Kinetic analysis reveals that independent nucleation events determine the progression of polyglutamine aggregation in C. elegans.

Protein aggregation is associated with a wide range of degenerative human diseases with devastating consequences, as exemplified by Alzheimer's, Parkinson's, and Huntington's diseases. In vitro kinetic studies have provided a mechanistic understanding of the aggregation process at the molecular level. However, it has so far remained largely unclear to what extent the biophysical principles of amyloid formation learned in vitro translate to the complex environment of living organisms. Here, we take advantage of the unique properties of a Caenorhabditis elegans model expressing a fluorescently tagged polyglutamine (polyQ) protein, which aggregates into discrete micrometer-sized inclusions that can be directly visualized in real time. We provide a quantitative analysis of protein aggregation in this system and show that the data are described by a molecular model where stochastic nucleation occurs independently in each cell, followed by rapid aggregate growth. Global fitting of the image-based aggregation kinetics reveals a nucleation rate corresponding to 0.01 h-1 per cell at 1 mM intracellular protein concentration, and shows that the intrinsic molecular stochasticity of nucleation accounts for a significant fraction of the observed animal-to-animal variation. Our results highlight how independent, stochastic nucleation events in individual cells control the overall progression of polyQ aggregation in a living animal. The key finding that the biophysical principles associated with protein aggregation in small volumes remain the governing factors, even in the complex environment of a living organism, will be critical for the interpretation of in vivo data from a wide range of protein aggregation diseases.

Biophysical studies of protein misfolding and aggregation in in vivo models of Alzheimer's and Parkinson's diseases.

Neurodegenerative disorders, including Alzheimer's (AD) and Parkinson's diseases (PD), are characterised by the formation of aberrant assemblies of misfolded proteins. The discovery of disease-modifying drugs for these disorders is challenging, in part because we still have a limited understanding of their molecular origins. In this review, we discuss how biophysical approaches can help explain the formation of the aberrant conformational states of proteins whose neurotoxic effects underlie these diseases. We discuss in particular models based on the transgenic expression of amyloid-ß (Aß) and tau in AD, and α-synuclein in PD. Because biophysical methods have enabled an accurate quantification and a detailed understanding of the molecular mechanisms underlying protein misfolding and aggregation in vitro, we expect that the further development of these methods to probe directly the corresponding mechanisms in vivo will open effective routes for diagnostic and therapeutic interventions.

Membrane-Catalyzed Aggregation of Islet Amyloid Polypeptide Is Dominated by Secondary Nucleation.

Type II diabetes is characterized by the loss of pancreatic ß-cells. This loss is thought to be a consequence of membrane disruption, caused by the aggregation of islet amyloid polypeptide (IAPP) into amyloid fibrils. However, the molecular mechanisms of IAPP aggregation in the presence of membranes have remained unclear. Here, we use kinetic analysis to elucidate the aggregation mechanism of IAPP in the presence of mixed zwitterionic and anionic lipid membranes. The results converge to a model in which aggregation on the membrane is strongly dominated by secondary nucleation, that is, the formation of new nuclei on the surface of existing fibrils. The critical nucleus consists of a single IAPP molecule, and anionic lipids catalyze both primary and secondary nucleation, but not elongation. The fact that anionic lipids promote secondary nucleation implies that these events take place at the interface between the membrane and existing fibrils, demonstrating that fibril growth occurs at least to some extent on the membrane surface. These new insights into the mechanism of IAPP aggregation on membranes may help to understand IAPP toxicity and will be important for the development of therapeutics to prevent ß-cell death in type II diabetes.

Challenging Proteostasis: Role of the Chaperone Network to Control Aggregation-Prone Proteins in Human Disease.

Protein homeostasis (Proteostasis) is essential for correct and efficient protein function within the living cell. Among the critical components of the Proteostasis Network (PN) are molecular chaperones that serve widely in protein biogenesis under physiological conditions, and prevent protein misfolding and aggregation enhanced by conditions of cellular stress. For Alzheimer's, Parkinson's, Huntington's diseases and ALS, multiple classes of molecular chaperones interact with the highly aggregation-prone proteins amyloid-ß, tau, α-synuclein, huntingtin and SOD1 to influence the course of proteotoxicity associated with these neurodegenerative diseases. Accordingly, overexpression of molecular chaperones and induction of the heat shock response have been shown to be protective in a wide range of animal models of these diseases. In contrast, for cancer cells the upregulation of chaperones has the undesirable effect of promoting cellular survival and tumor growth by stabilizing mutant oncoproteins. In both situations, physiological levels of molecular chaperones eventually become functionally compromised by the persistence of misfolded substrates, leading to a decline in global protein homeostasis and the dysregulation of diverse cellular pathways. The phenomenon of chaperone competition may underlie the broad pathology observed in aging and neurodegenerative diseases, and restoration of physiological protein homeostasis may be a suitable therapeutic avenue for neurodegeneration as well as for cancer.

