五味子醇甲对APP/PS1小鼠学习记忆和NF-κB p65的影响

目的研究五味子醇甲(SCH)对APP/PS1双转基因痴呆小鼠行为学和NF-κB p65的影响。方法将35只APP/PS1双转基因痴呆小鼠随机分为模型(APP/PS1)组、五味子醇甲(SCH+APP/PS1)组;另以同背景同月龄的C 57 BL/6 J阴性小鼠为空白(WT)组。SCH+APP/PS1组给予SCH悬浊液灌胃(2.6 mg·kg^-1·d^-1),模型组和空白组给予等量蒸馏水灌胃。各组灌胃30 d进行Mirror水迷宫空间探索试验后取材。HE染色法观察皮层及海马CA1区神经细胞的形态结构。运用DCFH-DA法检测脑组织活性氧(ROS)含量。Western印迹检测法检验磷酸化核转录因子(NF-κB p65,pp65)的表达。结果与APP/PS 1组相比,APP/PS 1+SCH组有效区停留距离及时间明显延长,差异有统计学意义(P<0.05,P<0.01);APP/PS 1+SCH组穿越有效区次数和平台区停留距离增加,均差异有统计学意义(P<0.01)。APP/PS 1+SCH组HE染色观察皮层有部分神经细胞萎缩、减少,较APP/PS1组明显好转,海马细胞排列整齐度尚可、较均匀。与APP/PS1组相比,APP/PS1+SCH组能降低ROS含量,差异有统计学意义(P<0.05)。APP/PS 1+SCH组皮层及海马pp65蛋白相对表达量下调(P<0.05,P<0.01)。结论SCH可能通过降低ROS含量和抑制pp65保护脑组织形态结构和提高APP/PS1鼠空间探索记忆能力。     

生慧汤对APP/PS1雄性AD小鼠海马内APP代谢产物的昼夜变化影响＊

目的研究生慧汤对APP/PS1（β-amyloid precursor protein/presenilin 1246E）小鼠海马内β淀粉样前体蛋白（Amyloid precursor protein，APP）代谢产物昼夜表达的影响。方法 将56只雄性APP/PS1双转基因痴呆模型小鼠随机分为4组，分别为：痴呆模型组、褪黑素治疗组（0.78 mg·kg^-1·d^-1）、生慧汤高剂量组（27.0 g·kg^-1·d^-1）、生慧汤低剂量组（13.5 g·kg^-1·d^-1），每组14只；对照组为14只雄性C57BL/6J小鼠，连续给药30天。采用Morris水迷宫检测小鼠学习记忆能力，采用酶联免疫吸附检测（ELISA）检测不同时间段取出的海马组织sAPPα含量。对海马β淀粉样蛋白_1-40、β淀粉样蛋白_1-42表达进行免疫印迹分析。免疫组化（IHC）检测海马CA1区Aβ_1-40蛋白的表达。结果 Morris水迷宫结果表明，与对照组相比，模型组上平台潜伏期明显延长、穿越平台次数明显减少（P<0.01）；与模型组相比，生慧汤不同剂量组上平台潜伏期明显缩短（P<0.01、P<0.05），穿越平台次数明显增加（P<0.05）。ELISA结果表明，与对照组相比，模型组的sAPPα总量、各时间段（12：00、24：00）sAPPα含量明显减少（P<0.01）；与模型组相比，生慧汤不同剂量组sAPPα总量明显增多（P<0.01、P<0.05），生慧汤高剂量组仅能增加12：00 sAPPα含量（P<0.01），生慧汤低剂量组仅能增加24：00 sAPPα含量（P<0.05）。对照组sAPPα含量具有明显昼夜差异（P<0.01）。模型组sAPPα含量的昼夜差异性消失（P>0.05）。生慧汤高剂量组sAPPα含量具有昼夜差异（P<0.01），生慧汤低剂量组sAPPα含量不具有昼夜差异（P>0.05）。Western blot结果显示，与对照组相比，模型组Aβ_1-40、Aβ_1-42蛋白表达明显增加（P<0.01）；与模型组相比，生慧汤高剂量组Aβ_1-40、Aβ_1-42蛋白表达减少（P<0.05、P<0.01）；生慧汤低剂量组仅能减少Aβ_1-40蛋白的表达（P<0.05），对Aβ_1-42蛋白表达无影响（P>0.05）。免疫组化结果表明，与对照组相比，模型组Aβ_1-40表达明显增加（P<0.01）；与模型组相比，生慧汤不同剂量组Aβ_1-40表达不同程度减少（P<0.01、P<0.05）。结论 生慧汤能够改善5月龄APP/PS1阿尔茨海默病模型小鼠的学习记忆障碍，其机制可能与恢复海马内APP代谢产物sAPPα的昼夜节律性表达、从而减少Aβ的沉积有关。     

Protective mechanism of Wnt4 gene on Parkinson’s disease (PD) transgenic Drosophila

Objective: The main pathological change of Parkinson’s disease (PD) is progressive degeneration and necrosis of dopaminergic neurons in the midbrain, forming a Lewy body in many of the remaining neurons. Studies have found that in transgenic Drosophila, mutations in the PTEN-inducible kinase 1 (PINK1) gene may cause indirect flight muscle defects in Drosophila, and mitochondrial structural dysfunction as well.

