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Genetic backgrounds influence cellular phenotypes, drug responses, and health outcomes, yet most human iPSC lines are derived from individuals of European descent, with lines from indigenous Africans particularly scarce. Addressing this gap, we generated iPSCs from dermal fibroblasts of a healthy 60-year-old indigenous Nigerian male of the Babur ethnic group using Sendai virus. The iPSC line displayed a normal karyotype, was characterized for pluripotency markers and differentiated into neural progenitor cells and astrocytes. To enhance African representation in research, this iPSC line will be available to the scientific community, with ongoing efforts focused on creating an open-access African iPSC biobank. Resource Table.
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Diferenciação Celular , Células-Tronco Pluripotentes Induzidas , Humanos , Células-Tronco Pluripotentes Induzidas/citologia , Células-Tronco Pluripotentes Induzidas/metabolismo , Masculino , Pessoa de Meia-Idade , Nigéria , Linhagem Celular , Fibroblastos/citologia , Fibroblastos/metabolismoRESUMO
INTRODUCTION: In tauopathies, altered tau processing correlates with impairments in synaptic density and function. Changes in hyperpolarization-activated cyclic nucleotide-gated (HCN) channels contribute to disease-associated abnormalities in multiple neurodegenerative diseases. METHODS: To investigate the link between tau and HCN channels, we performed histological, biochemical, ultrastructural, and functional analyses of hippocampal tissues from Alzheimer's disease (AD), age-matched controls, Tau35 mice, and/or Tau35 primary hippocampal neurons. RESULTS: Expression of specific HCN channels is elevated in post mortem AD hippocampus. Tau35 mice develop progressive abnormalities including increased phosphorylated tau, enhanced HCN channel expression, decreased dendritic branching, reduced synapse density, and vesicle clustering defects. Tau35 primary neurons show increased HCN channel expression enhanced hyperpolarization-induced membrane voltage "sag" and changes in the frequency and kinetics of spontaneous excitatory postsynaptic currents. DISCUSSION: Our findings are consistent with a model in which pathological changes in tauopathies impact HCN channels to drive network-wide structural and functional synaptic deficits. HIGHLIGHTS: Hyperpolarization-activated cyclic nucleotide-gated (HCN) channels are functionally linked to the development of tauopathy. Expression of specific HCN channels is elevated in the hippocampus in Alzheimer's disease and the Tau35 mouse model of tauopathy. Increased expression of HCN channels in Tau35 mice is accompanied by hyperpolarization-induced membrane voltage "sag" demonstrating a detrimental effect of tau abnormalities on HCN channel function. Tau35 expression alters synaptic organization, causing a loosened vesicle clustering phenotype in Tau35 mice.
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Doença de Alzheimer , Hipocampo , Canais Disparados por Nucleotídeos Cíclicos Ativados por Hiperpolarização , Camundongos Transgênicos , Sinapses , Proteínas tau , Animais , Canais Disparados por Nucleotídeos Cíclicos Ativados por Hiperpolarização/metabolismo , Hipocampo/metabolismo , Hipocampo/patologia , Humanos , Camundongos , Proteínas tau/metabolismo , Doença de Alzheimer/metabolismo , Doença de Alzheimer/patologia , Doença de Alzheimer/genética , Sinapses/patologia , Sinapses/metabolismo , Neurônios/metabolismo , Neurônios/patologia , Canalopatias/genética , Canalopatias/patologia , Masculino , Feminino , Idoso , Modelos Animais de Doenças , Tauopatias/patologia , Tauopatias/metabolismoRESUMO
Fibroins' transition from liquid to solid is fundamental to spinning and underpins the impressive native properties of silk. Herein, we establish a fibroin heavy chain fold for the Silk-I polymorph, which could be relevant for other similar proteins, and explains mechanistically the liquid-to-solid transition of this silk, driven by pH reduction and flow stress. Combining spectroscopy and modelling we propose that the liquid Silk-I fibroin heavy chain (FibH) from the silkworm, Bombyx mori, adopts a newly reported ß-solenoid structure. Similarly, using rheology we propose that FibH N-terminal domain (NTD) templates reversible higher-order oligomerization driven by pH reduction. Our integrated approach bridges the gap in understanding FibH structure and provides insight into the spatial and temporal hierarchical self-assembly across length scales. Our findings elucidate the complex rheological behaviour of Silk-I, solutions and gels, and the observed liquid crystalline textures within the silk gland. We also find that the NTD undergoes hydrolysis during standard regeneration, explaining key differences between native and regenerated silk feedstocks. In general, in this study we emphasize the unique characteristics of native and native-like silks, offering a fresh perspective on our fundamental understanding of silk-fibre production and applications.
