突触功能必需基因参与线虫衰老的调控

目的鉴定参与线虫衰老的神经内分泌调控的新基因。方法鉴于神经系统在衰老调控中的重要作用,通过寿命分析和脂褐质自发荧光的检测,从编码突触蛋白的遗传位点中筛选参与衰老调控的基因。我们还进一步检查了这些遗传位点相应的突变体的永久性幼虫形成情况,探讨它们是否可能受胰岛素样信号通路的调控。结果遗传任点unc-10,syd-2,hlb-1,dlk-1,mkk-4,scd-2,snb-1,ric-4,nrx-1,unc-13,sbt-1,unc-64可能参与线虫衰老的调控。而且在衰老的调控中,unc-10,syd-2,hlb-1,dlk-1,mkk-4,scd-2,snb-1,ric-4,nrx-1的功能可能与unc-13,sbt-1,unc-64相反。肠道脂褐质自发荧光的检测进一步证明了筛选出的各基因对应突变体的长寿或短寿表型,是由减慢或缩短的组织衰老所致。在筛选出的基因中,syd-2,hlb-1,mkk-4,scd-2,snb-1,ric-4,unc-64也参与了永久性幼虫形成的调控。另外,daf-2突变增强了syd-2和hlb-1的表达,降低了mkk-4,nrx-1,ric-4,sbt-1,rpm-1,unc-10,dlk-1,unc-13的表达。daf-16突变提高了syd-2和hlb-1的表达,降低了mkk-4,nrx-1,sbt-1,rpm-1,unc-10,dlk-1,unc-13的表达．结论突触功能可能在个体寿命和永久性幼虫形成的调控机制中具有重要的作用。     

质膜相关的SNAREs syntaxin/UNC-64与SNAP-25/RIC-4蛋白调控秀丽线虫的脂肪积累(英文)

目的研究神经突触组装与功能调控相关基因是否参与秀丽线虫的脂肪积累调节。方法分析神经突触组装与功能调控相关基因突变体的脂肪积累变化,进而观察SNAREs syntaxin/unc-64和SNAP-25/ric-4基因与daf-2、daf-7、nhr-49、sbp-1及mdt-15所介导的信号通路在调控脂肪积累上的遗传关系。对unc-64与ric-4基因进行组织特异性活性分析,以确定它们在神经系统与肠道内对脂肪积累的影响。结果突触前为神经突触组装所必需的基因的突变并未明显影响脂肪积累。对调控神经突触功能的基因进行分析的结果显示,SNAREssyntaxin/unc-64与SNAP-25/ric-4基因均参与了脂肪积累的调节。利用苏丹黑染色与尼罗红标记法观察到unc-64与ric-4突变体肠道中脂肪积累显著增加,而unc-64与ric-4突变体中积累的脂肪颗粒并未出现尺寸的显著变化。在神经系统与肠道中,unc-64基因的表达均能显著降低unc-64突变体动物的脂肪积累,而基因ric-4在神经系统的表达则可以完全恢复ric-4突变体动物的脂肪积累。遗传分析表明,unc-64与ric-4对脂肪积累的调控独立于daf-2...     

嗅觉神经元AWA功能必需基因以胰岛素信号依赖的方式调控秀丽线虫的衰老(英文)

目的研究嗅觉神经元AWA功能必需基因与胰岛素信号之间在调控秀丽线虫衰老上的关系。方法测定AWA、AWC、ASE与AFD感觉神经元的功能必需基因突变后线虫的寿命、咽泵运动速率、肠道荧光、永久性幼虫形成与后代数目的变化。此外,进行基因功能的恢复实验以确认AWA功能必需基因在调控衰老中的作用。最后,对AWA功能必需基因与胰岛素信号间在调控衰老上的遗传关系进行了分析。结果AWA功能必需基因odr-7、odr-2与odr-3突变能显著延长动物寿命,并在衰老过程中诱导产生相对于野生型更高的咽泵运动速率和更少的肠道脂褐质积累。然而,odr-7、odr-2与odr-3的基因突变并不影响线虫的后代数目与永久性幼虫的形成,且odr-7、odr-2与odr-3基因对于寿命的调控并未呈现出明显的温度依赖性。比较而言,感觉神经元ASE、AWC与AFD的功能必需基因的突变并未显著影响动物寿命。而且,在嗅觉神经元AWA表达odr-7、odr-2与odr-3基因可以完全或很大程度上恢复对应突变体的长寿表现型。进一步遗传分析表明,odr-7突变体的长寿表现型可被daf-16基因突变所抑制,被daf-2或age-1基因突变所增强,而mev-1与...     

