A familial case of Alzheimer's disease without tau pathology may be linked with chromosome 3 markers

Alzheimer's disease is the most common form of dementia that occurs in later years. The diagnosis is confirmed by the pathological findings of βA4-amyloid-containing neuritic plaques and neurofibrillary tangles, the former being present in sufficient quantity commensurate with age. Other forms of dementia are more difficult to diagnose clinically; their pathology is noted for the lack of plaques and tangles. A patient with a family history of dementia presented with the clinical signs of Alzheimer's disease which lasted for 13 years. At autopsy the brain tissue had βA4-amyloid-containing neuritic plaques, but no neurofibrillary tangles (i.e., the tissue was negative for staining with the tau antibody). Genetic analysis of DNA from family members revealed no linkage with chromosome 17 markers, indicating that this was not frontotemporal dementia. However, there was linkage with chromosome 3 markers. Thus, this form of Alzheimer's disease with a pathology of plaques only is linked with markers on chromosome 3. Electronic Publication     

Differential sensitivity of the microtubule-associated protein, tau, in Alzheimer's disease tissue to formalin fixation

Immunohistochemistry of formalin-fixed human Alzheimer's disease (AD) tissue using an anti-tau antibody (Tau-1) reveals staining of neurofibrillary tangles (NFTs) and neuritic plaques (NPs), whereas normal axonal staining is less apparent. In this study, we used a combined biochemical and histochemical approach to assess effects of formalin on immunoreactivity of AD tau. Nitrocellulose blots were treated with fixative to mimic conditions used with tissue sections, a method that might be generally useful for assessing antigen sensitivity to different fixatives. A progressive decrease in Tau-1 immunoreactivity of the tau bands on a Western blot was observed with increasing times of formalin fixation. Phosphatase-digested blots demonstrated an increase in Tau-1 immunoreactivity compared to control blots. These results mimic the phosphatase-sensitive Tau-1 immunohistochemical staining of formalin-fixed AD tissue slices previously reported. Fixation of AD tissue with periodate-lysine-paraformaldehyde (PLP) preserves axonal tau antigenicity. Phosphatase digestion of PLP-fixed AD tissue enhances Tau-1 immunoreactivity of NFTs and NPs but does not alter axonal staining. These results indicate that axonal form(s) of tau are more sensitive to formalin fixation than pathology-associated tau. In addition, a modification of AD tau in pathological structures may protect it from the effects of formalin with regard to Tau-1 antigenicity.     

Neighbored phosphorylation sites as PHF-tau specific markers in Alzheimer's disease

Neurofibrillary tangles, which represent a major pathological hallmark in Alzheimer's disease (AD), are deposits of the hyperphosphorylated microtubule-associated tau protein (PHF-tau). However, a link between the phosphorylation pattern and the cause or the progress of AD is still missing. The work reported here focused on PHF-tau specific local phosphorylation patterns at Thr212/Ser214 and Thr231/Ser235 using monoclonal antibodies (mAb) generated against correspondingly modified peptides. The binding motifs of the obtained six mAbs were characterized with non-, mono-, and double-phosphorylated peptides as well as terminally shortened sequences. Five mAbs stained neurofibrillary tangles, neuritic plaques, and neuropil threads from autoptic brains of AD cases. Four mAbs recognized PHF-tau without significant cross-reactivity towards normal human tau, bovine tau, and dephosphorylated PHF-tau in ELISA and Western blot analysis. Thus, double phosphorylation is sufficient to distinguish PHF-tau from all other tau versions and there is no need to postulate any PHF-tau specific conformation for this region.     

Tau epitope display in progressive supranuclear palsy and corticobasal degeneration

Filamentous aggregates of the protein tau are a prominent feature of Alzheimer's disease (AD), progressive supranuclear palsy (PSP) and corticobasal degeneration (CBD). However, the extent to which the molecular structure of the tau in these aggregates is similar or differs between these diseases is unclear. We approached this question by examining these disorders with a panel of antibodies that represent different structural, conformational, and cleavage-specific tau epitopes. Although each of these antibodies reveals AD pathology, they resolved into three classes with respect to PSP and CBD: AD2 and Tau-46.1 stained the most tau pathology in all cases; Tau-1, 2, 5, and 12 exhibited variable reactivity; and Tau-66 and MN423 did not reveal any tau pathology. In addition, hippocampal neurofibrillary tangles in these cases showed a predominantly PSP/CBD-like, rather than AD-like, staining pattern. These results indicate that the patterns of the tau epitopes represented by this panel that reside in the pathological aggregates of PSP and CBD are similar to each other but distinct from that of AD.     