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Bombyx , Fibroínas , Bombyx/metabolismo , Bombyx/química , Animais , Fibroínas/química , Fibroínas/metabolismo , Reologia , Seda/química , Seda/metabolismo , Concentração de Íons de HidrogênioRESUMO
Cellular compartments formed by biomolecular condensation are widespread features of cell biology. These organelle-like assemblies compartmentalize macromolecules dynamically within the crowded intracellular environment. However, the intermolecular interactions that produce condensed droplets may also create arrested states and potentially pathological assemblies such as fibers, aggregates, and gels through droplet maturation. Protein liquid-liquid phase separation is a metastable process, so maturation may be an intrinsic property of phase-separating proteins, where nucleation of different phases or states arises in supersaturated condensates. Here, we describe the formation of both phase-separated droplets and proteinaceous fibers driven by a de novo designed polypeptide. We characterize the formation of supramolecular fibers in vitro and in bacterial cells. We show that client proteins can be targeted to the fibers in cells using a droplet-forming construct. Finally, we explore the interplay between phase separation and fiber formation of the de novo polypeptide, showing that the droplets mature with a post-translational switch to largely ß conformations, analogous to models of pathological phase separation.
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Fenômenos Bioquímicos , Proteínas , Humanos , Proteínas/química , Peptídeos/metabolismo , Processamento de Proteína Pós-Traducional , Conformação MolecularRESUMO
Protein misfolding is common to neurodegenerative diseases (NDs) including Alzheimer's disease (AD), which is partly characterized by the self-assembly and accumulation of amyloid-beta in the brain. Lysosomes are a critical component of the proteostasis network required to degrade and recycle material from outside and within the cell and impaired proteostatic mechanisms have been implicated in NDs. We have previously established that toxic amyloid-beta oligomers are endocytosed, accumulate in lysosomes, and disrupt the endo-lysosomal system in neurons. Here, we use pioneering correlative cryo-structured illumination microscopy and cryo-soft X-ray tomography imaging techniques to reconstruct 3D cellular architecture in the native state revealing reduced X-ray density in lysosomes and increased carbon dense vesicles in oligomer treated neurons compared with untreated cells. This work provides unprecedented visual information on the changes to neuronal lysosomes inflicted by amyloid beta oligomers using advanced methods in structural cell biology.
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Peptídeos beta-Amiloides , Lisossomos , Neurônios , Lisossomos/metabolismo , Peptídeos beta-Amiloides/metabolismo , Peptídeos beta-Amiloides/química , Neurônios/metabolismo , Tomografia por Raios X/métodos , Animais , Humanos , Microscopia Crioeletrônica/métodosRESUMO
ß-Peptides have great potential as novel biomaterials and therapeutic agents, due to their unique ability to self-assemble into low dimensional nanostructures, and their resistance to enzymatic degradation in vivo. However, the self-assembly mechanisms of ß-peptides, which possess increased flexibility due to the extra backbone methylene groups present within the constituent ß-amino acids, are not well understood due to inherent difficulties of observing their bottom-up growth pathway experimentally. A computational approach is presented for the bottom-up modelling of the self-assembled lipidated ß3-peptides, from monomers, to oligomers, to supramolecular low-dimensional nanostructures, in all-atom detail. The approach is applied to elucidate the self-assembly mechanisms of recently discovered, distinct structural morphologies of low dimensional nanomaterials, assembled from lipidated ß3-peptide monomers. The resultant structures of the nanobelts and the twisted fibrils are stable throughout subsequent unrestrained all-atom molecular dynamics simulations, and these assemblies display good agreement with the structural features obtained from X-ray fiber diffraction and atomic force microscopy data. This is the first reported, fully-atomistic model of a lipidated ß3-peptide-based nanomaterial, and the computational approach developed here, in combination with experimental fiber diffraction analysis and atomic force microscopy, will be useful in elucidating the atomic scale structure of self-assembled peptide-based and other supramolecular nanomaterials.
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Simulação de Dinâmica Molecular , Nanoestruturas , Peptídeos , Nanoestruturas/química , Peptídeos/química , Lipídeos/química , Microscopia de Força AtômicaRESUMO
[This corrects the article DOI: 10.3389/fmolb.2021.779240.].
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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 Aß 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 Aß and tau function, aggregation, and toxicity. Here we review the current understanding of the role of DiY on Aß and tau gathered over the last 20 years since the first observation, and discuss the effect of this modification for Aß and tau aggregation, and its potential as a biomarker for AD.
