Three-/four-repeat-dependent aggregation profile of tau microtubule-binding domain clarified by dynamic light scattering analysis

The analysis of the self-assembly mechanism of the tau microtubule-binding domain (MBD) could provide the information needed to develop an effective method for the inhibition of the tau filament formation because of its core region that forms the filament. The MBD domain in the living body consists of similar three or four 31- to 32-residue repeats, namely 3RMBD (R134) and 4RMBD (R1234), respectively. The filament formation of the MBD has been mainly investigated by fluorescence spectroscopy utilizing the β-sheet structure-binding signal sensor thioflavin. This method observes the aggregation indirectly, and provides no information on the time-dependent change in aggregation size or volume. Thus, to determine the structure necessary for initiating MBD self-association, the dynamic light scattering (DLS) method was applied to the analysis of the aggregations of 3RMBD, 4RMBD and their component single repeats and shown to be a powerful tool for directly analyzing filament formation. DLS analysis clearly showed that the building unit for initiating the aggregation is the intermolecular R3-R3 disulfide-bonded dimer for 3RMBD and the intramolecular R2-R3 disulfide-bonded monomer for 4RMBD, and their aggregation processes under physiological condition differ from each other, which has not been clearly revealed by the conventional fluorescence method. The repeat-number-dependent aggregation model of MBD, together with the function of each repeat, reported in this paper should help to devise a method of preventing tau PHF formation.     

An SRp75/hnRNPG complex interacting with hnRNPE2 regulates the 5' splice site of tau exon 10, whose misregulation causes frontotemporal dementia

Tau is a neuronal-specific microtubule-associated protein that plays an important role in establishing neuronal polarity and maintaining the axonal cytoskeleton. Aggregated tau is the major component of neurofibrillary tangles (NFTs), structures present in the brains of people affected by neurodegenerative diseases called tauopathies. Tauopathies include Alzheimer's disease (AD), frontotemporal dementia with Parkinsonism (FTDP-17), the early onset dementia observed in Down syndrome (DS; trisomy 21) and the dementia component of myotonic dystrophy type 1 (DM1). Splicing misregulation of adult-specific exon 10, which codes for a microtubule binding domain, results in expression of abnormal ratios of tau isoforms, leading to FTDP-17. Positions 3 to 19 of the intron downstream of exon 10 define a hotspot of splicing regulation: the region diverges between humans and rodents, and point mutations within it result in tauopathies. In this study, we investigated three regulators of exon 10 splicing: serine/arginine-rich protein SRp75 and heterogeneous nuclear ribonucleoproteins hnRNPG and hnRNPE2. SRp75 and hnRNPG inhibit splicing of exon 10 whereas hnRNPE2 activates it. Using co-transfections, co-immunoprecipitations and RNAi we discovered that SRp75 binds to the proximal downstream intron of tau exon 10 at the FTDP-17 hotspot region; and that hnRNPG and hnRNPE2 interact with SRp75. Thus, increased exon 10 inclusion in FTDP mutants may arise from weakened SRp75 binding. This work provides insights into the splicing regulation of the tau gene and into possible strategies for correcting the imbalance in tauopathies caused by changes in the ratio of exon 10.     

DNA damage, prophage induction and mutation by furazolidone

Ultraviolet absorption data and thermal chromatography through hydroxyapatite (HAP) column revealed that furazolidone treatment of Vibrio cholerae cells produced more than 80% of DNA reversibly bihelical due to the formation of interstrand cross-links and the reaction obeyed a first order relation. Sensitivities of the Escherichia coli strains to the lethal action of the drug were in the order: AB 2480(uvr- rec-) greater than AB 2463(rec-) greater than AB 1886(uvr-) greater than AB 1157(repair proficient) or AB 4401(wild type). Furazolidone was 'Rec test' positive, produced dose-dependent prophage induction in E. coli cells and also dose-dependent streptomycin-resistance forward mutation in V. cholerae cells. The quantitative aspect and also the mode of furazolidone action on DNA were discussed.     

Biochemical characterization of tau protein and its associated syndapin 1 and protein kinase Cɛ for their functional regulation in rat brain

Background

We recently reported that both sulfatide and cholesterol-3-sulfate (SCS) function as potent stimulators for the GSK-3β-mediated phosphorylation of tau protein (TP) in vitro [J. Biochem. 143 (2008) 359–367].

Methods

By means of successive gel filtration on a Superdex 200 pg column and three distinct ion-exchange column chromatographies, TP and its associated proteins were highly purified from the extract of rat brain.

Results

We found that (i) syndapin 1 and novel protein kinase C? (nPKC?) were identified as the TP-associated proteins; (ii) SCS highly stimulated the phosphorylation of TP and syndapin 1 by nPKC? as well as CK1; (iii) the full phosphorylation of TP and syndapin 1 by nPKC? in the presence of sulfatide resulted in their dissociation; (iv) TP primed by CK1 functioned as an effective phosphate acceptor for GSK-3β; (v) syndapin 1 highly stimulated the GSK-3β-mediated phosphorylation of TP; and (vi) TP isoforms were highly expressed in aged brain, whereas syndapin 1 was consistently detected in adult brain, but not in newborn brain.

General significance

These results provided here suggest that (i) TP-associated nPKC? suppresses the GSK-3β-mediated phosphorylation of TP through the phosphorylation of GSK-3β by the kinase in vitro; and (ii) SCS act as effective sole mediators to induce the GSK-3β-mediated high phosphorylation of both TP and its associated syndapin 1 involved in the biochemical processes of neuronal diseases, including Alzheimer's disease.     

