Alzheimer's paired helical filaments: Amyloid precursor protein epitope mapping

Paired helical filaments (PHF) were electro-phoretically purified and solubilized from Alzheimer's neurofibrillary tangles and consisted of a primary 66 kDa protein on SDS-PAGE analysis. A panel of antibodies raised against restricted regions of the beta-amyloid precursor protein (APP) were employed for epitope mapping studies of this 66 kDa PHF protein. Western blot studies revealed that C-terminal APP antibodies were immunoreactive with the 66 kDa PHF protein. Further analysis revealed that only antisera raised against peptides that include the beta/A4-amyloid region within the C-terminal portion of APP were immunoreactive with PHF proteins. These data complement previous immunocytochemical studies which indicated that C-terminal APP antibodies preferentially label PHF-containing neurofibrillary tangles in Alzheimer's brain. The present data suggest a similarity of secondary or tertiary structure between beta/A4-amyloid and PHF which accounts for the cross-reactivity of beta/A4-amyloid antibodies with PHF proteins.     

Alzheimer paired helical filaments: Bulk isolation,solubility, and protein composition

Summary A method has been developed for the bulk isolation of Alzheimer neurofibrillary tangles (ANT) of paired helical filaments (PHF) from histopathologically confirmed cases of Alzheimer disease/senile dementia of the Alzheimer type (AD/SDAT). The fresh or frozen autopsied cerebral cortex affected with Alzheimer neurofibrillary changes is dissociated by homogenization and sieving through nylon bolting cloth and the ANT are separated by a combination of sucrose discontinuous density gradient centrifugation, glass bead column chromatography, and sodium dodecyl sulfate (SDS) treatment. The isolated ANT produce red-green birefringence when viewed through polarized light after staining with Congo red. Ultrastructurally, the isolated PHF are well preserved and have the dimensions of the PHF seen in situ. Two major Populations of ANT which exist in different proportions in AD/SDAT brains are identified on the basis of their solubility in SDS. The ANT I and the ANT II are soluble and insoluble respectively on treatment with 2% SDS at room temperature for 5 min. Solubilization of the ANT II requires several repeated extractions with a solution containing 10% each of SDS and -mercaptoethanol (BME) at 100°C for 10 min. Sonication of the ANT II greatly facilitates their solubilization. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) of the isolated ANT reveals the presence of two major polypeptides with molecular weights (MW) of 62,000 and 57,000, several minor polypeptides with MW below 57,000, and a significant amount of material not entering the stacking and the resolving gels. Re-electrophoresis of polypeptides extracted from various areas of the resolving gel or of the material which does not enter the gel generates the same polypeptide profile as on the first gel, suggesting that the PHF material which does not enter the gel may result from the reaggregation of the polypeptides that enter the resolving gel. None of the polpeptides observed in the isolated PHF comigrate in the SDS-PAGE with any of the neurofilament polypeptides, tubulin, actin, or myosin.Supported in part by NIH grants NS 17487 and NS 18105     

Paired helical filaments in astrocytes: electron microscopy and immunohistochemistry in a case of atypical Alzheimer's disease

Summary A 54-year-old man who had cerebellar ataxia and pseudobulbar palsy at the age of 29 years, and soon developed dementia, myoclonus and convulsions, died after about 20 years in a vegetative state. Histological examination of the extensively atrophic and devastated brain (680 g) revealed the almost total loss of cerebral cortical neurons associated with numerous -protein amyloid plaques, many extracellular tangles and a large number of hypertrophic astrocytes, and prominent amyloid angiopathy. The astrocytes were frequently immunopositive for anti-human tau antibody (anti-htau) and anti-ubiquitin antibody (anti-ubi). Double immunostaining with anti-htau and anti-glial fibrillary acidic protein (GFAP) antibody clearly demonstrated htau-positive domains within the GFAP-positive perikarya/and processes of several astrocytes. Electron microscopy of the hippocampal CA1, which was completely devoid of pyramidal neurons, revealed, in astrocytes, abnormal filaments indistinguishable from the paired helical filaments (PHFs) seen in neurons. On immunoelectron microscopy, the filaments were observed to be labeled with anti-htau and anti-ubi, exhibiting the same immunohistochemical features as neuronal PHFs. This is the first demonstration of clearly constricted and both tau- and ubiquitin-positive PHFs in astrocytes, indicating that, in some special conditions like in our case, processes similar to those that attack neurons also affect astrocytes and ultimately make the latter form PHFs.Supported in part by Grants-in-Aid for Scientific Research on Priority Areas, Nos. 01658005 and 02240104, from the Ministry of Education, Science and Culture, Japan     

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。     

pax1蛋白在子宫颈癌中的表达及与预后的关系

目的:研究配对盒基因1(pax1)在子宫颈组织中的蛋白表达及其临床意义,探讨pax1蛋白与子宫颈癌预后的关系。方法:采用免疫组化法检测中日友好医院2011年8月至2017年10月收治的101例子宫颈癌、120例子宫颈上皮内瘤变(CIN)及20例正常子宫颈组织(对照组)中pax1蛋白表达水平,分析其子宫颈癌临床病理参数,采用受试者工作曲线(ROC)评估其诊断效能并随访,探讨其与子宫颈鳞癌(SCC)及子宫颈腺癌(AC)预后的关系。结果:①对照组的pax1蛋白表达水平(90. 0%)高于CIN(73. 3%)及子宫颈癌(38. 6%),SCC患者pax1蛋白阳性表达率(20. 9%)明显低于AC(70. 0%)及特殊类型子宫颈癌(100. 0%),差异有统计学意义(P <0. 05)。②ROC曲线显示,pax1蛋白曲线下面积(AUC)为0. 829,诊断子宫颈癌的灵敏度90. 0%,特异度61. 3%。③pax1蛋白阳性表达率与子宫颈癌患者的HPV感染、分化程度、组织类型及脉管浸润有关(P <0. 05)。④SCC患者中pax1蛋白阳性组的生存时间明显高于阴性组(P=0. 040...     