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Two propeller-shaped chiral CoIII3YIII complexes built from fluorinated ligands are synthesized and characterized by single-crystal X-ray diffraction (SXRD), IR, UV-vis, circular dichroism (CD), elemental analysis, thermogravimetric analysis (TGA), electron spray ionization mass spectroscopy (ESI-MS), and NMR (1H, 13C, and 19F). This work explores the sensing and discrimination abilities of these complexes, thus providing an innovative sensing method using a 19F NMR chemosensory system and opening new directions in 3d/4f chemistry. Control experiments and theoretical studies shed light on the sensing mechanism, while the scope and limitations of this method are discussed and presented.
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Tau is a natively unfolded protein that contributes to the stability of microtubules. Under pathological conditions such as Alzheimer's disease (AD), tau protein misfolds and self-assembles to form paired helical filaments (PHFs) and straight filaments (SFs). Full-length tau protein assembles poorly and its self-assembly is enhanced with polyanions such as heparin and RNA in vitro, but a role for heparin or other polyanions in vivo remains unclear. Recently, a truncated form of tau (297-391) has been shown to self-assemble in the absence of additives which provides an alternative in vitro PHF model system. Here we describe methods to prepare in vitro PHFs and SFs from tau (297-391) named dGAE. We also discuss the range of biophysical/biochemical techniques used to monitor tau filament assembly and structure.
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Doença de Alzheimer , Proteínas tau , Humanos , Proteínas tau/metabolismo , Emaranhados Neurofibrilares/metabolismo , Doença de Alzheimer/metabolismo , Heparina/metabolismoRESUMO
Aggregation of the tau protein into fibrillar cross-ß 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 vitro in the absence of polyanionic co-factors often used for in vitro 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 ex vivo PHFs, making it a promising model for the study of AD tau filaments in vitro. 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.
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Hierarchical self-assembly is an effective means of preparing useful materials. However, control over assembly across length scales is a difficult challenge, often confounded by the perceived need to redesign the molecular building blocks when new material properties are needed. Here, we show that we can treat a simple dipeptide building block as a polyelectrolyte and use polymer physics approaches to explain the self-assembly over a wide concentration range. This allows us to determine how entangled the system is and therefore how it might be best processed, enabling us to prepare interesting analogues to threads and webs, as well as films that lose order on heating and "noodles" which change dimensions on heating, showing that we can transfer micellar-level changes to bulk properties all from a single building block.
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Tau is an intrinsically disordered protein that has the ability to self-assemble to form paired helical and straight filaments in Alzheimer's disease, as well as the ability to form additional distinct tau filaments in other tauopathies. In the presence of microtubules, tau forms an elongated form associated with tubulin dimers via a series of imperfect repeats known as the microtubule binding repeats. Tau has recently been identified to have the ability to phase separate in vitro and in cells. The ability of tau to adopt a wide variety of conformations appears fundamental both to its biological function and also its association with neurodegenerative diseases. The recently highlighted involvement of low-complexity domains in liquid-liquid phase separation provides a critical link between the soluble function and the insoluble dysfunctional properties of tau.
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Doença de Alzheimer , Proteínas tau , Humanos , Proteínas tau/química , Proteínas tau/metabolismo , Emaranhados Neurofibrilares/metabolismo , Doença de Alzheimer/metabolismo , Microtúbulos/metabolismo , Tubulina (Proteína)/metabolismoRESUMO
A characteristic hallmark of Alzheimer's Disease (AD) is the pathological aggregation and deposition of tau into paired helical filaments (PHF) in neurofibrillary tangles (NFTs). Oxidative stress is an early event during AD pathogenesis and is associated with tau-mediated AD pathology. Oxidative environments can result in the formation of covalent dityrosine crosslinks that can increase protein stability and insolubility. Dityrosine cross-linking has been shown in Aß plaques in AD and α-synuclein aggregates in Lewy bodies in ex vivo tissue sections, and this modification may increase the insolubility of these aggregates and their resistance to degradation. Using the PHF-core tau fragment (residues 297 - 391) as a model, we have previously demonstrated that dityrosine formation traps tau assemblies to reduce further elongation. However, it is unknown whether dityrosine crosslinks are found in tau deposits in vivo in AD and its relevance to disease mechanism is unclear. Here, using transmission electron microscope (TEM) double immunogold-labelling, we reveal that neurofibrillary NFTs in AD are heavily decorated with dityrosine crosslinks alongside tau. Single immunogold-labelling TEM and fluorescence spectroscopy revealed the presence of dityrosine on AD brain-derived tau oligomers and fibrils. Using the tau (297-391) PHF-core fragment as a model, we further showed that prefibrillar tau species are more amenable to dityrosine crosslinking than tau fibrils. Dityrosine formation results in heat and SDS stability of oxidised prefibrillar and fibrillar tau assemblies. This finding has implications for understanding the mechanism governing the insolubility and toxicity of tau assemblies in vivo.
