Modeling transthyretin (TTR) amyloid diseases, from monomer to amyloid fibrils.

ATTR amyloidosis is caused by deposition of large, insoluble aggregates (amyloid fibrils) of cross-ß-sheet TTR protein molecules on the intercellular surfaces of tissues. The process of amyloid formation from monomeric TTR protein molecules to amyloid deposits has not been fully characterized and is therefore modeled in this paper. Two models are considered: 1) TTR monomers in the blood spontaneously fold into a ß-sheet conformation, aggregate into short proto-fibrils that then circulate in the blood until they find a complementary tissue where the proto-fibrils accumulate to form the large, insoluble amyloid fibrils found in affected tissues. 2) TTR monomers in the native or ß-sheet conformation circulate in the blood until they find a tissue binding site and deposit in the tissue or tissues forming amyloid deposits in situ. These models only differ on where the selection for ß-sheet complementarity occurs, in the blood where wt-wt, wt-v, and v-v interactions determine selectivity, or on the tissue surface where tissue-wt and tissure-v interactions also determine selectivity. Statistical modeling in both cases thus involves selectivity in fibril aggregation and tissue binding. Because binding of protein molecules into fibrils and binding of fibrils to tissues occurs through multiple weak non-covalent bonds, strong complementarity between ß-sheet molecules and between fibrils and tissues is required to explain the insolubility and tissue selectivity of ATTR amyloidosis. Observation of differing tissue selectivity and thence disease phenotypes from either pure wildtype TTR protein or a mix of wildtype and variant molecules in amyloid fibrils evidences the requirement for fibril-tissue complementarity. Understanding the process that forms fibrils and binds fibrils to tissues may lead to new possibilities for interrupting the process and preventing or curing ATTR amyloidosis.

Amyloidosis and Amyloidogenesis: One Name, Many Diseases.

Amyloidosis is a heterogenous group of disorders, caused by the deposition of insoluble fibrils derived from misfolded proteins in the extracellular space of various organs. These proteins have an unstable structure that causes them to misfold, aggregate, and deposit as amyloid fibrils with the pathognomonic histologic property of green birefringence when viewed under cross-polarized light after staining with Congo red. Amyloid fibrils are insoluble and degradation-resistant; resistance to catabolism results in progressive tissue amyloid accumulation. The outcome of this process is organ disfunction independently from the type of deposited protein, however there can be organ that are specifically targeted from certain proteins.

Amyloidogenic regions in beta-strands II and III modulate the aggregation and toxicity of SOD1 in living cells.

Mutations in the protein superoxide dismutase-1 (SOD1) promote its misfolding and aggregation, ultimately causing familial forms of the debilitating neurodegenerative disease amyotrophic lateral sclerosis (ALS). Currently, over 220 (mostly missense) ALS-causing mutations in the SOD1 protein have been identified, indicating that common structural features are responsible for aggregation and toxicity. Using in silico tools, we predicted amyloidogenic regions in the ALS-associated SOD1-G85R mutant, finding seven regions throughout the structure. Introduction of proline residues into ß-strands II (I18P) or III (I35P) reduced the aggregation propensity and toxicity of SOD1-G85R in cells, significantly more so than proline mutations in other amyloidogenic regions. The I18P and I35P mutations also reduced the capability of SOD1-G85R to template onto previously formed non-proline mutant SOD1 aggregates as measured by fluorescence recovery after photobleaching. Finally, we found that, while the I18P and I35P mutants are less structurally stable than SOD1-G85R, the proline mutants are less aggregation-prone during proteasome inhibition, and less toxic to cells overall. Our research highlights the importance of a previously underappreciated SOD1 amyloidogenic region in ß-strand II (15QGIINF20) to the aggregation and toxicity of SOD1 in ALS mutants, and suggests that ß-strands II and III may be good targets for the development of SOD1-associated ALS therapies.

Exploration of neuroanatomical characteristics to differentiate prodromal Alzheimer's disease from cognitively unimpaired amyloid-positive individuals.

