Protein structure can be analysed using a range of different techniques. Many of the available principal techniques for assessing protein structure are developed and optimized for natively folded proteins that are soluble. Amyloids are an alternative protein fold, often associated with protein malfunction and disease but are also found as the functional fold for some proteins. While functional amyloids, just like soluble globular proteins, commonly attain one specific and precise fold to exert its function, disease associated amyloids are often poorly soluble and structurally highly variable. This is known as structural polymorphism. To investigate to what extent molecular biophysics techniques can be standardized for studies of amyloid fibrils, five sites within the Horizon 2020 funded project MOlecular-Scale Biophysics Research Infrastructure (MOSBRI) addressed this issue via a joint research activity. For this study we selected bovine insulin as a convenient, accessible, and distributable amyloid model system. The benchmark comparable techniques for observing formed amyloid fibrils at the different sites were fluorescence spectroscopy of two amyloid ligands (ThT and pFTAA) and negative stain transmission electron microscopy. The outcome of this study yielded results with a large variability between different insulin amyloid fibril preparations, between sites, and within chemically identical preparations from one site. We mainly attribute these hard to control differences to the intrinsic polymorphic behaviour of insulin amyloid fibrils. We also present a range of experimental measurement techniques to highlight their potential use for studying amyloid structure and amyloid polymorphism.

HSP10 is a well-known human co-chaperone that interacts with HSP60 to comprise the HSP60/10 chaperonin complex which upholds mitochondrial proteostasis. HSP10 also demonstrates independent roles in binding to misfolded proteins and interacts with several amyloidogenic client proteins. Using a variety of biophysical and biochemical methods, we studied the interactions of HSP10 with the amyloidogenic protein α-synuclein (α-syn) associated with Parkinson's disease. HSP10 efficiently inhibited fibril formation of wild type (WT) and disease-mutant A30P α-syn at sufficient concentrations of chaperone by both binding to α-syn monomers and by blocking secondary nucleation on fibril surfaces. However, under sub-stoichiometric conditions, below 1:5 (HSP10:α-syn), the chaperone sequestered multiple A30P α-syn monomers and thereby promoted nucleation of fibril formation with a magnitude comparable to the efficacy of seeding with preformed fibrils. The fibril formation acceleration effect of the HSP10 chaperone was client-specific as it was observed for A30P but not WT α-syn. Our results broaden the scope of HSP10 chaperone activity and can have implications for disease onset in synucleinopathies.

A(3-amyloid plaques and cerebral amyloid angiopathy (CAA) in the brain are pathological hallmarks of Alzheimer's disease (AD) and vascular dementia. The spreading of A(3 amyloidosis in the brain appears to be mediated by a seeding mechanism, where preformed fibrils (called seeds) accelerate A(3 fibril formation by bypassing the rate-determining nucleation step. Several studies have demonstrated that A(3 amyloidosis can be induced in transgenic mice, producing human A(3, by injecting A(3-rich brain extracts (seeds) derived from transgenic mice and human AD brains. However, studies on recombinant seeds are limited. Therefore, we investigated the seeding activity of pure recombinant human A(3 fibrils of different compositions. Seeds were inoculated into APP23 mice at the age of 3 months and were analyzed after 6 months of incubation. Recombinant fibril seeds made from A(3-peptides with an N-terminal methionine (i.e. (preformed fibrils from A(3M1-42, A(3M1-40, and A(3M1-40 +A(3M1-42) accelerated A(3-amyloid plaque formation in vivo compared to non-inoculated transgenic control mice of the same age. In addition, all seeds induced CAA pathology. Interestingly, A(3M1-42 containing seeds produced significantly more CAA and amyloid plaques than seeds containing pure A(3M1-40, which was surprising given that APP23 mice produce approximately four-fold more A(31-40 substrate than A(31-42. This study showed that A(3M1-42 fibrils are highly potent in seeding CAA and implies that conformational templating occurs in amyloid plaque as deduced by comparative amyloid ligand staining. Our results verify that recombinant A(3 fibrils are transmissible amyloids, and that in vivo seeding can accelerate, and redirect A(3 amyloidosis patterns compared to spontaneous age dependent amyloidosis. (c) 2024 The Author(s). Published by Elsevier Ltd. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/).

