ABSTRACT
The capsaicin receptor TRPV1 has been widely characterized in the sensory system as a key component of pain and inflammation. A large amount of evidence shows that TRPV1 is also functional in the brain although its role is still debated. Here we report that TRPV1 is highly expressed in microglial cells rather than neurons of the anterior cingulate cortex and other brain areas. We found that stimulation of microglial TRPV1 controls cortical microglia activation per se and indirectly enhances glutamatergic transmission in neurons by promoting extracellular microglial microvesicles shedding. Conversely, in the cortex of mice suffering from neuropathic pain, TRPV1 is also present in neurons affecting their intrinsic electrical properties and synaptic strength. Altogether, these findings identify brain TRPV1 as potential detector of harmful stimuli and a key player of microglia to neuron communication.
Subject(s)
Biomarkers/metabolism , Encephalitis/metabolism , Neuralgia/metabolism , TRPV Cation Channels/metabolism , Animals , Cell-Derived Microparticles , Excitatory Postsynaptic Potentials , Male , Mice, Inbred C57BL , Mice, Knockout , Mice, Transgenic , Microglia/metabolism , Neurons/metabolism , Neurons/physiology , Synaptic Transmission , TRPV Cation Channels/geneticsABSTRACT
Osteopontin is a pleiotropic cytokine that is involved in several diseases including multiple sclerosis. Secreted osteopontin is cleaved by few known proteases, modulating its pro-inflammatory activities. Here we show by in vitro experiments that secreted osteopontin can be processed by extracellular proteasomes, thereby producing fragments with novel chemotactic activity. Furthermore, osteopontin reduces the release of proteasomes in the extracellular space. The latter phenomenon seems to occur in vivo in multiple sclerosis, where it reflects the remission/relapse alternation. The extracellular proteasome-mediated inflammatory pathway may represent a general mechanism to control inflammation in inflammatory diseases.
Subject(s)
Multiple Sclerosis/metabolism , Osteopontin/metabolism , Proteasome Endopeptidase Complex/metabolism , Amino Acid Sequence , Chemotaxis/immunology , Endothelial Cells/metabolism , Extracellular Space/metabolism , Extracellular Vesicles/immunology , Extracellular Vesicles/metabolism , Humans , Lymphocytes/immunology , Lymphocytes/metabolism , Models, Molecular , Multiple Sclerosis/immunology , Osteopontin/chemistry , Osteopontin/immunology , Proteasome Endopeptidase Complex/immunology , Protein Conformation , Structure-Activity RelationshipABSTRACT
Growing evidence indicates that sphingosine-1-P (S1P) upregulates glutamate secretion in hippocampal neurons. However, the molecular mechanisms through which S1P enhances excitatory activity remain largely undefined. The aim of this study was to identify presynaptic targets of S1P action controlling exocytosis. Confocal analysis of rat hippocampal neurons showed that S1P applied at nanomolar concentration alters the distribution of Synapsin I (SynI), a presynaptic phosphoprotein that controls the availability of synaptic vesicles for exocytosis. S1P induced SynI relocation to extrasynaptic regions of mature neurons, as well as SynI dispersion from synaptic vesicle clusters present at axonal growth cones of developing neurons. S1P-induced SynI relocation occurred in a Ca(2+)-independent but ERK-dependent manner, likely through the activation of S1P3 receptors, as it was prevented by the S1P3 receptor selective antagonist CAY1044 and in neurons in which S1P3 receptor was silenced. Our recent evidence indicates that microvesicles (MVs) released by microglia enhance the metabolism of endogenous sphingolipids in neurons and stimulate excitatory transmission. We therefore investigated whether MVs affect SynI distribution and whether endogenous S1P could be involved in the process. Analysis of SynI immunoreactivity showed that exposure to microglial MVs induces SynI mobilization at presynaptic sites and growth cones, whereas the use of inhibitors of sphingolipid cascade identified S1P as the sphingolipid mediating SynI redistribution. Our data represent the first demonstration that S1P induces SynI mobilization from synapses, thereby indicating the phosphoprotein as a novel target through which S1P controls exocytosis. SIGNIFICANCE STATEMENT: Growing evidence indicates that the bioactive lipid sphingosine and its metabolite sphingosine-1-P (S1P) stimulate excitatory transmission. While it has been recently clarified that sphingosine influences directly the exocytotic machinery by activating the synaptic vesicle protein VAMP2 to form SNARE fusion complexes, the molecular mechanism by which S1P promotes neurotransmission remained largely undefined. In this study, we identify Synapsin I, a presynaptic phosphoprotein involved in the control of availability of synaptic vesicles for exocytosis, as the key target of S1P action. In addition, we provide evidence that S1P can be produced at mature axon terminals as well as at immature growth cones in response to microglia-derived signals, which may be important to stabilize nascent synapses and to restore or potentiate transmission.