Monoclonal Antibodies Against the Human Metalloprotease EC 3.4.24.15 Label Neurofibrillary Tangles in Alzheimer's Disease Brain

Abstract: Alzheimer's disease is characterized neuropathologically by the presence of neuritic and amyloid plaques, vascular amyloid, and neurofibrillary tangles in specific brain areas. The main constituent of amyloid deposits is amyloid β protein, a 40–42 amino acid proteolytic product of the amyloid β-precursor protein. In our search for proteases that can generate the N-terminus of amyloid β protein (β-secretases), we discovered a thiol-dependent metalloprotease that was identified, by peptide sequencing, as metalloendopeptidase EC 3.4.24.15. In vitro, the metalloprotease cleaves the methionine-aspartic acid bond in a 10 amino acid synthetic peptide, indicating that it could generate the N-terminus of amyloid β protein, and generates amyloidogenic fragments from full-length recombinant amyloid β-precursor protein. Mouse monoclonal antibodies produced against a unique synthetic peptide from the metalloprotease labeled various monkey tissues as detected by western blots and immunohistochemistry. Unexpectedly, two monoclonal antibodies, IVD6 and IIIF3, immunolabeled strongly intracellular neurofibrillary tangles, neurites of senile plaques, and neuropil threads, but not "ghost" tangles or amyloid in sections taken from Alzheimer's disease brain. This finding provides further evidence for the metalloprotease's relevance to Alzheimer's disease pathology, although the connection between tangle staining and the formation of amyloid β protein remains to be elucidated.     

Tau-66: evidence for a novel tau conformation in Alzheimer's disease

We have characterized a novel monoclonal antibody, Tau-66, raised against recombinant human tau. Immunohistochemistry using Tau-66 reveals a somatic-neuronal stain in the superior temporal gyrus (STG) that is more intense in Alzheimer's disease (AD) brain than in normal brain. In hippocampus, Tau-66 yields a pattern similar to STG, except that neurofibrillary lesions are preferentially stained if present. In mild AD cases, Tau-66 stains plaques lacking obvious dystrophic neurites (termed herein 'diffuse reticulated plaques') in STG and the hippocampus. Enzyme-linked immunosorbent assay (ELISA) analysis reveals that Tau-66 is specific for tau, as there is no cross-reactivity with MAP2, tubulin, Abeta(1-40), or Abeta(1-42), although Tau-66 fails to react with tau or any other polypeptide on western blots. The epitope of Tau-66, as assessed by ELISA testing of tau deletion mutants, appears discontinuous, requiring residues 155-244 and 305-314. Tau-66 reactivity exhibits buffer and temperature sensitivity in an ELISA format and is readily abolished by SDS treatment. Taken together these lines of evidence indicate that the Tau-66 epitope is conformation-dependent, perhaps involving a close interaction of the proline-rich and the third microtubule-binding regions. This is the first indication that tau can undergo this novel folding event and that this conformation of tau is involved in AD pathology.     

Antigenic changes similar to those seen in neurofibrillary tangles are elicited by glutamate and Ca2+ influx in cultured hippocampal neurons.

In several neurological disorders including Alzheimer's disease, abnormal accumulations of cytoskeleton-associated proteins manifest as neurofibrillary tangles (NFTs) in vulnerable brain regions. Antibodies recognizing tau (5E2 and Alz-50) and ubiquitin epitopes in NFTs were used to examine the influence of glutamate and Ca2+ influx on antigen expression in cultured rat hippocampal neurons. Glutamate caused the degeneration of a subpopulation of pyramidal neurons, which exhibited increased immunostaining with all three antibodies. Subtoxic levels of glutamate also increased 5E2 and Alz-50 antigen levels in a subpopulation of neurons, particularly in the distal regions of the axons. Both glutamate-induced degeneration and increases in tau and ubiquitin immunostaining were prevented by removal of extracellular Ca2+. Increased immunostaining was also induced by Ca2+ ionophore A23187 or elevated levels of extracellular K+. The antigenic changes occurred within 1 hr of exposure to glutamate or A23187 and were not prevented by the protein synthesis inhibitor cycloheximide. These data indicate that Ca2+ influx caused by glutamate can lead to modifications of extant proteins similar to those seen in NFTs.     

