Benedetta Mannini

Systematic development of small molecules to inhibit specific microscopic steps of Aβ42 aggregation in Alzheimer’s disease

Significance The absence of fully reproducible protein aggregation assays has contributed to the systematic failures in clinical trials for Alzheimer’s disease (AD) of compounds targeting the aggregation process of the amyloid-β peptide (Aβ). To address this problem, we report the identification of a library of compounds against Aβ aggregation using a drug discovery strategy based on highly quantitative aggregation rate measurements. We then demonstrate, both in Caenorhabditis elegans and human cerebrospinal fluid, that this approach can systematically provide a rich variety of related small molecules to take forward into a drug discovery process. We therefore report an approach that should substantially help overcome the very high level of attrition associated with drug discovery programs for AD. The aggregation of the 42-residue form of the amyloid-β peptide (Aβ42) is a pivotal event in Alzheimer’s disease (AD). The use of chemical kinetics has recently enabled highly accurate quantifications of the effects of small molecules on specific microscopic steps in Aβ42 aggregation. Here, we exploit this approach to develop a rational drug discovery strategy against Aβ42 aggregation that uses as a read-out the changes in the nucleation and elongation rate constants caused by candidate small molecules. We thus identify a pool of compounds that target specific microscopic steps in Aβ42 aggregation. We then test further these small molecules in human cerebrospinal fluid and in a Caenorhabditis elegans model of AD. Our results show that this strategy represents a powerful approach to identify systematically small molecule lead compounds, thus offering an appealing opportunity to reduce the attrition problem in drug discovery.

SERS Detection of Amyloid Oligomers on Metallorganic-Decorated Plasmonic Beads

Protein misfolded proteins are among the most toxic endogenous species of macromolecules. These chemical entities are responsible for neurodegenerative disorders such as Alzheimers, Parkinsons, Creutzfeldt-Jakobs and different non-neurophatic amyloidosis. Notably, these oligomers show a combination of marked heterogeneity and low abundance in body fluids, which have prevented a reliable detection by immunological methods so far. Herein we exploit the selectivity of proteins to react with metallic ions and the sensitivity of surface-enhanced Raman spectroscopy (SERS) toward small electronic changes in coordination compounds to design and engineer a reliable optical sensor for protein misfolded oligomers. Our strategy relies on the functionalization of Au nanoparticle-decorated polystyrene beads with an effective metallorganic Raman chemoreceptor, composed by Al(3+) ions coordinated to 4-mercaptobenzoic acid (MBA) with high Raman cross-section, that selectively binds aberrant protein oligomers. The mechanical deformations of the MBA phenyl ring upon complexation with the oligomeric species are registered in its SERS spectrum and can be quantitatively correlated with the concentration of the target biomolecule. The SERS platform used here appears promising for future implementation of diagnostic tools of aberrant species associated with protein deposition diseases, including those with a strong social and economic impact, such as Alzheimers and Parkinsons diseases.

Chaperones as Suppressors of Protein Misfolded Oligomer Toxicity

Chaperones have long been recognized to play well defined functions such as to: (i) assist protein folding and promote formation and maintenance of multisubunit complexes; (ii) mediate protein degradation; (iii) inhibit protein aggregation; and (iv) promote disassembly of undesired aberrant protein aggregates. In addition to these well-established functions, it is increasingly clear that chaperones can also interact with aberrant protein aggregates, such as pre-fibrillar oligomers and fibrils, and inhibit their toxicity commonly associated with neurodegenerative diseases without promoting their disassembly. In particular, the evidence collected so far in different labs, exploiting different experimental approaches and using different chaperones and client aggregated proteins, indicates the existence of two distinct mechanisms of action mediated by the chaperones to neutralize the toxicity of aberrant proteins oligomers: (i) direct binding of the chaperones to the hydrophobic patches exposed on the oligomer/fibril surface, with resulting shielding or masking of the moieties responsible for the aberrant interactions with cellular targets; (ii) chaperone-mediated conversion of aberrant protein aggregates into large and more innocuous species, resulting in a decrease of their surface-to-volume ratio and diffusibility and in deposits more easily manageable by clearance mechanisms, such as autophagy. In this review article we will describe the in vitro and in vivo evidence supporting both mechanisms and how this results in a suppression of the detrimental effects caused by protein misfolded aggregates.

