Investigating the Aggregation of α-synuclein Variants and Their Interactions with a Molecular Tweezer

The protein α-synuclein (α-syn) self-assembles under abnormal conditions into toxic aggregates thought to be a central cause of pathology in neurodegenerative diseases such as Parkinson’s Disease (PD). A promising approach for treating PD is to inhibit the abnormal self-assembly of α-syn in the brain by using small molecules called “molecular tweezers” that the Bitan laboratory has been developing. Molecular tweezers bind to lysine (Lys) residues and prevent both hydrophobic and electrostatic interactions that are key to abnormal self-assembly. The molecular tweezer CLR01 inhibits the self-assembly of α-syn in vitro and in vivo by preferentially binding to Lys at positions 10 and/or 12 and at the region spanning residues 43-58. This leads me to investigate how effective CLR01 is in preventing aggregation when the amino acid at the binding site is substituted, by using Lys to Ala variants of α-syn at positions 43 and 58. I hypothesize that CLR01 will prevent more effectively aggregation in the wild type (WT) than it will in the two variants because essential binding sites will be missing in these variants. As my main experimental method, I use the Thioflavin-T fluorescence assay to measure the amount and kinetics of -pleated sheet formation, which is analogous to α-syn aggregation regardless of the absence or concentration of CLR01. This study will provide insight into the preferred binding site of CLR01 and the behavior of α-syn containing amino acid substitutions and thus will increase our understanding of this important mechanism underlying PD and potentially direct future drug development efforts

Molecular Lysine Tweezers Counteract Aberrant Protein Aggregation.

Molecular tweezers (MTs) are supramolecular host molecules equipped with two aromatic pincers linked together by a spacer (Gakh, 2018). They are endowed with fascinating properties originating from their ability to hold guests between their aromatic pincers (Chen and Whitlock, 1978; Zimmerman, 1991; Harmata, 2004). MTs are finding an increasing number of medicinal applications, e.g., as bis-intercalators for DNA such as the anticancer drug Ditercalinium (Gao et al., 1991), drug activity reverters such as the bisglycoluril tweezers Calabadion 1 (Ma et al., 2012) as well as radioimmuno detectors such as Venus flytrap clusters (Paxton et al., 1991). We recently embarked on a program to create water-soluble tweezers which selectively bind the side chains of lysine and arginine inside their cavity. This unique recognition mode is enabled by a torus-shaped, polycyclic framework, which is equipped with two hydrophilic phosphate groups. Cationic amino acid residues are bound by the synergistic effect of disperse, hydrophobic, and electrostatic interactions in a kinetically fast reversible process. Interactions of the same kind play a key role in numerous protein-protein interactions, as well as in pathologic protein aggregation. Therefore, these particular MTs show a high potential to disrupt such events, and indeed inhibit misfolding and self-assembly of amyloidogenic polypeptides without toxic side effects. The mini-review provides insight into the unique binding mode of MTs both toward peptides and aggregating proteins. It presents the synthesis of the lead compound CLR01 and its control, CLR03. Different biophysical experiments are explained which elucidate and help to better understand their mechanism of action. Specifically, we show how toxic aggregates of oligomeric and fibrillar protein species are dissolved and redirected to form amorphous, benign assemblies. Importantly, these new chemical tools are shown to be essentially non-toxic in vivo. Due to their reversible moderately tight binding, these agents are not protein-, but rather process-specific, which suggests a broad range of applications in protein misfolding events. Thus, MTs are highly promising candidates for disease-modifying therapy in early stages of neurodegenerative diseases. This is an outstanding example in the evolution of supramolecular concepts toward biological application

Aptamers targeting amyloidogenic proteins and their emerging role in neurodegenerative diseases

Aptamers are oligonucleotides selected from large pools of random sequences based on their affinity for bioactive molecules and are used in similar ways to antibodies. Aptamers provide several advantages over antibodies, including their small size, facile, large-scale chemical synthesis, high stability, and low immunogenicity. Amyloidogenic proteins, whose aggregation is relevant to neurodegenerative diseases, such as Alzheimer’s, Parkinson’s, and prion diseases, are among the most challenging targets for aptamer development due to their conformational instability and heterogeneity, the same characteristics that make drug development against amyloidogenic proteins difficult. Recently, chemical tethering of aptagens (equivalent to antigens) and advances in high-throughput sequencing-based analysis have been used to overcome some of these challenges. In addition, internalization technologies using fusion to cellular receptors and extracellular vesicles have facilitated central nervous system (CNS) aptamer delivery. In view of the development of these techniques and resources, here we review antiamyloid aptamers, highlighting preclinical application to CNS therapy

Photo-Induced Cross-Linking of Unmodified Proteins (PICUP) Applied to Amyloidogenic Peptides

