RESUMO
Stathmin is a ubiquitous cytosolic protein which undergoes extensive phosphorylation in response to a variety of external signals. It is highly abundant in developing neurons. The use of antisense oligonucleotides which selectively block stathmin expression has allowed us to study directly its role in rat PC12 cells. We show that stathmin depletion prevents nerve growth factor (NGF)-stimulated differentiation of PC12 cells into sympathetic-like neurons although the expression of several NGF-inducible genes was not affected. Furthermore, we found that stathmin phosphorylation in PC12 cells which is induced by NGF depends on mitogen-activated protein kinase (MAPK) activity. We conclude that stathmin is an essential component of the NGF-induced MAPK signaling pathway and performs a key role during differentiation of developing neurons.
Assuntos
Glicoproteínas de Membrana , Proteínas dos Microtúbulos , Fatores de Crescimento Neural/fisiologia , Neurônios/citologia , Fosfoproteínas/fisiologia , Animais , Proteínas Quinases Dependentes de Cálcio-Calmodulina/antagonistas & inibidores , Proteínas Quinases Dependentes de Cálcio-Calmodulina/fisiologia , Proteínas de Transporte , Diferenciação Celular/efeitos dos fármacos , Diferenciação Celular/fisiologia , Inibidores Enzimáticos/farmacologia , Proteínas do Olho/biossíntese , Flavonoides/farmacologia , Expressão Gênica/efeitos dos fármacos , Proteínas de Filamentos Intermediários/biossíntese , Proteínas de Membrana , Fatores de Crescimento Neural/biossíntese , Fatores de Crescimento Neural/farmacologia , Proteínas do Tecido Nervoso/biossíntese , Neuritos , Neuropeptídeos , Oligonucleotídeos Antissenso/farmacologia , Células PC12 , Periferinas , Fosfoproteínas/biossíntese , Fosfoproteínas/genética , Fosfoproteínas/metabolismo , Fosforilação , Ratos , Transdução de Sinais/fisiologia , Estatmina , Tionucleotídeos/farmacologiaRESUMO
SNAP-25 is known as a neuron specific molecule involved in the fusion of small synaptic vesicles with the presynaptic plasma membrane. By immunolocalization and Western blot analysis, it is now shown that SNAP-25 is also expressed in pancreatic endocrine cells. Botulinum neurotoxins (BoNT) A and E were used to study the role of SNAP-25 in insulin secretion. These neurotoxins inhibit transmitter release by cleaving SNAP-25 in neurons. Cells from a pancreatic B cell line (HIT) and primary rat islet cells were permeabilized with streptolysin-O to allow toxin entry. SNAP-25 was cleaved by BoNT/A and BoNT/E, resulting in a molecular mass shift of approximately 1 and 3 kD, respectively. Cleavage was accompanied by an inhibition of Ca(++)-stimulated insulin release in both cell types. In HIT cells, a concentration of 30-40 nM BoNT/E gave maximal inhibition of stimulated insulin secretion of approximately 60%, coinciding with essentially complete cleavage of SNAP-25. Half maximal effects in terms of cleavage and inhibition of insulin release were obtained at a concentration of 5-10 nM. The A type toxin showed maximal and half-maximal effects at concentrations of 4 and 2 nM, respectively. In conclusion, the results suggest a role for SNAP-25 in fusion of dense core secretory granules with the plasma membrane in an endocrine cell type- the pancreatic B cell.
