Department of Chemical and Structural Biology, Weizmann Institute of Science, Israel; Department of Chemical Research Support, Weizmann Institute of Science, Israel;

The microtubule-associated protein, tau, is the major subunit of neurofibrillary tangles associated with neurodegenerative conditions, such as Alzheimer"s disease. In the cell, however, tau aggregation can be prevented by a class of proteins known as molecular chaperones. While numerous chaperones are known to interact with tau, though, little is known regarding the mechanisms by which these prevent tau aggregation. Here, we describe the effects of ATP-independent Hsp40 chaperones, DNAJA2 and DNAJB1, on tau amyloid-fiber formation and compare these to the small heat shock protein HSPB1. We find that the chaperones play complementary roles, with each preventing tau aggregation differently and interacting with distinct sets of tau species. Whereas HSPB1 only binds tau monomers, DNAJB1 and DNAJA2 recognize aggregation-prone conformers and even mature fibers. In addition, we find that both Hsp40s bind tau seeds and fibers via their C-terminal domain II (CTDII), with DNAJA2 being further capable of recognizing tau monomers by a second, distinct site in CTDI. These results lay out the mechanisms by which the diverse members of the Hsp40 family counteract the formation and propagation of toxic tau aggregates and highlight the fact that chaperones from different families/classes play distinct, yet complementary roles in preventing pathological protein aggregation.

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Several neurological conditions, such as Alzheimer’s and Parkinson’s disease, are characterized by the build-up of protein clumps known as aggregates. In the case of Alzheimer’s disease, a key protein, called tau, aggregates to form fibers that are harmful to neuronal cells in the brain. One of the ways our cells can prevent this from occurring is through the action of proteins known as molecular chaperones, which can bind to tau proteins and prevent them from sticking together.

Tau can take on many forms. For example, a single tau protein on its own, known as a monomer, is unstructured. In patients with Alzheimer’s, these monomers join together into small clusters, known as seeds, that rapidly aggregate and accumulate into rigid, structured fibers. One chaperone, HSPB1, is known to bind to tau monomers and prevent them from being incorporated into fibers. Recently, another group of chaperones, called J-domain proteins, was also found to interact with tau. However, it was unclear how these chaperones prevent aggregation and whether they bind to tau in a similar manner as HSPB1.

To help answer this question, Irwin, Faust et al. studied the effect of two J-domain proteins, as well as the chaperone HSBP1, on tau aggregation. This revealed that, unlike HSBP1, the two J-domain proteins can bind to multiple forms of tau, including when it has already aggregated in to seeds and fibers. This suggests that these chaperones can stop the accumulation of fibers at several different stages of the aggregation process. Further experiments examining which sections of the J-domain proteins bind to tau, showed that both attach to fibers via the same region. However, the two J-domain proteins are not identical in their interaction with tau. While one of them uses a distinct region to bind to tau monomers, the other does not bind to single tau proteins at all.

These results demonstrate how different cellular chaperones can complement one another in order to inhibit harmful protein aggregation. Further studies will be needed to understand the full role of J-domain proteins in preventing tau from accumulating into fibers, as well as their potential as drug targets for developing new treatments.

Tau is an intrinsically disordered protein (IDP) that is highly expressed in neurons and plays essential roles in microtubule self-assembly and stability (Mandelkow and Mandelkow, 2012), axonal transport (Gustke et al., 1994), and neurite outgrowth (Biernat and Mandelkow, 1999). Tau binds to microtubules via its central microtubule-binding repeat (MTBR) domain (Figure 1a), an interaction that is modulated by post-translational modifications (PTMs). Aberrant PTMs such as hyperphosphorylation and acetylation (Hanger et al., 2009; Morris et al., 2015; Cook et al., 2014) were suggested to decrease the affinity of tau to microtubules, thus subsequently reducing microtubule stability. In addition, when tau dissociates from microtubules, it can form oligomers with the potential to disrupt cellular membranes, thereby impairing synaptic and mitochondrial functions, before ultimately forming amyloid fibers (Shafiei et al., 2017).

Interaction of chaperones with monomeric tau.
(A) Domain organization of the longest splice isoform of tau protein (2N4R) and the short variant (4R/K18) containing only the microtubule binding repeats (MBTRs). The location of the two N-terminal inserts (N1 and N2) and the polyproline region (PPR) is indicated. The MTBR region of tau consists of four partially repeated sequences, R1 to R4, with the PHF6* and PHF6 aggregation-driving hexapeptides highlighted in gray. (B, C) ThT-based aggregation assay of 2N4R (B) and 4R (C) tau variants (10 µM) in the presence of 5 µM HSPB1 (green), DNAJA2 (purple), or DNAJB1 (blue) chaperones. The inset shows tau aggregation profiles in the presence of twofold excess (20 µM) of the same chaperones, showing complete inhibition over the course of the experiment. Representative data from three independent experiments is shown. (D) Tau4R binding profiles to HSPB1 (green), DNAJA2 (purple), and DNAJB1 (blue) chaperones, probed by NMR. Changes in NMR intensity ratios (I/I0) upon addition of twofold excess of each chaperone are plotted as a function of tau4R residue number. The gray boxes represent the positions of the tau PHF6* and PHF6 aggregation-prone motifs. Values lower than 0.5 indicate intermolecular interactions.

