Arbitrary template docking fails to recapitulate early amyloid fibril oligomers in neurodegenerative diseases

The formation of protein aggregates and inclusions in the brain and spinal cord is a common neuropathological feature of a number of neurodegenerative diseases including Alzheimer's disease (AD), Parkinson's disease (PD), amyotrophic lateral sclerosis (ALS), and many others. These are commonly referred as neurodegenerative proteinopathies or protein-misfolding diseases. The main characteristic of protein aggregates in these disorders is the fact that they are enriched in amyloid fibrils. Since protein aggregation is considered to play a central role for the onset of neurodegenerative proteinopathies, research is ongoing to develop strategies aimed at preventing or removing protein aggregation in the brain of affected patients. Numerous studies have shown that small molecule-based approaches may be potentially the most promising for halting protein aggregation in neurodegenerative diseases. Indeed, several of these compounds have been found to interact with intrinsically disordered proteins and promote their clearing in experimental models. This notwithstanding, at present small molecule inhibitors still awaits achievements for clinical translation. Hopefully, if we determine whether the formation of insoluble inclusions is effectively neurotoxic and find a valid biomarker to assess their protein aggregation-inhibitory activity in the human central nervous system, the use of small molecule inhibitors will be considered as a cure for neurodegenerative protein-misfolding diseases.

In this thesis, we study the relationship between proteins and neurodegenerative disease using different computational and experimental approaches. We focus on two disease mechanisms: changes in protein abundance and changes in protein structure. Additionally, we investigate the difference in unfolding and refolding profile between native and denatured proteins. Changes in protein structure are especially important in neurodegenerative disease, where large β-stranded aggregates called amyloid fibrils are thought to play a major role. Some amyloid fibrils are known to denature at low temperatures. In Chapter 2, we delineate the entropic and enthalpic contributions of the temperature dependence of hydrophobicity in relation to cold denaturation. We use Monte Carlo simulations on a 3D lattice to gain mechanistic insight into cold denaturation of amyloid fibrils. Additionally, we confirm our results using isothermal titration calorimetry experiments on different types of amyloid fibrils in vitro. We identify three necessary conditions for cold denaturation of amyloid fibrils. We conclude that while heat denaturation of amyloid fibrils is mostly driven by the chain entropy, cold denaturation of fibrils is mostly driven by the hydrophobic contribution to the enthalpy. In Chapter 3, we define three measures for protein surface hydrophobicity and develop a tool to calculate surface hydrophobicity from 3D protein structure. Additionally, we try to predict these measures from sequence and use these predictions to investigate the abundance of protein surface hydrophobicity in the human proteome. We find that proteins in the human brain are relatively hydrophobic, which could explain why protein aggregation diseases are mostly found in the nervous system. In Chapter 4, we use a combination of Quartz Crystal Microbalance experimental measurements with kinetic models to study the effect of disordered flanks on fibril formation of α-synuclein. We find that these flanks have an inhibitory effect on amyloid fibril formation. Without the flanks, amyloid fibril growth was significantly increased. The mathematical models suggest that the increased growth rate is due to secondary nucleation, a process by which monomers bind to the amyloid fibril surface, eventually leading to the growth of new fibrils away from the surface of the initial fibril. In Chapter 5, we investigate the interaction between nanoPET and α-synuclein. Nanoplastics are commonly found as pollutants in the environment. We show that nanoPET may be able to enhance amyloid fibril formation of α-synuclein, and thus exposure to these compounds might stimulate amyloidoses. Because misfolded proteins can play a role in disease, it would be valuable to be able to distinguish native from misfolded proteins in protein samples. In Chapter 6, we combine steered MD simulations with AFM experiments to study the difference in unfolding pattern between native and denatured Hemoglobin protease (Hbp) proteins at single-molecule level. Additionally, we show that the stability of Hbp comes from a stack of hydrophobic residues in the core of the protein, and study the refolding ability of Hbp after being unfolded. In Chapter 7, we use a proteomics workflow to investigate the molecular pathways affected in frontotemporal dementia, including a novel method to validate the results. We identify and validate several modules of co-abundant proteins affected in FTLD-tau. Specifically, PTBP1 is a protein that plays an important role in the alternative splicing of MAPT, and could be an interesting therapeutic target for diagnosis or treatment of FTD. In conclusion, we have studied the relationship between protein aggregation, hydrophobicity and neurodegenerative disease. In this thesis, we use different experimental and computational techniques to delineate physical factors affecting amyloid fibril formation and growth. We identify hydrophobicity as one of the major physical properties important for aggregation. Additionally, we identify molecular pathways affected in frontotemporal dementia through a novel proteomics workflow.