Assuntos
Insulina/metabolismo , Ilhotas Pancreáticas/metabolismo , Proteínas de Membrana , Proteínas do Tecido Nervoso/biossíntese , Animais , Toxinas Botulínicas/farmacologia , Cálcio/farmacologia , Células Cultivadas , Secreção de Insulina , Proteínas do Tecido Nervoso/efeitos dos fármacos , Ratos , Proteína 25 Associada a SinaptossomaRESUMO
To gain insights into transcription factors defining neuronal identity, we generated transgenic mice carrying a 1.8 kb rat neuron-specific enolase (NSE) promoter fragment fused to an E. coli lacZ gene. Four of seven transgenic families expressed transgene RNA in the nervous system but not in most other tissues. Histochemical analysis of adult brain from the two lines with highest lacZ mRNA levels showed neuron-specific, pan-neuronal beta-galactosidase activity. Developmental RNA and histochemical analyses showed parallel onset of transgene and endogenous NSE gene expression in various neuronal cell types, although the magnitude of NSE mRNA accumulation later in development was not matched by the transgene. These results suggest that cis-acting regulatory elements, subject to neuron-specific control, are located within 1.8 kb upstream from the NSE gene.
Assuntos
Regulação da Expressão Gênica , Neurônios/enzimologia , Fosfopiruvato Hidratase/genética , Regiões Promotoras Genéticas , Animais , Sequência de Bases , Northern Blotting , Southern Blotting , Encéfalo/metabolismo , Clonagem Molecular , Genes , Óperon Lac , Masculino , Camundongos , Camundongos Transgênicos , Dados de Sequência Molecular , Sequências Reguladoras de Ácido Nucleico , Testículo/metabolismo , beta-Galactosidase/genética , beta-Galactosidase/metabolismoRESUMO
Naturally occurring cell death (NOCD) is a prominent feature of the developing nervous system. During this process, neurons express bcl-2, a major regulator of cell death whose expression may determine whether a neuron dies or survives. To gain insight into the possible role of bcl-2 during NOCD in vivo, we generated lines of transgenic mice in which neurons overexpress the human BCL-2 protein under the control of the neuron-specific enolase (NSE) or phosphoglycerate kinase (PGK) promoters. BCL-2 overexpression reduced neuronal loss during the NOCD period, which led to hypertrophy of the nervous system. For instance, the facial nucleus and the ganglion cell layer of the retina had, respectively, 40% and 50% more neurons than normal. Consistent with this finding, more axons than normal were found in the facial and optic nerves. We also tested whether neurons overexpressing BCL-2 were more resistant to permanent ischemia induced by middle cerebral artery occlusion; in transgenic mice, the volume of the brain infarction was reduced by 50% as compared with wild-type mice. These animals represent an invaluable tool for studying the effects of increased neuronal numbers on brain function as well as the mechanisms that control the survival of neurons during development and adulthood.
Assuntos
Morte Celular , Expressão Gênica , Ataque Isquêmico Transitório/patologia , Neurônios/fisiologia , Proteínas Proto-Oncogênicas/genética , Proteínas Proto-Oncogênicas/fisiologia , Animais , Encéfalo/metabolismo , Nervo Facial/patologia , Gânglios Espinais , Humanos , Camundongos , Camundongos Transgênicos , Neurônios Motores/fisiologia , Nervo Óptico/patologia , Fosfoglicerato Quinase/genética , Fosfopiruvato Hidratase/genética , Regiões Promotoras Genéticas , Proteínas Proto-Oncogênicas c-bcl-2 , Medula Espinal/metabolismo , Distribuição TecidualRESUMO
Microtubule-associated protein 1B is an essential protein during brain development and neurite outgrowth and was studied by several assays to further characterize actin as a major interacting partner. Tubulin and actin co-immunoprecipitated with MAP1B at similar ratios throughout development. Their identity was identified by mass spectrometry and was confirmed by Western blots. In contrast to previous reports, the MAP1B-actin interaction was not dependent on the MAP1B phosphorylation state, since actin was precipitated from brain tissue throughout development at similar ratios and equal amounts were precipitated before and after dephosphorylation with alkaline phosphatase. MAP1B heavy chain was able to bind actin directly and therefore the N-terminal part of MAP1B heavy chain must also contain an actin-binding site. The binding force of this interaction was measured by atomic force microscopy and values were in the same range as those of MAP1B binding to tubulin or that measured in MAP1B self-aggregation. Aggregation was confirmed by negative staining and electron microscopy. Experiments including COS-7 cells, PC12 cells, cytochalasin D and immunocytochemistry with subsequent confocal laser microscopy, suggested that MAP1B may bind to actin but has no obvious microfilament stabilizing effect. We conclude, that the MAP1B heavy chain has a microtubule-stabilization effect, and contains an actin-binding site that may play a role in the crosslinking of actin and microtubules, a function that may be important in neurite elongation.
