Identification of cells expressing galectin-1, a galactose-binding receptor,in the rat olfactory system

Interactions between carbohydrate ligands and their receptors play an important role in cell adhesion and migration in many tissues. Cell-surface carbohydrates that contain terminal galactose have previously been implicated in primary sensory axon growth in the rodent olfactory system. The aim of the present study was to determine whether galectin-1, a galactose-binding receptor, was expressed within the rat primary olfactory pathway. Immunohistochemical and in situ hybridisation analyses revealed expression of galectin-1 by primary sensory olfactory neurons during the major embryonic period of axonogenesis as well as in maturity. In the adult olfactory bulb, galectin-1 was expressed by both second-order projection neurons and interneurons and was selectively localised to the synaptic neuropil layers. Mitral cells, the principal postsynaptic target of primary olfactory axons, began expressing this lectin soon after genesis and maintained high levels into adulthood. The expression of galectin-1 in the primary olfactory pathway and olfactory bulb neuropil suggests a role for this lectin both in the initial formation and in the subsequent maintenance of neuronal connections between the peripheral and the central olfactory neurons as well as between neurons within the bulb. © 1995 Wiley-Liss, Inc.     

Induction of microtubule-associated protein 1B expression in Schwann cells during nerve regeneration

Microtubule-associated protein 1B (MAP1B) is expressed at high levels during development of the nervous system and is localized primarily in neurons while specific phosphorylated isoforms of MAP1B are localized exclusively in growing axons. The levels of MAP1B are down regulated in most regions of the adult CNS, but remain high in neurons and axons of the PNS. This study demonstrates that the expression of MAP1B is induced in adult Schwann cells following sciatic nerve lesion and regeneration. High levels of both mRNA and the MAP1B protein were detected in Schwann cells associated with the axotomized distal stump. Expression of MAP1B was also observed in cultured primary Schwann cells from neonatal rats. The properties of the MAP1B protein in cultured Schwann cells were further characterized by Western blot analysis using specific antibodies that recognize the N-terminal, middle and C-terminal domains of MAP1B. All of these antibodies detected a protein of 320-340 kDa demonstrating that MAP1B expressed by Schwann cells is very similar, or identical, to MAP1B expressed by neurons. The phosphorylation of MAP1B in Schwann cells was also studied using monoclonal antibodies (mAb) that recognize specific phosphorylation epitopes. The results indicated that the expression of MAP1B in Schwann cells exhibited a differential phosphorylation state that was recognized by mAb 1B6 but not by other mAbs, including 1B-P, 150 and RT97, that recognize phosphorylated MAP1B in growing axons. We therefore conclude that MAP1B is expressed in Schwann cells during both development and axonal regeneration, suggesting that the developmental pattern of MAP1B in these cells is recapitulated in adult Schwann cells during the early stages of regeneration and remyelination of injured peripheral axons. The presence of MAP1B in Schwann cells may support morphological changes of these cells, particularly the formation of processes prior to their differentiation into myelin forming Schwann cells.     

Dynamin-related protein 1 mediates mitochondria-dependent apoptosis in chlorpyrifos-treated SH-SY5Y cells

Recent studies have demonstrated that dynamin-related protein 1 (Drp1), a mitochondrial fission protein, mediates mitochondria-dependent apoptosis through mitochondrial division. However, little is known about the mechanism by which Drp1 modulates apoptosis in response to chlorpyrifos (CPF)-induced toxicity. In this study, we determined that CPF-induced mitochondrial apoptosis is mediated by Drp1 translocation in SH-SY5Y human neuroblastoma cells. Our results showed that CPF treatment induced intrinsic apoptosis by activating caspase-9, caspase-3, and cytochrome c release in SH-SY5Y cells. Cytosolic Drp1 translocated to the mitochondria in CPF-treated cells and was phosphorylated at Ser616. Treating cells with CPF induced the generation of reactive oxygen species (ROS) and activation of mitogen-activated protein kinases (MAPKs). Inhibiting this ROS generation and MAPK activation abolished CPF-induced expression of phospho-Drp1. Furthermore, Drp1 was required for p53 to translocate to the mitochondria under CPF-induced oxidative stress. Treating cells with mitochondrial-division inhibitor-1 (mdivi-1), which blocks Drp1 translocation, increased the viability of CPF-treated cells by abrogating Drp1 translocation and caspase-3 activation. Specifically, pretreating cells with mdivi-1 inhibited Bax translocation to the mitochondria by blocking p53 signaling. Taken together, these data reveal a novel mechanism by which Drp1 activates mitochondrial-dependent apoptosis and indicate that inhibiting Dpr1 function can protect against CPF-induced cytotoxicity. We propose that inhibiting Drp1 is a possible therapeutic approach for pesticide-induced toxicity when hyperactivated Drp1 contributes to pathology.     

