Moran Frenkel-Pinter

Inhibiting α-Synuclein Oligomerization by Stable Cell-Penetrating β-Synuclein Fragments Recovers Phenotype of Parkinson's Disease Model Flies

The intracellular oligomerization of α-synuclein is associated with Parkinsons disease and appears to be an important target for disease-modifying treatment. Yet, to date, there is no specific inhibitor for this aggregation process. Using unbiased systematic peptide array analysis, we indentified molecular interaction domains within the β-synuclein polypeptide that specifically binds α-synuclein. Adding such peptide fragments to α-synuclein significantly reduced both amyloid fibrils and soluble oligomer formation in vitro. A retro-inverso analogue of the best peptide inhibitor was designed to develop the identified molecular recognition module into a drug candidate. While this peptide shows indistinguishable activity as compared to the native peptide, it is stable in mouse serum and penetrates α-synuclein over-expressing cells. The interaction interface between the D-amino acid peptide and α-synuclein was mapped by Nuclear Magnetic Resonance spectroscopy. Finally, administering the retro-inverso peptide to a Drosophila model expressing mutant A53T α-synuclein in the nervous system, resulted in a significant recovery of the behavioral abnormalities of the treated flies and in a significant reduction in α-synuclein accumulation in the brains of the flies. The engineered retro-inverso peptide can serve as a lead for developing a novel class of therapeutic agents to treat Parkinsons disease.

A Blood-Brain Barrier (BBB) Disrupter Is Also a Potent α-Synuclein (α-syn) Aggregation Inhibitor: A NOVEL DUAL MECHANISM OF MANNITOL FOR THE TREATMENT OF PARKINSON DISEASE (PD)*

Background: α-syn aggregation is a main pathology of PD. Results: Mannitol interferes with α-syn aggregation in vitro and in vivo, whereas no adverse effects were observed in control animals. Conclusion: In addition to its BBB-disrupting properties, mannitol, a chemical chaperon, may serve as a potential drug. Significance: mannitol may serve as a basis for a dual mechanism therapeutic agent for treating PD. The development of disease-modifying therapy for Parkinson disease has been a main drug development challenge, including the need to deliver the therapeutic agents to the brain. Here, we examined the ability of mannitol to interfere with the aggregation process of α-synuclein in vitro and in vivo in addition to its blood-brain barrier-disrupting properties. Using in vitro studies, we demonstrated the effect of mannitol on α-synuclein aggregation. Although low concentration of mannitol inhibited the formation of fibrils, high concentration significantly decreased the formation of tetramers and high molecular weight oligomers and shifted the secondary structure of α-synuclein from α-helical to a different structure, suggesting alternative potential pathways for aggregation. When administered to a Parkinson Drosophila model, mannitol dramatically corrected its behavioral defects and reduced the amount of α-synuclein aggregates in the brains of treated flies. In the mThy1-human α-synuclein transgenic mouse model, a decrease in α-synuclein accumulation was detected in several brain regions following treatment, suggesting that mannitol promotes α-synuclein clearance in the cell bodies. It appears that mannitol has a general neuroprotective effect in the transgenic treated mice, which includes the dopaminergic system. We therefore suggest mannitol as a basis for a dual mechanism therapeutic agent for the treatment of Parkinson disease.

Enhanced bioavailability of polyaromatic hydrocarbons in the form of mucin complexes.

Increasing exposure of biological systems to large amounts of polycyclic aromatic hydrocarbons is of great public concern. Organisms have an array of biological defense mechanisms, and it is believed that mucosal gel (which covers the respiratory system, the gastrointestinal tract, etc.) provides an effective chemical shield against a range of toxic materials. However, in this work, we demonstrate, for the first time, that, upon complexation of polyaromatic hydrocarbons with mucins, enhanced bioavailability and, therefore, toxicity are obtained. This work was aimed to demonstrate how complexation of various highly hydrophobic polycyclic aromatic hydrocarbons with representative mucin glycoprotein could lead to the formation of previously undescribed materials, which exhibit increased toxicity versus pristine polycyclic aromatic hydrocarbons. In the present work, we show that a representative mucin glycoprotein, bovine submaxillary mucin, has impressive and unprecedented capabilities of binding and solubilizing water-insoluble materials in physiological solution. The complexes formed between the mucin and a series of polycyclic aromatic hydrocarbons were comprehensively characterized, and their toxicity was evaluated by both in vivo and in vitro assays. In addition, the bioavailability and membrane-penetration capabilities were tested using an internalization assay. Our results provide, for the first time, evidence of an unknown route by which hydrophobic materials may achieve higher bioavailability, penetrating some of the biological defense systems, in the form of water-soluble complexes with mucosal proteins.

