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Methods in Molecular Biology (Clifton,... 2022Synaptic degeneration is one of the earliest and phenotypically most significant features associated with numerous neurodegenerative conditions, including Alzheimer's...
Synaptic degeneration is one of the earliest and phenotypically most significant features associated with numerous neurodegenerative conditions, including Alzheimer's and Parkinson's diseases. Synaptic changes are also known to be important in neurocognitive disorders such as schizophrenia and autism spectrum disorders. Several labs, including ours, have demonstrated that conventional (fluorescence-based) flow cytometry of individual synaptosomes is a robust and reproducible method. However, the repertoire of probes needed to assess comprehensively the type of synapse, pathologic proteins (including protein products of risk genes discovered in GWAS), and markers of stress and injury far exceeds what is achievable with conventional flow cytometry. We recently developed a method that applies CyTOF (Cytometry by Time-Of-Flight mass spectrometry) to high-dimensional analysis of individual human synaptosomes, overcoming many of the multiplexing limitations of conventional flow cytometry. We call this new method Mass Synaptometry. Here we describe the preparation of synaptosomes from human and mouse brain, the generation and quality control of the "SynTOF" (Synapse by Time-Of-Flight mass spectrometry) antibody panel, the staining protocol, and CyTOF parameter setup for acquisition, post-acquisition processing, and analysis.
Topics: Animals; Flow Cytometry; Mass Spectrometry; Mice; Synapses; Synaptosomes
PubMed: 35099792
DOI: 10.1007/978-1-0716-1916-2_6 -
Neuroscience Letters Jul 2019Desoxypipradrol (2-DPMP), a new psychoactive substance (NPS), acts as a norepinephrine-dopamine reuptake inhibitor (NDRI). NDRIs can be addictive due to their action...
Desoxypipradrol (2-DPMP), a new psychoactive substance (NPS), acts as a norepinephrine-dopamine reuptake inhibitor (NDRI). NDRIs can be addictive due to their action mechanisms similar to cocaine and methamphetamine. However, there is a lack of scientific information regarding the exact dependency of 2-DPMP. Thus, the purpose of this study was to evaluate rewarding and reinforcing effects of 2-DPMP in rodents. The effective dose range of 2-DPMP was determined by climbing behavior test. To evaluate rewarding effects of 2-DPMP, conditioned place preference (CPP) test was performed at selected doses in mice. Self-administration (SA) test was then undertaken at two doses that caused the highest effects in the CPP test. Dopamine level changes were analyzed using synaptosomes in order to investigate effects of 2-DPMP on the central nervous system (CNS). Significant responses were observed in the climbing behavior test at doses of 0.1, 0.5, and 1 mg/kg by intraperitoneal injection (i.p.). In the CPP test, mice i.p. administered 2-DPMP at 1 mg/kg showed a significant preference in drug-paired compartment. In the SA test, mice intravenously given 0.1 mg/kg/infusion showed significantly higher active lever responses. Further, dopamine was increased in a dose-dependent manner. Taken together, these results suggest that 2-DPMP may act on the CNS and induce rewarding and reinforcing effects, indicating its dependence liability.
Topics: Animals; Behavior, Animal; Conditioning, Psychological; Dopamine; Dose-Response Relationship, Drug; Male; Mice; Piperidines; Reward; Self Administration; Synaptosomes
PubMed: 31005652
DOI: 10.1016/j.neulet.2019.04.037 -
Journal of Neurochemistry Oct 2016This Obituary honors Victor P. Whittaker, one of the pioneers in the field of neurochemistry. Victor Whittaker died on 5th July 2016 aged 97 in Cambridge (UK) after a...
This Obituary honors Victor P. Whittaker, one of the pioneers in the field of neurochemistry. Victor Whittaker died on 5th July 2016 aged 97 in Cambridge (UK) after a short illness. Victor is best known for his landmark advances in the subcellular fractionation of brain tissue which led to the isolation of synaptosomes and subsequently synaptic vesicles at the beginning of the 1960s and for the cellular and molecular analysis of the cholinergic synapse.
Topics: Brain Chemistry; Cholinesterases; England; Germany; History, 20th Century; History, 21st Century; Neurochemistry; Synaptosomes
PubMed: 27538027
DOI: 10.1111/jnc.13778 -
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 -
Molecular Brain Feb 2019We aimed to test the therapeutic effects of baicalin on attention deficit hyperactivity disorder (ADHD) in an animal model and to explain the potential mechanism. We...
