Alzheimer Paired Helical Filaments Research Articles

Distinct differences in prion-like seeding and aggregation between Tau protein variants provide mechanistic insights into tauopathies

The accumulation of aberrantly aggregated MAPT (microtubule-associated protein Tau) defines a spectrum of tauopathies, including Alzheimer's disease. Mutations in the MAPT gene cause frontotemporal dementia with Parkinsonism linked to chromosome 17 (FTDP-17), characterized by neuronal pathological Tau inclusions in the form of neurofibrillary tangles and Pick bodies and in some cases glial Tau pathology. Increasing evidence points to the importance of prion-like seeding as a mechanism for the pathological spread in tauopathy and other neurodegenerative diseases. Herein, using a cell culture model, we examined a multitude of genetic FTDP-17 Tau variants for their ability to be seeded by exogenous Tau fibrils. Our findings revealed stark differences between FTDP-17 Tau variants in their ability to be seeded, with variants at Pro301 and Ser320 showing robust aggregation with seeding. Similarly, we elucidated the importance of certain Tau protein regions and unique residues, including the role of Pro301 in inhibiting Tau aggregation. We also revealed potential barriers in cross-seeding between three-repeat and four-repeat Tau isoforms. Overall, these differences alluded to potential mechanistic differences between wildtype and FTDP-17 Tau variants, as well as different Tau isoforms, in influencing Tau aggregation. Furthermore, by combining two FTDP-17 Tau variants (either P301L or P301S with S320F), we generated aggressive models of tauopathy that do not require exogenous seeding. These models will allow for rapid screening of potential therapeutics to alleviate Tau aggregation without the need for exogenous Tau fibrils. Together, these studies provide novel insights in the molecular determinants that modulate Tau aggregation.

High–Molecular-Weight Paired Helical Filaments from Alzheimer Brain Induces Seeding of Wild-Type Mouse Tau into an Argyrophilic 4R Tau Pathology in Vivo

In Alzheimer disease, the development of tau pathology follows neuroanatomically connected pathways, suggesting that abnormal tau species might recruit normal tau by passage from cell to cell. Herein, we analyzed the effect of stereotaxic brain injection of human Alzheimer high-molecular-weight paired helical filaments (PHFs) in the dentate gyrus of wild-type and mutant tau THY-Tau22 mice. After 3 months of incubation, wild-type and THY-Tau22 mice developed an atrophy of the dentate gyrus and a tau pathology characterized by Gallyas and tau-positive grain-like inclusions into granule cells that extended in the hippocampal hilus and eventually away into the alveus, and the fimbria. Gallyas-positive neuropil threads and oligodendroglial coiled bodies were also observed. These tau inclusions were composed only of mouse tau, and were immunoreactive with antibodies to 4R tau, phosphotau, misfolded tau, ubiquitin, and p62. Although local hyperphosphorylation of tau was increased in the dentate gyrus in THY-Tau22 mice, the development of neurofibrillary tangles made of mutant human tau was not accelerated in the hippocampus, indicating that wild-type human PHFs were inefficient in seeding tau aggregates made of G272V/P301S mutant human tau. Our results indicate thus that injection of human wild-type Alzheimer PHF seeded aggregation of wild-type murine tau into an argyrophilic 4R tau pathology, and constitutes an interesting model independent of expression of a mutant tau protein.

Embryonic Stem Cell–Based Modeling of Tau Pathology in Human Neurons

Alterations in the microtubule (MT)-associated protein, tau, have emerged as a pivotal phenomenon in several neurodegenerative disorders, including frontotemporal dementia and Alzheimer's disease. Although compelling lines of evidence from various experimental models suggest that hyperphosphorylation and conformational changes of tau can cause its aggregation into filaments, the actual tau species and effective mechanisms that conspire to trigger the degeneration of human neurons remain obscure. Herein, we explored whether human embryonic stem cell-derived neural stem cells can be exploited to study consequences of an overexpression of 2N4R tau (two normal N-terminal and four MT-binding domains; n-tau) versus pseudohyperphosphorylated tau (p-tau) directly in human neurons. Given the involvement of tau in MT integrity and cellular homeostasis, we focused on the effects of both tau variants on subcellular transport and neuronal survival. By using inducible lentiviral overexpression, we show that p-tau, but not n-tau, readily leads to an MC-1-positive protein conformation and impaired mitochondrial transport. Although these alterations do not induce cell death under standard culture conditions, p-tau-expressing neurons cultured under non-redox-protected conditions undergo degeneration with formation of axonal varicosities sequestering transported proteins and progressive neuronal cell death. Our data support a causative link between the phosphorylation and conformational state of tau, microtubuli-based transport, and the vulnerability of human neurons to oxidative stress. They further depict human embryonic stem cell-derived neurons as a useful experimental model for studying tau-associated cellular alterations in an authentic human system.

