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Notes: Abstract: Microtubule-associated protein τ is abnormally hyperphosphorylated and aggregated in affected neurons of Alzheimer disease brain. This hyperphosphorylated τ can be dephosphorylated at some of the abnormal phosphorylated sites by purified protein phosphatase-1, 2A, and 2B in vitro. In the present study, we have developed an assay to measure protein phosphatase activity toward τ-1 sites (Ser199/Ser202) using the hyperphosphorylated τ isolated from Alzheimer disease brain as substrate. Using this assay, we have identified that in normal brain, protein phosphatase-2A and 2B and, to a lesser extent, 1 are involved in the dephosphorylation of τ. The Km values of dephosphorylation of the hyperphosphorylated τ by protein phosphatase-2A and 2B are similar. The τ phosphatase activity is decreased by ∼30% in brain of Alzheimer disease patients compared with those of age-matched controls. These findings suggest that a defect of protein phosphatase could be the cause of the abnormal hyperphosphorylation of τ in Alzheimer disease.

Notes: Abstract: In Alzheimer's disease, paired helical filaments composed mainly of abnormally phosphorylated τ accumulate in certain selected neurons of the brain, and microtubules are rarely seen in the affected cells. In the present study, the binding of 32P-labeled 8-azidoguanosine triphosphate ([γ-32P]8N3GTP), the photoaffinity analogue of GTP, to the β-subunit of tubulin in brain homogenates was found to be markedly lower in patients with Alzheimer's disease than in aged control human cases. No significant differences were observed in the levels of the α- and β-subunits of tubulin between Alzheimer's disease and control brains obtained 2–7 h postmortem. In nine of 19 Alzheimer's disease and 11 of 12 control autopsied brains (2–7 h postmortem and stored at −75°C) tubulin was isolated successfully from brain cytosol by in vitro polymerization induced with DEAE-dextran. The GTP binding was observed in the two cycled assembled microtubule preparations from all the normal control, and in eight of nine Alzheimer's disease cases. Alzheimer's disease microtubule preparations contained varying amounts of abnormally phosphorylated τ, whereas no abnormal τ was detected in the control brain preparations. Addition of bovine τ to bovine, normal human, and Alzheimer's disease brain tubulin preparations markedly increased GTP binding to the β-subunit. An alkaline phosphatase-treated paired helical filament-enriched preparation increased by approximately twofold the GTP binding to bovine brain tubulin. GTP binding to tubulin prepared by phosphocellulose chromatography of two cycled microtubules from three Alzheimer's disease and three normal control brains, revealed insignificant differences between the two groups. These findings have suggested that (1) τ protein promotes the GTP binding to the β-subunit of tubulin, and (2) the breakdown of the microtubule system in brains of patients with Alzheimer's disease might in part be due to the abnormal phosphorylation of τ which depresses the GTP binding.

Notes: Abstract: Abnormally hyperphosphorylated τ is the major protein subunit of paired helical filaments in Alzheimer brains. We have examined its site-specific dephosphorylation by different protein phosphatases. Dephosphorylation of τ was monitored by its interaction with several phosphorylation-dependent antibodies. Alzheimer τ was dephosphorylated by brain protein phosphatase-2B at the abnormally phosphorylated sites Ser46, Ser199, Ser202, Ser235, Ser396, and Ser404, and its relative mobility on sodium dodecyl sulfate-polyacrylamide gel electrophoresis shifted to that of normal τ. Protein phosphatases-1 and -2A could dephosphorylate only some of the above six phosphorylation sites. These results indicate that protein phosphatase-2B might be involved in hyperphosphorylation of τ in Alzheimer's disease.

Notes: Abstract: Microtubule-associated protein τ is known to be hyperphosphorylated in Alzheimer disease brain and this abnormal hyperphosphorylation is associated with an inability of τ to promote the assembly of microtubule in the affected neurons. Our previous studies demonstrated that abnormally phosphorylated τ could be dephosphorylated after treatment with alkaline phosphatase, thereby suggesting that the abnormal phosphorylation of τ might in part be the result of a deficiency of the phosphoprotein phosphatase system in patients with Alzheimer disease. In the present study we used 32P-labeled phosphorylase kinase and poly(Glu.Tyr) 4:1 as substrates to measure phosphoprotein phosphatase activities in Alzheimer disease and control brains. The activities of phosphoseryl/ phosphothreonyl-protein phosphatase types 1, 2A, 2B, and 2C and of phosphotyrosyl-protein phosphatase in frontal gray and white matters from 13 Alzheimer brains were determined and compared with those from 12 age-matched control brains. The activities of type 1 phosphatase and phosphotyrosyl phosphatase in gray matter and of type 2A phosphatase in both gray and white matters were significantly lower in Alzheimer disease brains than in controls. These findings suggest that the hyperphosphorylation of τ in Alzheimer disease brain could result from a protein dephosphorylation defect in vivo. The decrease in the phosphatase activities in Alzheimer disease might also be involved in the formation of β-amyloid by augmenting the amyloidogenic pathway processing of β-amyloid precursor protein.

Notes: Abstract: Immunocytochemical and peptide sequencing studies indicate that the regulatory protein ubiquitin (Ub) is incorporated into the paired helical filaments (PHF) of Alzheimer disease. In this study, we showed that some antibodies raised to PHF recognize epitopes of Ub. Analysis of the Ub sequences recognized by the antibodies raised to PHF, along with the known specificity of several monoclonal antibodies raised to artificial Ub conjugates, indicates the immunochemical representation of Ub residues 34–76 in PHF. The Ub epitopes recognized by antibodies raised to PHF are distinct from those recognized by antibodies raised to artificial Ub conjugates in two respects. First, antibodies that are raised to PHF and that recognize Ub react with PHF equally, whether denatured or not, whereas those raised to artificial Ub conjugates show greater reaction after denaturation. Second, mapping of the epitopes recognized by two monoclonal antibodies to PHF onto Ub indicates a distinction in the Ub residues recognized, compared with monoclonal antibodies raised to artificial Ub conjugates. The proximity of their epitopes to the site of conjugation, as well as their affinity for PHF polypeptides, suggests that the PHF antibodies that recognize Ub may be directed specifically to Ub epitopes defined by the protein conjugated to Ub.

