Tissue specific and non-specific changes in gene expression by aging and by early stage CR

Aging alters the expression of a variety of genes. Calorie restriction (CR), which extends life span in laboratory rodents, also changes gene expression. This study investigated changes in gene expression across three different tissues from the same mouse to examine how aging and early stage CR influence gene expression in different tissues of an organism. Expression profiling of heart, liver, and hypothalamus tissues was done in young (4-6 months) ad libitum fed (AL), young CR (2.5-4.5 months of CR), and old (26-28 months) AL male C57BL/6 mice. Aging significantly altered the expressions of 309, 1819, and 1085 genes in heart, liver, and hypothalamus tissues, respectively. In nine genes, aging altered expression across all three tissues although the regulation directions did not agree across all three tissues for some genes. Early stage CR in young mice significantly changed the expressions of 192, 839, and 100 genes in heart, liver, and hypothalamus tissues, respectively, and seven genes altered expression across all three tissues; three were up regulated and four were down regulated. The results of Gene Ontology (GO) Biological Process analysis indicated up regulation of antigen processing/presentation genes by aging and down regulation of stress response genes by early stage CR in all three tissues. The comparison of the results of aging and short term CR studies showed there were 389 genes, 18 GO biological processes, and 20 GO molecular functions in common.     

Flies and their golden apples: the effect of dietary restriction on Drosophila aging and age-dependent gene expression

Reduced nutrient availability (dietary restriction) extends lifespan in species as diverse as yeast, nematode worms, Daphnia, Drosophila, and mammals. Recent demographic experiments have shown that moderate nutrient manipulation in adult Drosophila affects current mortality rate in a completely reversible manner, which suggests that dietary restriction in Drosophila increases lifespan through a reduction of the current risk of death rather than a slowing of aging-related damage. When examined in the light of the new demographic data, age-dependent changes in gene expression in normal and diet-restricted flies can provide unique insight into the biological processes affected by aging and may help identify molecular pathways that regulate it.     

Microarray workshop on aging

The advent of microarrays in studying gene expression in aging has created tremendous excitement due to its potential for uncovering molecular mechanisms of aging and age-related disease. However, the appropriate implementation of this technology in the science of aging requires serious attention to methodological detail and statistical rigor. This report highlights discussions from the microarray workshop on aging held at the First Conference on Functional Genomics of Aging in Seville, Spain. The topics discussed by the participants included technical issues relating to the printing of arrays, RNA isolation, cDNA labeling and hybridization, optimal design of microarray experiments, and statistical analysis of these data. Microarray analysis of complex tissues through the use of laser capture microdissection was also discussed.     

Chronic antidepressant treatment selectively increases expression of plasticity-related proteins in the hippocampus and medial prefrontal cortex of the rat

Antidepressants protect against hippocampal volume loss in humans and reverse stress-induced atrophic changes in animals thus supporting the hypothesis that the pathophysiology of stress-related disorders such as depression involves reductions in neuronal connectivity and this effect is reversible by antidepressant treatment. However, it is unclear which brain areas demonstrate such alterations in plasticity in response to antidepressant treatment. The aim of the present study was to examine the effect of antidepressant treatment on the expression of three plasticity-associated marker proteins, the polysialylated form of nerve cell adhesion molecule (PSA-NCAM), phosphorylated cyclic-AMP response element binding protein (pCREB) and growth-associated protein 43 (GAP-43), in the rat brain. To this end, rats were treated either acutely (60 min) or chronically (21 days) with imipramine (30 and 15 mg/kg, respectively) and the expression of PSA-NCAM, pCREB, and GAP-43 was assessed using immunohistochemistry. Initial mapping revealed that chronic imipramine treatment increased expression of these plasticity-associated proteins in the hippocampus, medial prefrontal cortex and piriform cortex but not in the other brain regions examined. Since PSA-NCAM and pCREB are expressed in recently-generated neurons in the dentate gyrus, it is likely that chronic imipramine treatment increased their expression in the hippocampus at least partially by increasing neurogenesis. In contrast, since chronic imipramine treatment is not associated with neurogenesis in the medial prefrontal cortex, increased expression of PSA-NCAM and pCREB in the prelimbic cortex implicates changes in synaptic connectivity in this brain region. Acute treatment with imipramine increased the number of pCREB positive nuclei in the hippocampus and the prefrontal cortex but did not alter expression of GAP-43 or PSA-NCAM in any of the brain regions examined. Taken together, the results of the present study suggest that antidepressant treatment increases synaptic plasticity and connectivity in brain regions associated with mood disorders.     

