Structure, microtubule interactions, and paired helical filament aggregation by tau mutants of frontotemporal dementias

We have studied biochemical and structural parameters of several missense and deletion mutants of tau protein (G272V, N279K, DeltaK280, P301L, V337M, R406W) found in frontotemporal dementia and parkinsonism linked to chromosome 17 (FTDP-17). The mutant proteins were expressed on the basis of both full-length tau (htau40) and constructs derived from the repeat domain. They were analyzed with respect to the capacity to enhance microtubule assembly, binding of tau to microtubules, secondary structure content, and aggregation into Alzheimer-like paired helical or straight filaments. We find that the mutations cause a moderate decrease in microtubule interactions and stabilization, and they show no gross structural changes compared with the natively unfolded conformation of the wild-type protein, but the aggregation into PHFs is strongly enhanced, particularly for the mutants DeltaK280 and P301L. This gain of pathological aggregation would be consistent with the autosomal dominant nature of the disease.     

Sensitive Detection of Proteopathic Seeding Activity with FRET Flow Cytometry

Increasing evidence supports transcellular propagation of toxic protein aggregates, or proteopathic seeds, as a mechanism for the initiation and progression of pathology in several neurodegenerative diseases, including Alzheimer''s disease and the related tauopathies. The potentially critical role of tau seeds in disease progression strongly supports the need for a sensitive assay that readily detects seeding activity in biological samples. By combining the specificity of fluorescence resonance energy transfer (FRET), the sensitivity of flow cytometry, and the stability of a monoclonal cell line, an ultra-sensitive seeding assay has been engineered and is compatible with seed detection from recombinant or biological samples, including human and mouse brain homogenates. The assay employs monoclonal HEK 293T cells that stably express the aggregation-prone repeat domain (RD) of tau harboring the disease-associated P301S mutation fused to either CFP or YFP, which produce a FRET signal upon protein aggregation. The uptake of proteopathic tau seeds (but not other proteins) into the biosensor cells stimulates aggregation of RD-CFP and RD-YFP, and flow cytometry sensitively and quantitatively monitors this aggregation-induced FRET. The assay detects femtomolar concentrations (monomer equivalent) of recombinant tau seeds, has a dynamic range spanning three orders of magnitude, and is compatible with brain homogenates from tauopathy transgenic mice and human tauopathy subjects. With slight modifications, the assay can also detect seeding activity of other proteopathic seeds, such as α-synuclein, and is also compatible with primary neuronal cultures. The ease, sensitivity, and broad applicability of FRET flow cytometry makes it useful to study a wide range of protein aggregation disorders.     

Structural and microtubule binding properties of tau mutants of frontotemporal dementias

Several mutations in the gene encoding the microtubule-associated protein tau are responsible for the formation of neurofibrillary inclusions in frontotemporal dementia with Parkinsonism linked to chromosome 17 (FTDP-17). Here we present the high-resolution characterization of the conformational properties of two FTDP-17 mutants of the four-repeat domain of tau, P301L and DeltaK280, and their properties for binding to polyanions and microtubules. Multidimensional NMR spectroscopy shows that the mutations do no lead to a significant increase in the level of beta-structure in their monomeric state, even though the mutations strongly promote beta-structure during aggregation. However, local structural changes are induced in the second repeat. These changes only weakly affect the binding to the polyanion heparin, which promotes paired helical filament formation. The extent of binding to microtubules, however, is strongly decreased. Our results demonstrate that the reversible binding of tau to microtubules involves specific interactions, which are not essential for binding to polyanions.     

Mutations of tau protein in frontotemporal dementia promote aggregation of paired helical filaments by enhancing local beta-structure

The microtubule-associated protein tau is a natively unfolded protein in solution, yet it is able to polymerize into the ordered paired helical filaments (PHF) of Alzheimer's disease. In the splice isoforms lacking exon 10, this process is facilitated by the formation of beta-structure around the hexapeptide motif PHF6 ((306)VQIVYK(311)) encoded by exon 11. We have investigated the structural requirements for PHF polymerization in the context of adult tau isoforms containing four repeats (including exon 10). In addition to the PHF6 motif there exists a related PHF6* motif ((275)VQIINK(280)) in the repeat encoded by the alternatively spliced exon 10. We show that this PHF6* motif also promotes aggregation by the formation of beta-structure and that there is a cross-talk between the two hexapeptide motifs during PHF aggregation. We also show that two of the tau mutations found in hereditary frontotemporal dementias, DeltaK280 and P301L, have a much stronger tendency for PHF aggregation which correlates with their high propensity for beta-structure around the hexapeptide motifs.     

