Home » AT2 Receptors » one-way ANOVA with Dunnett’s test was performed with = 18 for WT and = 3 for each tau mutant

one-way ANOVA with Dunnett’s test was performed with = 18 for WT and = 3 for each tau mutant

one-way ANOVA with Dunnett’s test was performed with = 18 for WT and = 3 for each tau mutant. 4). In addition, mutations are directly associated with frontotemporal dementia with parkinsonism linked to chromosome 17 (FTDP-17t) (5, 6). As an MT-associated protein, tau binds longitudinally along the MT surface, providing YKL-06-061 MT stability and promoting tubulin assembly (7,C10). Physiologically, tau is usually primarily expressed in neurons and is concentrated in the distal axon (11). In the human brain, tau protein is usually alternatively spliced into six major different isoforms based on inclusion or exclusion of exons 2, 3, and 10 (12, 13). Inclusion of one or two N-terminal domains generates 0N, 1N, and 2N isoforms due to alternative splicing of exons 2 and 3. Varied forms of tau also result from the presence of three (3R) or four (4R) MT-binding repeats of 31 or 32 amino acids due to alternative splicing of exon 10 (14, 15). More than Rabbit polyclonal to CapG 50 pathogenic mutations have been identified (5, 6, 16). Of note, many of these are intronic and silent mutations that affect exon 10 splicing and thus the ratio of 3R/4R tau isoforms expressed. Missense mutations directly alter the primary protein sequence, but in some cases they can also affect exon 10 splicing (6, 16). Most tau missense mutations are clustered within the MT-binding domain name (MTBD) (6, 17), suggesting that impairment of tauCMT interactions can be directly involved in pathogenesis. Loss of MT mass due to MT instability is usually a common feature of AD (18,C21). tauCMT dysfunction can affect synaptic plasticity and impair axonal transport of vesicles and other molecules, causing cognitive deficits in learning and memory (22,C25). Defects in tau can also activate MT-severing proteins such as katanin and cause degradation of MTs (26). Predominantly studies indicate that tau mutants can alter tubulin assembly and MT binding (17, 27). tau post-translational modifications such as phosphorylation can also decrease MT-binding activity (28,C30). Because of the progressive nature of tauopathies, tau aggregation has been hypothesized to propagate from neuron to neuron by a prion-like mechanism (31). In clinical staging of AD patients, it has been proposed that aggregated forms of tau may spread from the hippocampus to the entorhinal region, and eventually to the rest of the neocortex (32, 33). Experimentally, tau can transfer from cell to cell and can be seeded by preformed aggregated tau fibrils to induce aggregation (34,C36). Transgenic mouse models of tau can be injected with tau seeds of recombinant proteins, mouse brain lysate, and even human brain lysate to induce neurofibrillary tangles with tau fibrillar aggregates (37,C41). Although there is no clinical evidence of iatrogenic spread between AD patients (42), experimental studies support the hypothesis that tau can spread in a prion-like manner along anatomical connections YKL-06-061 to other neurons. A previous study used a cell-based assay to examine prion-like seeding in 19 missense pathogenic tau mutants and revealed that only mutants at the Pro-301 position were uniquely prone to seed induced YKL-06-061 aggregation (43). Building from this unexpected finding, we investigated and characterized an extensive series of tau mutants for MT binding using a mammalian cell-based assay, and we extended the previous series of pathogenic tau mutants for prion-like seeding. These studies show that most tau mutants share a common mechanism of impaired MT binding with only heterogeneous potential for aggregation. Results tau variants with mutations at the Pro-301 position severely impaired MT binding compared with WT tau and several other tau mutants in the R1 and R2 repeats Although tauCMT associations can be visualized in the cytoplasm by immunofluorescent labeling, the amount of tau that is directly bound to MTs cannot be quantified by this method (Fig. S1). A previously established cell-based MT-binding assay (44,C46) was performed on diverse tau missense mutants (Fig. 1) to assess changes in MT binding associated with a spectrum of tau variants with missense tau mutations. Furthermore, most previous studies investigated tau mutants with an MT-binding assay that used recombinant tau expressed from bacteria and tubulin assembled from bovine or porcine sources (Table 1). This cell-based MT-binding assay is usually more physiologically relevant as it, at least partially, incorporates the effects of post-translational modifications such as phosphorylation (Fig. S2), differential tau folding in mammalian cells, and interactions with human MT isotypes in HEK293T cells. The 0N4R human tau isoform was used for all.