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.     

Transglutaminase bonds in neurofibrillary tangles and paired helical filament tau early in Alzheimer's disease.

Transglutaminase-catalyzed epsilon(gamma-glutamyl)lysine cross-links exist in Alzheimer's disease (AD) paired helical filament (PHF) tau protein but not normal soluble tau. To test the hypothesis that these cross-links could play a role in the formation of neurofibrillary tangles (NFT), we used single- and double-label immunofluorescence confocal microscopy and immunoaffinity purification and immunoblotting to examine epsilon(gamma-glutamyl)lysine cross-links in AD and control brains. The number of neurons that are immunoreactive with an antibody directed at the epsilon-(gamma-glutamyl)lysine bond was significantly higher in AD cortex compared with age-matched controls and schizophrenics. PHF tau-directed antibodies AT8, MC-1 and PHF-1 co-localized with epsilon(gamma-glutamyl)lysine immunolabeling in AD NFT. Immunoaffinity purification and immunoblotting experiments demonstrated that PHF tau contains epsilon(gamma-glutamyl)lysine bonds in parietal and frontal cortex in AD. In control cases with NFT present in the entorhinal cortex and hippocampus, indicative of Braak and Braak stage II, epsilon(gamma-glutamyl)lysine bonds were present in PHF tau in parietal and frontal cortex, despite the lack of microscopically detectable NFT or senile plaques in these cortical regions. The presence of PHF tau with epsilon(gamma-glutamyl)lysine bonds in brain regions devoid of NFT in stage II (but regions, which would be expected to contain NFT in stage III) suggests that these bonds occur early in the formation of NFT.     

Immunocytochemical study at the ultrastructural level of neurofibrillary degeneration in Alzheimer's disease

Paired Helical Filaments (PHF), as demonstrated at the ultrastructure level, are one of the main pathological landmarks of Alzheimer's disease (AD). A polyclonal rabbit antiserum raised against PHF had been shown to label tangles and the periphery of plaques at the light microscope level. The same antiserum labels PHF specifically at the ultrastructure level, as demonstrated with a postembedding immunogold technique. Normal cytoskeleton constituents and plaque amyloid were not labelled. Specific labelling of a characteristic landmark may be a clue for a biological marker of AD.     

Improved detection of substantia nigra pathology in Alzheimer's disease.

The role of substantia nigra pathology in Alzheimer's disease (AD) is uncertain. Detection of pathology may be obscured by intraneuronal neuromelanin and influenced by stains. We determined methods for optimal visualization of nigral pathology in 45 cases of AD. For detection of Lewy bodies (LBs), we compared ubiquitin and alpha-synuclein immunostains to hematoxylin and eosin (H&E). For neurofibrillary tangles (NFTs) and neuropil threads (NTs), we compared Gallyas silver and paired helical filament (PHF) immunostains, after bleaching of melanin, to modified Bielschowsky, Gallyas, and PHF alone. The number of LB cases was not different using the three stains. However, more LBs per section were detected using alpha-synuclein (z=4.88, p<0.001). Twice the number of cases exhibited NFT (z=8.21; p<0.001) and the mean NFT number per section was 2.8-5.2-fold greater, using Gallyas and PHF after bleaching compared to without bleaching (chi(2)=142.17; p<0.001). More NTs (z=6.54; p<0.001) were observed with PHF and Gallyas after bleaching. With optimal methods, we found LBs in 27%, NFTs in 89%, and NTs in all 45 AD cases. We show that detection of nigra pathology is influenced by histological method. Clinicopathological studies using these methods are needed to determine the role of nigral pathology in AD.     

Brain levels of CDK5 activator p25 are not increased in Alzheimer's or other neurodegenerative diseases with neurofibrillary tangles

Elevated levels of p25 and constitutive activation of CDK5 have been observed in AD brains. This has led to the hypothesis that increased p25 levels could promote neurofibrillary tangles (NFT) through CDK5-mediated hyperphosphorylation of tau, the principal component of NFTs. We examined p25 immunoreactivity in brains from sporadic and familial AD cases, as well as other neurologic diseases that exhibit NFT, such as Down's syndrome (DS), Pick's disease (Pick), corticobasal degeneration (CBD), progressive supranuclear palsy (PSP), frontotemporal dementia (FTD). Neither the p25 immunoreactivity nor the p25/p35 ratio was elevated in the AD brains or in the other tauopathies (n = 34) compared with controls (n = 11). Although Abeta peptides have been suggested to activate calpain-mediated cleavage of p35 to p25 in cultured neurons, p25 levels in brains of TgCRND8 mice, which express high levels of brain Abeta peptides, were similar to those of non-Tg littermates. Our data suggest that high Abeta levels in brain do not activate p35 proteolysis, and p25 is unlikely to be a causative agent for NFT formation in AD or other tauopathies.     

