Most Synaptic Vesicles Isolated from Rat Brain Carry Three Membrane Proteins, SV2, Synaptophysin, and p65

We have prepared highly purified synaptic vesicles from rat brain by subjecting vesicles purified by our previous method to a further fractionation step, i.e., equilibrium centrifugation on a Ficoll gradient. Monoclonal antibodies to three membrane proteins enriched in synaptic vesicles--SV2, synaptophysin, and p65--each were able to immunoprecipitate specifically approximately 90% of the total membrane protein from Ficoll-purified synaptic vesicle preparations. Anti-SV2 precipitated 96% of protein, anti-synaptophysin 92%, and anti-p65 83%. These results demonstrate two points: (1) Ficoll-purified synaptic vesicles appear to be greater than 90% pure, i.e., less than 10% of membranes in the preparation do not carry synaptic vesicle-associated proteins. These very pure synaptic vesicles may be useful for direct biochemical analyses of mammalian synaptic vesicle composition and function. (2) SV2, synaptophysin, and p65 coexist on most rat brain synaptic vesicles. This result suggests that the functions of these proteins are common to most brain synaptic vesicles. However, if SV2, synaptophysin, or p65 is involved in synaptic vesicle dynamics, e.g., in vesicle trafficking or exocytosis, separate cellular systems are very likely required to modulate the activity of such proteins in a temporally or spatially specific manner.     

Glutamate immunoreactivity is enriched over pinealocytes of the gerbil pineal gland

Summary Mammalian pinealocytes have been shown to contain synaptic-like microvesicles with putative secretory functions. As a first step to elucidate the possibility that pinealocyte microvesicles store messenger molecules, such as neuroactive amino acids, we have studied the distributional pattern of glutamate immunoreactivity in the pineal gland of the Mongolian gerbil (Meriones unguiculatus) at both light- and electron-microscopic levels. In semithin sections of plastic-embedded pineals, strong glutamate immunoreactivity could be detected in pinealocytes throughout the pineal gland. The density of glutamate immunolabeling in pinealocytes varied among individual cells and was mostly paralled by the density of immunostaining for synaptophysin, a major integral membrane protein of synaptic and synaptic-like vesicles. Postembedding immunogold staining of ultrathin pineal sections revealed that gold particles were enriched over pinealocytes. In particular, a high degree of immunoreactivity was associated with accumulations of microvesicles that filled dilated process terminals of pinealocytes. A positive correlation between the number of gold particles and the packing density of microvesicles was found in three out of four process terminals analyzed. However, the level of glutamate immunoreactivity in pinealocyte process endings was lower than in presumed glutamatergic nerve terminals of the cerebellum and posterior pituitary. The present results provide some evidence for a microvesicular compartmentation of glutamate in pinealocytes. Our findings thus lend support to the hypothesis that glutamate serves as an intrapineal signal molecule of physiological relevance to the neuroendocrine functions of the gland.     

Neuronotypic Differentiation Results in Reduced Levels and Altered Distribution of Synaptophysin in PC 12 Cells

Abstract: Several synaptic vesicle proteins including synap-tophysin and p65/synaptotagmin are expressed by the pheochromocytoma cell line PC12. Stimulation of these cells with nerve growth factor for 7 days induces morphologic neuronotypic differentiation, but the levels of synaptophysin are markedly reduced. Stimulation with cyclic AMP analogs also produces neuronotypic differentiation of PC12 cells, and the degree of morphologic differentiation induced by these agents parallels their ability to effect reduction in synaptophysin levels. By contrast, levels of p65/synaptotagmin are increased following neuronotypic differentiation. The contrasting effects of neuronotypic differentiation on levels of synaptophysin and p65/synaptotagmin indicate potential differences in the regulation of these proteins in PC12 cells. Immunocytochemical labeling of undifferentiated PC12 cells reveals concentrations of synaptophysin in the perinuclear region. After neuronotypic differentiation, there is reduction in perinuclear labeling and concentration of label in swellings along PC12 cell processes. At the ultra-structural level, synaptophysin labeling is found on similar organelles in both undifferentiated and nerve growth factor-stimulated PC12 cells. Although the highest labeling densities were seen on small clear vesicles, specific labeling was also seen on dense core vesicles. The presence of synaptophysin on both small clear vesicles and dense core vesicles indicates potential functional similarities in these vesicle types. The changes in the levels and immunocytochemical distribution of synaptophysin after neuronotypic differentiation suggest possible functional heterogeneity among morphologically similar populations of small clear vesicles.     

