Lysosomes: Hotbeds Where Tau Oligomers Seed Aggregation?

How does a nice (that is, soluble and relatively unstructured) protein like tau end up rigidly locked into paired helical filaments and neurofibrillary tangles? For tau mutants, the process could start on lysosomes, according to new work from the labs of Eckhard and Eva-Maria Mandelkow at the Max Planck Institute in Hamburg, Germany, along with Ana Maria Cuervo at the Albert Einstein College of Medicine in New York. In a study published online August 4 in Human Molecular Genetics, the researchers tracked the proteolytic processing of tau in cells and discovered that a failure of chaperone-mediated breakdown of mutant tau leads to the production of amyloidogenic fragments and formation of small oligomers on the surface of lysosomes. Besides seeding toxic aggregation, the tau fragments appear to stall chaperone-mediated breakdown of other proteins, leaving cells without a vital defense against stress. The results raise the possibility that aberrant tau degradation in lysosomes could contribute to pathogenesis in tauopathies.

Previous work from the Mandelkows’ labs had shown that sequential cleavage of tau in Neuro2A cells overexpressing a truncated, mutant form of the protein (TauRDΔK280) produced fragments that can seed tau aggregation and promote cell toxicity (see ARF related news story on Wang et al., 2007). To identify the proteases responsible, first author Yipeng Wang and colleagues treated mutant tau with cell fractions derived from cytosol or lysosomes. The scientists found that a still-unknown cytosolic protease lopped off the N-terminus to produce the F1 fragment, while a lysosomal protease, cathepsin L, was responsible for the C-terminal cleavage that generates the amyloidogenic fragments F2 and F3.

From this, Cuervo told ARF, the German investigators supposed that F1 was subject to processing by macroautophagy, in which vesicles engulf cytosolic contents and deliver the whole mess to lysosomes for degradation. But when they blocked macroautophagy in the cell model, they found that while the degradation of tau and its aggregates indeed occurred via that pathway, the production of F2 and F3 did not.

That led them to consider other ways in which F1 might get into lysosomes, and an obvious candidate was chaperone-mediated autophagy (CMA). In this process, the Hsc70 chaperone binds to a recognition sequence on a target protein and escorts it to the lysosome. Whereas macroautophagy is responsible for bulk delivery of proteins via vesicle fusion, CMA is more selective: Specific proteins are tagged for delivery and cross into the lumen via the lysosome-associated membrane protein type 2A (LAMP-2A) receptor. The researchers noticed that the tau fragment had two potential CMA-targeting motifs and, using purified lysosomes, then showed that Hsc70 and LAMP-2A were required for F1’s delivery to lysosomes and cleavage there.

Once at the lysosome, though, tau behaved strangely. For starters, tau was never taken inside the lumen of the organelle. Even after cleavage, it remained on the outside. There the tau fragments began to aggregate, thanks to their local concentration, or the presence of negatively charged membrane lipids, or both. They formed soluble oligomers ranging from dimers up to tetramers or octamers that could be detected on SDS gels, the scientists report. The data also suggest that F2 and F3 can disrupt lysosomal membranes, leading to release of enzymes and tau fragments into the cytosol, where they can catalyze more degradation and seed the formation of higher aggregates, respectively.

Tau oligomers have been observed in brain in connection with early AD pathology, before the formation of neurofibrillary tangles (Maeda et al., 2006; Maeda et al., 2007), and they are viewed as potential seeds for fibril formation. “Some people think the oligomers are more toxic than aggregates, and we do see in vitro that they will break lysosomes. But the main idea is that the oligomers will be seeds for aggregates,” Cuervo says.

The most likely scenario accounting for tau’s odd behavior is that the F1 fragment sticks its C-terminal through the lysosomal membrane, where cathepsin L can cut it, but that the protein never fully translocates. This idea led the researchers to ask if mutant tau could inhibit clearance of other substrates of CMA, and the answer was, yes. That could contribute to the toxic effects of tau in cells, Cuervo says. “CMA is important for the cellular response to stress. If you block CMA in normal cells, in basal conditions they are okay, but with even minimal stress, they die.”

