大鼠海马tau蛋白过度磷酸化时细胞周期蛋白cyclin D1的表达

目的:探讨大鼠海马tau蛋白过度磷酸化时细胞周期蛋白cyclin D1的表达.方法:用cAMP-依赖性蛋白激酶 A (PKA) 的激动剂Forskolin注射大鼠侧脑室,免疫印迹和免疫组织化学技术检测大鼠海马tau蛋白的磷酸化水平,同时采用免疫印迹、免疫组织化学和RT-PCR方法检测海马cyclin D1和cyclin D1 mRNA.结果:侧脑室注射Forskolin 48h,大鼠海马tau蛋白Ser214、Ser396和Ser202/Thr205位点的磷酸化水平升高,同时检测到细胞周期蛋白cyclin D1和cyclin D1 mRNA.结论:侧脑室注射Forskolin诱导大鼠海马 tau蛋白过度磷酸化的同时,大鼠海马出现细胞周期蛋白cyclin D1的表达,提示了tau蛋白过度磷酸化与细胞周期相关蛋白cyclin D1之间可能的内在联系.     

神经生长因子与降钙素基因相关肽减轻局灶性脑缺血再灌注大鼠海马tau蛋白磷酸化

为探讨外源性神经生长因子(NGF)和降钙素基因相关肽(CGRP)对局灶性脑缺血再灌注大鼠海马神经元tau蛋白过度磷酸化的影响,用线栓法制作局灶性脑缺血再灌注模型,应用免疫组织化学SABC法、Western Blotting和图像分析方法检测大鼠海马tau蛋白在Ser199/202位点磷酸化程度和总tau蛋白表达,以及NGF与CGRP对tau蛋白过度磷酸化的影响。结果显示:缺血再灌注组同侧海马tau蛋白在Ser199/202位点磷酸化水平和总tau蛋白明显升高(P<0.05);NGF组及CGRP组大鼠海马tau蛋白在Ser199/202位点磷酸化水平明显低于缺血再灌注组,总tau也下降(P<0.05);NGF与CGRP合用组海马tau蛋白磷酸化水平进一步降低,分别低于NGF组和CGRP组(P<0.05)。以上结果表明NGF及CGRP明显减轻局灶性脑缺血再灌注大鼠海马tau蛋白磷酸化程度,二者合用作用更强,降低缺血神经元tau蛋白磷酸化水平可能对缺血神经元起保护作用。     

降钙素基因相关肽对局灶性脑缺血再灌注大鼠海马tau磷酸化影响

目的探讨降钙素基因相关肽(CGRP)对局灶性脑缺血再灌注大鼠海马神经元tau蛋白过度磷酸化的影响。方法应用免疫组织化学SABC法、Western Blotting和图像分析方法检测局灶性脑缺血再灌注大鼠海马CA1区tau蛋白在Ser199/202位点磷酸化程度和总tau蛋白表达、以及CGRP对缺血神经元tau蛋白过度磷酸化的影响。结果缺血再灌注大鼠海马CA1区tau蛋白在Ser199/202位点磷酸化水平和总tau表达水平明显增高,CGRP治疗后tau蛋白磷酸化水平和总tau表达水平明显低于缺血再灌注组(P<0.05)。结论CGRP明显减轻局灶性脑缺血再灌注大鼠海马tau蛋白磷酸化程度和总tau表达水平,可能对缺血神经元起保护作用。     

褪黑素对异丙肾上腺素诱导的大鼠海马tau蛋白过度磷酸化和空间记忆损伤的保护作用（英文）

目的：在大鼠整体研究褪黑素对异丙肾上腺素（IP）诱导的tau过度磷酸化和动物保留记忆损伤的保护作用。方法：大鼠海马双侧注射IP或同时注射褪黑素（0.1 μg，1μg），利用免疫组化、免疫印迹和Morris水迷宫等指标，观察褪黑素的效果。结果：0.1 μg和1 μg剂量的褪黑素明显降低IP引起的海马骨架蛋白Tau Ser396/404位点的过度磷酸化；1 μg褪黑素可明显改善IP引起的动物保留记忆损伤。结论：褪黑素对大鼠学习记忆和海马骨架蛋白Tau有一定的保护作用。     