In vitro generation of tau aggregates conformationally distinct from parent tau seeds of Alzheimer’s brain

Normal monomeric tau can be converted into pathogenic aggregates and acquire protease resistance in a prion-like manner. This acquisition of partial protease-resistance in tau aggregates has to date only been partially investigated in various studies exploring the prion-like properties of tau. In this study, we induced the aggregation of tau repeat domain (RD) in cultured cells using detergent insoluble fractions of Alzheimer’s brain tissue as seeds. The seeded aggregation of tau RD in cultured cells formed a ~7 kDa protease-resistant fragment in contrast to the ~12 kDa tau fragment characteristic of the AD seeds, suggesting that the in vitro generated tau aggregates were conformationally distinct from parent seeds.     

Effects of different anti-tau antibodies on tau fibrillogenesis: RTA-1 and RTA-2 counteract tau aggregation

Tau is the major antigenic component of neurofibrillary pathology in tauopathy, including Alzheimer's disease. Although conversion of soluble tau to an insoluble polymerized fibrillar form is a key factor in the pathogenesis of tauopathy, the mechanism of the change is unclear and no inhibitors of fibril formation are available. Monoclonal antibodies against the 1st or 2nd repeat of the microtubule binding domain, but not the C-terminal 16 residues, completely inhibited tau aggregation into PHF. Furthermore, they did not inhibit tau-induced tubulin assembly. Thus, they are useful to investigate tau protein conversion and will be useful therapeutic lead materials.     

Characterization of alpha-synuclein aggregation and synergistic toxicity with protein tau in yeast

A yeast model was generated to study the mechanisms and phenotypical repercussions of expression of alpha-synuclein as well as the coexpression of protein tau. The data show that aggregation of alpha-synuclein is a nucleation-elongation process initiated at the plasma membrane. Aggregation is consistently enhanced by dimethyl sulfoxide, which is known to increase the level of phospholipids and membranes in yeast cells. Aggregation of alpha-synuclein was also triggered by treatment of the yeast cells with ferrous ions, which are known to increase oxidative stress. In addition, data are presented in support of the hypothesis that degradation of alpha-synuclein occurs via autophagy and proteasomes and that aggregation of alpha-synuclein disturbs endocytosis. Reminiscent of observations in double-transgenic mice, coexpression of alpha-synuclein and protein tau in yeast cells is synergistically toxic, as exemplified by inhibition of proliferation. Taken together, the data show that these yeast models recapitulate major aspects of alpha-synuclein aggregation and cytotoxicity, and offer great potential for defining the underlying mechanisms of toxicity and synergistic actions of alpha-synuclein and protein tau.     

Cytotoxic tau released from lung microvascular endothelial cells upon infection with Pseudomonas aeruginosa promotes neuronal tauopathy

Patients who recover from nosocomial pneumonia oftentimes exhibit long-lasting cognitive impairment comparable with what is observed in Alzheimer’s disease patients. We previously hypothesized that the lung endothelium contributes to infection-related neurocognitive dysfunction, because bacteria-exposed endothelial cells release a form(s) of cytotoxic tau that is sufficient to impair long-term potentiation in the hippocampus. However, the full-length lung and endothelial tau isoform(s) have yet to be resolved and it remains unclear whether the infection-induced endothelial cytotoxic tau triggers neuronal tau aggregation. Here, we demonstrate that lung endothelial cells express a big tau isoform and three additional tau isoforms that are similar to neuronal tau, each containing four microtubule-binding repeat domains, and that tau is expressed in lung capillaries in vivo. To test whether infection elicits endothelial tau capable of causing transmissible tau aggregation, the cells were infected with Pseudomonas aeruginosa. The infection-induced tau released from endothelium into the medium-induced neuronal tau aggregation in reporter cells, including reporter cells that express either the four microtubule-binding repeat domains or the full-length tau. Infection-induced release of pathological tau variant(s) from endothelium, and the ability of the endothelial-derived tau to cause neuronal tau aggregation, was abolished in tau knockout cells. After bacterial lung infection, brain homogenates from WT mice, but not from tau knockout mice, initiated tau aggregation. Thus, we conclude that bacterial pneumonia initiates the release of lung endothelial-derived cytotoxic tau, which is capable of propagating a neuronal tauopathy.     