The aggregation of the protein α-synuclein (aSyn) into amyloid fibrils in the human brain is associated with the development of several neurodegenerative diseases, including Parkinson's disease. The previously observed prion-like spreading of aSyn aggregation throughout the brain and the finding that heterologous cross-seeding of amyloid aggregation occurs in vitro for some proteins suggest that exposure to amyloids in general may pose a risk for disease development. To elucidate which protein fibril characteristics determine if and how heterologous amyloid seeding can occur, we investigated the potential of amyloid fibrils formed from proteins found in food, hen egg white lysozyme, and bovine milk β-lactoglobulin to cross-seed aSyn aggregation in the test tube. We observed that amyloid fibrils from lysozyme, but not β-lactoglobulin, potently cross-seeded the aggregation of aSyn as indicated by a significantly shorter lag phase of aSyn aggregation in the presence of lysozyme fibrils. The cross-seeding effect of lysozyme was found to be primarily driven by a surface-mediated nucleation mechanism. The differential seeding effect of lysozyme and β-lactoglobulin on aSyn aggregation could be explained on the basis of binding affinity, binding site, and electrostatic interactions. Our results indicate that heterologous seeding of proteins may occur depending on the physicochemical characteristics of the seed protein fibril. Our findings suggest that heterologous seeding has the potential to determine the pathogenesis of neurodegenerative amyloid diseases.

From protective enzyme to facilitator of amyloid propagation: Cathepsin D-mediated amyloid fibril fragmentation.

BackgroundAmyloid fibrils associated with neurodegenerative diseases can be considered biologically relevant failures of cellular quality control mechanisms. It is known that in vivo human Tau protein, human prion protein, and human copper, zinc superoxide dismutase (SOD1) have the tendency to form fibril deposits in a variety of tissues and they are associated with different neurodegenerative diseases, while rabbit prion protein and hen egg white lysozyme do not readily form fibrils and are unlikely to cause neurodegenerative diseases. In this study, we have investigated the contrasting effect of macromolecular crowding on fibril formation of different proteins.Methodology/Principal FindingsAs revealed by assays based on thioflavin T binding and turbidity, human Tau fragments, when phosphorylated by glycogen synthase kinase-3β, do not form filaments in the absence of a crowding agent but do form fibrils in the presence of a crowding agent, and the presence of a strong crowding agent dramatically promotes amyloid fibril formation of human prion protein and its two pathogenic mutants E196K and D178N. Such an enhancing effect of macromolecular crowding on fibril formation is also observed for a pathological human SOD1 mutant A4V. On the other hand, rabbit prion protein and hen lysozyme do not form amyloid fibrils when a crowding agent at 300 g/l is used but do form fibrils in the absence of a crowding agent. Furthermore, aggregation of these two proteins is remarkably inhibited by Ficoll 70 and dextran 70 at 200 g/l.Conclusions/SignificanceWe suggest that proteins associated with neurodegenerative diseases are more likely to form amyloid fibrils under crowded conditions than in dilute solutions. By contrast, some of the proteins that are not neurodegenerative disease-associated are unlikely to misfold in crowded physiological environments. A possible explanation for the contrasting effect of macromolecular crowding on these two sets of proteins (amyloidogenic proteins and non-amyloidogenic proteins) has been proposed.