神经生长因子及降钙素基因相关肽减轻局灶性脑缺血再灌注大鼠顶叶皮质tau蛋白磷酸化

目的:探讨外源性神经生长凶子(NGF)和降钙素基因相关肽(CGRP)对局灶性脑缺血再灌注大鼠顶叶皮质神经元tau蛋白过度磷酸化的影响.方法:制作局灶性脑缺血再灌注模型,应用免疫组织化学SABC法、免疫印迹法和图像分析方法检测大鼠顶叶皮质tau蛋白在Ser199/202位点磷酸化程度和总tau蛋白表达,以及NGF与CGRP对tau蛋白过度磷酸化的影响.结果:缺血再灌注组同侧顶叶皮质tau蛋白在Scr199'202位点磷酸化水平和总tau蛋白明显升高;NGF组及CGRP组大鼠顶叶皮质tau蛋白在scr199'202位点磷酸化水平明显低于缺血再灌注组,总tau蛋白也下降;NGF与CGRP合用组顶叶皮质tau蛋白磷酸化水平进一步降低,分别低于NGF组和CGRP组.结论:NGF及CGRP明显减轻脑缺血再灌注大鼠脑tau蛋白磷酸化程度.两者合用作用更强.     

冈田酸诱导大鼠海马神经元Tau 蛋白过度磷酸化

目的研究蛋白磷酸酯酶抑制剂冈田酸(OA)对海马神经元微管相关蛋白(Tau)磷酸化的影响,建立Tau过度磷酸化的大鼠模型。方法实验随机分为正常组、二甲基亚砜(DMSO)对照组、OA模型组。模型组又分为OA12h、24h、48h和2周组。OA模型组大鼠海马CA1区背侧定向注射1.5μl溶于10%DMSO的OA,DMSO对照组注射1.5μl10%DMSO溶液。通过Bielschowski染色、免疫组织化学染色、蛋白免疫印迹分别观察海马神经元形态的改变和磷酸化Tau的表达水平;检测蛋白磷酸酶2A(PP2A)的活性,了解其动态变化与Tau磷酸化的关系。结果OA模型各组与正常组和DMSO对照组比较,Bielschowski染色示海马神经元胞体和突起着色较深,欠均匀,部分神经元轴丘处浓染成斑块状,但各模型组均未见到老年斑和神经元纤维缠结样改变;免疫组织化学染色示模型组海马神经元Thr231和Ser199202磷酸化Tau蛋白表达增加,与DMSO对照组相比具有显著意义(P<0.05);蛋白免疫印迹提示OA可引起Tau蛋白Thr231、Ser396和Ser199/202位点发生磷酸化,且不同位点磷酸化的稳定性不同,注射OA48h后PP2A的活性明显降低,其变化与Tau蛋白Thr231和Ser396位点的磷酸化改变相一致。结论海马CA1区背侧单次注射OA可诱导建立神经元Tau蛋白过度磷酸化的大鼠模型。     