Transglutaminase and the Neuronal Cytoskeleton in Alzheimer''s Disease

Transglutaminase [EC 2.3.2.13, (R)-glutaminyl-peptide:amine gamma-glutamyltransferase], an enzyme that catalyzes the introduction of glutamine-lysine cross-links into proteins, was purified. Neurofilament and microtubule proteins were substrates for this enzyme but the insoluble neurofibrillary tangles (NFT) isolated from Alzheimer's disease brain were not substrates. In vitro cross-linking of neurofilaments and microtubules by the enzyme did not produce paired helical filaments (PHF), which are the major ultrastructural component of NFT. These results make it unlikely that PHF are formed by the straightforward cross-linking of neurofilaments or microtubules.     

Carboxyl-terminal fragments of presenilin-1 are closely related to cytoskeletal abnormalities in Alzheimer's brains

To clarify the role of presenilin-1 (PS-1) in the pathology of Alzheimer's disease (AD), we tested four antisera to PS-1. The specific antisera to the N-terminus (HSN-2) and C-terminus (HS-C) of PS-1 detected a 44/40kD holoprotein, a 25kD N-terminal fragment (NTF) and a 16kD C-terminal fragment (CTF) of PS-1 in COS-7 cells. The 25kD NTF and 16kD CTF were observed in human brains, and their amounts were not significantly different between the control and AD brains. The antibody HS-C labeled extensive neurofibrillary tangles, dystrophic neurites and curly fibers in the AD brains. In the paired helical filament (PHF) fraction containing A68 protein from AD brains, a smear pattern of CTFs was revealed. Antisera (HS-L292 and HS-L300) to cleavage sites of PS-1 also revealed immunoreactive neurofibrillary tangles in the AD brain sections and the smear pattern of CTFs of A68 protein fraction. The CTFs of PS-1 accumulate with PHF tau, suggesting a close relationship between PS-1 and cytoskeletal abnormalities in AD brains.     

Immunolocalization of an Amino-Terminal Fragment of Apolipoprotein E in the Pick's Disease Brain

Although the risk factor for apolipoprotein E (apoE) polymorphism in Alzheimer''s disease (AD) has been well described, the role that apoE plays in other neurodegenerative diseases, including Pick''s disease, is not well established. To examine a possible role of apoE in Pick''s disease, an immunohistochemical analysis was performed utilizing a novel site-directed antibody that is specific for an amino-terminal fragment of apoE. Application of this antibody, termed the amino-terminal apoE cleavage fragment (nApoECF) antibody, consistently labeled Pick bodies within area CA1 of the hippocampus in 4 of the 5 cases examined. Co-localization of the nApoECF antibody with PHF-1, a general marker for Pick bodies, as well as with an antibody to caspase-cleaved tau (TauC3) was evident within the hippocampus. While staining of the nApoECF antibody was robust in area CA1, little co-localization with PHF-1 in Pick bodies within the dentate gyrus was observed. A quantitative analysis indicated that approximately 86% of the Pick bodies identified in area CA1 labeled with the nApoECF antibody. The presence of truncated apoE within Pick bodies suggests a broader role of apoE beyond AD and raises the question as to whether this protein contributes to pathogenesis associated with Pick''s disease.     

Analysis of N-glycans of pathological tau: possible occurrence of aberrant processing of tau in Alzheimer's disease

In a previous study [Wang et al. (1996) Nat. Med. 2, 871-875], Wang et al. found (i) that abnormally hyperphosphorylated tau (AD P-tau) isolated from Alzheimer's disease (AD) brain as paired helical filaments (PHF)-tau and as cytosolic AD P-tau but not tau from normal brain were stained by lectins, and (ii) that on in vitro deglycosylation the PHF untwisted into sheets of thin straight filaments, suggesting that tau only in AD brains is glycosylated. To elucidate the primary structure of N-glycans, we comparatively analyzed the N-glycan structures obtained from PHF-tau and AD P-tau. More than half of N-glycans found in PHF-tau and AD P-tau were different. High mannose-type sugar chains and truncated N-glycans were found in both taus in addition to a small amount of sialylated bi- and triantennary sugar chains. More truncated glycans were richer in PHF-tau than AD P-tau. This enrichment of more truncated glycans in PHF might be involved in promoting the assembly and or stabilizing the pathological fibrils in AD.     

