DnaJA1 antagonizes constitutive Hsp70-mediated stabilization of tau

Abstract

Tau aggregation and amyloidogenesis are common hallmarks for neurodegenerative disorders called tauopathies. The molecular chaperone network constitutes the cellular defense against insults such as tau aggregation. However, chaperone effects on tau are dichotomous. Loss of tau's microtubule-binding activity facilitates an inappropriate chaperone interaction that promotes an amyloidogenic tau conformation. Conversely, other chaperones are capable of promoting tau clearance. Here, we demonstrate that a critical contributor to tau triage is the DnaJ-binding domain of Hsp70 proteins. In particular, over-expression of the constitutive DnaJ, DnaJA1, mediated tau clearance, while knockdown facilitated tau accumulation. This clearance was not specific to distinct pathogenic tau species. The activity of DnaJA1 was attenuated by concomitant increases in Hsp70. Tau reductions facilitated by DnaJA1 were dependent on the integrity of lysines known to be poly-ubiquitinated in human Alzheimer's brain. In vivo, DnaJA1 and tau levels were inversely correlated. The effects of DnaJA1 were partially specific: DnaJA1 reduced the levels of a polyQ protein but had no significant effect on α-synuclein levels. These data suggest that DnaJA1 triages all tau species for ubiquitin-dependent clearance mechanisms. Moreover, the levels of DnaJA1 and Hsp70 seem to play against each other with regard to tau: as DnaJA1 levels increase, tau levels are reduced, but this can be prevented if Hsp70 levels are simultaneously induced. Thus, the DnaJ repertoire possibly represents a powerful set of genetic modifiers for tau pathogenesis. Further investigations could provide new insights about triage decisions that facilitate or prevent amyloidogenesis of tau and other proteins associated with neurodegenerative disease.

Figure 1. The DnaJ-binding domain on Hsp70 and its main DnaJ protein, DnaJA1, play a critical role in tau stability

HeLa cells stably over-expressing tau were transiently transfected with 2 µg of plasmid corresponding to flag-tagged DnaJ sequence (A), siRNA to DnaJA1 (B), 2 mg flag-tagged DnaJA1 (C and E), and 2 µg of either flag-tagged or untagged DnaJA1 (F). (A) Representative western blot of tau in cells over-expressing the DnaJ domain peptide. (B) Representative immunoblot of tau in cells transfected with siRNA for DnaJA1. (C) Representative western blot of tau in cells over-expressing DnaJA1. (D) Quantification graph of (C) indicating a 47% reduction in tau levels (*p< 0.05). (E) Sample western blot of pathogenic tau species in cells over-expressing DnaJA1. (F) Representative immunoblot of tau in cells overexpressing flag-tagged versus untagged DnaJA1.

Figure 2. DnaJA1 abrogates tau independently of Hsp70

(A) Tau-overexpressing HeLa cells were transfected with DnaJA1, tau was immuno-precipitated, and samples were subjected to western blot analyses. Representative western blot showing that tau immuno-precipitates with DnaJA1, and this association favors recruitment of Hsp70 by 2.76 fold. Input immunoblots confirm DnaJA1-dependent tau abrogation. HeLa cells stably overexpressing tau were transfected with either a control plasmid or Flag-tagged DnaJA1 and consequently immuno-fluorescently stained with anti-Flag and anti-tau antibodies as well as with Hoechst reagent to reveal cell nuclei (blue). (B) Low magnification (5×) images of cells; scale bar = 80 µm. (C) Images at higher magnification of an A1-transfected cell (scale bar = 25 µm). (D) Co-localization plot of 2C showing complete co-localization (Pearson coefficient > 0.999). (E) Diagram depicting three tau mutants used in figures 2F, 3B, and 3C. The tau mutant mτ-Ubq has K/A substitutions at 254, 311, and 353, thereby preventing ubiquitination at these sites. The mutant mτ-Hsp70 cannot bind to Hsp70 due to the deletion of I308 and V309. The tau mutant mτ-CMA has replacements of amino acids at 336, 337 and 350, 351 for alanine residues, targeting it for chaperone-mediated autophagy (CMA). (F) Representative western blots showing tau abrogation by DnaJA1 in M17 cells co-transfected with wildtype tau (wt-τ) or mτ-Hsp70; DnaJA1 cleared mτ-Hsp70 more potently than wt-τ. (G) Representative immunoblot showing that DnaJA1-mediated clearance of tau and pTau is reversed by co-expression of Hsp70. Samples were obtained from tau-overexpressing HeLa cells co-transfected with DnaJA1 alone or in combination with Hsp70.

Figure 3. DnaJA1-mediated abrogation of tau requires ubiquitination

M17 cells were transiently co-transfected with DnaJA1 and the indicated tau variants. Representative western blots showing that in the presence of DnaJA1, wt-τ (A) and mτ-CMA (B) were decreased, while mτ-Ubq (C) accumulated.

Figure 4. DnaJA1 levels and distribution are inversely correlated with AD pathology and tau aggregates

(A) Representative immunoblot of DnaJA1 levels in human AD and age-matched control brain tissue. (B) Quantitative analysis of (A) shows that DnaJA1 is reduced by 47% (*p<0.05). (C–E) Immunofluorescent staining of tau (red) and DnaJA1 (green), in the CA2-CA3 hippocampal region of nine-month old non-transgenic (NTG; C) and rTg4510 (Tg; D and E) mice. White dotted lines highlight the granular layer of the hippocampus. (C) DnaJA1 (green) is expressed in cells of the neuronal layer (arrowheads) in NTG mice, while tau (red) is virtually undetectable. (D) Distribution of DnaJA1 (green) remains unchanged in Tg mice. (E) Higher magnification images show no proximity of DnaJA1 with tau signal. (F) Co-localization plot shows inverse co-localization between tau and DnaJA1. Scale bar for 20× (C and D) and 63× (E) images equals 50 µm and 10 µm, respectively. Three mouse brains were analyzed for each condition.

Figure 5. A1 displays selectivity for some, but not all, disease-relevant, aggregate-prone clients

HeLa and M17 cells were co-transfected with DnaJA1 and either α-synuclein or 84-repeat poly-glutamine (polyQ84). Cells were harvested 48 h post-transfection. (A and B) Representative western blots of lysates from HeLa cells show that DnaJA1 did not significantly affect α-synuclein levels; in contrast, DnaJA1 significantly reduced polyQ84. (C) Quantification graph of (A and B) showing that DnaJA1 reduced α-synuclein and polyQ84 by 22% (p> 0.05) and 76% (*p< 0.05), respectively.

Figure 6. Schematic representation of DnaJA1-mediated abrogation of tau

Under normal conditions, tau provides stabilization to microtubules. However, in tauopathies and upon dissociation and re-conformation into an unfolded and toxic state, tau undergoes chaperone triage by DnaJA1. If Hsc/p70 protein is abundant, it will form a complex with tau and DnaJA1; this partnership will preserve tau from being degraded. On the other hand, in the absence of Hsc/p70, DnaJA1 chaperones tau for clearance via a ubiquitin-dependent pathway.