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An amendment to this paper has been published and can be accessed via a link at the top of the paper.
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An amendment to this paper has been published and can be accessed via a link at the top of the paper.
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The lateral habenula (LHb) is a key brain region involved in the pathophysiology of depression. It is activated by stimuli associated with negative experiences and is involved in encoding aversive signals. Hyperactivity of LHb is found in both rodent models of depression and human patients with depression. However, little is known about the underlying molecular mechanisms. Here we show that in LHb neurons, p11, a multifunctional protein implicated in depression, is significantly upregulated by chronic restraint stress. Knockdown of p11 expression in LHb alleviates the stress-induced depression-like behaviors. Moreover, chronic restraint stress induces bursting action potentials in LHb neurons, which are abolished by p11 knockdown. Overexpression of p11 in dopamine D2 receptor-containing LHb neurons of control mice induces depression-like behaviors. These results have identified p11 in LHb as a key molecular determinant regulating negative emotions, which may help to understand the molecular and cellular basis of depression.
Assuntos
Anexina A2/metabolismo , Depressão/metabolismo , Habenula/metabolismo , Proteínas S100/metabolismo , Animais , Anexina A2/genética , Depressão/genética , Depressão/fisiopatologia , Transtorno Depressivo/metabolismo , Transtorno Depressivo/fisiopatologia , Modelos Animais de Doenças , Técnicas de Silenciamento de Genes/métodos , Habenula/fisiologia , Masculino , Camundongos , Camundongos Transgênicos , Neurônios/metabolismo , Receptores de Dopamina D2/metabolismo , Proteínas S100/genética , Regulação para CimaRESUMO
The serotonergic neurotransmitter system has been widely implicated in the pathophysiology of mood-related disorders such as anxiety and major depressive disorder (MDD). The onset of therapeutic efficacy of traditional antidepressants is delayed by several weeks. The 5-HT4 receptor has emerged as a new therapeutic target since agonists of this receptor induce rapid antidepressant-like responses in rodents. Here we show that the 5-HT4 receptor is regulated by CK2, at transcriptional and post-transcriptional levels. We present evidence, in two different CK2α knockout mouse lines, that this regulation is region-specific, with the 5-HT4 receptor upregulated in prefrontal cortex (PFC) but not striatum or hippocampus where CK2α is also ablated. 5-HT4 receptor signaling is enhanced in vitro, as evidenced by enhanced cAMP production or receptor plasma membrane localization in the presence of CK2 inhibitor or shRNA targeting CK2α. In vivo, 5-HT4 receptor signaling is also upregulated since ERK activation is elevated and sensitive to the inverse agonist, GR113808 in the PFC of CK2α KO mice. Behaviorally, KO mice as well as mice with AAV-mediated deletion of CK2α in the PFC show a robust 'anti-depressed-like' phenotype and display an enhanced response to antidepressant treatment when tested in paradigms for mood and anxiety. Importantly, it is sufficient to overexpress the 5-HT4 receptor in the mPFC to generate mice with a similar 'anti-depressed-like' phenotype. Our findings identify the mPFC as the region that mediates the effect of enhanced 5-HT4 receptor activity and CK2 as modulator of 5-HT4 receptor levels in this brain region that regulates mood-related phenotypes.