SCG 10, A neuron-specific growth-associated protein in Alzheimer''s disease

Neuronal growth-associated proteins (nGAPs) are markers of neuronal process outgrowth and are associated with both degenerative and sprouting responses in Alzheimer's disease (AD) brain. To study possible involvement of SCG10, an nGA,P in AD, we cloned human SCG10 cDNA and analyzed SCG-10 at mRNA and protein levels in control and AD brains. The deduced amino acid sequence of human SCG10 was 69% identical to stathmin, another nGAP. By in situ hybridization, both SCG10 and stathmin mRNAs were detected in selected neuronal populations in aged human brains. Quantitative analysis by RNase protection revealed that levels of neither SCG10 nor stathmin mRNAs were significantly altered in AD. Using an SCG10-specific antibody Western blot analysis did not reveal any quantitative changes of SCG10 in AD. However, when the concentration of SCG10 protein was plotted against the number of tangles, a positive correlation was found. SCG10 levels did not correlate with plaque numbers. Furthermore, immunohistochemical study revealed that neuronal SCG10 protein accumulated in the cell bodies in AD-affected regions. Thus, SCG10 compartmentalization and metabolism may be altered in AD possibly due to mechanisms related to tangle formation in this disease.     

脊髓半横断损伤后大鼠皮层神经元细胞骨架蛋白基因表达的变化

目的　探讨神经再生过程中细胞骨架蛋白的作用机制 ,了解中枢和外周神经系统神经元应答神经损伤和再生的差异。　方法　用原位分子杂交方法观察了大鼠T10～ 11脊髓半横断损伤后 1、3、5、7、10、14、2 1、2 8、5 6d皮层感觉运动区皮层神经元中α 管蛋白和 3个神经丝蛋白亚基mRNA的表达变化。　结果　脊髓半横断损伤后损伤侧皮层感觉运动区皮层神经元中α 管蛋白、神经丝 L、 M、 HmRNA表达明显下调。α 管蛋白mRNA表达水平在损伤后 1d降低 ,而神经丝亚基mRNA表达水平直到下一个时间点 (损伤后 3d)才下调 ,至损伤后 5 6d均无恢复趋势。结论　皮层神经元损伤后α 管蛋白mRNA的表达被抑制 ,提示皮层神经元和外周神经元应答损伤的信号不同     

老年大鼠空间记忆减退与cNOS基因表达变化

目的：从基因表达角度研究原生型NOS（cNOS,包括神经元型nNOS和内应型eNOS）与老年记忆减退发生的关系。方法：用Morris水迷宫将老年大鼠筛选为记忆正常和记忆减退两组,以老年记忆减退大鼠作为老年记忆减退模型;用半定量反转录一聚合酶链反应（RT－PCR）方法测定nNOS和eNOS的mRNA含量。结果：（1）海马组织中老年记忆正常组和老年记忆减退组较青年组nNOS和eNOSmRNA含量均下降,nNOS以记忆减退组下降更为显著;（2）小脑组织中老年记忆减退组nNOS和eNOSmRNA含量的下降与老年记忆正常组和青年组均有显著差异。（3）额叶组织nNOSmRNA含量三组之间均无显著差异;eNOSmRNA含量老年记忆正常组和老年记忆减退组技青年组均下降,以记忆减退组下降更为显著。结论：老年记忆减退的发生可能与有关脑区cNOS基因表达下降有关。     

