ABSTRACT
The amyloid ß (Aß) peptide has a central role in Alzheimer's disease (AD) pathology. The peptide length can vary between 37 and 49 amino acids, with Aß1-42 being considered the most disease-related length. However, Aß1-40 is also found in Aß plaques and has shown to form intertwined fibrils with Aß1-42. The peptides have previously also shown to form different fibril conformations, proposed to be related to disease phenotype. To conduct more representative in vitro experiments, it is vital to uncover the impact of different fibril conformations on neurons. Hence, we fibrillized different Aß1-40:42 ratios in concentrations of 100:0, 90:10, 75:25, 50:50, 25:75, 10:90 and 0:100 for either 24 h (early fibrils) or 7 days (aged fibrils). These were then characterized based on fibril width, LCO-staining and antibody-staining. We further challenged differentiated neuronal-like SH-SY5Y human cells with the different fibrils and measured Aß content, cytotoxicity and autophagy function at three different time-points: 3, 24, and 72 h. Our results revealed that both Aß1-40:42 ratio and fibril maturation affect conformation of fibrils. We further show the impact of these conformation changes on the affinity to commonly used Aß antibodies, primarily affecting Aß1-40 rich aggregates. In addition, we demonstrate uptake of the aggregates by neuronally differentiated human cells, where aggregates with higher Aß1-42 ratios generally caused higher cellular levels of Aß. These differences in Aß abundance did not cause changes in cytotoxicity nor in autophagy activation. Our results show the importance to consider conformational differences of Aß fibrils, as this can have fundamental impact on Aß antibody detection. Overall, these insights underline the need for further exploration of the impact of conformationally different fibrils and the need to reliably produce disease relevant Aß aggregates.
ABSTRACT
Amyloid beta (Aß) deposition in the neocortex is a major hallmark of Alzheimer's disease (AD), but the extent of deposition does not readily explain phenotypic diversity and rate of disease progression. The prion strain-like model of disease heterogeneity suggests the existence of different conformers of Aß. We explored this paradigm using conformation-dependent immunoassay (CDI) for Aß and conformation-sensitive luminescent conjugated oligothiophenes (LCOs) in AD cases with variable progression rates. Mapping the Aß conformations in the frontal, occipital, and temporal regions in 20 AD patients with CDI revealed extensive interindividual and anatomical diversity in the structural organization of Aß with the most significant differences in the temporal cortex of rapidly progressive AD. The fluorescence emission spectra collected in situ from Aß plaques in the same regions demonstrated considerable diversity of spectral characteristics of two LCOs-quatroformylthiophene acetic acid and heptaformylthiophene acetic acid. Heptaformylthiophene acetic acid detected a wider range of Aß deposits, and both LCOs revealed distinct spectral attributes of diffuse and cored plaques in the temporal cortex of rapidly and slowly progressive AD and less frequent and discernible differences in the frontal and occipital cortex. These and CDI findings indicate a major conformational diversity of Aß accumulating in the neocortex, with the most notable differences in temporal cortex of cases with shorter disease duration, and implicate distinct Aß conformers (strains) in the rapid progression of AD.
Subject(s)
Alzheimer Disease/metabolism , Amyloid beta-Peptides/metabolism , Neocortex/metabolism , Plaque, Amyloid/metabolism , Alzheimer Disease/pathology , Humans , Male , Neocortex/pathology , Plaque, Amyloid/pathologyABSTRACT
SARS-CoV-2 infection is associated with a surprising number of morbidities. Uncanny similarities with amyloid-disease associated blood coagulation and fibrinolytic disturbances together with neurologic and cardiac problems led us to investigate the amyloidogenicity of the SARS-CoV-2 spike protein (S-protein). Amyloid fibril assays of peptide library mixtures and theoretical predictions identified seven amyloidogenic sequences within the S-protein. All seven peptides in isolation formed aggregates during incubation at 37 °C. Three 20-amino acid long synthetic spike peptides (sequence 192-211, 601-620, 1166-1185) fulfilled three amyloid fibril criteria: nucleation dependent polymerization kinetics by ThT, Congo red positivity, and ultrastructural fibrillar morphology. Full-length folded S-protein did not form amyloid fibrils, but amyloid-like fibrils with evident branching were formed during 24 h of S-protein coincubation with the protease neutrophil elastase (NE) in vitro. NE efficiently cleaved S-protein, rendering exposure of amyloidogenic segments and accumulation of the amyloidogenic peptide 194-203, part of the most amyloidogenic synthetic spike peptide. NE is overexpressed at inflamed sites of viral infection. Our data propose a molecular mechanism for potential amyloidogenesis of SARS-CoV-2 S-protein in humans facilitated by endoproteolysis. The prospective of S-protein amyloidogenesis in COVID-19 disease associated pathogenesis can be important in understanding the disease and long COVID-19.
