Contribution of the 12-17 hydrophobic region of islet amyloid polypeptide in self-assembly and cytotoxicity.

The islet amyloid polypeptide (IAPP) is a 37-residue aggregation-prone peptide hormone whose deposition as insoluble fibrils in the islets of Langerhans is associated with type II diabetes. Therapeutic interventions targeting IAPP amyloidogenesis, which contributes to pancreatic ß-cell degeneration, remain elusive owing to the lack of understanding of the self-assembly mechanisms and of the quaternary proteospecies mediating toxicity. While countless studies have investigated the contributions of the 20-29 amyloidogenic core in self-assembly, IAPP central region, i.e. positions 11 to 19, has been less studied, notwithstanding its potential key role in oligomerization. In this context, the present study aimed at investigating the physicochemical and conformational properties driving IAPP self-assembly and associated cytotoxicity. Computational tools and all-atom molecular dynamics simulation suggested that the hydrophobic 12-17 segment promotes IAPP self-recognition and aggregation. Alanine scanning revealed that the hydrophobic side chains of Leu12, Phe15 and Val17 are critical for amyloid fibril formation. Destabilization of the α-helical folding by Pro substitution enhanced self-assembly when the pyrrolidine ring was successively introduced at positions Ala13, Asn14 and Phe15, in comparison to respective Ala-substituted counterparts. Modulating the peptide backbone flexibility at position Leu16 through successive incorporation of Pro, Gly and α-methylalanine, inhibited amyloid formation and reduced cytotoxicity, while the isobutyl side chain of Leu16 was not critical for self-assembly and IAPP-mediated toxicity. These results highlight the importance of the 12-17 hydrophobic region of IAPP for self-recognition, ultimately supporting the development of therapeutic approaches to prevent oligomerization and/or fibrillization.

Cell surface glycosaminoglycans exacerbate plasma membrane perturbation induced by the islet amyloid polypeptide.

Glycosaminoglycans (GAGs) are long and unbranched anionic heteropolysaccharides that have been associated with virtually all amyloid deposits. Soluble sulfated GAGs are known for their propensity to promote the self-assembly of numerous amyloidogenic proteins and to modulate their cytotoxicity. Nonetheless, although GAGs are prevalent on the outer leaflet of eukaryotic cell plasma membrane as part of proteoglycans, their contributions in the perturbation of lipid bilayer induced by amyloid polypeptides remain unknown. Herein, we investigate the roles of GAGs in the cytotoxicity and plasma membrane perturbation induced by the islet amyloid polypeptide (IAPP), whose deposition in the pancreatic islets is associated with type II diabetes. Cellular assays using GAG-deficient cells reveal that GAGs exacerbate IAPP-induced cytotoxicity and permeabilization of the plasma membrane. Confocal microscopy and flow cytometry analyses show that IAPP sequestration at the cell surface is dependent of GAGs and of the aggregation propensity of the peptide. Using giant plasma membrane vesicles (GPMVs) prepared from GAG-deficient cells, we investigate the direct contributions of membrane-embedded proteoglycans in IAPP-induced membrane disassembly. In sharp contrast to soluble sulfated GAGs, kinetics of amyloid self-assembly expose that the presence of GAGs on GPMVs does not significantly modulate in vitro amyloid formation. Overall, this study indicates that cell surface GAGs increase the local concentration of IAPP in the vicinity of the plasma membrane, promoting lipid bilayer perturbation and cell death.

Spatial learning by mice in three dimensions.

We tested whether mice can represent locations distributed throughout three-dimensional space, by developing a novel three-dimensional radial arm maze. The three-dimensional radial maze, or "radiolarian" maze, consists of a central spherical core from which arms project in all directions. Mice learn to retrieve food from the ends of the arms without omitting any arms or re-visiting depleted ones. We show here that mice can learn both a standard working memory task, in which all arms are initially baited, and also a reference memory version in which only a subset are ever baited. Comparison with a two-dimensional analogue of the radiolarian maze, the hexagon maze, revealed equally good working-memory performance in both mazes if all the arms were initially baited, but reduced working and reference memory in the partially baited radiolarian maze. This suggests intact three-dimensional spatial representation in mice over short timescales but impairment of the formation and/or use of long-term spatial memory of the maze. We discuss potential mechanisms for how mice solve the three-dimensional task, and reasons for the impairment relative to its two-dimensional counterpart, concluding with some speculations about how mammals may represent three-dimensional space.