Insight into the conformational stability of membrane-embedded BamA using a combined solution and solid-state NMR approach.

The ß-barrel assembly machinery (BAM) is involved in folding and insertion of outer membrane proteins in Gram-negative bacteria, a process that is still poorly understood. With its 790 residues, BamA presents a challenge to current NMR methods. We utilized a "divide and conquer" approach in which we first obtained resonance assignments for BamA's periplasmic POTRA domains 4 and 5 by solution NMR. Comparison of these assignments to solid-state NMR (ssNMR) data obtained on two BamA constructs including the transmembrane domain and one or two soluble POTRA domains suggested that the fold of POTRA domain 5 critically depends on the interface with POTRA 4. Using specific labeling schemes we furthermore obtained ssNMR resonance assignments for residues in the extracellular loop 6 that is known to be crucial for BamA-mediated substrate folding and insertion. Taken together, our data provide novel insights into the conformational stability of membrane-embedded, non-crystalline BamA.

An Efficient Labelling Approach to Harness Backbone and Side-Chain Protons in (1) H-Detected Solid-State NMR Spectroscopy.

(1) H-detection can greatly improve spectral sensitivity in biological solid-state NMR (ssNMR), thus allowing the study of larger and more complex proteins. However, the general requirement to perdeuterate proteins critically curtails the potential of (1) H-detection by the loss of aliphatic side-chain protons, which are important probes for protein structure and function. Introduced herein is a labelling scheme for (1) H-detected ssNMR, and it gives high quality spectra for both side-chain and backbone protons, and allows quantitative assignments and aids in probing interresidual contacts. Excellent (1) H resolution in membrane proteins is obtained, the topology and dynamics of an ion channel were studied. This labelling scheme will open new avenues for the study of challenging proteins by ssNMR.

Proton clouds to measure long-range contacts between nonexchangeable side chain protons in solid-state NMR.

We show that selective labeling of proteins with protonated amino acids embedded in a perdeuterated matrix, dubbed 'proton clouds', provides general access to long-range contacts between nonexchangeable side chain protons in proton-detected solid-state NMR, which is important to study protein tertiary structure. Proton-cloud labeling significantly improves spectral resolution by simultaneously reducing proton line width and spectral crowding despite a high local proton density in clouds. The approach is amenable to almost all canonical amino acids. Our method is demonstrated on ubiquitin and the ß-barrel membrane protein BamA.

N-terminal domain of human Hsp90 triggers binding to the cochaperone p23.

The molecular chaperone Hsp90 is a protein folding machine that is conserved from bacteria to man. Human, cytosolic Hsp90 is dedicated to folding of chiefly signal transduction components. The chaperoning mechanism of Hsp90 is controlled by ATP and various cochaperones, but is poorly understood and controversial. Here, we characterized the Apo and ATP states of the 170-kDa human Hsp90 full-length protein by NMR spectroscopy in solution, and we elucidated the mechanism of the inhibition of its ATPase by its cochaperone p23. We assigned isoleucine side chains of Hsp90 via specific isotope labeling of their δ-methyl groups, which allowed the NMR analysis of the full-length protein. We found that ATP caused exclusively local changes in Hsp90's N-terminal nucleotide-binding domain. Native mass spectrometry showed that Hsp90 and p23 form a 22 complex via a positively cooperative mechanism. Despite this stoichiometry, NMR data indicated that the complex was not fully symmetric. The p23-dependent NMR shifts mapped to both the lid and the adenine end of Hsp90's ATP binding pocket, but also to large parts of the middle domain. Shifts distant from the p23 binding site reflect p23-induced conformational changes in Hsp90. Together, we conclude that it is Hsp90's nucleotide-binding domain that triggers the formation of the Hsp90(2)p23(2) complex. We anticipate that our NMR approach has significant impact on future studies of full-length Hsp90 with cofactors and substrates, but also for the development of Hsp90 inhibiting anticancer drugs.

mNeonGreen aggregates when overexpressed in C. elegans neurons.