The assembly of amyloidogenic proteins into toxic oligomers is a seminal event in the pathogenesis of protein misfolding diseases, including Alzheimer's, Parkinson's, and Huntington's diseases, hereditary amyotrophic lateral sclerosis, and type 2 diabetes. Owing to the metastable nature of these protein assemblies, it is difficult to assess their oligomer size distribution quantitatively using classical methods, such as electrophoresis, chromatography, fluorescence, or dynamic light scattering. Oligomers of amyloidogenic proteins exist as metastable mixtures, in which the oligomers dissociate into monomers and associate into larger assemblies simultaneously. PICUP stabilizes oligomer populations by covalent cross-linking and when combined with fractionation methods, such as sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) or size-exclusion chromatography (SEC), PICUP provides snapshots of the oligomer size distributions that existed before cross-linking. Hence, PICUP enables visualization and quantitative analysis of metastable protein populations and can be used to monitor assembly and decipher relationships between sequence modifications and oligomerization1. Mechanistically, PICUP involves photo-oxidation of Ru2+ in a tris(bipyridyl)Ru(II) complex (RuBpy) to Ru3+ by irradiation with visible light in the presence of an electron acceptor. Ru3+ is a strong one-electron oxidizer capable of abstracting an electron from a neighboring protein molecule, generating a protein radical1,2. Radicals are unstable, highly-reactive species and therefore disappear rapidly through a variety of intra- and intermolecular reactions. A radical may utilize the high energy of an unpaired electron to react with another protein monomer forming a dimeric radical, which subsequently loses a hydrogen atom and forms a stable, covalently-linked dimer. The dimer may then react further through a similar mechanism with monomers or other dimers to form higher-order oligomers. Advantages of PICUP relative to other photo- or chemical cross-linking methods3,4 include short (≤1 s) exposure to non-destructive visible light, no need for pre facto modification of the native sequence, and zero-length covalent cross-linking. In addition, PICUP enables cross-linking of proteins within wide pH and temperature ranges, including physiologic parameters. Here, we demonstrate application of PICUP to cross-linking of three amyloidogenic proteins the 40- and 42-residue amyloid β-protein variants (Aβ40 and Aβ42), and calcitonin, and a control protein, growth-hormone releasing factor (GRF)

CNS-Derived Blood Exosomes as a Promising Source of Biomarkers: Opportunities and Challenges.

Eukaryotic cells release different types of extracellular vesicles (EVs) including exosomes, ectosomes, and microvesicles. Exosomes are nanovesicles, 30-200 nm in diameter, that carry cell- and cell-state-specific cargo of proteins, lipids, and nucleic acids, including mRNA and miRNA. Recent studies have shown that central nervous system (CNS)-derived exosomes may carry amyloidogenic proteins and facilitate their cell-to-cell transfer, thus playing a critical role in the progression of neurodegenerative diseases, such as tauopathies and synucleinopathies. CNS-derived exosomes also have been shown to cross the blood-brain-barrier into the bloodstream and therefore have drawn substantial attention as a source of biomarkers for various neurodegenerative diseases as they can be isolated via a minimally invasive blood draw and report on the biochemical status of the CNS. However, although isolating specific brain-cell-derived exosomes from the blood is theoretically simple and the approach has great promise, practical details are of crucial importance and may compromise the reproducibility and utility of this approach, especially when different laboratories use different protocols. In this review we discuss the role of exosomes in neurodegenerative diseases, the usefulness of CNS-derived blood exosomes as a source of biomarkers for these diseases, and practical challenges associated with the methodology of CNS-derived blood exosomes and subsequent biomarker analysis

RNA aptamers generated against oligomeric Abeta40 recognize common amyloid aptatopes with low specificity but high sensitivity.

Aptamers are useful molecular recognition tools in research, diagnostics, and therapy. Despite promising results in other fields, aptamer use has remained scarce in amyloid research, including Alzheimer's disease (AD). AD is a progressive neurodegenerative disease believed to be caused by neurotoxic amyloid beta-protein (Abeta) oligomers. Abeta oligomers therefore are an attractive target for development of diagnostic and therapeutic reagents. We used covalently-stabilized oligomers of the 40-residue form of Abeta (Abeta40) for aptamer selection. Despite gradually increasing the stringency of selection conditions, the selected aptamers did not recognize Abeta40 oligomers but reacted with fibrils of Abeta40, Abeta42, and several other amyloidogenic proteins. Aptamer reactivity with amyloid fibrils showed some degree of protein-sequence dependency. Significant fibril binding also was found for the naïve library and could not be eliminated by counter-selection using Abeta40 fibrils, suggesting that aptamer binding to amyloid fibrils was RNA-sequence-independent. Aptamer binding depended on fibrillogenesis and showed a lag phase. Interestingly, aptamers detected fibril formation with > or =15-fold higher sensitivity than thioflavin T (ThT), revealing substantial beta-sheet and fibril formation undetected by ThT. The data suggest that under physiologic conditions, aptamers for oligomeric forms of amyloidogenic proteins cannot be selected due to high, non-specific affinity of oligonucleotides for amyloid fibrils. Nevertheless, the high sensitivity, whereby aptamers detect beta-sheet formation, suggests that they can serve as superior amyloid recognition tools

Despite its role in assembly, methionine 35 is not necessary for amyloid β-protein toxicity