重组腺相关病毒介导的VLDLR基因转移改善2型糖尿病大鼠大脑海马tau蛋白Alzheimer病样改变

目的:Tau蛋白过度磷酸化是Alzheimer病(AD)的重要特征,在2型糖尿病中tau蛋白呈AD样过度磷酸化改变。本研究运用极低密度脂蛋白受体(VLDLR)基因转移干预2型糖尿病大鼠(T2DM),观察海马tau蛋白磷酸化修饰水平的变化。方法:将8周龄雄性Wistar大鼠随机分为3组,正常对照组(CTL)以普通饮食喂养,2型糖尿病组(T2DM)及2型糖尿病处理组(T2DM+VLDLR)。葡萄糖氧化酶法检测血浆血糖,放免法检测血浆胰岛素,Western blotting分析海马内总tau、tau蛋白上部分位点磷酸化及VLDLR水平。[γ-32P]标记的ATP和特异性底物肽检测海马内胰岛素信号转导系统中的关键酶糖原合成酶激酶-3β(GSK-3β)活性。结果:T2DM组大鼠经VLDLR基因处理之后(T2DM+VLDLR)与对照组(CTL)海马回总tau蛋白水平无差异,T2DM组tau(pS214)、tau(pS396)、tau(pS422)及tau(pSpS199/202)位点上的磷酸化水平显著高于CTL组,而在tau-1位点免疫反应显著弱于CTL组;运用VLDLR基因处理之后,tau蛋白上tau(pT217)、tau(pS396)及tau(pSpS199/202)位点磷酸化水平显著下降,tau-1位点显著增强,但tau(pS214)及tau(pS422)位点磷酸化水平无显著下降。免疫组化结果检测到T2DM组大鼠海马区tau蛋白在Ser214位点上磷酸化程度较CTL组显著增高,运用VLDLR基因处理之后,tau蛋白磷酸化程度逆转不明显。3组大鼠海马细胞内总GSK-3β无显著差异,但T2DM组GSK-3β磷酸化程度显著下降,运用VLDLR基因处理后GSK-3β磷酸化程度显著上升。结论:重组腺相关病毒介导的VLDLR基因处理可能是通过抑制GSK-3β活性来改善2型糖尿病大鼠海马tau蛋白上部分位点的Alzheimer病样过度磷酸化状态。     

半枝莲黄酮抑制复合Aβ所致大鼠皮层细胞NFT沉积及其调节机制

目的:探讨半枝莲黄酮(SBF)对复合Aβ,即β淀粉样蛋白25-35(Aβ25-35)联合三氯化铝(AlCl_3)和重组人类转化生长因子-β1(RHTGF-β1)所致大鼠脑内神经元纤维缠结(NFT)沉积,tau蛋白磷酸化的影响及糖原合成激酶(GSK)3β和蛋白磷酸酶(PP)2A的调节机制。方法:雄性SD大鼠,脑室注射复合Aβ建立拟阿尔茨海默病(AD)大鼠记忆障碍模型,Morris水迷宫进行记忆障碍模型筛选,模型成功大鼠灌胃35、70和140 mg/kg的SBF和140 mg/kg的阳性对照药银杏叶黄酮(GLF),持续37 d。硝酸银法测定大鼠大脑皮层的NFT,Western blot法检测大鼠海马、皮层中总tau蛋白、Ser199和Ser214位点磷酸化的tau蛋白水平以及相关GSK3β和PP2A蛋白的表达水平。RT-PCR法检测海马、皮层中GSK3β和PP2A的mRNA水平。结果:大鼠脑室注射复合Aβ可以引起大鼠脑内NFT生成增加、Ser199和Ser214位点磷酸化tau蛋白、GSK3β蛋白和mRNA表达水平皆明显增加,PP2A的蛋白和mRNA表达水平明显降低。3种剂量的SBF灌胃37 d不同程度地逆转复合Aβ所致大鼠脑内上述异常改变。GLF也表现出与SBF相似的结果。结论:SBF能够抑制复合Aβ所致大鼠脑内NFT沉积,该作用可能是通过抑制GSK3β活性、增加PP2A活性从而降低tau蛋白磷酸化而实现的。     