Mechanisms and consequences of impaired lipid trafficking in Niemann–Pick type C1-deficient mammalian cells

Niemann–Pick C disease is a fatal progressive neurodegenerative disorder caused in 95% of cases by mutations in the NPC1 gene; the remaining 5% of cases result from mutations in the NPC2 gene. The major biochemical manifestation of NPC1 deficiency is an abnormal sequestration of lipids, including cholesterol and glycosphingolipids, in late endosomes/lysosomes (LE/L) of all cells. In this review, we summarize the current knowledge of the NPC1 protein in mammalian cells with particular focus on how defects in NPC1 alter lipid trafficking and neuronal functions. NPC1 is a protein of LE/L and is predicted to contain thirteen transmembrane domains, five of which constitute a sterol-sensing domain. The precise function of NPC1, and the mechanism by which NPC1 and NPC2 (both cholesterol binding proteins) act together to promote the movement of cholesterol and other lipids out of the LE/L, have not yet been established. Recent evidence suggests that the sequestration of cholesterol in LE/L of cells of the brain (neurons and glial cells) contributes to the widespread death and dysfunction of neurons in the brain. Potential therapies include treatments that promote the removal of cholesterol and glycosphingolipids from LE/L. Currently, the most promising approach for extending life-span and improving the quality of life for NPC patients is a combination of several treatments each of which individually modestly slows disease progression.     

Effects of different anti-tau antibodies on tau fibrillogenesis: RTA-1 and RTA-2 counteract tau aggregation

Tau is the major antigenic component of neurofibrillary pathology in tauopathy, including Alzheimer's disease. Although conversion of soluble tau to an insoluble polymerized fibrillar form is a key factor in the pathogenesis of tauopathy, the mechanism of the change is unclear and no inhibitors of fibril formation are available. Monoclonal antibodies against the 1st or 2nd repeat of the microtubule binding domain, but not the C-terminal 16 residues, completely inhibited tau aggregation into PHF. Furthermore, they did not inhibit tau-induced tubulin assembly. Thus, they are useful to investigate tau protein conversion and will be useful therapeutic lead materials.     

The missing link in the amyloid cascade of Alzheimer’s disease – Metal ions

Progressive deposition of amyloid beta (Aβ) peptides into amyloid plaques is the pathological hallmark of Alzheimer’s disease (AD). The amyloid cascade hypothesis pins this deposition as the primary cause of the disease, but the mechanisms that causes this deposition remain elusive. An increasing amount of evidence shows that biometals Zn(II) and Cu(II) can interact with Aβ, thus influencing the fibrillization and toxicity. This review focuses on the role of Zn(II) and Cu(II) in AD, and revisits the amyloid cascade hypothesis demonstrating the possible roles of Zn(II) and Cu(II) in the disease pathogenesis.     

Lipid peroxidation and neurodegenerative disease

Lipid peroxidation is a complex process involving the interaction of oxygen-derived free radicals with polyunsaturated fatty acids, resulting in a variety of highly reactive electrophilic aldehydes. Since 1975, lipid peroxidation has been extensively studied in a variety of organisms. As neurodegenerative diseases became better understood, research establishing a link between this form of oxidative damage, neurodegeneration, and disease has provided a wealth of knowledge to the scientific community. With the advent of proteomics in 1995, the identification of biomarkers for neurodegenerative disorders became of paramount importance to better understand disease pathogenesis and develop potential therapeutic strategies. This review focuses on the relationship between lipid peroxidation and neurodegenerative diseases. It also demonstrates how findings in current research support the common themes of altered energy metabolism and mitochondrial dysfunction in neurodegenerative disorders.     

O-GlcNAc Modification of tau Directly Inhibits Its Aggregation without Perturbing the Conformational Properties of tau Monomers

The aggregation of the microtubule-associated protein tau into paired helical filaments to form neurofibrillary tangles constitutes one of the pathological hallmarks of Alzheimer's disease. Tau is post-translationally modified by the addition of N-acetyl-d-glucosamine O-linked to several serine and threonine residues (O-GlcNAc). Previously, increased O-GlcNAcylation of tau has been shown to block the accumulation of tau aggregates within a tauopathy mouse model. Here we show that O-GlcNAc modification of full-length human tau impairs the rate and extent of its heparin-induced aggregation without perturbing its activity toward microtubule polymerization. O-GlcNAcylation, however, does not impact the “global-fold” of tau as measured by a Förster resonance energy transfer assay. Similarly, nuclear magnetic resonance studies demonstrated that O-GlcNAcylation only minimally perturbs the local structural and dynamic features of a tau fragment (residues 353–408) spanning the last microtubule binding repeat to the major GlcNAc-acceptor Ser400. These data indicate that the inhibitory effects of O-GlcNAc on tau aggregation may result from enhanced monomer solubility or the destabilization of fibrils or soluble aggregates, rather than by altering the conformational properties of the monomeric protein. This work further underscores the potential of targeting the O-GlcNAc pathway for potential Alzheimer's disease therapeutics.     

Synthesis and evaluation of 1,2,3,4-tetrahydro-1-acridone analogues as potential dual inhibitors for amyloid-beta and tau aggregation

Amyloid-β (Aβ) and tau protein are two crucial hallmarks in Alzheimer’s disease (AD). Their aggregation forms are thought to be toxic to the neurons in the brain. A series of new 1,2,3,4-tetrahydro-1-acridone analogues were designed, synthesized, and evaluated as potential dual inhibitors for Aβ and tau aggregation. In vitro studies showed that compounds 25–30 (20?μM) with N-methylation of the quinolone ring effectively inhibited Aβ_1-42 aggregation by 84.7%–99.5% and tau aggregation by 71.2%–101.8%. Their structure-activity relationships are discussed. In particular, 30 could permeate the blood-brain barrier, bind to Aβ_1-42 and tau, inhibit Aβ_1-42 β-sheets formation, and prevent tau aggregation in living cells.