-
Biomolecules Aug 2021In the brain, polyamines are mainly synthesized in neurons, but preferentially accumulated in astrocytes, and are proposed to be involved in...
BACKGROUND
In the brain, polyamines are mainly synthesized in neurons, but preferentially accumulated in astrocytes, and are proposed to be involved in neurodegenerative/neuroinflammatory disorders and neuron injury. A transgenic mouse overexpressing spermine oxidase (SMOX, which specifically oxidizes spermine) in the neocortex neurons (Dach-SMOX mouse) was proved to be a model of increased susceptibility to excitotoxic injury.
METHODS
To investigate possible alterations in synapse functioning in Dach-SMOX mouse, both cerebrocortical nerve terminals (synaptosomes) and astrocytic processes (gliosomes) were analysed by assessing polyamine levels, ezrin and vimentin content, glutamate AMPA receptor activation, calcium influx, and catalase activity.
RESULTS
The main findings are as follows: (i) the presence of functional calcium-permeable AMPA receptors in synaptosomes from both control and Dach-SMOX mice, and in gliosomes from Dach-SMOX mice only; (ii) reduced content of spermine in gliosomes from Dach-SMOX mice; and (iii) down-regulation and up-regulation of catalase activity in synaptosomes and gliosomes, respectively, from Dach-SMOX mice.
CONCLUSIONS
Chronic activation of SMOX in neurons leads to major changes in the astrocyte processes including reduced spermine levels, increased calcium influx through calcium-permeable AMPA receptors, and stimulation of catalase activity. Astrocytosis and the astrocyte process alterations, depending on chronic activation of polyamine catabolism, result in synapse dysregulation and neuronal suffering.
Topics: Animals; Astrocytes; Calcium; Catalase; Cerebral Cortex; Cytoskeletal Proteins; Disease Models, Animal; Gliosis; Mice; Mice, Transgenic; Nerve Endings; Neuroglia; Neurons; Oxidative Stress; Oxidoreductases Acting on CH-NH Group Donors; Polyamines; Receptors, AMPA; Spermine; Synaptosomes; Vimentin; Polyamine Oxidase
PubMed: 34572487
DOI: 10.3390/biom11091274 -
The Journal of Neuroscience : the... Jan 2020Individual variation in the addiction liability of amphetamines has a heritable genetic component. We previously identified (heterogeneous nuclear ribonucleoprotein H1)...
Individual variation in the addiction liability of amphetamines has a heritable genetic component. We previously identified (heterogeneous nuclear ribonucleoprotein H1) as a quantitative trait gene underlying decreased methamphetamine-induced locomotor activity in mice. Here, we showed that mice (both females and males) with a heterozygous mutation in the first coding exon of (H1) showed reduced methamphetamine reinforcement and intake and dose-dependent changes in methamphetamine reward as measured via conditioned place preference. Furthermore, H1 mice showed a robust decrease in methamphetamine-induced dopamine release in the NAc with no change in baseline extracellular dopamine, striatal whole-tissue dopamine, dopamine transporter protein, dopamine uptake, or striatal methamphetamine and amphetamine metabolite levels. Immunohistochemical and immunoblot staining of midbrain dopaminergic neurons and their forebrain projections for TH did not reveal any major changes in staining intensity, cell number, or forebrain puncta counts. Surprisingly, there was a twofold increase in hnRNP H protein in the striatal synaptosome of H1 mice with no change in whole-tissue levels. To gain insight into the mechanisms linking increased synaptic hnRNP H with decreased methamphetamine-induced dopamine release and behaviors, synaptosomal proteomic analysis identified an increased baseline abundance of several mitochondrial complex I and V proteins that rapidly decreased at 30 min after methamphetamine administration in H1 mice. In contrast, the much lower level of basal synaptosomal mitochondrial proteins in WT mice showed a rapid increase. We conclude that H1 decreases methamphetamine-induced dopamine release, reward, and reinforcement and induces dynamic changes in basal and methamphetamine-induced synaptic mitochondrial function. Methamphetamine dependence is a significant public health concern with no FDA-approved treatment. We discovered a role for the RNA binding protein hnRNP H in methamphetamine reward and reinforcement. mutation also blunted methamphetamine-induced dopamine release in the NAc, a key neurochemical event contributing to methamphetamine addiction liability. Finally, mutants showed a marked increase in basal level of synaptosomal hnRNP H and mitochondrial proteins that decreased in response to methamphetamine, whereas WT mice showed a methamphetamine-induced increase in synaptosomal mitochondrial proteins. Thus, we identified a potential role for hnRNP H in basal and dynamic mitochondrial function that informs methamphetamine-induced cellular adaptations associated with reduced addiction liability.