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Doença de Alzheimer , Emaranhados Neurofibrilares , Tirosina , Proteínas tau , Doença de Alzheimer/metabolismo , Humanos , Emaranhados Neurofibrilares/química , Conformação Proteica em alfa-Hélice , Tirosina/análogos & derivados , Tirosina/química , alfa-Sinucleína/química , Proteínas tau/químicaRESUMO
A functionalised dipeptide that self-assembles to form wormlike micelles at high pH can be treated as a surfactant. By varying salt concentration, the self-assembled structures and interactions between them change, resulting in solutions with very different shear and extensional viscosity. From these, gel noodles with different mechanical properties can be prepared.
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Micelas , Tensoativos , Tensoativos/química , ViscosidadeRESUMO
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.
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Doença de Alzheimer , Amiloide , Amiloidose , Doença de Alzheimer/patologia , Amiloide/química , Proteínas Amiloidogênicas/química , Amiloidose/patologia , Microscopia Crioeletrônica/métodos , Humanos , Microscopia de Força Atômica , Estrutura Secundária de ProteínaRESUMO
Tau35 is a truncated form of tau found in human brain in a subset of tauopathies. Tau35 expression in mice recapitulates key features of human disease, including progressive increase in tau phosphorylation, along with cognitive and motor dysfunction. The appearance of aggregated tau suggests that Tau35 may have structural properties distinct from those of other tau species that could account for its pathological role in disease. To address this hypothesis, we performed a structural characterization of monomeric and aggregated Tau35 and compared the results to those of two longer isoforms, 2N3R and 2N4R tau. We used small angle X-ray scattering to show that Tau35, 2N3R and 2N4R tau all behave as disordered monomeric species but Tau35 exhibits higher rigidity. In the presence of the poly-anion heparin, Tau35 increases thioflavin T fluorescence significantly faster and to a greater extent than full-length tau, demonstrating a higher propensity to aggregate. By using atomic force microscopy, circular dichroism, transmission electron microscopy and X-ray fiber diffraction, we provide evidence that Tau35 aggregation is mechanistically and morphologically similar to previously reported tau fibrils but they are more densely packed. These data increase our understanding of the aggregation inducing properties of clinically relevant tau fragments and their potentially damaging role in the pathogenesis of human tauopathies.
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We report the rational design of a tunable Cu(II) chelating scaffold, 2-(((2-((pyridin-2-ylmethyl)amino)ethyl)amino)methyl)phenol, Salpyran (HL). This tetradentate ligand is predicated to have suitable permeation, has an extremely high affinity for Cu compared to clioquinol (pCu7.4 = 10.65 vs 5.91), and exhibits excellent selectivity for Cu(II) over Zn(II) in aqueous media. Solid and solution studies corroborate the formation of a stable [Cu(II)L]+ monocationic species at physiological pH values (7.4). Its action as an antioxidant was tested in ascorbate, tau, and human prion protein assays, which reveal that Salpyran prevents the formation of reactive oxygen species from the binary Cu(II)/H2O2 system, demonstrating its potential use as a therapeutic small molecule metal chelator.
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Antioxidantes/farmacologia , Quelantes/farmacologia , Complexos de Coordenação/farmacologia , Cobre/farmacologia , Espécies Reativas de Oxigênio/antagonistas & inibidores , Antioxidantes/síntese química , Antioxidantes/química , Quelantes/síntese química , Quelantes/química , Complexos de Coordenação/síntese química , Complexos de Coordenação/química , Cobre/química , Cristalografia por Raios X , Humanos , Concentração de Íons de Hidrogênio , Modelos Moleculares , Estrutura Molecular , Espécies Reativas de Oxigênio/metabolismo , TermodinâmicaAssuntos
Proteínas/química , Animais , Humanos , Aprendizado de Máquina , Conformação Proteica , Dobramento de Proteína , SoftwareRESUMO
A key hallmark of Alzheimer's disease is the extracellular deposition of amyloid plaques composed primarily of the amyloidogenic amyloid-ß (Aß) peptide. The Aß peptide is a product of sequential cleavage of the Amyloid Precursor Protein, the first step of which gives rise to a C-terminal Fragment (C99). Cleavage of C99 by γ-secretase activity releases Aß of several lengths and the Aß42 isoform in particular has been identified as being neurotoxic. The misfolding of Aß leads to subsequent amyloid fibril formation by nucleated polymerisation. This requires an initial and critical nucleus for self-assembly. Here, we identify and characterise the composition and self-assembly properties of cell-derived hexameric Aß42 and show its assembly enhancing properties which are dependent on the Aß monomer availability. Identification of nucleating assemblies that contribute to self-assembly in this way may serve as therapeutic targets to prevent the formation of toxic oligomers.