Pure, postmitotic, polarized human neurons derived from NTera 2 cells provide a system for expressing exogenous proteins in terminally differentiated neurons.

NTera 2/cl.D1 (NT2) cells, a human teratocarcinoma cell line, were manipulated following retinoic acid treatment to yield greater than 95% pure cultures of neuronal cells (NT2-N cells). The commitment of NT2-N cells to a stable neuronal phenotype is irreversible as judged by the lack of mitotic activity or phenotypic reversion over a period of 2 months in culture. Furthermore, NT2-N cells express a variety of neuronal markers including many neuronal cytoskeletal proteins, secretory markers, and surface markers. NT2-N cells resemble primary neuronal cultures from rodents morphologically and in density of process outgrowth and, like primary neurons, go on to elaborate processes that differentiate into axons and dendrites. This culture method yields sufficient highly differentiated postmitotic NT2-N cells for both biochemical and molecular biological studies. Indeed, when undifferentiated NT2 cells were stably transfected with a beta-galactosidase (beta-gal) expression plasmid, beta-gal expression was shown to be present in both undifferentiated NT2 and postmitotic NT2-N cells. Thus, the ability to transfect expression plasmids into undifferentiated NT2 cells will allow the introduction of normal and mutant gene products into cells that can then be induced to become stable, postmitotic human neurons. We conclude that NT2 cells and NT2-N cells represent a unique model system for studies of human neurons, and a novel vehicle for the expression of diverse gene products in terminally differentiated polarized neurons.     

Function of death‐associated protein 1 in proliferation,differentiation, and apoptosis of chicken satellite cells

Introduction: Muscle growth and regeneration are processes closely associated with proliferation, differentiation, and apoptosis of muscle cells. Death‐associated protein 1 (DAP1) has been identified as a negative regulator of autophagy. Little is known about the function of DAP1 in the regulation of myogenesis and satellite cells. Methods: Chicken satellite cells were transfected with DAP1 cloned into the pCMS–enhanced green fluorescent protein vector or pcDNA3.1 vector, or a small interference RNA against the endogenous DAP1 gene. The cells were assayed for proliferation, differentiation, and apoptosis. Results: The overexpression of DAP1 increased proliferation, differentiation, and myotube diameter, but it had no effect on satellite cell apoptosis. In contrast, knockdown of DAP1 significantly decreased proliferation, differentiation, and number of nuclei per myotube, and it increased apoptosis of the cells. Conclusion: DAP1 is required for regulating myogenesis and apoptosis of satellite cells, which may affect muscle mass accretion and regeneration, and ameliorate muscle sarcopenia. Muscle Nerve 48:777–790, 2013     

Akt induces apoptosis in neuroblastoma cells expressing a C98X vasopressin mutant following autophagy suppression