Selective Inhibition of Aggregation and Toxicity of a Tau-Derived Peptide using Its Glycosylated Analogues.

Protein glycosylation is a ubiquitous post-translational modification that regulates the folding and function of many proteins. Misfolding of protein monomers and their toxic aggregation are the hallmark of many prevalent diseases. Thus, understanding the role of glycans in protein aggregation is highly important and could contribute both to unraveling the pathology of protein misfolding diseases as well as providing a means for modifying their course for therapeutic purposes. Using β-O-linked glycosylated variants of the highly studied Tau-derived hexapeptide motif VQIVYK, which served as a simplified amyloid model, we demonstrate that amyloid formation and toxicity can be strongly attenuated by a glycan unit, depending on the nature of the glycan itself. Importantly, we show for the first time that not only do glycans hinder self-aggregation, but the glycosylated peptides are capable of inhibiting aggregation of the non-modified corresponding amyloid scaffold.

Naphthoquinone-Tryptophan Hybrid Inhibits Aggregation of the Tau-Derived Peptide PHF6 and Reduces Neurotoxicity.

Tauopathies, such as Alzheimers disease (AD), are a group of disorders characterized neuropathologically by intracellular toxic accumulations of abnormal protein aggregates formed by misfolding of the microtubule-associated protein tau. Since protein self-assembly appears to be an initial key step in the pathology of this group of diseases, intervening in this process can be both a prophylactic measure and a means for modifying the course of the disease for therapeutic purposes. We and others have shown that aromatic small molecules can be effective inhibitors of aggregation of various protein assemblies, by binding to the aromatic core in aggregation-prone motifs and preventing their self-assembly. Specifically, we have designed a series of small aromatic naphthoquinone-tryptophan hybrid molecules as candidate aggregation inhibitors of β -sheet based assembly and demonstrated their efficacy toward inhibiting aggregation of the amyloid-β peptide, another culprit of AD, as well as of various other aggregative proteins involved in other protein misfolding diseases. Here we tested whether a leading naphthoquinone-tryptophan hybrid molecule, namely NQTrp, can be repurposed as an inhibitor of the aggregation of the tau protein in vitro and in vivo. We show that the molecule inhibits the in vitro assembly of PHF6, the aggregation-prone fragment of tau protein, reduces hyperphosphorylated tau deposits and ameliorates tauopathy-related behavioral defect in an established transgenic Drosophila model expressing human tau. We suggest that NQTrp, or optimized versions of it, could act as novel disease modifying drugs for AD and other tauopathies.

A Novel, Sensitive Assay for Behavioral Defects in Parkinson's Disease Model Drosophila

Parkinsons disease is a common neurodegenerative disorder with the pathology of α-synuclein aggregation in Lewy bodies. Currently, there is no available therapy that arrests the progression of the disease. Therefore, the need of animal models to follow α-synuclein aggregation is crucial. Drosophila melanogaster has been researched extensively as a good genetic model for the disease, with a cognitive phenotype of defective climbing ability. The assay for climbing ability has been demonstrated as an effective tool for screening new therapeutic agents for Parkinsons disease. However, due to the assays many limitations, there is a clear need to develop a better behavioral test. Courtship, a stereotyped, ritualized behavior of Drosophila, involves complex motor and sensory functions in both sexes, which are controlled by large number of neurons; hence, behavior observed during courtship should be sensitive to disease processes in the nervous system. We used a series of traits commonly observed in courtship and an additional behavioral trait—nonsexual encounters—and analyzed them using a data mining tool. We found defective behavior of the Parkinsons model male flies that were tested with virgin females, visible at a much younger age than the climbing defects. We conclude that this is an improved behavioral assay for Parkinsons model flies.