Prion particles (prion) are proteinaceous infectious particles and it has been proved that there are two different structures exist for prion proteins. The normal cellular prion protein, denoted as PrPC, may convert into an abnormal scrapie cellular prion, denoted as PrPSc, through a process whereby a portion of its α-helical structure is refolded into β-sheet. The presence of PrPSc will catalyze the transformation of PrPC to PrPSc by the misfolding process. When PrPSc forms aggregate through hydrogen bonds and Van der Waals interactions, the amyloid fibril will be formed and cause the neurodegenerative disease. The transmissible spongiform encephalopathies (TSE) is one of the neurodegenerative diseases that caused by the amyloid fibril depositions. Different protein sequences may have different pathogenicities and distinctive amyloid fibril structures. Therefore, understanding structural conversion of amyloid fibril is very important. Owing to the low solublity and non-crystalline characteristics of amyloid fibrils, it is difficult to use conventional experimental techniques such as solution-state NMR and XRD to analyze the structure of amyloid fibrils. Hence, solid-state NMR is still the most suitable method to characterize structures of this kind. In this study, we mutated the 117 position of Syrian hamster prion protein 109-122 fragment (SHaPrP109-122, A117I, Ac-MKHMAGAAIAGAVV-NH2) from Ala to Ile. The idea is to compare the structural difference induced by mutation on the aligned position of β-sheet in the native SHaPrP109-122. We have incubated the amyloid fibril formed by SHaPrP109-122, A117I successfully. We report the ThT fluorescence, TEM, AFM experiments to observe the formation of amyloid fibrils. From the TEM and AFM images, we have measured the matured fibrils about 24 nm in width, 1.1 nm in height and 380 nm in length. From the isotope-edited FTIR study, the fibril has an anti-parallel β-strand secondary structure and showed an obvious alignment at I117. The chemical shift and linewidth data obtained from ssNMR showed that β-sheet structure exists in the core region. The distance about 4.9±0.2A between the two β layers is determined by fpRFDR-CT experiment. From the experimental data it has been inferred that the fibrils formed by A117I SHaPrP109-122 has the steric zipper structure. Finally, a molecular model for the fibrils was constructed by molecular dynamics simulations incorporated with structural constraints obtained from ssNMR measurements. The results point out that the fibrils still maintain the steric zipper structure after mutating alanine at residue 117 to isoleucine, which has a very bulky side chain. In addition, we have also investigated the amyloid fibrils formed by the residues 127-147 of the human prion protein (HuPrP127-147, Ac-GYMLGSAMSRPIIHFGSDYED-NH2) by TEM to trace the initial stages of amyloid fibrils formation and observe the morphology of the fibril and spherical aggregates. The fibrils are about 6 nm in width from the sample incubated for 5 minutes only. The distance between the β-strands was determined by ssNMR to be 5±0.1 A at the proline residue. It demonstrates that the side chain structure of P137 does not disrupt the β-sheet structure.

Neurodegenerative diseases (NDs) such as Alzheimer’s disease (AD) and Parkinson’s disease (PD) constitute a spectrum of diseases characterized by the abnormal aggregation of specific amyloid fibrillar proteins; these include β-amyloid (Aβ) and tau in the form of the extracellular Aβ plaques and neuronal neurofibrillary tangles in AD and fibrillar α-synuclein aggregation in the form of Lewy bodies and Lewy neurites in PD. Transmembrane protein 106B (TMEM106B) is a type II transmembrane lysosomal protein that participates in lysosome morphology, localization, acidification, and trafficking; t is involved in the pathogenesis of several NDs, especially frontotemporal lobular degeneration with TAR DNA-binding protein immunoreactive inclusions (FTLD-TDP). Studies from four independent research groups revealed that the luminal domain of TMEM106B (120-254aa) forms amyloid fibrils in several brain regions in patients with a series of NDs and neurologically normal older adults. Given its potentially critical roles in the pathogenesis of NDs and brain aging, this surprising finding has focused attention on TMEM106B and suggested that it is nearly as fundamental as other pathogenic amyloid proteins (e.g., Aβ, tau, α-syn); nevertheless, new questions surrounding TMEM106B must be asked. In this review,we firstly introduce the physiological function of TMEM106B and its involvement in NDs. Then, we elucidate the identification and cryo-electronic microscopic structure of TMEM106B fibrils and analyze the factors that contribute to the polymorphism of TMEM106B fibrils. Finally, the potential pathogenic role of TMEM106B fibrils is discussed, and the future directions for TMEM106 research in NDs are briefly summarized.