Long COVID, or postacute sequelae of COVID-19 from SARS-CoV-2 infection, is a persistent debilitating disease affecting multiple systems and organs. Long COVID pathophysiology is a complex and not fully established process. One prevailing theory is that the formation of fibrin amyloid microclots (fibrinaloids), due to SARS-CoV-2 infection, can induce persistent inflammation and capillary blockage. An association between the amyloidogenic Spike protein of SARS-CoV-2 and impaired fibrinolysis was made when it was observed that fibrin clots formed in the presence of a mixture of amyloid fibrils from the spike protein mediated resistance to plasmin lysis. Here, we use purified components from the coagulation cascade to investigate the molecular processes of impaired fibrinolysis using seven amyloidogenic SARS-COV-2 Spike peptides. Five of seven Spike amyloid fibrils appeared not to substantially interfere with the fibrinogen-fibrin-fibrinolysis process in vitro, while two spike fibrils were active in different ways. Spike601 amyloid fibrils (sequence 601-620) impaired thrombin-mediated fibrin formation by binding and sequestering fibrinogen but did not affect fibrinolysis. On the contrary, fibrin clots formed in the presence of Spike685 amyloid fibrils (sequence 685-701) exhibited a marked resistance to plasmin-mediated fibrinolysis. We conclude that Spike685 amyloid fibrils can induce dense fibrin clot networks as well as incorporate fibrin into aggregated structures that resist fibrinolysis. Our study proposes a molecular mechanism for how the Spike protein of SARS-CoV-2 could contribute to the formation of fibrinolysis-resistant microclots observed in long COVID.

The existence of transmissible amyloid fibril strains has long intrigued the scientific community. The strain theory originates from prion disorders, but here, we provide evidence of strains in systemic amyloidosis. Human AA amyloidosis manifests as two distinct clinical phenotypes called common AA and vascular AA. Glomerular amyloid deposition of the kidney defines the common form, while in the vascular type amyloid deposits are massive in the renal medulla and in arteries throughout the body, while glomeruli are spared. By electron microscopy the two types appeared morphologically different. The common type was composed of dispersed fibrils which tended to be clustered whereas the vascular type was composed of longer and more distinct less clustered fibrils. Staining with fluorescent amyloid binding ligands analyzed by hyperspectral microscopy showed differential staining patterns between the two groups supporting the notion of human AA amyloid strains. AA amyloid staining was significantly different from systemic AL amyloid. Both types of AA (common and vascular) and AL amyloid fibrils were isolated and used to seed mouse AA amyloid in groups of inflamed NMRI mice (n = 9–10 per group). All but two mice showed amyloid deposits in the spleen induced by the human seeds. Amyloid binding ligand analysis was applied on the splenic amyloid deposits and revealed no clear significant difference between mice seeded with AA fibrils from different donors being vascular or common, but the AA deposits of mice given AL fibrils showed significantly different amyloid fluorescent signals compared to all groups of mice receiving AA fibrils. The combined results support the hypothesis that AA amyloid fibril structures can vary depending on the seed and may manifest as amyloid strains.

Different types of deposits comprised of amyloid-β (Aβ) peptides are one of the pathological hallmarks of Alzheimer's disease (AD) and novel methods that enable identification of a diversity of Aβ deposits during the AD continuum are essential for understanding the role of these aggregates during the pathogenesis. Herein, different combinations of five fluorescent thiophene-based ligands were used for detection of Aβ deposits in brain tissue sections from transgenic mouse models with aggregated Aβ pathology, as well as brain tissue sections from patients affected by sporadic or dominantly inherited AD. When analyzing the sections with fluorescence microscopy, distinct ligand staining patterns related to the transgenic mouse model or to the age of the mice were observed. Likewise, specific staining patterns of different Aβ deposits were revealed for sporadic versus dominantly inherited AD, as well as for distinct brain regions in sporadic AD. Thus, by using dual-staining protocols with multiple combinations of fluorescent ligands, a chronological and spatial histological designation of different Aβ deposits could be achieved. This study demonstrates the potential of our approach for resolving the role and presence of distinct Aβ aggregates during the AD continuum and pinpoints the necessity of using multiple ligands to obtain an accurate assignment of different Aβ deposits in the neuropathological evaluation of AD, as well as when evaluating therapeutic strategies targeting Aβ aggregates.

The amyloidogenic homotetrameric plasma protein transthyretin (TTR) has an affinity for bicyclic small molecule ligands in its two thyroxine (T4) binding sites. We have shown that native tetrameric TTR binds to amyloid ligands based on the trans-stilbene scaffold. The fluorescent Congo-red analogue, X34, is a symmetric bi-trans-stilbene that contains two salicylic acid motifs. We used fluorescence spectroscopy methods to interrogate X34 binding to the TTR tetramer and fibril. We discovered two binding sites in both TTR forms by tryptophan FRET, ligand self-quenching, Stern-Volmer plots and binding curves, for the latter including the competitive ligand diflunisal. X34 binds with the similar affinity as diflunisal in the first binding site (Kd1=150 nM), and negative cooperativity renders the binding to the second site with lower affinity very similar compared to diflunisal (Kd2= 1.1 mu M). This behavior is coherent with the salicylic acid moiety of diflunisal binding into the binding pocket of TTR (reverse mode). Interestingly X34 binding to TTR fibrils was also well fitted to two binding sites, however with overall lower affinity (Kd1=1.2 mu M; Kd2=2.1 mu M) compared to binding to the native tetramer. X34 fluorescence when bound to TTR-fibrils was significantly blue shifted compared to binding to the TTR-tetramer.

