阿尔茨海默病脑脊液磷酸化神经丝及双螺旋丝免疫活性

目的探讨阿尔茨海默病（Ａｌｚｈｅｉｍｅｒｓｄｉｓｅａｓｅ,ＡＤ）患者脑脊液（ＣＳＦ）磷酸化神经丝（ＰＮＦ）和双螺旋丝（ＰＨＦ）免疫活性的变化及临床意义。方法采用竞争抑制型ＥＬＩＳＡ检测ＡＤ的ＣＳＦ中ＰＮＦ和ＰＨＦ的免疫活性,并与对照组即多发梗死性痴呆（ＭＩＤ）、其他神经疾病（ＯＤＣ）、精神分裂症（ＳＣＨ）及正常人（ＮＣ）进行比较。结果ＡＤ组ＣＳＦ中ＰＮＦ活性与ＭＩＤ、ＮＣ、ＳＣＨ无显著性差异。ＡＤ组ＣＳＦ中ＰＨＦ活性高于各对照组,而其ＰＮＦ／ＰＨＦ比值低于各对照组,有显著性意义。ＰＨＦ值与对照组个体间重叠的４例ＡＤ中仅１例其ＰＮＦ／ＰＨＦ值与相应的ＭＩＤ有重叠。结论单纯测定ＣＳＦ中ＰＮＦ免疫活性对ＡＤ的鉴别诊断无意义,测定ＣＳＦ中ＰＨＦ活性与ＰＮＦ／ＰＨＦ比值相结合可提高ＡＤ的诊断正确率,有可能发展为有临床意义的实验室诊断试验。     

ELISA测定Alzheimer病脑脊液磷酸化神经丝和双螺旋丝抗原活性

应用160～200KD磷酸化神经丝(PNF)和双螺旋丝(PHF)单克隆抗体建立竞争抑制ELISA。并检测了21例Alzheimer病(AD)、17例多发梗塞性痴呆、33例其他神经疾病、33例精神分裂症及40例正常人的CSF中PNF和PHF抗原活性。结果AD组CSF中PHF抗原活性明显高于其他各组;而其PNF/PHF比值明显低于其他各组。本法有可能成为诊断AD的实验室方法。     

老年阿尔茨海默病患者尿液中神经丝蛋白含量的研究

目的探讨尿液中阿尔茨海默病(AD)相关神经丝蛋白(AD7c-NTP)含量在老年AD诊断中的意义。方法选择老年AD患者67例(AD组)和认知功能正常老年人(对照组)85名,应用简易精神状态检查(MMSE)量表、日常生活活动(ADL)量表和临床痴呆评定(CDR)量表进行精神状态评价,采用酶联免疫吸附测定(ELISA)法检测尿AD7c-NTP含量。结果尿AD7c-NTP含量AD组为(2.34±1.59)μg·L-1,对照组为(1.04±0.39)μg·L-1,两组间差异有统计学意义(t=-6.59,P=0.000)。经相关性分析表明AD7c-NTP水平与MMSE、FT4值呈负相关(R=-0.689,P=0.000;R=-0.211,P=0.009);与ADL、CDR值呈正相关(R=0.267,P=0.001;R=0.52,P=0.001)。根据ROC曲线,确定1.492μg·L-1为界定值,其对应的敏感度和特异度分别为82.1%和95.3%,ROC曲线下面积为0.924,95%CI:0.88~0.97。结论尿AD7c-NTP含量检测可作为老年AD患者辅助诊断的生物学指标。     