Differentiating clinical stages based solely on positive findings from amyloid PET is challenging. We aimed to investigate the neuroanatomical characteristics at the whole-brain level that differentiate prodromal Alzheimer's disease (AD) from cognitively unimpaired amyloid-positive individuals (CU A+) in relation to amyloid deposition and regional atrophy. We included 45 CU A+ participants and 135 participants with amyloid-positive prodromal AD matched 1:3 by age, sex, and education. All participants underwent 18F-florbetaben positron emission tomography and 3D structural T1-weighted magnetic resonance imaging. We compared the standardized uptake value ratios (SUVRs) and volumes in 80 regions of interest (ROIs) between CU A+ and prodromal AD groups using independent t-tests, and employed the least absolute selection and shrinkage operator (LASSO) logistic regression model to identify ROIs associated with prodromal AD in relation to amyloid deposition, regional atrophy, and their interaction. After applying False Discovery Rate correction at < 0.1, there were no differences in global and regional SUVR between CU A+ and prodromal AD groups. Regional volume differences between the two groups were observed in the amygdala, hippocampus, entorhinal cortex, insula, parahippocampal gyrus, and inferior temporal and parietal cortices. LASSO logistic regression model showed significant associations between prodromal AD and atrophy in the entorhinal cortex, inferior parietal cortex, both amygdalae, and left hippocampus. The mean SUVR in the right superior parietal cortex (beta coefficient = 0.0172) and its interaction with the regional volume (0.0672) were also selected in the LASSO model. The mean SUVR in the right superior parietal cortex was associated with an increased likelihood of prodromal AD (Odds ratio [OR] 1.602, p = 0.014), particularly in participants with lower regional volume (OR 3.389, p < 0.001). Only regional volume differences, not amyloid deposition, were observed between CU A+ and prodromal AD. The reduced volume in the superior parietal cortex may play a significant role in the progression to prodromal AD through its interaction with amyloid deposition in that region.

Physiological and pathological functions of TMEM106B in neurodegenerative diseases.

As an integral lysosomal transmembrane protein, transmembrane protein 106B (TMEM106B) regulates several aspects of lysosomal function and is associated with neurodegenerative diseases. The TMEM106B gene mutations lead to lysosomal dysfunction and accelerate the pathological progression of Neurodegenerative diseases. Yet, the precise mechanism of TMEM106B in Neurodegenerative diseases remains unclear. Recently, different research teams discovered that TMEM106B is an amyloid protein and the C-terminal domain of TMEM106B forms amyloid fibrils in various Neurodegenerative diseases and normally elderly individuals. In this review, we discussed the physiological functions of TMEM106B. We also included TMEM106B gene mutations that cause neurodegenerative diseases. Finally, we summarized the identification and cryo-electronic microscopic structure of TMEM106B fibrils, and discussed the promising therapeutic strategies aimed at TMEM106B fibrils and the future directions for TMEM106B research in neurodegenerative diseases.

Autophagy preferentially degrades non-fibrillar polyQ aggregates.

Aggregation of proteins containing expanded polyglutamine (polyQ) repeats is the cytopathologic hallmark of a group of dominantly inherited neurodegenerative diseases, including Huntington's disease (HD). Huntingtin (Htt), the disease protein of HD, forms amyloid-like fibrils by liquid-to-solid phase transition. Macroautophagy has been proposed to clear polyQ aggregates, but the efficiency of aggrephagy is limited. Here, we used cryo-electron tomography to visualize the interactions of autophagosomes with polyQ aggregates in cultured cells in situ. We found that an amorphous aggregate phase exists next to the radially organized polyQ fibrils. Autophagosomes preferentially engulfed this amorphous material, mediated by interactions between the autophagy receptor p62/SQSTM1 and the non-fibrillar aggregate surface. In contrast, amyloid fibrils excluded p62 and evaded clearance, resulting in trapping of autophagic structures. These results suggest that the limited efficiency of autophagy in clearing polyQ aggregates is due to the inability of autophagosomes to interact productively with the non-deformable, fibrillar disease aggregates.

Ligand Profiling as a Diagnostic Tool to Differentiate Patient-Derived α-Synuclein Polymorphs.