Effect of molecular chaperones on aberrant protein oligomers in vitro: Super-versus sub-stoichiometric chaperone concentrations

Abstract Living systems protect themselves from aberrant proteins by a network of chaperones. We have tested in vitro the effects of different concentrations, ranging from 0 to 16 μm, of two molecular chaperones, namely αB-crystallin and clusterin, and an engineered monomeric variant of transthyretin (M-TTR), on the morphology and cytotoxicity of preformed toxic oligomers of HypF-N, which represent a useful model of misfolded protein aggregates. Using atomic force microscopy imaging and static light scattering analysis, all were found to bind HypF-N oligomers and increase the size of the aggregates, to an extent that correlates with chaperone concentration. SDS-PAGE profiles have shown that the large aggregates were predominantly composed of the HypF-N protein. ANS fluorescence measurements show that the chaperone-induced clustering of HypF-N oligomers does not change the overall solvent exposure of hydrophobic residues on the surface of the oligomers. αB-crystallin, clusterin and M-TTR can diminish the cytotoxic effects of the HypF-N oligomers at all chaperone concentration, as demonstrated by MTT reduction and Ca2+ influx measurements. The observation that the protective effect is primarily at all concentrations of chaperones, both when the increase in HypF-N aggregate size is minimal and large, emphasizes the efficiency and versatility of these protein molecules.

Delivery of Native Proteins into C. elegans Using a Transduction Protocol Based on Lipid Vesicles

The nematode worm Caenorhabditis elegans (C. elegans) is a versatile and widely used animal model for in vivo studies of a broad range of human diseases, in particular for understanding their genetic origins and for screening drug candidates. Nevertheless, the challenges associated with the administration of native proteins to C. elegans have limited the range of applications of this animal model in protein-based drug discovery programs. Here, we describe a readily usable protocol for the transduction of native proteins in C. elegans, which is based on the encapsulation of the proteins of interest within cationic lipid vesicles, prior to their administration to worms. This procedure limits the degradation of the proteins in the guts of the animals, and promotes their adsorption into body tissues. To illustrate the efficacy of this approach we apply it to deliver an antibody designed to inhibit α-synuclein aggregation, and show that it can lead to the rescue of the disease phenotype in a C. elegans model of Parkinson’s disease. As this transduction protocol is fast and inexpensive, we anticipate that it will be readily applicable to protein-based drug discovery studies that utilize C. elegans as a model organism.

Multistep Inhibition of α-Synuclein Aggregation and Toxicity in Vitro and in Vivo by Trodusquemine

The aggregation of α-synuclein, an intrinsically disordered protein that is highly abundant in neurons, is closely associated with the onset and progression of Parkinsons disease. We have shown previously that the aminosterol squalamine can inhibit the lipid induced initiation process in the aggregation of α-synuclein, and we report here that the related compound trodusquemine is capable of inhibiting not only this process but also the fibril-dependent secondary pathways in the aggregation reaction. We further demonstrate that trodusquemine can effectively suppress the toxicity of α-synuclein oligomers in neuronal cells, and that its administration, even after the initial growth phase, leads to a dramatic reduction in the number of α-synuclein inclusions in a Caenorhabditis elegans model of Parkinsons disease, eliminates the related muscle paralysis, and increases lifespan. On the basis of these findings, we show that trodusquemine is able to inhibit multiple events in the aggregation process of α-synuclein and hence to provide important information about the link between such events and neurodegeneration, as it is initiated and progresses. Particularly in the light of the previously reported ability of trodusquemine to cross the blood-brain barrier and to promote tissue regeneration, the present results suggest that this compound has the potential to be an important therapeutic candidate for Parkinsons disease and related disorders.

