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Methods in Molecular Biology (Clifton,... 2023In addition to microvilli's role as structural scaffold for TCR clustering, we recently discovered a novel function as message senders. We found that microvilli are...
In addition to microvilli's role as structural scaffold for TCR clustering, we recently discovered a novel function as message senders. We found that microvilli are separated from the T cell body shortly upon TCR stimulation and vesiculated to form T cell microvilli particles (TMPs), a new type of membrane vesicles. TMPs and synaptic ectosomes, which bud from the synaptic cleft, constitute "T cell immunological synaptosomes (TISs)" and act as conveyors of T cell messages or traits to cognate antigen-presenting cells. In practice, it is almost impossible to distinguish between TMPs and synaptic ectosomes. Here, we describe a newly developed protocol to isolate TISs from activated T cells using antibody-immobilized agarose beads and density gradient ultracentrifugation. We further describe the methods for TIS quantification with flow cytometry and to evaluate TIS efficacy on dendritic cells.
Topics: T-Lymphocytes; Synaptosomes; Antigen-Presenting Cells; Cell-Derived Microparticles; Receptors, Antigen, T-Cell
PubMed: 37106184
DOI: 10.1007/978-1-0716-3135-5_13 -
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 -
Molecular Neurobiology Jan 2019The synthesis of brain metabolic DNA (BMD) is modulated by learning and circadian oscillations and is not involved in cell division or DNA repair. Data from rats have...
The synthesis of brain metabolic DNA (BMD) is modulated by learning and circadian oscillations and is not involved in cell division or DNA repair. Data from rats have highlighted its prevalent association with the mitochondrial fraction and its lack of identity with mtDNA. These features suggested that BMD could be localized in synaptosomes that are the major contaminants of brain mitochondrial fractions. The hypothesis has been examined by immunochemical analyses of the large synaptosomes of squid optic lobes that are readily prepared and identified. Optic lobe slices were incubated with 5-bromo-2-deoxyuridine (BrdU) and the isolated synaptosomal fraction was exposed to the green fluorescent anti-BrdU antibody. This procedure revealed that newly synthesized BrdU-labeled BMD is present in a significant percent of the large synaptosomes derived from the nerve terminals of retinal photoreceptor neurons and in synaptosomal bodies of smaller size. Synaptosomal BMD synthesis was strongly inhibited by actinomycin D. In addition, treatment of the synaptosomal fraction with Hoechst 33258, a blue fluorescent dye specific for dsDNA, indicated that native DNA was present in all synaptosomes. The possible role of synaptic BMD is briefly discussed.
Topics: Animals; DNA; Dactinomycin; Decapodiformes; Synaptosomes
PubMed: 29675577
DOI: 10.1007/s12035-018-1071-3 -
Nature Methods Aug 2014
Topics: Animals; Brain; Presynaptic Terminals; Synaptic Vesicles; Synaptosomes; Vesicular Transport Proteins
PubMed: 25229096
DOI: 10.1038/nmeth.3057 -
The International Journal of... Feb 2023We aimed to investigate whether m-calpain (a Ca2+-dependent neutral cysteine protease) is released from synaptosomes. This research was carry on Wistar male rats and...
We aimed to investigate whether m-calpain (a Ca2+-dependent neutral cysteine protease) is released from synaptosomes. This research was carry on Wistar male rats and isolated nerve endings - synaptosomes. The synaptosomal integrity was checked by the method of measuring LDH activity. Activity of calpains was measured by the casein zymography in gel and in solution. Extracellular calpain was detected by immunoprecipitation and immunoblotting procedures Prediction of secreted proteins peptide on a protein sequence through a local version of the PrediSi tool (http://www.predisi.de). The probability of calpain isoform nonclassical secretion was analyzed by using SecretomeP (http://www.cbs.dtu.dk/services/SecretomeP2.0) software. It has been shown that calcium- and time-dependent m-calpain is released from synaptosomes in an activated form or in a form capable of activation, and this process is not a result of a violation of the integrity of synaptosomes. Analysis of the probability of secretion of the small catalytic subunit of rat m-calpain along a nonclassical pathway showed a high probability of its secretion. Additionally, the release of calpain from synaptosomes revealed by us is suppressed by the addition of glyburide, an ABC transporter inhibitor, to the incubation medium. Among extracellular proteins, potential substrates of calpains are of calpains are found, for example, matrix metalloprotease-2 and -9, alpha-synuclein, etc. Active m-calpain is present in the media generated from striatal synaptosomes. Glyburide prevents m-calpain release from striatal synaptosomes.