Subject(s)
Amyloid , COVID-19 , Spike Glycoprotein, Coronavirus , Amino Acid Sequence , Amyloid/chemistry , Amyloidogenic Proteins/chemistry , COVID-19/complications , Humans , Leukocyte Elastase , Peptides/chemistry , Prospective Studies , Protein Structure, Secondary , SARS-CoV-2 , Spike Glycoprotein, Coronavirus/chemistry , Post-Acute COVID-19 SyndromeABSTRACT
Amyloid-ß (Aß) pathology in Alzheimer's disease (AD) is characterized by the formation of polymorphic deposits comprising diffuse and cored plaques. Because diffuse plaques are predominantly observed in cognitively unaffected, amyloid-positive (CU-AP) individuals, pathogenic conversion into cored plaques appears to be critical to AD pathogenesis. Herein, we identified the distinct Aß species associated with amyloid polymorphism in brain tissue from individuals with sporadic AD (s-AD) and CU-AP. To this end, we interrogated Aß polymorphism with amyloid conformation-sensitive dyes and a novel in situ MS paradigm for chemical characterization of hyperspectrally delineated plaque morphotypes. We found that maturation of diffuse into cored plaques correlated with increased Aß1-40 deposition. Using spatial in situ delineation with imaging MS (IMS), we show that Aß1-40 aggregates at the core structure of mature plaques, whereas Aß1-42 localizes to diffuse amyloid aggregates. Moreover, we observed that diffuse plaques have increased pyroglutamated Aßx-42 levels in s-AD but not CU-AP, suggesting an AD pathology-related, hydrophobic functionalization of diffuse plaques facilitating Aß1-40 deposition. Experiments in tgAPPSwe mice verified that, similar to what has been observed in human brain pathology, diffuse deposits display higher levels of Aß1-42 and that Aß plaque maturation over time is associated with increases in Aß1-40. Finally, we found that Aß1-40 deposition is characteristic for cerebral amyloid angiopathy deposition and maturation in both humans and mice. These results indicate that N-terminal Aßx-42 pyroglutamation and Aß1-40 deposition are critical events in priming and maturation of pathogenic Aß from diffuse into cored plaques, underlying neurotoxic plaque development in AD.
Subject(s)
Alzheimer Disease/pathology , Amyloid beta-Peptides/metabolism , Peptide Fragments/metabolism , Plaque, Amyloid/metabolism , Pyrrolidonecarboxylic Acid/metabolism , Alzheimer Disease/metabolism , Amyloid beta-Peptides/chemistry , Amyloid beta-Peptides/genetics , Animals , Disease Progression , Humans , Male , Mice , Mice, Transgenic , Models, Animal , Peptide Fragments/chemistry , Peptide Fragments/genetics , Protein Conformation , Spectrometry, Mass, Matrix-Assisted Laser Desorption-IonizationABSTRACT
Microtubule Associated Protein Tau (MAPT) forms proteopathic aggregates in several diseases. The G273R tau mutation, located in the first repeat region, was found by exome sequencing in a patient who presented with dementia and parkinsonism. We herein return to pathological examination which demonstrated tau immunoreactivity in neurons and glia consistent of mixed progressive supranuclear palsy (PSP) and corticobasal degeneration (CBD) features. To rationalize the pathological findings, we used molecular biophysics to characterize the mutation in more detail in vitro and in Drosophila. The G273R mutation increases the aggregation propensity of 4-repeat (4R) tau and alters the tau binding affinity towards microtubules (MTs) and F-actin. Tau aggregates in PSP and CBD are predominantly 4R tau. Our data suggest that the G273R mutation induces a shift in pool of 4R tau by lower F-actin affinity, alters the conformation of MT bound 4R tau, while increasing chaperoning of 3R tau by binding stronger to F-actin. The mutation augmented fibrillation of 4R tau initiation in vitro and in glial cells in Drosophila and showed preferential seeding of 4R tau in vitro suggestively causing a late onset 4R tauopathy reminiscent of PSP and CBD.
