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[
Proc Natl Acad Sci U S A,
2021]
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 <i>Caenorhabditis elegans</i> 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<sup>-1</sup> 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.
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[
J Vis Exp,
2018]
Caenorhabditis elegans is a well-established animal model in biomedical research, widely employed in functional genomics and ageing studies. To assess the health and fitness of the animals under study, one typically relies on motility readouts, such as the measurement of the number of body bends or the speed of movement. These measurements usually involve manual counting, making it challenging to obtain good statistical significance, as time and labor constraints often limit the number of animals in each experiment to 25 or less. Since high statistical power is necessary to obtain reproducible results and limit false positive and negative results when weak phenotypic effects are investigated, efforts have recently been made to develop automated protocols focused on increasing the sensitivity of motility detection and multi-parametric behavioral profiling. In order to extend the limit of detection to the level needed to capture the small phenotypic changes that are often crucial in genetic studies and drug discovery, we describe here a technological development that enables the study of up to 5,000 individual animals simultaneously, increasing the statistical power of the measurements by about 1,000-fold compared to manual assays and about 100-fold compared to other available automated methods.
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[
Nat Protoc,
2020]
Caenorhabditis elegans is a valuable model organism in biomedical research that has led to major discoveries in the fields of neurodegeneration, cancer and aging. Because movement phenotypes are commonly used and represent strong indicators of C. elegans fitness, there is an increasing need to replace manual assessments of worm motility with automated measurements to increase throughput and minimize observer biases. Here, we provide a protocol for the implementation of the improved wide field-of-view nematode tracking platform (WF-NTP), which enables the simultaneous analysis of hundreds of worms with respect to multiple behavioral parameters. The protocol takes only a few hours to complete, excluding the time spent culturing C. elegans, and includes (i) experimental design and preparation of samples, (ii) data recording, (iii) software management with appropriate parameter choices and (iv) post-experimental data analysis. We compare the WF-NTP with other existing worm trackers, including those having high spatial resolution. The main benefits of WF-NTP relate to the high number of worms that can be assessed at the same time on a whole-plate basis and the number of phenotypes that can be screened for simultaneously.
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[
Sci Adv,
2017]
Antibodies targeting A42 are under intense scrutiny because of their therapeutic potential for Alzheimer's disease. To enable systematic searches, we present an "antibody scanning" strategy for the generation of a panel of antibodies against A42. Each antibody in the panel is rationally designed to target a specific linear epitope, with the selected epitopes scanning the A42 sequence. By screening in vitro the panel to identify the specific microscopic steps in the A42 aggregation process influenced by each antibody, we identify two antibodies that target specifically the primary and the secondary nucleation steps, which are key for the production of A42 oligomers. These two antibodies act, respectively, to delay the onset of aggregation and to block the proliferation of aggregates, and correspondingly reduce the toxicity in a Caenorhabditis elegans model overexpressing A42. These results illustrate how the antibody scanning method described here can be used to readily obtain very small antibody libraries with extensive coverage of the sequences of target proteins.
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[
ACS Chem Neurosci,
2018]
Small oligomers formed during the aggregation of certain peptides and proteins are highly cytotoxic in numerous neurodegenerative disorders. Because of their transient nature and conformational heterogeneity, however, the structural and biological features of these oligomers are still poorly understood. Here we describe a method of generating stable oligomers formed by the Alzheimer's A40 peptide by carrying out an aggregation reaction in the presence of zinc ions. The resulting oligomers are amenable to detailed biophysical and biological characterization, which reveals a homogenous population with small size, high cross- sheet structure content and extended hydrophobic surface patches. We also show that these oligomers decrease the viability of neuroblastoma cells and impair the motility of C. elegans. The availability of these oligomers offers novel opportunities for studying the mechanisms of A40 toxicity in vitro and in cellular and animal models of Alzheimer's disease.
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Perni M, Dobson CM, Knowles TPJ, Vendruscolo M, Corzana F, Jimenez-Oses G, Bertoldo JB, Lindstedt PR, Bernardim B, Peter Q, Bernardes GJL, Aprile FA, Matos MJ
[
ACS Cent Sci,
2019]
Protein behavior is closely regulated by a plethora of post-translational modifications (PTMs). It is therefore desirable to develop approaches to design rational PTMs to modulate specific protein functions. Here, we report one such method, and we illustrate its successful implementation by potentiating the anti-aggregation activity of a molecular chaperone. Molecular chaperones are a multifaceted class of proteins essential to protein homeostasis, and one of their major functions is to combat protein misfolding and aggregation, a phenomenon linked to a number of human disorders. In this work, we conjugated a small-molecule inhibitor of the aggregation of -synuclein, a process associated with Parkinson's disease (PD), to a specific cysteine residue on human Hsp70, a molecular chaperone with five free cysteines. We show that this regioselective conjugation augments in vitro the anti-aggregation activity of Hsp70 in a synergistic manner. This Hsp70 variant also displays in vivo an enhanced suppression of -synuclein aggregation and its associated toxicity in a <i>Caenorhabditis elegans</i> model of PD.
