Distinct FTDP-17 Missense Mutations in Tau Produce Tau Aggregates and Other Pathological Phenotypes in Transfected CHO Cells

Abstract

Multiple tau gene mutations are pathogenic for hereditary frontotemporal dementia and parkinsonism linked to chromosome 17 (FTDP-17), with filamentous tau aggregates as the major lesions in the CNS of these patients. Recent studies have shown that bacterially expressed recombinant tau proteins with FTDP-17 missense mutations cause functional impairments, i.e., a reduced ability of mutant tau to bind to or promote the assembly of microtubules. To investigate the biological consequences of FTDP-17 tau mutants and assess their ability to form filamentous aggregates, we engineered Chinese hamster ovary cell lines to stably express tau harboring one or several different FTDP-17 mutations and showed that different tau mutants produced distinct pathological phenotypes. For example, delta K, but not several other single tau mutants (e.g., V337 M, P301L, R406W), developed insoluble amorphous and fibrillar aggregates, whereas a triple tau mutant (VPR) containing V337M, P301L, and R406W substitutions also formed similar aggregates. Furthermore, the aggregates increased in size over time in culture. Significantly, the formation of aggregated delta K and VPR tau protein correlated with reduced affinity of these mutants to bind microtubules. Reduced phosphorylation and altered proteolysis was also observed in R406W and delta K tau mutants. Thus, distinct pathological phenotypes, including the formation of insoluble filamentous tau aggregates, result from the expression of different FTDP-17 tau mutants in transfected Chinese hamster ovary cells and implies that these missense mutations cause diverse neurodegenerative FTDP-17 syndromes by multiple mechanisms.

Seeding of Normal Tau by Pathological Tau Conformers Drives Pathogenesis of Alzheimer-like Tangles

“…This is because Tau is a highly soluble and naturally unfolded protein, and therefore, it has been notoriously difficult to make it aggregate in cultured cells. Strategies have been used in the past with minimal success to produce Tau tangles in cells, including overexpression of aggregation-prone Tau mutants (22), overexpression of truncated Tau that fibrillizes more readily than fulllength Tau (41, 42), addition of pre-aggregated amyloid-␤ fibrils (43), treatment conditions that promote Tau phosphorylation (44), and the addition of aromatic dyes such as Congo Red to overcome the kinetic barrier of fibrillization (45). In our optimized system, however, Tau aggregation could be rapidly induced within hours after the introduction of small quantities of misfolded Tau pffs and result in the accumulation of large amounts of insoluble Tau fibrils.…”

“…However, because Tau is a highly soluble protein, overexpressed Tau resists aggregation despite spontaneous hyperphosphorylation in most cell lines. Moreover, high expression of Tau overstabilizes MTs and inhibits cell division, and it is therefore not well tolerated by dividing cultured cells (21,22). Thus, our current understanding of Tau fibrillization has relied on studies using cell-free systems in which the formation of Tau amyloid fibrils is greatly enhanced by polyanionic factors (23)(24)(25).…”

Trans-cellular Propagation of Tau Aggregation by Fibrillar Species

“…We engineered various mutations known to increase Tau aggregation into a four-repeat domain (RD) protein: ⌬K280 (termed ⌬K), P301L, and V337M. The P301L and V337M mutants were combined in one protein (termed LM) to create a mutant form of RD with strongly increased aggregation potential, similar to what has been described previously (28). This "non-physiologic" mutant facilitates assays of transfer events and transcellular propagation of misfolding that depend on efficient formation of intracellular aggregates and complements similar but less robust aggregation phenotypes of the "physiologic" ⌬K mutant.…”

Retarded Axonal Transport of R406W Mutant Tau in Transgenic Mice with a Neurodegenerative Tauopathy

“…Some of these mutations (e.g., P301L) also facilitate the pathological fibrillogenesis of tau (Hasegawa et al, 1998;Hong et al, 1998;Bugiani et al, 1999;D'Souza et al, 1999;Murrell et al, 1999;Rizzu et al, 1999;Barghorn et al, 2000;Pickering-Brown et al, 2000;Rizzini et al, 2000). Finally, several tau gene mutations, most notably R406W, alter the phosphorylation state and solubility of tau, thereby contributing to the formation of fibrillary tau aggregates (Lee et al, 1991;Crowther and Goedert, 2000;Perez et al, 2000;Sahara et al, 2000;Vogelsberg-Ragaglia et al, 2000;Connell et al, 2001;Delobel et al, 2002).…”

“…The RW mutation, located on exon 13 of the tau gene, was identified in three distinct FTDP-17 kindreds (Hutton et al, 1998;Rizzu et al, 1999;Van Swieten et al, 1999). The RW mutation reduces the ability of tau to bind MTs (Hong et al, 1998) and impairs the assembly as well as the stability of MTs in vitro and in cell culture systems Barghorn et al, 2000;Vogelsberg-Ragaglia et al, 2000). Because RW tau Tg mice express tau much more prominently in the somato-dendritic compartment than in axons compared with hWT tau Tg mice, we speculated that this may result from a mutation-induced reduced binding of tau to MTs and therefore retardation of the slow axonal transport of RW tau.…”