Subject(s)
Brain/pathology , Neurons/metabolism , Supranuclear Palsy, Progressive/metabolism , Tauopathies/pathology , Animals , Basal Ganglia Diseases/metabolism , Brain/metabolism , Drosophila , Mutation/genetics , Neuroglia/metabolismABSTRACT
The molecular architecture of amyloids formed in vivo can be interrogated using luminescent conjugated oligothiophenes (LCOs), a unique class of amyloid dyes. When bound to amyloid, LCOs yield fluorescence emission spectra that reflect the 3D structure of the protein aggregates. Given that synthetic amyloid-ß peptide (Aß) has been shown to adopt distinct structural conformations with different biological activities, we asked whether Aß can assume structurally and functionally distinct conformations within the brain. To this end, we analyzed the LCO-stained cores of ß-amyloid plaques in postmortem tissue sections from frontal, temporal, and occipital neocortices in 40 cases of familial Alzheimer's disease (AD) or sporadic (idiopathic) AD (sAD). The spectral attributes of LCO-bound plaques varied markedly in the brain, but the mean spectral properties of the amyloid cores were generally similar in all three cortical regions of individual patients. Remarkably, the LCO amyloid spectra differed significantly among some of the familial and sAD subtypes, and between typical patients with sAD and those with posterior cortical atrophy AD. Neither the amount of Aß nor its protease resistance correlated with LCO spectral properties. LCO spectral amyloid phenotypes could be partially conveyed to Aß plaques induced by experimental transmission in a mouse model. These findings indicate that polymorphic Aß-amyloid deposits within the brain cluster as clouds of conformational variants in different AD cases. Heterogeneity in the molecular architecture of pathogenic Aß among individuals and in etiologically distinct subtypes of AD justifies further studies to assess putative links between Aß conformation and clinical phenotype.
Subject(s)
Alzheimer Disease/metabolism , Amyloid beta-Peptides/chemistry , Amyloid/chemistry , Plaque, Amyloid/metabolism , Protein Aggregates , Alzheimer Disease/classification , Alzheimer Disease/genetics , Alzheimer Disease/pathology , Amyloid/classification , Amyloid/ultrastructure , Amyloid beta-Peptides/metabolism , Amyloid beta-Protein Precursor/genetics , Amyloid beta-Protein Precursor/metabolism , Animals , Disease Models, Animal , Female , Fluorescent Dyes/chemistry , Frontal Lobe/chemistry , Frontal Lobe/metabolism , Frontal Lobe/pathology , Gene Expression , Humans , Male , Mice , Occipital Lobe/chemistry , Occipital Lobe/metabolism , Occipital Lobe/pathology , Peptide Hydrolases/chemistry , Plaque, Amyloid/classification , Plaque, Amyloid/genetics , Plaque, Amyloid/pathology , Presenilin-1/genetics , Presenilin-1/metabolism , Protein Binding , Protein Conformation , Proteolysis , Spectrometry, Fluorescence , Temporal Lobe/chemistry , Temporal Lobe/metabolism , Temporal Lobe/pathology , Thiophenes/chemistryABSTRACT
Amyloid plaque formation constitutes one of the main pathological hallmarks of Alzheimer's disease (AD) and is suggested to be a critical factor driving disease pathogenesis. Interestingly, in patients that display amyloid pathology but remain cognitively normal, Aß deposits are predominantly of diffuse morphology suggesting that cored plaque formation is primarily associated with cognitive deterioration and AD pathogenesis. Little is known about the molecular mechanism responsible for conversion of monomeric Aß into neurotoxic aggregates and the predominantly cored deposits observed in AD. The structural diversity among Aß plaques, including cored/compact- and diffuse, may be linked to their distinct Aß profile and other chemical species including neuronal lipids. We developed a novel, chemical imaging paradigm combining matrix assisted laser desorption/ionization imaging mass spectrometry (MALDI IMS) and fluorescent amyloid staining. This multimodal imaging approach was used to probe the lipid chemistry associated with structural plaque heterogeneity in transgenic AD mice (tgAPPSwe) and was correlated to Aß profiles determined by subsequent laser microdissection and immunoprecipitation-mass spectrometry. Multivariate image analysis revealed an inverse localization of ceramides and their matching metabolites to diffuse and cored structures within single plaques, respectively. Moreover, phosphatidylinositols implicated in AD pathogenesis, were found to localize to the diffuse Aß structures and correlate with Aß1-42. Further, lysophospholipids implicated in neuroinflammation were increased in all Aß deposits. The results support previous clinical findings on the importance of lipid disturbances in AD pathophysiology and associated sphingolipid processing. These data highlight the potential of multimodal imaging as a powerful technology to probe neuropathological mechanisms.