Subject(s)
Lysophospholipids/physiology , Presynaptic Terminals/metabolism , Sphingosine/analogs & derivatives , Synapses/metabolism , Synapsins/biosynthesis , Animals , Cells, Cultured , Female , Hippocampus/chemistry , Hippocampus/cytology , Hippocampus/metabolism , Lysophospholipids/analysis , Male , Mice , Mice, Inbred C57BL , Mice, Knockout , Presynaptic Terminals/chemistry , Rats , Rats, Sprague-Dawley , Sphingosine/analysis , Sphingosine/physiology , Synapses/chemistry , Synapsins/analysisABSTRACT
Endocannabinoids primarily influence neuronal synaptic communication within the nervous system. To exert their function, endocannabinoids need to travel across the intercellular space. However, how hydrophobic endocannabinoids cross cell membranes and move extracellularly remains an unresolved problem. Here, we show that endocannabinoids are secreted through extracellular membrane vesicles produced by microglial cells. We demonstrate that microglial extracellular vesicles carry on their surface N-arachidonoylethanolamine (AEA), which is able to stimulate type-1 cannabinoid receptors (CB1), and inhibit presynaptic transmission, in target GABAergic neurons. This is the first demonstration of a functional role of extracellular vesicular transport of endocannabinoids.
Subject(s)
Endocannabinoids/metabolism , Microglia/metabolism , Animals , Arachidonic Acids , Cells, Cultured , Exosomes/metabolism , Polyunsaturated Alkamides , Presynaptic Terminals/metabolism , Rats , Receptors, Cannabinoid/metabolism , Synaptic Transmission/physiologyABSTRACT
Microglia/macrophages (M) are major contributors to postinjury inflammation, but they may also promote brain repair in response to specific environmental signals that drive classic (M1) or alternative (M2) polarization. We investigated the activation and functional changes of M in mice with traumatic brain injuries and receiving intracerebroventricular human bone marrow mesenchymal stromal cells (MSCs) or saline infusion. MSCs upregulated Ym1 and Arginase-1 mRNA (p < 0.001), two M2 markers of protective M polarization, at 3 and 7 d postinjury, and increased the number of Ym1(+) cells at 7 d postinjury (p < 0.05). MSCs reduced the presence of the lysosomal activity marker CD68 on the membrane surface of CD11b-positive M (p < 0.05), indicating reduced phagocytosis. MSC-mediated induction of the M2 phenotype in M was associated with early and persistent recovery of neurological functions evaluated up to 35 days postinjury (p < 0.01) and reparative changes of the lesioned microenvironment. In vitro, MSCs directly counteracted the proinflammatory response of primary murine microglia stimulated by tumor necrosis factor-α + interleukin 17 or by tumor necrosis factor-α + interferon-γ and induced M2 proregenerative traits, as indicated by the downregulation of inducible nitric oxide synthase and upregulation of Ym1 and CD206 mRNA (p < 0.01). In conclusion, we found evidence that MSCs can drive the M transcriptional environment and induce the acquisition of an early, persistent M2-beneficial phenotype both in vivo and in vitro. Increased Ym1 expression together with reduced in vivo phagocytosis suggests M selection by MSCs towards the M2a subpopulation, which is involved in growth stimulation and tissue repair.