Topics: Animals; Anxiety; Corpus Striatum; Dopamine; Dopaminergic Neurons; Exons; Exploratory Behavior; Female; Heterogeneous-Nuclear Ribonucleoprotein Group F-H; Heterogeneous-Nuclear Ribonucleoproteins; Heterozygote; Male; Mesencephalon; Methamphetamine; Mice; Mice, Inbred C57BL; Mitochondria; Mitochondrial Proteins; Mutation; Reflex, Startle; Reinforcement, Psychology; Reward; Rotarod Performance Test; Substance-Related Disorders; Synaptosomes
PubMed: 31704785
DOI: 10.1523/JNEUROSCI.1808-19.2019 -
Molecular Neurobiology Sep 2018Excitotoxic stress has been associated with several different neurological disorders, and it is one of the main causes of neuronal degeneration and death. To identify...
Excitotoxic stress has been associated with several different neurological disorders, and it is one of the main causes of neuronal degeneration and death. To identify new potential proteins that could represent key factors in excitotoxic stress and to study the relationship between polyamine catabolism and excitotoxic damage, a novel transgenic mouse line overexpressing spermine oxidase enzyme in the neocortex (Dach-SMOX) has been engineered. These transgenic mice are more susceptible to excitotoxic injury and display a higher oxidative stress, highlighted by 8-Oxo-2'-deoxyguanosine increase and activation of defense mechanisms, as demonstrated by the increase of nuclear factor erythroid 2-related factor 2 (Nrf-2) in the nucleus. In Dach-SMOX astrocytes and neurons, an alteration of the phosphorylated and non-phosphorylated subunits of glutamate receptors increases the kainic acid response in these mice. Moreover, a decrease in excitatory amino acid transporters and an increase in the system x transporter, a Nrf-2 target, was observed. Sulfasalazine, a system x transporter inhibitor, was shown to revert the increased susceptibility of Dach-SMOX mice treated with kainic acid. We demonstrated that astrocytes play a crucial role in this process: neuronal spermine oxidase overexpression resulted in an alteration of glutamate excitability, in glutamate uptake and efflux in astrocytes involved in the synapse. Considering the involvement of oxidative stress in many neurodegenerative diseases, Dach-SMOX transgenic mouse can be considered as a suitable in vivo genetic model to study the involvement of spermine oxidase in excitotoxicity, which can be considered as a possible therapeutic target.
Topics: 8-Hydroxy-2'-Deoxyguanosine; Amino Acid Transport System y+; Animals; Behavior, Animal; Brain; Deoxyguanosine; Epilepsy; Excitatory Amino Acid Transporter 1; Excitatory Amino Acid Transporter 2; Glutamic Acid; Mice, Transgenic; Models, Biological; NF-E2-Related Factor 2; Neuroglia; Neurotoxins; Oxidoreductases Acting on CH-NH Group Donors; Protein Subunits; Protein Transport; Receptors, AMPA; Sulfasalazine; Synaptosomes; Polyamine Oxidase
PubMed: 29397558
DOI: 10.1007/s12035-017-0864-0 -
Methods in Molecular Biology (Clifton,... 2018Characterizing the detailed structure of the mammalian synapse is of crucial importance to understand its mechanisms of function. Here I describe a protocol to study...
Characterizing the detailed structure of the mammalian synapse is of crucial importance to understand its mechanisms of function. Here I describe a protocol to study synaptic architecture by cryo-electron tomography (cryo-ET), a powerful electron microscopy technique that enables 3D visualization of unstained, fully hydrated cellular structures at molecular resolution. The protocol focuses on purified synaptic terminals ("synaptosomes"), currently the most suitable preparation to analyze mammalian synaptic architecture by cryo-ET.
Topics: Animals; Brain; Electron Microscope Tomography; Mice; Synapses; Synaptosomes
PubMed: 30129020
DOI: 10.1007/978-1-4939-8719-1_16 -
International Journal of Molecular... Jul 2019Synaptosomes are used to decipher the mechanisms involved in chemical transmission, since they permit highlighting the mechanisms of transmitter release and confirming... (Review)
Review
Synaptosomes are used to decipher the mechanisms involved in chemical transmission, since they permit highlighting the mechanisms of transmitter release and confirming whether the activation of presynaptic receptors/enzymes can modulate this event. In the last two decades, important progress in the field came from the observations that synaptosomes retain changes elicited by both "in vivo" and "in vitro" chemical stimulation. The novelty of these studies is the finding that these adaptations persist beyond the washout of the triggering drug, emerging subsequently as functional modifications of synaptosomal performances, including release efficiency. These findings support the conclusion that synaptosomes are plastic entities that respond dynamically to ambient stimulation, but also that they "learn and memorize" the functional adaptation triggered by exposure to chemical agents. This work aims at reviewing the results so far available concerning this form of synaptosomal learning, also highlighting the role of these chemical adaptations in pathological conditions.