阿尔茨海默病患者神经丝轻链蛋白水平与临床特点相关性分析

目的 探讨阿尔茨海默病患者神经丝轻链蛋白（neurofilament light chain,NfL）与临床特点相关性。方法 分析2020年6月—2022年12月宁波市第二医院收治的58例阿尔茨海默病患者的临床资料为研究对象。检测58例阿尔茨海默病患者的神经丝轻链蛋白水平。Simoa法检测阿尔茨海默病患者神经丝轻链蛋白水平,探索神经丝轻链蛋白表达与临床因素的关联性。结果 58例患者中女性患者33例,NfL水平为（24.93±17.07） pg/ml;男性患者24例,NfL水平为（28.14±17.98）pg/ml,差异无统计学意义。不同文化程度与NfL水平分别为：文盲[（27.87±14.75）pg/ml(7/58)]、小学[（15.22±9.62）pg/ml(7/58)]、初中[（27.95±15.60）pg/ml (14/58)]、高中[（12.84±2.97）pg/ml(4/58)]、中专[（37.35±26.63）pg/ml(7/58)]、大专[（33.79±21.81）pg/ml(5/58)]、大学[（15.22±9.62） pg/ml(7/58)],结果无统计学差异。用相关...     

尿沉渣指标对阿尔茨海默病相关神经丝蛋白检测水平的影响

目的 探讨不同性别人群尿常规指标对阿尔茨海默病相关神经丝蛋白（AD7c-NTP）检测水平的影响。方法 纳入 2021 年 7— 10 月在桂林医学院附属医院进行健康体检的 314 名受试者,采集尿液标本进行 AD7c-NTP 酶联免疫吸附剂测定,收集年龄、性别及尿沉渣检测指标数据,分析不同性别人群的尿沉渣指标对 AD7c-NTP 水平的影响。结果 不同红细胞、白细胞、上皮细胞、小圆上皮细胞、管型、类酵母菌、结晶水平的男性受试者尿液中的 AD7c-NTP 水平比较,差异无统计学意义（P＞ 0.05）;男性受试者低电导率、中电导率和高电导率之间的 AD7c-NTP 水平比较［（0.68（0.48,1.40））ng/ml 比 0.50（0.32,1.06）ng/ml 比 0.34（0.31,0.51）ng/ml］,差异有统计学意义（Z=-3.823,P＜ 0.05）。不同红细胞、白细胞、上皮细胞、小圆上皮细胞、管型、类酵母菌、电导率水平的女性受试者尿液中的 AD7c-NTP 水平比较,差异无统计学意义（P＞ 0.05）;高结晶水平的女性受试者尿液中的 AD7c-NTP 水平低于低结晶水平的女性受试者［0.51（0.33,0.62）ng/ml 比 0.65（0.42,1.18）ng/ml］,差异有统计学意义（Z=-2.331,P＜ 0.05）。结论 男性尿液电导率和女性尿液结晶可能对 AD7c-NTP 检测水平产生影响。     

组蛋白去乙酰化酶6在阿尔茨海默病中的作用

组蛋白去乙酰化酶6(HDAC6)是HDAC家族中Ⅱb类成员之一,具有去乙酰化酶活性和参与细胞内异常蛋白降解的功能。近来研究发现HDAC6参与调节Tau蛋白磷酸化、糖原合成酶激酶3β(GSK3β)影响线粒体运输等有关的生化过程,提示HDAC6可能与AD的发生有关,是治疗AD的潜在靶点。本文论述了HDAC6的结构与功能、HDAC6的选择性抑制剂以及在AD中的作用。     