Stages and Conformations of the Tau Repeat Domain during Aggregation and Its Effect on Neuronal Toxicity

Several neurodegenerative diseases are characterized by the aggregation and posttranslational modifications of Tau protein. Its “repeat domain” (TauRD) is mainly responsible for the aggregation properties, and oligomeric forms are thought to dominate the toxic effects of Tau. Here we investigated the conformational transitions of this domain during oligomerization and aggregation in different states of β-propensity and pseudo-phosphorylation, using several complementary imaging and spectroscopic methods. Although the repeat domain generally aggregates more readily than full-length Tau, its aggregation was greatly slowed down by phosphorylation or pseudo-phosphorylation at the KXGS motifs, concomitant with an extended phase of oligomerization. Analogous effects were observed with pro-aggregant variants of TauRD. Oligomers became most evident in the case of the pro-aggregant mutant TauRDΔK280, as monitored by atomic force microscopy, and the fluorescence lifetime of Alexa-labeled Tau (time-correlated single photon counting (TCSPC)), consistent with its pronounced toxicity in mouse models. In cell models or primary neurons, neither oligomers nor fibrils of TauRD or TauRDΔK280 had a toxic effect, as seen by assays with lactate dehydrogenase and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide, respectively. However, oligomers of pro-aggregant TauRDΔK280 specifically caused a loss of spine density in differentiated neurons, indicating a locally restricted impairment of function.     

The core of tau-paired helical filaments studied by scanning transmission electron microscopy and limited proteolysis

In Alzheimer's disease and frontotemporal dementias the microtubule-associated protein tau forms intracellular paired helical filaments (PHFs). The filaments formed in vivo consist mainly of full-length molecules of the six different isoforms present in adult brain. The substructure of the PHF core is still elusive. Here we applied scanning transmission electron microscopy (STEM) and limited proteolysis to probe the mass distribution of PHFs and their surface exposure. Tau filaments assembled from the three repeat domain have a mass per length (MPL) of approximately 60 kDa/nm and filaments from full-length tau (htau40DeltaK280 mutant) have approximately 160 kDa/nm, compared with approximately 130 kDa/nm for PHFs from Alzheimer's brain. Polyanionic cofactors such as heparin accelerate assembly but are not incorporated into PHFs. Limited proteolysis combined with N-terminal sequencing and mass spectrometry of fragments reveals a protease-sensitive N-terminal half and semiresistant PHF core starting in the first repeat and reaching to the C-terminus of tau. Continued proteolysis leads to a fragment starting at the end of the first repeat and ending in the fourth repeat. PHFs from tau isoforms with four repeats revealed an additional cleavage site within the middle of the second repeat. Probing the PHFs with antibodies detecting epitopes either over longer stretches in the C-terminal half of tau or in the fourth repeat revealed that they grow in a polar manner. These data describe the physical parameters of the PHFs and enabled us to build a model of the molecular arrangement within the filamentous structures.     

Structure,stability, and aggregation of paired helical filaments from tau protein and FTDP-17 mutants probed by tryptophan scanning mutagenesis

By using tryptophan scanning mutagenesis, we observed the kinetics and structure of the polymerization of tau into paired helical filaments (PHFs) independently of exogenous reporter dyes. The fluorescence exhibits pronounced blue shifts due to burial of the residue inside PHFs, depending on Trp position. The effect is greatest near the center of the repeat domain, showing that the packing is tightest near the beta-structure inducing hexapeptide motifs. The tryptophan response allows measurement of PHF stability made by different tau isoforms and mutants. Unexpectedly, the stability of PHFs is quite low (denaturation half-points approximately 1.0 m GdnHCl), implying that incipient aggregation should be reversible and that the observed high stability of Alzheimer PHFs is due to other factors. The stability increases with the number of repeats and with tau mutants promoting beta-structure, arguing for a gain of toxic function in frontotemporal dementias. Fluorescence resonance energy transfer (FRET) was used to analyze the distances of Tyr(310) to tryptophans in different positions. The degree of FRET in the soluble protein was position-dependent, with highest signals within the second and third repeats but low or no signals further away. In PHFs most mutants showed FRET, indicating that tight packing results from assembly of tau into PHFs.     

