-
Mitochondrion Nov 2023Mitochondrial function at synapses can be assessed in isolated nerve terminals. Synaptosomes are structures obtained in vitro by detaching the nerve endings from...
Mitochondrial function at synapses can be assessed in isolated nerve terminals. Synaptosomes are structures obtained in vitro by detaching the nerve endings from neuronal bodies under controlled homogenization conditions. Several protocols have been described for the preparation of intact synaptosomal fractions. Herein a fast and economical method to obtain synaptosomes with optimal intrasynaptic mitochondria functionality was described. Synaptosomal fractions were obtained from mouse brain cortex by differential centrifugation followed by centrifugation in a Ficoll gradient. The characteristics of the subcellular particles obtained were analyzed by flow cytometry employing specific tools. Integrity and specificity of the obtained organelles were evaluated by calcein and SNAP-25 probes. The proportion of positive events of the synaptosomal preparation was 75 ± 2 % and 48 ± 7% for calcein and Synaptosomal-Associated Protein of 25 kDa (SNAP-25), respectively. Mitochondrial integrity was evaluated by flow cytometric analysis of cardiolipin content, which indicated that 73 ± 1% of the total events were 10 N-nonylacridine orange (NAO)-positive. Oxygen consumption, ATP production and mitochondrial membrane potential determinations showed that mitochondria inside synaptosomes remained functional after the isolation procedure. Mitochondrial and synaptosomal enrichment were determined by measuring synaptosomes/ homogenate ratio of specific markers. Functionality of synaptosomes was verified by nitric oxide detection after glutamate addition. As compared with other methods, the present protocol can be performed briefly, does not imply high economic costs, and provides an useful tool for the isolation of a synaptosomal preparation with high mitochondrial respiratory capacity and an adequate integrity and function of intraterminal mitochondria.
Topics: Mice; Animals; Synaptosomes; Mitochondria; Energy Metabolism; Brain; Cerebral Cortex
PubMed: 37944836
DOI: 10.1016/j.mito.2023.10.002 -
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 -
Journal of Neurochemistry Dec 1986Mouse cortical synaptosomal structure and function are altered when exposed to hypoxanthine/xanthine oxidase (HPX/XOD)-generated active oxygen/free radical species. The...
Mouse cortical synaptosomal structure and function are altered when exposed to hypoxanthine/xanthine oxidase (HPX/XOD)-generated active oxygen/free radical species. The structure of both the synaptic vesicle and plasma membrane systems are altered by HPX/XOD treatment. The alteration of synaptic vesicle structure is exhibited by a significant increase in the cumulative length of nonsynaptic vesicle membrane per nerve terminal. With respect to the nerve terminal plasma membrane, the length of the perimeter of the synaptosome is increased as the membrane pulls away from portions of the terminal in blebs. The functional lesion generated by HPX/XOD treatment results in a reduction in selective high-affinity gamma-[14C]aminobutyric acid (GABA) uptake. Kinetic analysis of the reduction in high-affinity uptake reveals that the Vmax is significantly altered whereas the Km is not. Preincubation with specific active oxygen/free radical scavengers indicates that the super-oxide radical is directly involved. This radical, most probably in the protonated perhydroxyl form, initiates lipid peroxidative damage of the synaptosomal membrane systems. Low-affinity [14C]GABA transport is unaltered by the HPX/XOD treatment. The apparent ineffectiveness of free radical exposure on low-affinity [14C]GABA transport coupled with its effectiveness in reducing high-affinity transport supports the idea that two separate and different amino acid uptake systems exist in CNS tissue, with the high-affinity being more sensitive (lipid-dependent) and/or more energy-dependent (Na+,K+-ATPase) than the low-affinity system.
Topics: Animals; Cerebral Cortex; Free Radicals; Kinetics; Mice; Mice, Inbred BALB C; Microscopy, Electron; Superoxides; Synaptic Membranes; Synaptosomes; Xanthine Oxidase; gamma-Aminobutyric Acid
PubMed: 3021906
DOI: 10.1111/j.1471-4159.1986.tb13092.x -
Biochemical and Biophysical Research... Jan 2023ALS2/alsin, the causative gene product for a number of juvenile recessive motor neuron diseases, acts as a guanine nucleotide exchange factor (GEF) for Rab5, regulating...
ALS2/alsin, the causative gene product for a number of juvenile recessive motor neuron diseases, acts as a guanine nucleotide exchange factor (GEF) for Rab5, regulating early endosome trafficking and maturation. It has been demonstrated that ALS2 forms a tetramer, and this oligomerization is essential for its GEF activity and endosomal localization in established cancer cells. However, despite that ALS2 deficiency is implicated in neurological diseases, neither the subcellular distribution of ALS2 nor the form of its complex in the central nervous system (CNS) has been investigated. In this study, we showed that ALS2 in the brain was enriched both in synaptosomal and cytosolic fractions, while those in the liver were almost exclusively present in cytosolic fraction by differential centrifugation. Gel filtration chromatography revealed that cytosolic ALS2 prepared both from the brain and liver formed a tetramer. Remarkably, synaptosomal ALS2 existed as a high-molecular weight complex in addition to a tetramer. Such complex was also observed not only in embryonic brain but also several neuronal and glial cultures, but not in fibroblast-derived cell lines. Thus, the high-molecular weight ALS2 complex represents a unique form of ALS2-homophilic oligomers in the CNS, which may play a role in the maintenance of neural function.