中重度阿尔茨海默病与血管性痴呆患者脑脊液tau蛋白的预研究

背景：阿尔茨海默病（Alzheimer’s disease,AD）患病率高、医疗费用高、照料困难,已成为老龄化社会面临的严重社会、经济问题。迄今,AD的诊断尚无可靠的客观诊断指标。近年来,国内外对AD患者脑脊液生物学标志物的研究甚多,AD患者脑脊液T-tau、P-tau升高已是不争的事实。但是,这些标志物与痴呆严重程度以及随病程进展的关系还有待进一步深入研究。
　　目标：比较中重度阿尔茨海默病（Alzheimer’s dis-ease,AD）与血管性痴呆（vascular demenita,VD）患者脑脊液（cerebrospinal lfuid,CSF）总tau蛋白（total tau,T-tau）和第231位苏氨酸磷酸化tau蛋白（P-tau231）的基线浓度,随访6个月,观察它们在AD患者与对照组之间的差异以及随病程进展的变化。
　　方法：基线期中度AD患者11例（10≤ MMSE ≤20）,重度AD患者10例（MMSE ≤9）以及年龄匹配的重度VD患者7例,其中7例AD与6例VD完成了6个月的随访。用双抗体夹心ELISA检测脑脊液T-tau, P-tau231的浓度。
　　结果：基线期AD组与VD组患者CSF T-tau的浓度分别为470.08（263.58）pg/mL、208.76(42.24）pg/mL,差异有统计学意义（Z=-3.369,p <0.001）;CSF P-tau231的浓度分别为90.94(49.86）pg/mL、42.96（13.10） pg/mL,差异有统计学意义（Z=-3.237,p<0.001）。重度AD与重度VD相比,CSF T-tau（Z=-2.830,p =0.005）、CSF P-tau231（Z =-2.392,p=0.017）的浓度仍有统计学差异,重度AD患者 CSF T-tau、CSF P-tau231的浓度比重度VD患者高。中度AD患者CSF P-tau231（Z=-2.605, p=0.009）的浓度显著高于重度AD患者。6个月随访期间,两组患者脑脊液T-tau、P-tau231浓度的变化均无统计学意义。
　　结论：AD患者脑脊液T-tau、P-tau231与VD相比显著升高。中度AD患者CSF P-tau231显著高于重度AD患者。6个月随访期间两组患者脑脊液T-tau、P-tau231浓度的变化均无统计学意义。     

MAPK信号通路与阿尔茨海默病中tau蛋白磷酸化的关系

阿尔茨海默病(AD)是一类神经退行性疾病,tau蛋白过度磷酸化形成的神经原纤维缠结为其主要病理特征之一,是AD发病的重要因素.促分裂原活化蛋白激酶(MAPK)是一类脯氨酸依赖的蛋白激酶,在AD病人体内参与诱导tau蛋白的过度磷酸化.MAPK的三条途径ERK、JNK、p38都参与诱导tau蛋白过度磷酸化,且与Aβ、氧化应激、炎性因子及蛋白磷酸酯酶等因素相关,由此阐述MAPK在AD进程中的重要作用,并提示MAPK可成为AD治疗中的新靶点.     

阿尔茨海默病发病机制中β淀粉样蛋白和Tau蛋白过度磷酸化的相互关系

阿尔茨海默病(Alzheimer's disease,AD)是发生于老年 期或老年前期的慢性进行性神经退行性疾病,表现为大脑普遍萎缩,神经突触和神经元缺失为最早出现的改变,其病理 特征为以神经细胞内神经纤维缠结(neurofibrillary tangles, NFTs)、细胞外β淀粉样蛋白(β-amyloid proteion,Aβ)沉积 所导致的老年斑(senile plaques,SP)形成及神经元缺失为主要表现[1].     

阿尔茨海默病患者尿中AD7c-NTP含量的研究

目的探讨尿中阿尔茨海默病相关神经丝蛋白(AD7c-NTP)含量在阿尔茨海默病(AD)诊断中的意义。方法采用直接竞争性酶联免疫吸附测定法(ELISA),检测218例老年人,其中AD组46例、血管性痴呆组(VD组)50例、智能正常老年对照组122例尿液中AD7c-NTP含量,所有数据均经过SPSS软件进行统计学处理。结果AD组、VD组及智能正常老年对照组尿液中AD7c-NTP含量分别为33.35±1.61、18.19±1.41、18.30±1.45μg/ml,AD组明显高于其他两组,而VD组与智能正常老年对照组差异无统计学意义(P>0.05);91.4%的AD病例AD7c-NTP含量升高((22μg/ml),非AD病例中90.7%均为正常(?22μg/ml)。结论尿液中AD7c-NTP含量检测作为无创性检查,在AD诊断中具有重要的临床参考价值。     