Figure 1—source data 1

Aggregation assay of 2N4R and 4R tau variants.
Figure 1—source data 2

NMR binding experiments of tau4R to HSPB1, DNAJB1, and DNAJA2 chaperones.

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These abnormal forms of tau are thought to play a key role in the pathogenesis of various human tauopathies, including Alzheimer’s disease (AD), frontotemporal dementias, and progressive supranuclear palsy (Ballatore et al., 2007). In such cases, tau forms large intracellular aggregates, termed neurofibrillary tangles, whose abundance and localization in the brain correlates with cognitive decline (Ballatore et al., 2007; Brunello et al., 2020). It is still unclear, however, if the fibrils themselves are the neurotoxic species or whether prefibrillar soluble aggregates and oligomers of tau promote neuronal death by spreading tau pathogenicity from cell to cell in a prion-like manner.

Chaperone machineries, and in particular members of the Hsp70 and the ATP-independent, small heat shock protein (sHSP) families, engage with tau during these pathogenic events, counteracting its aggregation into amyloids and targeting the misfolded species for degradation (Dou et al., 2003; Voss et al., 2012; Petrucelli et al., 2004; Mok et al., 2018; Caballero et al., 2021). Moreover, in various cellular models, increased chaperone levels were shown to play an important role in tau cellular homeostasis by promoting tau solubility and microtubule binding while reducing the levels of pathological tau species (Dou et al., 2003; Perez et al., 1991; Renkawek et al., 1994; Dabir et al., 2004; Sahara et al., 2007; Jinwal et al., 2013; Shimura et al., 2004). The chaperones do so by specifically recognizing the six-residue aggregation-prone regions located at the start of the second and third repeats in the MTBR domain (Mok et al., 2018; Freilich et al., 2018; Baughman et al., 2018; Weickert et al., 2020; Figure 1A). These two motifs, 275VQIINK280 and 306VQIVYK311 (also called PHF6* and PHF6, respectively), are susceptible to the formation of β-sheet structures that have been found to be a prerequisite for tau aggregation (von Bergen et al., 2000; Mukrasch et al., 2005).

Despite both Hsp70 and HSPB1 chaperones recognizing the same regions, their tau aggregation-prevention mechanisms have been found to be markedly different. Hsp70 interaction with tau suppresses the formation of aggregation-prone tau nuclei and sequesters tau oligomers and fibrils (Baughman et al., 2018; Patterson et al., 2011), thereby neutralizing their ability to damage membranes and seed further tau aggregation (Kundel et al., 2018a). In contrast, HSPB1 was shown to delay tau fiber formation by weakly interacting with early species in the aggregation reaction (Baughman et al., 2018). Thus, it would appear that different cellular chaperones can bind to distinct tau species and affect tau homeostasis in different ways.

Excitingly, though, HSPB1 is not the only ATP-independent chaperone reported to interfere with the tau aggregation pathway – members of the Hsp40 family (also known as J-domain proteins ) have also recently been reported to affect tau aggregation in the cell (Mok et al., 2018; Brehme et al., 2014; Fontaine et al., 2015; Hou et al., 2020).

JDPs are a diverse group of proteins that function as co-chaperones of the Hsp70 machinery, and are responsible both for selecting and delivering clients to the chaperones, and stimulating Hsp70 ATPase activity, thereby activating the chaperone cycle. These multidomain proteins are all structurally characterized by the conserved signature J-domain, essential for stimulation of Hsp70 ATPase activity (Kityk et al., 2018). In addition, canonical class A and B JDPs also comprise a regulatory glycine‐rich (GF) region adjacent to the N-terminal J-domain (Faust et al., 2020; Karamanos et al., 2019), two structurally similar C-terminal β‐barrel domains (CTDI and CTDII) containing the substrate binding region, and a dimerization domain (Kampinga and Craig, 2010; Rosenzweig et al., 2019). Class A JDPs further contain a zinc-finger-like region (ZFLR) protruding from CTDI.

Recently, several studies have indicated that JDPs can also function as bona fide chaperones, utilizing holdase activity to prevent the aggregation of their client proteins (Ayala Mariscal and Kirstein, 2021).

DNAJA2, a member of this JDP family, was recently identified as a potent suppressor of tau aggregation, capable of effectively preventing the seeding of tau and formation of amyloids in cells (Mok et al., 2018; Abisambra et al., 2012), with DNAJA2 levels being selectively increased in AD patient neuronal cells (Mok et al., 2018). Additionally, it was recently shown that, along with the Hsp70 system, DNAJB1, a class B JDP, can break apart tau amyloid fibers extracted from AD brain tissues (Nachman et al., 2020).

Little is known, however, regarding how these co-chaperones interact with tau or the mechanism by which they modify tau disease-related amyloid states.

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We therefore used NMR spectroscopy, in combination with kinetic aggregation assays, to elucidate the effect of the DNAJA2 and DNAJB1 chaperones on tau aggregation. We found that the aggregation-prevention mechanisms of DNAJB1 and DNAJA2 are strikingly different from that of HSPB1 holdase chaperone. Moreover, we found that the two Hsp40 family members also diverge in their interactions with tau, whereas DNAJA2 interacts with all species along the tau aggregation pathway, including inert tau monomers, DNAJB1 only interacts with aggregation-prone tau conformers, such as seeding competent species or mature fibers.