Subject(s)
Brain Injuries/metabolism , Brain Injuries/therapy , Brain/metabolism , Cell Polarity , Mesenchymal Stem Cells/metabolism , Microglia/metabolism , Animals , Cells, Cultured , Cytokines/metabolism , Humans , Injections, Intraventricular , Intercellular Signaling Peptides and Proteins/metabolism , Macrophages/metabolism , Male , Mesenchymal Stem Cell Transplantation , Mice , Mice, Inbred C57BL , Motor Activity , RNA, Messenger/metabolismABSTRACT
Activation of protein kinase A (PKA) pathway at presynaptic terminals plays a crucial role in the supply of synaptic vesicles (SVs) from the reserve pool, affecting the steady-state level of activity and the reconstitution of the readily releasable pool after intense stimulation. However, the identity of the stimuli activating this pathway is undefined. Using fluorescence resonance energy transfer and molecular genetic, we show that kainate, through the activation of presynaptic kainate receptors, induces PKA activation and enhances synapsin I phosphorylation at PKA-specific residues. This leads to a dispersion of synapsin I immunoreactivity, which is accompanied by a PKA-dependent increase in the rate of SV recycling at the growth cone and by an enhanced miniature excitatory postsynaptic currents frequency in mature networks. Selective activation of this pathway is induced by the native neurotransmitter glutamate, when applied in the high nanomolar range. These data identify glutamate, specifically acting on KARs, as one of the stimuli able to induce phosphorylation of synapsin at PKA sites, both at the axonal growth cone and at the mature synapse, thus increasing SV availability and contributing to plasticity phenomena.
Subject(s)
Cyclic AMP-Dependent Protein Kinases/metabolism , Growth Cones/physiology , Neuronal Plasticity/physiology , Receptors, Kainic Acid/metabolism , Synaptic Vesicles/physiology , Animals , Cells, Cultured , Enzyme Activation/physiology , Excitatory Amino Acid Agonists/metabolism , Excitatory Amino Acid Agonists/pharmacology , Excitatory Postsynaptic Potentials/physiology , Female , Fluorescence Resonance Energy Transfer , Glutamic Acid/metabolism , Hippocampus/physiology , Immunohistochemistry , Kainic Acid/metabolism , Kainic Acid/pharmacology , Neurons/physiology , Patch-Clamp Techniques , RNA, Small Interfering , Rats , Rats, Sprague-Dawley , Synapsins/metabolismABSTRACT
OBJECTIVE: Microvesicles (MVs) have been indicated as important mediators of intercellular communication and are emerging as new biomarkers of tissue damage. Our previous data indicate that reactive microglia/macrophages release MVs in vitro. The aim of the study was to evaluate whether MVs are released by microglia/macrophages in vivo and whether their number varies in brain inflammatory conditions, such as multiple sclerosis (MS). METHODS: Electron and fluorescence microscopy and flow cytometry were used to detect myeloid MVs in the cerebrospinal fluid (CSF) of healthy controls, MS patients, and rodents affected by experimental autoimmune encephalomyelitis (EAE), the animal model of MS. RESULTS: Myeloid MVs were detected in CSF of healthy controls. In relapsing and remitting EAE mice, the concentration of myeloid MVs in the CSF was significantly increased and closely associated with disease course. Analysis of MVs in the CSF of 28 relapsing patients and 28 patients with clinical isolated syndrome from 2 independent cohorts revealed higher levels of myeloid MVs than in 13 age-matched controls, indicating a clinical value of MVs as a companion tool to capture disease activity. Myeloid MVs were found to spread inflammatory signals both in vitro and in vivo at the site of administration; mice impaired in MV shedding were protected from EAE, suggesting a pathogenic role for MVs in the disease. Finally, FTY720, the first approved oral MS drug, significantly reduced the amount of MVs in the CSF of EAE-treated mice. INTERPRETATION: These findings identify myeloid MVs as a marker and therapeutic target of brain inflammation.