Subject(s)
Amyloid beta-Peptides/chemistry , Lipid Metabolism , Multimodal Imaging , Plaque, Amyloid/diagnostic imaging , Plaque, Amyloid/metabolism , Protein Aggregates , Amyloid beta-Peptides/metabolism , Animals , Male , Mice , Neurons/metabolism , Spectrometry, Mass, Matrix-Assisted Laser Desorption-IonizationABSTRACT
We revisited the Congo red analogue 2,5-bis(4'-hydroxy-3'-carboxy-styryl)benzene (X-34) to develop this highly fluorescent amyloid dye for imaging Alzheimer's disease (AD) pathology comprising Aß and Tau fibrils. A selection of ligands with distinct optical properties were synthesized by replacing the central benzene unit of X-34, with other heterocyclic moieties. Full photophysical characterization was performed, including recording absorbance and fluorescence spectra, Stokes shift, quantum yield and fluorescence lifetimes. All ligands displayed high affinity towards recombinant amyloid fibrils of Aß1-42 (13-300â nm Kd ) and Tau (16-200â nm Kd ) as well as selectivity towards the corresponding disease-associated protein aggregates in AD tissue. We observed that these ligands efficiently displaced X-34, but not Pittsburgh compound B (PiB) from recombinant Aß1-42 amyloid fibrils, arguing for retained targeting of the Congo red type binding site. We foresee that the X-34 scaffold offers the possibility to develop novel high-affinity ligands for Aß pathology found in human AD brain in a different mode compared with PiB, potentially recognizing different polymorphs of Aß fibrils.
Subject(s)
Alkenes/chemistry , Amyloid beta-Peptides/chemistry , Amyloid/chemistry , Amyloid/metabolism , Aniline Compounds/chemistry , Benzoates/chemistry , Fluorescent Dyes/chemistry , Thiazoles/chemistry , tau Proteins/chemistry , Amyloid beta-Peptides/metabolism , HumansABSTRACT
Two analogues to the fluorescent amyloid probe 2,5-bis(4'-hydroxy-3'-carboxy-styryl)benzene (X-34) were synthesized based on the trans-stilbene pyrene scaffold (Py1SA and Py2SA). The compounds show strikingly different emission spectra when bound to preformed Aß1-42 fibrils. This remarkable emission difference is retained when bound to amyloid fibrils of four distinct proteins, suggesting a common binding configuration for each molecule. Density functional theory calculations show that Py1SA is twisted, while Py2SA is more planar. Still, an analysis of the highest occupied molecular orbitals (HOMOs) and lowest unoccupied molecular orbitals (LUMOs) of the two compounds indicates that the degree of electronic coupling between the pyrene and salicylic acid (SA) moieties is larger in Py1SA than in Py2SA. Excited state intramolecular proton transfer (ESIPT) coupled-charge transfer (ICT) was observed for the anionic form in polar solvents. We conclude that ICT properties of trans-stilbene derivatives can be utilized for amyloid probe design with large changes in emission spectra and decay times from analogous chemical structures depending on the detailed physical nature of the binding site.
Subject(s)
Amyloid beta-Peptides/chemistry , Peptide Fragments/chemistry , Protons , Pyrenes/chemistry , Salicylates/chemistry , Stilbenes/chemistry , Density Functional Theory , Fluorescence , Fluorescent Dyes/chemical synthesis , Fluorescent Dyes/chemistry , Fluorescent Dyes/radiation effects , Light , Models, Chemical , Molecular Structure , Protein Multimerization , Pyrenes/chemical synthesis , Pyrenes/radiation effects , Salicylates/chemical synthesis , Salicylates/radiation effects , Stilbenes/chemical synthesis , Stilbenes/radiation effectsABSTRACT
The accumulation of protein aggregates is associated with many devastating neurodegenerative diseases and the existence of distinct aggregated morphotypes has been suggested to explain the heterogeneous phenotype reported for these diseases. Thus, the development of molecular probes able to distinguish such morphotypes is essential. We report an anionic tetrameric oligothiophene compound that can be utilized for spectral assignment of different morphotypes of ß-amyloid or tau aggregates present in transgenic mice at distinct ages. The ability of the ligand to spectrally distinguish between the aggregated morphotypes was reduced when the spacing between the anionic substituents along the conjugated thiophene backbone was altered, which verified that specific molecular interactions between the ligand and the protein aggregate are necessary to detect aggregate polymorphism. Our findings provide the structural and functional basis for the development of new fluorescent ligands that can distinguish between different morphotypes of protein aggregates.