Crystallization and preliminary X-ray diffraction analysis of two peptides from Alzheimer PHF in complex with the MN423 antibody Fab fragment.

The major constituent of the Alzheimer's disease paired helical filaments (PHF) core is the intrinsically disordered protein (IDP) tau. Globular binding partners, e.g. monoclonal antibodies, can stabilize the fold of disordered tau in complexes. A previously published structure of a proteolytically generated tau fragment in a complex with the PHF-specific monoclonal antibody MN423 revealed a turn-like structure of the PHF core C-terminus [Sevcik et al. (2007). FEBS Lett. 581, 5872-5878]. To examine the structures of longer better-defined PHF segments, crystals of the MN423 Fab fragment were grown in the presence of two synthetic peptides derived from the PHF core C-terminus. For each, X-ray diffraction data were collected at 100 K at a synchrotron source and initial phases were obtained by molecular replacement.

Structural Polymorphism of 441-Residue Tau at Single Residue Resolution

Alzheimer disease is characterized by abnormal protein deposits in the brain, such as extracellular amyloid plaques and intracellular neurofibrillary tangles. The tangles are made of a protein called tau comprising 441 residues in its longest isoform. Tau belongs to the class of natively unfolded proteins, binds to and stabilizes microtubules, and partially folds into an ordered β-structure during aggregation to Alzheimer paired helical filaments (PHFs). Here we show that it is possible to overcome the size limitations that have traditionally hampered detailed nuclear magnetic resonance (NMR) spectroscopy studies of such large nonglobular proteins. This is achieved using optimal NMR pulse sequences and matching of chemical shifts from smaller segments in a divide and conquer strategy. The methodology reveals that 441-residue tau is highly dynamic in solution with a distinct domain character and an intricate network of transient long-range contacts important for pathogenic aggregation. Moreover, the single-residue view provided by the NMR analysis reveals unique insights into the interaction of tau with microtubules. Our results establish that NMR spectroscopy can provide detailed insight into the structural polymorphism of very large nonglobular proteins.

Structure Solution of Misfolded Conformations Adopted by Intrinsically Disordered Alzheimers Tau Protein

Until now it was impossible to obtain atomic structure of intrinsically disordered protein (IDP) tau and/or its assembly in Alzheimer's paired helical filaments as neither of them could have been prepared in the form amenable to X-ray or NMR techniques. Using IDP tau property to attain regular tertiary structure after binding events during self-assembly or when complexed with its target we propose monoclonal antibodies as surrogate tau protein binding partners to form complexes and crystals for structure solution by X-ray technique.

Structure and regulation of MARK, a kinase involved in abnormal phosphorylation of Tau protein

Protein kinases of the MARK family phosphorylate tau protein in its repeat domain and thereby regulate its affinity for microtubules and affect the aggregation of tau into Alzheimer paired helical filaments. We are searching for low molecular weight compounds to interfere with the activity of MARK and its pathways. Here we summarize structural features of MARK and cellular pathways of regulation.

Characterization of Alzheimer’s-like Paired Helical Filaments from the Core Domain of Tau Protein Using Solid-State NMR Spectroscopy

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 beta-strands. Combination of through-bond and through-space ssNMR transfer methods with water-edited ((15)N, (13)C) and ((13)C, (13)C) 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.

Phenylthiazolyl-Hydrazide and Its Derivatives Are Potent Inhibitors of τ Aggregation and Toxicity in Vitro and in Cells