Topics: Animals; Mice; Amyotrophic Lateral Sclerosis; Brain; Central Nervous System; Endosomes; Guanine Nucleotide Exchange Factors; Molecular Weight; Synaptosomes
PubMed: 36459881
DOI: 10.1016/j.bbrc.2022.11.061 -
Biochemistry and Cell Biology =... Jan 1990To study lipid breakdown in brain membranes following hemorrhage, synaptosome and myelin fractions isolated from rat brain were incubated with rat serum. After 3 h in...
To study lipid breakdown in brain membranes following hemorrhage, synaptosome and myelin fractions isolated from rat brain were incubated with rat serum. After 3 h in vitro at 37 degrees C, 0.43 and 0.26 mumol of fatty acid were released in incubations containing synaptosomes (1.37 mumols phospholipid) or myelin (1.23 mumols phospholipid), respectively, in the presence of 0.25 mL serum. Less than 0.05 mumol of fatty acid was liberated in incubations containing only serum, synaptosomes, or myelin. For synaptosomes and serum, docosahexaenoate was the principal fatty acid released (28 mol% of total) after 3 h of incubation. This fatty acid and arachidonate made up 43 mol% of the liberated fatty acid. The presence of free docosahexaenoate was of interest, as this fatty acid is particularly enriched in phosphatidylserine and phosphatidylethanolamine, phospholipids found in the cytoplasmic half of the synaptosomal plasma membrane and in interior synaptosomal membranes. In incubations of serum and myelin, oleate was the major free fatty acid produced in 30 min to 3 h of incubation (29-35 mol% of total). After 3 h, docosahexaenoate contributed 20 mol% to the total. The release of fatty acids from the membranes may be mediated by serum phospholipase(s) or possibly by activated endogenous lipolytic activities.
Topics: Animals; Blood Physiological Phenomena; Brain; Fatty Acids; In Vitro Techniques; Kinetics; Male; Myelin Proteins; Rats; Rats, Inbred Strains; Synaptosomes
PubMed: 2350483
DOI: 10.1139/o90-020 -
Neurobiology of Aging Aug 2017Normal aging is associated with impairments in cognitive functions. These alterations are caused by diminutive changes in the biology of synapses, and ineffective...
Normal aging is associated with impairments in cognitive functions. These alterations are caused by diminutive changes in the biology of synapses, and ineffective neurotransmission, rather than loss of neurons. Hitherto, only a few studies, exploring molecular mechanisms of healthy brain aging in higher vertebrates, utilized synaptosomal fractions to survey local changes in aging-related transcriptome dynamics. Here we present, for the first time, a comparative analysis of the synaptosomes transcriptome in the aging mouse brain using RNA sequencing. Our results show changes in the expression of genes contributing to biological pathways related to neurite guidance, synaptosomal physiology, and RNA splicing. More intriguingly, we also discovered alterations in the expression of thousands of novel, unannotated lincRNAs during aging. Further, detailed characterization of the cleavage and polyadenylation factor I subunit 1 (Clp1) mRNA and protein expression indicates its increased expression in neuronal processes of hippocampal stratum radiatum in aging mice. Together, our study uncovers a new layer of transcriptional regulation which is targeted by aging within the local environment of interconnecting neuronal cells.
Topics: Aging; Animals; Brain; Cognition; Gene Expression; Hippocampus; Mice, Inbred C57BL; Nuclear Proteins; Phosphotransferases; Polyadenylation; RNA Splicing; RNA, Long Noncoding; RNA, Messenger; RNA, Untranslated; Sequence Analysis, RNA; Synaptic Transmission; Synaptosomes; Transcription Factors; Transcriptome
PubMed: 28499146
DOI: 10.1016/j.neurobiolaging.2017.04.005 -
Current Microbiology Sep 2016It is well established now that neuronal dysfunction rather than structural damage may be responsible for the development of rabies. In order to explore the underlying...
It is well established now that neuronal dysfunction rather than structural damage may be responsible for the development of rabies. In order to explore the underlying mechanisms in rabies virus (RABV) and synaptic dysfunctions, a quantitative proteome profiling was carried out on synaptosome samples from mice hippocampus. Synaptosome samples from mice hippocampus were isolated and confirmed by Western blot and transmission electron microscopy. Synaptosome protein content changes were quantitatively detected by Nano-LC-MS/MS. Protein functions were classified by the Gene Ontology (GO) and KEGG pathway. PSICQUIC was used to create a network. MCODE algorithm was applied to obtain subnetworks. Of these protein changes, 45 were upregulated and 14 were downregulated following RABV infection relative to non-infected (mock) synaptosomes. 28 proteins were unique to mock treatment and 12 were unique to RABV treatment. Proteins related to metabolism and synaptic vesicle showed the most changes in expression levels. Furthermore, protein-protein interaction (PPI) networks revealed that several key biological processes related to synaptic functions potentially were modulated by RABV, including energy metabolism, cytoskeleton organization, and synaptic transmission. These data will be useful for better understanding of neuronal dysfunction of rabies and provide the foundation for future research.