Topics: Rats; Male; Animals; Calpain; Synaptosomes; Glyburide; Rats, Wistar
PubMed: 33688783
DOI: 10.1080/00207454.2021.1901697 -
JAMA Psychiatry Jan 2020Findings from unbiased genetic studies have consistently implicated synaptic protein networks in schizophrenia, but the molecular pathologic features within these...
IMPORTANCE
Findings from unbiased genetic studies have consistently implicated synaptic protein networks in schizophrenia, but the molecular pathologic features within these networks and their contribution to the synaptic and circuit deficits thought to underlie disease symptoms remain unknown.
OBJECTIVE
To determine whether protein levels are altered within synapses from the primary auditory cortex (A1) of individuals with schizophrenia and, if so, whether these differences are restricted to the synapse or occur throughout the gray matter.
DESIGN, SETTING, AND PARTICIPANTS
This paired case-control study included tissue samples from individuals with schizophrenia obtained from the Allegheny County Office of the Medical Examiner. An independent panel of health care professionals made consensus DSM-IV diagnoses. Each tissue sample from an individual with schizophrenia was matched by sex, age, and postmortem interval with 1 sample from an unaffected control individual. Targeted mass spectrometry was used to measure protein levels in A1 gray matter homogenate and synaptosome preparations. All experimenters were blinded to diagnosis. Mass spectrometry data were collected from September 26 through November 4, 2016, and analyzed from November 3, 2016, to July 15, 2019.
MAIN OUTCOMES AND MEASURES
Primary measures were homogenate and synaptosome protein levels and their coregulation network features. Hypotheses generated before data collection were (1) that levels of canonical postsynaptic proteins in A1 synaptosome preparations would differ between individuals with schizophrenia and controls and (2) that these differences would not be explained by changes in total A1 homogenate protein levels.
RESULTS
Synaptosome and homogenate protein levels were investigated in 48 individuals with a schizophrenia diagnosis and 48 controls (mean age in both groups, 48 years [range, 17-83 years]); each group included 35 males (73%) and 13 females (27%). Robust alterations (statistical cutoff set at an adjusted Limma P < .05) were observed in synaptosome levels of canonical mitochondrial and postsynaptic proteins that were highly coregulated and not readily explained by postmortem interval, antipsychotic drug treatment, synaptosome yield, or underlying alterations in homogenate protein levels.
CONCLUSIONS AND RELEVANCE
These findings suggest a robust and highly coordinated rearrangement of the synaptic proteome. In line with unbiased genetic findings, alterations in synaptic levels of postsynaptic proteins were identified, providing a road map to identify the specific cells and circuits that are impaired in individuals with schizophrenia A1.