Assuntos
Caseína Quinase II/metabolismo , Depressão/metabolismo , Receptores 5-HT4 de Serotonina/metabolismo , Animais , Antidepressivos/farmacologia , Ansiedade/metabolismo , Encéfalo/metabolismo , Caseína Quinase II/genética , Corpo Estriado/metabolismo , Depressão/tratamento farmacológico , Transtorno Depressivo Maior/tratamento farmacológico , Transtorno Depressivo Maior/metabolismo , Hipocampo/metabolismo , Indóis/farmacologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Córtex Pré-Frontal/metabolismo , Córtex Pré-Frontal/fisiologia , Agonistas do Receptor 5-HT4 de Serotonina/farmacologia , Transdução de Sinais/efeitos dos fármacos , Sulfonamidas/farmacologiaRESUMO
Chronic stress has a crucial role in the development of psychiatric diseases, such as anxiety and depression. Dysfunction of the medial prefrontal cortex (mPFC) has been linked to the cognitive and emotional deficits induced by stress. However, little is known about the molecular and cellular determinants in mPFC for stress-associated mental disorders. Here we show that chronic restraint stress induces the selective loss of p11 (also known as annexin II light chain, S100A10), a multifunctional protein binding to 5-HT receptors, in layer II/III neurons of the prelimbic cortex (PrL), as well as depression-like behaviors, both of which are reversed by selective serotonin reuptake inhibitors (SSRIs) and the tricyclic class of antidepressant (TCA) agents. In layer II/III of the PrL, p11 is highly concentrated in dopamine D2 receptor-expressing (D2+) glutamatergic neurons. Viral expression of p11 in D2+ PrL neurons alleviates the depression-like behaviors exhibited by genetically manipulated mice with D2+ neuron-specific or global deletion of p11. In stressed animals, overexpression of p11 in D2+ PrL neurons rescues depression-like behaviors by restoring glutamatergic transmission. Our results have identified p11 as a key molecule in a specific cell type that regulates stress-induced depression, which provides a framework for the development of new strategies to treat stress-associated mental illnesses.
Assuntos
Anexina A2/metabolismo , Depressão/metabolismo , Proteínas S100/metabolismo , Estresse Psicológico/metabolismo , Animais , Anexina A2/genética , Anexina A2/fisiologia , Transtornos de Ansiedade/metabolismo , Transtornos de Ansiedade/fisiopatologia , Doença Crônica , Depressão/fisiopatologia , Transtorno Depressivo/metabolismo , Transtorno Depressivo/fisiopatologia , Emoções/efeitos dos fármacos , Humanos , Camundongos , Camundongos Transgênicos , Neurônios/metabolismo , Córtex Pré-Frontal/metabolismo , Córtex Pré-Frontal/fisiopatologia , Receptores de Dopamina D2/metabolismo , Receptores de Serotonina/metabolismo , Proteínas S100/genética , Proteínas S100/fisiologia , Inibidores Seletivos de Recaptação de Serotonina/farmacologia , Estresse Psicológico/fisiopatologiaRESUMO
The most recent genome-wide association studies (GWAS) of schizophrenia (SCZ) identified hundreds of risk variants potentially implicated in the disease. Further, novel statistical methodology designed for polygenic architecture revealed more potential risk variants. This can provide a link between individual genetic factors and the mechanistic underpinnings of SCZ. Intriguingly, a large number of genes coding for ionotropic and metabotropic receptors for various neurotransmitters-glutamate, γ-aminobutyric acid (GABA), dopamine, serotonin, acetylcholine and opioids-and numerous ion channels were associated with SCZ. Here, we review these findings from the standpoint of classical neurobiological knowledge of neuronal synaptic transmission and regulation of electrical excitability. We show that a substantial proportion of the identified genes are involved in intracellular cascades known to integrate 'slow' (G-protein-coupled receptors) and 'fast' (ionotropic receptors) neurotransmission converging on the protein DARPP-32. Inspection of the Human Brain Transcriptome Project database confirms that that these genes are indeed expressed in the brain, with the expression profile following specific developmental trajectories, underscoring their relevance to brain organization and function. These findings extend the existing pathophysiology hypothesis by suggesting a unifying role of dysregulation in neuronal excitability and synaptic integration in SCZ. This emergent model supports the concept of SCZ as an 'associative' disorder-a breakdown in the communication across different slow and fast neurotransmitter systems through intracellular signaling pathways-and may unify a number of currently competing hypotheses of SCZ pathophysiology.
