淀粉样前体蛋白基因的分子遗传学和分子生物学

淀粉样前体蛋白 (amyloid precusor protein,APP)是 Aβ(β- am yloid peptide,Aβ)的前体蛋白。后者是阿尔茨海默疾病 (Alzheimer's disease,AD)最主要的病理标志。 APP突变后增加神经毒性蛋白 Aβ42 / 43的表达量 ,从而导致家族性 AD(FAD)的发生。本文综述了 APP的基因结构、功能、突变体的类型及基因突变的病理学后果 ,同时追踪了如下两方面的研究进展 :1Aβ产生的途径 ;2 APP代谢的调控机制     

β-淀粉样前体蛋白分子生物学研究进展

β-淀粉样前体蛋白基因定位于21号染色体长臂,具“管家基因”结构特点。在组织中广泛表达但有一定的特异性。其近侧启动子区(-96～+101)为转录活性所必需,研究发现β-淀粉样前体蛋白基因表达调控由多种顺式元件和反式因子的相互作用所决定。β-淀粉样前体蛋白呈细胞表面受体构象,具有多种生物学功能。业已证明其分子生物学功能的改变与Alzheimer's病的发生、发展有着十分密切的关系。     

β淀粉样蛋白1~42诱导原代皮质神经元损伤过程中的淀粉样前体蛋白信号通路*☆

背景：近年有研究表明纤维状β淀粉样蛋白能够促进细胞表面淀粉样前体蛋白在细胞外的积聚,导致神经损伤。 目的：分析淀粉样前体蛋白信号通路在纤维状β淀粉样蛋白1~42诱导神经元损伤机制中的作用。 方法：体外分离培养孕17.0~18.0 d SD大鼠皮质神经元,培养7 d后加入0(正常对照),0.05,0.5,5 mol/L纤维状β淀粉样蛋白1~42,孵育8 h建立毒性损伤模型,采用生化方法检测神经元细胞培养上清中的Calcein释放,分别用免疫荧光双标、Western blotting方法检测淀粉样前体蛋白和Fe65的表达。 结果与结论：与正常对照组比较,加入不同浓度纤维状β淀粉样蛋白1~42诱导损伤8 h后,神经元培养上清中Calcein释放增加(P < 0.05或P < 0.01),Western blotting和免疫荧光方法分别检测到淀粉样前体蛋白和Fe65的表达及共定位增加。说明纤维状β淀粉样蛋白1~42可诱导原代培养皮质神经元的毒性损伤,淀粉样前体蛋白-Fe65信号通路可能是其损伤机制之一。      

淀粉样前体蛋白在痴呆模型大鼠背海马结构内的表达

研究用ＡｌＣｌ３灌胃，建立鼠老年性痴呆模型。用免疫细胞化学ＡＢＣ法，观察了淀粉样前体蛋白在背海马结构的表达。结果显示在痴呆模型鼠背海马结构双侧各部均可见较强的淀粉样前体蛋白表达，且与对照组间有显著性差异。光镜结果，阳性反应产物主要位于神经元核周质和突起及其分支。细胞大小不等，多数淀粉样前体蛋白样免疫反应阳性神经元胞体为梭形和锥形。背海马结构内淀粉样前体蛋白样免疫反应神经元主要分布在ＣＡＩ、ＣＡ２、     

β-淀粉样前体蛋白转基因小鼠发育过程中海马 CAI区神经细胞凋亡变化

目的:探讨瑞典家系β-淀粉样前体蛋白(APPSWE)转基因小鼠发育过程中海马 Cal 区神经细胞凋亡规律.方法:取不同发育时间(P0、P7、P14、P30、P90、P180)APPSWE 转基因模型鼠与同时间点对照鼠,Nissl 染色观察海马结构和锥体细胞形态,免疫组织化学方法观察海马细胞内 Caspase-3 表达变化,RT-PCR 检测 Caspase-3 mRNA 表达变化.结果:随着小鼠的生长发育,P14 时间点以后,模型组 Cal 区神经元 Caspase-3 阳性细胞密度比对照组高;RT-PCR 检测结果与Caspase-3 免疫组织化学结果基本一致.结论:APPSWE 转基因小鼠发育中的海马神经细胞过度凋亡可能与阿尔茨海默病的发生、发展具有联系.     