Cl-NQTrp Alleviates Tauopathy Symptoms in a Model Organism through the Inhibition of Tau Aggregation-Engendered Toxicity

Alzheimers disease (AD) is the most abundant tauopathy and is characterized by Aβ-derived plaques and tau-derived tangles, resulting from the unfolding of the corresponding monomeric subunits into ordered β-sheet oligomers and fibrils. Intervening in the toxic aggregation process is a promising therapeutic approach, but, to date, a disease-modifying therapy is neither available for AD nor for other tauopathies. Along these lines, we have previously demonstrated that a small naphthoquinone-tryptophan hybrid, termed NQTrp, is an effective modulator of tauopathy in vitro and in vivo. However, NQTrp is difficult to synthesize and is not very stable. Therefore, we tested whether a more stable and easier-to-synthesize modified version of NQTrp, containing a Cl ion, namely Cl-NQTrp, is also an effective inhibitor of tau aggregation in vitro and in vivo. Cl-NQTrp was previously shown to efficiently inhibit the aggregation of various amyloidogenic proteins and peptides. We demonstrate that Cl-NQTrp inhibits the in vitro assembly of PHF6, the aggregation-prone fragment of tau, and alleviates tauopathy symptoms in a transgenic Drosophila model through the inhibition of tau aggregation-engendered toxicity. These results suggest that Cl-NQTrp could be a unique potential therapeutic for AD since it targets aggregation of both Aβ and tau.

Interplay between protein glycosylation pathways in Alzheimer’s disease

Glycome analysis of Alzheimer’s patients reveals interplay between glycosylation pathways and suggests novel biomarkers. Deviations from the normal nucleoplasmic protein O-GlcNAcylation, as well as from normal protein sialylation and N-glycosylation in the secretory pathway, have been reported in Alzheimer’s disease (AD). However, the interplay between the cytoplasmic protein O-GlcNAcylation and the secretory N-/O-glycosylation in AD has not been described. We present a comprehensive analysis of the N-, O-, and O-GlcNAc–glycomes in AD-affected brain regions as well as in AD patient serum. We detected marked differences in levels of glycan involved in both protein O-GlcNAcylation and N-/O-glycosylation between patients and healthy individuals and revealed brain region–specific glycosylation-related pathology in patients. These alterations are not general for other neurodegenerative conditions, such as frontotemporal dementia and corticobasal degeneration. The alterations in the AD glycome in the serum could potentially lead to novel glyco-based biomarkers for AD progression. Strikingly, negative interrelationship was found between the pathways of protein O-GlcNAcylation and N-/O-glycosylation, suggesting a novel intracellular cross-talk.

Inhibition of the Aggregation and Toxicity of the Minimal Amyloidogenic Fragment of Tau by Its Pro-Substituted Analogues

Inhibiting the toxic aggregation of amyloid-β and the tau protein, the key pathological agents involved in Alzheimers, is a leading approach in modulating disease progression. Using an aggregative tau-derived model peptide, Ac-PHF6-NH2 , the substitution of its amino acids with proline, a known efficient β-breaker, is shown to reduce self-assembly. This effect is attributed to the steric hindrance created by the proline substitution, which results in disruption of the β-sheet formation process. Moreover, several of the proline-substituted peptides inhibit the aggregation of Ac-PHF6-NH2 amyloidogenic peptide. Two of these modified inhibitors also disassemble pre-formed Ac-PHF6-NH2 fibrils and one inhibits induced cytotoxicity of the fibrils. Taken together, these lead β-breaker peptides may be developed into novel Alzheimers disease therapeutics.

Distinct Effects of O-GlcNAcylation and Phosphorylation of a Tau-Derived Amyloid Peptide on Aggregation of the Native Peptide

Protein phosphorylation and O-GlcNAcylation are very common nucleoplasmic post-translational modifications. Mono-addition of either the phosphate or the O-GlcNAc group were shown to inhibit the self-aggregation of amyloidogenic proteins and peptides, which is the hallmark of various protein misfolding diseases. However, their comparable effect upon co-incubation with a native non-modified amyloid scaffold has not been reported. O-linked glycans and phosphate variants of the tau protein-derived VQIVYK hexapeptide motif were generated as a simplified amyloid scaffold model and demonstrate that, while self-aggregation can be attenuated by either a single glycan or a phosphate unit, only co-incubation with the O-GlcNAc variant inhibits aggregation of the native peptide. These results shed light on the role of post-translational modifications in protein aggregation and suggest a novel therapeutic approach to protein misfolding diseases.