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.
Background: Tau oligomers are thought to play key roles in Alzheimer disease.
Results: Oligomers of the Tau repeat domain do not cause gross neuronal toxicity but decrease spine density.
Conclusion: Synaptic toxicity from extracellular Tau oligomers is local, without general loss of viability.
Significance: Extracellular Tau oligomers are not likely to explain Tau-mediated neurodegeneration but may cause functional impairment by spine reduction.
Microtubule-dependent transport of vesicles and organelles appears saltatory because particles switch between periods of rest, random Brownian motion, and active transport. The transport can be ...regulated through motor proteins, cargo adaptors, or microtubule tracks. We report here a mechanism whereby microtubule associated proteins (MAPs) represent obstacles to motors which can be regulated by microtubule affinity regulating kinase (MARK)/Par-1, a family of kinases that is known for its involvement in establishing cell polarity and in phosphorylating tau protein during Alzheimer neurodegeneration. Expression of MARK causes the phosphorylation of MAPs at their KXGS motifs, thereby detaching MAPs from the microtubules and thus facilitating the transport of particles. This occurs without impairing the intrinsic activity of motors because the velocity during active movement remains unchanged. In primary retinal ganglion cells, transfection with tau leads to the inhibition of axonal transport of mitochondria, APP vesicles, and other cell components which leads to starvation of axons and vulnerability against stress. This transport inhibition can be rescued by phosphorylating tau with MARK.
Aggregation of the microtubule-associated protein tau into neurofibrillary tangles is the pathological hallmark of a variety of dementias. For reasons not yet known, tau becomes excessively ...phosphorylated in Alzheimer’s brains and as a result no longer binds properly to microtubules. Here we studied the impact of phosphorylation on the conformational and binding properties of the repeat region of tau (K18) that is necessary for microtubule assembly and forms the core of paired helical filaments. To mimic phosphorylation, we introduced four mutations of serine to glutamate residues at positions 262, 293, 324, and 356. NMR spectroscopy demonstrates that pseudophosphorylation at these sites modifies the structural properties in repeats 1 and 2, in particular for Gln265−Lys267. Gln265−Lys267 are in close proximity to Ser262, the phosphorylation site that most strongly attenuates binding to microtubules. In contrast, the pseudophosphorylation mimic of tau efficiently interacts with the polyanion heparin. Thus, phosphorylation of the repeat region of natively unfolded tau induces specific conformational changes that have a strong impact on its biological function and involvement in disease.
The abnormal aggregation of tau protein into paired helical filaments (PHFs) is one of the hallmarks of Alzheimer's disease. Aggregation takes place in the cytoplasm and could therefore be cytotoxic ...for neurons. To find inhibitors of PHF aggregation we screened a library of 200,000 compounds. The hits found in the PHF inhibition assay were also tested for their ability to dissolve preformed PHFs. The results were obtained using a thioflavin S fluorescence assay for the detection and quantification of tau aggregation in solution, a tryptophan fluorescence assay using tryptophan-containing mutants of tau, and confirmed by a pelleting assay and electron microscopy of the products. Here we demonstrate the feasibility of the approach with several compounds from the family of anthraquinones, including emodin, daunorubicin, adriamycin, and others. They were able to inhibit PHF formation with IC50 values of 1–5 μm and to disassemble preformed PHFs at DC50 values of 2–4 μm. The compounds had a similar activity for PHFs made from different tau isoforms and constructs. The compounds did not interfere with the stabilization of microtubules by tau. Tau-inducible neuroblastoma cells showed the formation of tau aggregates and concomitant cytotoxicity, which could be prevented by inhibitors. Thus, small molecule inhibitors could provide a basis for the development of tools for the treatment of tau pathology in AD and other tauopathies.