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Vendruscolo M, Aprile FA, Sormanni P, Middel C, Heller GT, Michaels TCT, Ikenoue T, Haas CP, Knowles TPJ, Perni M, Ruggeri FS, Dobson CM, Limbocker R, Mannini B
[
Sci Rep,
2020]
Bicyclic peptides have great therapeutic potential since they can bridge the gap between small molecules and antibodies by combining a low molecular weight of about 2kDa with an antibody-like binding specificity. Here we apply a recently developed in silico rational design strategy to produce a bicyclic peptide to target the C-terminal region (residues 31-42) of the 42-residue form of the amyloid peptide (A42), a protein fragment whose aggregation into amyloid plaques is linked with Alzheimer's disease. We show that this bicyclic peptide is able to remodel the aggregation process of A42 in vitro and to reduce its associated toxicity in vivo in a C. elegans worm model expressing A42. These results provide an initial example of a computational approach to design bicyclic peptides to target specific epitopes on disordered proteins.
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Michaels TCT, Lohr T, Mannini B, Aprile FA, Heller GT, Felli IC, Ruggeri FS, Limbocker R, De Simone A, Camilloni C, Pierattelli R, Bonomi M, Perni M, Knowles TPJ, Vendruscolo M, Dobson CM
[
Sci Adv,
2020]
Disordered proteins are challenging therapeutic targets, and no drug is currently in clinical use that modifies the properties of their monomeric states. Here, we identify a small molecule (10074-G5) capable of binding and sequestering the intrinsically disordered amyloid- (A) peptide in its monomeric, soluble state. Our analysis reveals that this compound interacts with A and inhibits both the primary and secondary nucleation pathways in its aggregation process. We characterize this interaction using biophysical experiments and integrative structural ensemble determination methods. We observe that this molecule increases the conformational entropy of monomeric A while decreasing its hydrophobic surface area. We also show that it rescues a <i>Caenorhabditis elegans</i> model of A-associated toxicity, consistent with the mechanism of action identified from the in silico and in vitro studies. These results illustrate the strategy of stabilizing the monomeric states of disordered proteins with small molecules to alter their behavior for therapeutic purposes.
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Challa PK, Muller T, Habchi J, Koopman M, Vecchi G, Knowles TPJ, Dobson CM, Nollen EAA, Casford S, Perni M, Vendruscolo M, Hardenberg MC, Sormanni P, Roode LWY, Kirkegaard JB, Limbocker R, Saar KL, Fernando NW
[
J Neurosci Methods,
2018]
BACKGROUND: The nematode worm C. elegans is a model organism widely used for studies of genetics and of human disease. The health and fitness of the worms can be quantified in different ways, such as by measuring their bending frequency, speed or lifespan. Manual assays, however, are time consuming and limited in their scope providing a strong motivation for automation. NEW METHOD: We describe the development and application of an advanced machine vision system for characterizing the behaviour of C. elegans, the Wide Field-of-view Nematode Tracking Platform (WF-NTP), which enables massively parallel data acquisition and automated multi-parameter behavioural profiling of thousands of worms simultaneously. RESULTS: We screened more than a million worms from several established models of neurodegenerative disorders and characterised the effects of potential therapeutic molecules against Alzheimer's and Parkinson's diseases. By using very large numbers of animals we show that the sensitivity and reproducibility of behavioural assays is very greatly increased. The results reveal the ability of this platform to detect even subtle phenotypes. COMPARISON WITH EXISTING METHODS: The WF-NTP method has substantially greater capacity compared to current automated platforms that typically either focus on characterising single worms at high resolution or tracking the properties of populations of less than 50 animals. CONCLUSIONS: The WF-NTP extends significantly the power of existing automated platforms by combining enhanced optical imaging techniques with an advanced software platform. This approach will further extend the scope and utility of C. elegans as a model organism.
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Needham LM, Salinas PC, Ruggeri FS, Podpolny M, Heller GT, Mannini B, Lee SF, Sormanni P, Chhangur S, Limbocker R, Vendruscolo M, Scheidt T, Dobson CM, Habchi J, Aprile FA, Knowles TPJ, Perni M, Sneideris T
[
Proc Natl Acad Sci U S A,
2020]
Protein misfolding and aggregation is the hallmark of numerous human disorders, including Alzheimer's disease. This process involves the formation of transient and heterogeneous soluble oligomers, some of which are highly cytotoxic. A major challenge for the development of effective diagnostic and therapeutic tools is thus the detection and quantification of these elusive oligomers. Here, to address this problem, we develop a two-step rational design method for the discovery of oligomer-specific antibodies. The first step consists of an "antigen scanning" phase in which an initial panel of antibodies is designed to bind different epitopes covering the entire sequence of a target protein. This procedure enables the determination through in vitro assays of the regions exposed in the oligomers but not in the fibrillar deposits. The second step involves an "epitope mining" phase, in which a second panel of antibodies is designed to specifically target the regions identified during the scanning step. We illustrate this method in the case of the amyloid (A) peptide, whose oligomers are associated with Alzheimer's disease. Our results show that this approach enables the accurate detection and quantification of A oligomers in vitro, and in <i>Caenorhabditis elegans</i> and mouse hippocampal tissues.