线虫中影响化学传感器结构与功能的遗传位点及其可能对应的microRNA的生物信息学分析

目的为了系统评价microRNA（miRNA）在调控化学传感器结构和功能方面的重要作用,我们进行了线虫中有关影响化学传感器结构与功能的遗传位点以及和其可能对应的miRNA的生物信息学分析。方法我们选择了55个与化学传感器结构与功能相关的遗传位点,并且进一步通过评价序列比对后的E值等手段来分析和确认其可能对应的miRNA。结果在55个遗传位点中,我们发现其中30个遗传位点存在与其可能对应的miRNA。这些所鉴定的基因在功能上几乎涵盖了化学传感器结构和功能调控的诸多方面。而且,我们的数据还表明化学传感器结构和功能可能受到一系列网状交联的上游信号通路所调节。另外,对应miRNA在这30个基因的3’端非编码区上的分布也呈现出各自特有的模式。结论我们勾勒出了可能存在的由miRNA介导的通过控制相应感觉神经元与中间神经元功能从而调控化学趋向性与温度趋向性的信号通路。本研究将有助于我们更好的了解线虫中可能由miRNA调控的化学趋向性和温度趋向性行为。     

线虫中影响活动区形成遗传位点的遗传筛选(英文)

目的筛选与鉴定线虫中影响神经突触活动区形成的遗传位点。方法我们通过构建SYD-2::GFP转基因动物作为活动区的标记进行正向遗传筛选,以鉴定影响活动区形成的系列遗传位点。结果从15 000筛选群体中,我们分离出8个感兴趣的遗传位点。其突变体的表现型主要反映在SYD-2::GFP荧光标记数目、形态与分布的改变以及大缺口的出现。一些突变体还表现出明显的行为缺陷以及针对涕灭威(aldicarb)的抗性或者敏感性。结论这些突变体为我们进一步系统研究活动区的形成与神经递质释放的机制提供了重要前提。     

β-catenin/Tcf-4转录调控AKT2基因表达促进人脑胶质瘤细胞恶性转化的研究

目的 探究胶质瘤中β- catenin/Tcf -4转录调控AKT2基因以促进其恶性表型转化的机制.方法 CHIP - PCR及荧光素酶实验确立AKT2启动子区的Tcf -4结合位点及活性.阿司匹林( ASA)阻断β- catenin/Tcf -4转录复合物活性及恢复AKT2基因的表达后,流式细胞术检测细胞周期,Annexin V检测凋亡及Transwell实验检测侵袭能力.结果 CHIP - PCR表明,AKT2基因启动子区存在两个Tcf -4结合位点- 349/- 343 bp(位点1)和- 3491/- 3497 bp(位点2).荧光素酶实验结果显示位点2的活性比位点1的强.抑制β - catenin/Tcf -4转录活性后,细胞周期阻滞于G0/G1期,侵袭能力下降;恢复AKT2的表达后,细胞S期比例增加,侵袭能力增强.结论 β- catenin/Tcf -4可以直接转录调控AKT2来促进胶质瘤细胞的增殖和侵袭.     

