突变型早老素-1致维甲酸诱导PC12细胞ERp29表达增强及其意义

目的 探讨突变型早老素-1(PS-1)对维甲酸(RA)诱导PC12细胞基因表达的影响,寻找与阿尔茨海默病发病机制相关的基因.方法 分别建立表达突变型和野生型PS-1的经RA诱导的基因工程化PC12细胞,应用银染mRNA差异显示技术研究突变型PS-1对RA诱导的PC12细胞基因差异表达的影响.结果 成功建立了表达突变型和野生型PS-1的经RA诱导的基因工程化PC12细胞后,应用银染mRNA差异显示技术发现29型内质网蛋白(ERp29)在表达突变型PS-1的基因工程化PC12细胞中高表达,并与细胞凋亡密切相关.结论 突变型PS-1致细胞内ERp29的表达增强可能是内质网应激的结果,ERp29表达增强与阿尔茨海默病的发生与发展可能存在着密切关系.     

2002/原花青素对β-淀粉样肽（25-35）诱导去血清培养PC12细胞细胞周期分布的影响

目的 探讨原花青素 (Procyanidins,PC) 对β-淀粉样肽（25-35）[βamyloid peptide -(25-35),Aβ25-35] 诱导去血清培养PC12细胞的凋亡及细胞周期分布的影响。方法 流式细胞仪检测细胞周期分布情况及细胞凋亡率;RT-PCR 检测 p53基因 mRNA 表达;Western blot检测 P53蛋白表达。 结果 30mg/L PC预处理 PC12 细胞 1 h ,可降低Aβ25-35引起的去血清培养PC12细胞凋亡率,逆转Aβ25-35引起的去血清培养PC12细胞的细胞周期S期阻滞,降低 p53 mRNA及P53蛋白表达。 结论 PC可对抗Aβ25-35 诱导的去血清培养 PC12 细胞的凋亡及细胞周期S期阻滞,其机制可能与下调p53基因表达有关。     

奥氮平对无血清诱导PC12细胞凋亡的影响

目的:探讨奥氮平对无血清诱导PC12细胞凋亡的保护作用及其机制。方法:以神经生长因子(NGF)诱导后的PC12细胞作为细胞模型,采用无血清培养诱导细胞凋亡。给予奥氮平后,采用MTT法检测细胞活性,流式细胞仪检测细胞凋亡率、细胞周期以及Hoechst33342染色观察细胞形态学的改变。结果:奥氮平(100μM)组培养72h的细胞活性与对照组相比差异显著(P<0·05),奥氮平(12·5、25、50、200μM)组与对照组无明显差异;而各浓度氟哌啶醇组的细胞活性均低于对照组;流式细胞仪结果显示血清组、奥氮平组、氟哌啶醇组、对照组的调亡率依次是17·9%、36·6%、59·8%、51·9%,其中对照组、氟哌啶醇组大部分细胞滞留于G1期;奥氮平组Hoechst33342染色偶见凋亡细胞,以核浓缩为主,而对照组、氟哌啶醇组多见核碎裂。结论:奥氮平能保护PC12细胞免于无血清培养诱导的凋亡,可能是其神经保护作用机制之一。     

含巯基抗氧化剂对多巴胺诱导PC12细胞凋亡的保护作用

目的 观察不同的抗氧化剂对多巴胺诱导的PC12细胞凋亡的保护作用,探讨帕金森病(PD)神经元的死亡机制.方法 应用TUNEL染色及电泳技术,观察4种不同的抗氧化剂对多巴胺(DA)诱导的PC12细胞凋亡的保护性作用. 结果 适当浓度的多巴胺可诱导PC12细胞凋亡,抗氧化剂GSH及N-AC在10mmol/L浓度下能显著抑制DA诱导的PC12细胞凋亡(P<0.05),而相同浓度的维生素C及维生素E则无保护作用.结论 细胞凋亡可能参与了PD的发病过程,适当的抗氧化剂对于DA诱导的细胞凋亡具有保护作用.     