Mutations in the arginine vasopressin (AVP)-neurophysin II (NP-II) gene that affect the folding and transport of the prohormone result in loss of secretion of the anti-diuretic hormone AVP from pituitary nerve terminals and cause autosomal dominant familial neurohypophyseal diabetes insipidus (adFNDI). One such mutation consists of the replacement of a Cys residue at position 98 with a stop codon (C98X) in the AVP precursor (corresponding to C67X in NP domain). In neuroblastoma cells over-expressing this truncated AVP precursor autophagy, a macromolecular degradation process, was shown to be essential for assuring cell survival. In the present study, we investigated the role of the Akt pro-survival signalling in the regulation of autophagy and of apoptosis linked with the handling of C98X AVP. Impairing autophagy-lysosomal sequestration or cathepsin D (CD)-mediated proteolysis triggered the activation of the intrinsic death pathway of apoptosis in C98X-expressing cells, but not in the wild-type -AVP-expressing cells. This was shown by the expression of a Vps34 dominant negative, which down-regulates the PI3k class III-dependent signalling needed for autophagosome (APH) formation, by genetic silencing as a result of RNA interference (RNAi) of Lamp2, a protein indispensable for the fusion of APHs with lysosomes, and by RNAi silencing of the lysosomal protease CD. Ectopic expression of either the wild-type or the mutated C98X AVP altered neither the expression nor the phosphorylation of the pro-survival signalling molecule Akt. Strikingly, the ectopic adenoviral-directed expression of a constitutively active Akt, instead of preserving cell survival, resulted in the suppression of autophagy, and precipitated Bax-mediated cell death. The present data demonstrate the need for autophagy-mediated degradation of mutated C98X peptides, which otherwise become toxic to the cell, and suggest that, in the presence of mis-folded proteins, the stimulation of the Akt signalling counteracts the beneficial effects of autophagy and precipitates cell death. It follows that growth factors impinging on the Akt pathway may have deleterious effect in neurones expressing mutant neuropeptides. This can provide an explanation for the late onset and progressive neuronal cell loss observed in hypothalamic magnocellular neurones of adFNDI patients.     

Chinese herbal medicine Yougui Pill reduces exogenous glucocorticoid-induced apoptosis in anterior pituitary cells

Long-term glucocorticoid use may result in sustained suppression of one or more secreted components from the hypothalamo-pituitary-adrenal axis, and often results in apoptosis. Yougui Pill(YGP), a 10-component traditional Chinese herbal medicine, has been shown to be clinically effective for glucocorticoid-induced suppression of the hypothalamo-pituitary-adrenal axis. However, the pharmacological and molecular mechanisms remain unclear. We hypothesized that YGP would exert an anti-apoptosis effect on dexamethasone-treated anterior pituitary cells. In vivo experiments showed that YGP significantly reduced the number of apoptotic cells, down-regulated m RNA expression of cytochrome c, caspase-3, and caspase-9, and up-regulated m RNA expression of Bcl-2. These findings suggest that YGP reduced glucocorticoid-induced apoptosis in rat anterior pituitary cells by regulating the mitochondria-mediated apoptosis pathway.     

抑制磷酸酶和tau过度磷酸化诱导神经细胞凋亡

目的探讨磷酸酶活性抑制、tau蛋白异常磷酸化与神经细胞变性死亡的关系.方法磷酸酶(PP)-2A/PP-1抑制剂岗田酸(okadaicacid,OA)10nmol/L与成神经细胞瘤细胞(SH-SY5Y)共培养,DNA-梯带和末端转移酶标记技术.结果用10nmol/LOA与SY5Y细胞共培养24h和48h均可引起DNA-梯带出现,此时末端转移酶标记显示阳性细胞数由2.16%±0.94%分别增多至18.05%±3.57%(P＜0.01)和22.52%±4.78%(P＜0.01).结论由于上述检测指标均与细胞凋亡有关,提示抑制PP-2A和部分抑制PP-1引起tau蛋白异常磷酸化可能通过神经细胞凋亡而引起AD患者神经细胞丢失.     