One of the antigenic determinants of paired helical filaments is related to tau protein

Paired helical filaments (PHF) are unusual neuronal fibers which accumulate progressively in the brain in Alzheimer's disease (AD). The insolubility of PHF in various kinds of solvents enabled us to obtain highly purified PHF, but prevented the application of conventional analytical methods to identify their components. Here we report that antibodies against purified PHF recognize tau protein, a brain-specific microtubule-associated protein, suggesting that a portion of PHF is tau protein.     

Two-Dimensional Characterization of Paired Helical Filament-Tau from Alzheimer's Disease: Demonstration of an Additional 74-kDa Component and Age-Related Biochemical Modifications

Abstract: PHF-tau proteins are the major components of the paired helical filament (PHF) from Alzheimer's disease (AD) neurofibrillary lesions. They differ both qualitatively and quantitatively in their degree of phosphorylation when compared with native tau proteins. However, little is known about the extent and heterogeneity of phosphorylated sites or the isoform composition and the isoelectric variants of PHF-tau. Therefore, we have characterized PHF-tau proteins from cortical brain tissue homogenates of 13 AD patients using two-dimensional gel electrophoresis. Whatever the topographical origin of brain tissue homogenates, PHF-tau proteins shared the same two-dimensional gel electrophoresis profile made of a tau triplet of 55, 64, and 69 kDa. A 74-kDa hyperphosphorylated tau component was detected particularly in the youngest and most severely affected AD patients. This additional component of hyperphosphorylated tau was shown to correspond to the longest brain tau isoform. Furthermore, the isoelectric points of PHF-tau from older AD patients were significantly more basic, indicating a lower degree of phosphorylation. These results show that the severity of neurofibrillary degeneration of AD is modulated by age.     

APOE polymorphism and clinical duration determine regional neuropathology in Swedish APP(670, 671) mutation carriers: implications for late-onset Alzheimer's disease

Neurofibrillary changes throughout the brain were investigated for three relatives who carried the Swedish APP_670,671 mutation which causes overproduction of Aβ40 and Aβ42. They differed in terms of APOE genotype, age at the onset of dementia, and disease duration (P1: ɛ2/3, age 57, 11 years; P2: ɛ2/3, age 61, 5 years; P3: ɛ4/4, age 44, 12 years). For each subject, paraffin-embedded sections from diverse anatomically and cytoarchitectonically well-preserved regions were stained using the modified Bielschowsky method. Neurofibrillary tangles (NFT) and neuritic plaques (NP) were counted, and the area occupied by plaque estimated (%NP). In addition, sections from the medial frontal gyrus were stained with monoclonal antibodies to APOE. The regional patterns of neurofibrillary changes were consistent with those for late-onset AD. Longer disease duration was associated with further accumulations in earlier-affected areas, with superficial cortical layers consistently containing higher %NP than deep layers. APOE ɛ4/4 was associated with deeper limbic and frontal NFT, with an excess of NP (especially in the outer parietal cortex) which stained heavily for APOE - as well as with very early onset. APP_670,671 mutation carriers demonstrate regional brain neurofibrillary changes characteristic of late-onset Alzheimer's disease with evidence for more Aβ deposition for ɛ4/4 than ɛ2/3. This raises the possibility that early Braak Stage I-II lesions might also follow this pattern of promotion.     

Alzheimer's Disease Neurofibrillary Tangles Contain Mitosis-Specific Phosphoepitopes

