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Amino Acids Dec 2020Synaptosomes are frequently used research objects in neurobiology studies focusing on synaptic transmission as they mimic several aspects of the physiological synaptic...
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 homogenization of nervous tissue in the isoosmotic milieu and can be isolated from the homogenate by various approaches. Each enrichment method has its own benefits and drawbacks and there is not a single method that is optimal for all research purposes. For a proper proteomic experiment, it is desirable to preserve the native synaptic structure during the isolation procedure and keep the degree of contamination from other organelles or cell types as low as possible. In this article, we examined five synaptosome isolation methods from a proteomic point of view by the means of electron microscopy, Western blot, and liquid chromatography-mass spectrometry to compare their efficiency in the isolation of synaptosomes and depletion of contaminating subcellular structures. In our study, the different isolation procedures led to a largely overlapping pool of proteins with a fairly similar distribution of presynaptic, active zone, synaptic vesicle, and postsynaptic proteins; however, discrete differences were noticeable in individual postsynaptic proteins and in the number of identified transmembrane proteins. Much pronounced variance was observed in the degree of contamination with mitochondrial and glial structures. Therefore, we suggest that in selecting the appropriate isolation method for any neuroproteomics experiment carried out on synaptosomes, the degree and sort/source of contamination should be considered as a primary aspect.
Topics: Animals; Brain; Chromatography, Liquid; Humans; Mass Spectrometry; Membrane Potentials; Membrane Proteins; Microscopy, Electron; Mitochondria; Presynaptic Terminals; Proteomics; Rats; Synapses; Synaptic Transmission; Synaptosomes
PubMed: 33211194
DOI: 10.1007/s00726-020-02912-6 -
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 -
International Journal of Molecular... Aug 2021Retinitis pigmentosa (RP) is a family of inherited disorders caused by the progressive degeneration of retinal photoreceptors. There is no cure for RP, but recent...
Retinitis pigmentosa (RP) is a family of inherited disorders caused by the progressive degeneration of retinal photoreceptors. There is no cure for RP, but recent research advances have provided promising results from many clinical trials. All these therapeutic strategies are focused on preserving existing photoreceptors or substituting light-responsive elements. Vision recovery, however, strongly relies on the anatomical and functional integrity of the visual system beyond photoreceptors. Although the retinal structure and optic pathway are substantially preserved at least in early stages of RP, studies describing the visual cortex status are missing. Using a well-established mouse model of RP, we analyzed the response of visual cortical circuits to the progressive degeneration of photoreceptors. We demonstrated that the visual cortex goes through a transient and previously undescribed alteration in the local excitation/inhibition balance, with a net shift towards increased intracortical inhibition leading to improved filtering and decoding of corrupted visual inputs. These results suggest a compensatory action of the visual cortex that increases the range of residual visual sensitivity in RP.
Topics: Animals; Female; Male; Mice; Mice, Inbred C57BL; Neurotransmitter Agents; Photoreceptor Cells, Vertebrate; Retinitis Pigmentosa; Synaptosomes; Visual Cortex
PubMed: 34502320
DOI: 10.3390/ijms22179412 -
The Journal of Pharmacology and... Nov 2022Using synaptosomes purified from the brains of two transgenic mouse models overexpressing mutated human tau (TgP301S and Tg4510) and brains of patients with sporadic...
Using synaptosomes purified from the brains of two transgenic mouse models overexpressing mutated human tau (TgP301S and Tg4510) and brains of patients with sporadic Alzheimer's disease, we showed that aggregated and hyperphosphorylated tau was both present in purified synaptosomes and released in a calcium- and synaptosome-associated protein of 25 kDa (SNAP25)-dependent manner. In all mouse and human synaptosomal preparations, tau release was inhibited by the selective metabotropic glutamate receptor 2/3 (mGluR2/3) agonist LY379268, an effect prevented by the selective mGlu2/3 antagonist LY341495. LY379268 was also able to block pathologic tau propagation between primary neurons in an in vitro microfluidic cellular model. These novel results are transformational for our understanding of the molecular mechanisms mediating tau release and propagation at synaptic terminals in Alzheimer's disease and suggest that these processes could be inhibited therapeutically by the selective activation of presynaptic G protein-coupled receptors. SIGNIFICANCE STATEMENT: Pathological tau release and propagation are key neuropathological events underlying cognitive decline in Alzheimer's disease patients. This paper describes the role of regulated exocytosis, and the soluble N-ethylmaleimide-sensitive factor attachment receptor (SNARE) protein SNAP25, in mediating tau release from rodent and human synaptosomes. This paper also shows that a selective mGluR2/3 agonist is highly effective in blocking tau release from synaptosomes and tau propagation between neurons, opening the way to the discovery of novel therapeutic approaches to this devastating disease.