We aimed to test the therapeutic effects of baicalin on attention deficit hyperactivity disorder (ADHD) in an animal model and to explain the potential mechanism. We investigated the therapeutic effects and mechanisms of baicalin in a spontaneously hypertensive rat (SHR) model of ADHD depending on the dopamine (DA) deficit theory. In this study, fifty SHRs were randomly divided into five groups: methylphenidate (MPH), baicalin (50 mg/kg, 100 mg/kg, or 150 mg/kg), and saline-treated. Ten Wistar Kyoto (WKY) rats were used as controls. All rats were orally administered the treatment for four weeks. Motor activity, spatial learning and memory ability were assessed with the open-field and Morris water-maze tests. The mRNA and protein levels of tyrosine hydroxylase (TH), vesicular monoamine transporter 2 (VMAT2), synaptosomal-associated protein of molecular mass 25kD (SNAP25) and synataxin 1a in synaptosomes were detected with real-time polymerase chain reaction (PCR) and Western blot. In addition, DA levels were measured in the prefrontal cortex and striatum. The results indicated that both MPH and baicalin at doses of 150 mg/kg and 100 mg/kg significantly decreased the hyperactivity and improved the spatial learning memory deficit in the SHRs and increased the synaptosomal mRNA and protein levels of TH, SNAP25, VMAT2 and synataxin 1a compared with saline treatment. MPH significantly increased DA levels in both the prefrontal cortex (PFC) and striatum, while baicalin significantly increased DA levels only in the striatum. The results of the present study showed that baicalin treatment was effective for controlling the core symptoms of ADHD. Baicalin increased DA levels only in the striatum, which suggested that baicalin may target the striatum. The increased DA levels may partially be attributed to the increased mRNA and protein expression of TH, SNAP25, VMAT2, and syntaxin 1a. Therefore, these results suggested that the pharmacological effects of baicalin were associated with the synthesis, vesicular localization, and release of DA and might be effective in treating ADHD. However, further studies are required to better understand the molecular mechanisms underlying these findings.
Topics: Animals; Attention Deficit Disorder with Hyperactivity; Body Weight; Dopamine; Eating; Flavonoids; Growth and Development; Male; Maze Learning; Memory; Motor Activity; Movement; Neostriatum; Nerve Tissue Proteins; Prefrontal Cortex; RNA, Messenger; Rats, Inbred SHR; Reaction Time; Spatial Learning; Swimming; Synaptosomes
PubMed: 30736828
DOI: 10.1186/s13041-019-0428-5 -
Human Molecular Genetics Sep 2022Large-scale genomic studies of schizophrenia implicate genes involved in the epigenetic regulation of transcription by histone methylation and genes encoding components...
Large-scale genomic studies of schizophrenia implicate genes involved in the epigenetic regulation of transcription by histone methylation and genes encoding components of the synapse. However, the interactions between these pathways in conferring risk to psychiatric illness are unknown. Loss-of-function (LoF) mutations in the gene encoding histone methyltransferase, SETD1A, confer substantial risk to schizophrenia. Among several roles, SETD1A is thought to be involved in the development and function of neuronal circuits. Here, we employed a multi-omics approach to study the effects of heterozygous Setd1a LoF on gene expression and synaptic composition in mouse cortex across five developmental timepoints from embryonic day 14 to postnatal day 70. Using RNA sequencing, we observed that Setd1a LoF resulted in the consistent downregulation of genes enriched for mitochondrial pathways. This effect extended to the synaptosome, in which we found age-specific disruption to both mitochondrial and synaptic proteins. Using large-scale patient genomics data, we observed no enrichment for genetic association with schizophrenia within differentially expressed transcripts or proteins, suggesting they derive from a distinct mechanism of risk from that implicated by genomic studies. This study highlights biological pathways through which SETD1A LOF may confer risk to schizophrenia. Further work is required to determine whether the effects observed in this model reflect human pathology.
Topics: Animals; Epigenesis, Genetic; Histone Methyltransferases; Histone-Lysine N-Methyltransferase; Histones; Humans; Mice; Synaptosomes; Transcriptome
PubMed: 35531971
DOI: 10.1093/hmg/ddac105 -
Molecular Neurobiology Jun 2021Neurons are highly compartmentalized cells that depend on local protein synthesis. Messenger RNAs (mRNAs) have thus been detected in neuronal dendrites, and more...
Neurons are highly compartmentalized cells that depend on local protein synthesis. Messenger RNAs (mRNAs) have thus been detected in neuronal dendrites, and more recently in the pre- and postsynaptic compartments as well. Other RNA species such as microRNAs have also been described at synapses where they are believed to control mRNA availability for local translation. A combined dataset analyzing the synaptic coding and non-coding RNAome via next-generation sequencing approaches is, however, still lacking. Here, we isolate synaptosomes from the hippocampus of young wild-type mice and provide the coding and non-coding synaptic RNAome. These data are complemented by a novel approach for analyzing the synaptic RNAome from primary hippocampal neurons grown in microfluidic chambers. Our data show that synaptic microRNAs control almost the entire synaptic mRNAome, and we identified several hub microRNAs. By combining the in vivo synaptosomal data with our novel microfluidic chamber system, our findings also support the hypothesis that part of the synaptic microRNAome may be supplied to neurons via astrocytes. Moreover, the microfluidic system is suitable for studying the dynamics of the synaptic RNAome in response to stimulation. In conclusion, our data provide a valuable resource and point to several important targets for further research.