Assuntos
Receptores Ionotrópicos de Glutamato/genética , Receptores de Glutamato Metabotrópico/genética , Esquizofrenia/genética , Encéfalo/metabolismo , Dopamina/metabolismo , Fosfoproteína 32 Regulada por cAMP e Dopamina/metabolismo , Predisposição Genética para Doença/genética , Estudo de Associação Genômica Ampla , Genótipo , Humanos , Herança Multifatorial/genética , Polimorfismo de Nucleotídeo Único/genética , Receptores Ionotrópicos de Glutamato/metabolismo , Receptores de Glutamato Metabotrópico/metabolismo , Fatores de Risco , Transdução de Sinais/genética , Transmissão Sináptica/genética , Ácido gama-Aminobutírico/metabolismoRESUMO
Mood disorders and antidepressant therapy involve alterations of monoaminergic and glutamatergic transmission. The protein S100A10 (p11) was identified as a regulator of serotonin receptors, and it has been implicated in the etiology of depression and in mediating the antidepressant actions of selective serotonin reuptake inhibitors. Here we report that p11 can also regulate depression-like behaviors via regulation of a glutamatergic receptor in mice. p11 directly binds to the cytoplasmic tail of metabotropic glutamate receptor 5 (mGluR5). p11 and mGluR5 mutually facilitate their accumulation at the plasma membrane, and p11 increases cell surface availability of the receptor. Whereas p11 overexpression potentiates mGluR5 agonist-induced calcium responses, overexpression of mGluR5 mutant, which does not interact with p11, diminishes the calcium responses in cultured cells. Knockout of mGluR5 or p11 specifically in glutamatergic neurons in mice causes depression-like behaviors. Conversely, knockout of mGluR5 or p11 in GABAergic neurons causes antidepressant-like behaviors. Inhibition of mGluR5 with an antagonist, 2-methyl-6-(phenylethynyl)pyridine (MPEP), induces antidepressant-like behaviors in a p11-dependent manner. Notably, the antidepressant-like action of MPEP is mediated by parvalbumin-positive GABAergic interneurons, resulting in a decrease of inhibitory neuronal firing with a resultant increase of excitatory neuronal firing. These results identify a molecular and cellular basis by which mGluR5 antagonism achieves its antidepressant-like activity.
Assuntos
Anexina A2/metabolismo , Depressão/etiologia , Receptor de Glutamato Metabotrópico 5/metabolismo , Proteínas S100/metabolismo , Animais , Neurônios GABAérgicos/metabolismo , Ácido Glutâmico/metabolismo , Células HEK293 , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Inibição Neural , Neurônios/metabolismo , Parvalbuminas/metabolismo , Receptor de Glutamato Metabotrópico 5/antagonistas & inibidores , Transdução de Sinais , SinapsesRESUMO
Cognitive impairments are common in depression and involve dysfunctional serotonin neurotransmission. The 5-HT1B receptor (5-HT(1B)R) regulates serotonin transmission, via presynaptic receptors, but can also affect transmitter release at heterosynaptic sites. This study aimed at investigating the roles of the 5-HT(1B)R, and its adapter protein p11, in emotional memory and object recognition memory processes by the use of p11 knockout (p11KO) mice, a genetic model for aspects of depression-related states. 5-HT(1B)R agonist treatment induced an impairing effect on emotional memory in wild type (WT) mice. In comparison, p11KO mice displayed reduced long-term emotional memory performance. Unexpectedly, 5-HT(1B)R agonist stimulation enhanced memory in p11KO mice, and this atypical switch was reversed after hippocampal adeno-associated virus mediated gene transfer of p11. Notably, 5-HT(1B)R stimulation increased glutamatergic neurotransmission in the hippocampus in p11KO mice, but not in WT mice, as measured by both pre- and postsynaptic criteria. Magnetic resonance spectroscopy demonstrated global hippocampal reductions of inhibitory GABA, which may contribute to the memory enhancement and potentiation of pre- and post-synaptic measures of glutamate transmission by a 5-HT(1B)R agonist in p11KO mice. It is concluded that the level of hippocampal p11 determines the directionality of 5-HT(1B)R action on emotional memory processing and modulates hippocampal functionality. These results emphasize the importance of using relevant disease models when evaluating the role of serotonin neurotransmission in cognitive deficits related to psychiatric disorders.