淀粉样前体蛋白在因的分子遗传学和分子生物学

淀粉样前体蛋白(amyloid precusor protein,APP）是Aβ（β－amyloid peptide,Aβ）的前体蛋白,后是阿尔蒋海墨疾病（Alzheimer's disease,AD)最主要的病理标志,APP突变后增加神经毒性蛋白Aβ42／43的表达量,从而导致家族性AD（FAD）的发生,本综述了APP的基因结构,功能,突变体的类型及基因突变的病理学后果,同时追踪了如下两方面的研究进展：（1）Aβ产生的途径,（2）APP代放的调控机制。     

大鼠原发性脑于损伤延脑淀粉样β蛋白及其蛋白前体的表达

目的 探讨早期诊断原发性脑干损伤(PBSI)中弥散性轴索损伤(DAI)的敏感标记.方法 应用免疫组织化学SP法及RT-PCR技术研究在大鼠原发性脑干损伤模型中,延脑DAI与淀粉样β蛋白(Aβ蛋白)的表达及淀粉样β前体蛋白(β-APP)mRNA的半定量分析,以此来评估进行DAI早期诊断的有效性.结果 免疫组化Aβ染色神经轴突测量3～24 h损伤组,轴突明显增粗,并且稳步增加,与对照组相比差异均有统计学意义,并且与时间呈正相关,β-APPmRNA表达增加最早从1 h损伤组开始,并于3 h达到高峰.结论 β-APP是DAI的敏感性和特异性标记,RT-PCR技术是一种快速的和敏感的方法,再结合免疫组化技术检测Aβ蛋白,可能对早期诊断DAI是非常有效的.     

β-淀粉样前体蛋白转基因小鼠发育过程中海马CA1区神经细胞凋亡变化

目的：探讨瑞典家系β-淀粉样前体蛋白（APPSWE）转基因小鼠发育过程中海马CA1区神经细胞凋亡规律。方法：取不同发育时间（P0、P7、P14、P30、P90、P180）APPSWE转基因模型鼠与同时间点对照鼠，Nissl染色观察海马结构和锥体细胞形态，免疫组织化学方法观察海马细胞内Caspase-3表达变化，RT-PCR检测Caspase-3mRNA表达变化。结果：随着小鼠的生长发育，P14时间点以后，模型组CA1区神经元Caspase-3阳性细胞密度比对照组高；RT-PCR检测结果与Caspase-3免疫组织化学结果基本一致。结论：APPSWE转基因小鼠发育中的海马神经细胞过度凋亡可能与阿尔茨海默病的发生、发展具有联系。     

携带淀粉样前体蛋白瑞典型突变基因转基因小鼠的制备及体内表达研究

目的 建立携带人类淀粉样前体蛋白瑞典型突变（Swedish mutation of amyloid precursor protein,APPSWE)基因的转基因小鼠模型。方法 采用受精卵原核显微注射法，将人类APPSWE转基因导入C57及昆明种小鼠受精卵内，然后将注射后保持完整的受精卵移植到假孕母鼠的输卵管内，然后应用聚合酶链反应（polymerase chain reaction,PCR)、荧光原位杂交及逆转录PCR分析子代小鼠中外源基因的整合及表达情况。结果 注射后卵的存活率和幼子出现率分别为76.62%和10.38%；外源基因整合率为35.29%；共获得6只首建小鼠，已稳定传3代，PCR检测共55只阳性；提取阳性小鼠心、脑、肝、肾组织及骨骼肌以人类APPSWE基因外显子特异的引物进行逆转录PCR分析，结果发现其心、脑组织及骨骼肌中具有人类APPSWE基因的表达。结论 说明携带淀粉样前体蛋白瑞典型突变基因的转基因小鼠模型已制备成功。     

胆碱能前体细胞移植入β-淀粉样前体蛋白转基因小鼠脑内的存活和迁移

目的 观察新生小鼠海马原代培养的胆碱能神经前体细胞移植人淀粉样前体蛋白(amyloid precusor protein,APP)转基因小鼠脑内以后的存活和迁移状况,以探索胆碱能神经前体细胞对阿尔茨海默病的治疗前景.方法 取新生24h以内增强型绿色荧光蛋白(enhanced green fluorescent protein,EGFP)转基因小鼠,以原代细胞分离培养方法获得海马胆碱能神经前体细胞,以前体细胞特异性标记抗巢蛋白(Nestin)和胆碱能细胞特异性标记胆碱乙酰基转移酶(cholineacetyltransferase, ChAT)做细胞免疫组化鉴定细胞特性.细胞培养成功后,收集细胞,在小鼠脑立体定位仪的固定下,参照小鼠脑立体定位图谱对APP转基因小鼠施行脑内注射.移植1周以后灌注取移植鼠脑组织行冰冻切片,在共聚焦显微镜下通过外源细胞的绿色自发荧光观察移植细胞的存活和迁移情况.结果 在移植位点,外源性绿色荧光细胞存活良好并以移植点为中心向外迁移,且呈继续迁移的趋势.结论 新生小鼠海马原代细胞培养以后呈现神经前体细胞的特性并且胆碱能阳性,移植入APP转基因小鼠脑内以后,这些胆碱能阳性的神经前体细胞存活良好并呈现较强迁移能力,为进一步探索该细胞对阿尔茨海默病的治疗可能性提供了依据.     

