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J. Biol. Chem., Vol. 282, Issue 22, 16454-16464, June 1, 2007

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Tau Aggregation and Toxicity in a Cell Culture Model of Tauopathy^*

Bhaswati Bandyopadhyay¹, Guibin Li¹, Haishan Yin¹², and Jeff Kuret³

From the Center for Molecular Neurobiology, the Department of Molecular and Cellular Biochemistry, and the Ohio State Biochemistry Program, Ohio State University, Columbus, Ohio 43210

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 small-molecule agonist of the tau aggregation reaction, Congo red, was used to drive aggregation within HEK-293 cells expressing full-length tau isoform htau40. Formation of detergent-insoluble aggregates was both time and agonist concentration dependent. At 10 µM 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 30% 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 Ser³⁹⁶/Ser⁴⁰⁴. 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.

Received for publication, January 8, 2007 , and in revised form, April 3, 2007.

^* This work was supported by National Institutes of Health Grant AG14452 and the Alzheimer Association (to J. K.), with equipment access through center Grant P30-NS045758. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

¹ These authors contributed equally to this work.

² Current address: Dept. of Neuropathology, The Johns Hopkins University, Baltimore, MD.

³ To whom correspondence should be addressed: 1060 Carmack Rd., Columbus, OH 43210. Tel.: 614-688-5899; Fax: 614-292-5379; E-mail: kuret.3{at}osu.edu.

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