Rabbit IgG cross-reacts with Alzheimer neurofibrillary tangles

Biochemical studies have demonstrated that the paired helical filaments (PHF) of Alzheimer neurofibrillary tangles are mostly made up of tau and to a lesser degree of ubiquitin and other proteins. In addition, immunocytochemical labeling of tangles with antibodies to various other neuronal proteins has been shown previously. We report here the labeling of the locations of PHF, i.e., Alzheimer neurofibrillary tangles, neuropil threads and plaque neurites in tissue sections with a goat antiserum to rabbit IgG (GAR-T). The labeling is comparable in strength and distribution to that of tau and ubiquitin antibodies. The PHF-staining antibodies could be removed by absorption with native rabbit IgG but not with human IgG, IgG-depleted rabbit serum, rabbit IgG heavy chains or light chains eluted from nitrocellulose membranes. Furthermore, the PHF reactivity was obliterated by absorption with brain homogenate and a fraction enriched in soluble abnormally phosphorylated tau, but not with purified bovine tau or SDS-washed preparations of the relatively insoluble population of PHF. On immunoblots of both normal human tau and Alzheimer abnormally phosphorylated tau-enriched preparations, GAR-T labeled a set of three to five polypeptides in the tau region. Some of these polypeptides co-migrated with the tau bands. These results indicate (i) that PHF in Alzheimer's disease brain cross-react with a structural epitope/s present on native rabbit IgG, and (ii) that the cross-reactivity with PHF is probably due to tau.Supported in part by the New York State Office of Mental Retardation and Developmental Disabilities and grants NS 18105, AG04220, AG05892 and AG08076 from the National Institutes of Health     

Alzheimer neurofibrillary tangles: Monoclonal antibodies to inherent antigen(s)

Summary Using isolated Alzheimer neurofibrillary tangles as the immunogen, nine mouse hybridomas were generated which produced antibodies to the tangles as tested in both tissue sections and isolated neurons from Alzheimer brain. Extraction of isolated neurofibrillary tangles with 2% SDS could not remove the antigen(s) with which these monoclonal antibodies reacted. Immunocytochemical study revealed that each of the monoclonal antibodies reacted with one or more of other tissue antigens in addition to the Alzheimer tangles. However, no reaction with either neurofilaments or microtubules was observed with any one of these antibodies. This is the first demonstration of monoclonal antibodies which have been generated against isolated Alzheimer neurofibrillary tangles; these antibodies react with antigen(s) inherent to the tangles.Supported in part by NIH grants NS 18105 and NS 17487     

Dystrophic neurites infiltrate extracellular neurofibrillary tangles in Alzheimer disease

The neurotrophic activity of β-amyloid protein (β-AP) has been suggested to be responsible for the dystrophic neurites that surround β-AP deposits in senile plaques of Alzheimer disease. The recent finding that neurofibrillary tangles (NFT) that remain as remnants in the extracellular space (E-NFT) after the death of the neuron contain β-AP, suggested that dystrophic neurites might also be associated with E-NFT. In this study, we use a probe for E-NFT, basic fibroblast growth factor (bFGF)-binding to show that E-NFT do contain dystrophic neurites. Since these neurites contain the amyloid precursor protein whose cleavage can lead to β-AP, they may also play a role in further β-AP deposition in the E-NFT.     

Substructures of paired helical filaments from alzheimer's disease neurofibrillary tangles

Summary Presented studies reveal that each of the approximately 10 nm filaments forming the paired helical filaments (PHF) is made up of four protofilaments. Each of the protofilaments is a beaded structure, consisting of globules connected by longitudinal bars. A cross-view of PHF shows eight globules linked by transverse bars. The transverse bars are shorter than the longitudinal bars. Comparison between PHF and neurofilament protofilaments indicates structural differences between these profiles, i.e., the globules making the PHF protofilaments are larger and the longitudinal bars are longer than those in the normal neurofilaments. A three-dimensional diagram of PHF structure is presented.Supported in part by grants from the NIA (1P01 AG/NS 04220) and from the Aluminium Association (914-1065A)     

Alzheimer neurofibrillary tangles contain 2.1 nm filaments structurally identical to the microtubule-associated protein τ: a high-resolution transmission electron microscope study of tangles and senile plaque core amyloid