The optical transparency of the nematode Caenorhabditis elegans makes it possible to monitor the behaviour of fluorescently labelled proteins in a living multicellular organism. This study investigates the suitability of mNeonGreen as a fluorescent tag for studying proteins of interest in the nervous system of adult C. elegans . Despite its reported brightness, stability, and monomeric nature, our findings reveal that mNeonGreen forms solid aggregates in C. elegans neurons, particularly upon plasmid overexpression. We anticipate that this property may lead to artefacts when analysing for example the subcellular distribution or turnover of a tagged protein of interest.

Islet amyloid polypeptide tagged with green fluorescent protein localises to mitochondria and forms filamentous aggregates in Caenorhabditis elegans.

Type 2 diabetes (T2D) is the most common form of diabetes and represents a growing health concern. A characteristic feature of T2D is the aggregation of islet amyloid polypeptide (IAPP), which is thought to be associated with the death of pancreatic ß-cells. Inhibiting IAPP aggregation is a promising therapeutic avenue to treat T2D, but the mechanisms of aggregation and toxicity are not yet fully understood. Caenorhabditis elegans is a well-characterised multicellular model organism that has been extensively used to study protein aggregation diseases. In this study, we aimed to develop a simple in vivo model to investigate IAPP aggregation and toxicity based on expression in the C. elegans body wall muscle cells. We show that IAPP tagged with green fluorescent protein (GFP) localises to mitochondria not only in muscle cells but also when expressed in the intestine, in line with previous observations in mouse and human pancreatic ß-cells. The IAPP-GFP fusion protein forms solid aggregates, which have a filamentous appearance as seen by electron microscopy. However, the animals expressing IAPP-GFP in the body wall muscle cells do not display a strong motility phenotype, suggesting that the IAPP-GFP aggregates are not considerably toxic. Nevertheless, the mitochondrial localisation and aggregate formation may be useful read-outs to screen for IAPP-solubilizing compounds as a therapeutic strategy for T2D.

Fibril elongation by human islet amyloid polypeptide is the main event linking aggregation to membrane damage.

The aggregation of human islet amyloid polypeptide (hIAPP) is linked to the death of pancreatic ß-cells in type II diabetes. The process of fibril formation by hIAPP is thought to cause membrane damage, but the precise mechanisms are still unclear. Previously, we showed that the aggregation of hIAPP in the presence of membranes containing anionic lipids is dominated by secondary nucleation events, which occur at the interface between existing fibrils and the membrane surface. Here, we used vesicles with different lipid composition to explore the connection between hIAPP aggregation and vesicle leakage. We found that different anionic lipids promote hIAPP aggregation to the same extent, whereas remarkably stochastic behaviour is observed on purely zwitterionic membranes. Vesicle leakage induced by hIAPP consists of two distinct phases for any of the used membrane compositions: (i) an initial phase in which hIAPP binding causes a certain level of leakage that is strongly dependent on osmotic conditions, membrane composition and the used dye, and (ii) a main leakage event that we attribute to elongation of hIAPP fibrils, based on seeded experiments. Altogether, our results shed more light on the relationship between hIAPP fibril formation and membrane damage, and strongly suggest that oligomeric intermediates do not considerably contribute to vesicle leakage.

Molecular mechanisms of amyloid formation in living systems.

Fibrillar protein aggregation is a hallmark of a variety of human diseases. Examples include the deposition of amyloid-ß and tau in Alzheimer's disease, and that of α-synuclein in Parkinson's disease. The molecular mechanisms by which soluble proteins form amyloid fibrils have been extensively studied in the test tube. These investigations have revealed the microscopic steps underlying amyloid formation, and the role of factors such as chaperones that modulate these processes. This perspective explores the question to what extent the mechanisms of amyloid formation elucidated in vitro apply to human disease. The answer is not yet clear, and may differ depending on the protein and the associated disease. Nevertheless, there are striking qualitative similarities between the aggregation behaviour of proteins in vitro and the development of the related diseases. Limited quantitative data obtained in model organisms such as Caenorhabditis elegans support the notion that aggregation mechanisms in vivo can be interpreted using the same biophysical principles established in vitro. These results may however be biased by the high overexpression levels typically used in animal models of protein aggregation diseases. Molecular chaperones have been found to suppress protein aggregation in animal models, but their mechanisms of action have not yet been quantitatively analysed. Several mechanisms are proposed by which the decline of protein quality control with organismal age, but also the intrinsic nature of the aggregation process may contribute to the kinetics of protein aggregation observed in human disease.

