The constituent paired helical filaments (PHFs) in neurofibrillary tangles are insoluble intracellular deposits central to the development of Alzheimer’s disease (AD) and other tauopathies. ...Full‐length tau requires the addition of anionic cofactors such as heparin to enhance assembly. We have shown that a fragment from the proteolytically stable core of the PHF, tau 297‐391 known as ‘dGAE’, spontaneously forms cross‐β‐containing PHFs and straight filaments under physiological conditions. Here, we have analysed and compared the structures of the filaments formed by dGAE in vitro with those deposited in the brains of individuals diagnosed with AD. We show that dGAE forms PHFs that share a macromolecular structure similar to those found in brain tissue. Thus, dGAEs may serve as a model system for studying core domain assembly and for screening for inhibitors of tau aggregation.
Truncated tau (297‐391) is the principal component of the core of paired helical filaments (PHFs) in neurofibrillary tangles isolated from Alzheimer’s brain tissue. Truncated tau forms filaments in vitro that share macromolecular characteristics with PHFs found in vivo. Truncated tau, without modification, is sufficient for the assembly of PHFs.
Tau protein, found in both neuronal and non-neuronal cells, forms aggregates in neurons that constitutes one of the hallmarks of Alzheimer's disease (AD). For nearly four decades, research efforts ...have focused more on tau's role in physiology and pathology in the context of the microtubules, even though, for over three decades, tau has been localised in the nucleus and the nucleolus. Its nuclear and nucleolar localisation had stimulated many questions regarding its role in these compartments. Data from cell culture, mouse brain, and the human brain suggests that nuclear tau could be essential for genome defense against cellular distress. However, its nature of translocation to the nucleus, its nuclear conformation and interaction with the DNA and other nuclear proteins highly suggest it could play multiple roles in the nucleus. To find efficient tau-based therapies, there is a need to understand more about the functional relevance of the varied cellular distribution of tau, identify whether specific tau transcripts or isoforms could predict tau's localisation and function and how they are altered in diseases like AD. Here, we explore the cellular distribution of tau, its nuclear localisation and function and its possible involvement in neurodegeneration.
Aggregation of the tau protein into fibrillar cross-β aggregates is a hallmark of Alzheimer's diseases (AD) and many other neurodegenerative tauopathies. Recently, several core structures of ...patient-derived tau paired helical filaments (PHFs) have been solved revealing a structural variability that often correlates with a specific tauopathy. To further characterize the dynamics of these fibril cores, to screen for strain-specific small molecules as potential biomarkers and therapeutics, and to develop strain-specific antibodies, recombinant in-vitro models of tau filaments are needed. We recently showed that a 95-residue fragment of tau (from residue 297 to 391), termed dGAE, forms filaments
in the absence of polyanionic co-factors often used for
aggregation of full-length tau. Tau(297-391) was identified as the proteolytic resistant core of tau PHFs and overlaps with the structures characterized by cryo-electron microscopy in
PHFs, making it a promising model for the study of AD tau filaments
. In the present study, we used solid-state NMR to characterize tau(297-391) filaments and show that such filaments assembled under non-reducing conditions are more dynamic and less ordered than those made in the presence of the reducing agent DTT. We further report the resonance assignment of tau(297-391)+DTT filaments and compare it to existing core structures of tau.
Oxidative stress is a significant source of damage that accumulates during aging and contributes to Alzheimer's disease (AD) pathogenesis. Oxidation of proteins can give rise to covalent links ...between adjacent tyrosines known as dityrosine (DiY) cross-linking, amongst other modifications, and this observation suggests that DiY could serve as a biomarker of accumulated oxidative stress over the lifespan. Many studies have focused on understanding the contribution of DiY to AD pathogenesis and have revealed that DiY crosslinks can be found in both Aβ and tau deposits - the two key proteins involved in the formation of amyloid plaques and tau tangles, respectively. However, there is no consensus yet in the field on the impact of DiY on Aβ and tau function, aggregation, and toxicity. Here we review the current understanding of the role of DiY on Aβ and tau gathered over the last 20 years since the first observation, and discuss the effect of this modification for Aβ and tau aggregation, and its potential as a biomarker for AD.
