The presence of aggregation-prone, neurotoxic fragments of tau has previously been shown (Zilka et al., 2006; Hrnkova et al., 2007). A C-terminal truncation of tau by caspase-3 produces a fragment that promotes aggregation, and is present in AD brain (Gamblin et al., 2003).

The new results in the May 29 online PNAS show that tau is processed in cultured human cells to small fragments of the tau repeat region that then seed the aggregation of full-length protein. In vitro, the fragments induce tau to assume paired helical filament structure, a process that normally requires chemical enhancers of fibrillization. The results suggest that little snippets of the tau repeat domain may be a template for tangle formation, and raise the question of whether protease inhibitors could present a new approach to treating tauopathies.

In the study, first author Yipeng Wang and colleagues characterized the proteolysis and aggregation of tau in human N2a cells engineered for inducible tau expression, a model developed in the Mandelkow lab for studying tau aggregation and neurotoxicity (see ARF Paper Alert). Expression of a tau repeat domain fragment (specifically, the four-repeat isoform Q244-E372 with the FTDP17 mutation δK280) resulted in the appearance of three cleavage products. The protein was first clipped at the N-terminus by a thrombin-like protease and then underwent two more truncations at the C-terminus by an unknown activity. The N-terminal truncated fragment (F1) was soluble in the cells, while the shorter products (F2, F3) turned up in the sarcosyl centrifugation pellet as insoluble proteins along with the aggregated parent protein.

The production of C-terminal truncations (F2, F3) was required to see aggregation of the tau repeat fragment in the cells. When the researchers prevented N-terminal processing by mutating the cleavage site, tau was no longer processed at the N- or C-terminal, and there was no aggregation of the precursor.

Further, the scientists showed that expression of a short F3 fragment nucleates aggregation of coexpressed full-length wild-type tau, a form that does not normally aggregate. F3 also induced aggregation of the FTDP17 mutants δK280 and P301L. Thioflavin S staining and electron microscopy confirmed a paired helical filament structure. The aggregation was recapitulated in vitro, where F3 readily seeded the aggregation of full-length tau in solution into thioflavin-positive aggregates.

The researchers also looked at the role of phosphorylation. By analyzing phosphoepitopes in the expressed proteins, they found that repeat region sites were phosphorylated mainly in the soluble protein fraction, consistent with that modification inhibiting aggregation. Phosphorylation in the C-terminal PHF1 epitope was seen in both the soluble and aggregated full-length proteins, suggesting it may have a positive effect on aggregation. The ability of F3 to nucleate fibril formation of full-length tau will allow further studies on the effect of phosphorylation sites throughout the protein.

To look at the toxicity of tau, the investigators induced tau expression in cells and measured the release of lactate dehydrogenase (LDH) as an indicator of cell membrane dysfunction and cell death. In cells containing unaggregated forms of tau, no LDH was released, while expression of the F3 fragment led to both aggregation and LDH release. By simultaneously staining cells for tau aggregates and DNA fragmentation, the researchers revealed a correlation between the extent of aggregation and toxicity. This result is at odds with recent in-vivo work that shows cognitive defects in mice are unrelated to the presence of tau tangles (see ARF related news story), and the suggestion that other tau species are the toxic ones. Experiments aimed at manipulating tau proteolysis in vivo may shed some light on the potential pathological role of this new pathway to aggregation.—Pat McCaffrey.

Reference:
Wang YP, Biernat J, Pickhardt M, Mandelkow E, Mandelkow EM. Stepwise proteolysis liberates tau fragments that nucleate the Alzheimer-like aggregation of full-length tau in a neuronal cell model. Proc Natl Acad Sci U S A. 2007 May 29; [Epub ahead of print] Abstract