Fig. 8

Mutant Tau^AT does not aggregate, adopts a pathological conformation and anti-aggregation compounds do not rescue the paralysis. a Sequential extraction of tau from aged animals (mixed stage consisting worms of mostly > 3 days old). CK10 line transgenic for htauV337M (1N4R) accumulates detergent insoluble tau (insoluble aggregated tau); while no insoluble tau is detected in Tau^wt or Tau^AT lines. 2N4R-tau (single arrow) in Tau^wt and Tau^AT lines migrates slower than the 1N4R-tau (double arrow) in CK10 worms. With age, more Tau accumulates in the detergent soluble fraction (representing Tau bound to membranous structures) (see also Additional file 13A for day-1 old animals). b Bar diagram of anti-aggregant Tau construct Tau^AT+PP with A152T mutation plus two additional proline substitutions in the hexapeptide motifs (htau40A152TI ²⁷⁷ PI ³⁰⁸ P). c 30 day-1 adult animals from non-tg, Ex[Tau^wt], Ex[Tau^AT] and its anti-aggregant variant Ex[Tau^AT+PP] after lysis in 1 x sample buffer, then subjected to 10 % PAGE and subsequent western blot analysis using K9JA-pan-tau antibody. Ex[Tau^wt], Ex[Tau^AT] and its anti-aggregant variant Ex[Tau^AT+PP] carrying the respective Tau transgenes as extrachromosomal arrays (Ex) show comparable levels of Tau expression. Tubulin serves as loading control. d Micrographs of the worms. The anti-aggregant variant of mutant Tau^AT (Tau^AT+PP) is equally toxic and produces a similar paralytic phenotype as the single mutant Tau^AT. Note the absence of tracks and coiled body in Ex[Tau^AT+PP] similar to Ex[Tau^AT]. e Mean thrashing assay of day-1 old adult animals carrying Tau^wt, Tau^AT or its anti-aggregant variant (Tau^AT+PP) transgenes as extrachromosomal arrays. Ex[Tau^AT+PP] shows less thrashes than the non-tg (~5 % of non-tg) or Ex[Tau^wt] worms (~9 % of Tau^wt worms). But there is no difference between the Ex[Tau^AT] and its anti-aggregant variant Ex[Tau^AT+PP], indicating that the toxicity does not depend on amyloidogenic aggregation. Non-tg strain serves as control. Error bars denote SEM, n ≥ 30. ***P < 0.001, ns., not significant. One-way ANOVA with Tukey’s test applied for multiple comparisons. f Immunostaining of day-1 old Tau^wt-lo, Tau^wt-hi and Tau^AT-lo with conformation-specific antibody MC1. Tau in Tau^AT-lo worms adopts a pathological state as seen by dense staining in the nerve ring and ventral cord. White arrows show stained neuronal processes either in the nerve ring or ventral cord region. Tau^wt (-lo and -hi) show only mild staining occasionally. g Worm extracts prepared from mixed stage adults in buffer C, resolved by native PAGE and immunoblotted with K9JA show Tau enriched in soluble high molecular weight complexes in Tau^AT worm extracts. In addition to two bands common to both Tau^wt-lo and Tau^AT-lo lysates (~170 KDa and >250 KDa marked by asterisks), a smear in the range of 72-95 KDa corresponding to lower oligomeric species and a higher band (> > 250 KDa, black arrowhead) can be seen in the Tau^AT-lo lysate. h Mean number of bends per 30 s of Tau^AT-lo worms treated either with DMSO (solvent control) or with 50 or 100 μM concentrations each of known aggregation inhibitors of Tau (Rhodanine compound bb14, PTH compound BSc3094 in DMSO). The lack of rescue indicates that the toxicity of Tau^AT is based on some mechanism distinct from aggregation (for comparison see [17]). Error bars denote SEM. One-way ANOVA with Tukey’s test applied for multiple comparisons (ns., not significant)