Assuntos
Citoesqueleto de Actina/metabolismo , Actinas/metabolismo , Encéfalo/metabolismo , Proteínas Associadas aos Microtúbulos/metabolismo , Microtúbulos/metabolismo , Neuritos/metabolismo , Animais , Animais Recém-Nascidos , Sítios de Ligação/fisiologia , Encéfalo/crescimento & desenvolvimento , Células COS , Chlorocebus aethiops , Substâncias Macromoleculares/química , Substâncias Macromoleculares/metabolismo , Espectrometria de Massas , Camundongos , Microscopia de Força Atômica , Microscopia Eletrônica , Proteínas Associadas aos Microtúbulos/química , Microtúbulos/ultraestrutura , Neuritos/ultraestrutura , Células PC12 , Fosforilação , Ligação Proteica/fisiologia , Subunidades Proteicas/química , Subunidades Proteicas/metabolismo , Ratos , Frações SubcelularesRESUMO
Transmitter release and membrane expansion involves the fusion of specialized vesicles to their target membranes. The mechanisms that regulate these fusion events might contribute to short- and long-term changes of synaptic efficiency that are associated with learning. A series of recently described protein-protein interactions has shed new light on vesicle binding to the cytoskeleton, vesicle docking to the target membranes and, finally, vesicle fusion and membrane retrieval. Specific steps in this pathway might be key sites for modulating the strength of synaptic connections that underlie the molecular basis of learning.
Assuntos
Aprendizagem/fisiologia , Terminações Nervosas/fisiologia , Proteínas do Tecido Nervoso/fisiologia , Vesículas Sinápticas/fisiologia , Animais , Humanos , Fusão de Membrana/fisiologia , Terminações Nervosas/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Plasticidade Neuronal/fisiologiaRESUMO
This study examines the role of glucose and lactate as energy substrates to sustain synaptic vesicle cycling. Synaptic vesicle turnover was assessed in a quantitative manner by fluorescence microscopy in primary cultures of mouse cortical neurons. An electrode-equipped perfusion chamber was used to stimulate cells both by electrical field and potassium depolarization during image acquisition. An image analysis procedure was elaborated to select in an unbiased manner synaptic boutons loaded with the fluorescent dye N-(3-triethylammoniumpropyl)-4-(4-(dibutylamino)styryl)pyridinium dibromide (FM1-43). Whereas a minority of the sites fully released their dye content following electrical stimulation, others needed subsequent K(+) depolarization to achieve full release. This functional heterogeneity was not significantly altered by the nature of metabolic substrates. Repetitive stimulation sequences of FM1-43 uptake and release were then performed in the absence of any metabolic substrate and showed that the number of active sites dramatically decreased after the first cycle of loading/unloading. The presence of 1 mM glucose or lactate was sufficient to sustain synaptic vesicle cycling under these conditions. Moreover, both substrates were equivalent for recovery of function after a phase of decreased metabolic substrate availability. Thus, lactate appears to be equivalent to glucose for sustaining synaptic vesicle turnover in cultured cortical neurons during activity.
