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Biophysical Studies of Protein Misfolding and Aggregation in In Quarterly Reviews of Biophysical studies of protein misfolding and Biophysics aggregation in in vivo models of Alzheimer’s cambridge.org/qrb and Parkinson’s diseases Tessa Sinnige* , Karen Stroobants , Christopher M. Dobson Major Review and Michele Vendruscolo *Current address: Department of Molecular Department of Chemistry, Centre for Misfolding Diseases, University of Cambridge, Lensfield Road, Cambridge Biosciences, Northwestern University, 2205 CB2 1EW, UK Tech Drive, Evanston, IL 60208-3500, USA. Cite this article: Sinnige T, Stroobants K, Abstract Dobson CM, Vendruscolo M (2020). Biophysical Neurodegenerative disorders, including Alzheimer’s (AD) and Parkinson’s diseases (PD), are studies of protein misfolding and aggregation characterised by the formation of aberrant assemblies of misfolded proteins. The discovery of in in vivo models of Alzheimer’s and Parkinson’s diseases. Quarterly Reviews of disease-modifying drugs for these disorders is challenging, in part because we still have a lim- Biophysics 53, e10, 1–43. https://doi.org/ ited understanding of their molecular origins. In this review, we discuss how biophysical 10.1017/S0033583520000025 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 Received: 28 February 2020 β β α Revised: 19 April 2020 transgenic expression of amyloid- (A ) and tau in AD, and -synuclein in PD. Because bio- Accepted: 20 April 2020 physical methods have enabled an accurate quantification and a detailed understanding of the molecular mechanisms underlying protein misfolding and aggregation in vitro, we expect that Key words: the further development of these methods to probe directly the corresponding mechanisms ’ Alzheimer s disease; biophysical methods; in vivo will open effective routes for diagnostic and therapeutic interventions. in vivo models; Parkinson’s disease; protein misfolding Authors for correspondence: Tessa Sinnige, Table of contents E-mail: [email protected]; Michele Vendruscolo, E-mail: [email protected] Introduction 1 Determination of protein aggregation mechanisms using in vitro biophysical methods 2 Aβ aggregation 3 Tau aggregation 5 α-Synuclein aggregation 6 In vivo models of protein misfolding diseases 7 Aβ models 7 Tau models 19 Models combining Aβ and tau 22 α-Synuclein models 24 Identification of biological pathways using in vivo models 26 Biological pathways in Alzheimer’s disease models 27 Biological pathways in Parkinson’s disease models 30 Methodological challenges 31 Conclusions 32 Introduction © The Author(s), 2020. Published by Protein misfolding and aggregation are associated with a wide variety of human diseases Cambridge University Press. This is an Open (Hardy and Selkoe, 2002; Eisenberg and Jucker, 2012; Jucker and Walker, 2013; Knowles, Access article, distributed under the terms of Vendruscolo and Dobson, 2014; Chiti and Dobson, 2017). These diseases have been linked the Creative Commons Attribution licence (http://creativecommons.org/licenses/by/4.0/), to a progressive failure of the protein homoeostasis system, which controls protein folding, which permits unrestricted re-use, trafficking and degradation (Balch et al., 2008; Kim et al., 2013). The capacity of this system distribution, and reproduction in any medium, has been shown to decline with age both in model organisms and in humans, which can result provided the original work is properly cited. in the accumulation of aberrant protein aggregates (Labbadia and Morimoto, 2015). Neurons appear to be particularly vulnerable to such events (Freer et al., 2016; Surmeier et al., 2017; Luna et al., 2018;Fuet al., 2019), and the vast majority of neurodegenerative diseases are accompanied by the formation of protein deposits, including those of amyloid-β (Aβ) and tau in Alzheimer’s disease (AD), and of α-synuclein in Parkinson’s disease (PD) (Hardy Downloaded from https://www.cambridge.org/core. IP address: 170.106.203.244, on 04 Oct 2021 at 20:11:07, subject to the Cambridge Core terms of use, available at https://www.cambridge.org/core/terms. https://doi.org/10.1017/S0033583520000025 2 Tessa Sinnige et al. Table 1. Commonly used biophysical methods for the study of protein misfolding in vivo and in vitro Methods and tools to probe protein misfolding and aggregation in vivo and in situ Amyloid binding dyes ThS, Congo red, NIAD-4, X-34, silver stain (tau tangles) Microscopy EM, immunohistochemistry, fluorescence microscopy including FLIM and FRAP Conformation-specific antibodies A11 (oligomers), MC1 (misfolded tau), TOC1 (tau oligomers), FILA-1 (α-synuclein oligomers), DesAbs (designed antibodies) Detergent solubility Sarkosyl, SDS Size determination SEC, sucrose gradient, SDS-PAGE, western