PubMed: 29024336

Interplay of pathogenic forms of human tau with different autophagic pathways.
Aging cell
Caballero B | Cuervo AM | Diaz A | Juste YR | Mandelkow E | Mandelkow EM | Stiller B | Tasset I | Wang Y

Evidence d5a4b04154

Cells expressing WT tau behave as control cells and display a dose-dependent increase in CMA activity upon exposure to paraquat (Fig. 3f) or thapsigargin (Fig. 3g)

Evidence 52ae4b1f81

We found that WT tau was taken up and efficiently degraded by LE (Fig. 2c,d)

Evidence 7fcadf543f

This process was significantly impaired for tau-P301L and, to higher extent, for tau- A152T (Fig. 2c,d)

Evidence cbab779a03

In agreement with our previous observations (Wang et al., 2009), we found that lysosomes contribute to degradation of WT tau (reflected as an increase in tau levels upon blockage of lysosomal proteolysis with inhibitors)

Evidence 7940b438fa

In contrast, once the first N-terminal insert is lost (in 0N3R tau), we observed a very pronounced decrease in tau uptake (Fig. 5b,c)

Evidence 617c0ea471

Absence of the second N-terminal insert (in 1N3R tau) did not reduce CMA of tau, but instead this isoform displayed faster internalization (lower binding because of more efficient uptake) (Fig. 5b,c)

Evidence 7943a1a2f6

Absence of the second N-terminal insert also significantly reduced e-MI of tau (Fig. 5e,f)

Evidence 236f8d84ed

Analysis of their uptake by isolated CMA-active lysosomes revealed that 2N3R tau behaved similarly to 2N4R tau (which we have used in the rest of the study as control)

Evidence 4f74ca6679

In this study, we analyzed the contribution of three different types of autophagy, macroautophagy, chaperone-mediated autophagy, and endosomal microautophagy to the degradation of tau protein variants and tau mutations associated with this agerelated disease. We have found that the pathogenic P301L mutation inhibits degradation of tau by any of the three autophagic pathways, whereas the risk-associated tau mutation A152T reroutes tau for degradation through a different autophagy pathway

Evidence bf59587c3f

Taken together, our in vitro and cell-based studies argue that these two point mutations, A152T and P301L, reduce the normal degradation of tau by CMA, although the P301L mutation has a more pronounced inhibitory effect

Evidence b308153292

Our previous studies and data presented in this work support substantial contribution of CMA to the degradation of wild-type unmodified tau (Wang et al., 2009)

Evidence e00b8a9040

This lysosomal degradation occurred, in large part, through CMA, as genetic blockage of this pathway almost completely abolished lysosomal degradation of WT tau and led to its accumulation (Fig. 1a,b; GAPDH is shown as an example of a well-characterized CMA substrate (Aniento et al., 1993) known to accumulate intracellularly upon blockage of CMA (Schneider et al., 2014))

Evidence 3bf7a5acfd

Consistent with our previous findings (Wang et al., 2009), WT tau is a very efficient CMA substrate, to the point that binding is almost undetectable because the protein is rapidly internalized (Fig. 1c,d)

Evidence b7092f285f

Tau-A152T displayed very similar degradation dynamics, although this mutation slightly reduced tau’s rates of lysosomal degradation (20% inhibition) when compared with WT tau (Fig. 1a,b).

Evidence 5600c66d37

Blockage of CMA in cells expressing tau-A152T also resulted in significant accumulation of this variant and ablated its lysosomal degradation, suggesting preferential degradation of A152T by CMA (Fig. 1a,b)

Evidence dfb32f57df

In the case of tau-A152T, the dynamics of internalization/degradation through CMA were comparable to WT tau (Fig. 1c,d), in agreement with our studies in intact cells in culture (Fig. 1a, b), but we found a significantly higher amount of tau-A152T bound to the membrane of CMA-active lysosomes (Fig. 1c,d)

Evidence bfd7a52495

Interestingly, although tau-P301L was not degraded in lysosomes, blockage of CMA promoted accumulation of this protein variant, albeit at significantly lower levels than WT and A152T. We propose that overall loss of proteostasis as a consequence of CMA blockage could indirectly affect clearance of tau-P301L through other systems

Evidence 3fcacb5c72

The most notable difference between the two tau mutants was the inability of P301L to undergo degradation by CMA or by macroautophagy

Evidence 6a78a32e24

Interestingly, although judging by the studies in intact cells the contribution of e-MI to tau degradation is small (Fig. 2a), our in vitro studies with isolated LE revealed a high efficiency for e-MI of tau (Fig. 2c)

Evidence b48ed4a4e4

Contrary to WT tau, which accumulates in e- MI-defective cells, intracellular levels of A152T and P301L tau did not change in cells knocked down for Vps4, suggesting that both point mutations in tau compromise its ability to undergo degradation by this pathway (Fig. 2a,b)

Evidence 0dd74cb70c

Previous studies have demonstrated that oxidized proteins accumulate inside multivesicular bodies (Cannizzo et al., 2012), suggesting that oxidation may be a prerequisite to complete internalization of tau by e-MI, and that the LE environment may contribute to that modification

Evidence 6d95f4e066

Loss of neuronal proteostasis, a common feature of the aging brain, is accelerated in neurodegenerative disorders, including different types of tauopathies