The effect of a DeltaK280 mutation on the unfolded state of a microtubule-binding repeat in Tau

Tau is a natively unfolded protein that forms intracellular aggregates in the brains of patients with Alzheimer's disease. To decipher the mechanism underlying the formation of tau aggregates, we developed a novel approach for constructing models of natively unfolded proteins. The method, energy-minima mapping and weighting (EMW), samples local energy minima of subsequences within a natively unfolded protein and then constructs ensembles from these energetically favorable conformations that are consistent with a given set of experimental data. A unique feature of the method is that it does not strive to generate a single ensemble that represents the unfolded state. Instead we construct a number of candidate ensembles, each of which agrees with a given set of experimental constraints, and focus our analysis on local structural features that are present in all of the independently generated ensembles. Using EMW we generated ensembles that are consistent with chemical shift measurements obtained on tau constructs. Thirty models were constructed for the second microtubule binding repeat (MTBR2) in wild-type (WT) tau and a DeltaK280 mutant, which is found in some forms of frontotemporal dementia. By focusing on structural features that are preserved across all ensembles, we find that the aggregation-initiating sequence, PHF6*, prefers an extended conformation in both the WT and DeltaK280 sequences. In addition, we find that residue K280 can adopt a loop/turn conformation in WT MTBR2 and that deletion of this residue, which can adopt nonextended states, leads to an increase in locally extended conformations near the C-terminus of PHF6*. As an increased preference for extended states near the C-terminus of PHF6* may facilitate the propagation of beta-structure downstream from PHF6*, these results explain how a deletion at position 280 can promote the formation of tau aggregates.     

Huntington toxicity in yeast model depends on polyglutamine aggregation mediated by a prion-like protein Rnq1

The cause of Huntington's disease is expansion of polyglutamine (polyQ) domain in huntingtin, which makes this protein both neurotoxic and aggregation prone. Here we developed the first yeast model, which establishes a direct link between aggregation of expanded polyQ domain and its cytotoxicity. Our data indicated that deficiencies in molecular chaperones Sis1 and Hsp104 inhibited seeding of polyQ aggregates, whereas ssa1, ssa2, and ydj1-151 mutations inhibited expansion of aggregates. The latter three mutants strongly suppressed the polyQ toxicity. Spontaneous mutants with suppressed aggregation appeared with high frequency, and in all of them the toxicity was relieved. Aggregation defects in these mutants and in sis1-85 were not complemented in the cross to the hsp104 mutant, demonstrating an unusual type of inheritance. Since Hsp104 is required for prion maintenance in yeast, this suggested a role for prions in polyQ aggregation and toxicity. We screened a set of deletions of nonessential genes coding for known prions and related proteins and found that deletion of the RNQ1 gene specifically suppressed aggregation and toxicity of polyQ. Curing of the prion form of Rnq1 from wild-type cells dramatically suppressed both aggregation and toxicity of polyQ. We concluded that aggregation of polyQ is critical for its toxicity and that Rnq1 in its prion conformation plays an essential role in polyQ aggregation leading to the toxicity.     