Tau protein plays an important role in neuronal physiology and Alzheimer’s neurodegeneration. Its abilities to aggregate abnormally, to bind to microtubules (MTs), and to promote MT assembly are all ...influenced by phosphorylation. Phosphorylation of serine residues in the KXGS motifs of Tau’s repeat domain, crucial for MT interactions and aggregation, is facilitated most efficiently by microtubule-associated protein/microtubule affinity-regulating kinases (MARKs). Here we applied high-resolution nuclear magnetic resonance analysis to study the kinetics of phosphorylation of Tau by MARK2 and its impact on the structure and microtubule binding of Tau. We demonstrate that MARK2 binds to the N-terminal tail of Tau and selectively phosphorylates three major and five minor serine residues in the repeat domain and C-terminal tail. Structural changes induced by phosphorylation of Tau by MARK2 are highly localized in the proximity of the phosphorylation site and do not affect the global conformation, in contrast to phosphorylation in the proline-rich region. Furthermore, single-residue analysis of binding of Tau to MTs provides support for a model in which Tau’s hot spots of MT interaction bind independently of each other and are differentially affected by phosphorylation.
The polymerization of the microtubule-associated protein tau into paired helical filaments (PHFs) represents one of the hallmarks of Alzheimer’s disease. We employed solid-state nuclear magnetic ...resonance (NMR) to investigate the structure and dynamics of PHFs formed in vitro by the three-repeat-domain (K19) of protein tau, representing the core of Alzheimer PHFs. While N and C termini of tau monomers in PHFs are highly dynamic and solvent-exposed, the rigid segment consists of three major β-strands. Combination of through-bond and through-space ssNMR transfer methods with water-edited (15N,13C) and (13C,13C) correlation experiments suggests the existence of a fibril core that is largely built by repeat unit R3, flanked by surface-exposed units R1 and R4. Solid-state NMR, circular dichroism, and the fibrillization behavior of a K19 mutant furthermore indicate that electrostatic interactions play a central role in stabilizing the K19 PHFs.
Tau, a natively unstructured protein that regulates the organization of neuronal microtubules, is also found in high concentrations in neurofibrillary tangles of Alzheimer's disease and other ...neurodegenerative disorders. The conformational transition between these vastly different healthy and pathological forms remains poorly understood. We have measured residual dipolar couplings (RDCs), J-couplings, and nuclear Overhauser enhancement (NOE) in construct K18 of tau, containing all four repeat domains R1-R4. NHN RDCs were compared with prediction on the basis of a statistical model describing the intrinsic conformational sampling of unfolded proteins in solution. While local variation and relative amplitude of RDCs agrees with propensity-based prediction for most of the protein, homologous sequences in each repeat domain (DLKN, DLSN, DLSK, and DKFD in repeats R1−R4) show strong disagreement characterized by inversion of the sign of the central couplings. Accelerated molecular dynamic simulations (AMD) in explicit solvent revealed strong tendencies to form turns, identified as type I β-turns for repeats R1−R3. Incorporation of the backbone dihedral sampling resulting from AMD into the statistical coil model closely reproduces experimental RDC values. These localized sequence-dependent conformational tendencies interrupt the propensity to sample more extended conformations in adjacent strands and are remarkably resistant to local environmental factors, as demonstrated by the persistence of the RDC signature even under harsh denaturing conditions (8 M urea). The role that this specific conformational behavior may play in the transition to the pathological form is discussed.