One of the key pathological features of Alzheimer's disease is the aggregation of tau protein. We are therefore searching for compounds capable of inhibiting this reaction. On the basis of an initial screen of 200000 compounds [Pickhardt, M., Gazova, Z., von Bergen, M., Khlistunova, I., Wang, Y., Hascher, A., Mandelkow, E. M., Biernat, J., and Mandelkow, E. (2005) Anthraquinones inhibit tau aggregation and dissolve Alzheimer's paired helical filaments in vitro and in cells, J. Biol. Chem. 280, 3628-3635], we performed an in silico screen and predicted a new phenylthiazolyl-hydrazide (PTH) compound as a possible hit [Larbig, G., Pickhardt, M., Lloyd, D. G., Schmidt, B., and Mandelkow, E. (2007) Screening for inhibitors of tau protein aggregation into Alzheimer paired helical filaments: A ligand based approach results in successful scaffold hopping. Curr. Alzheimer Res. 4 (3), 315-323.]. Synthesis of this compound showed that it was indeed active in terms of inhibiting de novo tau aggregation and disassembling preformed aggregates (IC50 = 7.7 microM and DC50 = 10.8 microM). We have now synthesized 49 similar structures and identified the core of the PTHs to be crucial for activity, thus representing a lead structure. Analysis of the binding epitope by saturation transfer difference NMR shows strong interactions between the tau protein and the ligand in the aromatic regions of the inhibitor. By chemical variation of the core, we improved the inhibitory potency five-fold. The compounds showed a low toxicity as judged by an N2A cell model of tau aggregation and lend themselves for further development.

Inducible Expression of Tau Repeat Domain in Cell Models of Tauopathy: AGGREGATION IS TOXIC TO CELLS BUT CAN BE REVERSED BY INHIBITOR DRUGS

We generated several cell models of tauopathy in order to study the mechanisms of neurodegeneration in diseases involving abnormal changes of tau protein. N2a neuroblastoma cell lines were created that inducibly express different variants of the repeat domain of tau (tau(RD)) when exposed to doxycycline (Tet-On system). The following three constructs were chosen: (i) the repeat domain of tau that coincides with the core of Alzheimer paired helical filaments; (ii) the repeat domain with the deletion mutation DeltaK280 known from frontotemporal dementia and highly prone to spontaneous aggregation; and (iii) the repeat domain with DeltaK280 and two proline point mutations that inhibit aggregation. The comparison of wild-type, pro-aggregation, and anti-aggregation mutants shows the following. (a) Aggregation of tau(RD) is toxic to cells. (b) The degree of aggregation and toxicity depends on the propensity for beta-structure. (c) Soluble mutants of tau(RD) that cannot aggregate are not toxic. (d) Aggregation is preceded by fragmentation. (e) Fragmentation of tau(RD) in cells is initially due to a thrombin-like protease activity. (f) Phosphorylation of tau(RD) (at KXGS motifs) precedes aggregation but is not correlated with the degree of aggregation. (g) Aggregates of tau(RD) disappear when the expression is silenced, showing that aggregation is reversible. (h) Aggregation can be prevented by drugs and even pre-formed aggregates can be dissolved again by drugs. Thus, the cell models open up new insights into the relationship between the structure, expression, phosphorylation, aggregation, and toxicity of tau(RD) that can be used to test current hypotheses on tauopathy and to develop drugs that prevent the aggregation and degeneration of cells.

Characterization of Alzheimer paired helical filaments by electron microscopy

We show how electron microscopy can be used to answer several critical issues in neurodegenerative disorders that course with the formation of aberrant filamentous structures. Thus, electron microscopy is a useful technique to study in vitro assembly of pathogenic proteins, to map the regions involved in filament formation, as well as to detect by immunoelectron microscopy which proteins bind to the filaments. Furthermore, electron microscopy is the main technique used to discover if an animal model develops fibrillar pathology and if those filaments are similar to those found in human patients. This review focuses on Alzheimer's disease and related tauopathies, although similar studies have been done with other neurodegenerative disorders as, for example, Huntington's disease.

S3-02-01 Principles of assembly of tau protein into Alzheimer paired helical filaments

S3-02-01 Principles of assembly of tau protein into Alzheimer paired helical filaments

P3-225 Conformation and stability of tau and Alzheimer paired helical filaments probed by tryptophan scanning mutagenesis

P3-225 Conformation and stability of tau and Alzheimer paired helical filaments probed by tryptophan scanning mutagenesis

Expression of an altered form of tau in Sf9 insect cells results in the assembly of polymers resembling Alzheimer's paired helical filaments

Tau is the main component of the paired helical filaments (PHFs), aberrant structures that develop in the brain of Alzheimer's disease (AD) patients and other tauopathies like frontotemporal dementia and parkinsonism associated to chromosome 17 (FTDP-17). Previous work has shown that tau overexpression in Sf9 insect cells results in the formation of long cytoplasmatic extensions as a consequence of microtubule stabilization and bundling. Throughout this work, we have taken studies in this system further by overexpression of an altered form of tau characteristic of FTDP-17, which includes three mutations (G272V, P301L and R406W) and biochemically behaves as a hyperphosphorylated form of the protein, with the aim of developing an in vitro model which would favour the formation of tau aggregates. Our results indicate that filaments resembling PHFs assemble when Sf9 cells overexpress FTDP-17 tau. The amount of these polymers is reduced in lithium treated cells which suggests that phosphorylation of FTDP-17 tau by GSK3 induces a conformational change favouring the formation of fibrillar polymers.