Author Manuscript 2011 June 1.An important component of the pathologic process underlying Alzheimer’s disease is oxidative stress. Met[superscript 35] in amyloid β-protein (Aβ) is prone to participating in redox reactions promoting oxidative stress, and therefore is believed to contribute significantly Aβ-induced toxicity. Thus, substitution of Met[superscript 35] by residues that do not participate in redox chemistry would be expected to decrease Aβ toxicity. Indeed, substitution of Met[superscript 35] by norleucine (Nle) was reported to reduce Aβ toxicity. Surprisingly, however, substitution of Met[superscript 35] by Val was reported to increase toxicity. Aβ toxicity is known to be strongly related to its self-assembly. However, neither substitution is predicted to affect Aβ assembly substantially. Thus, the effect of these substitutions on toxicity is difficult to explain. We revisited this issue and compared Aβ40 and Aβ42 with analogs containing Met[superscript 35]→Nle or Met[superscript 35]→Val substitutions using multiple biophysical and toxicity assays. We found that substitution of Met[superscript 35] by Nle or Val had moderate effects on Aβ assembly. Surprisingly, despite these effects, neither substitution changed Aβ neurotoxicity significantly in three different assays. These results suggest that the presence of Met[superscript 35] in Aβ is not important for Aβ toxicity, challenging to the prevailing paradigm, which suggests that redox reactions involving Met35 contribute substantially to Aβ-induced toxicity.Alzheimer's Association (Grant IIRG- 07-5833)National Institutes of Health (U.S.) (Grant AG027818

Biomarkers for parkinsonian disorders in CNS-originating EVs: promise and challenges

Extracellular vesicles (EVs), including exosomes, microvesicles, and oncosomes, are nano-sized particles enclosed by a lipid bilayer. EVs are released by virtually all eukaryotic cells and have been shown to contribute to intercellular communication by transporting proteins, lipids, and nucleic acids. In the context of neurodegenerative diseases, EVs may carry toxic, misfolded forms of amyloidogenic proteins and facilitate their spread to recipient cells in the central nervous system (CNS). CNS-originating EVs can cross the blood–brain barrier into the bloodstream and may be found in other body fluids, including saliva, tears, and urine. EVs originating in the CNS represent an attractive source of biomarkers for neurodegenerative diseases, because they contain cell- and cell state-specific biological materials. In recent years, multiple papers have reported the use of this strategy for identification and quantitation of biomarkers for neurodegenerative diseases, including Parkinson’s disease and atypical parkinsonian disorders. However, certain technical issues have yet to be standardized, such as the best surface markers for isolation of cell type-specific EVs and validating the cellular origin of the EVs. Here, we review recent research using CNS-originating EVs for biomarker studies, primarily in parkinsonian disorders, highlight technical challenges, and propose strategies for overcoming them

Different Amyloid-β Self-Assemblies Have Distinct Effects on Intracellular Tau Aggregation.

Alzheimer's disease (AD) pathology is characterized by the aggregation of beta-amyloid (Aβ) and tau in the form of amyloid plaques and neurofibrillary tangles in the brain. It has been found that a synergistic relationship between these two proteins may contribute to their roles in disease progression. However, how Aβ and tau interact has not been fully characterized. Here, we analyze how tau seeding or aggregation is influenced by different Aβ self-assemblies (fibrils and oligomers). Our cellular assays utilizing tau biosensor cells show that transduction of Aβ oligomers into the cells greatly enhances seeded tau aggregation in a concentration-dependent manner. In contrast, transduced Aβ fibrils slightly reduce tau seeding while untransduced Aβ fibrils promote it. We also observe that the transduction of α-synuclein fibrils, another amyloid protein, has no effect on tau seeding. The enhancement of tau seeding by Aβ oligomers was confirmed using tau fibril seeds derived from both recombinant tau and PS19 mouse brain extracts containing human tau. Our findings highlight the importance of considering the specific form and cellular location of Aβ self-assembly when studying the relationship between Aβ and tau in future AD therapeutic development

Rapid Photochemical Cross-Linking — A New Tool for Studies of Metastable, Amyloidogenic Protein Assemblies

Amyloidoses comprise a class of diseases characterized pathologically by the presence of deposits of fibrillar, aberrantly folded proteins, known as amyloids. Historically, these deposits were considered the key factors causing disease. However, recent evidence suggests that soluble protein oligomers, which are precursors for amyloid fibrils, are the primary toxic effectors responsible for the disease process. Understanding the mechanism by which these oligomers exert their toxicity requires knowledge of the structure, kinetics, and thermodynamics of their formation and conversion into larger assemblies. Such studies have been difficult due to the metastable nature of the oligomers. For the amyloid beta-protein (Abeta), a consensus about the size and relative abundance of small oligomers has not been achieved. We describe here the application of the method Photoinduced Cross-Linking of Unmodified Proteins (PICUP) to the study of Abeta oligomerization. This approach distinguishes oligomerization patterns of amyloidogenic and nonamyloidogenic proteins, allows quantification of each component in oligomer mixtures, and provides a means of correlating primary structure modifications with assembly characteristics. PICUP thus is a powerful tool for the investigation of small, metastable protein oligomers. The method provides essential insights into the factors that control the assembly of pathogenic protein oligomers, facilitating efforts toward the development of therapeutic agents