Secretion from bovine chromaffin cells acutely expressing exogenous proteins using a recombinant Semliki Forest virus containing an EGFP reporter

Acute expression of recombinant proteins throughout a population of postmitotic bovine chromaffin cells was achieved using the Semliki Forest virus expression system (P. Liljestrom and H. Garoff (1991) Biotechnology 9:1356-1361). The virus was modified to express a green fluorescent protein, which faithfully reported the expression of the recombinant proteins. Two types of reporting virus were constructed: the first included a second subgenomic element, and the second an internal ribosome entry site. Both were used to express the recombinant proteins beta-galactosidase, 5HT3 receptor, or tetanus toxin light chain. Beta-galactosidase was used to quantify the rate of expression of recombinant protein in chromaffin cells, the 5HT3 receptor to trigger secretion, and the toxin to block secretion. The experiments clearly show that infection and expression of recombinant proteins throughout a population of chromaffin cells do not, per se, affect the rate and extent of triggered exocytosis, endocytosis, or membrane recycling pathways. The catecholamine content of the cell is unaltered, and the secretory mechanism can be accessed within a few hours after infection. This noncytopathic method of acutely expressing specific proteins at physiological levels in chromaffin cells offers a powerful new tool for dissecting the roles of many proteins implicated in exo- and endocytosis.     

外源性TRAIL基因转染对胶质瘤C6细胞凋亡的实验研究

目的探讨外源性肿瘤坏死因子相关凋亡诱导配体（TRAIL）基因对大鼠C6胶质瘤细胞的凋亡作用。方法C6细胞在杜伯改良的Eagle培养基（DMEM）,37℃,5％CO2条件下培养;质粒提取与转染;用HoeChst试剂盒检测C6细胞凋亡。结果转染重组质粒PDC315-TRAIL的C6细胞组凋亡率明显高于未转染的C6细胞组及转染空载体PDC315的C6细胞组（P〈0．05）。结论外源性TRAIL基因可引起C6细胞凋亡。     

Effects of exogenous GM1 and GD1a on S20Y neuroblastoma cells

The effects of exogenous GM1 and GD1a on S20Y murine neuroblastoma cells were assessed by monitoring morphology, tumorigenicity, mitotic index, and plating efficiency. S20Y cells were seeded at a density equivalent to 5 X 10(4) cells per 35-mm tissue culture dish; 38-42 hr after seeding (preconfluent stage) the cells were treated for 12 hr with 100 micrograms of ganglioside per ml of medium in which the serum content was reduced from 10% to 0.5%. Analysis of the cell lipids indicated that added ganglioside became tightly associated with the membrane during the 12-hr exposure. GM1 treatment resulted in increased projections on the cell surface and fine structures projecting from the cell processes. GD1a treatment resulted in a reduction in the cellular mitotic index. Plating efficiency was reduced by both GM1 and GD1a. Neither ganglioside affected tumorigenicity of the S20Y cells. Twelve hours after removal of the added ganglioside and exposure of the cells to normal medium, the ganglioside composition of the membranes from treated cells approached that of the controls, and the ganglioside-induced effects had been reversed. These results suggest that addition of specific gangliosides induces different cellular responses and that these changes are dependent upon the continued presence of the ganglioside.     

Infection of human oligodendroglioma cells by a recombinant measles virus expressing enhanced green fluorescent protein

One of the hallmarks of the human CNS disease subacute sclerosing panencephalitis (SSPE) is a high level of measles virus (MV) infection of oligodendrocytes. It is therefore surprising that there is only one previous report of MV infection of rat oligodendrocytes in culture and no reports of human oligodendrocyte infection in culture. In an attempt to develop a model system to study MV infection of oligodendrocytes, time-lapse confocal microscopy, immunocytochemistry, and electron microscopy (EM) were used to study infection of the human oligodendroglioma cell line, MO3.13. A rat oligodendrocyte cell line, OLN-93, was also studied as a control. MO3.13 cells were shown to be highly susceptible to MV infection and virus budding was observed from the surface of infected MO3.13 cells by EM. Analysis of the infection in real time and by immunocytochemistry revealed that virus spread occurred by cell-to-cell fusion and was also facilitated by virus transport in cell processes. MO3.13 cells were shown to express CD46, a MV receptor, but were negative for the recently discovered MV receptor, signaling leucocyte activation molecule (SLAM). Immunohistochemical studies on SSPE tissue sections demonstrated that CD46 was also expressed on populations of human oligodendrocytes. SLAM expression was not detected on oligodendrocytes. These studies, which are the first to show MV infection of human oligodendrocytes in culture, show that the cells are highly susceptible to MV infection and this model cell line has been used to further our understanding of MV spread in the CNS.     