Topics: Adolescent; Adult; Aged; Aged, 80 and over; Animals; Auditory Cortex; Case-Control Studies; Female; Gray Matter; Humans; Male; Mass Spectrometry; Mice; Mice, Inbred C57BL; Middle Aged; Mitochondrial Proteins; Protein Interaction Maps; Proteome; Schizophrenia; Synapses; Synaptosomes; Young Adult
PubMed: 31642882
DOI: 10.1001/jamapsychiatry.2019.2974 -
Frontiers in Immunology 2020Microvilli are finger-like membrane protrusions, supported by the actin cytoskeleton, and found on almost all cell types. A growing body of evidence suggests that the... (Review)
Review
Microvilli are finger-like membrane protrusions, supported by the actin cytoskeleton, and found on almost all cell types. A growing body of evidence suggests that the dynamic lymphocyte microvilli, with their highly curved membranes, play an important role in signal transduction leading to immune responses. Nevertheless, challenges in modulating local membrane curvature and monitoring the high dynamicity of microvilli hampered the investigation of the curvature-generation mechanism and its functional consequences in signaling. These technical barriers have been partially overcome by recent advancements in adapted super-resolution microscopy. Here, we review the up-to-date progress in understanding the mechanisms and functional consequences of microvillus formation in T cell signaling. We discuss how the deformation of local membranes could potentially affect the organization of signaling proteins and their biochemical activities. We propose that curved membranes, together with the underlying cytoskeleton, shape microvilli into a unique compartment that sense and process signals leading to lymphocyte activation.
Topics: Actin Cytoskeleton; Animals; Carrier Proteins; Cell Line; Cell Membrane; Cell Shape; Cyclodextrins; Cytokines; Glycocalyx; Humans; Lymphocyte Activation; Membrane Lipids; Membrane Proteins; Mice; Microfilament Proteins; Microscopy, Electron, Scanning; Microvilli; Receptors, Antigen, T-Cell; Signal Transduction; Stress, Mechanical; Surface Properties; Synaptosomes; T-Lymphocytes
PubMed: 33013920
DOI: 10.3389/fimmu.2020.02187 -
Translational Psychiatry Dec 2022Early-life stress (ELS) leads to increased vulnerability to psychiatric disorders including depression later in life. Neuroinflammatory processes have been implicated in...
Early-life stress (ELS) leads to increased vulnerability to psychiatric disorders including depression later in life. Neuroinflammatory processes have been implicated in ELS-induced negative health outcomes, but how ELS impacts microglia, the main tissue-resident macrophages of the central nervous system, is unknown. Here, we determined the effects of ELS-induced by limited bedding and nesting material during the first week of life (postnatal days [P]2-9) on microglial (i) morphology; (ii) hippocampal gene expression; and (iii) synaptosome phagocytic capacity in male pups (P9) and adult (P200) mice. The hippocampus of ELS-exposed adult mice displayed altered proportions of morphological subtypes of microglia, as well as microglial transcriptomic changes related to the tumor necrosis factor response and protein ubiquitination. ELS exposure leads to distinct gene expression profiles during microglial development from P9 to P200 and in response to an LPS challenge at P200. Functionally, synaptosomes from ELS-exposed mice were phagocytosed less by age-matched microglia. At P200, but not P9, ELS microglia showed reduced synaptosome phagocytic capacity when compared to control microglia. Lastly, we confirmed the ELS-induced increased expression of the phagocytosis-related gene GAS6 that we observed in mice, in the dentate gyrus of individuals with a history of child abuse using in situ hybridization. These findings reveal persistent effects of ELS on microglial function and suggest that altered microglial phagocytic capacity is a key contributor to ELS-induced phenotypes.
Topics: Animals; Child; Male; Mice; Adverse Childhood Experiences; Child Abuse; Transcriptome; Humans; Microglia; Phagocytosis; Synaptosomes; Hippocampus; Dentate Gyrus
PubMed: 36481769
DOI: 10.1038/s41398-022-02265-6 -
Cellular and Molecular Neurobiology Aug 2022Alzheimer's disease (AD) is the most common age-associated dementia with complex pathological hallmarks. Mitochondrion, synaptosome, and myelin sheath appear to be...
Proteomic Profiling of Cerebrum Mitochondria, Myelin Sheath, and Synaptosome Revealed Mitochondrial Damage and Synaptic Impairments in Association with 3 × Tg-AD Mice Model.
