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Intracellular aggregation of the microtubule-associated protein tau into filamentous inclusions is a defining characteristic of Alzheimer disease. Because appearance of tau-aggregate bearing lesions correlates with both cognitive decline and neurodegeneration, it has been hypothesized that tau aggregation may be directly toxic to cells that harbor them. Testing this hypothesis in cell culture has been complicated by the resistance of full-length tau isoforms to aggregation over experimentally tractable time periods. To overcome this limitation, a smallmolecule agonist of the tau aggregation reaction, Congo red, was used to drive aggregation within HEK-293 cells expressing fulllength tau isoform htau40. Formation of detergent-insoluble aggregates was both time and agonist concentration dependent. At 10 M Congo red, detergent-insoluble aggregates appeared with pseudo-first order kinetics and a half-life of approximately 5 days. By 7 days in culture, total tau levels increased 2-fold, with ϳ30% of total tau converted into detergent-insoluble aggregates. Agonist addition also led to rapid losses in the tubulin binding activity of tau, although tau was not hyperphosphorylated as judged by occupancy of phosphorylation sites Ser 396 / Ser 404 . Tau aggregation was associated with decreased viability as detected by ToPro-3 uptake. The results, which establish a new approach for analysis of tau aggregation in cells independent of tau hyperphosphorylation, suggest that conformational changes associated with aggregation are incompatible with microtubule binding, and that toxicity associated with intracellular tau aggregation is not acute but develops over a period of days.

Alzheimer's disease is characterized in part by the aggregation of tau protein into filamentous inclusions. Because tau filaments form in brain regions associated with memory retention, and because their appearance correlates well with the degree of dementia, they have emerged as robust markers of disease progression. Yet the discovery that mutations in tau protein can lead directly to filament and tangle formation in humans, and that filament formation is linked to neurodegeneration in model biological systems, suggests that tau aggregation may also contribute directly to degeneration in affected neurons. In this context, the mechanism of tau filament formation and its modulation by mutation and posttranslational modification is of fundamental importance. Here, recent progress on the molecular mechanisms underlying tau aggregation deduced from in vivo and in vitro experimentation is reviewed and a model rationalizing the effect of posttranslational and other structural modifications on assembly kinetics and thermodynamics is presented. We hypothesize that tau aggregation can be described as a heterogeneous nucleation reaction, where exogenous effectors, tau gene mutations, or other modifications that stabilize assembly-competent conformations of tau act to trigger the fibrillization reaction. In contrast, those that modulate postnuclear equilibria can enhance fibrillization by increasing the free energy difference between polymers and unincorporated monomers, resulting in stabilization of filaments at low bulk protein concentrations.

New methods for analyzing tau fibrillization have yielded insights into the biochemical transitions involved in the process. Here we review the parallels between the sequential progression of tau fibrillization observed macroscopically in Alzheimer's disease (AD) lesions and the pathway of tau aggregation observed in vitro with purified tau preparations. In addition, pharmacological agents for further dissection of fibrillization mechanism and lesion formation are discussed.

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Casein kinase-1 is a family of ubiquitous eukaryotic protein kinases that frequently function in tandem with the ubiquitin modification system to modulate protein turnover and trafficking. In Alzheimer's disease, these enzymes colocalize with ubiquitinated lesions, including neurofibrillary tangles and granulovacuolar degeneration bodies, suggesting they also play a role in disease pathogenesis. To identify binding partners that potentially regulate or recruit these enzymes toward disease lesions, a Sos-recruitment yeast two-hybrid screen was performed with human Ckidelta (the casein kinase-1 isoform most closely linked to granulovacuolar degeneration bodies) and a human brain cDNA library. All interacting clones contained a single open reading frame termed casein kinase-1 binding protein (CK1BP). On the basis of sequence alignments, CK1BP was a structural homologue of the acidic domain of dysbindin, a component of the dystrophin-associated protein complex and the biogenesis of lysosome-related organelles complex-1. CK1BP interacted with full-length Ckidelta, the isolated Ckidelta catalytic domain, Ckigamma2, -gamma3, and -epsilon in the yeast two-hybrid system, and bound Ckidelta and -epsilon in pulldown assays but did not interact with Ckialpha. Interaction with the Ckidelta catalytic domain led to concentration-dependent inhibition of protein kinase activity in the presence of protein substrates tau and alpha-synuclein. Although intact dysbindin did not bind any CK1 isoform, deletion of its coiled-coil domain yielded a protein fragment that behaved much like CK1BP in two-hybrid screens. These data suggest that the acidic domain of dysbindin and its paralogs in humans may function to recruit casein kinase-1 isoforms to protein complexes involved in multiple biological functions.

The composite membranes based on APEEK and SPEEK with good proton conductivity, excellent mechanical and thermal stabilities were prepared successfully.

No abstract