Assuntos
Córtex Cerebral/citologia , Glucose/farmacologia , Ácido Láctico/farmacologia , Neurônios/fisiologia , Vesículas Sinápticas/efeitos dos fármacos , Animais , Células Cultivadas , Distribuição de Qui-Quadrado , Diagnóstico por Imagem/métodos , Relação Dose-Resposta a Droga , Interações Medicamentosas , Embrião de Mamíferos , Imunofluorescência/métodos , Camundongos , Neurônios/citologia , Neurônios/efeitos dos fármacos , Neurônios/efeitos da radiação , Potássio/farmacologia , Compostos de Piridínio/farmacocinética , Compostos de Amônio Quaternário/farmacocinética , Vesículas Sinápticas/metabolismo , Fatores de TempoRESUMO
Glucose can react nonenzymatically with free amino groups on proteins and form advanced glycosylation end-products (AGEPs), that have been previously isolated and characterised in aging human connective tissues. In this study, we used immunocytochemistry to examine the distribution of AGEPs in the aging human brain. Our findings show that the pyramidal neurons selectively accumulate AGEP-containing vesicles in an age-dependent manner. In addition, our results demonstrate that AGEPs accumulate in the same type of neuron that degenerates in Alzheimer's disease.
Assuntos
Envelhecimento/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Células Piramidais/metabolismo , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Doença de Alzheimer/metabolismo , Doença de Alzheimer/patologia , Western Blotting , Encéfalo/metabolismo , Encéfalo/patologia , Criança , Pré-Escolar , Ensaio de Imunoadsorção Enzimática , Glicosilação , Humanos , Técnicas Imunoenzimáticas , Pessoa de Meia-Idade , Degeneração Neural/fisiologia , Emaranhados Neurofibrilares/metabolismo , Emaranhados Neurofibrilares/patologiaRESUMO
In neurons, synaptic vesicle exocytosis involves the formation of a core complex particle including syntaxin-1, synaptosomal-associated protein of 25 kDa (SNAP-25) and vesicle-associated membrane protein (VAMP)-2/synaptobrevin. The expression of these proteins was investigated in a panel of cell lines, including lines of endocrine and intestinal origin, by Western blotting and/or immunocytochemistry. The three core complex proteins were detected in the enteroendocrine, cholecystokinin (CCK)-secreting, cell lines STC-1 and GLUTag, and in the endocrine non-intestinal cell lines CA-77 and HIT-T15. In contrast, SNAP-25 and syntaxin-1 were undetected in the intestinal non-endocrine cell lines IEC-6, HT-29 and Caco-2, whereas a slight expression of VAMP-2 was documented in IEC-6 and HT-29 cells. Co-immunoprecipitation experiments indicated that syntaxin-1, SNAP-25 and VAMP-2 were present in a complex similar to that identified in brain. In the STC-1 cell line, treatment of streptolysin-O-permeabilized cells with tetanus toxin (Tetx) selectively cleaved VAMP-2 and VAMP-3/cellubrevin, and simultaneously abolished Ca2+-induced CCK secretion (IC50 approximately 12 nM). These results show that endocrine cell lines of intestinal origin express syntaxin-1, SNAP-25 and VAMP-2, and suggest a key role for a Tetx-sensitive protein (for example VAMP-2 and/or VAMP-3) in the CCK secretion by STC-1 cells.
Assuntos
Colecistocinina/metabolismo , Glândulas Endócrinas/metabolismo , Mucosa Intestinal/metabolismo , Proteínas de Membrana/metabolismo , Toxina Tetânica/farmacologia , Animais , Cálcio/metabolismo , Linhagem Celular , Cricetinae , Glândulas Endócrinas/citologia , Humanos , Hidrólise , Intestinos/citologia , Camundongos , RatosRESUMO
Synaptosomal associated protein of 25 kDa (SNAP-25) has been implicated in the membrane fusion machinery of neurotransmitter release and axonal growth. Using immunocytochemistry, we have analyzed the distribution and ultrastructural localization of SNAP-25 in selected areas of the central and peripheral nervous systems of adult rats. We show that the protein is specifically expressed in the trans face of the Golgi apparatus and in the axonal compartment. In axons and nerve endings, SNAP-25 is localized to discrete areas of the membranes of most organelles such as the axoplasmic reticulum, the axolemma, the outer membrane of mitochondria and synaptic vesicles. This wide distribution of SNAP-25 suggests that the protein is involved in the fusion of membranes in the whole axonal compartment of neurons.
