the combination of the classical subcellular fractionation techniques for the isolation of synaptosomes from the brain with the various proteomic analyses has facilitated this effort. This has resulted from the enrichment of many low abundant proteins comprising the fundamental structure and molecular machinery of brain neurotransmission and neuroplasticity. The analysis of various subproteomes obtained from the synapse, such as synaptic vesicles, synaptic membranes, presynaptic particles, synaptodendrosomes, and postsynaptic densities (PSD) holds great promise for improving our understanding of the temporal and spatial processes that coordinate synaptic proteins in closely related complexes under both normal and diseased states. This chapter will summarize a selection of recent studies that have drawn upon established and emerging proteomic technologies, along with fractionation techniques that are essential to the isolation and analysis of specific synaptic components, in an effort to understand the complexity and plasticity of the synapse proteome.
Topics: Animals; Humans; Protein Processing, Post-Translational; Proteomics; Synaptosomes
PubMed: 17953392
DOI: 10.1007/978-1-4020-5943-8_6 -
STAR Protocols Mar 2023We describe here a time-efficient, in-house protocol for synaptosome isolation and enrichment of the post-synaptic density (PSD) from hiPSC-derived motor neurons. By...
We describe here a time-efficient, in-house protocol for synaptosome isolation and enrichment of the post-synaptic density (PSD) from hiPSC-derived motor neurons. By using biochemical sub-cellular fractionation, the crude synaptosome is first isolated from the cytosol and is then further separated into the synaptic cytosol and the enriched PSD fraction. The protocol can also potentially be adapted to other hiPSC-derived neuronal types, with necessary changes made to cell seeding density and buffer volumes.
Topics: Synaptosomes; Induced Pluripotent Stem Cells; Post-Synaptic Density; Motor Neurons
PubMed: 36853677
DOI: 10.1016/j.xpro.2023.102061 -
International Journal of Molecular... Dec 2021The assembly of synaptic protein-DNA complexes by specialized proteins is critical for bringing together two distant sites within a DNA molecule or bridging two DNA...
The assembly of synaptic protein-DNA complexes by specialized proteins is critical for bringing together two distant sites within a DNA molecule or bridging two DNA molecules. The assembly of such synaptosomes is needed in numerous genetic processes requiring the interactions of two or more sites. The molecular mechanisms by which the protein brings the sites together, enabling the assembly of synaptosomes, remain unknown. Such proteins can utilize sliding, jumping, and segmental transfer pathways proposed for the single-site search process, but none of these pathways explains how the synaptosome assembles. Here we used restriction enzyme SfiI, that requires the assembly of synaptosome for DNA cleavage, as our experimental system and applied time-lapse, high-speed AFM to directly visualize the site search process accomplished by the SfiI enzyme. For the single-site SfiI-DNA complexes, we were able to directly visualize such pathways as sliding, jumping, and segmental site transfer. However, within the synaptic looped complexes, we visualized the threading and site-bound segment transfer as the synaptosome-specific search pathways for SfiI. In addition, we visualized sliding and jumping pathways for the loop dissociated complexes. Based on our data, we propose the site-search model for synaptic protein-DNA systems.
Topics: Binding Sites; Chromosome Pairing; DNA; DNA Restriction Enzymes; Plasmids; Protein Binding; Proteins; Synaptosomes
PubMed: 35008637
DOI: 10.3390/ijms23010212 -
Molecular Brain Feb 2021The SH3 and multiple ankyrin repeat domains 3 (Shank3) protein is a core organizer of the macromolecular complex in excitatory postsynapses, and its defects cause...
The SH3 and multiple ankyrin repeat domains 3 (Shank3) protein is a core organizer of the macromolecular complex in excitatory postsynapses, and its defects cause numerous synaptopathies, including autism spectrum disorders. Although the function of Shank3 as a postsynaptic scaffold is adequately established, other potential mechanisms through which Shank3 broadly modulates the postsynaptic proteome remain relatively unexplored. In our previous quantitative proteomic analysis, six up-regulated ribosomal proteins were identified in the striatal synaptosome of Shank3-overexpressing transgenic (TG) mice. In the present study, we validated the increased levels of RPLP1 and RPL36A in synaptosome, but not in whole lysate, of the TG striatum. Moreover, protein synthesis and extracellular signaling-regulated kinase (ERK) activity were enhanced in the TG striatal synaptosome. To understand the potential contribution of increased protein synthesis to the proteomic change in the TG striatal synaptosome, we performed RNA-sequencing analyses on both whole synaptosomal and synaptic polysome-enriched fractions. Comparative analyses showed a positive correlation only between the polysome-associated transcriptome and up-regulated proteome in the TG striatal synaptosome. Our findings suggest a novel mechanism through which Shank3 may remodel the postsynaptic proteome by regulating synaptic protein synthesis, whose dysfunction can be implicated in SHANK3-associated synaptopathies.