Alz 50, a monoclonal antibody to Alzheimer's disease antigen, cross-reacts with tau proteins from bovine and normal human brain

Microtubule-associated protein tau from bovine brain reacted on immunoblots and on enzyme-linked immunosorbent assay with a monoclonal antibody, Alz 50, which has previously been found to bind to an Alzheimer disease-specific antigen. The apparent affinity of binding of Alz 50 to tau was 2.1 X 10(-9) M on competitive enzyme-linked immunosorbent assay, and it was in the same range as for Tau-1 (0.5 X 10(-9) M), an antibody raised against purified bovine tau proteins. Immunoblotting of trypsin-digested tau revealed differences between Alz 50 and Tau-1 binding sites. The binding of both antibodies to tau was not affected by prior treatment with phosphatase, indicating that the cross-reactivity of Alz 50 with tau is due to the presence of phosphate-independent epitope. This epitope then differs from phosphate-dependent tau epitopes often shared with other cytoskeletal proteins. Alz 50 and Tau-1 binding sites were present in all isoelectric (pI 6-8) and molecular weight variants of tau. In contrast, phosphate-dependent epitopes recognized by another tau-reactive antibody (NP14) were found mostly in acidic tau variants. Similarly to tau proteins from bovine brain, tau-enriched preparations from normal human brain contained Alz 50 and Tau-1 reactive sites in all isoelectric (pI 6.5-8.5) and molecular weight variants. Our observation of Alz 50 cross-reactivity with tau suggests a relationship between tau and the novel protein identified recently in Alzheimer brains.     

Amyloid Angiopathy of Alzheimer''s Disease: Amino Acid Composition and Partial Sequence of a 4,200-Dalton Peptide Isolated from Cortical Microvessels

The cardinal lesions of Alzheimer's disease are neurofibrillary tangles, senile neuritic plaques, and vascular amyloid, the latter generally involving cortical arteries and small arterioles. All three lesions are composed of amyloid-like, beta-pleated sheet fibrils. Recently, a 4,200-dalton peptide has been isolated from extraparenchymal meningeal vessels, neuritic plaques, and neurofibrillary tangles. The assumption of N-terminal homogeneity in vascular amyloid has been used as an argument for a neuronal (versus blood) origin of the peptide. However, intracortical microvessels from Alzheimer's disease have not been previously isolated. The present studies describe the isolation of a microvessel fraction from Alzheimer's disease and control fresh autopsy human brain. Alzheimer's disease isolated brain microvessels that were extensively laden with amyloid and control microvessels were solubilized in 90% formic acid and analyzed by urea sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The arteriole fraction from the Alzheimer's subject with extensive amyloid angiopathy contained a unique 4,200-dalton peptide, whereas the arterioles or capillaries isolated from two controls and two Alzheimer's disease subjects without angiopathy did not. This peptide was purified by HPLC and amino acid composition analysis showed the peptide is nearly identical to the 4,200-dalton peptide recently isolated from neuritic plaques or from neurofibrillary tangles. Sequence analysis revealed N-terminal heterogeneity. The N-terminal sequence was: Asp-Ala-Glu-Phe-Arg-His-Asp-Ser-Gly-Tyr, which is identical to the N-terminal sequence of the 4,200-dalton peptide isolated previously from extraparenchymal meningeal vessels and neuritic plaques.(ABSTRACT TRUNCATED AT 250 WORDS)     