Monitoring Protein Aggregation Kinetics In Vivo using Automated Inclusion Counting in Caenorhabditis elegans.

Protein aggregation into insoluble inclusions is a hallmark of a variety of human diseases, many of which are age-related. The nematode Caenorhabditis elegans is a well-established model organism that has been widely used in the field to study protein aggregation and toxicity. Its optical transparency enables the direct visualization of protein aggregation by fluorescence microscopy. Moreover, the fast reproductive cycle and short lifespan make the nematode a suitable model to screen for genes and molecules that modulate this process. However, the quantification of aggregate load in living animals is poorly standardized, typically performed by manual inclusion counting under a fluorescence dissection microscope at a single time point. This approach can result in high variability between observers and limits the understanding of the aggregation process. In contrast, amyloid-like protein aggregation in vitro is routinely monitored by thioflavin T fluorescence in a highly quantitative and time-resolved fashion. Here, an analogous method is presented for the unbiased analysis of aggregation kinetics in living C. elegans, using a high-throughput confocal microscope combined with custom-made image analysis and data fitting. The applicability of this method is demonstrated by monitoring inclusion formation of a fluorescently labeled polyglutamine (polyQ) protein in the body wall muscle cells. The image analysis workflow allows the determination of the numbers of inclusions at different timepoints, which are fitted to a mathematical model based on independent nucleation events in individual muscle cells. The method described here may prove useful to assess the effects of proteostasis factors and potential therapeutics for protein aggregation diseases in a living animal in a robust and quantitative manner.

Observation of an α-synuclein liquid droplet state and its maturation into Lewy body-like assemblies.

Misfolded α-synuclein is a major component of Lewy bodies, which are a hallmark of Parkinson's disease (PD). A large body of evidence shows that α-synuclein can aggregate into amyloid fibrils, but the relationship between α-synuclein self-assembly and Lewy body formation remains unclear. Here, we show, both in vitro and in a Caenorhabditis elegans model of PD, that α-synuclein undergoes liquid‒liquid phase separation by forming a liquid droplet state, which converts into an amyloid-rich hydrogel with Lewy-body-like properties. This maturation process towards the amyloid state is delayed in the presence of model synaptic vesicles in vitro. Taken together, these results suggest that the formation of Lewy bodies may be linked to the arrested maturation of α-synuclein condensates in the presence of lipids and other cellular components.

Comparative Studies in the A30P and A53T α-Synuclein C. elegans Strains to Investigate the Molecular Origins of Parkinson's Disease.

The aggregation of α-synuclein is a hallmark of Parkinson's disease (PD) and a variety of related neurological disorders. A number of mutations in this protein, including A30P and A53T, are associated with familial forms of the disease. Patients carrying the A30P mutation typically exhibit a similar age of onset and symptoms as sporadic PD, while those carrying the A53T mutation generally have an earlier age of onset and an accelerated progression. We report two C. elegans models of PD (PDA30P and PDA53T), which express these mutational variants in the muscle cells, and probed their behavior relative to animals expressing the wild-type protein (PDWT). PDA30P worms showed a reduced speed of movement and an increased paralysis rate, control worms, but no change in the frequency of body bends. By contrast, in PDA53T worms both speed and frequency of body bends were significantly decreased, and paralysis rate was increased. α-Synuclein was also observed to be less well localized into aggregates in PDA30P worms compared to PDA53T and PDWT worms, and amyloid-like features were evident later in the life of the animals, despite comparable levels of expression of α-synuclein. Furthermore, squalamine, a natural product currently in clinical trials for treating symptomatic aspects of PD, was found to reduce significantly the aggregation of α-synuclein and its associated toxicity in PDA53T and PDWT worms, but had less marked effects in PDA30P. In addition, using an antibody that targets the N-terminal region of α-synuclein, we observed a suppression of toxicity in PDA30P, PDA53T and PDWT worms. These results illustrate the use of these two C. elegans models in fundamental and applied PD research.

Expression of the amyloid-ß peptide in a single pair of C. elegans sensory neurons modulates the associated behavioural response.