Assuntos
Sistema Nervoso Periférico/ultraestrutura , Proteínas/ultraestrutura , Medula Espinal/ultraestrutura , Sinaptossomos/ultraestrutura , Animais , Axônios/ultraestrutura , Gânglios Espinais/ultraestrutura , Imuno-Histoquímica , Masculino , Microscopia Eletrônica , Mitocôndrias/ultraestrutura , Proteínas/imunologia , Ratos , Ratos Wistar , Sinapses/ultraestrutura , Sinaptossomos/imunologiaRESUMO
We have studied the age-dependence of the effects of kainate (KA) on the chick retina as a prelude to the accompanying paper on the effects of target-removal on the isthmo-optic nucleus. KA was injected into the eyes of chick embryos and chicks at different ages, and the retinas were fixed a few hours or several days later. The former group of retinas was scanned for pyknotic cells. The earliest age at which KA caused pyknosis was embryonic day 10 (E10), when pyknotic cells appeared in a ventrotemporal patch in the amacrine sublayer near the fundus. Over the next two days the sensitive region expanded tangentially, reaching the periphery first temporally, then nasally. Only after E12 did the KA cause pyknotic cells to occur also in the bipolar sublayer, where the sensitivity spread in the same spatiotemporal sequence as the initial wave, but two days later. Cell loss was examined in embryos that survived a week or more after the KA injection. Substantial cell depletion was found in both the inner nuclear and ganglion cell layers, but only when the injection had been made after E12. With progressively later injections, the depleted zone expanded in the same spatiotemporal sequence as described above, until at E15 the injections caused depletion throughout the entire extent of the retina. The reasons for the lack of cell depletion after KA injections made before E12 are discussed. Cell counts in the ganglion cell layer and studies of anterograde transport of intravitreally injected peroxidase along the retinofugal fibers showed that about half the ganglion cells (including the displaced ganglion cells) pass through a period of vulnerability to the KA injections, to which they subsequently become sensitive.
Assuntos
Animais Recém-Nascidos/fisiologia , Galinhas/fisiologia , Ácido Caínico/farmacologia , Retina/efeitos dos fármacos , Animais , Animais Recém-Nascidos/crescimento & desenvolvimento , Encéfalo/fisiologia , Embrião de Galinha/fisiologia , Transmissão Sináptica , Fatores de Tempo , Vias Visuais/fisiologiaRESUMO
We have examined the capacity of neurons in the chick isthmo-optic nucleus (ION) to survive when their target neurons in the contralateral retinal are destroyed by intraocular injections of kainate (KA) at different stages in development. The retinal vulnerability to KA builds up progressively from embryonic day 10 (E10) until a plateau is reached at E15 (see accompanying paper); and the effects on the ION increase in parallel, almost all the ION neurons being rapidly lost after the E15 injections. KA injection before E15 lesioned only part of the retina and caused degeneration only in the topographically corresponding region of the ION. Near the end of the natural cell death period in the ION (E17), this initial dependence on the target cells is rapidly lost. Already at E16 the injections kill less ION neurons, and by E19 they kill none of them. The ION neurons have become completely insensitive to the KA injections and appear normal more than 4 months later, although axotomy (by eye removal) at a similar age would by then have killed them. The ectopic ION neurons, scattered outside the ION but projecting to the retina, are never affected by KA injections at any age.