Subject(s)
Amyloid beta-Peptides/chemistry , Anions/chemistry , Luminescent Agents/chemistry , Proteins/chemistry , Thiophenes/chemistry , Alzheimer Disease/pathology , Amyloid beta-Peptides/metabolism , Animals , Brain/pathology , Fluorescent Dyes/chemistry , Humans , Ligands , Luminescent Agents/pharmacology , Mice , Molecular Probes , Neurodegenerative Diseases/genetics , Neurodegenerative Diseases/pathologyABSTRACT
Amyloid plaques composed of fibrils of misfolded Aß peptides are pathological hallmarks of Alzheimer's disease (AD). Aß fibrils are polymorphic in their tertiary and quaternary molecular structures. This structural polymorphism may carry different pathologic potencies and can putatively contribute to clinical phenotypes of AD. Therefore, mapping of structural polymorphism of Aß fibrils and structural evolution over time is valuable to understanding disease mechanisms. Here, we investigated how Aß fibril structures in situ differ in Aß plaque of different mouse models expressing familial mutations in the AßPP gene. We imaged frozen brains with a combination of conformation-sensitive luminescent conjugated oligothiophene (LCO) ligands and Aß-specific antibodies. LCO fluorescence mapping revealed that mouse models APP23, APPPS1, and AppNL-F have different fibril structures within Aß-amyloid plaques depending on the AßPP-processing genotype. Co-staining with Aß-specific antibodies showed that individual plaques from APP23 mice expressing AßPP Swedish mutation have two distinct fibril polymorph regions of core and corona. The plaque core is predominantly composed of compact Aß40 fibrils, and the corona region is dominated by diffusely packed Aß40 fibrils. Conversely, the AßPP knock-in mouse AppNL-F, expressing the AßPP Iberian mutation along with Swedish mutation has tiny, cored plaques consisting mainly of compact Aß42 fibrils, vastly different from APP23 even at elevated age up to 21 months. Age-dependent polymorph rearrangement of plaque cores observed for APP23 and APPPS1 mice >12 months, appears strongly promoted by Aß40 and was hence minuscule in AppNL-F. These structural studies of amyloid plaques in situ can map disease-relevant fibril polymorph distributions to guide the design of diagnostic and therapeutic molecules.
Subject(s)
Amyloid beta-Peptides , Amyloid beta-Protein Precursor , Plaque, Amyloid , Animals , Humans , Mice , Aging/metabolism , Aging/pathology , Alzheimer Disease/metabolism , Alzheimer Disease/pathology , Alzheimer Disease/genetics , Amyloid beta-Peptides/metabolism , Amyloid beta-Peptides/genetics , Amyloid beta-Protein Precursor/genetics , Amyloid beta-Protein Precursor/metabolism , Brain/metabolism , Brain/pathology , Disease Models, Animal , Mice, Transgenic , Mutation , Plaque, Amyloid/metabolism , Plaque, Amyloid/pathology , Protein ConformationABSTRACT
The role of the polymorphism Met or Val in position 129 in the human prion protein is well documented regarding disease susceptibility and clinical manifestations. However, little is known about the molecular background to this phenomenon. We investigated herein the conformational stability, amyloid fibrillation kinetics, and seeding propensity of different 129 mutants, located in ß-strand 1 of PrP (Met(129) (WT), M129A, M129V, M129L, M129W, M129P, M129E, M129K, and M129C) in HuPrP(90-231). The mutations M129V, M129L, M129K, and M129C did not affect stability (midpoints of thermal denaturation, T(m) = 65-66 °C), whereas the mutants M129A and M129E and the largest side chain M129W were destabilized by 3-4 °C. The most destabilizing substitution was M129P, which lowered the T(m) by 7.2 °C. All mutants, except for M129C, formed amyloid-like fibrils within hours during fibril formation under near physiological conditions. Fibril-forming mutants showed a sigmoidal kinetic profile and showed shorter lag times during seeding with preformed amyloid fibrils implicating a nucleated polymerization reaction. In the spontaneous reactions, the lag time of fibril formation was rather uniform for the mutants M129A, M129V, and M129L resembling the wild type. When the substituted amino acid had a distinct feature discriminating it from the wild type, such as size (M129W), charge (M129E, M129K), or rotational constraint (M129P), the fibrillation was impeded. M129C did not form ThT/Congo red-positive fibrils, and non-reducing SDS-PAGE of M129C during fibrillation conditions at different time points revealed covalent dimer formation already 15 min after fibrillation reaction initiation. Position 129 appears to be a key site for dictating PrP receptiveness toward recruitment into the amyloid state.