Subject(s)
Biomarkers/cerebrospinal fluid , Central Nervous System Diseases/cerebrospinal fluid , Central Nervous System Diseases/drug therapy , Inflammation/cerebrospinal fluid , Inflammation/drug therapy , Spinal Cord/metabolism , Animals , Blotting, Western , Calcium Signaling/physiology , Cell Communication , Cells, Cultured , Encephalitis/cerebrospinal fluid , Encephalitis/pathology , Flow Cytometry , Lentivirus/genetics , Mice , Mice, Inbred C57BL , Mice, Knockout , Microscopy, Electron , Microscopy, Fluorescence , Multiple Sclerosis/pathology , Nervous System Autoimmune Disease, Experimental/cerebrospinal fluid , Nervous System Autoimmune Disease, Experimental/drug therapy , Neuroglia/metabolism , Neuroglia/physiology , Rats , Rats, Inbred Lew , Rats, Sprague-Dawley , Real-Time Polymerase Chain Reaction , Sphingomyelin Phosphodiesterase/genetics , Sphingomyelin Phosphodiesterase/physiologyABSTRACT
Microvesicles (MVs) released into the brain microenvironment are emerging as a novel way of cell-to-cell communication. We have recently shown that microglia, the immune cells of the brain, shed MVs upon activation but their possible role in microglia-to-neuron communication has never been explored. To investigate whether MVs affect neurotransmission, we analysed spontaneous release of glutamate in neurons exposed to MVs and found a dose-dependent increase in miniature excitatory postsynaptic current (mEPSC) frequency without changes in mEPSC amplitude. Paired-pulse recording analysis of evoked neurotransmission showed that MVs mainly act at the presynaptic site, by increasing release probability. In line with the enhancement of excitatory transmission in vitro, injection of MVs into the rat visual cortex caused an acute increase in the amplitude of field potentials evoked by visual stimuli. Stimulation of synaptic activity occurred via enhanced sphingolipid metabolism. Indeed, MVs promoted ceramide and sphingosine production in neurons, while the increase of excitatory transmission induced by MVs was prevented by pharmacological or genetic inhibition of sphingosine synthesis. These data identify microglia-derived MVs as a new mechanism by which microglia influence synaptic activity and highlight the involvement of neuronal sphingosine in this microglia-to-neuron signalling pathway.
Subject(s)
Microglia/metabolism , Neurons/physiology , Secretory Vesicles/metabolism , Sphingolipids/metabolism , Synapses/metabolism , Animals , Cerebral Cortex/cytology , Cerebral Cortex/physiology , Neurons/metabolism , RatsABSTRACT
BACKGROUND INFORMATION: ATP is the main transmitter stored and released from astrocytes under physiological and pathological conditions. Morphological and functional evidence suggest that besides secretory granules, secretory lysosomes release ATP. However, the molecular mechanisms involved in astrocytic lysosome fusion remain still unknown. RESULTS: In the present study, we identify tetanus neurotoxin-insensitive vesicle-associated membrane protein (TI-VAMP, also called VAMP7) as the vesicular SNARE which mediates secretory lysosome exocytosis, contributing to release of both ATP and cathepsin B from glial cells. We also demonstrate that fusion of secretory lysosomes is triggered by slow and locally restricted calcium elevations, distinct from calcium spikes which induce the fusion of glutamate-containing clear vesicles. Downregulation of TI-VAMP/VAMP7 expression inhibited the fusion of ATP-storing vesicles and ATP-mediated calcium wave propagation. TI-VAMP/VAMP7 downregulation also significantly reduced secretion of cathepsin B from glioma. CONCLUSIONS: Given that sustained ATP release from glia upon injury greatly contributes to secondary brain damage and cathepsin B plays a critical role in glioma dissemination, TI-VAMP silencing can represent a novel strategy to control lysosome fusion in pathological conditions.
Subject(s)
Adenosine Triphosphate/metabolism , Astrocytes/metabolism , Calcium/metabolism , Cathepsin B/metabolism , Lysosomes/metabolism , R-SNARE Proteins/metabolism , Animals , Astrocytes/cytology , Cerebral Cortex/cytology , Cerebral Cortex/embryology , Cerebral Cortex/metabolism , Down-Regulation , Embryo, Mammalian , Exocytosis , Glioma/metabolism , Glioma/pathology , Hippocampus/cytology , Hippocampus/embryology , Hippocampus/metabolism , Humans , Membrane Fusion , Neuroglia/cytology , Neuroglia/metabolism , Primary Cell Culture , Protein Binding , R-SNARE Proteins/antagonists & inhibitors , R-SNARE Proteins/genetics , RNA, Small Interfering/genetics , Rats , Signal Transduction , TransfectionABSTRACT
The enantiopure diastereomeric Δ2-isoxazoline derivatives (2S,5'R)-5a-10a and (2S,5'S)-5b, (2S,5'S)-9b, (2S,5'S)-11b, which are structural analogues of both ABT-418 2 and oxyimino ethers (S)-3 and (Z)-(S)-4, were synthesized through cycloaddition reactions involving nitrile oxides as 1,3-dipoles and (S)-N-Boc-2-vinylpyrrolidine-13 as the dipolarophile. The absolute configuration was unequivocally assigned to target compounds by means of an X-ray analysis. The derivatives under study were assayed at neuronal acetylcholine nicotinic receptors (nAChRs), where they showed a meaningful reduction in affinity at the heteromeric α4ß2 subtype when compared to the reference molecules. Conversely, anti (2S,5'S)-5b and syn (2S,5'R)-10a isomers showed an affinity for the α7 nAChRs comparable to that observed for the model compound ABT-418.