Abstract: Paired helical filaments (PHFs) are the major components of neurofibrillary lesions present in Alzheimer's disease (AD). PHFs are composed of the microtubule-associated protein (MAP) τ, which is abnormally phosphorylated in AD. Normal fetal τ is also phosphorylated and shares certain phosphoepitopes with PHF-τ. The abnormal phosphorylation of PHF-τ is considered to be involved in the formation of PHFs and subsequent degeneration of AD neurons. We have previously shown that other neuronal MAPs, such as MAP1B, contain mitosis-specific phosphoepitopes. In addition to mitotic cells, these epitopes are also expressed in fetal brain and PC12 cells during differentiation and neurite outgrowth. One hypothesis regarding the etiology of AD involves the reactivation of a fetal-like state and mitotic conditions in selected neurons. To determine if similar mitosis-associated phosphoepitopes appeared in AD, sections of hippocampal tissue were stained for immunoreactivity with antibodies recognizing both τ and mitotic phosphoepitopes. Both the MPM2 mitotic phosphoepitope antibody and the AT8 PHF-τ antibody stained neurofibrillary lesions and colocalized to pyramidal neurons in AD samples. In addition, PHFs isolated from an AD brain reacted with both antibodies. The MPM2 antibody specifically reacted with τ in the isolated PHF fraction but not normal adult τ. In addition, MPM2 failed to react with normal fetal or adult τ obtained from rat brains. The MPM2 antibody also recognized human MAP1B; however, MAP1B was not present in the PHF fraction. Our results indicate that MPM2 recognized a phosphoepitope present on PHF-τ. Because normal fetal or adult rat brain τ did not express the MPM2 epitope, it is likely that this phosphoepitope is specific for the disease state.     

Polymer hollow fiber-encapsulated peripheral nerve extracts change their activity towards injured hippocampal neurites in rats

The regeneration of the adult mammalian central nervous system (CNS) requires changes of the nonpromising environment. Applying peripheral nerve grafts and their extracts are both the useful method to induce regeneration of injured CNS neurites. Our previous reports showed that degeneration of peripheral nerves enhanced their neurotrophic activity in a time-dependent manner. Electrophoretical analysis of proteins obtained from degenerating sciatic nerves revealed significant changes in fractions of low molecular mass. The aim of the present work was to examine the influence of fractionated extracts from 7-day-predegenerated and non-predegenerated peripheral nerves upon injured hippocampal neurites in adult rats. The extracts were closed in fibrin-filled connective tissue chambers (CTC) or within CTC-wrapped polymer hollow fibers (PHF) of 30 kDa cut-off. The cell bodies of regrowing fibers were labeled with FITC-HRP. The CTCs appeared to be useful tool for implantation of artificial grafts into mammalian CNS. Full-spectrum nerve extracts induced strong regeneration of injured hippocampal neurites. The number of labeled cells within hippocampus was significantly lower in PHF groups than in CTC ones, indicating that low-mass proteins present in peripheral nerve extracts are not sufficient to induce successful regeneration.     

配对螺旋样纤维(PHF)-Tau与神经细胞的死亡

神经纤维缠结(neurofibrillary tangles, NFT)是老年性痴呆病的重要病理特征,人类神经tau分子聚集形成的配对螺旋样纤维(paired helical filaments, PHF)是NFT的主要成分.最近研究表明,PHF与神经细胞的坏死和程序化死亡密切相关,其作用机理可能是诱发细胞内氧化自由基系统的启动而导致神经细胞死亡的.     

4-Hydroxynonenal-Derived Advanced Lipid Peroxidation End Products Are Increased in Alzheimer's Disease

Abstract: Recent studies have demonstrated oxidative damage is one of the salient features of Alzheimer's disease (AD). In these studies, glycoxidation adduction to and direct oxidation of amino acid side chains have been demonstrated in the lesions and neurons of AD. To address whether lipid damage may also play an important pathogenic role, we raised rabbit antisera specific for the lysine-derived pyrrole adducts formed by lipid peroxidation-derived 4-hydroxynonenal (HNE). These antibodies were used in immunocytochemical evaluation of brain tissue from AD and age-matched control patients. HNE-pyrrole immunoreactivity not only was identified in about half of all neurofibrillary tangles, but was also evident in neurons lacking neurofibrillary tangles in the AD cases. In contrast, few senile plaques were labeled, and then only the dystrophic neurites were weakly stained, whereas the amyloid-β deposits were unlabeled. Age-matched controls showed only background HNE-pyrrole immunoreactivity in hippocampal or cortical neurons. In addition to providing further evidence that oxidative stress-related protein modification is a pervasive factor in AD, the known neurotoxicity of HNE suggests that lipid peroxidation may also play a role in the neuronal death in AD that underlies cognitive deficits.     