胰腺干细胞转分化为胰岛样细胞与天然胰岛细胞的基因差异性

背景：目前胰腺干细胞在体外诱导转分化效率和分化成熟度较低，哪些基因在转分化中起关键性调控作用尚不清楚。 目的：通过基因表达谱芯片分析转分化的胰岛样细胞与天然胰岛细胞在成熟度上的基因差异，筛选对转分化起关键性调节作用的基因。 设计、时间及地点：基因水平的对照实验，于2004-07/2006-04在暨南大学第二临床医学院（深圳市人民医院）临床医学研究中心实验室完成。 材料：SD大鼠胰腺分离的胰腺干细胞转分化的胰岛样细胞团，SD大鼠分离的天然胰岛。 方法：分离和消化SD大鼠胰腺，用差异性贴壁、低血清培养等方法纯化出大鼠胰腺导管上皮细胞，利用免疫组化方法鉴定CK-19和PDX-1。通过含有exendin-4、角质细胞生长因子、尼克酰胺等细胞因子和葡萄糖的培养基，使之转分化为胰岛样细胞团，应用双硫腙染色和胰岛素免疫荧光染色鉴定。提取转分化的胰岛样细胞和天然胰岛细胞RNA，利用大鼠22000位点寡核苷酸芯片进行杂交，筛选差异性表达的基因，并进一步采用反转录-聚合酶链反应验证13个相关基因的表达。 主要观察指标：转分化的胰岛样细胞团和天然胰岛鉴定，差异性基因筛选和鉴定。 结果：筛选出差异表达基因1 072个，其中表达上调的基因397个，上调10倍以上的基因35个；表达下调的基因有675个，其中下调20倍以上的基因37个。上调10倍以上的差异性基因中与细胞组织和生物发生相关基因最多，下调20倍以下的基因中与发育相关基因最多。对选定的13个与胰岛细胞发育密切相关的基因PDX-1, PDX-4, PDX-6, Nkx2.2, Nkx6.1，Nkx6.2，Ptfla，Isll，Ngn3，Myt1，Ptf1a，capn5，Ppy，通过反转录-聚合酶链反应进一步验证与芯片检测结果一致。 结论：转分化的胰岛样细胞与天然胰岛细胞基因表达差异显著，参与胰岛发育的关键基因表达明显下调。     

对秀丽线虫模型NCS-1调控的温度感知长时程记忆的评价研究

目的 评价线虫温度趋向性模型是否适用于长时程记忆的研究.方法 在20℃含有新鲜食物过夜培养的训练条件下,考察在不同时间点执行温度趋向性(isothermal tracking,IT)行为的虫体百分比.结果 训练后36与48h,线虫的IT百分数、温度趋向性模型内外的头部摆动频率、以及AFD和AIY神经元的表达模式与对照组相似,而训练后60、72和84 h的线虫则表现出运动行为上的缺陷,表明温度趋向性模型适合于在训练36和48 h后的线虫中进行长时程记忆的考察.此外,与野生型N2相比,神经元钙传感蛋白(neuronal calcium sensor-1,NCS-1)的突变能够降低训练后18、36和48 h的虫体的IT百分数.而铅暴露可以显著抑制训练后18、36和48 h野生型和ncs-1突变体动物在不同时间点下的IT百分数.结论 温度趋向性模型适用于评价秀丽线虫中NCS-1参与调控的温度感知长时程记忆,且该模型可用于针对线虫中参与调节或影响记忆行为的某一特定基因或刺激信号功能的揭示.     

线虫中有关影响突触结构与功能的遗传位点及其对应miRNA的生物信息学分析

目的为系统分析miRNA在调控神经突触组装和功能方面的重要功能,我们进行了线虫中有关影响突触结构和功能的遗传位点以及和其可能对应的miRNA的生物信息学分析。方法我们选择了198个与突触结构和功能相关的遗传位点,并且通过评价序列比对后的E值等手段来分析和确认其可能对应的miRNA。结果在198个遗传位点中,我们发现其中163遗传位点存在与其对应的miRNA,这些位点涵盖了大部分和突触结构和功能所必需的遗传位点。进一步通过对编码神经突触蛋白的38个遗传位点的分析,发现其中22个遗传位点很有可能受到miRNA的分子调控。此外,对应miRNA在这22基因的3’端非编码区（untranslated region,UTR）上的分布也呈现出各自特有的模式。结论我们提供了针对影响神经突触结构和功能所必须的遗传位点及其对应可能结合的miRNA的生物信息学系统分析结果。这些数据为我们进一步系统地开展线虫中miRNA在神经突触组装和功能上的分子调控机制研究提供了根本前提。     