Alzheimer neurofibrillary tangles (NFT) and senile plaque core amyloid (SPCA) isolated from the brain of patients with Alzheimer's disease were freeze-dried and replicated with a new platinum-carbon (Pt-C) vertical deposition method for high-resolution transmission electron microscopy (TEM). The resolution of this vertical Pt-C replication method is superior to either of the more conventional 20° rotary replication or 45° unidirectional replication methods and is dependent on the Pt-C film thickness coating the specimen. The paired helical filaments (PHF) observed within the tangles were right-handed helices with a fairly regular twist period averaging 79.3 ± 5.9 nm and a fairly regular maximum width averaging 14.9 ± 1.0 nm. The PHF regions of minimum width were not regular and fell into three size categories: 2.4 ± 0.3 nm, 4.9 ± 0.6 nm and 9.6 ± 1.4 nm. In addition to the PHF found in the tangles, a new filament was found within all the tangles. These 2.1 ± 0.2 nm diameter filaments were triple-stranded left helices with 1.0 ± 0.2 nm diameter strands with a structure identical to bovine τ. Like bovine τ polymer a number of filaments (1.30 nm to 2.38 nm) were longer than a fully strectched τ monomer of 96 nm. Images of neuritic senile plaque core amyloid (SPCA) showed that amyloid had a more solid appearance than the NFT and its branched filament structures were unlike the 2.1 nm diameter filaments or the PHF found in NFT.     

Induction of epitopes associated with neurofibrillary tangles in clonal mouse neuroblastoma (S20Y) cells

Summary Accumulation of paired helical filaments (PHF) in neurofibrillary tangles is a key neuropathological hallmark in Alzheimer's disease (AD). To date, PHF have been found primarily in humans. Cultured murine cholinergic neuroblastoma (S20Y) cells, following exposure to a serum-free medium or a differentiation medium, developed immunoreactivity to anti-PHF antibodies, and to the Alz-50 by immunocytochemical and immunoblot analyses. Electron microscopic examination revealed abundant fascicles of 10-nm filaments coursing tortuously amongst organelles, such as mitochondria, endoplasmic reticulum and dense-core vesicles, in perikarya and in neuritic extensions. However, subcellular structures identical or similar to PHF could not be found in these non-human cells. This convenient cell culture model may prove to be useful for studying certain aspects of the mechanisms underlying the abnormal cytoskeletal alterations which are characteristic of AD and related neurodegenerative disorders.Supported by grants from the Overbrook Foundation, the Will Rogers Institute, the Dr. I. Fund Foundation, the Winifred Masterson Burke Relief Foundation, the Alzheimer's Disease Research Program of the American Health Assistance Foundation and the National Institute of Aging (AG03853)     

Solubility of neurofibrillary tangles and ultrastructure of paired helical filaments in sodium dodecylsulphate

Summary Temporal cortex from 14 cases of Alzheimer-type dementia and 6 cases of Down's syndrome, all selected for severe Alzheimer pathology, was homogenised in distilled water, NaOH, or sodium dodecylsulphate (SDS) containing 0.1% -mercaptoethanol. The homogenates were stained with Congo red, and the neurofibrillary tangles and plaque cores were counted under crossed-polarisation microscopy. The number of tangles and plaque cores in the water-treated extracts was not related to age, sex, postmortem interval or duration of dementia. The number of tangles after extraction in SDS or NaOH, as a percentage of tangles in water-treated extracts, was 57±25 (mean±SD) for 1% SDS, 43±17 for 5% SDS and 37±22 for 0.2 M NaOH. Plaque cores were essentially insoluble in all three agents. The percentage of tangles insoluble in 1% SDS did not correlated with age or post-mortem interval but decreased with increasing duration of dementia. Enhanced tangle solubility with increasing duration of dementia suggests that the nature of tangles changes with time; one possibility is that this reflects transformation of intracellular to extracellular tangles. Paired helical filament (PHF) length and the number of repeats per PHF were measured in electron micrographs of PHF prepared with and without treatment by 1% SDS. There was no significant multimodality of PHF length to suggest that PHF broke at regular intervals. The mean repeat length (PHF length/number of repeats) was greater for PHF isolated in the presence of 1% SDS than in its absence, showing that SDS affects ultrastructure by untwisting PHF. An untwisting process may also occur in vivo producing the straight filaments found, together with PHF, in tangles and neurites.Supported by Miss E. Buchan (to the MRC Brain Metabolism Unit) and the British Foundation for Age Research and the Wellcome Trust (to P. A. M. Eagles). S. Hussey was in receipt of an MRC Partnership Award.     