Assuntos
Anexina A2/fisiologia , Aprendizagem da Esquiva/fisiologia , Emoções/fisiologia , Hipocampo/fisiologia , Memória/fisiologia , Receptor 5-HT1B de Serotonina/fisiologia , Proteínas S100/fisiologia , Animais , Anexina A2/deficiência , Anexina A2/genética , Aprendizagem da Esquiva/efeitos dos fármacos , Depressão/fisiopatologia , Modelos Animais de Doenças , Comportamento Exploratório/efeitos dos fármacos , Comportamento Exploratório/fisiologia , Feminino , Genes Reporter , Ácido Glutâmico/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Ressonância Magnética Nuclear Biomolecular , Fosforilação/efeitos dos fármacos , Terminações Pré-Sinápticas/metabolismo , Processamento de Proteína Pós-Traducional/efeitos dos fármacos , Piridinas/farmacologia , Tempo de Reação , Receptores de AMPA/metabolismo , Proteínas Recombinantes de Fusão/metabolismo , Proteínas S100/deficiência , Proteínas S100/genética , Agonistas do Receptor 5-HT1 de Serotonina/farmacologia , Transmissão Sináptica/efeitos dos fármacos , Transmissão Sináptica/fisiologia , Transdução GenéticaRESUMO
Alzheimer's disease (AD) is characterized by the accumulation of cerebral plaques composed of 40- and 42-amino acid beta-amyloid (Abeta) peptides, and autosomal dominant forms of AD appear to cause disease by promoting brain Abeta accumulation. Recent studies indicate that postmenopausal estrogen replacement therapy may prevent or delay the onset of AD. Here we present evidence that physiological levels of 17beta-estradiol reduce the generation of Abeta by neuroblastoma cells and by primary cultures of rat, mouse and human embryonic cerebrocortical neurons. These results suggest a mechanism by which estrogen replacement therapy can delay or prevent AD.
Assuntos
Peptídeos beta-Amiloides/biossíntese , Precursor de Proteína beta-Amiloide/biossíntese , Córtex Cerebral/citologia , Estradiol/farmacologia , Neurônios/fisiologia , Doença de Alzheimer , Animais , Células Cultivadas , Técnicas de Cocultura , Embrião de Mamíferos , Feto , Humanos , Camundongos , Neuroblastoma , Neurônios/citologia , Neurônios/efeitos dos fármacos , Fragmentos de Peptídeos/biossíntese , Ratos , Proteínas Recombinantes/biossíntese , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz , Transfecção , Células Tumorais CultivadasAssuntos
Anexina A2/metabolismo , Antidepressivos/uso terapêutico , Citalopram/uso terapêutico , Transtorno Depressivo Maior/tratamento farmacológico , Células Matadoras Naturais/metabolismo , Monócitos/metabolismo , Proteínas S100/metabolismo , Adulto , Biomarcadores Farmacológicos/metabolismo , Transtorno Depressivo Maior/metabolismo , Feminino , Citometria de Fluxo , Humanos , MasculinoRESUMO
Synapsin I, a major neuron-specific phosphoprotein, is localized on the cytoplasmic surface of small synaptic vesicles to which it binds with high affinity. It contains a collagenase-resistant head domain and a collagenase-sensitive elongated tail domain. In the present study, the interaction between synapsin I and phospholipid vesicles has been characterized, and the protein domains involved in these interactions have been identified. When lipid vesicles were prepared from cholesterol and phospholipids using a lipid composition similar to that found in native synaptic vesicle membranes (40% phosphatidylcholine, 32% phosphatidylethanolamine, 12% phosphatidylserine, 5% phosphatidylinositol, 10% cholesterol, wt/wt), synapsin I bound with a dissociation constant of 14 nM and a maximal binding capacity of about 160 fmol of synapsin I/microgram of phospholipid. Increasing the ionic strength decreased the affinity without greatly affecting the maximal amount of synapsin I bound. When vesicles containing cholesterol and either phosphatidylcholine or phosphatidylcholine/phosphatidylethanolamine were tested, no significant binding was detected under any conditions examined. On the other hand, phosphatidylcholine vesicles containing either phosphatidylserine or phosphatidylinositol strongly interacted with synapsin I. The amount of synapsin I maximally bound was directly proportional to the percentage of acidic phospholipids present in the lipid bilayer, whereas the Kd value was not affected by varying the phospholipid composition. A study of synapsin I fragments obtained by cysteine-specific cleavage showed that the collagenase-resistant head domain actively bound to phospholipid vesicles; in contrast, the collagenase-sensitive tail domain, though strongly basic, did not significantly interact. Photolabeling of synapsin I was performed with the phosphatidylcholine analogue 1-palmitoyl-2-[11-[4-[3-(trifluoromethyl)diazirinyl]phenyl] [2-3H]undecanoyl]-sn-glycero-3-phosphocholine; this compound generates a highly reactive carbene that selectively interacts with membrane-embedded domains of membrane proteins. Synapsin I was significantly labeled upon photolysis when incubated with lipid vesicles containing acidic phospholipids and trace amounts of the photoactivatable phospholipid. Proteolytic cleavage of photolabeled synapsin I localized the label to the head domain of the molecule.(ABSTRACT TRUNCATED AT 400 WORDS)