学习不良初中生心理健康状况的配对研究

目的　探讨学习不良初中生的心理健康状况及其与学习不良的关系。方法　采用 1∶ 1配对的对照研究方法 ,应用中学生心理健康量表 ( MSSMHS)对 1 1 0名初中生进行测查。结果　MSSMHS测查中 ,学习不良组的心理异常检出率高于对照组 ,差异有显著性意义 ( P<0 .0 5 ) ;学习不良组共有 9项指标高于对照组 ,差异有显著性意义 ( P<0 .0 5 ) ;学习不良组强迫、人际关系敏感、焦虑、学习压力及情绪不平衡因子轻度异常。结论　学习不良初中生的心理健康问题不容忽视     

利用CRISPR/Cas9技术制备成对框基因2敲除小鼠

目的 利用成簇规律间隔短回文重复序列（CRISPR）/重组CRISPR相关核酸酶9（Cas9）技术双切口法制备成对框基因2（Pax2）敲除小鼠,为探讨Pax2基因在多个系统发育的作用提供动物模型。方法 根据Pax2基因序列设计sgRNA,设计出的sgRNA和Cas9体外转录后显微注射到C57BL/6J 小鼠的受精卵中,F0代小鼠出生后取其基因DNA测序鉴定基因型。共获得8只F0 代小鼠,使敲除成功的F0代小鼠与野生C57BL/6J 小鼠交配,获得F1代小鼠,后均采用基因成功敲除的小鼠与C57BL/6J 小鼠进行交配,可获得稳定的Pax2基因敲除小鼠。结果 成功获得可稳定繁殖的Pax2杂合子基因敲除小鼠,其Pax2基因缺失1628 bp;组织HE染色显示,敲除小鼠的肾小球数量明显减少;Western blotting结果显示,敲除小鼠的肾皮质Pax2蛋白表达较野生型小鼠减少。结论 利用CRISPR/Cas9技术可成功构建Pax2杂合子基因敲除小鼠,为进一步研究Pax2基因的作用奠定基础。     

p73 haploinsufficiency causes tau hyperphosphorylation and tau kinase dysregulation in mouse models of aging and Alzheimer's disease

Haploinsufficiency for the p53 family member p73 causes behavioral and neuroanatomical correlates of neurodegeneration in aging mice, including the appearance of aberrant phospho-tau-positive aggregates. Here, we show that these aggregates and tau hyperphosphorylation, as well as a generalized dysregulation of the tau kinases GSK3β, c-Abl, and Cdk5, occur in the brains of aged p73+?− mice. To investigate whether p73 haploinsufficiency therefore represents a general risk factor for tau hyperphosphorylation during neurodegeneration, we crossed the p73+?− mice with 2 mouse models of neurodegeneration, TgCRND8+?Ø mice that express human mutant amyloid precursor protein, and Pin1−?− mice. We show that haploinsufficiency for p73 leads to the early appearance of phospho-tau-positive aggregates, tau hyperphosphorylation, and activation of GSK3β, c-Abl, and Cdk5 in the brains of both of these mouse models. Moreover, p73+?−;TgCRND8+?Ø mice display a shortened lifespan relative to TgCRND8+?Ø mice that are wild type for p73. Thus, p73 is required to protect the murine brain from tau hyperphosphorylation during aging and degeneration.     

Effects of memantine and galantamine on cognitive performance in aged rhesus macaques

Current pharmacotherapies for Alzheimer's disease (AD) are focused on improving performance of daily activities, personal care, and management of problematic behaviors. Both memantine, a noncompetitive N-methyl-D-aspartate channel blocker and galantamine, a selective acetylcholinesterase inhibitor, are currently prescribed as symptomatic therapies for AD. However, drugs that progressed directly from testing in rodent models to testing in AD patients in clinical trials failed to demonstrate consistent effects on cognitive symptoms. Considering the lack of nonhuman primate data on the effects of memantine and galantamine alone or in combination on cognitive dysfunction in aged nonhuman primates, the present study examined how closely data derived from aged nonhuman primates reflects data obtained in humans. Mild beneficial effects on aspects of cognitive performance in aged primates were found, in general agreement with the human clinical experience with these drugs but in contrast to the more positive effects reported in the rodent literature. These data suggest that the nonhuman primate might have more predictive validity for drug development in this area than comparable rodent assays.     

Neuritic plaques in the Lewy body variant of Alzheimer disease lack paired helical filaments

The Lewy body variant of Alzheimer disease (LBV) is a distinct category of dementia which, unlike pure diffuse Lewy body disease (DLBD), meets clinical and neuropathologic criteria for Alzheimer disease (AD) but differs from pure AD in having a neocortical predominance of diffuse and neuritic plaques (NP), with very few neurofibrillary tangles (NFT). We investigated the immunoreactivity of NP with a monoclonal antibody against paired helical filaments (PHF) composed of phosphorylated microtubule associated protein tau. With routine thioflavin-S preparations, 12 LBV and 14 AD cases had similar numbers of NP, but the LBV had significantly (P<0.05) fewer NFT per microscopic field in the midfrontal cortex. Among subjects with midfrontal NFT, 81–84% of NP in AD and LBV were anti-PHF positive. Among subjects without midfrontal NFT, 52% of NP in AD but only 12% of NP in LBV cases were anti-PHF positive (P<0.05). LBV differs from AD in that its NP generally do not contain PHF, unless they are accompanied by neocortical NFT.