Methods: In this study, Wnt4 gene overexpression and knockdown were performed in PINK1 mutant PD transgenic Drosophila, and the protective effect of Wnt4 gene on PD transgenic Drosophila and its possible mechanism were explored. The Wnt4 gene was screened in the previous experiment; And by using the PD transgenic Drosophila model of the MHC-Gal4/UAS system, the PINK1 gene could be specifically activated in the Drosophila muscle tissue.

Results: In PINK1 mutation transgenic fruit flies, the Wnt4 gene to study its implication on PD transgenic fruit flies’ wing normality and flight ability. We found that overexpression of Wnt4 gene significantly reduced abnormality rate of PD transgenic Drosophila and improved its flight ability, and then, increased ATP concentration, enhanced mitochondrial membrane potential and normalized mitochondrial morphology were found. All of these findings suggested Wnt4 gene may have a protective effect on PD transgenic fruit flies. Furthermore, in Wnt4 gene overexpression PD transgenic Drosophila, down-regulation autophagy and apoptosis-related proteins Ref(2)P, Pro-Caspase3, and up-regulation of Beclin1, Atg8a, Bcl2 protein were confirmed by Western Blotting.

Conclusion: The results imply that the restoring of mitochondrial function though Wnt4 gene overexpression in the PINK1 mutant transgenic Drosophila may be related to autophagy and/or apoptosis.     

A robust method for assessing chemically induced mutagenic effects in the oral cavity of transgenic Big Blue® rats

The Big Blue® (BB) in vivo mutation assay uses transgenic rodents to measure treatment‐induced mutations in virtually any tissue. The BB assay can be conducted in rats or mice and is ideal for investigating tissue‐specific mutagenic mode of action of tumor induction. Some tissues such as oral mucosa have not been thoroughly studied. Due to the small quantity and cartilaginous nature of oral cavity tissues, development of special prosection and DNA isolation methods was required to permit robust analysis of mutations in these tissues. Improved surgical methods permitted collection of adequate and reproducible quantities of tissue (～45 mg gingiva/buccal and ～30 mg gingiva/palate). Optimized DNA isolation methods included use of liquid nitrogen pulverization, homogenization, nuclei pelleting, digestion, and phenol/chloroform extraction, to yield sufficient quantities of DNA from these tissues. In preliminary optimization work, mutant frequency (MF) in tongue and gingiva was increased in rats exposed to the promutagen, benzo[a]pyrene, and the direct mutagen, N‐ethyl‐N‐nitrosourea. The oral cavity carcinogen, 4‐nitroquinoline‐1‐oxide (4‐NQO; 10 ppm in drinking water; 28 days), was qualified as a positive control for mutagenesis in oral tissues since it caused significant increases in cII MFs in gingiva/palate (50.2‐fold) and gingiva/buccal tissues (21.3‐fold), but not in liver or bone marrow (0.9‐ and 1.4‐fold, respectively). These results are consistent with the observation that 4‐NQO primarily induces tumors in oral cavity. Results also demonstrate the utility of the BB rat mutation assay and optimized methods for investigation of oral cavity mutagenicity, and by extension, analysis of other small and cartilaginous tissues. Environ. Mol. Mutagen. 56:629–636, 2015. © 2015 Wiley Periodicals, Inc.     

Establishment of a specific cell death induction system in Bombyx mori by a transgene with the conserved apoptotic regulator,mouse Bcl‐2‐associated X protein (mouse Bax)

The induction of apoptosis in vivo is a useful tool for investigating the functions and importance of particular tissues. B‐cell leukaemia/lymphoma 2‐associated X protein (Bax) functions as a pro‐apoptotic factor and induces apoptosis in several organisms. The Bax‐mediated apoptotic system is widely conserved from Caenorhabditis elegans to humans. In order to establish a tissue‐specific cell death system in the domestic silkworm, Bombyx mori, we constructed a transgenic silkworm that overexpressed mouse Bax (mBax) in particular tissues by the Gal4‐upstream activation sequence system. We found that the expression of mBax induced specific cell death in the silk gland, fat body and sensory cells. Fragmentation of genomic DNA was observed in the fat body, which expressed mBax, thereby supporting apoptotic cell death in this tissue. Using this system, we also demonstrated that specific cell death in sensory cells attenuated the response to the sex pheromone bombykol. These results show that we successfully established a tissue‐specific cell death system in vivo that enabled specific deficiencies in particular tissues. The inducible cell death system may provide useful means for industrial applications of the silkworm and possible utilization for other species.     