Subject(s)
Isoxazoles/chemical synthesis , Isoxazoles/pharmacology , Neurons/metabolism , Pyrrolidines/chemistry , Receptors, Nicotinic/metabolism , Crystallography, X-Ray , Isoxazoles/chemistry , Isoxazoles/metabolism , Models, Molecular , Molecular Conformation , Neurons/drug effects , Stereoisomerism , Structure-Activity RelationshipABSTRACT
The availability of drug affecting neuronal nicotinic acetylcholine receptors (nAChRs) may have important therapeutic potential for the treatment of several CNS pathologies. Pursuing our efforts on the systematic structural modification of cytisine and N-arylalkyl and N-aroylalkyl cytisines were synthesized and tested for the displacement of [(3)H]-epibatidine and [(125)I]-alpha-bungarotoxin from the most widespread brain nAChRs subtypes alpha(4)beta(2) and alpha(7), respectively. While the affinity for alpha(7) subtype was rather poor (K(i) from 0.4 to >50 microM), the affinity for alpha(4)beta(2) subtype was very interesting, with nanomolar K(i) values for the best compounds. The N-substituted cytisines were docked into the rat and human alpha(4)beta(2) nAChR models based on the extracellular domain of a molluscan acetylcholine binding protein. The docking results agreed with the binding data, allowing the detection of discrete amino acid residues of the alpha and beta subunits essential for the ligand binding on rat and human nAChRs, providing a novel structural framework for the development of new alpha(4)beta(2) selective ligands.
Subject(s)
Alkaloids/chemical synthesis , Alkaloids/metabolism , Models, Molecular , Receptors, Nicotinic/metabolism , Alkaloids/chemistry , Alkaloids/pharmacology , Animals , Azocines/chemical synthesis , Azocines/chemistry , Azocines/metabolism , Azocines/pharmacology , Cell Line , Humans , Ligands , Nicotinic Agonists/chemical synthesis , Nicotinic Agonists/chemistry , Nicotinic Agonists/metabolism , Nicotinic Agonists/pharmacology , Nicotinic Antagonists/chemical synthesis , Nicotinic Antagonists/chemistry , Nicotinic Antagonists/metabolism , Nicotinic Antagonists/pharmacology , Protein Binding , Protein Conformation , Quinolizines/chemical synthesis , Quinolizines/chemistry , Quinolizines/metabolism , Quinolizines/pharmacology , Rats , Receptors, Nicotinic/chemistry , ThermodynamicsABSTRACT
We have earlier shown that microglia, the immune cells of the CNS, release microparticles from cell plasma membrane after ATP stimulation. These vesicles contain and release IL-1beta, a crucial cytokine in CNS inflammatory events. In this study, we show that microparticles are also released by astrocytes and we get insights into the mechanism of their shedding. We show that, on activation of the ATP receptor P2X7, microparticle shedding is associated with rapid activation of acid sphingomyelinase, which moves to plasma membrane outer leaflet. ATP-induced shedding and IL-1beta release are markedly reduced by the inhibition of acid sphingomyelinase, and completely blocked in glial cultures from acid sphingomyelinase knockout mice. We also show that p38 MAPK cascade is relevant for the whole process, as specific kinase inhibitors strongly reduce acid sphingomyelinase activation, microparticle shedding and IL-1beta release. Our results represent the first demonstration that activation of acid sphingomyelinase is necessary and sufficient for microparticle release from glial cells and define key molecular effectors of microparticle formation and IL-1beta release, thus, opening new strategies for the treatment of neuroinflammatory diseases.