Neonatal handling and environmental enrichment increase the expression of GAP-43 in the hippocampus and promote cognitive abilities in prenatally stressed rat offspring

Neonatal handling and environmental enrichment have been used to aid the treatment and recovery of a diverse variety of brain dysfunctions. However, the underlying mechanism and the effects on cognitive function following neonatal handling and environmental enrichment are still unclear. In this study, we investigated GAP-43 protein levels in the hippocampus of prenatally stressed rat pups by Western blot on postnatal day (P) 10, P20 and P45. The cognitive ability of prenatally stressed rat pups was tested by using the Morris water maze on P45. GAP-43 protein levels were upregulated on P10 in the prenatal restraint stress (RS) group and the prenatal restraint stress plus neonatal handling and environmental enrichment (RE) group compared to the negative control (NC) group. However, the expression of GAP-43 in RS pups was lower on P20 and P45 than that in NC and RE pups. Exposure to prenatal stress prolonged average latency and total swim distance, but neonatal handling and environmental enrichment could reverse the change. Differences were also observed in the selection of search strategies. These results indicate that neonatal handling and environmental enrichment can improve the spatial learning and memory ability of prenatally stressed offspring, and the possible mechanism is the upregulation of GAP-43.     

Prenatal stress alters presynaptic marker proteins in the hippocampus of rat pups

Exposure to stress during critical periods of an organism's maturation can result in permanent behavioral changes and induced hyper-responsive to aversive stimuli as adult. Hippocampus is a plastic and vulnerable brain structure that is susceptible to damage during aging and repeated stress. The present study examines the effect of maternal restraint stress on the level of GAP-43, pGAP-43 and synaptophysin in the hippocampus of rat pups. Prenatal stress (PS) causes a significant increase of GAP-43 and pGAP-43 (p ≤ 0.001) in the pup's hippocampus during postnatal days 7 and 14, but not at later ages. Up-regulation of GAP-43 and pGAP-43 may alter the pattern of axonal growth and synapses’ formation in the pup's brain since the first two postnatal weeks are correlated with peak period of synaptogenesis in the rat brain. We also examined the level of synaptophysin, a synaptic vesicle membrane protein, in the pup's brain. Our finding revealed that, PS causes a significant decrease of synaptophysin in the pup's hippocampus as compared to control (p ≤ 0.001). These changes are due to the direct effects of maternal stress hormone since repeated injection with corticosterone (CORT, 40 mg/kg) to pregnant rat during gestation days (GDs) 14–21 also gave the same results. Abnormal axonal sprouting and reorganization together with the alterations in synaptic vesicle membrane protein during the critical period of synaptogenesis may lead to a defect in synapse formation and axonal pruning in the hippocampus. These changes may be associated with stress-induced impairment of hippocampal function that occurs in later life of the offspring.     

Distribution of non-phosphorylated and phosphorylated neurofilament proteins in the spinal cord of an anuran amphibian during development and regeneration

The changes in the neurofilament medium and high molecular weight subunits (NF150 and NF200) in the developing and transected spinal cords of bullfrog tadpoles were studied. A monoclonal antibody recognizing the nonphosphorylated epitope of NF150, NF150D, stained the neuronal cell bodies and axons, whereas other antibodies against the phosphorylated NFs, NF150P or NF200P, labeled chiefly the axons. During development, the intensity of axonal staining by the anti-NF150D in the ventral fasciculi in younger tadpoles appeared stronger than older animals, but the reverse was seen for NF150P and NF200P. Complete signal transection of stage IV tadpoles resulted in degeneration and then regeneration of the cord tissue of both cut ends. Each stump lengthened by about 350 m in the 4 weeks after the lesion. In the proximal stumps, the levels of NF150P or NF200P in the ventral axons at 550-350 m proximal to the transection site increased notably by about 24–73% of the control value 7–28 days post-transection; however, the content of NF150D was decreased. The densities of NF150D and NF150P protein spots on the Coomassie blue-stained two-dimensional gels of the normal and injured cords also displayed alterations similar to the immunocytochemical data. Intense labeling by the anti-NF150P or NF200P was present in the cell bodies of axotomized motor neurons in the ventral horn. The results suggest that central axonal regeneration may be accompanied by upregulated phosphorylated neurofilament proteins.     