突触囊泡蛋白在ALS转基因鼠海马的表达变化

目的为了为揭示肌萎缩脊髓侧索硬化症（amyotrophic lateral sclerosis,ALS）认知功能障碍的机制提供依据,观察不同年龄ALS转基因小鼠海马中突触囊泡蛋白（synaptophysin,Syp）的表达情况。方法取95d、108d和122dALS转基因鼠海马,应用免疫荧光、Westernblot、RT-PCR技术检测Syp在海马中的表达变化。结果与同窝野生型鼠比较,Syp蛋白和mRNA表达水平在95d龄ALS转基因鼠海马中无明显变化,在108d与122d龄ALS转基因鼠海马中明显降低。结论Syp在ALS转基因鼠海马中表达减少表明,突触可塑性降低是ALS学习记忆能力下降的重要病理学基础。     

戊四氮点燃癫痫大鼠空间学习记忆改变及海马突触素、突触后致密物PSD-95表达的变化

目的探讨戊四氮点燃癫痫对大鼠空间学习记忆的影响及可能的分子机制。方法戊四氮(pentylenetet-razol,PTZ)点燃建立慢性癫痫(chronic epileptic,CEP)模型,Morris水迷宫进行行为学检测,免疫组织化学方法观察大鼠海马CA1、CA3区突触素(synaptophysin,P38)和突触后致密物95(postsynaptic density 95,PSD-95)的表达,并用计算机图像分析系统对免疫反应结果进行处理。结果水迷宫试验检测癫痫组大鼠空间学习记忆能力受损;免疫组化结果表明其海马CA1、CA3区P38和PSD-95免疫反应产物较对照组明显减少(P<0.01,P<0.05)。结论戊四氮点燃癫痫大鼠伴有学习记忆功能减退,其海马神经元P38和PSD-95的表达减少可能参与了空间学习记忆受损。     

The effects of early life lead exposure on the expression of P2X7 receptor and synaptophysin in the hippocampus of mouse pups

The present study was undertaken to investigate the effects of maternal lead exposure on expression of P2X7 receptor and synaptophysin in the hippocampus of mice offspring. Lead exposure initiated from beginning of gestation to weaning. Lead acetate administered in drinking solutions was dissolved in distilled deionized water at the concentrations of 0.1%, 0.5% and 1% groups, respectively. On the 21st postnatal day, the Pb levels were also determined by graphite furnace atomic absorption spectrometry. The expression of P2X7 receptor and synaptophysin in hippocampus was examined by immunohistochemistry and Western blotting. The lead levels in blood and hippocampus of all lead exposure groups were significantly higher than that of the control group (P < 0.05). Compared with the control group, the expression of P2X7 receptor was increased in lead exposed groups (P < 0.05), but the expression of synaptophysin was decreased (P < 0.05). The high expression of P2X7 receptor and low expression of synaptophysin in the hippocampus of pups may contribute to the neurotoxicity associated with maternal Pb exposure.     

Peroxynitrite Induces Tyrosine Nitration and Modulates Tyrosine Phosphorylation of Synaptic Proteins

Peroxynitrite, the product of the radical-radical reaction between nitric oxide and superoxide anion, is a potent oxidant involved in tissue damage in neurodegenerative disorders. We investigated the modifications induced by peroxynitrite in tyrosine residues of proteins from synaptosomes. Peroxynitrite treatment (> or =50 microM) induced tyrosine nitration and increased tyrosine phosphorylation. Synaptophysin was identified as one of the major nitrated proteins and pp60src kinase as one of the major phosphorylated substrates. Further fractionation of synaptosomes revealed nitrated synaptophysin in the synaptic vesicles, whereas phosphorylated pp60src was enriched in the postsynaptic density fraction. Tyrosine phosphorylation was increased by treatment with 50-500 microM peroxynitrite and decreased by higher concentrations, suggesting a possible activation/inactivation of kinases. Immunocomplex kinase assay proved that peroxynitrite treatment of synaptosomes modulated the pp60src autophosphorylation activity. The addition of bicarbonate (CO2 1.3 mM) produced a moderate enhancing effect on some nitrated proteins but significantly protected the activity of pp60src against peroxynitrite-mediated inhibition so that at 1 mM peroxynitrite, the kinase was still more active than in untreated synaptosomes. The phosphotyrosine phosphatase activity of synaptosomes was inhibited by peroxynitrite (> or =50 microM) but significantly protected by CO2. Thus, the increase of phosphorylation cannot be attributed to peroxynitrite-mediated inhibition of phosphatases. We suggest that peroxynitrite may regulate the posttranslational modification of tyrosine residues in pre- and postsynaptic proteins. Identification of the major protein targets gives insight into the pathways possibly involved in neuronal degeneration associated with peroxynitrite overproduction.     