嗅鞘细胞移植入淀粉样前体蛋白转基因小鼠脑内降低β淀粉样蛋白沉积

目的 观察嗅鞘细胞移植入淀粉样前体蛋白(APP)转基因小鼠脑内后的存活和迁移状况及其对模型β淀粉样蛋白沉积的作用,以进一步探索嗅鞘细胞移植对阿尔茨海默病的治疗效果.方法 取自发绿色荧光蛋白的新生3d龄增强型绿色荧光蛋白(EGFP)转基因小鼠,体外分离培养嗅球嗅鞘细胞,在小鼠脑立体定位仪的固定下,参照AP小鼠脑立体定位图谱,将收集的嗅鞘细胞移植入APP转基因小鼠脑内.移植1个月后,取脑组织冷冻切片,在双光子共焦显微镜下观察细胞存活和迁移状况并采集图像.移植1个月后,行荧光免疫组织化学和免疫印迹法检测淀粉样蛋白沉积,每组4只.结果 嗅鞘细胞移植入APP转基因小鼠脑内存活良好,并以移植点为中心向外迁移,部分绿色荧光细胞呈现成血管现象,移植之后APP转基因小鼠脑内的β淀粉样蛋白沉积减少,APP表达明显减少(P<0.05).结论 嗅鞘细胞对减少APP转基因小鼠脑内淀粉样蛋白沉积有一定帮助.     

Huperzine A regulates amyloid precursor protein processing via protein kinase C and mitogen-activated protein kinase pathways in neuroblastoma SK-N-SH cells over-expressing wild type human amyloid precursor protein 695

Alpha-secretase (alpha-secretase), cleaves the amyloid precursor protein (APP) within the amyloid-beta (Abeta) sequence, resulting in the release of a secreted fragment of APP (alphaAPPs) and precluding Abeta generation. We investigated the effects of the acetylcholinesterase inhibitor, huperzine A (Hup A), on APP processing and Abeta generation in human neuroblastoma SK-N-SH cells overexpressing wild-type human APP695. Hup A dose-dependently (0-10 microM) increased alphaAPPs release. Therefore, we evaluated two alpha-secretase candidates, a disintegrin and metalloprotease (ADAM) 10 and ADAM17 in Hup A-induced non-amyloidogenic APP metabolism. Hup A enhanced the level of ADAM10, and the inhibitor of tumor necrosis factor-alpha converting enzyme (TACE)/ADAM17 inhibited the Hup A-induced rise in alphaAPPs levels, further suggesting Hup A directed APP metabolism toward the non-amyloidogenic alpha-secretase pathway. Hup A had no effect on Abeta generation in this cell line. The steady-state levels of full-length APP and cell viability were unaffected by Hup A. Alpha-APPs release induced by Hup A treatment was significantly reduced by muscarinic acetylcholine receptor antagonists (particularly by an M1 antagonist), protein kinase C (PKC) inhibitors, GF109203X and calphostin C, and the mitogen-activated kinase kinase (MEK) inhibitors, U0126 and PD98059. Furthermore, Hup A markedly increased the phosphorylation of p44/p42 mitogen-activated protein (MAP) kinase, which was blocked by treatment with U0126 and PD98059. In addition, Hup A inhibited acetylcholinesterase activity by 20% in neuroblastoma cells. Our results indicate that the activation of muscarinic acetylcholine receptors, PKC and MAP kinase may be involved in Hup A-induced alphaAPPs secretion in neuroblastoma cells and suggest multiple pharmacological mechanisms of Hup A regarding the treatment of Alzheimer's disease (AD).     