Smart grids are proposed to enable the integration of renewables and facilitate the energy transition. Households have been pointed out as a significant resource for demand response, that is to adapt their electricity consumption based on the status in the grid. This article analyses narrative mismatches in the context of smart grid implementation in Sweden. We compare policy narratives on the role of homes in the future energy system with home personas, emerging from interviews with households. The policy narratives envision households to become either actively engaged in time-shifting motivated by information and incentives, or bypassed through automation. The home personas, although seemingly similar, show great diversity, being well informed about their electricity use, concerned regarding the safety of technology, preferring to manage flexibility themselves, and reluctant to give up control. Several dissonances are identified between narratives and the home personas regarding smart meter communication, energy awareness, trust, agency, and control, that need further attention for demand response to be realised. The analysis illustrates how policy visions of the home in the future grid would encounter severe challenges in living up to values and characteristics of real households. Policy thus needs to acknowledge households as a diverse group to ensure a sustainable and democratic energy transition. We encourage the use of home personas to substantiate this diversity.

The amyloid beta (A beta) peptide has a central role in Alzheimer's disease (AD) pathology. The peptide length can vary between 37 and 49 amino acids, with A beta 1-42 being considered the most disease-related length. However, A beta 1-40 is also found in A beta plaques and has shown to form intertwined fibrils with A beta 1-42. 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 A beta 1-40:42 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 A beta content, cytotoxicity and autophagy function at three different time-points: 3, 24, and 72 h. Our results revealed that both A beta 1-40:42 ratio and fibril maturation affect conformation of fibrils. We further show the impact of these conformation changes on the affinity to commonly used A beta antibodies, primarily affecting A beta 1-40 rich aggregates. In addition, we demonstrate uptake of the aggregates by neuronally differentiated human cells, where aggregates with higher A beta 1-42 ratios generally caused higher cellular levels of A beta. These differences in A beta abundance did not cause changes in cytotoxicity nor in autophagy activation. Our results show the importance to consider conformational differences of A beta fibrils, as this can have fundamental impact on A beta 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 A beta aggregates.image

Amyloid plaques composed of fibrils of misfolded Aβ peptides are pathological hallmarks of Alzheimer's disease (AD). Aβ 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 Aβ fibrils and structural evolution over time is valuable to understanding disease mechanisms. Here, we investigated how Aβ fibril structures in situ differ in Aβ plaque of different mouse models expressing familial mutations in the AβPP gene. We imaged frozen brains with a combination of conformation-sensitive luminescent conjugated oligothiophene (LCO) ligands and Aβ-specific antibodies. LCO fluorescence mapping revealed that mouse models APP23, APPPS1, and AppNL-F have different fibril structures within Aβ-amyloid plaques depending on the AβPP-processing genotype. Co-staining with Aβ-specific antibodies showed that individual plaques from APP23 mice expressing AβPP Swedish mutation have two distinct fibril polymorph regions of core and corona. The plaque core is predominantly composed of compact Aβ40 fibrils, and the corona region is dominated by diffusely packed Aβ40 fibrils. Conversely, the AβPP knock-in mouse AppNL-F, expressing the AβPP Iberian mutation along with Swedish mutation has tiny, cored plaques consisting mainly of compact Aβ42 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 Aβ40 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.

The smart grid is expected to encompass the overall electrification of society, while simultaneously managing increasing amounts of renewable energy. This could significantly impact how everyday life will be organized. However, previous research has shown that the envisioned role of households in the future energy system remains obscure and even contradictory. In this article we further examine and critically analyse how households as a demand response resource are imagined in the Swedish future smart grid. We focus on policy documents from the Swedish energy sector. To guide our analysis, we use the critical policy analysis framework of “What's the problem represented to be?” (WPR) for inspiration. Aligning with prior research, our results show that households tend to be framed as an untapped flexibility resource where heating and smart home technologies are to be controlled automatically or through remote control, which households allow for, motivated by consumption feedback, price signals or other incentives. Topics silenced in the problem representation include: The paradox in transitioning into a sustainable energy system while simultaneously sustaining unsustainable norms; a lack of diversity and acknowledgement of non-technical households; how trust and control need renegotiation and alternative ways for citizen participation in the energy transition.

