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.

Reference:
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 Aug 4. Abstract