Amyloid fibrils are characteristic of many neurodegenerative diseases, including Alzheimer's and Parkinson's diseases. While different diseases may have fibrils formed of the same protein, the supramolecular morphology of these fibrils is disease-specific. Here, a method is reported to distinguish eight morphologically distinct amyloid fibrils based on differences in ligand binding properties. Eight fibrillar polymorphs of α-synuclein (αSyn) were investigated: five generated de novo using recombinant αSyn and three generated using protein misfolding cyclic amplification (PMCA) of recombinant αSyn seeded with brain homogenates from deceased patients diagnosed with Parkinson's disease (PD), multiple system atrophy (MSA), and dementia with Lewy bodies (DLB). Fluorescence binding assays were carried out for each fibril using a toolkit of six different ligands. The fibril samples were separated into five categories based on a binary classification of whether they bound specific ligands or not. Quantitative binding measurements then allowed every fibrillar polymorph to be uniquely identified, and the PMCA fibrils derived from PD, MSA, and DLB patients could be unambiguously distinguished. This approach constitutes a novel and operationally simple method to differentiate amyloid fibril morphologies and to identify disease states using PMCA fibrils obtained by seeding with patient samples.

Gut microbiota produces biofilm-associated amyloids with potential for neurodegeneration.

Age-related neurodegenerative diseases involving amyloid aggregation remain one of the biggest challenges of modern medicine. Alterations in the gastrointestinal microbiome play an active role in the aetiology of neurological disorders. Here, we dissect the amyloidogenic properties of biofilm-associated proteins (BAPs) of the gut microbiota and their implications for synucleinopathies. We demonstrate that BAPs are naturally assembled as amyloid-like fibrils in insoluble fractions isolated from the human gut microbiota. We show that BAP genes are part of the accessory genomes, revealing microbiome variability. Remarkably, the abundance of certain BAP genes in the gut microbiome is correlated with Parkinson's disease (PD) incidence. Using cultured dopaminergic neurons and Caenorhabditis elegans models, we report that BAP-derived amyloids induce α-synuclein aggregation. Our results show that the chaperone-mediated autophagy is compromised by BAP amyloids. Indeed, inoculation of BAP fibrils into the brains of wild-type mice promote key pathological features of PD. Therefore, our findings establish the use of BAP amyloids as potential targets and biomarkers of α-synucleinopathies.

Computerized decision support is an effective approach to select memory clinic patients for amyloid-PET.

BACKGROUND: The use of amyloid-PET in dementia workup is upcoming. At the same time, amyloid-PET is costly and limitedly available. While the appropriate use criteria (AUC) aim for optimal use of amyloid-PET, their limited sensitivity hinders the translation to clinical practice. Therefore, there is a need for tools that guide selection of patients for whom amyloid-PET has the most clinical utility. We aimed to develop a computerized decision support approach to select patients for amyloid-PET. METHODS: We included 286 subjects (135 controls, 108 Alzheimer's disease dementia, 33 frontotemporal lobe dementia, and 10 vascular dementia) from the Amsterdam Dementia Cohort, with available neuropsychology, APOE, MRI and [18F]florbetaben amyloid-PET. In our computerized decision support approach, using supervised machine learning based on the DSI classifier, we first classified the subjects using only neuropsychology, APOE, and quantified MRI. Then, for subjects with uncertain classification (probability of correct class (PCC) < 0.75) we enriched classification by adding (hypothetical) amyloid positive (AD-like) and negative (normal) PET visual read results and assessed whether the diagnosis became more certain in at least one scenario (PPC≥0.75). If this was the case, the actual visual read result was used in the final classification. We compared the proportion of PET scans and patients diagnosed with sufficient certainty in the computerized approach with three scenarios: 1) without amyloid-PET, 2) amyloid-PET according to the AUC, and 3) amyloid-PET for all patients. RESULTS: The computerized approach advised PET in n = 60(21%) patients, leading to a diagnosis with sufficient certainty in n = 188(66%) patients. This approach was more efficient than the other three scenarios: 1) without amyloid-PET, diagnostic classification was obtained in n = 155(54%), 2) applying the AUC resulted in amyloid-PET in n = 113(40%) and diagnostic classification in n = 156(55%), and 3) performing amyloid-PET in all resulted in diagnostic classification in n = 154(54%). CONCLUSION: Our computerized data-driven approach selected 21% of memory clinic patients for amyloid-PET, without compromising diagnostic performance. Our work contributes to a cost-effective implementation and could support clinicians in making a balanced decision in ordering additional amyloid PET during the dementia workup.

Amyloid, Crohn's disease, and Alzheimer's disease - are they linked?