在体转染GSK-3β引起tau蛋白在PHF-1位点的过度磷酸化

目的：探讨在体转染GSK-3β对tau蛋白在PHF-1位点磷酸化的影响。 方法： 21只大鼠随机分为GSK-3β转染组、空载体组和空白对照组3组：0.1 μg/3 μL GSK-3β-HA质粒和空载体分别注射入大鼠大脑，对照组大鼠不作处理，应用免疫印迹和免疫组织化学检测GSK-3β的表达，并应用磷酸化位点特异性抗体PHF-1检测tau蛋白的磷酸化水平。 结果： 转染48 h后，GSK-3β-HA表达在转染组；并且在转染区域的神经元内异常过度磷酸化tau蛋白（在PHF-1表位）聚积；异常过度磷酸化的tau蛋白与GSK-3β共定位。 结论： 在体转染GSK-3β引起导致神经退行性疾病发生机制相关的tau蛋白异常过度磷酸化，这进一步证明了GSK-3β是tau蛋白异常过度磷酸化的一个关键激酶，并且可作为一个防治与tau相关的神经退行性疾病的靶点。     

远志皂苷减轻Aβ1-40诱导的AD大鼠脑神经元tau蛋白Ser396位点的过度磷酸化

目的:探讨远志皂苷对β-淀粉样肽_1-40(Aβ_1-40)诱导的阿尔茨海默病(AD)大鼠脑神经元tau蛋白过度磷酸化的影响。方法:大鼠右侧海马CA1区注射Aβ_1-40建立AD模型,并用远志皂苷(18.5 mg/kg、37.0 mg/kg和74.0 mg/kg)对大鼠进行灌胃治疗;免疫组织化学染色法观察大脑神经元中总tau蛋白、p-tau(Ser³⁹⁶)、蛋白激酶A(PKA)和蛋白磷酸酶2A(PP2A)蛋白的表达;蛋白免疫印迹技术检测大脑神经元中总tau蛋白含量、tau蛋白Ser³⁹⁶位点磷酸化以及PKA、PP2A蛋白的表达水平。结果:与对照组相比,Aβ_1-40组大脑神经元中总tau蛋白含量、tau蛋白Ser³⁹⁶位点磷酸化水平和PKA蛋白的表达水平显著升高,而PP2A蛋白的表达水平明显降低。与Aβ_1-40组相比,远志皂苷各治疗组大鼠大脑神经元中总tau蛋白含量、tau蛋白Ser³⁹⁶位点磷酸化水平和PKA蛋白表达水平下降明显,而PP2A蛋白表达水平显著升高。结论:远志皂苷可能是通过下调PKA蛋白表达量,上调PP2A蛋白表达量,减轻AD大鼠脑神经元中tau蛋白Ser³⁹⁶位点的过度磷酸化,使神经细胞免遭Aβ_1-40的毒害。     

Nicotine modifies in vivo and in vitro rat hippocampal amyloid precursor protein processing in young but not old rats

Abnormally hyperphosphorylated microtubule-associated protein tau is the main component of the neurofibrillary tangles (NFT), a hallmark pathological feature of Alzheimer's disease (AD). A lot of studies suggested that there is highly neurobiological correlation between olfactory dysfunction and AD-like pathology, but the effect of the odor stimulation on tau phosphorylation remains unknown. Here, we examined the effect of short-term and long-term enriched odor exposure on the alterations of tau phosphorylation at multiple sites in the rat brains. We found that short-term odor enrichment did not affect the phosphorylation of tau, while long-term odor enrichment dramatically reduce the phosphorylation level of tau at Ser198/199/202, Thr231, Ser396, and Ser404 sites both in the hippocampus and cortex. These data suggest that long-term odor exposure prevent tau phosphorylation and may be a new therapeutic strategy of AD.     

Dynamic association of tau with neuronal membranes is regulated by phosphorylation

Tau is an abundant cytosolic protein which regulates cytoskeletal stability by associating with microtubules in a phosphorylation-dependent manner. We have found a significant proportion of tau is located in the membrane fraction of rat cortical neurons and is dephosphorylated, at least at Tau-1 (Ser199/Ser202), AT8 (Ser199/Ser202/Thr205) and PHF-1 (Ser396/Ser404) epitopes. Inhibition of tau kinases casein kinase 1 (CK1) or glycogen synthase kinase-3 decreased tau phosphorylation and significantly increased amounts of tau in the membrane fraction. Mutation of serine/threonine residues to glutamate to mimic phosphorylation in the N-terminal, but not C-terminal, region of tau prevented its membrane localization in transfected cells, demonstrating that the phosphorylation state of tau directly impacts its localization. Inhibiting CK1 in neurons lacking the tyrosine kinase fyn also induced tau dephosphorylation but did not affect its membrane association. Furthermore, inhibition of CK1 increased binding of neuronal tau to the fyn-SH3 domain. We conclude that trafficking of tau between the cytosol and the neuronal membrane is dynamically regulated by tau phosphorylation through a mechanism dependent on fyn expression.     