褪黑素对异丙肾上腺素诱导大鼠tau蛋白过度磷酸化的预防作用

异常过度磷酸化的微管相关蛋白tau是阿尔茨海默病(Alzheimer's disease,AD)患者大脑中神经原纤维缠结的主要组成部分.迄今为止,尚无有效的措施阻止tau蛋白的过度磷酸化.为探讨褪黑素(melatonin,Mel)对AD样tau蛋白过度磷酸化的预防作用,我们以β受体激动剂异丙肾上腺素(isoproterenol,IP)来复制AD样tau蛋白过度磷酸化的动物模型,在大鼠双侧海马注射IP前,以褪黑素作为保护组药物,于腹腔连续注射5 d.应用磷酸化位点特异性抗体(PHF-1和Tau-1)作免疫印迹和免疫组织化学检测tau蛋白的磷酸化水平,并用非磷酸化依赖的总tau蛋白抗体(111e)进行标准化.免疫印迹结果显示在注射IP 48 h后,tau蛋白在PHF-1表位的免疫反应显著增强,在Tau-1表位显著减弱,表明tau蛋白在Ser396/Ser404(PHF-1)和Ser199/Ser202(Tau-1)位点有过度磷酸化.免疫组织化学染色结果与免疫印迹结果相似,主要检测到在大鼠海马CA3区的神经纤维有tau蛋白过度磷酸化.褪黑素预处理大鼠可有效地阻止IP诱导tau蛋白在Tau-1和PHF-1位点的过度磷酸化.上述结果提示褪黑素可预防大鼠脑组织中由异丙肾上腺素引起的AD样tau蛋白的过度磷酸化.     

Proline-directed phosphorylation of human Tau protein.

The primary sequence of the microtubule-associated protein tau contains multiple repeats of the sequence -X-Ser/Thr-Pro-X-, the consensus sequence for the proline-directed protein kinase (p34cdc2/p58cyclin A). When phosphorylated by proline-directed protein kinase in vitro, tau was found to incorporate up to 4.4 mol of phosphate/mol of protein. Isoelectric focusing of the tryptic phosphopeptides demonstrated the presence of five distinct peptides with pI values of approximately 6.9, 6.5, 5.6-5.9, 4.7, and 3.6. Mapping of the tryptic phosphopeptides by high performance liquid chromatography techniques demonstrated three distinct peaks. Data from gas phase sequencing, amino acid analysis, and phosphoamino acid analysis suggest that proline-directed protein kinase phosphorylates tau at four sites. Each site demonstrates the presence of a proline residue on the carboxyl-terminal side of the phosphorylated residue. Two phosphorylation sites are located adjacent to the three-repeat microtubule-binding domain that has been found to be required for the in vivo co-localization of tau protein to microtubules. Two other putative phosphorylation sites are located within the identified epitope of the monoclonal antibody Tau-1. Phosphorylation of these sites altered the immunoreactivity of tau to Tau-1 antibody. Since the neuronal microtubule-associated protein tau is multiply phosphorylated in Alzheimer's disease, and Tau-1 immunoreactivity is similarly reduced in neurofibrillary tangles and enhanced after dephosphorylation, phosphorylation at one or more of these sites may correlate with abnormally phosphorylated sites in tau protein in Alzheimer's disease.     

Basic fibroblast growth factor binding is a marker for extracellular neurofibrillary tangles in Alzheimer disease

Neurofibrillary tangles (NFT) are abnormal filamentous inclusions that develop in neurons in Alzheimer disease and other disorders. When neurons die, the neurofibrillary tangles that persist in the extracellular space show ultrastructural and antigenic changes. Both intra- and extracellular NFT have recently been shown to contain heparan sulfate proteoglycans (HSPGs). HSPGs are also present in other amyloid deposits in the brain and in systemic amyloidoses. Basic fibroblast growth factor (bFGF) is a heparin binding growth factor which is involved in angiogenesis and also has neurite promoting activity. We now report that bFGF binds avidly to extracellular NFT. Alz-50, a monoclonal antibody (MAb) to an abnormal form of tau and bFGF binding label mutually exclusive subpopulations of neurofibrillary tangles. bFGF binding is abolished by heparinase or heparitinase treatment and therefore is most likely based on the presence of HSPG. Binding of bFGF is a specific and sensitive morphological method to distinguish intra- from extracellular NFT. As intracellular NFT, which also contain HSPGs, are not labeled by bFGF binding, this finding also suggests that HSPGs are modified when the NFT become extracellular.     

