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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 -
Journal of Neurocytology Sep 1993Detached synapses (synaptosomes), first isolated by the author in 1958 and identified as such in 1960, are sealed presynaptic nerve terminals often with a portion of the... (Review)
Review
Detached synapses (synaptosomes), first isolated by the author in 1958 and identified as such in 1960, are sealed presynaptic nerve terminals often with a portion of the target cell--sometimes amounting to a complete dendritic spine--adhering to their external surface. They can be prepared in high yield from brain tissue and also in decreasing yield from spinal cord, retina, sympathetic ganglia, myenteric plexus and electric organs. They are sealed structures which, under metabolizing conditions, respire, take up oxygen and glucose, extrude Na+, accumulate K+, maintain a normal membrane potential and, on depolarization, release transmitter in a Ca(2+)-dependent manner. They thus provide an excellent preparation with which to investigate synaptic function without the complications encountered with synapses in situ. They also serve as the parent fraction for preparations of synaptic vesicles and other synaptic components.
Topics: Animals; Centrifugation, Density Gradient; Models, Neurological; Neurotransmitter Agents; Research; Synapses; Synaptosomes
PubMed: 7903689
DOI: 10.1007/BF01181319 -
Journal of Neurochemistry Jan 1988
Comparative Study
Topics: Cell Fractionation; Centrifugation, Density Gradient; Synaptosomes
PubMed: 3335849
DOI: 10.1111/j.1471-4159.1988.tb13270.x -
Journal of Neurochemistry Mar 2020Increasing evidence suggests that both synaptic loss and neuroinflammation constitute early pathologic hallmarks of Alzheimer's disease. A downstream event during...
Increasing evidence suggests that both synaptic loss and neuroinflammation constitute early pathologic hallmarks of Alzheimer's disease. A downstream event during inflammatory activation of microglia and astrocytes is the induction of nitric oxide synthase type 2, resulting in an increased release of nitric oxide and the post-translational S-nitrosylation of protein cysteine residues. Both early events, inflammation and synaptic dysfunction, could be connected if this excess nitrosylation occurs on synaptic proteins. In the long term, such changes could provide new insight into patho-mechanisms as well as biomarker candidates from the early stages of disease progression. This study investigated S-nitrosylation in synaptosomal proteins isolated from APP/PS1 model mice in comparison to wild type and NOS2 mice, as well as human control, mild cognitive impairment and Alzheimer's disease brain tissues. Proteomics data were obtained using an established protocol utilizing an isobaric mass tag method, followed by nanocapillary high performance liquid chromatography tandem mass spectrometry. Statistical analysis identified the S-nitrosylation sites most likely derived from an increase in nitric oxide (NO) in dependence of presence of AD pathology, age and the key enzyme NOS2. The resulting list of candidate proteins is discussed considering function, previous findings in the context of neurodegeneration, and the potential for further validation studies.
Topics: Aged; Aged, 80 and over; Alzheimer Disease; Animals; Brain; Disease Models, Animal; Female; Humans; Male; Mice; Mice, Inbred C57BL; Mice, Transgenic; Nerve Tissue Proteins; Nitric Oxide; Nitric Oxide Synthase Type II; Proteomics; Signal Transduction; Synaptosomes
PubMed: 31520481
DOI: 10.1111/jnc.14870 -
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 -
Cold Spring Harbor Protocols May 2015Alongside rodent brain slices and primary neuronal cultures, synaptosomes (isolated nerve terminals) have been an important model system for studying the molecular...
Alongside rodent brain slices and primary neuronal cultures, synaptosomes (isolated nerve terminals) have been an important model system for studying the molecular mechanisms of synaptic function in the brain. Synaptosomes were first prepared in the late 1950s by Whittaker and colleagues and were instrumental in studying synaptic structure and defining the functional components of the synapse, including the identity of the major neurotransmitters and their uptake mechanisms. Synaptosomes can also be stimulated to release neurotransmitters and were used to discover a number of regulatory signaling pathways that fine-tune synaptic transmission. In the past decade, landmark proteomic studies of synaptosomes and synaptic vesicle preparations have further dissected the protein composition of the synapse. This introduction briefly describes the history of the synaptosome preparation and highlights how it continues to be relevant as our focus in the neuroscience community centers on synaptic dysfunction in aging and neurological disease.
Topics: Animals; Brain; Cell Fractionation; Chromosome Pairing; Models, Biological; Rodentia; Synaptosomes
PubMed: 25934942
DOI: 10.1101/pdb.top074450 -
ENeuro 2019Flow cytometry and fluorescence-activated sorting are powerful techniques that hold great promise for studying heterogeneous populations of submicron particles such as...