Sequence of a human MAP-2 region sharing epitopes with Alzheimer neurofibrillary tangles

Microtubule-associated protein 2 (MAP-2), an abundant neuronal protein, consists of a short microtubule-binding domain and a long projection arm. MAP-2 shares epitopes with Alzheimer neurofibrillary tangles (NFT). However, most anti-MAP-2 antibodies do not stain detergent-extracted NFT, and the role of MAP-2 in NFT formation has therefore been unclear. We have determined the sequence of a 1.7 kb partial MAP-2 cDNA encoding at least three NFT epitopes. The epitopes are not removed by detergent extraction of tangle preparations, suggesting that they are integral components of NFT. Expression vectors containing restriction fragments of the cDNA were used to assign the epitopes to a 51-amino-acid region near the end of the MAP-2 projection arm, distal to the microtubule.     

Amyloid-like (Congophilic) neurofibrillary tangles do not react with neurofilament antisera in Alzheimer's cerebral cortex

Summary The immunohistological properties of Alzheimer's neurofibrillary tangles (NFT) were studied by immunofluorescence with neurofilament (NF) antisera and with antiserum raised to paired helical filaments (PHF) in NFT preparations, brain smears, and cryostat sections. NFT decorated by NF antisera were Congo red-negative. Conversely, PHF antisera stained Congo red-positive NFT but failed to decorate NF-positive NFT. It is concluded that NF do not cross react with typical NFT, i.e., NFT displaying amyloid-like birefringence, under the conditions reported in this study.Supported by the Veterans Administration, by NIH grants NS 13034 and AG 01307 (DJS)     

Alzheimer paired helical filaments: Cross-reacting polypeptide/s normally present in brain

Summary Antisera to microtubule-enriched fraction fron normal human brain (anti-MT sera) label neurofibrillary tangles and neurites of neuritic (senile) plaques in brain sections of cases with Alzheimer disease/senile demintia of the Alzheimer type (AD/SDAT); the plaque core amyloid is not labeled. These anti-MT sera label both tangles in tissue sections and smears of isolated tangles which had been extracted with sodium dodecyl sulfate (SDS) to remove impurities trapped in between the paired helical filaments (PHF). The tangle labeling of anti-MT sera is eliminated on their absorption both with microtubule-enriched fractions from human and animal brain and with the isolated PHF. Neurofilament triplet, actin, myosin, keratin, or fibroblasts do not absorb the tangles staining antibodies. Furthermore, antisera containing antibodies to tubulin, microtubule-associated high mol. wt. polypeptides (MAPS), neurofilament triplet, and the 50,000 mol. wt. contaminant of CNS neurofilament preparations do not label tangles. On immunoblots of SDS-polyacrylamide gels of isolated PHF anti-MT sera label some of the same polypeptides identified with antisera to PHF; affinity-purified antibodies to tubulin used as a control do not label any PHF polypeptide on the immunoblots. The anti-MT sera, when preabsorbed with the PHF polypeptides eluted from SDS-polyacrylamide gels, do not label tangles. These studies demonstrate that a polypeptide/s cross-reactive with Alzheimer PHF is indeed normally present in brain and that it is different from tubulin, neurofilament triplet, actin, myosin, vimentin, and keratin.Parts of this paper were reported at the XIIth International Congress of Gerontology, Satellite Symposium, Heidelberg, Federal Republic of Germany, 1981; at the IXth International Congress of Neuropathology, Vienna, Austria, 1982; and at the 60th Annual Meeting of the American Association of Neuropathologists, San Diego, CA, USA, 1984Supported by NIH grants NS 18105 and NS 17487