blot Visualisation of deposits CLARITY, 3DISCO, Scale, MRI, PET Biochemical assays ELISA, SIMOA, amyloid-seeding assay, PMCA, RT-QuIC Methods to probe cellular pathways in vivo and in situ Genetic screens RNAi, random mutagenesis Transcriptomics RNA microarrays, next-generation sequencing (RNAseq) Proteomics TMT, iTRAQ, label-free mass spectrometry Methods to probe protein aggregation in vitro Aggregation kinetics ThT fluorescence, quartz crystal microbalance, filter trap assay Secondary structure determination CD spectroscopy, FTIR spectroscopy Size/shape determination DLS, SEC-MALS, smFRET, SAXS, AFM, super-resolution microscopy 3D structure determination Cryo-EM, solid-state NMR spectroscopy, X-ray diffraction The application of these methods to study protein aggregation and the associated references are mentioned throughout the main text. and Selkoe, 2002; Eisenberg and Jucker, 2012; Jucker and Walker, disease phenotypes better, and are typically used in preclinical 2013; Knowles et al., 2014; Chiti and Dobson, 2017). studies to examine efficacy and safety prior to testing in humans Although mature Aβ fibrils are in many cases relatively inert, (Jucker, 2010; Sasaguri et al., 2017). studies in cell culture and animal models have revealed that In general, in vivo models of disease can be studied at different misfolded oligomers, which are formed during the process of fibril levels. Organismal and cellular phenotypes can be related to formation, possess cytotoxic properties (Bucciantini et al., 2002; the aggregation states of disease-associated proteins at the Baglioni et al., 2006;HaassandSelkoe,2007; Bemporad and Chiti, molecular level by biochemical and biophysical tools (Table 1). 2012;Benilovaet al., 2012; Chiti and Dobson, 2017). Several mech- Correspondingly, at the system level, disrupted molecular path- anisms have been proposed to account for the toxicity of these olig- ways can be comprehensively monitored by genomics, transcrip- omers, including disruption of lipid membranes, transcriptional tomics and proteomics techniques (Barabási et al., 2011). With deregulation, mitochondrial dysfunction, oxidative stress, protea- these approaches, the association of a perturbation (e.g. the some inhibition and disruption of synaptic plasticity (Haass and expression of a mutant gene) with a phenotype (i.e. an aspect Selkoe, 2007;Benilovaet al., 2012; Roberts and Brown, 2015;De of the disease) is translated into knowledge of specific biochemical et al., 2019). Some of these mechanisms affect components of the processes associated with the disease under investigation. Finally, protein homoeostasis system, resulting in a collapse of protein qual- a comparison with in vitro studies can contribute to deciphering ity control and a further increase in protein misfolding and toxicity the molecular mechanisms of these processes, and represents a (Labbadia and Morimoto, 2015). On the whole, however, the molec- powerful strategy for revealing the molecular origins of a disease, ular mechanisms whereby protein misfolding gives rise to toxicity and for identifying possible targets for therapeutic interventions are not yet understood to the extent needed to enable the develop- (Knowles et al., 2014; Chiti and Dobson, 2017). ment of disease-modifying treatments for the vast majority of Here, we provide an overview of the analysis of protein mis- these disorders. folding in transgenic models of AD and PD based on the trans- In this context, in vivo models can both increase our under- genic expression of Aβ or tau, and of α-synuclein, respectively. standing of the mechanisms of disease and provide a platform We furthermore discuss methodological challenges in developing for drug screening and preclinical development (Dawson et al., biophysical tools to probe in vivo the aggregation process and the 2018; Götz et al., 2018). A variety of model organisms has been resulting aggregate species, which are required to provide quanti- exploited to probe protein misfolding diseases, ranging from sim- tative assessments of cause–effect relationships in disease and of ple unicellular systems such as yeast, to more complex ones such the efficacy of potential therapies. as rodents and primates. Simple organisms lack specific aspects of human biology, but can be faster and more cost-effective to work Determination of protein aggregation mechanisms using with, making them particularly suitable for high-throughput in vitro biophysical methods screens, and for the study of highly conserved cellular pathways (Khurana and Lindquist, 2010; Rincon-Limas et al., 2012; Sin Biophysical studies of protein aggregation in vitro enable the iden- et al., 2014). More complex organisms can recapitulate human tification
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