Subject(s)
Cell-Derived Microparticles/enzymology , Neuroglia/metabolism , Sphingomyelin Phosphodiesterase/metabolism , Adenosine Triphosphate/analogs & derivatives , Adenosine Triphosphate/metabolism , Adrenergic Uptake Inhibitors/metabolism , Affinity Labels/metabolism , Animals , Brain/cytology , Brain/metabolism , Brain/pathology , Cell-Derived Microparticles/ultrastructure , Cells, Cultured , Enzyme Activation , Imipramine/metabolism , Interleukin-1beta/metabolism , Mice , Mice, Knockout , Models, Biological , Neuroglia/cytology , Particle Size , Receptors, Purinergic P2/metabolism , Receptors, Purinergic P2X7 , Signal Transduction/physiology , Sphingomyelin Phosphodiesterase/genetics , p38 Mitogen-Activated Protein Kinases/metabolism , src-Family Kinases/metabolismABSTRACT
A group of novel 4,5-dihydro-3-methylisoxazolyl derivatives, structurally related to epiboxidine (=(1R,4S,6S)-6-(3-methylisoxazol-5-yl)-7-azabicyclo[2.2.1]heptane), was prepared via 1,3-dipolar cycloaddition of acetonitrile oxide to different olefins. Target compounds 1a and 1b, 2a and 2b, 3, 4, and 5 were tested for affinity at neuronal nicotinic heteromeric (alpha4beta2) and homomeric (alpha7) acetylcholine receptors. Notably, diastereoisomers 1a and 1b were characterized by a massive drop of the affinity at the alpha4beta2 subtypes (K(i) values spanning the range 4.3-126 microM), when compared with that of epiboxidine (K(i)=0.6 nM). Therefore, the replacement of the 3-methylisoxazole ring of epiboxidine with the 4,5-dihydro-3-methylisoxazole nucleus is detrimental for the affinity at alpha4beta2 receptors. A comparable lack of affinity/selectivity for the two nAChR subtypes under study was evidenced for the remaining epiboxidine-related dihydroisoxazole derivatives 2a and 2b, and 3-5. Diastereoisomers 1a and 1b, and spirocyclic derivative 3 were docked into molecular models of the receptor subtypes under study, and their binding mode was compared with that of reference ligands endowed with high binding affinity.
Subject(s)
Isoxazoles/chemistry , Receptors, Nicotinic/metabolism , Isoxazoles/chemical synthesis , Kinetics , Models, Molecular , Protein Binding , Structure-Activity RelationshipABSTRACT
Neuronal nicotinic acetylcholine receptors (nAChRs) are a family of cation channels widely distributed in the brain, whose subunit composition and biophysical properties vary depending on the subtype and the area of the brain in which they are found. Brain nAChRs are also the target of nicotine, the most widespread drug of abuse. Chronic nicotine exposure differentially affects the number, subunit composition, stoichiometry and functional state of some nAChR subtypes, leaving others substantially unaffected. In this review, we will summarise recent data concerning the nAChR subtypes expressed in the CNS, and how they are regulated by means of chronic nicotine and/or nicotinic drugs. We will particularly focus on the possible mechanisms involved in the up-regulation of nAChRs.
Subject(s)
Gene Expression Regulation/physiology , Neurons/metabolism , Receptors, Nicotinic/metabolism , Animals , Brain/cytology , Gene Expression Regulation/drug effects , Nicotinic Antagonists/pharmacology , Protein Transport/drug effects , Protein Transport/physiology , Receptors, Nicotinic/chemistry , Receptors, Nicotinic/geneticsABSTRACT
The RS and SR enantiomers of 2-oxazolidinone and 1,4-benzodioxane bearing a 2-pyrrolidinyl substituent at the 5- and 2-position, respectively, were synthesized as candidate nicotinoids. One of the two benzodioxane stereoisomers reasonably fits the pharmacophore elements of (S)-nicotine and binds at alpha4beta2 nicotinic acetylcholine receptor with submicromolar affinity. Interestingly, both the synthesized pyrrolidinylbenzodioxanes exhibit analogous affinity at alpha(2) adrenergic receptor resembling the behaviour of some known alpha(2)-AR ligands recently proved to possess neuronal nicotinic affinity.