Notes: Abstract: The microtubule-associated protein τ is abnormally hyperphosphorylated in Alzheimer's disease. Both proline-dependent protein kinases (PDPKs) and non-PDPKs are involved in this hyperphosphorylation of τ. Several PDPKs can phosphorylate τ in vitro and induce Alzheimer-like epitopes to many phosphorylation-dependent antibodies. A similar induction has not been reported with non-PDPKs. In this study we have evaluated six non-PDPKs [cyclic AMP-dependent (A-kinase), calcium/phospholipid-dependent (C-kinase), casein kinase-1 (CK-1), casein kinase-2 (CK-2), calcium/calmodulin-dependent protein kinase II, and calcium/calmodulin-dependent protein kinase from rat cerebellum] for their abilities to induce Alzheimer-like epitopes on τ. Such epitopes were induced by A-kinase, C-kinase, CK-1, and CK-2, but the degree of induction achieved by CK-1 was much greater than with the other kinases. These results suggest that CK-1 may play an important role in the conversion of τ from the normal to the abnormal phosphorylation state in Alzheimer's disease.

Notes: Abstract: The microtubule-associated protein τ which stimulates the assembly of α-β tubulin heterodimers into microtubules, is abnormally phosphorylated in Alzheimer's disease (AD) brain and is the major component of paired helical filaments. In the present study, the levels of τ and abnormally phosphorylated τ were determined in brain homogenates of AD and age-matched control cases. A radioimmuno-slot-blot assay was developed, using a primary monoclonal antibody, Tau-1, and a secondary antibody, antimouse 125I-immunoglobulin G. To assay the abnormally phosphorylated τ, the blots were treated with alkaline phosphatase before immunolabeling. The levels of total τ were about eightfold higher in AD (7.3 ± 2.7 ng/μg of protein) than in control cases (0.9 ± 0.2 ng/μg), and this increase was in the form of the abnormally phosphorylated protein. These studies indicate that the abnormal phosphorylation—not a decrease in the level of τ—is a likely cause of neurofibrillary degeneration in AD.

Notes: Abstract— Oligodendroglia were isolated from fresh and frozen human autopsied brains by a modification of our technique for isolation of neuronal perikarya and astroglia (Iqbal & Tellez-Nagel, 1972). Cerebral white matter was minced in a hypertonic hexose-Ficoll buffered medium, passed through successive screens of decreasing pore size, and the cells were then separated on a discontinuous sucrose density gradient using low speed centrifugation. Cells could also be isolated by substituting sucrose for hexoses in the cell isolation medium. About 95% of the isolated cells had morphology characteristic of oligodendroglia. The usual contaminants in this fraction were capillary fragments, red blood cells, and a few astrocytes. Free myelin was only sparsely seen. About 5–10% of the isolated oligodendroglia had one or more loops of loose membranes extending from the cell plasma membrane. These membraneous loops resembled myelin. The ultrastructural preservation of the cells was poor. The average yield per gram of wet tissue was 52 million cells amounting to 2.4 mg protein, 283 μg DNA, and 94 μg RNA. The protein, DNA, and RNA contents per average cell were 47, 5.3, and 1.8 pg respectively. About 50% of the tissue DNA was recovered as isolated cells, suggesting a larger proportion of oligodendroglia to astroglia, the other principal cell type in human white matter.Comparison of the total protein profiles of the isolated oligodendroglia with those of the purified human myelin on sodium dodecyl sulfate-polyacrylamide gels revealed the presence of all human myelin constituent proteins in the isolated cells except the intermediate (or DM-20) myelin proteins. Densitometry of the gels stained with Fast Green showed that the ratio of protein bands corresponding to myelin encephalitogenic basic protein (BP) to myelin proteolipid protein (PLP) in the isolated cells was three-fold as high as in purified human myelin. The oligodendroglial BP which constituted about 14% of the total cell protein contents, also coelectrophoresed with the myelin BP on pH 4.3 polyacrylamide gels. Furthermore, the two dimensional peptide maps of the tryptic digest of the cell BP labelled with 125I were also identical to the similarly treated myelin BP. The data suggest that the BP can be used as a chemical marker for oligodendroglia.

Notes: Protein phosphatase 5 (PP5) is a 58-kDa novel phosphoseryl/phosphothreonyl protein phosphatase. It is ubiquitously expressed in all mammalian tissues examined, with a high level in the brain, but little is known about its physiological substrates. We found that this phosphatase dephosphorylated recombinant tau phosphorylated with cAMP-dependent protein kinase and glycogen synthase kinase-3β, as well as abnormally hyperphosphorylated tau isolated from brains of patients with Alzheimer's disease. The specific activity of PP5 toward tau was comparable to those reported with other protein substrates examined to date. The PP5 activity toward tau was stimulated by arachidonic acid by 30- to 45-fold. Immunostaining demonstrated that PP5 was primarily cytoplasmic in PC12 cells and in neurons of postmortem human brain tissue. A small pool of PP5 associated with microtubules. Expression of active PP5 in PC12 cells resulted in reduced phosphorylation of tau, suggesting that PP5 can also dephosphorylate tau in cells. These results suggest that PP5 plays a role in the dephosphorylation of tau and might be involved in the molecular pathogenesis of Alzheimer's disease.