臂丛损伤再生中GAP-43 mRNA及其蛋白的表达

目的：分析臂丛损伤后脊髓前角运动神经元表达GAP-43 mRNA及其蛋白的变化规律，探讨神经损伤再生的机制。方法：建立3种臂丛损伤模型：右C₇前根撕脱（A组）；右C₇前根撕脱＋同侧C5-T₁后根断离（B组）；右C₇前根撕脱+右C₅C₆间脊髓半横断（C组）。用荧光定量RT-PCR方法检测术后14 d时 C₇前角GAP-43 mRNA的表达量。用免疫组化方法检测术后1、 3、 7、14 d脊髓前角GAP-43免疫阳性运动神经元的表达。结果：对照组C₇前角GAP-43 mRNA呈低表达，损伤组GAP-43 mRNA表达显著上调。损伤组术后1 d、3 d时均未见C₇前角 GAP-43免疫阳性神经元，术后7 d各损伤组GAP-43免疫阳性神经元开始出现，14 d时免疫阳性神经元数目达到高峰。3组间比较，C组表达量最高，B组最低，A组居中。结论：臂丛损伤诱导运动神经元GAP-43 mRNA及其蛋白表达上调，GAP-43合成增加是神经元蛋白重组所致，与轴索再生和神经功能重建有关。     

TRAIL胞外段基因克隆及其表达与纯化

目的克隆TNF相关的诱导凋亡配体胞膜外段基因(the extracellular region of the human TRAIL cDNA,TPAILex),构建表达载体并在大肠杆菌DH5α中高效表达有生物学活性的TRAIL蛋白胞膜外段.方法抗CD3McAb刺激正常人外周血淋巴细胞,用RT-PCR、巢式-PCR方法从活化的淋巴细胞中获得人TRAIL基因及其胞膜外段cDNA,并克隆到pGEM-T Easy载体中,DNA测序鉴定.将TRAIL的胞膜外段基因插入表达载体pGEX-2T,构建重组表达质粒pGEX/TRAILex,用IPTG诱导表达TRAIL蛋白的胞膜外段,GST-Agarose 4B层析柱纯化GST-TRAILex融合蛋白,Westem-blot鉴定纯化产物.结果抗CD3 McAb能诱导正常人外周血单个核细胞TRAIL的高表达,成功地克隆了TRAIL胞膜外段基因,构建了重组表达载体pGEX/TRAILex,IPTG诱导后得到高效表达的TRAIL蛋白胞膜外段,经12%SDS-PAGE分析,可观察到一分子量和理论值相符(约54kD)的诱导表达带.Kodak软件分析表面GST-TRAIL胞膜外段融合蛋白的表达量占菌体蛋白总量的28%,进一步分析证实TRAIL蛋白的胞膜外段主要以可溶性的形式表达,用GST-Agarose4B层析柱纯化超声破菌后的上清,每升细菌培养液可得到9.8 mg纯度的95%以上的GST-Tex.Western-blot结果证实54KD的表达带可与鼠抗TRAIL McAb起特异性反应.结论成功地克隆了人TRAIL基因,并应用基因工程技术在大肠杆菌中高效表达人TRAIL蛋白的胞膜外段,为进一步研究TRAIL的功能及其在肿瘤生物治疗中的应用奠定了基础.