Genetically augmenting tau levels does not modulate the onset or progression of Abeta pathology in transgenic mice

The two hallmark pathologies of Alzheimer's disease (AD) are amyloid plaques, composed of the small amyloid-beta (Abeta) peptide, and neurofibrillary tangles, comprised aggregates of the microtubule binding protein, tau. The molecular linkage between these two lesions, however, remains unknown. Based on human and mouse studies, it is clear that the development of Abeta pathology can trigger tau pathology, either directly or indirectly. However, it remains to be established if the interaction between Abeta and tau is bidirectional and whether the modulation of tau will influence Abeta pathology. To address this question, we used the 3xTg-AD mouse model, which is characterized by the age-dependent buildup of both plaques and tangles. Here we show that genetically augmenting tau levels and hyperphosphorylation in the 3xTg-AD mice has no effect on the onset and progression of Abeta pathology. These data suggest that the link between Abeta and tau is predominantly if not exclusively unidirectional, which is consistent with the Abeta cascade hypothesis and may explain why tauopathy-only disorders are devoid of any Abeta pathology.     

Yeast surface display of full-length human microtubule-associated protein tau

Microtubule-associated protein tau is an intrinsically disordered, highly soluble protein found primarily in neurons. Under normal conditions, tau regulates the stability of axonal microtubules and intracellular vesicle transport. However, in patients of neurodegeneration such as Alzheimer's disease (AD), tau forms neurofibrillary deposits, which correlates well with the disease progression. Identifying molecular signatures in tau, such as posttranslational modification, truncation, and conformational change has great potential to detect earliest signs of neurodegeneration and develop therapeutic strategies. Here, we show that full-length human tau, including the longest isoform found in the adult brain, can be robustly displayed on the surface of yeast Saccharomyces cerevisiae. Yeast-displayed tau binds to anti-tau antibodies that cover epitopes ranging from the N-terminus to the 4R repeat region. Unlike tau expressed in the yeast cytosol, surface-displayed tau was not phosphorylated at sites found in AD patients (probed by antibodies AT8, AT270, AT180, and PHF-1). However, yeast-displayed tau showed clear binding to paired helical filament (PHF) tau conformation-specific antibodies Alz-50, MC-1, and Tau-2. Although the tau possessed a conformation found in PHFs, oligomerization or aggregation into larger filaments was undetected. Taken together, yeast-displayed tau enables robust measurement of protein interactions and is of particular interest for characterizing conformational change.     

The pathology of the neuronal cytoskeleton in Alzheimer's disease.

The two characteristic neuropathological lesions of Alzheimer's disease are the neurofibrillary tangles and the senile plaques. Neurofibrillary tangles are made of abnormal filaments (PHF) accumulating in neurons and mainly composed of a modified form of the microtubule-associated protein tau (PHF-tau). Senile plaques are composed of a cluster of dystrophic neurites surrounding an extracellular deposit of amyloid fibers made of a 42 amino-acid peptide (beta-amyloid peptide). The abnormal filaments contain the complete sequences of the different tau isoforms. The PHF-tau proteins can be distinguished from the normal tau proteins by the presence of several phosphorylated sites. One of these sites is phosphorylated by a calcium-calmodulin-dependent kinase. The relationship between PHF-tau and the cytoskeletal pathology in Alzheimer's disease is further discussed.     

Abeta immunotherapy leads to clearance of early, but not late, hyperphosphorylated tau aggregates via the proteasome

Amyloid-beta (Abeta) plaques and neurofibrillary tangles are the hallmark neuropathological lesions of Alzheimer's disease (AD). Using a triple transgenic model (3xTg-AD) that develops both lesions in AD-relevant brain regions, we determined the consequence of Abeta clearance on the development of tau pathology. Here we show that Abeta immunotherapy reduces not only extracellular Abeta plaques but also intracellular Abeta accumulation and most notably leads to the clearance of early tau pathology. We find that Abeta deposits are cleared first and subsequently reemerge prior to the tau pathology, indicative of a hierarchical and direct relationship between Abeta and tau. The clearance of the tau pathology is mediated by the proteasome and is dependent on the phosphorylation state of tau, as hyperphosphorylated tau aggregates are unaffected by the Abeta antibody treatment. These findings indicate that Abeta immunization may be useful for clearing both hallmark lesions of AD, provided that intervention occurs early in the disease course.     

