Neurofibrillary Tangles In Sections Research Articles

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 Aβ1–42 aggregates, SBIM-3 showed higher affinity for the tau aggregates than Aβ1–42 aggregates (ratio of Kd 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 2min after the injection) and rapid clearance from (0.12–0.33%ID/g at 60min 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.

Phenyldiazenyl benzothiazole derivatives as probes for in vivo imaging of neurofibrillary tangles in Alzheimer's disease brains

This paper describes the synthesis and biological evaluation of novel phenyldiazenyl benzothiazole (PDB) derivatives as probes for imaging neurofibrillary tangles (NFTs) in patients with Alzheimer's disease (AD). We successfully synthesized three PDB derivatives using a diazo coupling reaction. In binding experiments in vitro, the compounds displayed higher affinity for tau aggregates than for Aβ aggregates. In fluorescent staining experiments using AD brain sections, 9 visualized NFTs clearly. No-carrier-added radioiodinated PDB derivatives were successfully prepared through an iododestannylation reaction from the corresponding tributyltin derivatives. [125I]9 labeled NFTs in sections of brain tissue from a patient with AD, but not a control. In biodistribution experiments using normal mice, the PDB derivatives displayed an uptake into the brain, sufficient for imaging NFTs, ranging from 0.94 to 3.2% ID g−1, but a relatively slow washout. Although further modifications are necessary to improve the pharmacokinetics in the brain, PDB with high affinity for tau aggregates may be useful as a backbone structure to develop agents for imaging NFTs in AD brains.

Immunocytochemistry of neurofibrillary tangles with antibodies to subregions of tau protein: identification of hidden and cleaved tau epitopes and a new phosphorylation site.

Antibodies to multiple epitopes spanning the length of the tau molecule were used to study Alzheimer neurofibrillary tangles (NFT) using immunocytochemical methods and several different methods of fixation and tissue processing, including staining of vibratome sections, hydrated autoclaving of paraffin sections and immunofluorescence of NFT isolated from fresh brain tissue. Smears and sections were pretreated with trypsin and/or phosphatase to further characterize antibody binding. In tissue fixed briefly in periodate-lysine-paraformaldehyde, tau immunoreactivity was detected in astrocytes, but only a few tau epitopes were detected in NFT with this fixation method. In contrast, all tau epitopes were detected in NFT in tissue fixed in formaldehyde for prolonged periods of time. In the hippocampus, the number of NFT detected in the dentate fascia was in proportion to the duration of dementia, as we previously noted. Dentate fascia NFT were intracellular (i-NFT) and were reactive with antibodies recognizing epitopes in both the carboxy- and amino-terminal regions of tau, but not the microtubule-binding domain of tau, suggesting that microtubule-binding domain epitopes are hidden in i-NFT. In contrast, NFT in the subiculum and layer II of the parahippocampal cortex were mostly extracellular (e-NFT), especially in severe cases of long duration. e-NFT were immunoreactive with antibodies to the microtubule-binding domain, but only weakly reactive with antibodies to carboxy- or amino-terminal epitopes, suggesting that e-NFT may contain fragments of tau. In both isolated NFT and NFT in sections, amino-terminal epitopes, including the Alz-50 epitope, were sensitive to trypsin proteolysis, which suggests that the lack of staining of e-NFT by antibodies to the amino-terminal regions of tau is due to proteolysis. Antibodies reactive with amino-terminal epitopes also stained fewer NFT following hydrated autoclaving, while those reacting with the carboxy half of tau stained more NFT after hydrated autoclaving. Thus, although carboxy-terminal regions are not detected in e-NFT, they are probably masked, rather than proteolytically cleaved, since they can be revealed by hydrated autoclaving. Finally, phosphatase treatment of isolated NFT revealed enhanced immunostaining not only with Tau-1, as in previous studies demonstrating abnormal phosphorylation of tau proteins in NFT, but also with an antibody to exon 2, which reveals yet another phosphorylation site in tau of NFT.

Selective Expression of Epitopes in Multiphosphorylation Repeats of the High and Middle Molecular Weight Neurofilament Proteins in Alzheimer Neurofibrillary Tangles

Here we review our recent "epitope analyses" of a few of the fibrous intraneuronal inclusions that are distinctive hallmarks of human neurodenerative conditions using a large library of monoclonal antibodies (MAbs) raised to normal neuronal cytoskeletal proteins. Analyses of the low (NF-L), middle (NF-M), and high (NF-M), and high (NF-H) molecular weight neurofilament (NF) proteins with greater than 500 MAbs enumerated epitopes shared by NF proteins and the intraneuronal neurofibrillary tangles (NFTs) that occur in the hippocampus and brainstem of Alzheimer's disease (AD) subjects. We identified the NF-H multi-phosphorylation repeat domain, i.e. repeats of Lys-Ser-Pro-X (where X is a small uncharged amino acid and Ser acts as a phosphate acceptor), as the determinant recognized by 15/16 MAbs that detected NFTs in sections of AD hippocampus, and 11 of the same 16 MAbs recognised NF-M multi-phosphorylation repeats. Further, the antigen binding regions of these MAbs were shown to comprise 13 separate classes based on their differential binding to 12 synthetic peptides derived from the NF-H and NF-M multi-phosphorylation sites, NF subunits of 10 diverse mammalian and sub-mammalian species, and normal human tau (tau). None of these anti-NF MAbs recognized NFTs in the brainstem of subjects with progressive supranuclear palsy (PSP), but NFTs in AD brainstem sections were reactive with five of these MAbs. Both PSP and AD brainstem NFTs were recognized by MAbs specific for tau and paired helical filament antigens.(ABSTRACT TRUNCATED AT 400 WORDS)

Ubiquitin-protein conjugates in Alzheimer's lesions

The ubiquitin-dependent protein degradation system plays a major role in the removal of abnormal and denatured proteins which may form insoluble aggregates in pathological conditions or during other cellular stress. Neuritic plaques and neurofibrillary tangles in sections of Alzheimer's cortex contain insoluble aggregates of proteins and are shown here to specifically immunostain with an antiserum to ubiquitin-protein conjugates. Plaque core amyloid and normal neurons do not immunostain and sodium dodecyl sulphate (SDS)-insoluble tangle preparations are not ubiquitin-positive on slot blots. The possible role and consequences of ubiquitination in tangle and plaque production in Alzheimer's disease are discussed.