Subject(s)
Dioxanes/chemical synthesis , Dioxanes/metabolism , Oxazolidinones/chemical synthesis , Oxazolidinones/metabolism , Receptors, Nicotinic/metabolism , Protein BindingABSTRACT
Antibodies raised against human alpha2-6 and beta2-4 nicotinic receptor subunits were utilized to fractionate (3)H-epibatidine binding in human temporal cortex and striatum. The predominant receptor subtypes in both regions contained alpha4 and beta2 subunits. In normal cortex, 10% of binding was also associated with alpha2 subunits, whereas in the striatum, contributions by alpha6 (17%) and beta3 (23%) were observed. Minimal binding (< or =5%) was associated with alpha3. In Alzheimer's disease and dementia with Lewy bodies, cortical loss of binding was associated with reductions in alpha4 (50%, P < 0.01) and beta2 (30-38%, P < 0.05). In Parkinson's disease and dementia with Lewy bodies, striatal deficits in alpha6 (91 and 59% respectively, P < 0.01) and beta3 (72 and 75%, P < 0.05) tended to be greater than for alpha4 and beta2 (50-58%, P < 0.05). This study demonstrates distinct combinations of subunits contributing to heteromeric nicotinic receptor binding in the human brain that are area/pathway specific and differentially affected by neurodegeneration.
Subject(s)
Alzheimer Disease/pathology , Corpus Striatum/pathology , Lewy Bodies/pathology , Parkinson Disease/pathology , Receptors, Nicotinic/deficiency , Temporal Lobe/pathology , Aged , Aged, 80 and over , Amino Acid Sequence , Autopsy , Female , Humans , Male , Molecular Sequence Data , Peptide Fragments/immunology , Receptors, Nicotinic/chemistry , Receptors, Nicotinic/geneticsABSTRACT
Nicotinic drug treatment can affect the expression of neuronal nicotinic acetylcholine receptors (nAChR) both in vivo and in vitro through molecular mechanisms not fully understood. The present study investigated the effect of the novel cytisine dimer 1,2-bisN-cytisinylethane (CC4) on nAChR natively expressed by SH-SY5Y neuroblastoma cells in culture. CC4 lacked the agonist properties of cytisine and was a potent antagonist (IC50=220 nM) on nAChRs. Chronic treatment of SH-SY5Y cells with 1 mM CC4 for 48 h increased the expression of 3H-epibatidine (3H-Epi; 3-4-fold) or 125I-alpha-bungarotoxin (125I-alphaBgtx; 1.2-fold) sensitive receptors present on the cell membrane and in the intracellular pool. Comparable data were obtained with nicotine or cytisine, but not with carbamylcholine, d-tubocurarine, di-hydro-beta-erythroidine or hexametonium. Immunoprecipitation and immunopurification studies showed that the increase in 3H-Epi-binding receptors was due to the enhanced expression of alpha3beta2 and alpha3beta2beta4 subtypes without changes in subunit mRNA transcription or receptor half-life. The upregulation was not dependent on agonist/antagonist properties of the drugs, and did not concern muscarinic or serotonin receptors. Whole-cell patch clamp analysis of CC4-treated cells demonstrated larger nicotine-evoked inward currents with augmented sensitivity to the blockers alpha-conotoxin MII or methyllycaconitine. In conclusion, chronic treatment with CC4 increased the number of nAChRs containing beta2 and alpha7 subunits on the plasma membrane, where they were functionally active. In the case of beta2-containing receptors, we propose that CC4, by binding to intracellular receptors, triggered a conformational reorganisation of intracellular subunits that stimulated preferential assembly and membrane-directed trafficking of beta2-containing receptor subtypes..