Although the aggregation of the amyloid-ß peptide (Aß) into amyloid fibrils is a well-established hallmark of Alzheimer's disease, the complex mechanisms linking this process to neurodegeneration are still incompletely understood. The nematode worm C. elegans is a valuable model organism through which to study these mechanisms because of its simple nervous system and its relatively short lifespan. Standard Aß-based C. elegans models of Alzheimer's disease are designed to study the toxic effects of the overexpression of Aß in the muscle or nervous systems. However, the wide variety of effects associated with the tissue-level overexpression of Aß makes it difficult to single out and study specific cellular mechanisms related to the onset of Alzheimer's disease. Here, to better understand how to investigate the early events affecting neuronal signalling, we created a C. elegans model expressing Aß42, the 42-residue form of Aß, from a single-copy gene insertion in just one pair of glutamatergic sensory neurons, the BAG neurons. In behavioural assays, we found that the Aß42-expressing animals displayed a subtle modulation of the response to CO2, compared to controls. Ca2+ imaging revealed that the BAG neurons in young Aß42-expressing nematodes were activated more strongly than in control animals, and that neuronal activation remained intact until old age. Taken together, our results suggest that Aß42-expression in this very subtle model of AD is sufficient to modulate the behavioural response but not strong enough to generate significant neurotoxicity, suggesting that slightly more aggressive perturbations will enable effectively studies of the links between the modulation of a physiological response and its associated neurotoxicity.

Fast Fluorescence Lifetime Imaging Reveals the Aggregation Processes of α-Synuclein and Polyglutamine in Aging Caenorhabditis elegans.

The nematode worm Caenorhabditis elegans has emerged as an important model organism in the study of the molecular mechanisms of protein misfolding diseases associated with amyloid formation because of its small size, ease of genetic manipulation, and optical transparency. Obtaining a reliable and quantitative read-out of protein aggregation in this system, however, remains a challenge. To address this problem, we here present a fast time-gated fluorescence lifetime imaging (TG-FLIM) method and show that it provides functional insights into the process of protein aggregation in living animals by enabling the rapid characterization of different types of aggregates. Specifically, in longitudinal studies of C. elegans models of Parkinson's and Huntington's diseases, we observed marked differences in the aggregation kinetics and the nature of the protein inclusions formed by α-synuclein and polyglutamine. In particular, we found that α-synuclein inclusions do not display amyloid-like features until late in the life of the worms, whereas polyglutamine forms amyloid characteristics rapidly in early adulthood. Furthermore, we show that the TG-FLIM method is capable of imaging live and non-anaesthetized worms moving in specially designed agarose microchambers. Taken together, our results show that the TG-FLIM method enables high-throughput functional imaging of living C. elegans that can be used to study in vivo mechanisms of protein aggregation and that has the potential to aid the search for therapeutic modifiers of protein aggregation and toxicity.

Intrinsically aggregation-prone proteins form amyloid-like aggregates and contribute to tissue aging in Caenorhabditis elegans.

Reduced protein homeostasis leading to increased protein instability is a common molecular feature of aging, but it remains unclear whether this is a cause or consequence of the aging process. In neurodegenerative diseases and other amyloidoses, specific proteins self-assemble into amyloid fibrils and accumulate as pathological aggregates in different tissues. More recently, widespread protein aggregation has been described during normal aging. Until now, an extensive characterization of the nature of age-dependent protein aggregation has been lacking. Here, we show that age-dependent aggregates are rapidly formed by newly synthesized proteins and have an amyloid-like structure resembling that of protein aggregates observed in disease. We then demonstrate that age-dependent protein aggregation accelerates the functional decline of different tissues in C. elegans. Together, these findings imply that amyloid-like aggregates contribute to the aging process and therefore could be important targets for strategies designed to maintain physiological functions in the late stages of life.

Formation of the ß-barrel assembly machinery complex in lipid bilayers as seen by solid-state NMR.

The ß-barrel assembly machinery (BAM) is a pentameric complex (BamA-E), which catalyzes the essential process of ß-barrel protein insertion into the outer membrane of E. coli. Thus far, a detailed understanding of the insertion mechanism has been elusive but recent results suggest that local protein motion, in addition to the surrounding membrane environment, may be of critical relevance. We have devised a high-sensitivity solid-state NMR approach to directly probe protein motion and the structural changes associated with BAM complex assembly in lipid bilayers. Our results reveal how essential BamA domains, such as the interface formed by the polypeptide transport associated domains P4 and P5 become stabilized after complex formation and suggest that BamA ß-barrel opening and P5 reorientation is directly related to complex formation in membranes. Both the lateral gate, as well as P5, exhibit local dynamics, a property that could play an integral role in substrate recognition and insertion.