Topics: Alzheimer Disease; Animals; Calcium; Exocytosis; Humans; Mice; N-Ethylmaleimide-Sensitive Proteins; Receptors, Metabotropic Glutamate; SNARE Proteins; Synaptosomes; tau Proteins
PubMed: 36116796
DOI: 10.1124/jpet.122.001307 -
Methods in Molecular Biology (Clifton,... 2017For several decades, neurobiologists have used subcellular fractionation methods to analyze the molecular structure and some functional features of the cells in the...
For several decades, neurobiologists have used subcellular fractionation methods to analyze the molecular structure and some functional features of the cells in the central nervous system. Indeed, brain tissue contains a complex intermingled network of neuronal, glial, and vascular cells. To reduce this complexity biochemists have optimized fractionation protocols that enrich in specific compartments such as synapses (called "synaptosomes") and synaptic vesicles, for example. However, recently, these approaches suffered from a lack of specificity and purity. In a recent effort, we extended the conventional synaptosome preparation to purify fluorescent synaptosomes on a cell sorter. We could prove that our method allows for the steep enrichment in fluorescent excitatory VGLUT1 synaptosomes containing the presynaptic element and the tip of the post-synaptic element and a strong depletion in neuronal and glial contaminants. Here, we propose a detailed procedure for the implementation of Fluorescence Activated Synaptosome Sorting.
Topics: Animals; Brain; Cell Fractionation; Flow Cytometry; Mice; Synapses; Synaptic Transmission; Synaptic Vesicles; Synaptosomes; Ultracentrifugation
PubMed: 27943188
DOI: 10.1007/978-1-4939-6688-2_10 -
International Journal of Biological... Nov 2019Naja spp. venom is a natural source of active compounds with therapeutic application potential. Phospholipase A (PLA) is abundant in the venom of Naja spp. and can... (Review)
Review
Naja spp. venom is a natural source of active compounds with therapeutic application potential. Phospholipase A (PLA) is abundant in the venom of Naja spp. and can perform neurotoxicity, cytotoxicity, cardiotoxicity, and hematological disorders. The PLAs from Naja spp. venoms are Asp 49 isoenzymes with the exception of PLA Cys 49 from Naja sagittifera. When looking at the functional aspects, the neurotoxicity occurs by PLA called β-toxins that have affinity for phosphatidylcholine in nerve endings and synaptosomes membranes, and by α-toxins that block the nicotinic acetylcholine receptors in the neuromuscular junctions. In addition, these neurotoxins may inhibit K and Ca channels or even interfere with the Na/K/ATPase enzyme. The disturbance in the membrane fluidity also results in inhibition of the release of acetylcholine. The PLA can act as anticoagulants or procoagulant. The cytotoxicity exerted by PLAs result from changes in the cardiomyocyte membranes, triggering cardiac failure and hemolysis. The antibacterial activity, however, is the result of alterations that decrease the stability of the lipid bilayer. Thus, the understanding of the structural and functional aspects of PLAs can contribute to studies on the toxic and therapeutic mechanisms involved in the envenomation by Naja spp. and in the treatment of pathologies.
Topics: Animals; Cobra Neurotoxin Proteins; Naja; Neuromuscular Junction; Phosphatidylcholines; Phospholipases A2; Structure-Activity Relationship; Synaptosomes
PubMed: 31421173
DOI: 10.1016/j.ijbiomac.2019.08.125 -
Journal of Visualized Experiments : JoVE Sep 2022Synaptic terminals are the primary sites of neuronal communication. Synaptic dysfunction is a hallmark of many neuropsychiatric and neurological disorders. The...
Synaptic terminals are the primary sites of neuronal communication. Synaptic dysfunction is a hallmark of many neuropsychiatric and neurological disorders. The characterization of synaptic sub-compartments by biochemical isolation is, therefore, a powerful method to elucidate the molecular bases of synaptic processes, both in health and disease. This protocol describes the isolation of synaptic terminals and synaptic sub-compartments from mouse brains by subcellular fractionation. First, sealed synaptic terminal structures, known as synaptosomes, are isolated following brain tissue homogenization. Synaptosomes are neuronal pre- and post-synaptic compartments with pinched-off and sealed membranes. These structures retain a metabolically active state and are valuable for studying synaptic structure and function. The synaptosomes are then subjected to hypotonic lysis and ultracentrifugation to obtain synaptic sub-compartments enriched for synaptic vesicles, synaptic cytosol, and synaptic plasma membrane. Fraction purity is confirmed by electron microscopy and biochemical enrichment analysis for proteins specific to sub-synaptic compartments. The presented method is a straightforward and valuable tool for studying the structural and functional characteristics of the synapse and the molecular etiology of various brain disorders.