去除蛋白激酶A磷酸化位点的线粒体融合素2基因与血管平滑肌细胞增殖

背景: 线粒体融合素2基因作用于血管平滑肌细胞Ras蛋白,通过胞外信号调节蛋白激酶1/2通路抑制细胞增殖。线粒体融合素2基因氨基酸序列第442位丝氨酸为蛋白激酶A磷酸化位点,与其磷酸化状态密切相关,可能参与其功能调控。 目的：观察大鼠线粒体融合素2基因在去除蛋白激酶A磷酸化位点后对大鼠血管平滑肌细胞增殖的影响及其相关信号通路。 方法：利用已构建的携带绿色荧光蛋白基因、线粒体融合素2基因和去除蛋白激酶A磷酸化位点的线粒体融合素2基因的3种重组腺病毒,感染大鼠主动脉血管平滑肌细胞,将其传代培养3~10代后以抽签法随机分为4组：①不加干预的对照组。②感染携带绿色荧光蛋白的对照组(Adv-GFP组)。③感染携带线粒体融合素2基因的实验组(Adv-Mfn2组)。④感染携带去除蛋白激酶A磷酸化位点的线粒体融合素2基因的实验组(Adv- Mfn2-PKA(△)组)。激光共聚焦显微镜观察完整的和去除蛋白激酶A磷酸化位点的线粒体融合素2基因在细胞中的定位。Western blot检测p-ERK1/2表达水平及完整的和去除蛋白激酶A磷酸化位点的线粒体融合素2基因在血管平滑肌细胞中的表达。MTT法绘制细胞生长曲线。 结果与结论：完整的和去除蛋白激酶A磷酸化位点的线粒体融合素2基因在血管平滑肌细胞中均表达蛋白特异性条带。两种基因表达产物都主要分布于线粒体外膜。与对照组和Adv-GFP组相比,Adv-Mfn2组吸光度值在第3,4,5,6天都显著降低(P < 0.01),Adv-Mfn2-PKA(△)组吸光度值无明显变化。与对照组和Adv-GFP组相比,Adv-Mfn2组p-ERK1/2表达水平显著降低(P < 0.01),Adv-Mfn2-PKA(△)组无明显变化。提示去除蛋白激酶A磷酸化位点的线粒体融合素2基因定位于线粒体外膜,对血管平滑肌细胞的增殖无拮抗作用,对胞外信号调节蛋白激酶1/2通路无抑制作用。     

Regulation of aging by unc-13 and sbt-1 in Caenorhabditis elegans is temperature-dependent

Objective

To investigate the role of environmental factor—temperature in the regulation of aging process by unc-13 and sbt-1 in Caenorhabditis elegans.

Methods

The lifespan, the speed of pharynx pumping, and the intestinal autofluorescence of unc-13 and sbt-1 mutants were examined at different temperature conditions. In addition, to exclude the possible influences from other factors in unc-13 and sbt-1 mutants, the dauer formation, the thermotaxis, the brood size and the population percentage of the mutants expressing hsp16.2-gfp were further investigated.

Results

Mutations of unc-13 and sbt-1 significantly increased the mean and the maximum lifespans of nematodes cultured at 20 °C and 25 °C, while no noticeable increase was found at 15 °C in either the mean or the maximum lifespan. Investigations on the speed of pharynx pumping and the intestinal autofluorescence suggested that at 20 °C and 25 °C, mutations of unc-13 and sbt-1 could slow the aging process and delay the accumulation of aging-related cellular damage. Meanwhile, mutations of unc-13 or sbt-1 did not affect the dauer formation or the thermotaxis to different temperatures in nematodes. In contrast, at 20 °C and 25 °C conditions, mutations of unc-13 and sbt-1 significantly decreased the brood size and the percentage of nematodes expressing hsp16.2-gfp, while no such differences were detected at 15 °C. Moreover, the thermotolerance of unc-13 and sbt-1 mutants could be greatly strengthened after the 16-h heat shock at 35 °C.