p53基因、PTEN基因协同对胶质瘤U251细胞系生长抑制作用的研究

目的探讨p53基因和PTEN基因在脑胶质瘤细胞系U251发生发展过程中的作用机制。方法用不同MOI的p53腺病毒表达载体pAdCMV-p53及空载体pAdCMV-lacZ分别感染表达野生型PTEN基因和突变型PTEN基因的细胞系,RT-PCR及Westernblot方法检测转染效率;并通过MTT检测生长抑制率、流式细胞仪检测细胞周期及TUNEL检测分析细胞凋亡等指标观察p53基因及PTEN基因对U251细胞生长的影响。结果MOI为100时,p53基因可引起U251细胞G0G1期阻滞、诱导细胞凋亡,生长抑制;MOI为50时,U251-p53 PTEN生长抑制率明显高于U251-p53,并能出现细胞凋亡,而U251-p53仅出现少量细胞凋亡。结论p53基因可以通过细胞周期G0G1期阻滞及诱导细胞凋亡抑制胶质瘤细胞系U251的生长;PTEN基因可以促进p53基因对胶质瘤细胞系U251的生长抑制作用,并能增加U251细胞对p53基因诱导凋亡的敏感性。     

多巴胺抑制PC12细胞增殖和诱导凋亡作用的研究

目的　研究多巴胺(DA)对PC12细胞的增殖抑制和诱导凋亡的作用,探讨帕金森病(PD)神经元的死亡机制。方法　应用免疫组织化学、流式细胞仪、电镜及电泳技术,研究DA对大鼠嗜铬细胞瘤PC12细胞的增殖抑制及诱导凋亡的作用。结果　适当浓度(0.5mmol/L)DA能显著抑制PC12细胞的生长并诱导其凋亡,在作用时间较短时(<12h)表现为对PC12细胞的生长抑制,此时流式细胞仪检测未见凋亡峰,但细胞周期显示S期细胞明显抑制。此时Bcl-2染色呈强阳性,电镜下细胞形态基本正常,可见线粒体、内质网肿胀及核分裂相减少。当作用时间延长时(>24h),流式细胞仪可见典型亚二倍体凋亡峰,此时电泳可见典型DNA“阶梯状”电泳带,电镜可见核浓缩、染色体边聚等凋亡特征性核结构改变,Bcl-2染色阳性率降低。结论　DA具有抑制PC12细胞增殖和诱导凋亡作用,细胞凋亡参与了PD的病变过程。     

谷氨酸诱导转Bax基因PCl2细胞凋亡及神经生长因子的保护作用

目的 探讨谷氨酸诱导转Bax基因PC12细胞细胞凋亡及神经生长因子（NerveGrowth Factor,NGF)的保护作用。方法 应用脂质体介导细胞转染方法进行Bax基因转染PC12细胞,结合流式细胞术和TUNEL法检测了谷氨酸处理前后转Bax基因PC12细胞的凋亡及NGF的保护作用。结果 与对照组相比,谷氨酸处理转Bax基因PC12细胞凋亡峰增高,TUNEL阳性标记细胞增多。用NGF处理后该组细胞凋亡峰减低,TUNEL阳性标记细胞数减少,差异均有显著意义。结论 谷氧酸处理转染Bax基因PC12细胞凋亡增加,NGF可以保护谷氨酸诱导的转染Bax基因PC12细胞凋亡。     

99mTc-Annexin V检测早期凋亡多巴胺能神经元的实验研究

目的 研究放射性核素标记的膜联蛋白V(Annexin V)与凋亡的多巴胺能神经元的结合特性,探讨使用凋亡显像剂99mTc-Annexin V早期活体显像诊断帕金森病的可行性.方法 采用不同浓度的1-甲基-4-苯基吡啶离子(1-methyl-4-phenylpyridinium, MPP+)处理大鼠肾上腺嗜铬细胞瘤细胞(PC12)和人神经母细胞瘤细胞(SH-SY5Y)以诱导其凋亡(PC12 0～200 μm/L, SH-SY5Y 0～500 μm/L),FITC -Annexin V及碘化吡啶(propidiumiodide, PI)双染进行流式细胞仪凋亡检测.以99mTc-Annexin V与凋亡的细胞进行饱和结合实验及细胞摄取实验,研究凋亡细胞与Annexin V的亲和力及其摄取99mTc-Annexin V的动力学.结果 MPP+可诱导PC12细胞及SH-SY5Y细胞发生凋亡,并有明显的量效关系.凋亡细胞可特异性与99mTc-Annexin V结合,亲和力可达(7.16±1.78)nmol/L,每个凋亡的多巴胺能神经元表面的结合位点可达到(179±33)fmol/106cells (PC12)及(220±26)fmol/106cells (SH-SY5Y),且神经元的凋亡水平与其膜结合的99mTc-Annexin V放射性强度有相关性(P＜0.001).结论 凋亡的多巴胺能神经元与99mTc-Annexin V有高度亲和力,其所结合的99mTc-Annexin V放射性强度与细胞的凋亡水平相关,99mTc-Annexin V可用于检测多巴胺能神经元的早期凋亡.     