Hierarchical Phosphorylation of Recombinant Tau by the Paired-Helical Filament-Associated Protein Kinase Is Dependent on Cyclic AMP-Dependent Protein Kinase

Abstract : Immunoaffinity-purified paired helical filaments (PHFs) from Alzheimer's disease (AD) brain homogenates contain an associated protein kinase activity that is able to induce the phosphorylation of PHF proteins on addition of exogenous MgCl₂ and ATP. PHF kinase activity is shown to be present in immunoaffinity-purified PHFs from both sporadic and familial AD, Down's syndrome, and Pick's disease but not from normal brain homogenates. Although initial studies failed to show that the kinase was able to induce the phosphorylation of tau, additional studies presented in this article show that only cyclic AMP-dependent protein kinase-pretreated recombinant tau is a substrate for the PHF kinase activity. Deletional mutagenesis, phosphopeptide mapping, and site-directed mutagenesis have identified the PHF kinase phosphorylation sites as amino acids Thr³⁶¹ and Ser⁴¹² in htau40. In addition, the cyclic AMP-dependent protein kinase phosphorylation sites that direct the PHF kinase have been mapped to amino acids Ser³⁵⁶ and Ser⁴⁰⁹ in htau40. Additional data demonstrate that these hierarchical phosphorylations in the extreme C terminus of tau allow for the incorporation of recombinant tau into exogenously added AD-derived PHFs, providing evidence that certain unique phosphorylations of tau may play a role in the pathogenesis of neurofibrillary pathology in AD.     

Synthesis and biological evaluation of novel styryl benzimidazole derivatives as probes for imaging of neurofibrillary tangles in Alzheimer’s disease

This paper describes the synthesis and biological evaluation of styrylbenzimidazole (SBIM) derivatives as agents for imaging neurofibrillary tangles (NFT) in patients with Alzheimer’s disease (AD). SBIM derivatives were prepared with 4-iodobenzene-1,2-diamine and substituted cinnamaldehydes. In binding experiments using recombinant tau and Aβ_1–42 aggregates, SBIM-3 showed higher affinity for the tau aggregates than Aβ_1–42 aggregates (ratio of K_d values was 2.73). In in vitro autoradiography and fluorescent staining, [¹²⁵I]SBIM-3 (or SBIM-3) bound NFT in sections of AD brain tissue. In biodistribution experiments using normal mice, all [¹²⁵I]SBIM derivatives showed high initial uptake into (3.20–4.11%ID/g at 2 min after the injection) and rapid clearance from (0.12–0.33%ID/g at 60 min after the injection) the brain. In conclusion, appropriate structural modifications of SBIM derivatives could lead to more useful agents for the in vivo imaging of NFT in AD brains.     

A genomic screen for modifiers of tauopathy identifies puromycin-sensitive aminopeptidase as an inhibitor of tau-induced neurodegeneration

Neurofibrillary tangles (NFT) containing tau are a hallmark of neurodegenerative diseases, including Alzheimer's disease (AD). NFT burden correlates with cognitive decline and neurodegeneration in AD. However, little is known about mechanisms that protect against tau-induced neurodegeneration. We used a cross species functional genomic approach to analyze gene expression in multiple brain regions in mouse, in parallel with validation in Drosophila, to identify tau modifiers, including the highly conserved protein puromycin-sensitive aminopeptidase (PSA/Npepps). PSA protected against tau-induced neurodegeneration in vivo, whereas PSA loss of function exacerbated neurodegeneration. We further show that human PSA directly proteolyzes tau in vitro. These data highlight the utility of using both evolutionarily distant species for genetic screening and functional assessment to identify modifiers of neurodegeneration. Further investigation is warranted in defining the role of PSA and other genes identified here as potential therapeutic targets in tauopathy.     

Human fetal tau protein isoform: possibilities for Alzheimer's disease treatment

While early 1990s reports showed the phosphorylation pattern of fetal tau protein to be similar to that of tau in paired helical filaments (PHF) in Alzheimer's disease (AD), neither the molecular mechanisms of the transient developmental hyperphosphorylation of tau nor reactivation of the fetal plasticity due to re-expression of fetal protein kinases in the aging and AD human brain have been sufficiently investigated. Here, we summarize the current knowledge on fetal tau, adding new data on the specific patterns of tau protein and mRNA expression in the developing human brain as well as on change in tau phosphorylation in the perforant pathway after entorhinal cortex lesion in mice. As fetal tau isoform does not form PHF even in a highly phosphorylated state, understanding its expression and post-translational modifications represents an important avenue for future research towards the development of AD treatment and prevention.