Heat shock protein 70 prevents both tau aggregation and the inhibitory effects of preexisting tau aggregates on fast axonal transport

Aggregation and accumulation of the microtubule-associated protein tau are associated with cognitive decline and neuronal degeneration in Alzheimer's disease and other tauopathies. Thus, preventing the transition of tau from a soluble state to insoluble aggregates and/or reversing the toxicity of existing aggregates would represent a reasonable therapeutic strategy for treating these neurodegenerative diseases. Here we demonstrate that molecular chaperones of the heat shock protein 70 (Hsp70) family are potent inhibitors of tau aggregation in vitro, preventing the formation of both mature fibrils and oligomeric intermediates. Remarkably, addition of Hsp70 to a mixture of oligomeric and fibrillar tau aggregates prevents the toxic effect of these tau species on fast axonal transport, a critical process for neuronal function. When incubated with preformed tau aggregates, Hsp70 preferentially associated with oligomeric over fibrillar tau, suggesting that prefibrillar oligomeric tau aggregates play a prominent role in tau toxicity. Taken together, our data provide a novel molecular basis for the protective effect of Hsp70 in tauopathies.     

Competing interactions stabilize pro- and anti-aggregant conformations of human Tau

Aggregation of Tau into amyloid-like fibrils is a key process in neurodegenerative diseases such as Alzheimer. To understand how natively disordered Tau stabilizes conformations that favor pathological aggregation, we applied single-molecule force spectroscopy. Intramolecular interactions that fold polypeptide stretches of ~19 and ~42 amino acids in the functionally important repeat domain of full-length human Tau (hTau40) support aggregation. In contrast, the unstructured N terminus randomly folds long polypeptide stretches >100 amino acids that prevent aggregation. The pro-aggregant mutant hTau40ΔK280 observed in frontotemporal dementia favored the folding of short polypeptide stretches and suppressed the folding of long ones. This trend was reversed in the anti-aggregant mutant hTau40ΔK280/PP. The aggregation inducer heparin introduced strong interactions in hTau40 and hTau40ΔK280 that stabilized aggregation-prone conformations. We show that the conformation and aggregation of Tau are regulated through a complex balance of different intra- and intermolecular interactions.     

No full admission for tau to the exclusive prion club yet

Aggregation of the microtubule‐associated protein tau is a key feature of Alzheimer's disease and other so‐called tauopathies, yet what causes this protein to aggregate and what renders it toxic is only slowly being revealed. Because tau spreads in a stereotypical pattern through the diseased brain, it has been proposed that it possesses prion‐like properties, with aggregation‐prone tau facilitating the conversion of “naïve” tau into “toxic” forms. The current study by Wegmann et al (2015) addresses whether tau fulfils classical “prion criteria” by assessing its spreading and toxicity in the absence of endogenous tau. Using different transgenic and viral paradigms, the authors demonstrate that, although tau still propagates in this scenario, there is a decrease in its misfolding and neurotoxicity. They therefore conclude that tau is not a genuine prion, at least when the current definition of these infectious proteins is applied.     

Reduced binding of protein phosphatase 2A to tau protein with frontotemporal dementia and parkinsonism linked to chromosome 17 mutations

Coding region and intronic mutations in the tau gene cause frontotemporal dementia and parkinsonism linked to chromosome 17 (FTDP-17). We have previously reported that ABalphaC, a major form of protein phosphatase 2A (PP2A) in brain, binds tightly to tau protein in vitro and is a major tau phosphatase in vivo. Using in vitro assays, we show here that the FTDP-17 mutations G272V, DeltaK280, P301L, P301S, S305N, V337M, G389R, and R406W inhibit by approximately 20-95% the binding of recombinant three-repeat and four-repeat tau isoforms to the ABalphaC holoenzyme and the AC core enzyme of PP2A. Reduction in binding was maximal for tau proteins with the G272V, DeltaK280, and V337M mutations. We also show that tau protein can be specifically coimmunoprecipitated with endogenous PP2A from both rat brain and transfected cell extracts. It is significant that, by using similar coimmunoprecipitation assays, we show that all FTDP-17 mutations tested, including the N279K mutation, alter the ability of tau to associate with cellular PP2A. Taken together, these results indicate that FTDP-17 mutations induce a significant decrease in the binding affinity of tau for PP2A in vivo. We propose that altered protein-protein interactions between PP2A and tau may contribute to FTDP-17 pathogenesis.     