Cytoskeletal protein 4.1G is a binding partner of the metabotropic glutamate receptor subtype 1 alpha

Recent evidence suggests that cytoskeletal proteins play important roles in the clustering and anchoring of glutamate receptors to the cell surface membrane. To examine further this issue, we tested for direct interactions between the metabotropic glutamate receptor subtype 1alpha (mGlu1alpha) and 4.1G, which is a member of the erythrocyte membrane, cytoskeletal protein 4.1 family. First, co-localization of 4.1G and mGlu1alpha was observed in cultured hippocampal neurons. Second, in transiently transfected HEK 293 cells and in whole rat brain tissue, direct interactions between mGlu1alpha and 4.1G were observed. Third, we were able to identify the C-terminal tail of mGlu1alpha as an essential region for mGlu1alpha-4.1G interactions. Fourth, 4.1 G influences mGlu1alpha-mediated cAMP accumulation. Finally, we found that 4.1G increases the ligand-binding ability of mGlu1alpha and alters its cellular distribution. These observations identify 4.1G as a novel binding partner of mGlu1alpha that can regulate the action of mGlu1alpha.     

bFGF基因转染对放射诱导的C17.2神经干细胞凋亡的抑制作用

目的 观察外源性碱性成纤维细胞生长因子(bFGF)基因转染对放射诱导的C17.2神经干细胞(NSCs)凋亡的抑制作用,为探索治疗放射性脑损伤(RE)的新途径奠定实验基础.方法 以直线加速器照射C17.2 NSCs建立离体放射性损伤模型,流式细胞仪检测凋亡率和坏死率,MTT法检测细胞活性并拟合生存曲线.构建含bFGF片段的真核表达体pcDNA3.1-bFGF质粒后,脂质体法转染C17.2 NSCs,观察转染pcDNA3.1-bFGF质粒后细胞对放射的耐受情况,并与对照组比较.结果 转染pcDNA3.1-bFGF的C17.2 NSCs放射后凋亡率和死亡率均显著低于转染空质粒组和对照组(P＜0.05).结论 外源性bFGF基因转染对放射诱导的C17.2 NSCs凋亡具有明显抑制作用,对RE有潜在的治疗价值.     

过表达钙整合素结合蛋白CIB诱导SHG44胶质瘤细胞凋亡的实验研究

目的 观察CIB基因对人胶质瘤SHG44细胞体外生长和细胞凋亡的影响.方法 构建重组质粒pcDNA3-CIB,转染人胶质瘤细胞株SHG44,MTT观察各组细胞的体外生长情况,流式细胞术(FCM)测定各组细胞的细胞周期和凋亡细胞数量,Western-blot检测CIB转染后凋亡相关蛋白的表达变化.结果 RT-PCR、Western印迹显示pcDNA3-CIB转染组CIB的mRNA及CIB蛋白表达水平明显高于对照组;与对照组细胞相比,转染CIB的SHG44-CIB组细胞牛长明显减缓(P＜0.01),SHG44-CIB组细胞G_1、S期细胞减少,G_2期细胞增多,凋亡细胞增加至19.2%;凋亡相关蛋白Bcl-2表达下调,Bax表达上调.结论 CIB在体外明显抑制SHG44细胞的生长并诱导其发生凋亡,CIB诱导的凋亡可能与Bcl-2及Bax表达的变化相关.     