Assuntos
Bicamadas Lipídicas , Proteínas do Tecido Nervoso/metabolismo , Fragmentos de Peptídeos/metabolismo , Fosfolipídeos/metabolismo , Fosfoproteínas/metabolismo , Animais , Encéfalo/metabolismo , Bovinos , Cinética , Peso Molecular , Neurônios/metabolismo , Fosfatidilcolinas/metabolismo , Fosforilação , Ligação Proteica , SinapsinasRESUMO
Synapsin I is a major neuron-specific phosphoprotein that is specifically localized to the cytoplasmic surface of small synaptic vesicles. In the present study, the binding of synapsin I to small synaptic vesicles was characterized in detail. The binding of synapsin I was preserved when synaptic vesicles were solubilized and reconstituted in phosphatidylcholine. After separation of the protein and lipid components of synaptic vesicles under nondenaturing conditions, synapsin I bound to both components. The use of hydrophobic labeling procedures allowed the assessment of interactions between phospholipids and synapsin I in intact synaptic vesicles. Hydrophobic photolabeling followed by cysteine-specific cleavage of synapsin I demonstrated that the head domain of synapsin I penetrates into the hydrophobic core of the bilayer. The purified NH2-terminal fragment, derived from the head domain by cysteine-specific cleavage, bound to synaptic vesicles with high affinity confirming the results obtained from hydrophobic photolabeling. Synapsin I binding to synaptic vesicles could be inhibited by the entire molecule or by the combined presence of the NH2-terminal and tail fragments, but not by an excess of either NH2-terminal or tail fragment alone. The purified tail fragment bound with relatively high affinity to synaptic vesicles, though it did not significantly interact with phospholipids. Binding of the tail fragment was competed by holosynapsin I; was greatly decreased by phosphorylation; and was abolished by high ionic strength conditions or protease treatment of synaptic vesicles. The data suggest the existence of two sites of interaction between synapsin I and small synaptic vesicles: binding of the head domain to vesicle phospholipids and of the tail domain to a protein component of the vesicle membrane. The latter interaction is apparently responsible for the salt and phosphorylation dependency of synapsin I binding to small synaptic vesicles.
Assuntos
Proteínas do Tecido Nervoso/metabolismo , Fosfolipídeos/metabolismo , Fosfoproteínas/metabolismo , Vesículas Sinápticas/metabolismo , Animais , Sítios de Ligação , Encéfalo/metabolismo , Bovinos , Cinética , Bicamadas Lipídicas , Fragmentos de Peptídeos/metabolismo , Ligação Proteica , SinapsinasRESUMO
Synapsin I (formerly referred to as protein I) is the collective name for two almost identical phosphoproteins, synapsin Ia and synapsin Ib (protein Ia and protein Ib), present in the nervous system. Synapsin I has previously been shown by immunoperoxidase studies (De Camilli, P., T. Ueda, F. E. Bloom, E. Battenberg, and P. Greengard, 1979, Proc. Natl. Acad. Sci. USA, 76:5977-5981; Bloom, F. E., T. Ueda, E. Battenberg, and P. Greengard, 1979, Proc. Natl. Acad. Sci. USA 76:5982-5986) to be a neuron-specific protein, present in both the central and peripheral nervous systems and concentrated in the synaptic region of nerve cells. In those preliminary studies, the occurrence of synapsin I could be demonstrated in only a portion of synapses. We have now carried out a detailed examination of the distribution of synapsin I immunoreactivity in the central and peripheral nervous systems. In this study we have attempted to maximize the level of resolution of immunohistochemical light microscopy images in order to estimate the proportion of immunoreactive synapses and to establish their precise distribution. Optimal results were obtained by the use of immunofluorescence in semithin sections (approximately 1 micron) prepared from Epon-embedded nonosmicated tissues after the Epon had been removed. Our results confirm the previous observations on the specific localization of synapsin I in nerve cells and synapses. In addition, the results strongly suggest that, with a few possible exceptions involving highly specialized neurons, all synapses contain synapsin I. Finally, immunocytochemical experiments indicate that synapsin I appearance in the various regions of the developing nervous system correlates topographically and temporally with the appearance of synapses. In two accompanying papers (De Camilli, P., S. M. Harris, Jr., W. B. Huttner, and P. Greengard, and Huttner, W. B., W. Schiebler, P. Greengard, and P. De Camilli, 1983, J. Cell Biol. 96:1355-1373 and 1374-1388, respectively), evidence is presented that synapsin I is specifically associated with synaptic vesicles in nerve endings.