Does dysregulation of the Notch and wingless/Wnt pathways underlie the pathogenesis of Alzheimer's disease?

Alzheimer's disease is characterized by the presence of neurofibrillary tangles and senile neuritic plaques in the brain. Tangles are aggregates of paired helical filaments composed of the microtubule-associated protein, tau, in a hyperphosphorylated state. Senile plaques have a core of amyloid beta-peptide derived by proteolysis of the amyloid precursor protein. A major hurdle in defining the pathogenic mechanisms in Alzheimer's disease is to understand how both amyloid beta-peptide deposition and paired helical filament formation are biochemically linked. Recent genetic discoveries provide some clues, suggesting that components of two developmentally important signalling pathways, Notch and wingless, or the vertebrate homologue of wingless, Wnt, are involved.     

Alzheimer's disease: microtubule-associated proteins 2 (MAP 2) are not components of paired helical filaments

In Alzheimer's disease, the most characteristic neuropathological changes are the formation of neurofibrillary tangles (NFT) and neuritic plaques (NP) characterized by the presence of bundles of paired helical filaments (PHF) that accumulate in the degenerating neurites and neuronal cell bodies. Although the protein composition of the PHF is ill-defined, a number of microtubule-associated proteins have been implicated in these lesions. Here we report results with an antiserum monospecific for the microtubule-associated protein MAP 2 which does not cross-react with any other microtubular protein. Immunostaining with this antibody of sections from an Alzheimer's brain show a strong reactivity with NFT but no reactivity at the level of the NP. On the other hand, immunostaining of Alzheimer's brain sections with another antibody specific for the microtubule-associated protein tau shows strong staining of PHF on both NFT and NP. These findings confirm the presence of the tau proteins in the PHF and strongly suggest that MAP 2 may not be a main structural component of the PHF. Labelling of NFT with the anti-MAP 2 antiserum suggests a non-specific binding of MAP 2 to the PHF during the process of NFT formation.     

Monitoring protein aggregation and toxicity in Alzheimer's disease mouse models using in vivo imaging

Aggregation of amyloid beta peptide into senile plaques and hyperphosphorylated tau protein into neurofibrillary tangles in the brain are the pathological hallmarks of Alzheimer's disease. Despite over a century of research into these lesions, the exact relationship between pathology and neurotoxicity has yet to be fully elucidated. In order to study the formation of plaques and tangles and their effects on the brain, we have applied multiphoton in vivo imaging of transgenic mouse models of Alzheimer's disease. This technique allows longitudinal imaging of pathological aggregation of proteins and the subsequent changes in surrounding neuropil neurodegeneration and recovery after therapeutic interventions.     

Pin1 in Alzheimer's disease: multiple substrates, one regulatory mechanism?

Presence of neuritic plaques and neurofibrillary tangles in the brain are two neuropathological hallmarks of Alzheimer's disease (AD), although the molecular basis of their coexistence remains elusive. The neurofibrillary tangles are composed of microtubule binding protein Tau, whereas neuritic plaques consist of amyloid-beta peptides derived from amyloid precursor protein (APP). Recently, the peptidyl-prolyl cis/trans isomerase Pin1 has been identified to regulate the function of certain proteins after phosphorylation and to play an important role in cell cycle regulation and cancer development. New data indicate that Pin1 also regulates the function and processing of Tau and APP, respectively, and is important for protecting against age-dependent neurodegeneration. Furthermore, Pin1 is the only gene known so far that, when deleted in mice, can cause both Tau and Abeta-related pathologies in an age-dependent manner, resembling many aspects of human Alzheimer's disease. Moreover, in the human AD brain Pin1 is downregulated or inhibited by oxidative modifications and/or genetic changes. These results suggest that Pin1 deregulation may provide a link between formation of tangles and plaques in AD.