Topics: Animals; Brain; Cell Fractionation; Mice; Subcellular Fractions; Synaptic Membranes; Synaptic Vesicles; Synaptosomes
PubMed: 36190269
DOI: 10.3791/64574 -
Journal of Bioenergetics and... Apr 2015Mitochondria play multiple roles in the maintenance of neuronal function under physiological and pathological conditions. In addition to ATP generation, they can act as... (Review)
Review
Mitochondria play multiple roles in the maintenance of neuronal function under physiological and pathological conditions. In addition to ATP generation, they can act as major short-term calcium sinks and can both generate, and be damaged by, reactive oxygen species. Two complementary preparations have been extensively employed to investigate in situ neuronal mitochondrial bioenergetics, primary neuronal cultures and acutely isolated nerve terminals, synaptosomes. A major focus of the cell culture preparation has been the investigation of glutamate excitotoxicity. Oxidative phosphorylation, calcium transport and reactive oxygen species play complex interlocking roles in the life and death of the glutamate exposed neuron. Synaptosomes may be isolated from specific brain regions at any developmental stage and therefore provide a valuable ex vivo approach in studying mouse models. Recent advances have allowed synaptosomal bioenergetics to be studied on a microgram scale, and, in combination with approaches to correct for functional and transmitter heterogeneity, have allowed hypotheses concerning presynaptic mitochondrial dysfunction to be tested on a variety of genetic models of neurodegenerative disorders.
Topics: Animals; Cell Culture Techniques; Cell Survival; Energy Metabolism; Mice; Mitochondria; Neurons; Synaptosomes
PubMed: 25172197
DOI: 10.1007/s10863-014-9573-9 -
Journal of Chemical Neuroanatomy Nov 2020Alzheimer disease's (AD) is a neurodegenerative disease induced by amyloid-β (Aβ) aggregation and accumulation of neurotoxic metals in the brain. Fraxinus angustifolia...
Alzheimer disease's (AD) is a neurodegenerative disease induced by amyloid-β (Aβ) aggregation and accumulation of neurotoxic metals in the brain. Fraxinus angustifolia Vahl. (Oleaceae) is a Mediterranean plant traditionally used to treat several human problems as nervous system problems. This study aimed to evaluate the neuroprotective effects of F. angustifolia Vahl. bark extract (FAB) in vitro and in vivo against Aβ-aggregation and aluminium induced-neurotoxicity in mice. FAB was characterized by colorimetric methods and its individual compounds were identified and quantified by LC-MS. First, the neuroprotective effect of FAB was evaluated against Aβ-aggregation where it was directly incubated with Aβ and the kinetic of aggregation was measured by spectrophotometer at 200 nm. Then, the extract was tested against Aβ-induced cytotoxicity on PC12 cells and the cells viability was determined by MTT test. On the other hand, FAB (0.01-0.5 mg/mL) was tested against aluminium-activated lipid peroxidation in mice synaptosomal membranes, and in vivo against aluminium-caused neurotoxicity in male N.M.R.I. (Naval Medical Research Institute) mice; this test consisted of daily co-administration of the extract with Al for 60 days. At the end of the treatment, behavioral and memory tests (locomotor activity, black and white and Morris water maze tests) and histological analysis were realized. The identification and quantification of FAB phenolics revealed the presence of different phenolic classes with high concentration of phenylethanoids and hydroxycoumarins. FAB showed a high Aβ anti-aggregative effect and a dose dependent protective effect on PC12 cells. The extract also demonstrated a significant inhibition of lipid peroxidation and was found to prevent the Al harmful effects where it significantly increased the locomotor activity, decreased the anxiety, improved memory and reduced histological alterations. In conclusion, FAB is rich of bioactive compounds that gave it the ability to inhibit Aβ-aggregation and Al-caused neurotoxicity in mice.
Topics: Alzheimer Disease; Amyloid beta-Peptides; Animals; Cognition; Disease; Fraxinus; Lipid Peroxidation; Memory; Mice; Neuroprotective Agents; PC12 Cells; Peptide Fragments; Plant Extracts; Rats; Spatial Memory; Synaptosomes
PubMed: 32645433
DOI: 10.1016/j.jchemneu.2020.101848 -
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