Flow cytometry and fluorescence-activated sorting are powerful techniques that hold great promise for studying heterogeneous populations of submicron particles such as synaptosomes, but many technical challenges arise in these experiments. To date, most flow cytometry studies of synaptosomes have relied on particle detection using forward scatter (FSC) measurements and size estimation with polystyrene (PS) bead standards. However, these practices have serious limitations, and special care must be taken to overcome the poor sensitivity of conventional flow cytometers in the analysis of submicron particles. Technical artifacts can confound these experiments, especially the detection of multiple particles as a single event. Here, we compared analysis of P2 crude synaptosomal preparations from murine forebrain on multiple flow cytometers using both FSC-triggered and fluorescence-triggered detection. We implemented multicolor fluorescent dye-based assays to quantify coincident particle detection and aggregation, and we assessed the false colocalization of antigens in immunostaining analyses. Our results demonstrate that fluorescence triggering and proper dilution can control for coincident particle detection, but not particle aggregation. We confirmed previous studies showing that FSC-based size estimation with PS beads underestimates biological particle size, and we identified pervasive aggregation in the FSC range analyzed in most synaptosome flow cytometry studies. We found that analyzing P2 samples in sucrose/EDTA/tris (SET) buffer reduces aggregation compared to PBS, but does not completely eliminate the presence of aggregates, especially in immunostaining experiments. Our study highlights challenges and pitfalls in synaptosome flow cytometry and provides a methodological framework for future studies.
Topics: Animals; Artifacts; Flow Cytometry; Fluorescent Dyes; Male; Mice, Inbred C57BL; Particle Size; Polystyrenes; Prosencephalon; Reference Standards; Scattering, Radiation; Synaptosomes
PubMed: 31118205
DOI: 10.1523/ENEURO.0009-19.2019 -
Bioelectromagnetics Sep 2007The present study examined the effect on rat cortical synaptosomes of a 2 h exposure to 50-Hz electromagnetic fields (EMFs) with a peak magnetic field of 2 mT. We...
The present study examined the effect on rat cortical synaptosomes of a 2 h exposure to 50-Hz electromagnetic fields (EMFs) with a peak magnetic field of 2 mT. We measured modifications of synaptosomal mitochondrial respiration rate, ATP production, membrane potential, intrasynaptosomal Ca(2+) concentration and free iron release. The O(2) consumption remained unvaried in exposed synaptosomes at about 2 nM O(2)/min/mg proteins; ATP production was also unchanged. The intrasynaptosomal Ca(2+) concentration decreased slowly and there was a slight, but non-significant, depolarisation of the synaptosomal membrane. Finally, the free iron release by synaptosomal suspensions, a useful predictor of neuro-developmental outcome, remained unchanged after EMF exposure. On the whole, our results indicate that the physiological behaviour of cortical synaptosomes is not affected by weak pulsed EMFs.
Topics: Animals; Cells, Cultured; Dose-Response Relationship, Radiation; Electromagnetic Fields; Male; Mitochondria; Mitochondrial Membranes; Oxygen Consumption; Radiation Dosage; Rats; Rats, Sprague-Dawley; Synaptosomes
PubMed: 17497690
DOI: 10.1002/bem.20322 -
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 -
Glutamate treatment mimics LTP- and LTD-like biochemical activity in viable synaptosome preparation.Neurochemistry International Mar 2020Long-term potentiation (LTP) and long-term depression (LTD) are considered to be the cellular mechanisms behind the increase or decrease of synaptic strength...
Long-term potentiation (LTP) and long-term depression (LTD) are considered to be the cellular mechanisms behind the increase or decrease of synaptic strength respectively. Electrophysiologically induced LTP/LTD is associated with the activation of glutamate receptors in the synaptic terminals resulting in the initiation of biochemical processes in the postsynaptic terminals and thus propagation of synaptic activity. Isolated nerve endings i.e. synaptosome preparation was used to study here, the biochemical phenotypes of LTP and LTD, and glutamate treatment in varying concentration for different time was used to induce those biochemical phenomena. Treatment with 200 μM glutamate showed increased GluA1 phosphorylation at serine 831 and activation of CaMKIIα by phosphorylation at threonine 286 like LTP, whereas 100 μM glutamate treatment showed decrease in GluA1 phosphorylation level at both pGluA1(S831) and pGluA1(S845), and activation of GSK3β by de-phosphorylating pGSK3β at serine 9 like LTD. The 200 μM glutamate treatment was associated with an increase in the local translation of Arc, BDNF, CaMKIIα and Homer1, whereas 100 μM glutamate treatments resulted in decrease in the level of the said synaptic proteins and the effect was blocked by the proteasomal inhibitor, Lactasystin. Both, the local translation and local degradation was sensitive to the Ca chellator, Bapta-AM, indicating that both the phenomena were dependent on the rise in intra-synaptosomal Ca, like LTP and LTD. Overall the results of the present study suggest that synaptosomal preparations can be a viable alternative to study mechanisms underlying the biochemical activities of LTP/LTD in short term.
Topics: Animals; Excitatory Postsynaptic Potentials; Glutamic Acid; Long-Term Potentiation; Neuronal Plasticity; Presynaptic Terminals; Receptors, Glutamate; Synapses; Synaptic Transmission; Synaptosomes
PubMed: 31899196
DOI: 10.1016/j.neuint.2019.104655