Assuntos
Embrião de Galinha/fisiologia , Ácido Caínico , Neurônios/fisiologia , Nervo Óptico/fisiologia , Retina/fisiologia , Animais , Sobrevivência Celular/efeitos dos fármacos , Colchicina/farmacologia , Relação Dose-Resposta a Droga , Olho , Injeções , Ácido Caínico/farmacologia , Neurônios/efeitos dos fármacos , Nervo Óptico/citologia , Retina/citologia , Retina/efeitos dos fármacosRESUMO
In this article we study the relationship between the expression pattern of two recently identified isoforms of the 25-kD synaptosomal-associated protein (SNAP-25a and SNAP-25b) and the morphological changes inherent to neuronal plasticity during development and kainic acid treatment. SNAP-25 has been involved in vescicle fusion in the nerve terminal, and most likely participates in different membrane fusion-related processes, such as those involved in neurotransmitter release and axonal growth. In the adult brain, SNAP-25b expression exceeded SNAP-25a in distribution and intensity, being present in most brain structures . Moderate or high levels of SNAP-25a hybridization signal were found in neurons of the olfactory bulb, the layer Va of the frontal and parietal cortices, the piriform cortex, the subiculum and the hippocampal CA4 field, the substantia nigra/pars compacta, and the pineal gland, partially overlapping SNAP-25b mRNA distribution. In restricted regions of cerebral cortex, thalamus, mammillary bodies, substantia nigra, and pineal glands the two isoforms were distributed in reciprocal fashion. During development SNAP-25a mRNA was the predominant isoform, whereas SNAP-25b expression increased postnatally. The early expression of SNAP-25a in the embryo and the decrease after P21 is suggestive of a potential involvement of this isoform in axonal growth and/or synaptogenesis. This conclusion is indirectly supported by the observation that SNAP-25a mRNA, but not SNAP-25b mRNA, was upregulated in the granule cells of the adult dentate gyrus 48 hours after kainate-induced neurotoxic damage of the hippocampal CA3-CA4 regions. Increase of SNAP-25 immunoreactivity was observed as early as 4 days after kainate injection within the mossy fiber terminals of the CA3 region, and in the newly formed mossy fiber aberrant terminals of the supragranular layer. These data suggest an isoform-specific role of SNAP-25 in neural plasticity.
Assuntos
Encéfalo/metabolismo , Regulação da Expressão Gênica no Desenvolvimento/fisiologia , Proteínas de Membrana , Degeneração Neural/efeitos dos fármacos , Proteínas do Tecido Nervoso/genética , Plasticidade Neuronal/fisiologia , Animais , Sequência de Bases , Encéfalo/embriologia , Encéfalo/crescimento & desenvolvimento , Desenvolvimento Embrionário e Fetal/fisiologia , Ácido Caínico , Dados de Sequência Molecular , Sondas de Oligonucleotídeos , Ratos , Ratos Wistar , Proteína 25 Associada a SinaptossomaRESUMO
We have used the proteolytic properties of botulinum and tetanus neurotoxins (BoNT, TeNT) to cleave three proteins of the membrane fusion machinery, SNAP-25, VAMP/synaptobrevin, and syntaxin, in developing and differentiated rat central neurons in vitro. Then, we have studied the capacity of neurons to extend neurites, make synapses, and release neurotransmitters. All the toxins showed the expected specificity with the exception that BoNT/C cleaved SNAP-25 in addition to syntaxin and induced rapid neuronal death. In developing neurons, cleavage of SNAP-25 with BoNT/A inhibited axonal growth and prevented synapse formation. In contrast, cleavage of VAMP with TeNT or BoNT/B had no effects on neurite extension and synaptogenesis. All the toxins tested inhibited transmitter release in differentiated neurons, and cleavage of VAMP resulted in the strongest inhibition. These data indicate that SNAP-25 is involved in vesicle fusion for membrane expansion and transmitter release, whereas VAMP is selectively involved in transmitter release. In addition, our results support the hypothesis that synaptic activity is not essential for synapse formation in vitro.