Topics: Adaptation, Physiological; Animals; Disease Susceptibility; Glutamic Acid; Humans; Learning; Memory; Neurotransmitter Agents; Presynaptic Terminals; Receptors, Cell Surface; Synaptosomes
PubMed: 31349638
DOI: 10.3390/ijms20153641 -
British Journal of Pharmacology Mar 2021The existence of presynaptic, release-regulating NMDA receptors in the CNS has been long matter of discussion. Most of the reviews dedicated to support this conclusion... (Review)
Review
The existence of presynaptic, release-regulating NMDA receptors in the CNS has been long matter of discussion. Most of the reviews dedicated to support this conclusion have preferentially focussed on the results from electrophysiological studies, paying little or no attention to the data obtained with purified synaptosomes, even though this experimental approach has been recognized as providing reliable information concerning the presence and the role of presynaptic release-regulating receptors in the CNS. To fill the gap, this review is dedicated to summarising the results from studies with synaptosomes published during the last 40 years, which support the existence of auto and hetero NMDA receptors controlling the release of transmitters such as glutamate, GABA, dopamine, noradrenaline, 5-HT, acetylcholine and peptides, in the CNS of mammals. The review also deals with the results from immunochemical studies in isolated nerve endings that confirm the functional observations.
Topics: Animals; Dopamine; Nerve Endings; Norepinephrine; Presynaptic Terminals; Receptors, N-Methyl-D-Aspartate; Receptors, Presynaptic; Synaptosomes
PubMed: 33347605
DOI: 10.1111/bph.15349 -
Journal of Neurochemistry Oct 2021Methcathinone (MCAT) is a psychostimulant of abuse that can cause both persistent striatal dopaminergic and serotonergic, as well as hippocampal serotonergic, deficits....
Methcathinone (MCAT) is a psychostimulant of abuse that can cause both persistent striatal dopaminergic and serotonergic, as well as hippocampal serotonergic, deficits. Evidence suggests that the rapid effects of stimulants that are structurally and mechanistically similar to MCAT on monoamine transporter function may contribute to the abuse liability and/or persistent monoaminergic deficits caused by these agents. Thus, effects of MCAT on 1) striatal dopamine (DA) transporter (DAT); and 2) striatal and hippocampal serotonin transporter (SERT) function, as determined in tissues from adult male rats, were assessed. As reported previously, a single administration of MCAT rapidly (within 1 hr) decreases striatal [ H]DA uptake. Similarly, incubation of rat synaptosomes with MCAT at 37℃ (but not 4˚C) decreased striatal [ H]DA uptake. Incubation with MCAT likewise decreased [ H]5HT but not vesicular [ H]DA uptake. MCAT incubation in vitro was without effect on [ H]DA uptake in striatal synaptosomes prepared from MCAT-treated rats. The decrease in [ H]DA uptake caused by MCAT incubation: (a) reflected a decrease in V , with minimal change in K , and (b) was attenuated by co-incubation with the cell-permeable calcium chelator, N,N'-[1,2-ethanediylbis(oxy-2,1-phenylene)]bis[N-[2-[(acetyloxy)methoxy]-2-oxoethyl]-1,1'-bis[(acetyloxy)methyl] ester-glycine (BAPTA-AM), as well as the non-selective protein kinase-C (PKC) inhibitors bisindolylmaleimide-1 (BIM-1) and 2-[1-3(Aminopropyl)indol-3-yl]-3(1-methyl-1H-indol-3-yl)maleimide (or Bisindolylmaleimide VIII; Ro-31-7549). Taken together, these results suggest that in vitro MCAT incubation may model important aspects of MCAT administration in vivo, and that calcium and PKC contribute to the in vitro effects of MCAT on DAT.
Topics: Animals; Central Nervous System Stimulants; Corpus Striatum; Dopamine Plasma Membrane Transport Proteins; Male; Propiophenones; Protein Kinase C; Rats; Rats, Sprague-Dawley; Synaptosomes
PubMed: 34320222
DOI: 10.1111/jnc.15483 -
ELife Jan 2022Dopaminergic neurons modulate neural circuits and behaviors via dopamine (DA) release from expansive, long range axonal projections. The elaborate cytoarchitecture of...
Dopaminergic neurons modulate neural circuits and behaviors via dopamine (DA) release from expansive, long range axonal projections. The elaborate cytoarchitecture of these neurons is embedded within complex brain tissue, making it difficult to access the neuronal proteome using conventional methods. Here, we demonstrate APEX2 proximity labeling within genetically targeted neurons in the mouse brain, enabling subcellular proteomics with cell-type specificity. By combining APEX2 biotinylation with mass spectrometry, we mapped the somatodendritic and axonal proteomes of midbrain dopaminergic neurons. Our dataset reveals the proteomic architecture underlying proteostasis, axonal metabolism, and neurotransmission in these neurons. We find that most proteins encoded by DA neuron-enriched genes are localized within striatal dopaminergic axons, including ion channels with previously undescribed axonal localization. These proteomic datasets provide a resource for neuronal cell biology, and this approach can be readily adapted for study of other neural cell types.