Subject(s)
Amino Acid Substitution , Amyloid/chemistry , PrPC Proteins/chemistry , PrPC Proteins/genetics , Amyloid/genetics , Humans , Kinetics , Methionine/genetics , Mutagenesis, Site-Directed , Peptide Fragments/chemistry , Protein Stability , Protein Structure, Quaternary , Protein Structure, Secondary , Protein UnfoldingABSTRACT
Luminescent conjugated polymers (LCPs) interact with ordered protein aggregates and sensitively detect amyloids of many different proteins, suggesting that they may possess antiprion properties. Here, we show that a variety of anionic, cationic, and zwitterionic LCPs reduced the infectivity of prion-containing brain homogenates and of prion-infected cerebellar organotypic cultured slices and decreased the amount of scrapie isoform of PrP(C) (PrP(Sc)) oligomers that could be captured in an avidity assay. Paradoxically, treatment enhanced the resistance of PrP(Sc) to proteolysis, triggered the compaction, and enhanced the resistance to proteolysis of recombinant mouse PrP(23-231) fibers. These results suggest that LCPs act as antiprion agents by transitioning PrP aggregates into structures with reduced frangibility. Moreover, ELISA on cerebellar organotypic cultured slices and in vitro conversion assays with mouse PrP(23-231) indicated that poly(thiophene-3-acetic acid) may additionally interfere with the generation of PrP(Sc) by stabilizing the conformation of PrP(C) or of a transition intermediate. Therefore, LCPs represent a novel class of antiprion agents whose mode of action appears to rely on hyperstabilization, rather than destabilization, of PrP(Sc) deposits.
Subject(s)
Cerebellum/metabolism , Peptide Fragments/metabolism , Polymers/pharmacology , PrPSc Proteins/metabolism , Prions/metabolism , Proteolysis/drug effects , Thiophenes/pharmacology , Animals , Cerebellum/pathology , Mice , PrPSc Proteins/pathogenicity , Prions/pathogenicity , Protein Stability/drug effects , Protein Structure, TertiaryABSTRACT
The crosstalk between viral infections, amyloid formation and neurodegeneration has been discussed with varying intensity since the last century. Several viral proteins are known to be amyloidogenic. Post-acute sequalae (PAS) of viral infections is known for several viruses. SARS-CoV-2 and COVID-19 implicate connections between amyloid formation and severe outcomes in the acute infection, PAS and neurodegenerative diseases. Is the amyloid connection causation or just correlation? In this review we highlight several aspects where amyloids and viruses meet. The evolutionary driving forces that dictate protein amyloid formation propensity are different for viruses compared to prokaryotes and eukaryotes, while posttranslational endoproteolysis appears to be a common mechanism leading up to amyloid formation for both viral and human proteins. Not only do human and viral proteins form amyloid irrespective of each other but there are also several examples of co-operativity between amyloids, viruses and the inter-, and intra-host spread of the respective entity. Abnormal blood clotting in severe and long COVID and as a side effect in some vaccine recipients has been connected to amyloid formation of both the human fibrin and the viral Spike-protein. We conclude that there are many intersects between viruses and amyloids and, consequently, amyloid and virus research need to join forces here. We emphasize the need to accelerate development and implementation in clinical practice of antiviral drugs to preclude PAS and downstream neurological damage. There is also an ample need for retake on suitable antigen targets for the further development of next generation of vaccines against the current and coming pandemics.
Subject(s)
COVID-19 , Virus Diseases , Viruses , Humans , SARS-CoV-2 , Post-Acute COVID-19 Syndrome , Virus Diseases/complications , Amyloid , Viral ProteinsABSTRACT
Two different conformational isoforms or amyloid strains of insulin with different cytotoxic capacity have been described previously. Herein these filamentous and fibrillar amyloid states of insulin were investigated using biophysical and spectroscopic techniques in combination with luminescent conjugated oligothiophenes (LCO). This new class of fluorescent probes has a well defined molecular structure with a distinct number of thiophene units that can adopt different dihedral angles depending on its binding site to an amyloid structure. Based on data from surface charge, hydrophobicity, fluorescence spectroscopy and imaging, along with atomic force microscopy (AFM), we deduce the ultrastructure and fluorescent properties of LCO stained insulin fibrils and filaments. Combined total internal reflection fluorescence microscopy (TIRFM) and AFM revealed rigid linear fibrous assemblies of fibrils whereas filaments showed a short curvilinear morphology which assemble into cloudy deposits. All studied LCOs bound to the filaments afforded more blue-shifted excitation and emission spectra in contrast to those corresponding to the fibril indicating a different LCO binding site, which was also supported by less efficient hydrophobic probe binding. Taken together, the multi-tool approach used here indicates the power of ultrastructure identification applying AFM together with LCO fluorescence interrogation, including TIRFM, to resolve structural differences between amyloid states.