Cleavage and conformational changes of tau protein follow phosphorylation during Alzheimer's disease

Phosphorylation, cleavage and conformational changes in tau protein all play pivotal roles during Alzheimer's disease (AD). In an effort to determine the chronological sequence of these changes, in this study, using confocal microscopy, we compared phosphorylation at several sites (Ser(199/202/396/404/422)-Thr(205) and the second repeat domain), cleavage of tau (D(421)) and the canonical conformational Alz-50 epitope. While all of these posttranslational modifications are found in neurofibrillary tangles (NFTs) at all stages of the disease, we found significantly higher numbers of phospho-tau positive NFTs when compared with cleaved tau (P = 0.006 in Braak III; P = 0.002 in Braak IV; P = 0.012 in Braak V) or compared with the Alz-50 epitope (P < 0.05). Consistent with these findings, in a double transgenic mice model (Tet/GSK-3beta/VLW) overexpressing the enzyme glycogen synthase kinase-3beta (GSK-3beta) and tau with a triple FTDP-17 mutation (VLW) with AD-like neurodegeneration, phosphorylation at sites Ser(199/202)-Thr(205) was greater than truncated tau. Taken together, these data strongly support the notion that the conformational changes and truncation of tau occur after the phosphorylation of tau. We propose two probable pathways for the pathological processing of tau protein during AD, either phosphorylation and cleavage of tau followed by the Alz-50 conformational change or phosphorylation followed by the conformational change and cleavage as the last step.     

LPS- induced inflammation exacerbates phospho-tau pathology in rTg4510 mice

Inflammation and microglial activation are associated with Alzheimer's disease (AD) pathology. Somewhat surprisingly, injection of a prototypical inflammatory agent, lipopolysaccharide (LPS) into brains of amyloid precursor protein (APP) transgenic mice clears some of the pre-existing amyloid deposits. It is less well understood how brain inflammation modulates tau pathology in the absence of Aβ. These studies examined the role of LPS-induced inflammation on tau pathology. We used transgenic rTg4510 mice, which express the P301L mutation (4R0N TauP301L) and initiate tau pathology between 3-5 months of age. First, we found an age-dependent increase in several markers of microglial activation as these rTg4510 mice aged and tau tangles accumulated. LPS injections into the frontal cortex and hippocampus induced significant activation of CD45 and arginase 1 in rTg4510 and non-transgenic mice. In addition, activation of YM1 by LPS was exaggerated in transgenic mice relative to non-transgenic animals. Expression of Ser199/202 and phospho-tau Ser396 was increased in rTg4510 mice that received LPS compared to vehicle injections. However, the numbers of silver-positive neurons, implying presence of more pre- and mature tangles, was not significantly affected by LPS administration. These data suggest that inflammatory stimuli can facilitate tau phosphorylation. Coupled with prior results demonstrating clearance of Aβ by similar LPS injections, these results suggest that brain inflammation may have opposing effects on amyloid and tau pathology, possibly explaining the failures (to date) of anti-inflammatory therapies in AD patients.     

