BDNF对体外培养的大鼠脊髓前角神经元内突触素Ⅰ与突触囊泡素表达的影响

探讨BDNF对体外培养的大鼠脊髓前角神经元内突触素I与突触囊泡素(SYN)表达的影响。取孕14d大鼠子宫内胎鼠的脊髓腹侧部分神经元,体外有血清培养。在培养7d后,随机分成对照组、BDNF组和抗BDNF组。BDNF组培养液中加入BDNF(20ng/ml),抗BDNF组培养液中加入BDNF抗体(20μg/ml),对照组加入等量Hanks液。3d后在倒置显微镜下计数三组神经元存活数,并用NF200、MAP2、NSE的免疫组化反应对神经细胞进行鉴定。行突触素I与SYN免疫组化反应,对部分细胞行突触素ImRNA原位杂交反应,运用图像分析系统对突触素I与SYN免疫组织反应阳性产物以及突触素I原位杂交反应阳性产物作光密度分析。结果发现有血清培养时各组脊髓前角神经元的存活数无显著差异(P>0.05);BDNF组突触素I与SYN免疫反应阳性产物的平均光密度值高于其它两组,抗BDNF组最低(P<0.01)。BDNF组突触素ImRNA阳性产物的平均光密度值明显高于其它两组,抗BDNF组突触素ImRNA阳性产物的平均光密度值最低(P<0.01)。本研究结果提示BDNF对有血清培养时脊髓前角神经元的存活没有明显影响,但BDNF可明显上调培养的脊髓前角神经元内突触素I与SYN的表达。     

Characterization of synaptic vesicles and related neuronal features in nerve growth factor and ras oncogene differentiated PC12 cells

PC12 cells can differentiate into neuron-like cells after treatment with either nerve growth factor (NGF) or transduction with a retrovirus which expresses the K-ras oncogene. The concomitant treatment of NGF plus ras differentiates PC12 cells further than either agent alone with respect to neurite outgrowth, acetylcholinesterase levels, and most strikingly, the number of synaptic vesicle (SV) clusters. These SV clusters in PC12 cell neurites closely resemble those in the presynaptic terminals of neurons. Such SV clusters have not been described in cell lines previously. The SV clusters from all three differentiated groups (NGF, ras, and NGF plus ras) were similar in size, shape, and configuration, except that the ones in the doubly treated group occur in higher frequency and have more vesicles. The synaptic nature of these vesicle clusters was demonstrated by their regulated depletion after potassium stimulation. Furthermore, these vesicle clusters stained positively for two SV-associated proteins, synapsin I and synaptophysin, by EM immunocytochemistry (ICC). Such SV clusters in a cell line are very useful for characterizing the regulated release of SVs and the distribution of SV-related antigens in intact cells. Analysis by SDS-gel electrophoresis and immunoblotting indicated that synapsin I levels are higher in all three differentiated groups compared to untreated cells; whereas synaptophysin levels are lower in cells exposed to NGF alone or with NGF and ras double treatment. Possible convergence and/or divergence on the mechanisms of NGF and ras differentiation in PC12 cells are discussed. © 1995 Wiley-Liss, Inc.

1 This article is a US Government work and, as such, is in the public domain in the United States of America.

     

Synapsin-induced proliferation of T-cell lines against myelin basic protein obtained from rats with experimental autoimmune encephalomyelitis

We have previously described that antibodies and T cells against myelin basic protein (MBP) rose under conditions to induce acute experimental autoimmune encephalomyelitis (EAE) bind other proteins present in the synaptosomal fraction, some of them identified as synapsin I. The aim of this study was to evaluate whether anti-MBP T-cell lines can be also activated by synapsin. The analysis of rat anti-MBP T-cell lines cultured with each antigen showed that these cells responded also to purified rat synapsin and to the amino terminal portion of this protein. This recognition originated a proliferative response with a concomitant pattern of cytokine secretion similar to that induced by MBP itself implicating that this recognition would be mediated by the T-cell receptor. On the other hand, anti-synapsin T-cell lines were not capable of responding to MBP stimulation. Therefore, the immunological cross-reactivity between both proteins occurs only in one direction and these cross-reactive cells would be elicited only in animals sensitized with MBP. A possible implication of immunological agents against MBP cross-reactive with extra-myelin proteins in the process of EAE is considered.     