谷氨酸诱导转Bax基因PC12细胞凋亡及 神经生长因子的保护作用

目的探讨谷氨酸诱导转Bax基因PC12细胞凋亡及神经生长因子（NerveGrowthFactor,NGF）的保护作用。方法应用脂质体介导细胞转染方法进行Bax基因转染PC12细胞,结合流式细胞术和TUNEL法检测了谷氨酸处理前后转Bax基因PC12细胞的凋亡及NGF的保护作用。结果与对照组相比,谷氨酸处理转Bax基因PC12细胞凋亡峰增高,TUNEL阳性标记细胞增多;用NGF处理后该组细胞凋亡峰减低,TUNEL阳性标记细胞数减少。差异均有显著意义。结论谷氨酸处理转染Bax基因PC12细胞凋亡增加,NGF可以保护谷氨酸诱导的转染Bax基因PC12细胞凋亡。     

Superoxide mediates the cell-death-enhancing action of presenilin-1 mutations.

The mechanism whereby mutations in the presenilin-1 (PS-1) gene on chromosome 14 cause early-onset inherited Alzheimer's disease are unknown. We report that PC6 neural cells (a subclone of PC12 cells) expressing PS-1 mutations (M146V and L286V) exhibit increased superoxide production, nitrotyrosine accumulation, and membrane lipid peroxidation following exposure to amyloid beta-peptide 1-42 (Abeta). Mitochondrial calcium accumulation and membrane depolarization following exposure to Abeta were enhanced in cells expressing mutant PS-1. Overexpression of mitochondrial Mn-SOD greatly reduced superoxide production, nitrotyrosine formation, membrane lipid peroxidation, intramitochondrial calcium accumulation, and membrane depolarization following exposure to Abeta and conferred resistance to the apoptosis-enhancing action of the PS-1 mutations. Nitric oxide synthase inhibitors and the peroxynitrite scavenger uric acid blocked the apoptosis-enhancing action of PS-1 mutations. The data suggest pivotal roles for superoxide production and resulting peroxynitrite formation in the pathogenic mechanism of PS-1 mutations.     

Enhanced accumulation of phosphorylated alpha-synuclein in double transgenic mice expressing mutant beta-amyloid precursor protein and presenilin-1

A recent report showed that the accumulation of alpha-synuclein (alpha-syn) was detected in the brains of one-third of Alzheimer's disease and Down syndrome patients. However, the relationship between amyloid-beta protein (Abeta) and alpha-syn remains unclear. We analyzed the relation between the mutation of presenilin-1 (PS-1) and the pathological features of beta-amyloidosis and alpha-synucleinopathy. We generated doubly transgenic mice overexpressing mutant beta-amyloid precursor protein (betaAPP; Tg2576) and mutant PS-1 (PS1L286Vtg; line 198) and analyzed 19 double Tg betaAPP(+)/PS(+) mice at 5-23 months (young to old), 23 age-matched single Tg betaAPP(+)/PS(-) mice, and 11 non-Tg littermates. Immunohistochemical comparison was performed in these three groups by counting the area and the number of alpha-syn- or phosphorylated alpha-syn (palpha-syn)-positive dystrophic neurites per plaque (ASPDN, pASPDN). The acceleration of Abeta pathology was found with earlier onset and exaggerated numbers in double Tg betaAPP(+)/PS(+) compared with single Tg betaAPP(+)/PS(-) mouse brains. The accumulation of ASPDN and pASPDN was also accelerated in double Tg betaAPP(+)/PS(+) compared with single Tg betaAPP(+)/PS(-) mouse brains, especially in pASPDN. The number and area of alpha-syn and palpha-syn, and the ratio of palpha-syn positive neurites were significantly higher in double Tg betaAPP(+)/PS(+) than in single Tg betaAPP(+)/PS(-) mouse brains in middle-aged and old groups. Additional overexpression of mutant PS-1 accelerated Abeta-induced alpha-synucleinopathy and further facilitated the phosphorylation of alpha-syn, suggesting a direct association between mutant PS-1 and phosphorylation of alpha-syn.     