Conclusion

The regulation of aging by unc-13 and sbt-1 is temperaturedependent. And the alterations in reproduction capability and stress response may be associated with the formation of this temperature-dependent property.     

UNC-64 and RIC-4, the plasma membrane-associated SNAREs syntaxin and SNAP-25, regulate fat storage in nematode Caenorhabditis elegans

Objective

To investigate whether genes required for synaptogenesis and synaptic function are also involved in fat storage control in Caenorhabditis elegans.

Methods

Fat storage was examined in mutants of genes affecting the synaptogenesis and synaptic function. In addition, the genetic interactions of SNAREs syntaxin/unc-64 and SNAP-25/ric-4 with daf-2, daf-7, nhr-49, sbp-1 and mdt-15 in regulating fat storage were further investigated. The tissue-specific activities of unc-64 and ric-4 were investigated to study the roles of unc-64 and ric-4 in regulating fat storage in the nervous system and/or the intestine.

Results

Mutations of genes required for the formation of presynaptic neurotransmission site did not obviously influence fat storage. However, among the genes required for synaptic function, the plasma membrane-associated SNAREs syntaxin/unc-64 and SNAP-25/ric-4 genes were involved in the fat storage control. Fat storage in the intestinal cells was dramatically increased in unc-64 and ric-4 mutants as revealed by Sudan Black and Nile Red strainings, although the fat droplet size was not significantly changed. Moreover, in both the nervous system and the intestine, expression of unc-64 significantly inhibited the increase in fat storage observed in unc-64 mutant. And expression of ric-4 in the nervous system completely restored fat storage in ric-4 mutant. Genetic interaction assay further indicated that both unc-64 and ric-4 regulated fat storage independently of daf-2 [encoding an insulin-like growth factor-I (IGF-I) receptor], daf-7 [encoding a transforming growth factor-β (TGF-β) ligand], and nhr-49 (encoding a nuclear hormone receptor). Besides, mutation of daf-16 did not obviously affect the phenotype of increased fat storage in unc-64 or ric-4 mutant. Furthermore, unc-64 and ric-4 regulated fat storage probably through the ARC105/mdt-15- and SREBP/sbp-1-mediated signaling pathways. In addition, fat storage in unc-64; ric-4 was higher than that in either unc-64 or ric-4 single mutant nematodes, suggesting that unc-64 functions in parallel with ric-4 in regulating fat storage.

Conclusion

The plasma membrane-associated SNAREs syntaxin/unc-64 and SNAP-25/ric-4 function in parallel in regulating fat storage in C. elegans, probably through the ARC105/mdt-15- and SREBP/sbp-1-mediated signaling pathways.     

HLB-1 functions as a new regulator for the organization and function of neuromuscular junctions in nematode Caenorhabditis elegans

Objective To study the role of HLB-1 in regulating the organization and function of neuromuscular junctions in nematode Caenorhabditis elegans

Methods

To evaluate the functions of HLB-1 in regulating the organization and function of neuromuscular junctions, effects of hlb-1 mutation on the synaptic structures were revealed by uncovering the expression patterns of SNB-1::GFP and UNC-49::GFP, and pharmacologic assays with aldicarb and levamisole were also used to test the synaptic functions. Further rescue and mosaic analysis confirmed HLB-1’s role in regulating the organization and function of neuromuscular junctions.

