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Age-related changes in cellular metabolism can affect brain homeostasis, creating conditions that are permissive to the onset and progression of neurodegenerative disorders such as Alzheimer’s and Parkinson’s diseases. Although the roles of metabolites have been extensively studied with regard to cellular signaling pathways, their effects on protein aggregation remain relatively unexplored. By computationally analysing the Human Metabolome Database, we identified two endogenous metabolites, carnosine and kynurenic acid, that inhibit the aggregation of the amyloid beta peptide (Aβ) and rescue a C. elegans model of Alzheimer’s disease. We found that these metabolites act by triggering a cytosolic unfolded protein response through the transcription factor HSF-1 and downstream chaperones HSP40/J-proteins DNJ-12 and DNJ-19. These results help rationalise previous observations regarding the possible anti-ageing benefits of these metabolites by providing a mechanism for their action. Taken together, our findings provide a link between metabolite homeostasis and protein homeostasis, which could inspire preventative interventions against neurodegenerative disorders., Joshi et al. identify two human metabolites, carnosine and kynurenic acid, that rescue a C. elegans model of Alzheimer’s disease by inhibiting the aggregation of the amyloid beta peptide in vivo. They find that these metabolites trigger a cytosolic unfolded protein response through the transcription factor HSF-1 and molecular chaperones DNJ-12 and DNJ-19, thus providing mechanistic links between metabolite homeostasis and protein homeostasis to further insights into interventions against neurodegenerative diseases.

10 pags., 5 figs., The onset and progression of numerous protein misfolding diseases are associated with the presence of oligomers formed during the aberrant aggregation of several different proteins, including amyloid-β (Aβ) in Alzheimer’s disease and α-synuclein (αS) in Parkinson’s disease. These small, soluble aggregates are currently major targets for drug discovery. In this study, we show that trodusquemine, a naturally-occurring aminosterol, markedly reduces the cytotoxicity of αS, Aβ and HypF-N oligomers to human neuroblastoma cells by displacing the oligomers from cell membranes in the absence of any substantial morphological and structural changes to the oligomers. These results indicate that the reduced toxicity results from a mechanism that is common to oligomers from different proteins, shed light on the origin of the toxicity of the most deleterious species associated with protein aggregation and suggest that aminosterols have the therapeutically-relevant potential to protect cells from the oligomer-induced cytotoxicity associated with numerous protein misfolding diseases., This work was supported by the Cambridge Centre for Misfolding Diseases (R.L., B.M., F.S.R., C.K.X., M.P., S.C., S.W.C., J.H., T.K., J.R.K., T.P.J.K., M.V., and C.M.D.), the UK Biotechnology and Biochemical Sciences Research Council (M.V. and C.M.D.), the Wellcome Trust (203249/Z/16/Z to T.P.J.K and M.V.), the Frances and Augustus Newman Foundation (T.P.J.K.), the Regione Toscana – FAS Salute, project SUPREMAL (R.C., A.B., C.C., and F.C.), the Gates Cambridge Trust and St. John’s College Cambridge (R.L.), Darwin College Cambridge (F.S.R.), the Herchel Smith Fund (C.K.X.), a Faculty Development Research Fund grant from the United States Military Academy, West Point (R.L.) and a DTRA Service Academy Research Initiative grant (HDTRA1033862 to R.L.).