Presenilin-1 protects against neuronal apoptosis caused by its interacting protein PAG

Mutations in the presenilin-1 (PS-1) gene account for a significant fraction of familial Alzheimer's disease. The biological function of PS-1 is not well understood. We report here that the proliferation-associated gene (PAG) product, a protein of the thioredoxin peroxidase family, interacts with PS-1. Microinjection of a plasmid expressing PAG into superior cervical ganglion (SCG) sympathetic neurons in primary cultures led to apoptosis. Microinjection of plasmids expressing wild-type PS-1 or a PS-1 mutant with a deletion of exon 10 (PS1dE10) by themselves had no effect on the survival of primary SCG neurons. However, co-injection of wild-type PS-1 with PAG prevented neuronal death, whereas co-injection with the mutant PS-1 did not affect PAG-induced apoptosis. Furthermore, overexpression of PAG accelerated SCG neuronal death induced by nerve growth factor deprivation. This sensitizing effect was also blocked by wild-type PS-1, but not by PS1dE10. These results establish an assay for studying the function of PS-1 in primary neurons, reveal the neurotoxicity of a thioredoxin peroxidase, demonstrate a neuroprotective activity of the wild-type PS-1, and suggest possible involvement of defective neuroprotection by PS-1 mutants in neurodegeneration.     

Effects of RNAi-mediated silencing of PEN-2, APH-1a, and nicastrin on wild-type vs FAD mutant forms of presenilin 1

The gamma-secretase complex consists of PS1/PS2, nicastrin, APH-1a, and PEN-2. PS1 undergoes endoproteolytic processing to yield two fragments: PS1-NTF and PS1-CTF. Changes in PEN-2 levels have been shown previously to affect the endoproteolytic processing of wild-type (wt)-PS1. However, the effects of PEN-2 on the proteolytic processing of familial Alzheimer's disease (FAD) mutant forms of PS1 have not yet been reported. To determine whether PEN-2 affects the proteolytic processing of mutant PS1 in the same manner as that of wt-PS1, we established RNA interference (RNAi) for PEN-2 in H4 human neuroglioma cells stably transfected to express wt or FAD mutant forms of PS1 including L286V, A246E, and that lacking exon 9 (Delta9). As expected, in H4 cells expressing wt-PS1, RNAi for PEN-2 increased levels of PS1-FL and attenuated PS1 endoproteolysis. Likewise, in cells expressing PS1 with the FAD missense mutations, L286V and A246E, RNAi for PEN-2 increased PS1-FL and reduced PS1 endoproteolysis. However, in H4 cells stably transfected to express the FAD-linked Delta9 mutation (PS1 lacking exon 9), RNAi for PEN-2 did not increase but, instead, decreased PS1-FL. In contrast, RNAi for nicastrin and APH-1a decreased PS1-FL in H4 cells expressing either wt-PS1 or Delta9-PS1. In summary, the metabolism of wt-PS1 and FAD-linked Delta9-PS1 is specifically and differentially affected by loss of function of PEN-2.     

Herpes simplex virus type 1 ICP4 deletion mutant virus d120 infection failed to induce apoptosis in nerve growth factor-differentiated PC12 cells