Assuntos
Proteínas do Tecido Nervoso/análise , Fosfoproteínas/análise , Sinapses/análise , Animais , Química Encefálica , Embrião de Galinha , Galinhas , Feminino , Imunofluorescência , Imunodifusão , Microscopia de Fluorescência , Nervos Periféricos/análise , Coelhos , Ratos , Ratos Endogâmicos , Sinapsinas , Distribuição TecidualRESUMO
Recycling of synaptophysin (p38), a synaptic vesicle integral membrane protein, was studied by the use of antisera raised against the protein purified from frog brain. When frog cutaneous pectoris muscles were fixed at rest, a bright, specific immunofluorescent signal was observed in nerve-terminal regions only if their plasma membranes had been previously permeabilized. When muscles were fixed after they had been treated for 1 h with a low dose of alpha-latrotoxin in Ca2+-free medium, an equally intense fluorescence could be observed without previous permeabilization. Under this condition, alpha-latrotoxin depletes nerve terminals of their quantal store of acetylcholine and of synaptic vesicles. These results indicate that fusion of synaptic vesicles leads to the exposure of intravesicular antigenic determinants of synaptophysin on the outer surface of the axolemma, and provide direct support for the vesicle hypothesis of neurotransmitter release. After 1 h treatment with the same dose of alpha-latrotoxin in the presence of 1.8 mM extracellular Ca2+, immunofluorescent images were obtained only after permeabilization with detergents. Under this condition, the vesicle population was maintained by an active process of recycling and more than two times the initial store of quanta were secreted. Thus, despite the active turnover of synaptic vesicles and of quanta of neurotransmitter, no extensive intermixing occurs between components of the vesicle and presynaptic plasma membrane.
Assuntos
Axônios/metabolismo , Proteínas de Membrana/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Junção Neuromuscular/metabolismo , Animais , Autorradiografia , Eletroforese em Gel de Poliacrilamida , Exocitose , Imunofluorescência , Immunoblotting , Peso Molecular , Junção Neuromuscular/efeitos dos fármacos , Ranidae , Venenos de Aranha/farmacologia , Vesículas Sinápticas/metabolismo , SinaptofisinaRESUMO
Synapsin I (protein I) is a neuron-specific phosphoprotein, which is a substrate for cAMP-dependent and Ca/calmodulin-dependent protein kinases. In two accompanying studies (De Camilli, P., R. Cameron, and P. Greengard, and De Camilli, P., S. M. Harris, Jr., W. B. Huttner, and P. Greengard, 1983, J. Cell Biol. 96:1337-1354 and 1355-1373) we have shown, by immunocytochemical techniques at the light microscopic and electron microscopic levels, that synapsin I is present in the majority of, and possibly in all, nerve terminals, where it is primarily associated with synaptic vesicles. In the present study we have prepared a highly purified synaptic vesicle fraction from rat brain by a procedure that involves permeation chromatography on controlled-pore glass as a final purification step. Using immunological methods, synapsin I concentrations were determined in various subcellular fractions obtained in the course of vesicle purification. Synapsin I was found to copurify with synaptic vesicles and to represent approximately 6% of the total protein in the highly purified synaptic vesicle fraction. The copurification of synapsin I with synaptic vesicles was dependent on the use of low ionic strength media throughout the purification. Synapsin I was released into the soluble phase by increased ionic strength at neutral pH, but not by nonionic detergents. The highly purified synaptic vesicle fraction contained a calcium-dependent protein kinase that phosphorylated endogenous synapsin I in its collagenase-sensitive tail region. The phosphorylation of this region appeared to facilitate the dissociation of synapsin I from synaptic vesicles under the experimental conditions used.