16 pags., 6 figs., The self-assembly of α-synuclein (αS) into intraneuronal inclusion bodies is a key characteristic of Parkinson’s disease. To define the nature of the species giving rise to neuronal damage, we have investigated the mechanism of action of the main αS populations that have been observed to form progressively during fibril growth. The αS fibrils release soluble prefibrillar oligomeric species with cross-β structure and solvent-exposed hydrophobic clusters. αS prefibrillar oligomers are efficient in crossing and permeabilize neuronal membranes, causing cellular insults. Short fibrils are more neurotoxic than long fibrils due to the higher proportion of fibrillar ends, resulting in a rapid release of oligomers. The kinetics of released αS oligomers match the observed kinetics of toxicity in cellular systems. In addition to previous evidence that αS fibrils can spread in different brain areas, our in vitro results reveal that αS fibrils can also release oligomeric species responsible for an immediate dysfunction of the neurons in the vicinity of these species., This research was supported by the University of Florence (Fondi Ateneo to F.C. and C.C.), the Ministry of Education, Universities and Research of Italy (Progetto Dipartimento di Eccellenza “Gender Medicine” to C.C.), Parkinson’s UK (G-1508 to S.W.C. and C.M.D.), the Center for Misfolding Diseases of the University of Cambridge (S.W.C. and C.M.D.), the UK Medical Research Council (MR/N000676/1 to C.M.D.), the Agency of Science, Technology and Research of Singapore (to S.W.C.), and the Ministry of Economy and Competitiveness of Spain (MINECO RYC-2012-12068 and MINECO/FEDER EU BFU2015-64119-P to N.C.).

The aggregation of α-synuclein (αS), a protein abundant at presynaptic terminals, is associated with a range of highly debilitating neurodegenerative conditions, including Parkinson’s disease (PD), dementia with Lewy bodies (DLB) and multiple system atrophy (MSA). Emerging evidence indicates that the interaction of αS with lipid membranes defines both its physiological function and pathological effects. The characterisation of the modes of membrane binding by αS is therefore crucial to clarify the balance between normal and aberrant behaviour of this protein. Here we used solid-state nuclear magnetic resonance (ssNMR) spectroscopy to probe the nature of the N-terminally acetylated form of αS (NTAc-αS) bound to synaptic-like lipid vesicles. This post-translational modification is prevalent for the physiological form of αS and modulates the binding to lipid bilayers. By probing the structure, dynamics and membrane topology of NTAc-αS, we found that N-terminal acetylation does not alter significantly the conformational and topological properties of the membrane-bound state of αS, despite increasing its propensity for binding. Taken together, our data and previous characterisations of the cytosolic state of NTAc-αS clarify that the role of the N-terminal acetylation is to regulate the binding affinity of αS for synaptic vesicles without altering the structural properties of the bound state.

International audience; We describe an nMR approach based on the measurement of residual dipolar couplings (RDcs) to probe the structural and motional properties of the dynamic regions of the ribosome. Alignment of intact 70S ribosomes in filamentous bacteriophage enabled measurement of RDCs in the mobile C-terminal domain (CTD) of the stalk protein bL12. A structural refinement of this domain using the observed RDCs did not show large changes relative to the isolated protein in the absence of the ribosome, and we also found that alignment of the ctD was almost independent of the presence of the core ribosome particle, indicating that the inter-domain linker has significant flexibility. The nature of this linker was subsequently probed in more detail using a paramagnetic alignment strategy, which revealed partial propagation of alignment between neighbouring domains, providing direct experimental validation of a structural ensemble previously derived from SAXS and NMR relaxation measurements. Our results demonstrate the prospect of better characterising dynamical and functional regions of more challenging macromolecular machines and systems, for example ribosome-nascent chain complexes. In recent years, X-ray crystallography and cryo-electron microscopy (cryo-EM) have elucidated the details of high-resolution structures of ribosomes, revealing intricate mechanistic information about their function during the translation process 1,2. In parallel, NMR-based observations of nascent polypeptide chains emerging from the ribosome are providing unique structural and mechanistic insights into co-translational folding processes 3-5. In order to develop further solution-state NMR spectroscopy as a technique for structural studies of dynamic regions within large complexes, we have explored the measurement of residual dipolar couplings (RDCs) within intact ribosomes, focusing in particular on the mobile bL12 protein from the GTPase-associated region (GAR) of the prokaryotic 70S ribosome. RDCs have been used to characterise other macromolecular machines and assemblies, including HIV-1 capsid protein, bacterial Enzyme I, and the 20S proteasome 6-8. These developments are particularly relevant as macromolecular complexes tend to exhibit a wide variety of functional motions that are challenging to characterise by methods such as X-ray crystallography or cryoelectron microscopy. The GAR is a highly conserved region of both prokaryotic and eukaryotic ribosomes, and is so named to reflect its role in both the recruitment and the stimulation of the GTPase activity of several auxiliary factors associated with the key steps of protein synthesis: initiation (initiation factor 2, IF2), elongation (elongation factors EF-Tu and EF-G) and termination (release factor 3, RF3) 9. The prokaryotic GAR includes helices 42-44 and 95 of the 23S rRNA, and the ribosomal proteins bL10, bL11 and bL12 (Fig. 1a). bL12, the focus of the present work, is a 120 residue dimeric protein consisting of an N-terminal dimerisation domain (NTD), which binds to the extended bL10 helix of the core ribosome particle, and a C-terminal domain (CTD), which interacts with GTPase substrates to facilitate their recruitment to the ribosome. The bL12 CTD interacts with four major translational GTPases, IF2, EF-Tu, EF-G and RF3, through a highly conserved and positively charged binding site in helices 4 and 5, identified through NMR mapping and mutagenesis 10-14. GTPase binding has been found by mutagenesis to occur through the G4-G5 helix in the G domain of IF2 (and, by homology, likely also via the G domain in other GTPases), which presents a complementary negatively charged surface and results in extremely rapid binding driven by favourable electrostatics 14. The CTD is mobile in solution, being separated from the NTD by a flexible

