The microtubule-associated protein tau is a highly soluble protein that turns sticky in Alzheimer disease brain. Researchers have avidly studied the role of hyperphosphorylation in tau’s pathogenic conversion to neurofibrillary tangles, but other modifications have received less attention. Besides being phosphorylated, tau is proteolytically cleaved in neurons affected by AD, generating fragments that end up in tangles along with full-length tau. Those fragments could hold the key to tau aggregation, according to a new study from the labs of Eva-Maria and Eckhart Mandelkow at the Max Planck Unit for Structural Molecular Biology in Hamburg, Germany.

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

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  1. This eloquent study examines the post-translational modification of tau. The authors used a previously developed model of tauopathy by expressing the mutant tau repeat domain first discovered in FTP with PD linked to chromosome 17 (FTDP17). Several points of this study bear mentioning: first, the authors demonstrate that proteolytically cleaved fragments enhance the aggregation of full-length tau, a finding that has been previously reported following the caspase-cleavage of tau (1,2). Intriguing about this new study is that the identity of the proteases involved in cleaving tau is not known. Although tau is a well-known substrate for caspases and calpains, the role of other potential proteases has not been investigated. It will be interesting to follow whether the identity of these potential proteases will be revealed and what exactly is their role during the progression of AD.

    Another interesting finding of this study is the demonstration that aggregation of tau is modulated by phosphorylation but does not depend on it. One dilemma in this area of AD is how these two events, proteolytic processing of tau and hyperphosphorylation, are related (see 3 for a recent review).

    A question that remains to be fully answered is whether cleavage of tau precedes or follows phosphorylation. Several studies have suggested that the caspase cleavage of tau indeed precedes mature NFT formation (2,4) and is an early event associated with tangle pathology. In this manner, hyperphosphorylation may serve as a protective mechanism limiting the degree of aggregation of proteolytic fragments of tau together with full-length tau.

    Another important finding of the present study is the revelation that cellular toxicity was dependent upon the aggregation of tau, but also upon the proteolytically derived fragments. This is an interesting and important observation, as several studies have suggested that C-terminally derived fragments of tau following caspase cleavage are themselves proapoptotic (5,6). The findings suggest that, depending on which protease cleaves tau, divergent pathways of toxicity may be invoked that either arise from the fragments themselves or follow aggregation. Overall, the present study has revealed important findings regarding the turnover of tau. Post-translational modification of tau appears to be a mechanism that may promote the pathology associated with AD.

    View all comments by Troy Rohn

References

News Citations

  1. Paper Alert: Inducing and Reversing Cell-Based Tau Aggregation
  2. No Toxicity in Tau’s Tangles?

Paper Citations

  1. . Truncated tau from sporadic Alzheimer's disease suffices to drive neurofibrillary degeneration in vivo. FEBS Lett. 2006 Jun 26;580(15):3582-8. PubMed.
  2. . Neurodegeneration caused by expression of human truncated tau leads to progressive neurobehavioural impairment in transgenic rats. Brain Res. 2007 Jan 26;1130(1):206-13. PubMed.
  3. . Caspase cleavage of tau: linking amyloid and neurofibrillary tangles in Alzheimer's disease. Proc Natl Acad Sci U S A. 2003 Aug 19;100(17):10032-7. PubMed.
  4. . 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 Jun 12;104(24):10252-7. PubMed.

Further Reading

Papers

  1. . ERK1/2 activation mediates Abeta oligomer-induced neurotoxicity via caspase-3 activation and tau cleavage in rat organotypic hippocampal slice cultures. J Biol Chem. 2006 Jul 21;281(29):20315-25. PubMed.
  2. . 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 Jun 12;104(24):10252-7. PubMed.

Primary Papers

  1. . 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 Jun 12;104(24):10252-7. PubMed.