It has been suggested that terminally differentiated neuronal cells and mitotic cells respond differently in many aspects to herpes simplex virus type 1 (HSV-1) infection. The ICP4-deleted, Us3-defective, HSV-1 mutant strain d120 induces classical apoptosis in a variety of mitotic cell lines. Its behavior in postmitotic cells is not known. Here the authors report that mutant d120 virus failed to induce apoptosis in neuronal-like, nerve growth factor (NGF)-differentiated PC12 cells. More strikingly, rather than inducing apoptosis, d120 infection prolonged the life of nondividing NGF-differentiated PC12 cells in the culture flask. The virus genome had a half-life of 30 days. Unlike in other cells, such as Vero, neither wild-type nor d120 infection of NGF-differentiated PC12 cells induced the nuclear factor (NF)-kappa B p65 pathway, which has been associated with virus-induced apoptosis. Thus, the authors demonstrate, for the first time, that a potent apoptosis inducer mutant d120 failed to induce apoptosis in neuronal-like NGF-differentiated PC12 cells, unlike a number of other cell lines studied. The possible mechanisms involved in the failure of d120 to induce apoptosis in neuronal-like NGF-differentiated PC12 cells are discussed.     

Mutant presenilin-1 induces apoptosis and downregulates Akt/PKB.

Most early onset cases of familial Alzheimer's disease (AD) are caused by mutations in presenilin-1 (PS1) and presenilin-2 (PS2). These mutations lead to increased beta-amyloid formation and may induce apoptosis in some model systems. Using primary cultured hippocampal neurons (HNs) and rat pheochromocytoma (PC12) cells transiently transfected with replication-defective recombinant adenoviral vectors expressing wild-type or mutant PS1, we demonstrate that mutant PS1s induce apoptosis, downregulate the survival factor Akt/PKB, and affect several Akt/PKB downstream targets, including glycogen synthase kinase-3beta and beta-catenin. Expression of a constitutively active Akt/PKB rescues HNs from mutant PS1-induced neuronal cell death, suggesting a potential therapeutic target for AD. Downregulation of Akt/PKB may be a mechanism by which mutant PS1 induces apoptosis and may play a role in the pathogenesis of familial AD.     

Human wild presenilin-1 mimics the effect of the mutant presenilin-1 on the processing of Alzheimer's amyloid precursor protein in PC12D cells

Most familial early-onset Alzheimer's disease (FAD) is caused by mutations in the presenilin-1 (PS1) gene. Abeta 42 is derived from amyloid precursor protein (APP) and increased concentrations are widely believed to be a pathological hallmark of abnormal PS function. Thus, the interaction between PS1 and APP is central to the molecular mechanism of AD. To examine the effect of wild-type human PS1 on rat APP metabolism, we made several PC12D cell lines that expressed human wild or mutant PS1, and analyzed the processing of endogenous rat APP and the intracellular gamma-secretase activity. We found the ratio of Abeta 42/Abeta 40 increased in PC12D cells expressing wild-type human PS1. These changes were identical to those found in PC12D cells expressing human PS1 bearing the A260V mutation. These results suggest that APP metabolism is physiologically regulated by the PS1 and that loss of normal PS1 affects gamma-secretase activity.     

Object recognition memory and cholinergic parameters in mice expressing human presenilin 1 transgenes

Most autosomal dominant forms of Alzheimer disease (AD) are related to missense mutations in the human presenilin (PS) 1 gene. Although the underlying mechanisms associated with pathophysiology of AD have yet to be clearly established, pathogenic mutations in the PS1 gene influence the processing of beta-amyloid precursor protein, leading to increased production and deposition of highly fibrillogenic amyloid beta(1-42) peptide in the brains of AD patients. As cognitive dysfunction in AD is associated with a dramatic loss of cholinergic innervation particularly in the hippocampus and neocortex, we investigated learning and cholinergic neurochemistry in transgenic mice expressing pathogenic mutant L286V or wild-type(wt) human PS1 transgenes. Relative to wt, the L286V PS1 transgenic mice exhibited reduced sensorimotor activity and marked deterioration of object memory between 3 and 5 h after the first encounter. Activity of the biosynthetic enzyme choline acetyltransferase was not altered in the hippocampus, frontoparietal cortex, or striatum of mutant transgenic mice relative to wt transgenic or littermate nontransgenic controls. No differences in the densities of M1/[3H]pirenzepine, M2/[3H]AF-DX 384, or alpha(7) nicotinic/125I-alpha-bungarotoxin receptor binding sites were evident in any brain regions among L286V PS1 transgenic, wt PS1 transgenic, and littermate nontransgenic controls. These results suggest that overexpression of a mutated PS1 gene induces a subtle alteration in object memory without affecting cholinergic neurochemistry.