L-3-n-butylphthalide regulates amyloid precursor protein processing by PKC and MAPK pathways in SK-N-SH cells over-expressing wild type human APP695

Amyloid precursor protein (APP) is cleaved by α-secretase, within the amyloid-β (Aβ) sequence, resulting in the release of a secreted fragment (αAPPs) and precluding Aβ production. We investigated the effects of a promising anti-AD new drug, l-3-n-butylphthalide (L-NBP), on APP processing and Aβ generation in neuroblastoma SK-N-SH cells overexpressing wild-type human APP695. L-NBP significantly increased αAPPs release, and reduced Aβ generation. The steady-state full-length APP levels were unaffected by L-NBP. It suggested that L-NBP regulated APP processing towards to the non-amyloidogenic α-secretase pathway. Protein kinase C (PKC) and mitogen activated protein (MAP) kinase might be involved in L-NBP-induced αAPPs secretion. L-NBP significantly increased PKCα and ? activations, lowered PKCγ activation and increased the phosphorylation of p44/p42 MAPK. Furthermore, PKC and MAPK inhibitors partially reduced L-NBP-induced αAPPs secretion. The results suggested alternative pharmacological mechanisms of L-NBP regarding the treatment of Alzheimer's disease (AD).     

Investigation of an amyloid precursor protein protective mutation (A673T) in a North American case-control sample of late-onset Alzheimer's disease

A rare amyloid precursor protein gene variant, A673T (rs63750847) was recently reported to protect against Alzheimer's disease and age-related cognitive decline among Icelanders and the same rare variant was observed also in Finnish, Norwegian, and Swedish populations. We investigated this variant in 1674 late-onset Alzheimer's disease cases and 2644 elderly control subjects, all North American Whites (US Whites). We did not observe any example of the A673T variant in our large sample. Our findings suggest that this rare variant could be specific to the individuals of the origin from the Nordic countries.     

细胞外信号调节激酶在TPPB促进PC12产生可溶性淀粉样前体蛋白中的作用

目的：观察在蛋白激酶C(PKC)激动剂TPPB促进可溶性淀粉样前体蛋白(sAPPα)释放过程中参与的信号转导通路。方法:以1 μmol/L的TPPB作用于PC12细胞3 h，同时加入信号转导通路的抑制剂，Western印迹法检测上清液内sAPPα的含量和细胞外信号调节激酶(p42/44MAPK)及磷酸化的p42/44MAPK的表达。结果:1 μmol/L的TPPB作用于PC12细胞3 h可以显著增加上清液内sAPPα的含量，细胞外信号调节激酶抑制剂U0126、c-Jun氨基末端激酶抑制剂SP600125和蛋白酪氨酸激酶抑制剂genistein可以部分消除此作用；而p38MAPK抑制剂SB203580对sAPPα的含量无显著影响。1 μmol/L的TPPB可以使磷酸化的p42/44MAPK表达增加，而对总的p42/44MAPK无显著影响。结论:细胞外信号调节激酶、c-Jun氨基末端激酶和蛋白酪氨酸激酶可能参与TPPB促进sAPPα生成的过程。     

Aging and excitotoxic stress exacerbate neural circuit reorganization in amyloid precursor protein intracellular domain transgenic mice

The cleavage of amyloid precursor protein (APP) by presenilins simultaneously generates amyloid-β (Aβ) and APP intracellular Domain (AICD) peptides. Aβ plays a pivotal role in Alzheimer's disease (AD) pathology and recently AICD was also shown to contribute to AD. Transgenic mice overexpressing AICD show age-dependent tau phosphorylation and aggregation, memory deficits, and neurodegeneration. Moreover, these mice show aberrant electrical activity and silent seizures beginning at 3–4 months of age. Here we show that AICD mice also displayed abnormal mossy fiber sprouting beginning about the same time and that this sprouting intensified as the animals aged. Expression of neuropeptide Y was increased in mossy fiber terminals in aged but not young AICD mice. Importantly, young AICD mice injected with kainic acid showed similar pathology to that observed in aged AICD mice. These data show that elevated levels of AICD render neurons hypersensitive to stress and induce hippocampal circuit reorganization, which can further exacerbate hyperexcitability. These results further demonstrate that AICD, in addition to Aβ, can play a significant role in AD pathogenesis.     

The intracellular domain of amyloid precursor protein induces neuron-specific apoptosis

Sex differences are often reported in spatial abilities. However, some studies show conflicting results, which can be ascribed to the complexity of the variables involved in the visuo-spatial domain. Until a few years ago, it was widely accepted that men outperformed women on almost all spatial tasks. However, recently some studies [A. Postma, G. Jager, R.P.C. Kessels, H.P.F. Koppeschaar, J. van Honk, Sex differences for selective forms of spatial memory, Brain Cogn. 54 (2004) 24-34; D.H. McBurney, S.J.C. Gaulin, T. Devineni, C. Adams, Superior spatial memory of women: stronger evidence for the gathering hypothesis, Evol. Hum. Behav. 18 (1997) 165-174; Q. Rahman, G.D. Wilson, S. Abrahams, Sexual orientation related differences in spatial memory, J. Int. Neuropsychol. Soc. 9 (2003) 376-383] found sex differences for selective forms of spatial memory and described a female advantage in specific spatial abilities. In this paper, we studied sex differences by testing object locations and route memories with the Corsi Block-Tapping test (CBT), one of the non-verbal tasks most used in clinical settings, and its modified, large-scale version. Our results showed a performance advantage for males in both tests and a more homogeneous pattern of memory in females.     