Results

Loss of HLB-1 function did not result in defects in neuronal outgrowth or neuronal loss, but caused obvious defects of SNB-1::GFP and UNC-49::GFP puncta localization, suggesting the altered presynaptic and postsynaptic structures. The mutant animals exhibited severe defects in locomotion behaviors and altered responses to an inhibitor of acetylcholinesterase and a cholinergic agonist, indicating the altered presynaptic and postsynaptic functions. Rescue and mosaic analysis experiments suggested that HLB-1 regulated synaptic functions in a cell nonautonomously way. Moreover, HLB-1 expression was not required for the presynaptic active zone morphology. Genetic evidence further demonstrated that hlb-1 acted in a parallel pathway with syd-2 to regulate the synaptic functions.

Conclusion

HLB-1 appeared as a new regulator for the organization and function of neuromuscular junctions in C. elegans.     

Sensory control of dauer larva formation in Caenorhabditis elegans

As a sensory response to starvation or overcrowding, Caenorhabditis elegans second-stage larvae may molt into a developmentally arrested state called the dauer larva. When environmental conditions become favorable for growth, dauer larvae molt and resume development. Some mutants unable to form dauer larvae are simultaneously affected in a number of sensory functions, including chemotaxis and mating. The behavior and sensory neuroanatomy of three such mutants, representing three distinct genetic loci, have been determined and compared with wild-type strain. Morphological abnormalities in afferent nerve endings were detected in each mutant. Both amphid and outer labial sensilla are affected in the mutant CB1377 (daf-6)X, while another mutant, CB1387 (daf-10)IV, is abnormal in amphidial cells and in the tips of the cephalic neurons. The most pleiotropic mutant, CB1379 (che-3)I, exhibits gross abnormalities in the tips of virtually all anterior and posterior sensory neurons. The primary structural defect in CB1377 appears to be in the nonneuronal amphidial sheath cells. The disruption of neural organization in CB1377 is much greater in the adult than in the L2 stage. Of all the anterior sense organs examined, only the amphids are morphologically affected in all three mutants. Thus, one or more of the amphidial neurons may mediate the sensory signals for entry into the dauer larva stage in normal animals. Using temperature-sensitive mutants we determined that the same defects which block entry into the dauer stage also prevent recovery of dauer larvae.     

The Liprin homology domain is essential for the homomeric interaction of SYD-2/Liprin-α protein in presynaptic assembly

Synapses are asymmetric structures that are specialized for neuronal signal transduction. A unique set of proteins is present at the presynaptic active zone, which is a core structure essential for neurotransmitter release. In Caenorhabditis elegans HSN neurons, SYD-2, a Liprin-α family protein, acts together with a GAP protein SYD-1 to promote presynaptic assembly. Previous studies have shown that elevating the activity of syd-2 can bypass the requirement of syd-1. Liprin-α proteins are composed of coiled-coil-rich regions in the N-terminal half, which mediate interactions with adapter proteins at the presynaptic active zone, and three SAM domains in the C terminus, which bind proteins such as LAR receptor tyrosine phosphatase. To address the molecular mechanism by which SYD-2 activity is regulated, we performed structure-function studies. By monitoring the ability of SYD-2 transgenes to rescue syd-2(lf) and to suppress syd-1(lf) phenotypes in HSN neuron synapses, we identified the N-terminal half of SYD-2 as minimally required for rescuing syd-2(lf) phenotypes. A highly conserved short coiled-coil segment named Liprin Homology 1 (LH1) domain is both necessary and sufficient to suppress syd-1(lf) defects. We show that the LH1 domain forms a dimer and promotes further oligomerization and/or complex formation of SYD-2/Liprin-α proteins. The role of the LH1 domain in presynaptic assembly can be partially complemented by artificial dimerization. These findings suggest a model by which the self-assembly of SYD-2/Liprin-α proteins mediated by the coiled-coil LH1 domain is one of the key steps to the accumulation of presynaptic components at nascent synaptic junctions.     