An increasing number of reports suggest an association between COVID-19 infection and initiation or acceleration of neurodegenerative diseases (NDs) including Alzheimer’s disease (AD) and Creutzfeldt-Jakob disease (CJD). Both these diseases and several other NDs are caused by conversion of human proteins into a misfolded, aggregated amyloid fibril state. The fibril formation process is self-perpetuating by seeded conversion from preformed fibril seeds. We recently described a plausible mechanism for amyloid fibril formation of SARS-CoV-2 spike protein. Spike-protein formed amyloid fibrils upon cleavage by neutrophil elastase, abundant in the inflammatory response to COVID-19 infection. We here provide evidence of significant Spike-amyloid fibril seeded acceleration of amyloid formation of CJD associated human prion protein (HuPrP) using an in vitro conversion assay. By seeding the HuPrP conversion assay with other in vitro generated disease associated amyloid fibrils we demonstrate that this is not a general effect but a specific feature of spike-amyloid fibrils. We also showed that the amyloid fibril formation of AD associated Aβ1-42 was accelerated by Spike-amyloid fibril seeds. Of seven different 20-amino acid long peptides, Spike532 ( 532 NLVKNKCVNFNFNGLTGTGV 551 ) was most efficient in seeding HuPrP and Spike601 ( 601 GTNTSNQVAVLYQDVNCTEV 620 ) was most effective in seeding Aβ1-42, suggesting substrate dependent selectivity of the cross-seeding activity. Albeit purely in vitro , our data suggest that cross-seeding by Spike-amyloid fibrils can be implicated in the increasing number of reports of CJD, AD, and possibly other NDs in the wake of COVID-19.

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.

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 Aβ1–42. At sub-stoichiometric concentrations, eukaryotic HSP10s (human and Drosophila) significantly influenced the fibril formation process and the fibril structure of Aβ1–42, 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 Aβ1–42, 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.

Cerebrospinal fluid (CSF) biomarkers play an important role in diagnosing Alzheimer’s disease (AD) which is characterized by amyloid-β (Aβ) amyloidosis. Here, we used two App knock-in mouse models, App NL-F/NL-F and App NL-G-F/NL-G-F , exhibiting AD-like Aβ 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 App NL-F/NL-F and App NL-G-F/NL-G-F mice, strikingly already at three months of age in the App NL-F/NL-F mice and preclinical AD subjects having abnormal CSF-Aβ42 but normal cognition. Notably, in this group of subjects, CSF-decorin levels positively correlated with CSF-Aβ42 levels indicating that the change in CSF-decorin is associated with early Aβ 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 App NL-F/NL-F mice, increased CSF-decorin correlated with both Aβ plaque load and with decorin levels in choroid plexus. In addition, a low concentration of human Aβ42 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 Aβ amyloidosis in the brain may reflect pathological changes in choroid plexus, present in a subtype of AD subjects.

Aim: To propose a new multimodal imaging agent targeting amyloid-β (Aβ) 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 Aβ targeting in both transgenic mice and Aβ fibril-injected rats.

ABSTRACTSARS-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 191-210, 599-618, 1165-1184) 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 hours of S-protein co-incubation with the protease neutrophil elastase (NE) in vitro. NE efficiently cleaved S-protein rendering exposure of amyloidogenic segments and accumulation of the peptide 193-202, part of the most amyloidogenic synthetic Spike peptide. NE is overexpressed at inflamed sites of viral infection and at vaccine injection sites. Our data propose a molecular mechanism for amyloidogenesis of SARS-CoV-2 S-protein in humans facilitated by endoproteolysis. The potential implications of S-protein amyloidogenesis in COVID-19 disease associated pathogenesis and consequences following S-protein based vaccines should be addressed in understanding the disease, long COVID-19, and vaccine side effects.

Amyloid beta (Aβ) 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 Aβ. We explored this paradigm using conformation-dependent immunoassay (CDI) for Aβ and conformation-sensitive luminescent conjugated oligothiophenes (LCOs) in AD cases with variable progression rates. Mapping the Aβ 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 Aβ with the most significant differences in the temporal cortex of rapidly progressive AD. The fluorescence emission spectra collected in situ from Aβ 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 Aβ 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 Aβ accumulating in the neocortex, with the most notable differences in temporal cortex of cases with shorter disease duration, and implicate distinct Aβ conformers (strains) in the rapid progression of AD.