Carbon nanotubes (CNTs) have been extensively investigated and employed for industrial use because of their peculiar physical properties, which make them ideal for many industrial applications. However, rapid growth of CNT employment raises concerns about the potential risks and toxicities for public health, environment, and workers associated with the manufacture and use of these new materials. Here we investigate the main routes of entry following environmental exposure to multi-wall CNTs (MWCNTs; currently the most widely used in industry). We developed a novel murine model that could represent a surrogate of a workplace exposure to MWCNTs. We traced the localization of MWCNTs and their possible role in inducing an innate immune response, inflammation, macrophage recruitment, and inflammatory conditions. Following environmental exposure of CD1 mice, we observed that MWCNTs rapidly enter and disseminate in the organism, initially accumulating in lungs and brain and later reaching the liver and kidney via the bloodstream. Since recent experimental studies show that CNTs are associated with the aggregation process of proteins associated with neurodegenerative diseases, we investigated whether MWCNTs are able to induce amyloid fibril production and accumulation. Amyloid deposits in spatial association with macrophages and MWCNT aggregates were found in the brain, liver, lungs, and kidneys of exposed animals. Our data suggest that accumulation of MWCNTs in different organs is associated with inflammation and amyloid accumulation. In the brain, where we observed rapid accumulation and amyloid fibril deposition, exposure to MWCNTs might enhance progression of neurodegenerative and other amyloid-related diseases. Our data highlight the conclusion that, in a novel rodent model of exposure, MWCNTs may induce macrophage recruitment, activation, and amyloid deposition, causing potential damage to several organs.

Dipyridamole for tracking amyloidogenic proteins aggregation and enhancing polyubiquitination

A growing body of evidence indicates that small, soluble oligomeric species generated from a variety of proteins and peptides rather than mature amyloid fibrils are inherently highly cytotoxic. Here, we show for the first time that mature amyloid fibrils produced from full-length recombinant mammalian prion protein (rPrP) were highly toxic to cultured cells and primary hippocampal and cerebella neurons. Fibrils induced apoptotic cell death in a time- and dose-dependent manner. The toxic effect of fibrils was comparable with that exhibited by soluble small beta-oligomers generated from the same protein. Fibrils prepared from insulin were not toxic, suggesting that the toxic effect was not solely due to the highly polymeric nature of the fibrillar form. The cell death caused by rPrP fibrils or beta-oligomers was substantially reduced when expression of endogenous PrP(C) was down-regulated by small interfering RNAs. In opposition to the beta-oligomer and amyloid fibrils of rPrP, the monomeric alpha-helical form of rPrP stimulated neurite out-growth and survival of neurons. These studies illustrated that both soluble beta-oligomer and amyloid fibrils of the prion protein are intrinsically toxic and confirmed that endogenously expressed PrP(C) is required for mediating the toxicity of abnormally folded external PrP aggregates.