Interaction of PrP with NRAGE, a protein involved in neuronal apoptosis

Prion diseases involve the conversion of the endogenous prion protein, PrP(C), into a disease-associated form PrP(Sc). Reports show that a subset of PrP(C) is subject to degradation in the cytosol by the ubiquitin-proteasome system. Some studies show that cytosolic PrP(C) is neuroprotective, while others show that it is neurotoxic. Here, we report that cytosolic PrP(C) constructs interact with a pro-apoptotic protein, NRAGE (neurotrophin receptor interacting MAGE homolog). This novel interaction was identified in a yeast two-hybrid screen using PrP(C) as bait and confirmed by an in vitro binding assay and co-immunoprecipitations. Endogenous NRAGE accumulated in perinuclear aggregates following proteasome inhibition, and recombinant NRAGE and PrP(C)-EGFP co-localized in aggresomes after proteasome inhibition. Finally, co-expression of NRAGE and cytosolic PrP(C) affected mitochondrial membrane potential in neuroblastoma cells. Our results suggest that interaction of cytosolic PrP and NRAGE could affect neuronal viability.     

Neuropathology of NFHgp160 transgenic mice expressing HIV-1 env protein in neurons

The physiopathology of HIV-1 dementia remains largely hypothetical. Although several sets of evidence point towards an indirect multicellular inflammatory pathway, gp120, one of the HIV-1 env products, was shown to be very cytotoxic for neurons in vitro. To explore a direct pathway in the physiopathology of dementia in AIDS, we developed transgenic mouse models carrying the HIV-1 env proteins gp 120 and gp 41 (gp 160) under the control of the human light neurofilament and murine heavy neurofilament promoters. To date, this is the first mouse model in which the HIV-1 env protein can be detected in neurons by immunohistochemistry. The expression is found in several brainstem and spinal cord gray structures and in the cerebellum in one of the mouse lines bearing the NFHgp160 transgene. The morphological findings at 3 months are subtle and are dominated by a watery, dendritic degeneration and a reactive gliosis. At 12 months, the evidence of neuronal degeneration and loss is present along with various degenerative phenomena involving synapses, dendrites and axons, including axonal swellings. Cytoskeletal abnormalities were found by immunohistochemistry. Chronic inflammation was also observed in the leptomeninges of the spinal cord and brainstem and in the cerebellar white matter. These models thus offer an exciting sequence of morphological findings initiated by the neuronal expression of the HIV-1 env proteins and offer a different tool to explore the neuronal dysfunction in AIDS.     

外源性TRAIL基因转染联合氯喹诱导胶质瘤U251细胞凋亡

目的探讨外源性肿瘤坏死因子凋亡相关诱导配体(TRAIL)联合氯喹对U251细胞凋亡的作用。方法构建稳定表达TRAIL的质粒p EGFP-TRAIL,然后转染到胶质瘤细胞系U251细胞中(TRAIL组),以p EGFP-C1质粒为阴性对照,以不转染质粒为空白对照;将氯喹(50μmol/L)加入到转染p EGFP-TRAIL质粒U251细胞培养基中,作为联合组。共聚焦显微镜检测GFP-TRAIL蛋白表达,MTT法检测细胞抑制率,Annexin V-FITC/PI双染法检测细胞凋亡,免疫印迹法检测GFP-TRAIL和Cleaved caspases-8蛋白表达。结果质粒转染后,共聚焦荧光显微镜检测结果显示,p EGFP-C1在细胞质中成弥散分布;而GFP-TRAIL在细胞质中成聚点分布,且荧光表达可以持续48 h以上;免疫印迹法分析结果显示TRAIL组TRAIL蛋白表达水平明显高于空白对照组和阴性对照组(P0.05)。联合组细胞增殖抑制率[(47.22±0.15)%]明显高于阴性对照组[(3.21±0.04)%,P0.05]和TRAIL组[(23.88±0.22)%,P0.05]。联合组细胞凋亡率[(41.62±0.44)%]明显高于空白对照组[(2.14±0.09)%,P0.05]、阴性对照组[(3.46±0.17)%,P0.05]和TRAIL组[(22.48±0.43)%,P0.05]。联合组Cleaved caspases-8蛋白表达水平明显高于阴性对照组、TRAIL组(P0.05)。结论外源性TRAIL基因转染后可以在细胞中稳定表达并诱导细胞凋亡,与氯喹联合应用可以增强TRAIL诱导的细胞凋亡,其机制可能是氯喹抑制细胞自噬。