Jumping mode atomic force microscopy obtains reproducible images of Alzheimer paired helical filaments in liquids

Alzheimer paired helical filaments (PHF) have been investigated with atomic force microscopy (AFM) under physiological conditions. Dynamic mode (DM) and jumping mode (JM) AFM have been employed as imaging techniques. Data obtained in solution have been compared to data obtained in ambient air with DM. In liquids, PHF particles show distortion and irreversible damage when imaged with DM. On the contrary, JM images of PHF particles are reproducible and out of apparent damage. Dimensions of the PHF particles measured with JM, are in agreement with previously reported electron microscopy data. We have found that the forces involved in DM imaging are larger than those involved in JM imaging and hence we believe that this is the main reason of the damage caused by the tip when using DM in solution.

Characterization by Atomic Force Microscopy of Alzheimer Paired Helical Filaments under Physiological Conditions

Paired helical filaments (PHF) is an aberrant structure present in the brain of Alzheimer's disease patients which has been correlated with their degree of dementia. In order to determine the structure of PHF, several studies have been performed using atomic force microscopy (AFM). However, those studies have the limitation that they have not been done in solution and the sample could be far from the real physiological conditions. In this work we present an AFM analysis of PHF in liquid environment and we compare that analysis with that performed in dry conditions. PHF imaging in liquid was only possible by using jumping mode AFM as the imaging technique. Jumping mode AFM images of PHF in solution show first, a notable increase in the absolute values of the height of the filament, and second, a smaller ratio between the height measured at the upper and at the lower part of the PHF. Direct comparison of the experimental data with structural models has been performed. From this we conclude that the PHF structure is compatible with two coupled ribbons with an overall height of 20 nm and a width of 10 nm.

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 conformations of filamentous and soluble tau associated with Alzheimer paired helical filaments.

Paired helical filaments (PHF) occur in Alzheimer's diseased brains and are known to be composed of the microtubule-associated protein, tau. In the present report, circular dichroism (CD) spectroscopy and transmission electron microscopy (TEM) were used to characterize PHF suspended in Tris-buffered saline (TBS), sodium acetate buffer, and water. In TBS the CD spectrum of PHF was observed to have a spectral pattern consistent with 31-37% alpha-helix, 15-20% beta-sheet, 20-23% turn, and 26-29% unordered structure. The TBS sample was found to undergo a cooperative thermal transition between 70 and 75 degrees C, consistent with the changes observed in filament morphology, and it suggests that filamentous tau in the PHF (PHF-tau) makes a substantial contribution to the overall CD. Observed changes in the CD spectrum following removal of PHF by centrifugation suggest that PHF-tau possesses a higher fraction of alpha-helical structure than soluble tau. In acetate buffer, where only straight filaments were observed, the CD was consistent with a marked decrease in the fraction of alpha-helix and an increase in the fraction of beta-sheet relative to the sample in TBS. In water, where only rudimentary filaments remain, the CD was consistent with a Type II or II' beta-turn conformation. Only noncooperative thermal transitions were observed for the PHF samples in acetate buffer and water, consistent with the presence of a heterogeneous population of folded structures. Taken cumulatively, the results are consistent with immunological data showing the presence of folded forms of tau and suggest that phosphorylation or nonproteinaceous components are able to induce conformations of tau other than the random coil conformation previously reported for cloned or purified human tau.

Phosphorylation disrupts the central helix in Op18/stathmin and suppresses binding to tubulin.

Protein phosphorylation represents a ubiquitous control mechanism in living cells. The structural prerequisites and consequences of this important post-translational modification, however, are poorly understood. Oncoprotein 18/stathmin (Op18) is a globally disordered phosphoprotein that is involved in the regulation of the microtubule (MT) filament system. Here we document that phosphorylation of Ser63, which is located within a helix initiation site in Op18, disrupts the transiently formed amphipathic helix. The phosphoryl group reduces tubulin binding 10-fold and suppresses the MT polymerization inhibition activity of Op18's C-terminal domain. Op18 represents an example where phosphorylation occurs within a regular secondary structural element. Together, our findings have implications for the prediction of phosphorylation sites and give insights into the molecular behavior of a globally disordered protein.

Pathway of tau aggregation into Alzheimer paired helical filaments

Pathway of tau aggregation into Alzheimer paired helical filaments