Crohn's disease (CD) is a chronic inflammatory disease that most frequently affects part of the distal ileum, but it may affect any part of the gastrointestinal tract. CD may also be related to systemic inflammation and extraintestinal manifestations. Alzheimer's disease (AD) is the most common neurodegenerative disease, gradually worsening behavioral and cognitive functions. Despite the meaningful progress, both diseases are still incurable and have a not fully explained, heterogeneous pathomechanism that includes immunological, microbiological, genetic, and environmental factors. Recently, emerging evidence indicates that chronic inflammatory condition corresponds to an increased risk of neurodegenerative diseases, and intestinal inflammation, including CD, increases the risk of AD. Even though it is now known that CD increases the risk of AD, the exact pathways connecting these two seemingly unrelated diseases remain still unclear. One of the key postulates is the gut-brain axis. There is increasing evidence that the gut microbiota with its proteins, DNA, and metabolites influence several processes related to the etiology of AD, including ß-amyloid abnormality, Tau phosphorylation, and neuroinflammation. Considering the role of microbiota in both CD and AD pathology, in this review, we want to shed light on bacterial amyloids and their potential to influence cerebral amyloid aggregation and neuroinflammation and provide an overview of the current literature on amyloids as a potential linker between AD and CD.

Structural Insights into Seeding Mechanisms of hIAPP Fibril Formation.

The deposition of islet amyloid polypeptide (hIAPP) fibrils is a hallmark of ß-cell death in type II diabetes. In this study, we employ state-of-the-art MAS solid-state spectroscopy to investigate the previously elusive N-terminal region of hIAPP fibrils, uncovering both rigidity and heterogeneity. Comparative analysis between wild-type hIAPP and a disulfide-deficient variant (hIAPPC2S,C7S) unveils shared fibril core structures yet strikingly distinct dynamics in the N-terminus. Specifically, the variant fibrils exhibit extended ß-strand conformations, facilitating surface nucleation. Moreover, our findings illuminate the pivotal roles of specific residues in modulating secondary nucleation rates. These results deepen our understanding of hIAPP fibril assembly and provide critical insights into the molecular mechanisms underpinning type II diabetes, holding promise for future therapeutic strategies.

Characterization of amyloid-like metal-amino acid assemblies with remarkable catalytic activity.

While enzymes are potentially useful in various applications, their limited operational stability and production costs have led to an extensive search for stable catalytic agents that will retain the efficiency, specificity, and environmental-friendliness of natural enzymes. Despite extensive efforts, there is still an unmet need for improved enzyme mimics and novel concepts to discover and optimize such agents. Inspired by the catalytic activity of amyloids and the formation of amyloid-like assemblies by metabolites, our group pioneered the development of novel metabolite-metal co-assemblies (bio-nanozymes) that produce nanomaterials mimicking the catalytic function of common metalloenzymes that are being used for various technological applications. In addition to their notable activity, bio-nanozymes are remarkably safe as they are purely composed of amino acids and minerals that are harmless to the environment. The bio-nanozymes exhibit high efficiency and exceptional robustness, even under extreme conditions of temperature, pH, and salinity that are impractical for enzymes. Our group has recently also demonstrated the formation of ordered amino acid co-assemblies showing selective and preferential interactions comparable to the organization of residues in folded proteins. The identified bio-nanozymes can be used in various applications including environmental remediation, synthesis of new materials, and green energy.

Avoiding common pitfalls in designing kinetic protocols for catalytic amyloid studies.

Kinetic characterization of catalytic amyloids arguably presents a most challenging type of kinetic experiment where careful consideration of many factors is required. Here we outline common pitfalls in devising kinetic studies in such systems. Unlike the more specific protocols for various applications described in this volume, this chapter deals with general issues in setting up kinetic experiments that are incredibly important but often go without explicit mention in the specialized literature. The kinetic fundamentals described here can be also be of use to the enzymologists working with more traditional catalysts.

Catalytic amyloids for nucleotide hydrolysis.

The design of small peptides that assemble into catalytically active intermolecular structures has proven to be a successful strategy towards developing minimalistic catalysts that exhibit some of the unique functional features of enzymes. Among these, catalytic amyloids have emerged as a fruitful source to unravel many different activities. These assemblies can potentially have broad applications that range from biotechnology to prebiotic chemistry. Although many peptides that assemble into catalytic amyloids have been developed in recent years, the elucidation of convergent mechanistic aspects of the catalysis and the structure/function relationship is still a challenge. Novel catalytic activities are necessary to better address these issues and expand the current repertoire of applicability. In this chapter, we described a methodology to produce catalytic amyloids that are specifically active towards the hydrolysis of phosphoanhydride bonds of nucleotides. The design of potentially active amyloid-prone peptide sequences is explored using as template the active site of enzymes with nucleotidyltransferase activity. The procedures include an approach for sequence design, in vitro aggregation assays, morphological characterization of the amyloid state and a comprehensive methodology to measure activity in vitro using nucleoside and deoxynucleosides triphosphates as model substrates. The proposed strategy can also be implemented to explore different types of activities for the design of future catalytic amyloids.