海马sortilin在链脲佐菌素诱导的糖尿病认知损伤小鼠中的作用

目的 探讨海马sortilin在链脲佐菌素（STZ）诱导的糖尿病认知损伤小鼠中的作用。方法 24只成年雄性ICR小鼠,随机分为溶媒对照组（NS）和实验组（STZ）。STZ腹腔注射诱导糖尿病认知损伤动物模型,注射后用血糖仪检测血糖改变,注射后第8周采用新旧事物识别实验检测各组小鼠认知功能;免疫组织化学方法检测sortilin免疫阳性产物表达变化;Western blotting和Real-time PCR方法检测各组小鼠海马sortilin和脑源性神经营养因子（BDNF）蛋白及mRNA表达变化。结果 与NS组相比,STZ组小鼠空腹血糖显著增高（P<0.01）;在新旧事物识别实验中新事物辨别指数明显降低（P<0.05）;海马区sortilin的免疫阳性产物（P<0.05）、蛋白（P<0.01）以及mRNA（P<0.05）表达均显著下调;海马区BDNF mRNA（P<0.01）及蛋白（P<0.05）表达均明显降低。结论 STZ诱导的糖尿病小鼠认知损伤可能与海马sortilin的表达下调有关。     

Antibody binding to cell surface amyloid precursor protein induces neuronal injury by deregulating the phosphorylation of focal adhesion signaling related proteins

The biological function of full-length amyloid-β protein precursor (APP), the precursor of Aβ, is not fully understood. Mounting studies reported that antibody binding to cell surface APP causes neuronal injury. However, the mechanism of cell surface APP mediating neuronal injury remains to be determined. Colocalization of APP with integrin on cell surface leads us to suppose that focal adhesion (FA) related mechanism is involved in surface APP-mediated neuronal injury. In the present study, results demonstrated that primary cultured neurons treated with antibody against APP-N-terminal not only caused neuronal injury and aberrant morphologic changes of neurite, but also induced reaction of FA proteins appearing an acute increase then decrease pattern. Moreover, the elevation of tyrosine phosphorylation of FA proteins including paxillin and focal adhesion kinase (FAK), and down-regulated expression of protein tyrosine phosphatase (PTP1B) induced by APP antibody were prevented by inhibitor of Src protein kinases 4-amino-5-(4-chlorophenyl)-7(t-butyl) pyrazol (3,4-D) pyramide (PP2) and G protein inhibitor pertussis toxin (PTX), implying that Src family kinase and G protein play roles in APP-induced FA signals. In addition, pretreatment with PTX and PP2 was able to suppress APP-antibody induced neuronal injury. Taken together, the results suggest a novel mechanism for APP mediating neuronal injury through deregulating FA signals.     

Calcium regulates the interaction of amyloid precursor protein with Homer3 protein

Ca²⁺ dysregulation is an important factor implicated in Alzheimer's disease pathogenesis. The mechanisms mediating the reciprocal regulation of Ca²⁺ homeostasis and amyloid precursor protein (APP) metabolism, function, and protein interactions are not well known. We have previously shown that APP interacts with Homer proteins, which inhibit APP processing toward amyloid-β. In this study, we investigated the effect of Ca²⁺ homeostasis alterations on APP/Homer3 interaction. Influx of extracellular Ca²⁺ upon treatment of HEK293 cells with the ionophore A23187 or addition of extracellular Ca²⁺ in cells starved of calcium specifically reduced APP/Homer3 but not APP/X11a interaction. Endoplasmic reticulum Ca²⁺ store depletion by thapsigargin followed by store-operated calcium entry also decreased the interaction. Interestingly, application of a phospholipase C stimulator, which causes inositol 1,4,5-trisphosphate-induced endoplasmic reticulum Ca²⁺ release, caused dissociation of APP/Homer3 complex. In human neuroblastoma cells, membrane depolarization also disrupted the interaction. This is the first study showing that changes in Ca²⁺ homeostasis affect APP protein interactions. Our results suggest that Ca²⁺ and Homers play a significant role in the development of Alzheimer's disease pathology.