Toxic HypF-N Oligomers Selectively Bind the Plasma Membrane to Impair Cell Adhesion Capability

The deposition of fibrillar protein aggregates in human organs is the hallmark of several pathological states, including highly debilitating neurodegenerative disorders and systemic amyloidoses. It is widely accepted that small oligomers arising as intermediates in the aggregation process, released by fibrils, or growing in secondary nucleation steps are the cytotoxic entities in protein-misfolding diseases, notably neurodegenerative conditions. Increasing evidence indicates that cytotoxicity is triggered by the interaction between nanosized protein aggregates and cell membranes, even though little information on the molecular details of such interaction is presently available. In this work, we propose what is, to our knowledge, a new approach, based on the use of single-cell force spectroscopy applied to multifunctional substrates, to study the interaction between protein oligomers, cell membranes, and/or the extracellular matrix. We compared the interaction of single Chinese hamster ovary cells with two types of oligomers (toxic and nontoxic) grown from the N-terminal domain of the Escherichia coli protein HypF. We were able to quantify the affinity between both oligomer type and the cell membrane by measuring the mechanical work needed to detach the cells from the aggregates, and we could discriminate the contributions of the membrane lipid and protein fractions to such affinity. The fundamental role of the ganglioside GM1 in the membrane-oligomers interaction was also highlighted. Finally, we observed that the binding of toxic oligomers to the cell membrane significantly affects the functionality of adhesion molecules such as Arg-Gly-Asp binding integrins, and that this effect requires the presence of the negatively charged sialic acid moiety of GM1.

TARGETING AMYLOID FORMATION USING RATIONALLY DESIGNED ANTIBODIES

mutations within the Ab amino acid sequence for its aggregation. Methods:We used cryo-electron microscopy, solid state NMR spectroscopy and x-ray diffraction to obtain high resolution structural information of fibrils grown from recombinantly expressed Ab(142). Results: We determined the structure of an Ab(1-42) fibril composed of two intertwined protofilaments determined by cryoelectron microscopy (cryo-EM) to 4.0Angstrom resolution, complemented by solid-state nuclear magnetic resonance experiments [1]. The backbone of all 42 residues and nearly all side chains are well resolved in the EM density map, including the entire N terminus, which is part of the cross-b structure resulting in an overall “LS”-shaped topology of individual subunits. The dimer interface protects the hydrophobic C termini from the solvent. The characteristic staggering of the nonplanar subunits results in markedly different fibril ends, termed “groove” and “ridge,” leading to different binding pathways on both fibril ends, which has implications for fibril growth. The b strands are staggered with relation to one another in a zipper-like manner. At both fibril ends, the binding site for the addition of subunit i contains contributions of subunits i1, i-2, i-3, i-4, and i-5, or i+1, i+2, i+3, i+4, and i+5, respectively. Therefore, five Ab(1-42) subunits are required to provide the full interface for monomer addition. For a fragment of six subunits, the capping subunits would have the same full contact interface as those in an extended fibril. We define this structural element of six subunits as the minimal fibril unit. This minimal fibril unit may also be the minimal seed size for nucleation as suggested by results from Ab aggregation studies monitored by small angle neutron scattering and analytical ultracentrifugation. Conclusions: High resolution structural details help to understand the role of amino acid side chains within Ab for fibril formation and growth. [1] Gremer et al., Science 358, 116–119 (2017).

Stabilization and Characterization of Cytotoxic Aβ40 Oligomers Isolated from an Aggregation Reaction in the Presence of Zinc Ions

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 Alzheimers Aβ40 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 homogeneous population with small size, high cross-β 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 Aβ40 toxicity in vitro and in cellular and animal models of Alzheimers disease.