Topics: Animals; Brain; DNA-(Apurinic or Apyrimidinic Site) Lyase; Dopaminergic Neurons; Endonucleases; Female; Male; Mice; Multifunctional Enzymes; Proteomics; Synaptosomes
PubMed: 35098924
DOI: 10.7554/eLife.70921 -
Biochemical Pharmacology Apr 2020α6β2-Containing nicotinic acetylcholine receptors (α6β2* nAChRs) are predominantly expressed in midbrain dopaminergic neurons, including substantia nigra pars...
α6β2-Containing nicotinic acetylcholine receptors (α6β2* nAChRs) are predominantly expressed in midbrain dopaminergic neurons, including substantia nigra pars compacta (SNc) neurons and their projections to striatal regions, where they regulate dopamine release and nigrostriatal activity. It is well established that nAChR agonists exert protection against dopaminergic neurotoxicity in cellular assays and parkinsonian animal models. Historically, drug development in the nAChR field has been mostly focused on development of selective agonists and positive allosteric modulators (PAMs) for the predominant neuronal nAChRs, α7 and α4β2. Here, we report the discovery and characterization of AN6001, a novel selective α6β2* nAChR PAM. AN6001 mediated increases in both nicotine potency and efficacy at the human α6/α3β2β3 nAChR in HEK293 cells, and it positively modulated ACh-evoked currents through both α6/α3β2β3 and a concatenated β3-α6-β2-α6-β2 receptor in Xenopus oocytes, displaying EC values of 0.58 µM and 0.40 µM, respectively. In contrast, the compound did not display significant modulatory activity at α4β2, α3β4, α7 and muscle nAChRs. AN6001 also increased agonist-induced dopamine release from striatal synaptosomes and augmented agonist-induced global cellular responses and inward currents in dopaminergic neurons in SNc slices (measured by Ca imaging and patch clamp recordings, respectively). Finally, AN6001 potentiated the neuroprotective effect of nicotine at MPP-treated primary dopaminergic neurons. Overall, our studies demonstrate the existence of allosteric sites on α6β2* nAChRs and that positive modulation of native α6β2* receptors strengthens DA signaling. Hence, AN6001 represents an important tool for studies of α6β2* nAChRs and furthermore underlines the therapeutic potential in these receptors in Parkinson's disease.
Topics: Action Potentials; Allosteric Regulation; Animals; Brain; Calcium Signaling; Dopamine; Dopaminergic Neurons; HEK293 Cells; Humans; Mice; Neuroprotective Agents; Nicotine; Oocytes; Patch-Clamp Techniques; Rats, Sprague-Dawley; Rats, Wistar; Receptors, Nicotinic; Synaptosomes; Xenopus laevis
PubMed: 31887290
DOI: 10.1016/j.bcp.2019.113788 -
Journal of Neurochemistry Mar 2020Increasing evidence suggests that both synaptic loss and neuroinflammation constitute early pathologic hallmarks of Alzheimer's disease. A downstream event during...
Increasing evidence suggests that both synaptic loss and neuroinflammation constitute early pathologic hallmarks of Alzheimer's disease. A downstream event during inflammatory activation of microglia and astrocytes is the induction of nitric oxide synthase type 2, resulting in an increased release of nitric oxide and the post-translational S-nitrosylation of protein cysteine residues. Both early events, inflammation and synaptic dysfunction, could be connected if this excess nitrosylation occurs on synaptic proteins. In the long term, such changes could provide new insight into patho-mechanisms as well as biomarker candidates from the early stages of disease progression. This study investigated S-nitrosylation in synaptosomal proteins isolated from APP/PS1 model mice in comparison to wild type and NOS2 mice, as well as human control, mild cognitive impairment and Alzheimer's disease brain tissues. Proteomics data were obtained using an established protocol utilizing an isobaric mass tag method, followed by nanocapillary high performance liquid chromatography tandem mass spectrometry. Statistical analysis identified the S-nitrosylation sites most likely derived from an increase in nitric oxide (NO) in dependence of presence of AD pathology, age and the key enzyme NOS2. The resulting list of candidate proteins is discussed considering function, previous findings in the context of neurodegeneration, and the potential for further validation studies.
Topics: Aged; Aged, 80 and over; Alzheimer Disease; Animals; Brain; Disease Models, Animal; Female; Humans; Male; Mice; Mice, Inbred C57BL; Mice, Transgenic; Nerve Tissue Proteins; Nitric Oxide; Nitric Oxide Synthase Type II; Proteomics; Signal Transduction; Synaptosomes
PubMed: 31520481
DOI: 10.1111/jnc.14870