Subcellular Distribution of 65,000 Calmodulin-Binding Protein (p65) and Synaptophysin (p38) in Adrenal Medulla

Both neuronal and endocrine cells contain secretory vesicles that store and release neurotransmitters and peptides. Neuronal cells release their secretory material from both small synaptic vesicles and large dense-core vesicles (LDCVs), whereas endocrine cells release secretory products from LDCVs. Neuronal small synaptic vesicles are known to express three integral membrane proteins: 65,000 calmodulin-binding protein (65-CMBP) (p65), synaptophysin (p38), and SV2. A controversial question surrounding these three proteins is whether they are present in LDCV membranes of endocrine and neuronal cells. Sucrose density centrifugation of adrenal medulla was performed to study and compare the subcellular distribution of two of these small synaptic vesicle proteins (65-CMBP and synaptophysin). Subsequent immunoblotting and 125I-Protein A binding experiments performed on the fractions obtained from sucrose gradients showed that 65-CMBP was present in fractions corresponding to granule membranes and intact chromaffin granules. Similar immunoblotting and 125I-Protein A binding experiments with synaptophysin antibodies showed that this protein was also present in intact granules and granule membrane fractions. However, an additional membrane component, equilibrating near the upper portion of the sucrose gradient, also showed strong immunoreactivity with anti-synaptophysin and high 125I-Protein A binding activity. In addition, immunoblotting experiments on purified plasma and granule membranes demonstrated that 65-CMBP was a component of both membranes, whereas synaptophysin was only present in granule membranes. Thus, there appears to be a different subcellular localization between 65-CMBP and synaptophysin in the chromaffin cell.     

Synaptophysin immunoreactivity in the mammalian endocrine pancreas

Summary Synaptophysin, a major membrane glycoprotein of small presynaptic vesicles in neurons, has also been found in microvesicles of endocrine cells, e.g., of the endocrine pancreas. In the present study, the endocrine pancreas in 9 mammalian species (man, dog, mink, bovine, rabbit, guinea pig, rat, mouse, gerbil) has been investigated immunohistochemically for synaptophysin immunoreactivity. Synaptophysin-positive cells have been identified and localized on semithin plastic sections. Our study demonstrates that, in all species examined, all pancreatic endocrine cell types are consistently synaptophysin-positive independent of their location within the tissue, or the conditions of tissue processing. In addition, a few cells that cannot be hormonally identified show synaptophysin immunoreactivity. Hence, synaptophysin appears to be a regular constituent of all pancreatic endocrine cells in mammals. In several species, a subpopulation of endocrine cells, consisting of glucagon-containing and/or pancreatic-polypeptide-containing cells, exhibits a significantly higher degree of synaptophysin immunoreactivity. In the gerbil, this heterogeneity can readily be detected from the day of birth onwards. Our findings indicate that closely related endocrine cell types may differ with respect to the content of synaptophysin.     

突触素在人胎儿海马的表达与发育的研究

目的探讨突触素在不同周龄阶段人胎儿海马中的表达与胎儿海马发育的关系。方法采用免疫组织化学方法(S-P法),观察突触素在不同周龄的人胎儿海马中的表达水平,利用计算机图像分析技术测量不同周龄阶段海马突触素表达的平均光密度。结果光镜下突触素免疫反应产物主要以颗粒状、点状形式存在于海马各层,在各层中分布不均匀;16-20W胎儿海马中已出现了阳性产物,21-25W阳性产物表达增多明显(P<0.05),25-29W阳性产物表达有下降趋势(P<0.05),30-39W阳性产物表达又显著升高(P<0.05)。结论突触素的数量可以反映突触发育的程度,在人胎儿海马中突触素的数量随神经细胞的发育成熟而增加,其间突触素表达的波动可能与突触形成需经历的过度生长及重塑有关。