The presence of amyloid fibrils is a hallmark of more than 50 human disorders, including neurodegenerative diseases and systemic amyloidoses. A key unresolved challenge in understanding the ...involvement of amyloid in disease is to explain the relationship between individual structural polymorphs of amyloid fibrils, in potentially mixed populations, and the specific pathologies with which they are associated. Although cryo-electron microscopy (cryo-EM) and solid-state nuclear magnetic resonance (ssNMR) spectroscopy methods have been successfully employed in recent years to determine the structures of amyloid fibrils with high resolution detail, they rely on ensemble averaging of fibril structures in the entire sample or significant subpopulations. Here, we report a method for structural identification of individual fibril structures imaged by atomic force microscopy (AFM) by integration of high-resolution maps of amyloid fibrils determined by cryo-EM in comparative AFM image analysis. This approach was demonstrated using the hitherto structurally unresolved amyloid fibrils formed in vitro from a fragment of tau (297–391), termed ‘dGAE’. Our approach established unequivocally that dGAE amyloid fibrils bear no structural relationship to heparin-induced tau fibrils formed in vitro. Furthermore, our comparative analysis resulted in the prediction that dGAE fibrils are structurally closely related to the paired helical filaments (PHFs) isolated from Alzheimer’s disease (AD) brain tissue characterised by cryo-EM. These results show the utility of individual particle structural analysis using AFM, provide a workflow of how cryo-EM data can be incorporated into AFM image analysis and facilitate an integrated structural analysis of amyloid polymorphism.
Tau plays an important pathological role in a group of neurodegenerative diseases called tauopathies, including Alzheimer's disease, Pick's disease, chronic traumatic encephalopathy and corticobasal ...degeneration. In each disease, tau self-assembles abnormally to form filaments that deposit in the brain. Tau is a natively unfolded protein that can adopt distinct structures in different pathological disorders. Cryo-electron microscopy has recently provided a series of structures for the core of the filaments purified from brain tissue from patients with different tauopathies and revealed that they share a common core region, while differing in their specific conformation. This structurally resolvable part of the core is contained within a proteolytically stable core region from the repeat domain initially isolated from AD tau filaments. Tau has recently become an important target for therapy. Recent work has suggested that the prevention of tau self-assembly may be effective in slowing the progression of Alzheimer's disease and other tauopathies. Here we review the work that explores the importance of tau filament structures and tau self-assembly mechanisms, as well as examining model systems that permit the exploration of the mode of action of potential inhibitors.Tau plays an important pathological role in a group of neurodegenerative diseases called tauopathies, including Alzheimer's disease, Pick's disease, chronic traumatic encephalopathy and corticobasal degeneration. In each disease, tau self-assembles abnormally to form filaments that deposit in the brain. Tau is a natively unfolded protein that can adopt distinct structures in different pathological disorders. Cryo-electron microscopy has recently provided a series of structures for the core of the filaments purified from brain tissue from patients with different tauopathies and revealed that they share a common core region, while differing in their specific conformation. This structurally resolvable part of the core is contained within a proteolytically stable core region from the repeat domain initially isolated from AD tau filaments. Tau has recently become an important target for therapy. Recent work has suggested that the prevention of tau self-assembly may be effective in slowing the progression of Alzheimer's disease and other tauopathies. Here we review the work that explores the importance of tau filament structures and tau self-assembly mechanisms, as well as examining model systems that permit the exploration of the mode of action of potential inhibitors.
Background
Tauopathies are characterised by the accumulation of intracellular tau aggregates in the brain. Tau inclusions contain phosphorylated and truncated tau species, but the potential ...detrimental effects of these tau fragments are not well established. Tau35 is a C‐terminal fragment of wild‐type human tau, identified in tauopathy brain. Minimal expression of Tau35 in transgenic mice recapitulates key features of tauopathy, including cognitive and motor dysfunction.
Method
We used small angle X‐ray scattering (SAXS), circular dichroism (CD), transmission electron microscopy (TEM) and Thioflavin T (ThT) assays to characterise the conformations and assembly of recombinant Tau35 and full‐length (2N4R) human tau. To assess cellular effects of tau expression, differentiated SH‐SY5Y (dSH) cells and mouse primary cortical neurons were exposed to Cy5‐labelled aggregated recombinant tau and examined by immunofluorescence and on western blots.