Ultrastructural localization of active zone and synaptic vesicle proteins in a preassembled multi-vesicle transport aggregate

Although it has been suggested that presynaptic active zone (AZ) may be preassembled, it is still unclear which entities carry the various proteins to the AZ during synaptogenesis. Here, I propose that aggregates of dense core vesicles (DCV) and small clear vesicles in the axons of young rat hippocampal cultures are carriers containing preformed AZ and synaptic vesicle (SV) components on their way to developing synapses. The aggregates were positively labeled with antibodies against Bassoon and Piccolo (two AZ cytomatrix proteins), VAMP, SV2, synaptotagmin (three SV membrane proteins), and synapsin I (a SV-associated protein). Bassoon and Piccolo labeling were localized at dense material both in the aggregates and at the AZ. In addition to the SV at the synapses, the SV membrane proteins labeled the clear vesicles in the aggregate as well as many other SV-like and pleiomorphic vesicular structures in the axons, and synapsin I labeling was associated with the vesicles in the aggregates. In single sections, these axonal vesicle aggregates were approximately 0.22 by 0.13 microm in average dimensions and contain one to two DCV and five to six small clear vesicles. Serial sections confirmed that the aggregates were not synaptic junctions sectioned en face. Labeling intensities of Bassoon and Piccolo measured from serially sectioned transport aggregates and AZ were within range of each other, suggesting that one or a few aggregates, but not individual DCV, can carry sufficient Bassoon and Piccolo to form an AZ. The present findings provide the first ultrastructural evidence localizing various AZ and SV proteins in a preassembled multi-vesicle transport aggregate that has the potential to quickly form a functional active zone.     

Inflammation-like glial response in lead-exposed immature rat brain.

Numerous studies on lead (Pb) neurotoxicity have indicated this metal to be a dangerous toxin, particularly during developmental stages of higher organisms. Astrocytes are responsible for sequestration of this metal in brain tissue. Activation of astroglia may often lead to loss of the buffering function and contribute to pathological processes. This phenomenon is accompanied by death of neuronal cells and may be connected with inflammatory events arising from the production of a wide range of cytokines and chemokines. The effects of prolonged exposure to Pb upon glial activation are examined in immature rats to investigate this potential proinflammatory effect. When analyzed at the protein level, glial activation is observed after Pb exposure, as reflected by the increased level of glial fibrillary acidic protein and S-100beta proteins in all parts of the brain examined. These changes are associated with elevation of proinflammatory cytokines. Production of interleukin (IL)-1beta and tumor necrosis factor-alpha is observed in hippocampus, and production of IL-6 is seen in forebrain. The expression of fractalkine is observed in both hippocampus and forebrain but inconsiderably in the cerebellum. In parallel with cytokine expression, signs of synaptic damage in hippocampus are seen after Pb exposure, as indicated by decreased levels of the axonal markers synapsin I and synaptophysin. Obtained results indicate chronic glial activation with coexisting inflammatory and neurodegenerative features as a new mechanism of Pb neurotoxicity in immature rat brain.     

Transgenic expression of the protein-L-isoaspartyl methyltransferase (PIMT) gene in the brain rescues mice from the fatal epilepsy of PIMT deficiency

Protein-L-isoaspartyl methyltransfearase (PIMT) plays a physiological role in the repair of damaged proteins containing isoaspartyl residues. In previous studies, we showed that PIMT-deficient mice developed a fatal epileptic seizure associated with the accumulation of damaged proteins in the brain. The mutant mice also showed a neurodegenerative pathology in hippocampi and impaired spatial memory. Still undefined, however, is how the accumulation of isoaspartates leads to the death of PIMT-deficient mice. In the present study, we generated PIMT transgenic (Tg) mice to investigate whether the exogenous expression of PIMT could improve the symptoms associated with PIMT deficiency. Rescue experiments showed that Tg expression of PIMT driven by a prion promoter effectively cured the PIMT-deficient mice. Biochemically, a higher expression level of transgene led to the effective repair of damaged proteins in vivo. Although a lower level of expression caused an accumulation of damaged proteins in a partially rescued line, the mice survived. Interestingly, synapsin I, which was extensively modified posttranslationally in PIMT-deficient mice, was specifically repaired in a partially rescued, but symptom-improved, Tg line. Our results suggest that an overall accumulation of damaged proteins does not necessarily lead to a fatal epileptic seizure, whereas certain modifications, such as changes in synapsin I, may play a pivotal pathological role in epilepsy.     