Alzheimer's disease (AD) is the most common age-associated dementia with complex pathological hallmarks. Mitochondrion, synaptosome, and myelin sheath appear to be vulnerable and play a key role in the pathogenesis of AD. To clarify the early mechanism associated with AD, followed by subcellular components separation, we performed iTRAQ (isobaric tags for relative and absolute quantification)-based proteomics analysis to simultaneously investigate the differentially expressed proteins (DEPs) within the mitochondria, synaptosome, and myelin sheath in the cerebrum of the 6-month-old triple transgenic AD (3 × Tg-AD) and 6-month-old wild-type (WT) mice. A large number of DEPs between the AD and WT mice were identified. Most of them are related to mitochondria and synaptic dysfunction and cytoskeletal protein change. Differential expressions of Lrpprc, Nefl, and Sirpa were verified by Western blot analysis. The results suggest that decreased energy metabolism, impaired amino acid metabolism and neurotransmitter synthesis, increase compensatory fatty acid metabolism, up-regulated cytoskeletal protein expression, and oxidative stress are the early events of AD. Among these, mitochondrial damage, synaptic dysfunction, decreased energy metabolism, and abnormal amino acid metabolism are the most significant events. The results indicate that it is feasible to separate and simultaneously perform proteomics analysis on the three subcellular components.
Topics: Alzheimer Disease; Amino Acids; Animals; Cerebrum; Cytoskeletal Proteins; Disease Models, Animal; Mice; Mice, Transgenic; Mitochondria; Myelin Sheath; Proteomics; Synaptosomes
PubMed: 33560469
DOI: 10.1007/s10571-021-01052-z -
Molecular Neurodegeneration Jun 2022Apolipoprotein E4 (APOE4) is associated with a greater response to neuroinflammation and the risk of developing late-onset Alzheimer's disease (AD), but the mechanisms...
BACKGROUND
Apolipoprotein E4 (APOE4) is associated with a greater response to neuroinflammation and the risk of developing late-onset Alzheimer's disease (AD), but the mechanisms for this association are not clear. The activation of calcium-dependent cytosolic phospholipase A (cPLA2) is involved in inflammatory signaling and is elevated within the plaques of AD brains. The relation between APOE4 genotype and cPLA2 activity is not known.
METHODS
Mouse primary astrocytes, mouse and human brain samples differing by APOE genotypes were collected for measuring cPLA2 expression, phosphorylation, and activity in relation to measures of inflammation and oxidative stress.
RESULTS
Greater cPLA2 phosphorylation, cPLA2 activity and leukotriene B4 (LTB4) levels were identified in ApoE4 compared to ApoE3 in primary astrocytes, brains of ApoE-targeted replacement (ApoE-TR) mice, and in human brain homogenates from the inferior frontal cortex of persons with AD dementia carrying APOE3/4 compared to APOE3/3. Higher phosphorylated p38 MAPK but not ERK1/2 was found in ApoE4 primary astrocytes and mouse brains than that in ApoE3. Greater cPLA2 translocation to cytosol was observed in human postmortem frontal cortical synaptosomes with recombinant ApoE4 than ApoE3 ex vivo. In ApoE4 astrocytes, the greater levels of LTB4, reactive oxygen species (ROS), and inducible nitric oxide synthase (iNOS) were reduced after cPLA2 inhibition.
CONCLUSIONS
Our findings implicate greater activation of cPLA2 signaling system with APOE4, which could represent a potential drug target for mitigating the increased neuroinflammation with APOE4 and AD.
Topics: Alzheimer Disease; Animals; Apolipoprotein E3; Apolipoprotein E4; Apolipoproteins E; Calcium; Group IV Phospholipases A2; Humans; Leukotriene B4; Mice; Mice, Transgenic; Neuroinflammatory Diseases; Oxidative Stress; Phospholipases A2, Cytosolic; Synaptosomes
PubMed: 35705959
DOI: 10.1186/s13024-022-00549-5