Screening of oxidative behavior in catalytic amyloid assemblies.

Once considered a thermodynamic minimum of the protein fold or as simply by-products of a misfolding process, amyloids are increasingly showing remarkable potential for promoting enzyme-like catalysis. Recent studies have demonstrated a diverse range of catalytic behaviors that amyloids can promote way beyond the hydrolytic behaviors originally reported. We and others have demonstrated the strong propensity of catalytic amyloids to facilitate redox reactions both in the presence and in the absence of metal cofactors. Here, we present a detailed protocol for measuring the oxidative ability of supramolecular peptide assemblies.

Mining and engineering activity in catalytic amyloids.

This chapter describes how to test different amyloid preparations for catalytic properties. We describe how to express, purify, prepare and test two types of pathological amyloid (tau and α-synuclein) and two functional amyloid proteins, namely CsgA from Escherichia coli and FapC from Pseudomonas. We therefore preface the methods section with an introduction to these two examples of functional amyloid and their remarkable structural and kinetic properties and high physical stability, which renders them very attractive for a range of nanotechnological designs, both for structural, medical and catalytic purposes. The simplicity and high surface exposure of the CsgA amyloid is particularly useful for the introduction of new functional properties and we therefore provide a computational protocol to graft active sites from an enzyme of interest into the amyloid structure. We hope that the methods described will inspire other researchers to explore the remarkable opportunities provided by bacterial functional amyloid in biotechnology.

Stopped-flow measurement of CO2 hydration activity by catalytic amyloids.

With the ever-increasing rates of catalysis shown by catalytic amyloids, the use of faster characterization techniques is required for proper kinetic studies. The same is true for inherently fast chemical reactions. Carbon dioxide hydration is of significant interest to the field of enzyme design, given both carbonic anhydrases' status as a "perfect enzyme" and the central role carbonic anhydrase plays in the respiration and existence of all carbon-based life. Carbon dioxide is an underexplored hydrolysis substrate within the literature, and a lack of a direct spectroscopic marker for reaction monitoring can make studies more complex and require specialist equipment. Within this article we present a method for measuring the carbon dioxide hydration activity of amyloid fibrils.

Characterization of self-templating catalytic amyloids.

Amyloid aggregates with unique periodic structures have garnered significant attention due to their association with numerous diseases, including systemic amyloidoses and the neurodegenerative diseases Parkinson's, Alzheimer's, and Creutzfeld-Jakob. However, more recent investigations have expanded our understanding of amyloids, revealing their diverse functional biological roles. Amyloids have also been proposed to have played a significant role in prebiotic molecular evolution because of their exceptional stability, spontaneous formation in a prebiotic environment, catalytic and templating abilities, and cooperative interaction with fatty acids, polysaccharides, and nucleic acids. This chapter summarizes methods and techniques associated with studying short amyloidogenic peptides, including detailed procedures for investigating cross-templating and autocatalytic templating reactions. Since the work with amyloidogenic peptides and their aggregates present unique challenges, we have attempted to address these with essential details throughout the procedures. The lessons herein may be used in any amyloid-related research to ensure more reproducible results and reduce entrance barriers for researchers new to the field.

Catalytic physiological amyloids.

Amyloid fibrils have been identified in many protein systems, mostly linked to progression and cytotoxicity in neurodegenerative diseases and other pathologies, but have also been observed in normal physiological systems. A growing body of work has shown that amyloid fibrils can catalyze chemical reactions. Most studies have focused on catalysis by de-novo synthetic amyloid-like peptides; however, recent studies reveal that physiological, native amyloids are catalytic as well. Here, we discuss methodologies and major experimental aspects pertaining to physiological catalytic amyloids. We highlight analyzes of kinetic parameters related to the catalytic activities of amyloid fibrils, structure-function considerations, characterization of the catalytic active sites, and deciphering of catalytic mechanisms.