Functional characterization of calliphorid cell death genes and cellularization gene promoters for controlling gene expression and cell viability in early embryos

The New World screwworm fly, Cochliomyia hominivorax, and the Australian sheep blow fly, Lucilia cuprina, are major pests of livestock. The sterile insect technique was used to eradicate C. hominivorax from North and Central America. This involved area‐wide releases of male and female flies that had been sterilized by radiation. Genetic systems have been developed for making ‘male‐only’ strains that would improve the efficiency of genetic control of insect pests. One system involves induction of female lethality in embryos through activation of a pro‐apoptotic gene by the tetracycline‐dependent transactivator. Sex‐specific expression is achieved using an intron from the transformer gene, which we previously isolated from several calliphorids. In the present study, we report the isolation of the promoters from the C. hominivorax slam and Lucilia sericata bnk cellularization genes and show that these promoters can drive expression of a GFP reporter gene in early embryos of transgenic L. cuprina. Additionally, we report the isolation of the L. sericata pro‐apoptotic hid and rpr genes, identify conserved motifs in the encoded proteins and determine the relative expression of these genes at different stages of development. We show that widespread expression of the L. sericata pro‐apoptotic genes was lethal in Drosophila melanogaster. The isolated gene promoters and pro‐apoptotic genes could potentially be used to build transgenic embryonic sexing strains of calliphorid livestock pests.     

Evaluation of the antiviral potential of the soluble forms of glycoprotein D receptors on ocular herpes caused by HSV-1 and HSV-2 infections in a transgenic mouse model

Ocular herpes, caused by herpes simplex virus type 1 (HSV-1) and type 2 (HSV-2) infections, remains an important corneal disease, which may result in loss of vision. Because the frequency of acyclovir resistance in HSV has increased, novel antiviral agents are needed for therapeutic approaches to ocular herpes. Several studies have demonstrated that fusion proteins containing entire ectodomain of HSV glycoprotein D receptors, including herpesvirus entry mediator A (HVEM), nectin-1 and nectin-2, and the Fc portion of human IgG (HVEMIg, nectin-1Ig, and nectin-2Ig, respectively), can exert antiviral effects in vitro and in vivo. Here, to evaluate the antiviral potential of HVEMIg, nectin-1Ig, and nectin-2Ig against ocular infections with HSV, transgenic mice expressing these fusion proteins were ocularly inoculated with HSV-1 and HSV-2. Transgenic mouse lines expressing HVEMIg and nectin-1Ig showed marked resistance to ocular herpes; on the other hand, mouse lines expressing nectin-2Ig did not. Furthermore, to investigate the therapeutic effects of nectin-1Ig, which can neutralize HSVs in vitro against ocular disease, transgenic mouse serum containing nectin-1Ig was dropped into the eyes of wild-type mice after HSV infection. Reduction of severe symptoms could be observed in mice treated with nectin-1Ig serum. These results warrant further study of soluble HVEM and nectin-1 products as preventive and therapeutic agents against ocular herpes caused by HSV-1 and HSV-2 infections, especially nectin-1Ig as a new eye drop.     

Prion neurotoxicity

Although the mechanisms underlying prion propagation and infectivity are now well established, the processes accounting for prion toxicity and pathogenesis have remained mysterious. These processes are of enormous clinical relevance as they hold the key to identification of new molecular targets for therapeutic intervention. In this review, we will discuss two broad areas of investigation relevant to understanding prion neurotoxicity. The first is the use of in vitro experimental systems that model key events in prion pathogenesis. In this context, we will describe a hippocampal neuronal culture system we developed that reproduces the earliest pathological alterations in synaptic morphology and function in response to PrP^Sc. This system has allowed us to define a core synaptotoxic signaling pathway involving the activation of NMDA and AMPA receptors, stimulation of p38 MAPK phosphorylation and collapse of the actin cytoskeleton in dendritic spines. The second area concerns a striking and unexpected phenomenon in which certain structural manipulations of the PrP^C molecule itself, including introduction of N‐terminal deletion mutations or binding of antibodies to C‐terminal epitopes, unleash powerful toxic effects in cultured cells and transgenic mice. We will describe our studies of this phenomenon, which led to the recognition that it is related to the induction of large, abnormal ionic currents by the structurally altered PrP molecules. Our results suggest a model in which the flexible N‐terminal domain of PrP^C serves as a toxic effector which is regulated by intramolecular interactions with the globular C‐terminal domain. Taken together, these two areas of study have provided important clues to underlying cellular and molecular mechanisms of prion neurotoxicity. Nevertheless, much remains to be done on this next frontier of prion science.