Putative neurohemal release zones in the stomatogastric nervous system of decapod crustaceans

The stomatogastric nervous system (STNS) of decapod crustaceans has long been used to study the modulation of small neural circuits. Profiles in the sheath of the nerves and ganglia of the STNS, which contain only dense-core vesicles, have been described in electron microscopical studies (Friend [1976] Cell Tissue Res. 175:369-380; Kilman and Marder [1997] Soc Neurosci Abstr. 23:477; Skiebe and Ganeshina [2000] J Comp Neurol 420:373-397). These profiles resemble those found in neurohemal organs and suggest the presence of neurohemal release zones in the STNS. To map these putative neurohemal release zones, a combination of two antibodies was used in the present study. A synapsin antibody recognizing vesicle proteins of clear vesicles was combined with a synaptotagmin antibody recognizing vesicle proteins of clear and dense-core vesicles. Exclusive synaptotagmin-like staining, therefore, indicated the regions with only dense-core vesicles. Such a staining was found in a mesh in the perineural sheath of nerves in the STNS of all three species investigated. In the crayfish Cherax destructor and the lobster Homarus americanus, the stained mesh was located in the sheath of nerves connecting all four ganglia of the STNS, whereas in the crab Cancer pagurus it was found on different nerves, which are more directly exposed to the hemolymph in this species. Exclusive synaptotagmin-like staining was also found in a putative neurohemal release zone in the sheath of the circumoesophageal connectives and the postoesophageal commissure in C. destructor. These data suggest that an important source of modulation of the networks and the muscles of the stomach is a compartmentalized release of neurohormones from zones in the STNS.     

Synapsin III is a key component of α‐synuclein fibrils in Lewy bodies of PD brains

Lewy bodies (LB) and Lewy neurites (LN), which are primarily composed of α‐synuclein (α‐syn), are neuropathological hallmarks of Parkinson''s disease (PD) and dementia with Lewy bodies (DLB). We recently found that the neuronal phosphoprotein synapsin III (syn III) controls dopamine release via cooperation with α‐syn and modulates α‐syn aggregation. Here, we observed that LB and LN, in the substantia nigra of PD patients and hippocampus of one subject with DLB, displayed a marked immunopositivity for syn III. The in situ proximity ligation assay revealed the accumulation of numerous proteinase K‐resistant neuropathological inclusions that contained both α‐syn and syn III in tight association in the brain of affected subjects. Most strikingly, syn III was identified as a component of α‐syn‐positive fibrils in LB‐enriched protein extracts from PD brains. Finally, a positive correlation between syn III and α‐syn levels was detected in the caudate putamen of PD subjects. Collectively, these findings indicate that syn III is a crucial α‐syn interactant and a key component of LB fibrils in the brain of patients affected by PD.     

电针对血管性痴呆大鼠海马突触蛋白Ⅰ表达的影响

目的:探讨电针治疗对血管性痴呆大鼠学习记忆能力的影响及其作用机制。方法:采用双侧颈总动脉结扎法制作血管性痴呆大鼠模型,以电针治疗,Morris水迷宫检测大鼠的学习记忆能力,western blot检测海马突触蛋白Ⅰ的表达。结果:电针治疗可明显改善血管性痴呆大鼠的学习记忆能力,增加海马突触蛋白Ⅰ表达(P<0.05)。结论:电针治疗通过改善血管性痴呆大鼠海马突触蛋白Ⅰ表达,从而改善大鼠的学习记忆能力。     

Controlled contusion injury alters molecular systems associated with cognitive performance

We investigated whether a learning impairment after a controlled cortical impact (CCI) injury was associated with alterations in molecules involved in synaptic plasticity and learning and memory. Adult male rats with moderate CCI to the left parietal cortex, tested in a Morris water maze (MWM) beginning at postinjury day 10, showed impaired cognitive performance compared with sham-treated rats. Tissue was extracted for mRNA analysis on postinjury day 21. The expression of brain-derived neurotrophic factor (BDNF), synapsin I, cyclic-AMP response element binding protein (CREB), and calcium-calmodulin-dependent protein kinase II (alpha-CAMKII) were all significantly decreased compared with sham injury levels within the ipsilateral hippocampus after CCI. No significant molecular level changes were found in the contralateral hippocampus. Decreased expression of BDNF and synapsin I was also found within the ipsilateral parietal cortex of CCI-injured rats compared with shams. However, BDNF and synapsin I expressions were significantly increased in the contralateral parietal cortex of the CCI rats. CREB expression was significantly decreased within the contralateral cortex of the CCI group. These findings show enduring reductions in the expression of BDNF, synapsin I, CREB, and alpha-CAMKII ipsilateral to a CCI injury, which seem associated with the spatial learning deficits observed in this injury model. In addition, the delayed increase in the expression of BDNF and synapsin I within the cortex contralateral to CCI may reflect restorative processes in areas homotypical to the injury.