It remains to be seen how the processing of a truncated and mutated repeat-domain fragment that takes place in the cell model relates to in-vivo handling of full-length, wild-type tau and its toxicity in tauopathies. The cellular role of the lysosome bears an intriguing resemblance, however, to the case of α-synuclein. There, mutant or modified forms of the protein also end up at the lysosome via CMA without fully translocating into the lumen (see ARF related news story on Cuervo et al., 2004 and subsequent work by Martinez-Vincente et al., 2008); aberrant α-synuclein then blocks CMA to the detriment of neurons (see ARF related news story on Xiolouri et al., 2009). “In the case of tau, the additional feature is that the lysosomal-mediated cleavage favors formation of oligomeric structures directly at the lysosomal surface,” Cuervo said.—Pat McCaffrey

Comments

  1. In this manuscript, Yipeng Wang and colleagues showed lysosome/macroautophagy as a new tau degradation pathway in addition to the ubiquitin/proteasome pathway. Since an aggregated form of tau is thought to be involved in neuronal loss in AD, studies on identifying toxic aggregation, and removing or inhibiting it, are important in terms of future therapy for AD. In this manuscript, insoluble tau that is formed on the membrane of lysosomes exerts cytotoxicity. The tau repeat region is first cleaved at the N-terminus by an unidentified thrombin-like protease, then at the C-terminus by cathepsin L on the lysosomal membrane. The cleaved fragment is highly amyloidogenic and forms oligomers, which are thought to be a toxic tau aggregate. The toxic tau aggregate may be degraded by macroautophagy. Therefore, inhibition of cathepsin L, and/or activation of macroautophagy could become a potential therapy for AD.

    It is surprising that toxic tau oligomers form on the surface of lysosomes. The tau construct that the authors used in this study is the repeat region. It is conceivable that a small amount of toxic tau oligomers assembled by the repeat region fragments is enough to cause cellular dysfunction (neuronal toxicity), and these oligomers can seed tau aggregation (NFT formation); however, full-length hyperphosphorylated tau is still the dominant species existing in human tauopathies. We would be interested to know if full-length tau also forms toxic tau oligomers on this organelle.

    We have found granular tau oligomers as an intermediate form of tau fibrils in vitro and in human brain (e.g., Sahara et al., 2008). The increased granular tau oligomer is seen in Braak stage I of prefrontal cortex, and we also observed a significant inverse correlation between oligomers and heat shock proteins (HSPs) including Hsp90, Hsp40, Hsp27, α-crystallin, and CHIP. These results suggest that granular tau oligomer formation and corresponding changes in chaperone-responsible proteolytic systems occur long before NFT formation. If the toxic oligomer reported in this study and our granular tau oligomer are the same aggregate, then the granular tau oligomer we detect may have formed on lysosomes and may be a toxic form of tau aggregate. Overall, accumulated findings suggest that the tangles themselves might not be toxic, but that intermediate species are. Therefore, we need to identify a toxic form of tau aggregate, and find ways of inhibiting tau-induced neurotoxicity as a therapy for AD.

    References:

    Sahara N, Maeda S, Takashima A. Tau oligomerization: a role for tau aggregation intermediates linked to neurodegeneration. Curr Alzheimer Res. 2008 Dec;5(6):591-8. PubMed.

    View all comments by Naruhiko Sahara

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References

News Citations

  1. Does Tau Truncation Sow Seeds of Aggregation?
  2. Lysosomes and Proteasomes Compete for PD Researchers' Attention
  3. Tau Shows Subtle Hints of Genetic Association

Paper Citations

  1. 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 Jun 12;104(24):10252-7. PubMed.
  2. Maeda S, Sahara N, Saito Y, Murayama S, Ikai A, Takashima A. Increased levels of granular tau oligomers: an early sign of brain aging and Alzheimer's disease. Neurosci Res. 2006 Mar;54(3):197-201. PubMed.
  3. Maeda S, Sahara N, Saito Y, Murayama M, Yoshiike Y, Kim H, Miyasaka T, Murayama S, Ikai A, Takashima A. Granular tau oligomers as intermediates of tau filaments. Biochemistry. 2007 Mar 27;46(12):3856-61. PubMed.
  4. Cuervo AM, Stefanis L, Fredenburg R, Lansbury PT, Sulzer D. Impaired degradation of mutant alpha-synuclein by chaperone-mediated autophagy. Science. 2004 Aug 27;305(5688):1292-5. PubMed.
  5. Martinez-Vicente M, Talloczy Z, Kaushik S, Massey AC, Mazzulli J, Mosharov EV, Hodara R, Fredenburg R, Wu DC, Follenzi A, Dauer W, Przedborski S, Ischiropoulos H, Lansbury PT, Sulzer D, Cuervo AM. Dopamine-modified alpha-synuclein blocks chaperone-mediated autophagy. J Clin Invest. 2008 Feb;118(2):777-88. PubMed.
  6. Xilouri M, Vogiatzi T, Vekrellis K, Park D, Stefanis L. Abberant alpha-synuclein confers toxicity to neurons in part through inhibition of chaperone-mediated autophagy. PLoS One. 2009;4(5):e5515. PubMed.

Further Reading

Primary Papers

  1. Wang Y, Martinez-Vicente M, Krüger U, Kaushik S, Wong E, Mandelkow EM, Cuervo AM, Mandelkow E. Tau fragmentation, aggregation and clearance: the dual role of lysosomal processing. Hum Mol Genet. 2009 Nov 1;18(21):4153-70. PubMed.

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