Dysfunctional tau accumulation is a major contributing factor in tauopathies, and the heat‐shock protein 70 (Hsp70) seems to play an important role in this accumulation. Several reports suggest that ...Hsp70 proteins can cause tau degradation to be accelerated or slowed, but how these opposing activities are controlled is unclear. Here we demonstrate that highly homologous variants in the Hsp70 family can have opposing effects on tau clearance kinetics. When overexpressed in a tetracycline (Tet)‐based protein chase model, constitutive heat shock cognate 70 (Hsc70) and inducible Hsp72 slowed or accelerated tau clearance, respectively. Tau synergized with Hsc70, but not Hsp72, to promote microtubule assembly at nearly twice the rate of either Hsp70 homologue in reconstituted, ATP‐regenerating Xenopus extracts supplemented with rhodamine‐labeled tubulin and human recombinant Hsp72 and Hsc70. Nuclear magnetic resonance spectroscopy with human recombinant protein revealed that Hsp72 had greater affinity for tau than Hsc70 (I/I0 ratio difference of 0.3), but Hsc70 was 30 times more abundant than Hsp72 in human and mouse brain tissue. This indicates that the predominant Hsp70 variant in the brain is Hsc70, suggesting that the brain environment primarily supports slower tau clearance. Despite its capacity to clear tau, Hsp72 was not induced in the Alzheimer's disease brain, suggesting a mechanism for age‐associated onset of the disease. Through the use of chimeras that blended the domains of Hsp72 and Hsc70, we determined that the reason for these differences between Hsc70 and Hsp72 with regard to tau clearance kinetics lies within their C‐terminal domains, which are essential for their interactions with substrates and cochaperones. Hsp72 but not Hsc70 in the presence of tau was able to recruit the cochaperone ubiquitin ligase CHIP, which is known to facilitate the ubiquitination of tau, describing a possible mechanism of how the C‐termini of these homologous Hsp70 variants can differentially regulate tau triage. Thus, efforts to promote Hsp72 expression and inhibit Hsc70 could be therapeutically relevant for tauopathies.—Jinwal, U. K., Akoury, E., Abisambra, J. F., O'Leary, J. C., III, Thompson, A. D., Blair, L. J., Jin, Y., Bacon, J., Nordhues, B. A., Cockman, M., Zhang, J., Li, P., Zhang, B., Borysov, S., Uversky, V. N., Biernat, J., Mandelkow, E., Gestwicki, J. E., Zweckstetter, M., Dickey, C. A. Imbalance of Hsp70 family variants fosters tau accumulation. FASEB J. 27, 1450–1459 (2013). www.fasebj.org
The aggregation of Tau protein is a hallmark of neurodegenerative diseases including Alzheimer's disease. Previously, we generated a cell model of tauopathy based on the 4-repeat domain with the ...FTDP-17 mutation ΔK280 (Tau4RDΔK) which is expressed in a regulatable fashion (tet-on). The deletion variant ΔK280 is highly amyloidogenic and forms fibrous aggregates in neuroblastoma N2a cells staining with the reporter dye Thioflavin S. The aggregation of Tau4RDΔK is toxic, contrary to wildtype or anti-aggregant variants of the protein. Using a novel approach for monitoring in situ Tau aggregation and toxicity by combination of microscopic analysis with FACS and biochemical analysis of cells enabled the dissection of the aggregating species which cause a time-dependent increase of toxicity. The dominant initiating step is the dimerization of Tau4RDΔK which leads to further aggregation and induces a strong increase in reactive oxygen species (ROS) and cytoplasmic Ca2+ which damage the membranes and cause cell death. Tau-based treatments using Tau aggregation inhibitors reduce both soluble oligomeric and fully aggregated Tau species and decrease their toxicity.
Tau misfolding and assembly is linked to a number of neurodegenerative diseases collectively described as tauopathies, including Alzheimer’s disease (AD) and Parkinson’s disease. Anionic cellular ...membranes, such as the cytosolic leaflet of the plasma membrane, are sites that concentrate and neutralize tau, primarily due to electrostatic interactions with tau’s microtubule binding repeat domain (RD). In addition to electrostatic interactions with lipids, tau also has interactions with membrane proteins, which are important for tau’s cellular functions. Tau also interacts with lipid tails to facilitate direct translocation across the membrane and can form stable protein-lipid complexes involved in cell-to-cell transport. Concentrated tau monomers at the membrane surface can form reversible condensates, change secondary structures, and induce oligomers, which may eventually undergo irreversible crosslinking and fibril formation. These β-sheet rich tau structures are capable of disrupting membrane organization and are toxic in cell-based assays. Given the evidence for relevant membrane-based tau assembly, we review the emerging hypothesis that polyanionic membranes may serve as a site for phase-separated tau condensation. Membrane-mediated phase separation may have important implications for regulating tau folding/misfolding, and may be a powerful mechanism to spatially direct tau for native membrane-mediated functions.
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