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-β (Aβ) 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.

A small molecule binds to a disordered protein in its monomeric form, preventing its aggregation linked to Alzheimer’s disease., 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-β (Aβ) peptide in its monomeric, soluble state. Our analysis reveals that this compound interacts with Aβ 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 Aβ while decreasing its hydrophobic surface area. We also show that it rescues a Caenorhabditis elegans model of Aβ-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.

The aggregates of the Aβ peptide associated with Alzheimer's disease are able to both grow in size as well as generate, through secondary nucleation, new small oligomeric species, that are major cytotoxins associated with neuronal death. Despite the importance of these amyloid fibril-dependent processes, their structural and molecular underpinnings have remained challenging to elucidate. Here, we consider two molecular chaperones: the Brichos domain, which suppresses specifically secondary nucleation processes, and clusterin which our results show is capable of inhibiting, specifically, the elongation of Aβ fibrils at remarkably low substoichiometric ratios. Microfluidic diffusional sizing measurements demonstrate that this inhibition originates from interactions of clusterin with fibril ends with high affinity. Kinetic experiments in the presence of both molecular chaperones reveal that their inhibitory effects are additive and noncooperative, thereby indicating that the reactive sites associated with the formation of new aggregates and the growth of existing aggregates are distinct.

Engineered bacteria enable the screening of vast molecular libraries for discovering inhibitors of pathogenic protein aggregation., Protein misfolding and aggregation are associated with a many human disorders, including Alzheimer’s and Parkinson’s diseases. Toward increasing the effectiveness of early-stage drug discovery for these conditions, we report a bacterial platform that enables the biosynthesis of molecular libraries with expanded diversities and their direct functional screening for discovering protein aggregation inhibitors. We illustrate this approach by performing, what is to our knowledge, the largest functional screen of small-size molecular entities described to date. We generated a combinatorial library of ~200 million drug-like, cyclic peptides and rapidly screened it for aggregation inhibitors against the amyloid-β peptide (Aβ42), linked to Alzheimer’s disease. Through this procedure, we identified more than 400 macrocyclic compounds that efficiently reduce Aβ42 aggregation and toxicity in vitro and in vivo. Finally, we applied a combination of deep sequencing and mutagenesis analyses to demonstrate how this system can rapidly determine structure-activity relationships and define consensus motifs required for bioactivity.

