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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 -
Cytometry. Part a : the Journal of the... Sep 2021Mass-tag cell barcoding has increased the throughput, multiplexing, and robustness of multiple cytometry approaches. Previously, we adapted mass cytometry for cells to...
Mass-tag cell barcoding has increased the throughput, multiplexing, and robustness of multiple cytometry approaches. Previously, we adapted mass cytometry for cells to analyze synaptosome preparations (mass synaptometry or SynTOF), extending mass cytometry to these smaller, anuclear particles. To improve throughput and individual event resolution, we report here the application of palladium-based barcoding in human synaptosomes. Up to 20 individual samples, each with a unique combinatorial barcode, were pooled for labeling with an antibody cocktail. Our synaptosome protocol used six palladium-based barcoding reagents, and in combination with sequential gating increased the identification of presynaptic events approximately fourfold. These same parameters also efficiently resolved two other anuclear particles: human red blood cells and platelets. The addition of palladium-based mass-tag barcoding to our approach improves mass cytometry of synaptic particles.
Topics: Antibodies; Flow Cytometry; Humans; Synaptosomes
PubMed: 33818911
DOI: 10.1002/cyto.a.24340 -
The Journal of Biological Chemistry 2021The accurate retrieval of synaptic vesicle (SV) proteins during endocytosis is essential for the maintenance of neurotransmission. Synaptophysin (Syp) and...
The accurate retrieval of synaptic vesicle (SV) proteins during endocytosis is essential for the maintenance of neurotransmission. Synaptophysin (Syp) and synaptobrevin-II (SybII) are the most abundant proteins on SVs. Neurons lacking Syp display defects in the activity-dependent retrieval of SybII and a general slowing of SV endocytosis. To determine the role of the cytoplasmic C terminus of Syp in the control of these two events, we performed molecular replacement studies in primary cultures of Syp knockout neurons using genetically encoded reporters of SV cargo trafficking at physiological temperatures. Under these conditions, we discovered, 1) no slowing in SV endocytosis in Syp knockout neurons, and 2) a continued defect in SybII retrieval in knockout neurons expressing a form of Syp lacking its C terminus. Sequential truncations of the Syp C-terminus revealed a cryptic interaction site for the SNARE motif of SybII that was concealed in the full-length form. This suggests that a conformational change within the Syp C terminus is key to permitting SybII binding and thus its accurate retrieval. Furthermore, this study reveals that the sole presynaptic role of Syp is the control of SybII retrieval, since no defect in SV endocytosis kinetics was observed at physiological temperatures.
Topics: Endocytosis; Gene Knockout Techniques; Hippocampus; Neurons; Primary Cell Culture; SNARE Proteins; Synaptic Transmission; Synaptic Vesicles; Synaptophysin; Synaptosomes; Vesicle-Associated Membrane Protein 2
PubMed: 33769286
DOI: 10.1016/j.jbc.2021.100266 -
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 -
Biochimica Et Biophysica Acta. Proteins... Feb 2018Bartha, the pseudorabies virus (PRV) vaccine strain, is widely used in studies of neuronal circuit-tracing, due to its attenuated virulence and retrograde spreading....
Bartha, the pseudorabies virus (PRV) vaccine strain, is widely used in studies of neuronal circuit-tracing, due to its attenuated virulence and retrograde spreading. However, we know little regarding the molecular mechanisms of PRV infection and spreading between structurally connected neurons. In this study, we systematically analyzed the host brain proteomes after acute infection with PRV, attempting to identified the proteins involved in the processes. Mice were injected with PRV-Bartha and PRV-Becker (PRV-Bartha's wild-type parent strain) in the olfactory system, the proteomes of the brain and synaptosome were analyzed and compared at various infection intervals using mass spectrometry-based proteomics techniques. In all, we identified >100 PRV-infection regulated proteins at the whole-tissue level and the synaptosome level. While at whole-tissue level, bioinformatics analyses mapped most of the regulations to the inflammation pathways, at the synaptosome level, most of those to synaptic transmission, cargo transport and cytoskeleton organization. We established regulated protein networks demonstrating distinct cellular regulation pattern between the global and the synaptosome levels. Moreover, we identified a series of potentially PRV-strain-specific regulated proteins with diverse biological functions. This study may provide new clues for molecular mechanisms for PRV infection and spread.
Topics: Animals; Brain; Herpesvirus 1, Suid; Male; Mice; Nerve Tissue Proteins; Proteomics; Pseudorabies; Synaptosomes
PubMed: 29174846
DOI: 10.1016/j.bbapap.2017.11.010 -
Science (New York, N.Y.) Jul 2020Synapses connect neurons together to form the circuits of the brain, and their molecular composition controls innate and learned behavior. We analyzed the molecular and...
