Active Immunization Against hIAPP Oligomers Ameliorates the Diabetes- Associated Phenotype in a Transgenic Mice Model.

Type 2 diabetes is characterized by insulin tolerance in target cells followed by a reduction of pancreatic ß-cell mass. Islet amyloid polypeptide oligomeric assemblies were shown to contribute to ß-cell apoptosis by forming discrete pores that destabilize the cellular membrane. We previously characterized α-helical cytotoxic islet amyloid polypeptide oligomers which interact with cell membranes, following a complete internalization that leads to cellular apoptosis. Moreover, antibodies which bind the oligomers and neutralize the cytotoxicity were exclusively identified in the serum of type 2 diabetes patients. Here, we examined the usage of the newly characterized oligomers as an active immunization agent targeting amyloid self- assembly in a diabetes-associated phenotype transgenic mice model. Immunized transgenic mice showed an increase in hIAPP-antibody serum titer as well as improvement in diabetes-associated parameters. Lower fasting blood glucose levels, higher insulin, and lower islet amyloid polypeptide accumulation were observed. Furthermore, antibodies derived from the immunized mice reduced hIAPP oligomers cytotoxicity towards ß-cells in a dose-dependent manner. This study highlights the significance of targeting the early amyloid self-assembly events for potential disease management. Furthermore, it demonstrates that α-helical oligomers conformers are valid epitope for the development of future immunization therapy.

Single cell imaging and quantification of TDP-43 and α-synuclein intercellular propagation.

The intercellular spreading of protein assemblies is a major factor in the progression of neurodegenerative disorders. The quantitative study and visualization of cell-to-cell propagation using tagged-proteins is challenging due to the steric effect of relatively large fluorescence tags and the risk of 'false positive' identification when analyzing these rare transmission events. Here, we established a cell culture model to characterize the cell-to-cell transmission of TAR DNA-binding protein and α-synuclein, involved in amyotrophic lateral sclerosis and Parkinson's disease, respectively, using the small nine amino acid influenza hemagglutinin tag. The novel use of single cell resolution imaging flow cytometry allowed the visualization and quantification of all individual transmission events. Cell-level analysis of these events indicated that the degree of transfer is lower than previously reported based on conventional flow cytometry. Furthermore, our analysis can exclude 'false positive' events of cellular overlap and extracellular debris attachment. The results were corroborated by high-resolution confocal microscopy mapping of protein localization.

Total proteome turbidity assay for tracking global protein aggregation in the natural cellular environment.

Proteome homeostasis is crucial for optimal cellular function and survival in the face of various stressful impacts. This entails preservation of a balance between protein synthesis, folding, degradation, and trafficking collectively termed proteostasis. A hallmark of proteostasis failure, which underlies various diseases, is enhanced misfolding and aggregation of proteins. Here we adapted the measurement of protein turbidity, which is commonly used to evaluate aggregation of single purified proteins, for monitoring propensity for aggregation of the entire soluble cellular proteome incubated in vitro for several hours. We show that over-expression of an aggregation-prone protein or applying endoplasmic-reticulum (ER) stress to either cells in culture or to the intact organism, Drosophila, enhances the rise in turbidity of the global soluble proteome compared to untreated cells. Additionally, given that Alzheimer's disease (AD) is known to involve ER stress and aggregation of proteins, we demonstrate that the soluble fraction of brain extracts from AD patients displays markedly higher rise of global proteome turbidity than in healthy counterparts. This assay could be valuable for various biological, medical and biotechnological applications.

Orally administrated cinnamon extract reduces ß-amyloid oligomerization and corrects cognitive impairment in Alzheimer's disease animal models.

An increasing body of evidence indicates that accumulation of soluble oligomeric assemblies of ß-amyloid polypeptide (Aß) play a key role in Alzheimer's disease (AD) pathology. Specifically, 56 kDa oligomeric species were shown to be correlated with impaired cognitive function in AD model mice. Several reports have documented the inhibition of Aß plaque formation by compounds from natural sources. Yet, evidence for the ability of common edible elements to modulate Aß oligomerization remains an unmet challenge. Here we identify a natural substance, based on cinnamon extract (CEppt), which markedly inhibits the formation of toxic Aß oligomers and prevents the toxicity of Aß on neuronal PC12 cells. When administered to an AD fly model, CEppt rectified their reduced longevity, fully recovered their locomotion defects and totally abolished tetrameric species of Aß in their brain. Furthermore, oral administration of CEppt to an aggressive AD transgenic mice model led to marked decrease in 56 kDa Aß oligomers, reduction of plaques and improvement in cognitive behavior. Our results present a novel prophylactic approach for inhibition of toxic oligomeric Aß species formation in AD through the utilization of a compound that is currently in use in human diet.

Complete phenotypic recovery of an Alzheimer's disease model by a quinone-tryptophan hybrid aggregation inhibitor.

The rational design of amyloid oligomer inhibitors is yet an unmet drug development need. Previous studies have identified the role of tryptophan in amyloid recognition, association and inhibition. Furthermore, tryptophan was ranked as the residue with highest amyloidogenic propensity. Other studies have demonstrated that quinones, specifically anthraquinones, can serve as aggregation inhibitors probably due to the dipole interaction of the quinonic ring with aromatic recognition sites within the amyloidogenic proteins. Here, using in vitro, in vivo and in silico tools we describe the synthesis and functional characterization of a rationally designed inhibitor of the Alzheimer's disease-associated beta-amyloid. This compound, 1,4-naphthoquinon-2-yl-L-tryptophan (NQTrp), combines the recognition capacities of both quinone and tryptophan moieties and completely inhibited Abeta oligomerization and fibrillization, as well as the cytotoxic effect of Abeta oligomers towards cultured neuronal cell line. Furthermore, when fed to transgenic Alzheimer's disease Drosophila model it prolonged their life span and completely abolished their defective locomotion. Analysis of the brains of these flies showed a significant reduction in oligomeric species of Abeta while immuno-staining of the 3(rd) instar larval brains showed a significant reduction in Abeta accumulation. Computational studies, as well as NMR and CD spectroscopy provide mechanistic insight into the activity of the compound which is most likely mediated by clamping of the aromatic recognition interface in the central segment of Abeta. Our results demonstrate that interfering with the aromatic core of amyloidogenic peptides is a promising approach for inhibiting various pathogenic species associated with amyloidogenic diseases. The compound NQTrp can serve as a lead for developing a new class of disease modifying drugs for Alzheimer's disease.