Developmental alterations in sensory neuroanatomy of the Caenorhabditis elegans dauer larva

The anterior sensory ultrastructure of the C. elegans dauer larva was examined in several specimens and compared with that of the second-stage (L2) larva, which immediately precedes the dauer stage. In some instances comparisons were made with L3, postdauer L4, and adult stages. Whereas sensory structures in different nondauer stages closely resemble each other, including the inner labial sensilla, amphids, and deirids. The relative positions of the afferent tips of the two types of inner labial neurons are reversed in the dauer stage compared to the L2 and postdauer L4 stages. Inner labial neuron 1 rather than neuron 2 is more anterior in each of the six sensilla, and neuron 1 has an enlarged tip. The neuron 2 cilia are only one-third as long as those in the L2. Amphidial neurons c, d, g, and i and the amphidial sheath cell are altered in shape or position in the dauer stage. Neurons g and i are displaced posteriorly within the dauer amphidial channel. Neuron d has significantly more microvillar projections than do the d cells in L2, L3, or postdauer L4 larvae. Winglike processes of dauer neuron c form a 200 degrees-240 degrees arc in transverse section, including extensive overlap of the two cells. The arc in an L2 seldom spans more than 100 degrees, and overlap does not occur. While L2 larvae possess two separate bilateral amphidial sheath cells, the left and right sheath cells are often continuous in the dauer larva. Deirid sensory dendrites exhibit a dauer-specific structure and orientation. The tip of each neuron is attached to the body wall cuticle by a substructure not observed in L2 or postdauer L4 stages, and the neurons are oriented parallel to the longitudinal axis of the dauer larva. The deirid sensory terminals are oriented perpendicular to the cuticle in other stages. Reversible alterations in neural structure are discussed in the context of dauer-specific behavior.     

Visualization of integral and peripheral cell surface proteins in live Caenorhabditis elegans

To study the abundance of specific receptors and other cell surface proteins at synapses, it would be advantageous to specifically label these proteins only when inserted in the plasma membrane. We describe a method that allows to fluorescently label cell surface proteins in live and behaving animals, namely in the nematode Caenorhabditis elegans. Proteins such as subunits of the levamisole sensitive nicotinic acetylcholine receptor (nAChR) were epitope-tagged at their extracellular C-termini, and fluorescent antibodies against those tags were injected into the body fluid. These antibodies specifically labelled synaptic regions on the cell surface of muscles and neurons, and simultaneous use of different tags facilitated co-localization studies. Quantification of the fluorescence is possible, as verified by demonstrating that mutations in ric-3 and unc-38, which cause behavioural resistance to cholinergic agonists, strongly reduce or even abolish nAChR cell surface expression. We also used this method to visualize the extracellular peripheral membrane protein ODR-2, which is related to a neurotoxin-like protein regulating vertebrate neuronal nAChRs. Likewise, fluorescent alpha-bungarotoxin, when injected, bound to certain nAChRs in the pharynx and the nervous system. This showed that, theoretically, any molecular interaction of sufficient affinity may be used to specifically label cell surface structures in live nematodes.     

Increased chemokine gene expression during aging in the murine brain

Normal aging results in changes in the brain that contribute to the decline of various functions, including learning and memory. Mechanisms causing this decline have not been clearly established. Activation of microglia is associated with the normal aging process in rodents and primates. Microglial activation is regulated by chemokine gene expression, and activated microglia produce substances that can be detrimental to surrounding cells. In this study we determined whether changes in chemokine expression occur during normal aging in the mouse brain. RNA samples taken from the cortex, midbrain, hippocampus, and cerebellum of 4-, 10-, 21- and 30-month-old C57BL6/DBA2 mice were analyzed for changes in gene expression. RNase protection assays were used to examine a panel of chemokines. Increased expression of macrophage inflammatory protein (MIP)-1alpha, MIP-1beta, and RANTES occurred in all four regions of the brains in the oldest mice. These increases were first detectable at 21 months of age. Increases in MIP-1alpha, MIP-1beta, and RANTES protein levels were also detected in the brains of old mice, as measured by ELISA. Increased microglial activation in the brains of 30-month-old mice, as detected by immunohistochemistry using F4/80 antibodies, correlated with increases in chemokine expression. The observed increases in chemokine gene expression that occur in conjunction with increased microglial activation suggest that chemokines may contribute to the decreased brain function that occurs during normal aging.