Synapses connect neurons together to form the circuits of the brain, and their molecular composition controls innate and learned behavior. We analyzed the molecular and morphological diversity of 5 billion excitatory synapses at single-synapse resolution across the mouse brain from birth to old age. A continuum of changes alters synapse composition in all brain regions across the life span. Expansion in synapse diversity produces differentiation of brain regions until early adulthood, and compositional changes cause dedifferentiation in old age. The spatiotemporal synaptome architecture of the brain potentially accounts for life-span transitions in intellectual ability, memory, and susceptibility to behavioral disorders.
Topics: Animals; Atlases as Topic; Brain; Datasets as Topic; Longevity; Male; Mice; Neurons; Synapses; Synaptosomes
PubMed: 32527927
DOI: 10.1126/science.aba3163 -
Brain : a Journal of Neurology Jul 2015Endophilin-B1, also known as Bax-interacting factor 1 (Bif-1, and encoded by SH3GLB1), is a multifunctional protein involved in apoptosis, autophagy and mitochondrial...
Endophilin-B1, also known as Bax-interacting factor 1 (Bif-1, and encoded by SH3GLB1), is a multifunctional protein involved in apoptosis, autophagy and mitochondrial function. We recently described a unique neuroprotective role for neuron-specific alternatively spliced isoforms of endophilin-B1. To examine whether endophilin-B1-mediated neuroprotection could be a novel therapeutic target for Alzheimer's disease we used a double mutant amyloid precursor protein and presenilin 1 (APPswe/PSEN1dE9) mouse model of Alzheimer's disease and observed that expression of neuron-specific endophilin-B1 isoforms declined with disease progression. To determine if this reduction in endophilin-B1 has a functional role in Alzheimer's disease pathogenesis, we crossed endophilin-B1(-/-) mice with APPswe/PSEN1dE9 mice. Deletion of endophilin-B1 accelerated disease onset and progression in 6-month-old APPswe/PSEN1dE9/endophilin-B1(-/-) mice, which showed more plaques, astrogliosis, synaptic degeneration, cognitive impairment and mortality than APPswe/PSEN1dE9 mice. In mouse primary cortical neuron cultures, overexpression of neuron-specific endophilin-B1 isoforms protected against amyloid-β-induced apoptosis and mitochondrial dysfunction. Additionally, protein and mRNA levels of neuron-specific endophilin-B1 isoforms were also selectively decreased in the cerebral cortex and in the synaptic compartment of patients with Alzheimer's disease. Flow sorting of synaptosomes from patients with Alzheimer's disease demonstrated a negative correlation between amyloid-β and endophilin-B1 levels. The importance of endophilin-B1 in neuronal function was further underscored by the development of synaptic degeneration and cognitive and motor impairment in endophilin-B1(-/-) mice by 12 months. Our findings suggest that endophilin-B1 is a key mediator of a feed-forward mechanism of Alzheimer's disease pathogenesis where amyloid-β reduces neuron-specific endophilin-B1, which in turn enhances amyloid-β accumulation and neuronal vulnerability to stress.
Topics: Adaptor Proteins, Signal Transducing; Aged, 80 and over; Alzheimer Disease; Animals; Blotting, Western; Cells, Cultured; Disease Models, Animal; Female; Flow Cytometry; Fluorescent Antibody Technique; Humans; Immunoblotting; Male; Maze Learning; Mice; Mice, Inbred C57BL; Mice, Knockout; Neurons; Reverse Transcriptase Polymerase Chain Reaction; Synaptosomes
PubMed: 25981964
DOI: 10.1093/brain/awv128 -
Journal of Neuroscience Methods Jan 2019Synaptic alterations, especially presynaptic changes, are cardinal features of neurodegenerative diseases and strongly correlate with cognitive decline.
BACKGROUND
Synaptic alterations, especially presynaptic changes, are cardinal features of neurodegenerative diseases and strongly correlate with cognitive decline.
NEW METHOD
We report "Mass Synaptometry" for the high-dimensional analysis of individual human synaptosomes, enriched nerve terminals from brain. This method was adapted from cytometry by time-of-flight mass spectrometry (CyTOF), which is commonly used for single-cell analysis of immune and blood cells.
RESULT
Here we overcome challenges for single synapse analysis by optimizing synaptosome preparations, generating a 'SynTOF panel,' recalibrating acquisition settings, and applying computational analyses. Through the analysis of 390,000 individual synaptosomes, we also provide proof-of principle validation by characterizing changes in synaptic diversity in Lewy Body Disease (LBD), Alzheimer's disease and normal brain.
COMPARISON WITH EXISTING METHOD(S)
Current imaging methods to study synapses in humans are capable of analyzing a limited number of synapses, and conventional flow cytometric techniques are typically restricted to fewer than 6 parameters. Our method allows for the simultaneous detection of 34 parameters from tens of thousands of individual synapses.