Subject(s)
Insulin/chemistry , Protein Aggregates , Amyloid , Humans , Hydrophobic and Hydrophilic Interactions , Microscopy, Atomic Force , Spectrometry, Fluorescence , Thiophenes/chemistryABSTRACT
Neurodegenerative diseases (NDs) are associated with accumulated misfolded proteins (MPs). MPs oligomerize and form multiple forms of amyloid fibril polymorphs that dictate fibril propagation and cellular dysfunction. Protein misfolding processes that impair protein homeostasis are implicated in onset and progression of NDs. A wide variety of molecular chaperones safeguard the cell from MP accumulation. A rather overlooked molecular chaperone is HSP10, known as a co-chaperone for HSP60. Due to the ubiquitous presence in human tissues and protein overabundance compared with HSP60, we studied how HSP10 alone influences fibril formation in vitro of Alzheimer's disease-associated Aß1-42. At sub-stoichiometric concentrations, eukaryotic HSP10s (human and Drosophila) significantly influenced the fibril formation process and the fibril structure of Aß1-42, more so than the prokaryotic HSP10 GroES. Similar effects were observed for prion disease-associated prion protein HuPrP90-231. Paradoxically, for a chaperone, low concentrations of HSP10 appeared to promote fibril nucleation by shortened lag-phases, which were chaperone and substrate dependent. Higher concentrations of chaperone while still sub-stoichiometric extended the nucleation and/or the elongation phase. We hypothesized that HSP10 by means of its seven mobile loops provides the chaperone with high avidity binding to amyloid fibril ends. The preserved sequence of the edge of the mobile loop GGIM(V)L (29-33 human numbering) normally dock to the HSP60 apical domain. Interestingly, this segment shows sequence similarity to amyloidogenic core segments of Aß1-42, GGVVI (37-41), and HuPrP90-231 GGYML (126-130) likely allowing efficient competitive binding to fibrillar conformations of these MPs. Our results propose that HSP10 can function as an important molecular chaperone in human proteostasis in NDs.
ABSTRACT
Aim: To propose a new multimodal imaging agent targeting amyloid-ß (Aß) plaques in Alzheimer's disease. Materials & methods: A new generation of hybrid contrast agents, based on gadolinium fluoride nanoparticles grafted with a pentameric luminescent-conjugated polythiophene, was designed, extensively characterized and evaluated in animal models of Alzheimer's disease through MRI, two-photon microscopy and synchrotron x-ray phase-contrast imaging. Results & conclusion: Two different grafting densities of luminescent-conjugated polythiophene were achieved while preserving colloidal stability and fluorescent properties, and without affecting biodistribution. In vivo brain uptake was dependent on the blood-brain barrier status. Nevertheless, multimodal imaging showed successful Aß targeting in both transgenic mice and Aß fibril-injected rats.
The design and study of a new contrast agent targeting amyloid-ß (Aß) plaques in Alzheimer's disease (AD) is proposed. Aß plaques are the earliest pathological sign of AD, silently appearing in the brain decades before the symptoms of the disease are manifested. While current detection of Aß plaques is based on nuclear medicine (a technique using a radioactive agent), a different kind of contrast agent is here evaluated in animal models of AD. The contrast agent consists of a nanoparticle made of gadolinium and fluorine ions (core), and decorated with a molecule previously shown to bind to Aß plaques (grafting). The core is detectable with MRI and x-ray imaging, while the grafting molecule is detectable with fluorescence imaging, thus allowing different imaging methods to be combined to study the pathology. In this work, the structure, stability and properties of the contrast agent have been verified in vitro (in tubes and on brain sections). Then the ability of the contrast agent to bind to Aß plaques and provide a detectable signal in MRI, x-ray or fluorescence imaging has been demonstrated in vivo (in rodent models of AD). This interdisciplinary research establishes the proof of concept that this new class of versatile agent contrast can be used to target pathological processes in the brain.