Assuntos
Axônios/fisiologia , Fusão de Membrana/fisiologia , Proteínas de Membrana/fisiologia , Proteínas do Tecido Nervoso/metabolismo , Neurônios/ultraestrutura , Neurotransmissores/metabolismo , Animais , Células Cultivadas , Proteínas de Membrana/metabolismo , Neurônios/efeitos dos fármacos , Neurotoxinas/toxicidade , Proteínas Qa-SNARE , Proteínas R-SNARE , Ratos , Sinapses/efeitos dos fármacos , Proteína 25 Associada a Sinaptossoma , Toxina Tetânica/toxicidadeRESUMO
Age-related decline of cognition and memory, in humans and other animals, appears to be associated with neuronal loss. Experimental and clinical evidence has shown that the hippocampal formation is one of the brain regions most vulnerable to the ageing process. Because excess of glutamate is neurotoxic to hippocampal neurons, abnormalities in glutamate neurotransmitter function may play a crucial role in neurodegenerative disorders, especially in conjunction with brain ageing. We have used in situ hybridization to study the expression of the two major alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA)-selective glutamate receptor subunits, involved in the control of calcium permeability in the young adult and aged rat hippocampus. We show that the levels of messenger RNA encoding the AMPA-selective glutamate receptor subunit-1 (GluR1 or GluRA) and AMPA-selective glutamate receptor subunit-2 (GluR2 or GluRB) are highest in the dentate gyrus, followed by the CA1 and CA3 hippocampal subfields. We also show that the levels of both messenger RNAs decrease differentially with age in all subfields of the hippocampus. Finally, the GluR1/GluR2 messenger RNA ratios increase in the aged hippocampus, particularly in the CA3 subfield, suggesting that altered calcium homeostasis may contribute to age-related neuronal death.
Assuntos
Envelhecimento/metabolismo , Cálcio/metabolismo , Hipocampo/metabolismo , Neurônios/metabolismo , Receptores de AMPA/biossíntese , Animais , Autorradiografia , Morte Celular/fisiologia , Hipocampo/citologia , Hipocampo/crescimento & desenvolvimento , Hibridização In Situ , Masculino , Sondas de Oligonucleotídeos , RNA Mensageiro/biossíntese , Ratos , Ratos WistarRESUMO
The soluble N-ethylmaleimide-sensitive factor-attached protein receptor (SNARE) proteins syntaxin 1 and synaptosomal-associated protein-25 have been implicated in axonal outgrowth. Neuronal Sec1 (nSec1), also called murine unc18a (Munc18a), is a syntaxin 1-binding protein involved in the regulation of SNARE complex formation in synaptic vesicle membrane fusion. Here we analysed whether nSec1/Munc18a is involved in neurite formation. nSec1/Munc18a expressed under the control of an inducible promoter in differentiated PC12 cells as well as in hippocampal neurons appears first in the cell body, and at later times after induction along neurites and in growth cones. It is localised to distinct tubular and punctated structures. In addition, exogenous nSec1/Munc18a inhibited regulated secretion in PC12 cells. Overexpression in PC12 cells of nSec1/Munc18a or its homologue Munc18b, reduced the total length of neurites. This effect was enhanced with nSec1-T574A, a mutant that lacks a cyclin-dependent kinase 5 phosphorylation site and displays an increased binding to syntaxin 1. In contrast, in hippocampal neurons the total length of all primary neurites and branches was increased upon transfection of nSec1/Munc18a. Detailed morphometric analysis revealed that this was a consequence of an increased number of axonal side branches, while the average lengths in primary neurites and of side branches were not affected. From these results we suggest that nSec1/Munc18a is involved in the regulation of SNARE complex-dependent membrane fusion events implicated in the ramification of axonal processes in neurons.
Assuntos
Axônios/metabolismo , Hipocampo/metabolismo , Proteínas do Tecido Nervoso/biossíntese , Proteínas de Transporte Vesicular/biossíntese , Animais , Axônios/efeitos dos fármacos , Células Cultivadas , Hipocampo/citologia , Hipocampo/efeitos dos fármacos , Humanos , Proteínas Munc18 , Proteínas do Tecido Nervoso/farmacologia , Neurônios/citologia , Neurônios/efeitos dos fármacos , Neurônios/metabolismo , Células PC12 , Biossíntese de Proteínas , Proteínas/farmacologia , Ratos , Transfecção/métodos , Proteínas de Transporte Vesicular/farmacologiaRESUMO
Central nervous system structures containing neurons labeled by the fluorescent tracers Fast blue (FB), Diamidino yellow dihydrochloride (DY), Rhodamine B isothiocyanate (RITC) and Rhodamine-labeled latex microspheres (RLM) were processed with the Golgi method. The goal was to improve the visualization of the fluorescent labeled neurons and to allow their ultrastructural examination. While the fluorescence of FB and RITC is greatly attenuated by the Golgi method, RLM and DY are still visible in Golgi-impregnated neurons. However, it is usually necessary to remove the silver precipitate by gold-toning.