Topics: Animals; Corpus Striatum; MAP Kinase Signaling System; Mice, Transgenic; Microfilament Proteins; Nerve Tissue Proteins; Protein Biosynthesis; Receptors, Dopamine D1; Ribosomal Proteins; Synaptosomes
PubMed: 33622379
DOI: 10.1186/s13041-021-00756-z -
Journal of Nanobiotechnology Jan 2021Mitochondrial dysfunction is a critical factor in the onset and progression of neurodegenerative diseases. Recently, mitochondrial transplantation has been advised as an...
BACKGROUND
Mitochondrial dysfunction is a critical factor in the onset and progression of neurodegenerative diseases. Recently, mitochondrial transplantation has been advised as an innovative and attractive strategy to transfer and replace damaged mitochondria. Here we propose, for the first time, to use rat brain extracted synaptosomes, a subcellular fraction of isolated synaptic terminal that contains mitochondria, as mitochondrial delivery systems.
RESULTS
Synaptosome preparation was validated by the presence of Synaptophysin and PSD95. Synaptosomes were characterized in terms of dimension, zeta potential, polydispersity index and number of particles/ml. Nile Red or CTX-FITCH labeled synaptosomes were internalized in LAN5 recipient cells by a mechanism involving specific protein-protein interaction, as demonstrated by loss of fusion ability after trypsin treatment and using different cell lines. The loading and release ability of the synaptosomes was proved by the presence of curcumin both into synaptosomes and LAN5 cells. The vitality of mitochondria transferred by Synaptosomes was demonstrated by the presence of Opa1, Fis1 and TOM40 mitochondrial proteins and JC-1 measurements. Further, synaptosomes deliver vital mitochondria into the cytoplasm of neuronal cells as demonstrated by microscopic images, increase of TOM 40, cytochrome c, Hexokinase II mitochondrial proteins, and presence of rat mitochondrial DNA. Finally, by using synaptosomes as a vehicle, healthy mitochondria restored mitochondrial function in cells containing rotenone or CCCp damaged mitochondria.
CONCLUSIONS
Taken together these results suggest that synaptosomes can be a natural vehicle for the delivery of molecules and organelles to neuronal cells. Further, the replacement of affected mitochondria with healthy ones could be a potential therapy for treating neuronal mitochondrial dysfunction-related diseases.
Topics: Animals; Cytochromes c; DNA, Mitochondrial; Drug Delivery Systems; Homeostasis; Male; Membrane Potentials; Mitochondria; Protein Interaction Domains and Motifs; Rats; Subcellular Fractions; Synaptosomes
PubMed: 33407593
DOI: 10.1186/s12951-020-00748-6 -
The FEBS Journal Dec 2011Clostridium perfringens ε-toxin is produced by toxinotypes B and D strains. The toxin is the aetiological agent of dysentery in newborn lambs but is also associated... (Review)
Review
Clostridium perfringens ε-toxin is produced by toxinotypes B and D strains. The toxin is the aetiological agent of dysentery in newborn lambs but is also associated with enteritis and enterotoxaemia in goats, calves and foals. It is considered to be a potential biowarfare or bioterrorism agent by the US Government Centers for Disease Control and Prevention. The relatively inactive 32.9 kDa prototoxin is converted to active mature toxin by proteolytic cleavage, either by digestive proteases of the host, such as trypsin and chymotrypsin, or by C. perfringens λ-protease. In vivo, the toxin appears to target the brain and kidneys, but relatively few cell lines are susceptible to the toxin, and most work has been carried out using Madin-Darby canine kidney (MDCK) cells. The binding of ε-toxin to MDCK cells and rat synaptosomal membranes is associated with the formation of a stable, high molecular weight complex. The crystal structure of ε-toxin reveals similarity to aerolysin from Aeromonas hydrophila, parasporin-2 from Bacillus thuringiensis and a lectin from Laetiporus sulphureus. Like these toxins, ε-toxin appears to form heptameric pores in target cell membranes. The exquisite specificity of the toxin for specific cell types suggests that it binds to a receptor found only on these cells.
Topics: Animals; Bacterial Toxins; Clostridium perfringens; Humans; Models, Molecular; Protein Conformation; Rats; Synaptosomes
PubMed: 21518257
DOI: 10.1111/j.1742-4658.2011.08140.x -
FASEB Journal : Official Publication of... Aug 2022Vesicle-mediated transport is necessary for maintaining cellular homeostasis and proper signaling. The synaptosome-associated protein 23 (SNAP23) is a member of the...