CONCLUSION
We applied Mass Synaptometry to analyze 34 parameters simultaneously on more than 390,000 synaptosomes from 13 human brain samples. This new approach revealed regional and disease-specific changes in synaptic phenotypes, including validation of this method with the expected changes in the molecular composition of striatal dopaminergic synapses in Lewy body disease and Alzheimer's disease. Mass synaptometry enables highly parallel molecular profiling of individual synaptic terminals.
Topics: Alzheimer Disease; Brain; Computational Biology; Humans; Lewy Body Disease; Mass Spectrometry; Single-Cell Analysis; Synapses; Synaptosomes
PubMed: 30465796
DOI: 10.1016/j.jneumeth.2018.11.008 -
European Neuropsychopharmacology : the... Apr 2021Cinazepam CHBrClNO, ("Levana ІC") a partial GABA receptor agonist, and its active metabolite 3-hydroxyphenazepam CHBrClNO were comparatively assessed in vitro using...
Cinazepam CHBrClNO, ("Levana ІC") a partial GABA receptor agonist, and its active metabolite 3-hydroxyphenazepam CHBrClNO were comparatively assessed in vitro using nerve terminals isolated from rat cortex (synaptosomes). At the presynaptic site, cinazepam (100 and 200 µM) facilitated synaptosomal transporter-mediated [H]GABA uptake by enhancing both the initial rate and accumulation, and decreased the ambient level and transporter-mediated release of [H]GABA. Whereas, 3-hydroxyphenazepam decreased the uptake and did not change the ambient synaptosomal level and transporter-mediated release of [H]GABA. To exclude GABA transporter influence, NO-711, the transporter blocker, was applied and it was found that exocytotic release of [H]GABA decreased, whereas tonic release of [H]GABA was not changed in the presence of both cinazepam or 3-hydroxyphenazepam after treatment of synaptosomes with NO-711. In fluorimetric studies using potential- and pH-sensitive dyes rhodamine 6G and acridine orange, respectively, it was found that cinazepam hyperpolarized the synaptosomal plasma membrane, and increased synaptic vesicle acidification, whereas, 3-hydroxyphenazepam demonstrated opposite effects on these parameters. Therefore, action of cinazepam and its active metabolite 3-hydroxyphenazepam on GABAergic neurotransmission was different. Therapeutic effects of cinazepam can be associated with its ability to hyperpolarize the plasma membrane, to increase synaptic vesicle acidification and capacity of its active metabolite 3-hydroxyphenazepam to inhibit GABA transporter functioning.
Topics: Animals; Benzodiazepines; Benzodiazepinones; GABA Plasma Membrane Transport Proteins; GABA-A Receptor Agonists; Presynaptic Terminals; Rats; Rats, Wistar; Receptors, GABA-A; Synaptosomes; gamma-Aminobutyric Acid
PubMed: 33820715
DOI: 10.1016/j.euroneuro.2021.03.013 -
Journal of Neuroscience Methods Apr 2016Functional and structural properties of mitochondria are highly tissue and cell dependent, but isolation of highly purified human neuronal mitochondria is not currently...
BACKGROUND
Functional and structural properties of mitochondria are highly tissue and cell dependent, but isolation of highly purified human neuronal mitochondria is not currently available.
NEW METHOD
We developed and validated a procedure to isolate purified neuronal mitochondria from brain tissue. The method combines Percoll gradient centrifugation to obtain synaptosomal fraction with nitrogen cavitation mediated synaptosome disruption and extraction of mitochondria using anti mitochondrial outer membrane protein antibodies conjugated to magnetic beads. The final products of isolation are non-synaptosomal mitochondria, which are a mixture of mitochondria isolated from different brain cells (i.e. neurons, astrocytes, oligodendrocytes, microglia) and synaptic mitochondria, which are of neuronal origin. This method is well suited for preparing functional mitochondria from human cortex tissue that is surgically extracted.
RESULTS
The procedure produces mitochondria with minimal cytoplasmic contaminations that are functionally active based on measurements of mitochondrial respiration as well as mitochondrial protein import. The procedure requires approximately four hours for the isolation of human neuronal mitochondria and can also be used to isolate mitochondria from mouse/rat/monkey brains.
COMPARISON WITH EXISTING METHODS AND CONCLUSIONS
This method will allow researchers to study highly enriched neuronal mitochondria without the confounding effect of cellular and organelle contaminants.
Topics: Antibodies; Cell Fractionation; Cerebral Cortex; HLA Antigens; Humans; Membrane Potential, Mitochondrial; Mitochondria; Mitochondrial Membrane Transport Proteins; Mitochondrial Precursor Protein Import Complex Proteins; Mitochondrial Proteins; Neurons; Synaptosomes
PubMed: 26808294
DOI: 10.1016/j.jneumeth.2016.01.017