Topics: Animals; Hippocampus; Male; Mice, Inbred C57BL; MicroRNAs; Microfluidics; Neurons; RNA, Messenger; RNA, Untranslated; Synapses; Synaptosomes; Mice
PubMed: 33569760
DOI: 10.1007/s12035-021-02296-y -
Environmental Toxicology and... Nov 2020The exposure to environmental pollutants, such as fine and ultrafine particles (FP and UFP), has been associated with increased risk for Parkinson's disease, depression...
The exposure to environmental pollutants, such as fine and ultrafine particles (FP and UFP), has been associated with increased risk for Parkinson's disease, depression and schizophrenia, disorders related to altered dopaminergic transmission. The striatum, a neuronal nucleus with extensive dopaminergic afferents, is a target site for particle toxicity, which results in oxidative stress, inflammation, astrocyte activation and modifications in dopamine content and D receptor (DR) density. In this study we assessed the in vitro effect of the exposure to FP and UFP on dopaminergic transmission, by evaluating [H]-dopamine uptake and release by rat striatal isolated nerve terminals (synaptosomes), as well as modifications in the affinity and signaling of native and cloned DRs. FP and UFP collected from the air of Mexico City inhibited [H]-dopamine uptake and increased depolarization-evoked [H]-dopamine release in striatal synaptosomes. FP and UFP also enhanced DR affinity for dopamine in membranes from either rat striatum or CHO-K1 cells transfected with the long isoform of the human DR (hDR)2LR). In CHO-K1-hD2L In CHO-K1-hDR cells or striatal slices, FP and UFP increased the potency of dopamine or the DR agonist quinpirole, respectively, to inhibit forskolin-induced cAMP formation. The effects were concentration-dependent, with UFP being more potent than FP. These results indicate that FP and UFP directly affect dopaminergic transmission.
Topics: Air Pollutants; Animals; CHO Cells; Corpus Striatum; Cricetulus; Dopamine; In Vitro Techniques; Male; Mexico; Particulate Matter; Rats, Wistar; Receptors, Dopamine D2; Signal Transduction; Synaptosomes
PubMed: 32942001
DOI: 10.1016/j.etap.2020.103484 -
Experimental Neurology Nov 2021Methamphetamine (METH) is a highly addictive and powerful central nervous system psychostimulant with no FDA-approved pharmacotherapy. Parkin is a neuroprotective...
Methamphetamine (METH) is a highly addictive and powerful central nervous system psychostimulant with no FDA-approved pharmacotherapy. Parkin is a neuroprotective protein and its loss of function contributes to Parkinson's disease. This study used 3-month-old homozygous parkin knockout (PKO) rats to determine whether loss of parkin protein potentiates neurotoxicity of chronic METH to the nigrostriatal dopamine pathway. PKO rats were chronically treated with 10 mg/kg METH for 10 consecutive days and assessed for neurotoxicity markers in the striatum on the 5th and 10th day of withdrawal from METH. The PKO rats showed higher METH-induced hyperthermia; however, they did not display augmented deficits in dopaminergic and serotonergic neurotoxicity markers, astrocyte activation or decreased mitochondrial enzyme levels as compared to wild-type (WT) rats. Interestingly, saline-treated PKO rats had lower levels of dopamine (DA) as well as mitochondrial complex I and II levels while having increased basal levels of glial fibrillary acidic protein (GFAP), a marker of gliosis. These results indicate PKO display a certain resistance to METH neurotoxicity, possibly mediated by lowered DA levels and downregulated mitochondria.
Topics: Animals; Astrocytes; Central Nervous System Stimulants; Dopamine; Drug Administration Schedule; Hyperthermia, Induced; Locomotion; Male; Methamphetamine; Rats; Rats, Long-Evans; Rats, Transgenic; Synaptosomes; Ubiquitin-Protein Ligases
PubMed: 34298012
DOI: 10.1016/j.expneurol.2021.113811 -
ACS Chemical Neuroscience Jun 2017Synaptic vesicles measuring 30-50 nm in diameter containing neurotransmitters either completely collapse at the presynaptic membrane or dock and transiently fuse at the...
Synaptic vesicles measuring 30-50 nm in diameter containing neurotransmitters either completely collapse at the presynaptic membrane or dock and transiently fuse at the base of specialized 15 nm cup-shaped lipoprotein structures called porosomes at the presynaptic membrane of synaptosomes to release neurotransmitters. Recent study reports the unique composition of major lipids associated with neuronal porosomes. Given that lipids greatly influence the association and functions of membrane proteins, differences in lipid composition of synaptic vesicle and the synaptosome membrane was hypothesized. To test this hypothesis, the lipidome of isolated synaptosome, synaptosome membrane, and synaptic vesicle preparation were determined by using mass spectrometry in the current study. Results from the study demonstrate the enriched presence of triacyl glycerols and sphingomyelins in synaptic vesicles, as opposed to the enriched presence of phospholipids in the synaptosome membrane fraction, reflecting on the tight regulation of nerve cells in compartmentalization of membrane lipids at the nerve terminal.
Topics: Animals; Brain Chemistry; Cell Membrane; Mass Spectrometry; Membrane Lipids; Rats; Synaptic Vesicles; Synaptosomes
PubMed: 28244738
DOI: 10.1021/acschemneuro.7b00030