Assuntos
Proteínas do Tecido Nervoso/análise , Fosfoproteínas/análise , Vesículas Sinápticas/análise , Animais , Fracionamento Celular , Córtex Cerebral/análise , Córtex Cerebral/ultraestrutura , Clatrina , Proteínas de Membrana/análise , Microscopia Eletrônica , Fosforilação , Ratos , Ratos Endogâmicos , Solubilidade , Sinapsinas , Distribuição TecidualRESUMO
An intrinsic membrane protein of brain synaptic vesicles with Mr 38,000 (p38, synaptophysin) has recently been partially characterized (Jahn, R., W. Schiebler, C. Ouimet, and P. Greengard, 1985, Proc. Natl. Acad. Sci. USA, 83:4137-4141; Wiedenmann, B., and W. W. Franke, 1985, Cell, 41:1017-1028). We have now studied the presence of p38 in a variety of tissues by light and electron microscopy immunocytochemistry and by immunochemistry. Our results indicate that, within the nervous system, p38, like the neuron-specific phosphoprotein synapsin I, is present in virtually all nerve terminals and is selectively associated with small synaptic vesicles (SSVs). No p38 was detectable on large dense-core vesicles (LDCVs). p38 and synapsin I were found to be present in similar concentrations throughout the brain. Outside the nervous system, p38 was found in a variety of neuroendocrine cells, but not in any other cell type. In neuroendocrine cells p38 was localized on a pleiomorphic population of small, smooth-surfaced vesicles, which were interspersed among secretory granules and concentrated in the Golgi area, but not on the secretory granules themselves. Immunoblot analysis of endocrine tissues and cell lines revealed a band with a mobility slightly different from that of neuronal p38. This difference was attributable to a difference in glycosylation. The finding that p38, like synapsin I, is a component of SSVs of virtually all neurons, but not of LDCVs, supports the idea that SSVs and LDCVs are organelles of two distinct pathways for regulated neuronal secretion. In addition, our results indicate the presence in a variety of neuroendocrine cells of an endomembrane system, which is related to SSVs of neurons but is distinct from secretory granules.
Assuntos
Encéfalo/citologia , Proteínas de Membrana/análise , Vesículas Sinápticas/ultraestrutura , Animais , Anticorpos Monoclonais , Complexo Antígeno-Anticorpo , Encéfalo/ultraestrutura , Bovinos , Imunoglobulina G , Microscopia Eletrônica , Proteínas do Tecido Nervoso/análise , Ratos , Ratos Endogâmicos , Sinapsinas , Sinaptofisina , Distribuição TecidualRESUMO
Synapsin I is a synaptic vesicle-specific phosphoprotein composed of a globular and hydrophobic head and of a proline-rich, elongated and basic tail. Synapsin I binds with high affinity to phospholipid and protein components of synaptic vesicles. The head region of the protein has a very high surface activity, strongly interacts with acidic phospholipids and penetrates the hydrophobic core of the vesicle membrane. In the present paper, we have investigated the possible functional effects of the interaction between synapsin I and vesicle phospholipids. Synapsin I enhances both the rate and the extent of Ca(2+)-dependent membrane fusion, although it has no detectable fusogenic activity per se. This effect, which appears to be independent of synapsin I phosphorylation and localized to the head region of the protein, is attributable to aggregation of adjacent vesicles. The facilitation of Ca(2+)-induced liposome fusion is maximal at 50-80% of vesicle saturation and then decreases steeply, whereas vesicle aggregation does not show this biphasic behavior. Association of synapsin I with phospholipid bilayers does not induce membrane destabilization. Rather, 31P-nuclear magnetic resonance spectroscopy demonstrated that synapsin I inhibits the transition of membrane phospholipids from the bilayer (L alpha) to the inverted hexagonal (HII) phase induced either by increases in temperature or by Ca2+. These properties might contribute to the remarkable selectivity of the fusion of synaptic vesicles with the presynaptic plasma membrane during exocytosis.