Evidence 9ace79c3fe

It is well accepted that loss of proteostasis occurs gradually with age and underlies the basis of severe neurodegenerative disorders such as Parkinson’s disease, Huntington’s disease, Alzheimer’s disease, and other types of frontotemporal dementia (Prahlad & Morimoto, 2009; Voisine et al., 2010; Morimoto & Cuervo, 2014)

Evidence 946724e2b4

Macroautophagy blockage resulted in preferential accumulation of A152T, but not WT and P301L tau (Fig. 2e,f)

Evidence 205cb99fdd

In fact, increased macroautophagy may be responsible for the increase in the degradation of long-lived proteins that we observed for both mutant forms of tau under these conditions (Fig. 3b)

Evidence 2d1639b40c

This is in clear contrast to other pathogenic proteins such as mutant alpha-synuclein or mutant LRRK2 that bind to lysosomes but fail to translocate through CMA (Cuervo et al., 2004; Orenstein et al., 2013)

Evidence 978025e238

In the presence of any of the tau proteins, we found some sequestration of the probe in the multivesicular bodies, albeit significantly less in cells expressing the WT and A152T protein

Evidence d5382cd4fb

An increase in overall tau levels has been observed in brains from patients bearing either P301L or A152T mutation on tau (Torres et al., 1998)

Evidence d01a1e39ce

In summary, when comparing the pathogenic tau mutation P301L with the risk-associated mutation A152T, we found that both reduced normal turnover of tau by autophagy, but that the effect of the P301L mutation was more pronounced (summarized in Fig. 2g and Fig. S6, Supporting information)

Evidence 0236d31bc0

However, whereas the P301L mutation leads to tau aggregation into paired helical filaments (PHFs) (Barghorn et al., 2000), patients with the risk-associated A152T mutation display higher abundance of oligomers (Coppola et al., 2012)

Evidence 2316bda20a

Cells expressing tau-A152T displayed significantly higher rates of intracellular protein degradation than the other cells under basal conditions (Fig. 3a)

Evidence 299c4d0c97

A similar significant increase in protein degradation was observed in response to serum removal in cells expressing either of the mutants (Fig. 3b)

Evidence b648867274

We did however find that under serum deprivation conditions, tau-A152T-expressing cells displayed significantly higher CMA (Fig. 3d,e; 30% increase) than control cells

Evidence 9fec4d3a4d

We found that abundance of autophagic vacuoles (autophagosomes + autolysosomes) significantly increased in cells expressing either of the two tau mutants (Fig. 4d,e). This increase was mainly due to higher content of autolysosomes (red puncta) (Fig. 4d,e), in support of increased macroautophagic flux

Evidence 6d130e6c42

Reduced tau-P301L uptake is not caused by a problem in translocation across the lysosomal membrane, but rather by reduced targeting/binding to lysosomes, as we did not detect tau accumulation at the lysosomal membrane

Evidence 2a12f212d0

In contrast, the P301L mutation severely impaired lysosomal uptake of tau by CMA, resulting in a sixfold decrease in degradation when compared to WT tau protein (Fig. 1c,d)

Evidence 04e6d73ce7

Cells expressing tau-P301L displayed significant upregulation of CMA under basal conditions that was no longer observed upon serum removal (Fig. 3c–e)

Evidence 359ef73c6c

As expected, fluorescence puncta were visible in transduced control cells (Fig. 4a) and blockage of endo/lysosomal degradation with ammonium chloride and leupeptin significantly increased the number of fluorescent puncta by preventing their degradation (Fig. 4b)

Evidence 4421d900ff

However, cells expressing the mutant tau proteins failed to further upregulate CMA and even a decrease was noticeable in cells expressing tau-P301L (Fig. 3f,g)

Evidence 77d7138830

The two pseudophosphorylated tau proteins (hTau40 AT8/AT100/PHF-1 and hTau40 4xKXGE) displayed significantly decreased lysosomal uptake when compared to hTau40 WT Tau (Fig. 7a,b)

Evidence 28bf998049

Analysis of e-MI of the pseudophosphorylated forms of tau using LE showed reduced uptake/degradation of hTau40 AT8/AT100/PHF-1 when compared to WT tau (Fig. 7d,e)

Evidence 544d1ac51f

Interestingly, a deletion of lysine 280 (hTau40 DK280), known to lead to tau aggregation (Khlistunova et al., 2006), turned this protein into a very poor CMA substrate (Fig. 6a,b)

Evidence 9e465ba8ef

This conformational change, but not the aggregation itself, interferes also with tau degradation by e-MI, as we found a significant decrease in the uptake of both mutants by LE (Fig. 6d,e)

Evidence 72f11602cb

Thus, insertion of two prolines in the DK280 background (hTau40 DK280/2P), which prevents tau aggregation (Barghorn & Mandelkow, 2002), only partially rescued CMA uptake of the DK280 mutant (Fig. 6a,b)

Evidence 640c7ea1c3

Changing glutamic acid for alanine (hTau40 4xKXGA), which serves as a control for the pseudophosphorylation, partially rescued the uptake of this form of tau (Fig. 7a,b

Evidence 91a3b14c0a

This effect was more pronounced in the case of hTau40 4xKXGE, whose uptake and degradation by LE was completely abolished (Fig. 7d,e), suggesting that phosphorylation in the microtubule- binding domain diminishes its degradation by e-MI


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