Topics: Animals; Electrophoresis, Polyacrylamide Gel; Gene Expression Profiling; Hippocampus; Humans; Male; Mass Spectrometry; Mice; Mice, Inbred C57BL; Proteome; Rabies; Rabies virus; Stress, Physiological; Synaptosomes
PubMed: 27155843
DOI: 10.1007/s00284-016-1061-5 -
Brain & Development Apr 2008Specific aim of this study is to elucidate the direct effects of L-arginine on the synaptosomal neurotransmission related to the mitochondrial respiratory function.
OBJECTIVE
Specific aim of this study is to elucidate the direct effects of L-arginine on the synaptosomal neurotransmission related to the mitochondrial respiratory function.
METHODS
Using isolated endbrains from wild-type mice (ICR), crude synaptosome was analyzed for their concentration of gamma-aminobutyric acid (GABA) and glutamate (Glu) with/without addition of L-arginine. We analyzed the contents of releasing amino acids evoked by high potassium condition and uptake of them in three separated fractions (cytosol, vesicles, and intact mitochondria). The oxygen consumption was also measured by oxygen electrode.
RESULTS
The entire uptakes of GABA and Glu were inhibited by rotenone (about 30 nmol/mg protein) with dose-dependent manner and showed a plateau at about 70% of total uptake. L-arginine inhibited the uptake of Glu logarithmically, however it showed no change in uptake of GABA. The contents of GABA and Glu in synaptosome were decreased in the presence of L-arginine. L-arginine enhanced the respiration of state II by succinate on synaptosomal respiration, although the respiration of synaptosomal mitochondrial fraction and the respiratory chains enzyme activities were almost unaffected by L-arginine. In the presence of rotenone, L-arginine decreased the uptake of Glu without changing the uptake of GABA, increased the releasing of GABA, and may modulate the excitability of neuronal state on the cytosol, cytomembrane, and/or organelles except for mitochondria.
CONCLUSIONS
L-arginine may modulate excitation by neurotransmitters at nerve endings, in relation to potentiated respiratory metabolism of succinate in synaptosomes. Such effects might contribute to alleviation of stroke-like symptoms in MELAS.
Topics: Animals; Arginine; Dose-Response Relationship, Drug; Glutamic Acid; In Vitro Techniques; MELAS Syndrome; Mice; Mice, Inbred ICR; Mitochondria; Oxygen Consumption; Potassium; Succinic Acid; Synaptosomes; gamma-Aminobutyric Acid
PubMed: 17889473
DOI: 10.1016/j.braindev.2007.08.007 -
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 -
Alcohol (Fayetteville, N.Y.) Jun 2019Alcohol hangover (AH) has been associated with oxidative stress and mitochondrial dysfunction. We herein postulate that AH-induced mitochondrial alterations can be due...
Alcohol hangover (AH) has been associated with oxidative stress and mitochondrial dysfunction. We herein postulate that AH-induced mitochondrial alterations can be due to a different pattern of response in synaptosomes and non-synaptic (NS) mitochondria. Mice received intraperitoneal (i.p.) injections of ethanol (3.8 g/kg) or saline and were sacrificed 6 h afterward. Brain cortex NS mitochondria and synaptosomes were isolated by Ficoll gradient. Oxygen consumption rates were measured in NS mitochondria and synaptosomes by high-resolution respirometry. Results showed that NS-synaptic mitochondria from AH animals presented a 26% decrease in malate-glutamate state 3 respiration, a 64% reduction in ATP content, 28-37% decrements in ATP production rates (malate-glutamate or succinate-dependent, respectively), and 44% inhibition in complex IV activity. No changes were observed in mitochondrial transmembrane potential (ΔΨ) or in UCP-2 expression in NS-mitochondria. Synaptosome respiration driving proton leak (in the presence of oligomycin), and spare respiratory capacity (percentage ratio between maximum and basal respiration) were 30% and 15% increased in hangover condition, respectively. Synaptosomal ATP content was 26% decreased, and ATP production rates were 40-55% decreased (malate-glutamate or succinate-dependent, respectively) in AH mice. In addition, a 24% decrease in ΔΨ and a 21% increase in UCP-2 protein expression were observed in synaptosomes from AH mice. Moreover, mitochondrial respiratory complexes I-III, II-III, and IV activities measured in synaptosomes from AH mice were decreased by 18%, 34%, and 50%, respectively. Results of this study reveal that alterations in bioenergetics status during AH could be mainly due to changes in mitochondrial function at the level of synapses.
Topics: Alcoholic Intoxication; Animals; Binge Drinking; Cerebral Cortex; Energy Metabolism; Ethanol; Male; Mice; Mitochondria; Synaptosomes
PubMed: 30385200
DOI: 10.1016/j.alcohol.2018.10.010