C-H ... π interplay between Ile308 and Tyr310 residues in the third repeat of microtubule binding domain is indispensable for self-assembly of three- and four-repeat tau

Information on the structural scaffold for tau aggregation is important in developing a method of preventing Alzheimer's disease (AD). Tau contains a microtubule binding domain (MBD) consisting of three or four repeats of 31 and 32 similar residues in its C-terminal half. Although the key event in tau aggregation has been considered to be the formation of β-sheet structures from a short hexapeptide (306)VQIVYK(311) in the third repeat of MBD, its aggregation pathway to filament formation differs between the three- and four-repeated MBDs, owing to the intermolecular and intramolecular disulphide bond formations, respectively. Therefore, the elucidation of a common structural element necessary for the self-assembly of three-/four-repeated full-length tau is an important research subject. Expanding the previous results on the aggregation mechanism of MBD, in this paper, we report that the C-H … π interaction between the Ile308 and Tyr310 side chains in the third repeat of MBD is indispensable for the self-assembly of three-/four-repeated full-length tau, where the interaction provides a conformational seed for triggering the molecular association. On the basis of the aggregation behaviours of a series of MBD and full-length tau mutants, a possible self-association model of tau is proposed and the relationship between the aggregation form (filament or granule) and the association pathway is discussed.     

Neuroprotective activity and evaluation of Hsp90 inhibitors in an immortalized neuronal cell line

Alzheimer’s disease (AD) neuropathology is characterized by loss of synapses and neurons, neuritic plaques consisting of β-amyloid (Aβ) peptides, and neurofibrillary tangles consisting of intracellular aggregates of hyperphosphorylated tau protein in susceptible brain regions. Aβ oligomers trigger a cascade of pathogenic events including tau hyperphosphorylation and aggregation, inflammatory reactions, and excitotoxicity that contribute to the progression of AD. The molecular chaperone Hsp90 facilitates the folding of newly synthesized and denatured proteins and is believed to play a role in neurodegenerative disorders in which the defining pathology results in misfolded proteins and the accumulation of protein aggregates. Some agents that inhibit Hsp90 protect neurons against Aβ toxicity and tau aggregation, and assays for rapidly screening potential Hsp90 inhibitors are of interest. We used the release of the soluble cytosolic enzyme lactate dehydrogenase (LDH) as an indicator of the loss of cell membrane integrity and cytotoxicity resulting from exposure to Aβ peptides to evaluate the neuroprotective properties of novel novobiocin analogues and established Hsp90 inhibitors. Compounds were assessed for potency in protecting proliferating and differentiated SH-SY5Y neuronal cells against Aβ-induced cell death; the potential toxicity of each agent alone was also determined. The data indicated that several of the compounds decreased Aβ toxicity even at low nanomolar concentrations and, unexpectedly, were more potent in protecting the undifferentiated cells against Aβ. The novobiocin analogues alone were not toxic even up to 10 μM concentrations whereas GDA and the parent compound, novobiocin, were toxic at 1 and 10 μM, respectively. The results suggest that novobiocin analogues may provide novel leads for the development of neuroprotective drugs.     

Domain conformation of tau protein studied by solution small-angle X-ray scattering

Tau is one of the two main proteins involved in the pathology of Alzheimer's disease via formation of beta-sheet rich intracellular aggregates named paired helical filaments (PHFs). Given that tau is a natively unfolded protein with no folded core (even upon binding to physiological partners such as microtubules), its structural analysis by high-resolution techniques has been difficult. In this study, employing solution small-angle X-ray scattering from the full length isoforms and from a variety of deletion and point mutants the conformation of tau in solution is structurally characterized. A recently developed ensemble optimization method was employed to generate pools of random models and to select ensembles of coexisting conformations, which fitted simultaneously the scattering data from the full length protein and deletion mutants. The analysis of the structural properties of these selected ensembles allowed us to extract information about residual structure in different domains of the native protein. The short deletion mutants containing the repeat domain (considered the core constituent of the PHFs) are significantly more extended than random coils, suggesting an extended conformation of the repeat domain. The longer tau constructs are comparable in size with the random coils, pointing to long-range contacts between the N- and C-termini compensating for the extension of the repeat domain. Moreover, most of the aggregation-promoting mutants did not show major differences in structure from their wild-type counterparts, indicating that their increased pathological effect is triggered only after an aggregation core has been formed.