Assuntos
Bicamadas Lipídicas , Lipídeos de Membrana/metabolismo , Fosfolipídeos/metabolismo , Sinapsinas/metabolismo , Vesículas Sinápticas/metabolismo , Animais , Encéfalo/metabolismo , Cálcio , Bovinos , Colesterol , Espectroscopia de Ressonância Magnética/métodos , Miocárdio/metabolismo , Fósforo , Fosforilação , Ratos , Espectrometria de Fluorescência , Sinapsinas/química , Sinapsinas/isolamento & purificação , Vesículas Sinápticas/ultraestruturaRESUMO
Nerve endings of the posterior pituitary are densely populated by dense-core neurosecretory granules which are the storage sites for peptide neurohormones. In addition, they contain numerous clear microvesicles which are the same size as small synaptic vesicles of typical presynaptic nerve terminals. Several of the major proteins of small synaptic vesicles of presynaptic nerve terminals are present at high concentration in the posterior pituitary. We have now investigated the subcellular localization of such proteins. By immunogold electron microscopy carried out on bovine neurohypophysis we have found that three of these proteins, synapsin I, Protein III, and synaptophysin (protein p38) were concentrated on microvesicles but were not detectable in the membranes of neurosecretory granules. In addition, we have studied the distribution of the same proteins and of the synaptic vesicle protein p65 in subcellular fractions of bovine posterior pituitaries obtained by sucrose density centrifugation. We have found that the intrinsic membrane proteins synaptophysin and p65 had an identical distribution and were restricted to low density fractions of the gradient which contained numerous clear microvesicles with a size range the same as that of small synaptic vesicles. The peripheral membrane proteins synapsin I and Protein III exhibited a broader distribution extending into the denser part of the gradient. However, the amount of these proteins clearly declined in the fractions preceding the peak of neurosecretory granules. Our results suggest that microvesicles of the neurohypophysis are biochemically related to small synaptic vesicles of all other nerve terminals and argue against the hypothesis that such vesicles represent an endocytic byproduct of exocytosis of neurosecretory granules.
Assuntos
Proteínas de Membrana/análise , Neuro-Hipófise/análise , Vesículas Sinápticas/análise , Animais , Anticorpos Monoclonais , Western Blotting , Bovinos , Centrifugação com Gradiente de Concentração , Grânulos Citoplasmáticos/análise , Eletroforese em Gel de Poliacrilamida , Imunofluorescência , Immunoblotting , Terminações Nervosas/metabolismo , Proteínas do Tecido Nervoso/análise , Neuropeptídeos/análise , Neuro-Hipófise/inervação , Neuro-Hipófise/ultraestrutura , Ratos , Ratos Endogâmicos , Frações Subcelulares/análise , Sinapsinas , Vesículas Sinápticas/ultraestrutura , SinaptofisinaRESUMO
Synapsin I is a neuron-specific phosphoprotein that is concentrated in the presynaptic nerve terminal in association with the cytoplasmic surface of synaptic vesicles. It has been demonstrated to bundle F-actin in a phosphorylation-dependent manner in vitro, a property consistent with its proposed role in linking synaptic vesicles to the cytoskeleton and its involvement in the regulation of neurotransmitter release. Synapsin I is composed of two distinct domains, a COOH terminal, collagenase-sensitive, hydrophilic, and strongly basic tail region, and an NH2 terminal, collagenase-resistant head region relatively rich in hydrophobic amino acids. To elucidate the structural basis for the interactions between synapsin I and F-actin and how it relates to other characteristics of synapsin I, we have performed a structure-function analysis of fragments of synapsin I produced by cysteine-specific cleavage with 2-nitro-5-thiocyanobenzoic acid. The fragments were identified and aligned with the parent molecule using the deduced primary structure of synapsin I and the known phosphorylation sites as markers. We have purified these fragments and examined their interactions with F-actin. Two distinct fragments, a 29-kD NH2-terminal fragment and a 15-kD middle fragment, were shown to contain F-actin binding sites. A 51/54-kD middle/tail fragment retained the F-actin binding and bundling activity of synapsin I, but the isolated tail fragment did not retain either activity. In contrast to phosphorylation of sites two and three in intact synapsin I, which abolishes F-actin bundling activity, phosphorylation of these sites in the middle/tail fragment failed to abolish this activity. In conclusion, three domains of synapsin I appear to be involved in F-actin binding and bundling.