Vesicle-mediated transport is necessary for maintaining cellular homeostasis and proper signaling. The synaptosome-associated protein 23 (SNAP23) is a member of the SNARE protein family and mediates the vesicle docking and membrane fusion steps of secretion during exocytosis. Skeletal muscle has been established as a secretory organ; however, the role of SNAP23 in the context of skeletal muscle development is still unknown. Here, we show that depletion of SNAP23 in C2C12 mouse myoblasts reduces their ability to differentiate into myotubes as a result of premature cell cycle exit and early activation of the myogenic transcriptional program. This effect is rescued when cells are seeded at a high density or when cultured in conditioned medium from wild type cells. Proteomic analysis of collected medium indicates that SNAP23 depletion leads to a misregulation of exocytosis, including decreased secretion of the insulin-like growth factor 1 (IGF1), a critical protein for muscle growth, development, and function. We further demonstrate that treatment of SNAP23-depleted cells with exogenous IGF1 rescues their myogenic capacity. We propose that SNAP23 mediates the secretion of specific proteins, such as IGF1, that are important for achieving proper differentiation of skeletal muscle cells during myogenesis. This work highlights the underappreciated role of skeletal muscle as a secretory organ and contributes to the understanding of factors necessary for myogenesis.
Topics: Animals; Cell Differentiation; Mice; Muscle Development; Myoblasts; Proteomics; Qb-SNARE Proteins; Qc-SNARE Proteins; SNARE Proteins; Synaptosomes
PubMed: 35816155
DOI: 10.1096/fj.202101627RR -
Methods in Molecular Biology (Clifton,... 2022Synaptic degeneration is one of the earliest and phenotypically most significant features associated with numerous neurodegenerative conditions, including Alzheimer's...
Synaptic degeneration is one of the earliest and phenotypically most significant features associated with numerous neurodegenerative conditions, including Alzheimer's and Parkinson's diseases. Synaptic changes are also known to be important in neurocognitive disorders such as schizophrenia and autism spectrum disorders. Several labs, including ours, have demonstrated that conventional (fluorescence-based) flow cytometry of individual synaptosomes is a robust and reproducible method. However, the repertoire of probes needed to assess comprehensively the type of synapse, pathologic proteins (including protein products of risk genes discovered in GWAS), and markers of stress and injury far exceeds what is achievable with conventional flow cytometry. We recently developed a method that applies CyTOF (Cytometry by Time-Of-Flight mass spectrometry) to high-dimensional analysis of individual human synaptosomes, overcoming many of the multiplexing limitations of conventional flow cytometry. We call this new method Mass Synaptometry. Here we describe the preparation of synaptosomes from human and mouse brain, the generation and quality control of the "SynTOF" (Synapse by Time-Of-Flight mass spectrometry) antibody panel, the staining protocol, and CyTOF parameter setup for acquisition, post-acquisition processing, and analysis.
Topics: Animals; Flow Cytometry; Mass Spectrometry; Mice; Synapses; Synaptosomes
PubMed: 35099792
DOI: 10.1007/978-1-0716-1916-2_6 -
Molecular & Cellular Proteomics : MCP Dec 2013We present the first large scale study characterizing both N- and O-linked glycosylation in a site-specific manner on hundreds of proteins. We demonstrate that a...
We present the first large scale study characterizing both N- and O-linked glycosylation in a site-specific manner on hundreds of proteins. We demonstrate that a lectin-affinity fractionation step using wheat germ agglutinin enriches not only peptides carrying intracellular O-GlcNAc, but also those bearing ER/Golgi-derived N- and O-linked carbohydrate structures. Liquid chromatography-MS (LC/MS) analysis with high accuracy precursor mass measurements and high sensitivity ion trap electron-transfer dissociation (ETD) were utilized for structural characterization of glycopeptides. Our results reveal both the identity of the precise sites of glycosylation and information on the oligosaccharide structures possible on these proteins. We report a novel iterative approach that allowed us to interpret the ETD data set directly without making prior assumptions about the nature and distribution of oligosaccharides present in our glycopeptide mixture. Over 2500 unique N- and O-linked glycopeptides were identified on 453 proteins. The extent of microheterogeneity varied extensively, and up to 19 different oligosaccharides were attached at a given site. We describe the presence of the well-known mucin-type structures for O-glycosylation, an EGF-domain-specific fucosylation and a rare O-mannosylation on the transmembrane phosphatase Ptprz1. Finally, we identified three examples of O-glycosylation on tyrosine residues.
Topics: Amino Acid Motifs; Animals; Carbohydrate Sequence; Chemical Fractionation; Chromatography, Liquid; Fucose; Glycopeptides; Glycosylation; Mannose; Mice; Molecular Sequence Annotation; Molecular Sequence Data; Mucins; Protein Processing, Post-Translational; Proteomics; Synaptosomes; Tandem Mass Spectrometry; Wheat Germ Agglutinins
PubMed: 23816992
DOI: 10.1074/mcp.M113.030007