Result
SAXS analysis showed that both Tau35 and 2N4R tau display characteristics of intrinsically disordered monomeric proteins, with Tau35 exhibiting more restricted motions. ThT kinetics indicated that Tau35 is more prone to fibril formation than 2N4R tau, which was confirmed by TEM. CD showed an increase in beta content. Notably, both Tau35 and 2N4R tau were taken up by dSH cells and mouse cortical neurons, with Tau35 internalisation occurring at a faster rate. Tau35 and 2N4R tau intracellular accumulation in both cells increased over time, with both tau species entering the nucleus.
Conclusion
Our results demonstrate that Tau35, which is associated with human tauopathy, exhibits an increased propensity to aggregate in vitro. Our observation that Tau35 is both highly aggregating and rapidly internalised by neuronal cells, may promote its ability to propagate tau pathology in the tauopathies.
Alzheimer's disease is characterized by the self-assembly of tau and amyloid β proteins into oligomers and fibrils. Tau protein assembles into paired helical filaments (PHFs) that constitute the ...neurofibrillary tangles observed in neuronal cell bodies in individuals with Alzheimer's disease. The mechanism of initiation of tau assembly into PHFs is not well understood. Here we report that a truncated 95-amino-acid tau fragment (corresponding to residues 297–391 of full-length tau) assembles into PHF-like fibrils in vitro without the need for other additives to initiate or template the process. Using electron microscopy, circular dichroism and X-ray fiber diffraction, we have characterized the structure of the fibrils formed from truncated tau for the first time. To explore the contribution of disulfide formation to fibril formation, we have compared the assembly of tau(297–391) under reduced and non-reducing conditions and for truncated tau carrying a C322A substitution. We show that disulfide bond formation inhibits filament assembly and that the C322A variant rapidly forms long and highly ordered PHFs.
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•Truncated tau is a major component of paired helical filaments (PHF) in neurofibrillary tangles in Alzheimer's brain tissue and encompasses the core structure of PHF isolated from Alzheimer's disease brain.•Truncated tau (297–391) forms filaments that share fine structural characteristics with isolated PHF.•Reduction of the disulfide, or replacement of Cys with Ala in truncated tau, leads to enhanced filament formation.•Truncated unmodified tau is sufficient for the assembly of PHF found in Alzheimer's disease.
Apolipoprotein E4 (ApoE4) is one of three (E2, E3 and E4) human isoforms of an α-helical, 299-amino-acid protein. Homozygosity for the ε4 allele is the major genetic risk factor for developing ...late-onset Alzheimer's disease (AD). ApoE2, ApoE3 and ApoE4 differ at amino acid positions 112 and 158, and these sequence variations may confer conformational differences that underlie their participation in the risk of developing AD. Here, we compared the shape, oligomerization state, conformation and stability of ApoE isoforms using a range of complementary biophysical methods including small-angle x-ray scattering, analytical ultracentrifugation, circular dichroism, x-ray fiber diffraction and transmission electron microscopy We provide an in-depth and definitive study demonstrating that all three proteins are similar in stability and conformation. However, we show that ApoE4 has a propensity to polymerize to form wavy filaments, which do not share the characteristics of cross-β amyloid fibrils. Moreover, we provide evidence for the inhibition of ApoE4 fibril formation by ApoE3. This study shows that recombinant ApoE isoforms show no significant differences at the structural or conformational level. However, self-assembly of the ApoE4 isoform may play a role in pathogenesis, and these results open opportunities for uncovering new triggers for AD onset.
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•There are three apolipoprotein E isoforms, and E4E4 increases the risk for Alzheimer's disease.•ApoE2, E3 and E4 share a quaternary, tertiary and secondary structures, and stability.•ApoE4 forms non-amyloid-like filaments, while ApoE2 and E3 do not.•Assembly of E4 may play a role increased risk of late-onset Alzheimer's disease.
Most low molecular weight gelators are chiral, with racemic mixtures often unable to form gels. Here, we show an example where all enantiomers, diastereomers and racemates of a single functionalized ...dipeptide can form gels. At high pH, different self-assembled aggregates are formed and these directly template the structures formed in the gel. Hence, solutions and gels with different properties can be accessed simply by varying the chirality. This opens up new design rules for the field.
Different self-assembled structures can be formed by varying the chirality of a functionalised dipeptide allowing gels with different properties to be prepared.
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