Protein aggregation is a complex process resulting in the formation of heterogeneous mixtures of aggregate populations that are closely linked to neurodegenerative conditions, such as Alzheimer’s disease. Here, we find that soluble aggregates formed at different stages of the aggregation process of amyloid beta (Aβ42) induce the disruption of lipid bilayers and an inflammatory response to different extents. Further, by using gradient ultracentrifugation assay, we show that the smaller aggregates are those most potent at inducing membrane permeability and most effectively inhibited by antibodies binding to the C-terminal region of Aβ42. By contrast, we find that the larger soluble aggregates are those most effective at causing an inflammatory response in microglia cells and more effectively inhibited by antibodies targeting the N-terminal region of Aβ42. These findings suggest that different toxic mechanisms driven by different soluble aggregated species of Aβ42 may contribute to the onset and progression of Alzheimer’s disease., Amyloid beta (Aβ42) peptides form heterogeneous mixtures of aggregates, which are closely linked to Alzheimer’s disease. This study shows how different types of Aβ42 aggregates are associated with distinct mechanisms of toxicity

This study is supported by the Michael J. Fox Foundation (10200); The Royal Society with a University Research Fellowship (UF120277) (S.F.L.); a Marie‐Curie Individual Fellowship (S.D.); the Boehringer Ingelheim Fonds (P.F.), the Studienstiftung des Deutschen Volkes (P.F.); the UK Biotechnology and Biological Sciences Research Council (C.M.D.); the Wellcome Trust (C.M.D.); the Cambridge Centre for Misfolding Diseases (P.F. and C.M.D.), and the Royal Society and the European Research Council with an ERC Advanced Grant (669237) (D.K.). Small aggregates of misfolded proteins play a key role in neurodegenerative disorders. Such species have proved difficult to study due to the lack of suitable methods capable of resolving these heterogeneous aggregates, which are smaller than the optical diffraction limit. We demonstrate here an all-optical fluorescence microscopy method to characterise the structure of individual protein aggregates based on the fluorescence anisotropy of dyes such as thioflavin-T, and show that this technology is capable of studying oligomers in human biofluids such as cerebrospinal fluid. We first investigated in vitro the structural changes in individual oligomers formed during the aggregation of recombinant α-synuclein. By studying the diffraction-limited aggregates we directly evaluated their structural conversion and correlated this with the potential of aggregates to disrupt lipid bilayers. We finally characterised the structural features of aggregates present in cerebrospinal fluid of Parkinson's disease patients and age-matched healthy controls. Publisher PDF

5 pags, 3 figs, Oligomeric species populated during the aggregation process of a-synuclein have been linked to neuronal impairment in Parkinson's disease and related neurodegenerative disorders. By using solution and solid-state nuclear magnetic resonance techniques in conjunction with other structural methods, we identified the fundamental characteristics that enable toxic a-synuclein oligomers to perturb biological membranes and disrupt cellular function; these include a highly lipophilic element that promotes strong membrane interactions and a structured region that inserts into lipid bilayers and disrupts their integrity. In support of these conclusions, mutations that target the region that promotes strong membrane interactions by a-synuclein oligomers suppressed their toxicity in neuroblastoma cells and primary cortical neurons., This research was supported by Parkinson's UK (G-1508 to G.F., M.V., C.M.D., and A.D.); the UK Medical Research Council (MR/N000676/1 to G.F., M.V. C.M.D., and A.D.); the Wellcome Trust (104933/2/14E to kD.): the Leverhulme Trust (RPG-2015-350 to A.D. and RPG-2015-345 to L.Y.); the British Heart Foundation (PG/14/93/31237 to A.D. and PG/11/81/29130 to L.Y.); the UK Biotechnology and Biological Sciences Research Council (BB/M023923/1 to A.D. and BB/G00594X/1 to L.Y.); the Agency of Science, Technology and Research of Singapore (to S.W.C.); the Ministry of Economy, Industry, and Competitiveness of Spain (MINECO RYC-2012-12068 and MINECO/FEDER EU BEU2015-64119-P to N.C.); the Regione Toscana (SUPREMAL to E.C., C.C., and R.C); the University of Florence (Eondi di Ateneo to E.C. and. C.C); and the Centre for Misfolding Diseases of the University of Cambridge. The data supporting the findings of this study are available within the article and supplementary materials.