Assuntos
Corantes Fluorescentes , Neurônios/ultraestrutura , Coloração e Rotulagem/métodos , Amidinas , Animais , Gatos , Embrião de Galinha , Fixadores , Microscopia Eletrônica , Microesferas , RodaminasRESUMO
The neurons of the chick's isthmo-optic nucleus (ION) are known to innervate the retina. We here show that removing the retinal primordia causes the ION dendritic trees to be much less polarized than normal. Our observations were made at 11 embryonic days, which is before the isthmo-optic neurons become dependent on the retina for survival. Other parameters such as neuronal size were unchanged, so the effect seems to have been specific to dendritic shape. Our interpretation is that early target removal eliminates a retrograde signal that normally enhances dendritic polarization.
Assuntos
Encéfalo/anatomia & histologia , Dendritos/ultraestrutura , Animais , Encéfalo/crescimento & desenvolvimento , Encéfalo/ultraestrutura , Embrião de Galinha , Olho/crescimento & desenvolvimento , Vias Neurais/fisiologia , Retina/crescimento & desenvolvimentoRESUMO
Our basic question was whether the survival of developing neurons is critically dependent on the level of protein synthesis in the axonal target region. The experiments were carried out on the projection from the isthmo-optic nucleus (ION) to the contralateral retina in chick embryos. The ION is known to undergo almost 60% neuronal death between embryonic days (E) 12 and E17 and to be critically dependent on the retina for trophic support throughout this period and shortly afterwards. Various concentrations of the protein synthesis inhibitor cycloheximide were infused into one eye from E15 to E19. Moderate inhibition (up to about 40%) of retinal protein synthesis, which did not lead to retinal degeneration, had no detectable effects on the number of neurons, nor on the general morphology, in the ION. Only when the inhibition was as high as 50%, leading to widespread degeneration in the retina, did massive degeneration occur also in the ION. It was also shown that a single intraocular injection of cycloheximide at E15 that inhibited retinal protein synthesis by as much as 70-90% during the subsequent 24 h had little effect on the ION in embryos fixed at E19. These results indicate that although the ION neurons are critically dependent on the retina, they can resist major reductions in the level of retinal protein synthesis, which argues against the widespread belief that neuronal survival during development is regulated by the limited production of trophic molecules in the axonal target area. The data are, however, compatible with alternative hypotheses. Most plausibly, survival may be regulated by limited access to a nonlimiting supply of trophic molecules.
Assuntos
Neurônios/citologia , Biossíntese de Proteínas , Retina/embriologia , Animais , Morte Celular , Embrião de Galinha , Cicloeximida/farmacologia , Relação Dose-Resposta a Droga , Morfogênese/efeitos dos fármacos , Morfogênese/fisiologia , Neurônios/efeitos dos fármacos , Inibidores da Síntese de Proteínas/farmacologia , Retina/efeitos dos fármacosRESUMO
With the advent of modern molecular genetics and molecular biology, we will face more and more situations where novel gene products with unknown functions are identified. Genetic linkage analysis will allow the association of novel or known genes to Important diseases (1). Similarly, sensitlve differential cloning procedures will identify rare genes expressed in specific physiological or pathological situations (1, 3). In both cases, establishing the precise function of the identified gene is an essential step for the understanding of the cellular mechanisms that either lead to the disease or are pivotal in important physiological processes.