Emerging evidence from both clinical studies and animal models indicates the importance of the interaction between the gut microbiome and the brain in the pathogenesis of neurodegenerative diseases (NDs). Although how microbes modulate neurodegeneration is still mostly unclear, recent studies have started to probe into the mechanisms for the communication between microbes and hosts in NDs. In this review, we highlight the advantages of using Caenorhabditis elegans (C. elegans) to disentangle the microbe-host interaction that regulates neurodegeneration. We summarize the microbial pro- and anti-neurodegenerative factors identified using the C. elegans ND models and the effects of many are confirmed in mouse models. Specifically, we focused on the role of bacterial amyloid proteins, such as curli, in promoting proteotoxicity and neurodegeneration by cross-seeding the aggregation of endogenous ND-related proteins, such as α-synuclein. Targeting bacterial amyloid production may serve as a novel therapeutic strategy for treating NDs, and several compounds, such as epigallocatechin-3-gallate (EGCG), were shown to suppress neurodegeneration at least partly by inhibiting curli production. Because bacterial amyloid fibrils contribute to biofilm formation, inhibition of amyloid production often leads to the disruption of biofilms. Interestingly, from a list of 59 compounds that showed neuroprotective effects in C. elegans and mouse ND models, we found that about half of them are known to inhibit bacterial growth or biofilm formation, suggesting a strong correlation between the neuroprotective and antibiofilm activities. Whether these potential therapeutics indeed protect neurons from proteotoxicity by inhibiting the cross-seeding between bacterial and human amyloid proteins awaits further investigations. Finally, we propose to screen the long list of antibiofilm agents, both FDA-approved drugs and novel compounds, for their neuroprotective effects and develop new pharmaceuticals that target the gut microbiome for the treatment of NDs. To this end, the C. elegans ND models can serve as a platform for fast, high-throughput, and low-cost drug screens that target the microbe-host interaction in NDs.

Amyloid proteins, which are considered 'villains' in many neurodegenerative diseases, form enigmatic pathological strains that underlie disease pathogenesis and progression. Recent technical advances in cryogenic electron microscopy and solid-state NMR spectroscopy have enabled the high-resolution structures of full-length amyloid fibrils to be determined, initiating an era in which we have the opportunity to gain atomic-level structural understanding of pathogenic protein aggregation in neurodegenerative diseases. In this Review, we aim to explain the clinicopathological heterogeneity of neurodegenerative diseases by considering the polymorphic structures of amyloid fibrils. We decipher the structural basis for the generation of fibril polymorphs, how the fibril polymorphs differ in different disease contexts and how conformational changes alter the pathology caused by amyloid proteins during disease progression. Finally, we evaluate how this knowledge might aid clinical diagnostic and therapeutic strategies to treat neurodegenerative diseases.

Prions

The whole set of so-called >>conformational<< disorders, among them systemic amyloidoses, various dementias and other neurodegenerative diseases such as Parkinson's, Alzheimer's and amyotropic lateral sclerosis, may have similar molecular backgrounds: changes in protein conformation and aggregation lead to toxic amyloid oligomers and fibrils. The so called aggresomes in eukaryotes (equivalent to inclusion bodies in prokaryotes), located at the centriole by the nucleus and composed of aggregated proteins, are believed to sequester the toxic material. They eventually get cleared from the cell by autophagy. When the cell defence system fails due to continuous production of a mutated protein or to other damage to the cell such as oxidative stress or protein modification as part of normal aging, familial or sporadic neurodegenerative diseases develop. Initially - for years - they are silent with no or mild symptoms. It could well be that aggregates represent a response to some other trigger or even a means of defence. However, the inherited cases with mutations leading to increased aggregation suggest the opposite to be the case. Evidence has accumulated that the soluble oligomers of amyloidogenic proteins are themselves cytotoxic and trigger a cascade of detrimental events in the cell, as summarized in the "amyloid cascade hypothesis". Among other plausible hypotheses for the mechanism of toxicity is the "channel hypothesis", which states that the soluble oligomers interact with cell membranes, causing influx of Ca2+ ions, which is an early sign of pathology and contributes to uncontrolled neurotransmission. Another factor are metal ions, such as Zn(2+), Cu(2+), Fe(3+), Al(3+), etc., leading to the "metal hypothesis". The delicate balance of metal ions in the brain is important to prevent oxidative stress, which can itself modify proteins and make them aggregation-prone. The advances in molecular and cellular studies will hopefully lead to novel therapies and eventually to a cure.

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.