Subject(s)
Azocines/pharmacology , Nerve Tissue Proteins/metabolism , Neurons/drug effects , Nicotinic Antagonists/pharmacology , Quinolizines/pharmacology , Receptors, Nicotinic/metabolism , Alkaloids/pharmacology , Binding Sites , Bridged Bicyclo Compounds, Heterocyclic/pharmacology , Bungarotoxins/pharmacology , Cell Line, Tumor , Cell Membrane/drug effects , Cell Membrane/metabolism , Dose-Response Relationship, Drug , Humans , Inhibitory Concentration 50 , Membrane Potentials/drug effects , Nerve Tissue Proteins/drug effects , Neuroblastoma , Neurons/metabolism , Neurons/ultrastructure , Nicotine/pharmacology , Nicotinic Agonists/pharmacology , Protein Structure, Quaternary/drug effects , Protein Transport/drug effects , Pyridines/pharmacology , Receptors, Nicotinic/drug effects , Subcellular Fractions/drug effects , Subcellular Fractions/metabolism , Time Factors , Up-Regulation , alpha7 Nicotinic Acetylcholine ReceptorABSTRACT
mRNAs for the neuronal nicotinic acetylcholine receptor (nAChR) alpha6 and beta3 subunits are abundantly expressed and colocalized in dopaminergic cells of the substantia nigra and ventral tegmental area. Studies using subunit-null mutant mice have shown that alpha6- or beta3-dependent nAChRs bind alpha-conotoxin MII (alpha-CtxMII) with high affinity and modulate striatal dopamine release. This study explores the effects of beta3 subunit-null mutation on striatal and midbrain nAChR expression, composition, and pharmacology. Ligand binding and immunoprecipitation experiments using subunit-specific antibodies indicated that beta3-null mutation selectively reduced striatal alpha6* nAChR expression by 76% versus beta3(+/+) control. Parallel experiments showed a smaller reduction in both midbrain alpha3* and alpha6* nAChRs (34 and 42% versus beta3(+/+) control, respectively). Sedimentation coefficient determinations indicated that residual alpha6* nAChRs in beta3(-/-) striatum were pentameric, like their wild-type counterparts. Immunoprecipitation experiments on immunopurified beta3* nAChRs demonstrated that almost all wild-type striatal beta3* nAChRs also contain alpha4, alpha6, and beta2 subunits, although a small population of non-beta3 alpha6* nAChRs is also expressed. beta3 subunit incorporation seemed to increase alpha4 participation in alpha6beta2* complexes. (125)I-Epibatidine competition binding studies showed that the alpha-CtxMII affinity of alpha6* nAChRs from the striata of beta3(-/-) mice was similar to those isolated from beta3(+/+) animals. Together, the results of these experiments show that the beta3 subunit is important for the correct assembly, stability and/or transport of alpha6* nAChRs in dopaminergic neurons and influences their subunit composition. However, beta3 subunit expression is not essential for the expression of alpha6*, high-affinity alpha-CtxMII binding nAChRs.
Subject(s)
Corpus Striatum/metabolism , Gene Deletion , Protein Subunits/deficiency , Receptors, Nicotinic/biosynthesis , Receptors, Nicotinic/deficiency , Substantia Nigra/metabolism , Amino Acid Sequence , Animals , Gene Expression Regulation/physiology , Humans , Mice , Mice, Inbred C57BL , Mice, Knockout , Molecular Sequence Data , Protein Subunits/genetics , Rats , Receptors, Nicotinic/geneticsABSTRACT
By acting through retinal nicotinic acetylcholine receptors (nAChRs), acetylcholine plays an important role in the development of both the retina and central visual pathways. Ligand binding and immunoprecipitation studies with subunit-specific antibodies showed that the expression of alphaBungarotoxin (alphaBgtx) and high-affinity epibatidine (Epi) receptors is regulated developmentally and increases until postnatal day 21 (P21). The increase in Epi receptors is caused by a selective increase in the subtypes containing the alpha2, alpha4, alpha6, beta2, and beta3 subunits. Immunopurification studies revealed three major populations of Epi receptors on P21: alpha6(*) receptors (26%), which contain the alpha6beta3beta2, alpha6alpha4beta3beta2, and alpha6alpha3/alpha2beta3beta2 subtypes; alpha4(non-alpha6)(*) receptors (60%), which contain the alpha2alpha4beta2 and alpha4beta2 subtypes; and (non-alpha4/non-alpha6)(*) receptors (14%), which contain the alpha2beta2/beta4 and alpha3beta2/beta4 subtypes. These three populations can be pharmacologically discriminated using alphaconotoxin MII, which binds the alpha6(*) population with high affinity. In situ hybridization showed that the transcripts for all of the subunits are heterogeneously distributed throughout retinal neurons at P21, with alpha3, alpha6, and beta3 transcripts preferentially concentrated in the ganglion cell layer, alpha5 in the inner nuclear layer, and alpha4 and beta2 distributed rather homogeneously. To investigate whether nAChR expression is affected by visual experience, we also studied dark-reared P21 rats. Visual deprivation had no effect on the expression of alphaBgtx receptors or the developmentally regulated Epi receptors containing the alpha2, alpha6, and/or beta3 subunits but significantly increased the expression of the Epi receptors containing the alpha4 and beta2 subunits. Overall, this study demonstrates that the retina is the rat neural region that expresses the widest array of nAChR subtypes. These receptors have a specific distribution, and their expression is finely regulated during development and by visual experience.