叶酸和维生素B12减轻高同型半胱氨酸血症诱导的老年大鼠阿尔茨海默样病变

目的:探讨慢性高同型半胱氨酸血症对老年大鼠脑内tau蛋白结构和功能的影响以及其可能机制。方法:选取18月龄健康大鼠,尾静脉注射同型半胱氨酸(Hcy)生理盐水溶液(1.6mg·kg^-1·d^-1)制造高同型半胱氨酸血症模型组,在给予Hcy的同时通过饮用水给予叶酸(4mg·kg^-1·d^-1)和维生素B₁₂(VitB₁₂,250g·kg^-1·d^-1)作为治疗组,单纯注射生理盐水作为对照组,持续28周,Bielschowsky银染观察磷酸化tau蛋白的分布和聚集;Westernblotting检测磷酸化tau蛋白、糖原合酶激酶3β(GSK-3β)和蛋白磷酸酶2A(PP2A)的表达。结果:(1)血浆中Hcy水平增高导致神经原纤维样缠结;给予叶酸和VitB₁₂能够有效减轻缠结;(2)血浆高Hcy水平诱导老年大鼠脑内tau蛋白在Ser^199/202和Ser³⁹⁶位点磷酸化水平升高;(3)血浆高Hcy水平激活GSK-3β同时抑制PP2A的活性。补充叶酸和VitB₁₂有效地缓解了这些激酶和酯酶的活性改变,并下调了tau蛋白Ser^199/202和Ser³⁹⁶位点的磷酸化水平。结论:补充叶酸和VitB₁₂能够有效改善高同型半胱氨酸血症所诱导的tau蛋白阿尔茨海默样病理改变,其机制可能与其抑制GSK-3β活性和激活PP2A有关。     

小檗碱对冈田酸拟AD大鼠空间学习记忆的改善作用及其机制

目的:探讨小檗碱(berberine,Ber)对冈田酸拟阿尔茨海默病(Alzheimer’s disease,AD)大鼠学习记忆的改善作用及其可能机制。方法:36只雄性SD大鼠随机分为对照组、模型组和Ber组,用侧脑室微量注射冈田酸(okadaic acid,OA,0.4 mmol/L,1.5μl/次,共3次)建立AD模型,Ber组是在模型制作前后灌胃(100mg/kg/d,共4周);采用Morris水迷宫测试大鼠的空间学习记忆能力,用Bieischowsky镀银改良法观察脑内神经原纤维缠结(neurofibrillary tangle,NFTs)的变化;用免疫组织化学方法检测pTau-Ser396和pTau-Ser404蛋白的表达。结果:与对照组相比,模型组大鼠各时间段的逃避潜伏期均延长,脑内出现较多NFTs,皮层和海马神经元pTau-Ser396/404的表达明显增高(P<0.01);Ber组大鼠潜伏期较模型组明显缩短,脑内NFTs的数量减少,pTau-Ser396/404的表达明显下调(P<0.01,P<0.05)。结论:小檗碱可明显改善AD模型大鼠空间学习记忆障碍,其机制可能与降低Tau磷酸化水平,减少NFTs有关。     

Inhibition of protein phosphatase 2A- and protein phosphatase 1-induced tau hyperphosphorylation and impairment of spatial memory retention in rats

Tau hyperphosphorylation leads to formation of paired helical filament/neurofibrillary tangles, the hallmark lesion seen in Alzheimer's disease (AD) brain. An imbalanced regulation in protein kinases and protein phosphatases in the affected neurons is proposed to be a reasonable causative factor to the disease process. To verify the hypothesis, we have injected in the present study calyculin A, a potent and specific inhibitor of protein phosphatase (PP) 2A and PP1, into rat hippocampus bilaterally, thus reproduced an Alzheimer's-like deficiency in dephosphorylation system. It was found that calyculin A-injected rats developed lesions in spatial memory retention in Morris water maze test. At mean time, tau was hyperphosphorylated at Ser396/Ser404 (PHF-1) and Ser-262/Ser-356 (12E8) sites determined both by immunohistochemistry and Western blot. It is implicated that (1) PP2A and PP1 participate in the in vivo regulation of tau phosphorylation, and down-regulation of the two phosphatases will result in tau hyperphosphorylation; (2) hyperphosphorylation of tau at PHF-1 and 12E8 sites might be crucial to affect spatial memory in AD.