Subject(s)
Alzheimer Disease , Nanoparticles , Mice , Rats , Animals , Alzheimer Disease/diagnostic imaging , Tissue Distribution , Amyloid beta-Peptides/metabolism , Mice, Transgenic , Brain/diagnostic imaging , Brain/metabolism , Multimodal Imaging , Disease Models, AnimalABSTRACT
Cerebrospinal fluid (CSF) biomarkers play an important role in diagnosing Alzheimer's disease (AD) which is characterized by amyloid-ß (Aß) amyloidosis. Here, we used two App knock-in mouse models, AppNL-F/NL-F and AppNL-G-F/NL-G-F, exhibiting AD-like Aß pathology to analyze how the brain pathologies translate to CSF proteomes by label-free mass spectrometry (MS). This identified several extracellular matrix (ECM) proteins as significantly altered in App knock-in mice. Next, we compared mouse CSF proteomes with previously reported human CSF MS results acquired from patients across the AD spectrum. Intriguingly, the ECM protein decorin was similarly and significantly increased in both AppNL-F/NL-F and AppNL-G-F/NL-G-F mice, strikingly already at three months of age in the AppNL-F/NL-F mice and preclinical AD subjects having abnormal CSF-Aß42 but normal cognition. Notably, in this group of subjects, CSF-decorin levels positively correlated with CSF-Aß42 levels indicating that the change in CSF-decorin is associated with early Aß amyloidosis. Importantly, receiver operating characteristic analysis revealed that CSF-decorin can predict a specific AD subtype having innate immune activation and potential choroid plexus dysfunction in the brain. Consistently, in AppNL-F/NL-F mice, increased CSF-decorin correlated with both Aß plaque load and with decorin levels in choroid plexus. In addition, a low concentration of human Aß42 induces decorin secretion from mouse primary neurons. Interestingly, we finally identify decorin to activate neuronal autophagy through enhancing lysosomal function. Altogether, the increased CSF-decorin levels occurring at an early stage of Aß amyloidosis in the brain may reflect pathological changes in choroid plexus, present in a subtype of AD subjects.
Subject(s)
Alzheimer Disease , Amyloidosis , Alzheimer Disease/pathology , Amyloid beta-Peptides/metabolism , Amyloidosis/pathology , Animals , Brain/pathology , Decorin/cerebrospinal fluid , Decorin/metabolism , Humans , Mice , Plaque, Amyloid/pathology , Proteome/metabolismABSTRACT
Molecular probes for selective identification of protein aggregates are important to advance our understanding of the molecular pathogenesis underlying protein aggregation diseases. Here we report the chemical design of a library of anionic luminescent conjugated oligothiophenes (LCOs), which can be utilized as ligands for detection of protein aggregates. Certain molecular requirements were shown to be necessary for detecting (i) early non-thioflavinophilic protein assemblies of Aß1-42 and insulin preceding the formation of amyloid fibrils and (ii) for obtaining distinct spectral signatures of the two main pathological hallmarks observed in human Alzheimer's diease brain tissue (Aß plaques and neurofibrillary tangles). Our findings suggest that a superior anionic LCO-based ligand should have a backbone consisting of five to seven thiophene units and carboxyl groups extending the conjugated thiophene backbone. Such LCOs will be highly useful for studying the underlying molecular events of protein aggregation diseases and could also be utilized for the development of novel diagnostic tools for these diseases.
Subject(s)
Fluorescent Dyes/chemistry , Molecular Probes/chemistry , Proteins/analysis , Thiophenes/chemistry , Fluorescent Dyes/chemical synthesis , Ligands , Molecular Probes/chemical synthesis , Molecular Structure , Small Molecule Libraries , Thiophenes/chemical synthesisABSTRACT
Protein aggregation is associated with a wide range of diseases, and molecular probes that are able to detect a diversity of misfolded protein assemblies are of great importance. The identification of prefibrillar states preceding the formation of well-defined amyloid fibrils is of particular interest both because of their likely role in the mechanism of fibril formation and because of the growing awareness that these species are likely to play a critical role in the pathogenesis of protein deposition diseases. Herein, we explore the use of an anionic oligothiophene derivative, p-FTAA, for detection of prefibrillar protein aggregates during in vitro fibrillation of three different amyloidogenic proteins (insulin, lysozyme, and prion protein). p-FTAA generally detected prefibrillar protein aggregates that could not be detected by thioflavine T fluorescence and in addition showed high fluorescence when bound to mature fibrils. Second, the kinetics of protein aggregation or the formation of amyloid fibrils of insulin was not extensively influenced by the presence of various concentrations of p-FTAA. These results establish the use of p-FTAA as an additional tool for studying the process of protein aggregation.