Apoptosis induced by islet amyloid polypeptide soluble oligomers is neutralized by diabetes-associated specific antibodies

Abstract

Soluble oligomeric assemblies of amyloidal proteins appear to act as major pathological agents in several degenerative disorders. Isolation and characterization of these oligomers is a pivotal step towards determination of their pathological relevance. Here we describe the isolation of Type 2 diabetes-associated islet amyloid polypeptide soluble cytotoxic oligomers; these oligomers induced apoptosis in cultured pancreatic cells, permeated model lipid vesicles and interacted with cell membranes following complete internalization. Moreover, antibodies which specifically recognized these assemblies, but not monomers or amyloid fibrils, were exclusively identified in diabetic patients and were shown to neutralize the apoptotic effect induced by these oligomers. Our findings support the notion that human IAPP peptide can form highly toxic oligomers. The presence of antibodies identified in the serum of diabetic patients confirms the pathological relevance of the oligomers. In addition, the newly identified structural epitopes may also provide new mechanistic insights and a molecular target for future therapy.

Figure 1. Human IAPP oligomers Characterization.

(a) Western-Blot analysis under non-reducing conditions of human IAPP oligomers and a negative control of non amyloidogenic rat IAPP.(b) Oligomers stability assay, hIAPP oligomers were precipitated dialyzed against PBS buffer and incubated at 37°C oligomers association was monitored by ThT analysis and compared to hIAPP dissolved in PBS buffer. (c) Size exclusion chromatography (Superdex 75 10/300, PBS buffer pH 7.4) of hIAPP oligomers; I-monomer, I-dimer, III- trimer and IV- 90 kDa oligomer. (d) Atomic force microscopy (AFM) images of the ~90 kDa oligomers, AFM scale bar 600 nm. (e) CD spectroscopy of hIAPP and rIAPP, protein concentration of 5 μM. Each spectrum represents the average of three measurements.

Figure 2. Human IAPP oligomers Toxicity.

(a) Rin-m cells treated with hIAPP oligomers (grey) or with rIAPP (black) in diverse concentrations. Cells viability was estimated by MTT reduction assay (*P < 0.05, **P < 0.005). (b) Dye leakage from calcein containing liposomes. 1 μM hIAPP oligomers (green rectangle) or rIAPP (blue rhombus) were incubated with the liposomes and membrane damage was evaluated by increased fluorescence (excitation: 495, emission: 520) and compared to the control group (red square). (c) FACS results of the incubation of Rin-m cells with hIAPP oligomers at different concentrations. The Annexin V-FITC apoptosis detection kit was used for the detection of apoptotic cells. FL1-H is the fluorescence of V-FITC and FL2-H is the fluorescence of Annexin V-PE. I-cells in necrotic state, II- late apoptotic state, III- early apoptotic state and IV- viable cells (d) Diagram presentation of cell state dispersion of three FACS analysis assays, as presented in figure 2C, dark grey column represent viable cells, light grey column represent early and late apoptotic cells and black column represent necrotic cells.

Figure 3. hIAPP oligomers permeabilize cell membrane.

Confocal microscopic images of Rin-m cells incubated with 5 μM of hIAPP-Hiytelfluor 488 oligomers, cells were stained with phalloidin–tetramethylrhodamine. Incubation was preformed for one hour (I), two hours (II), four hours (IV) and eight hours (VIII). After one hour localization of hIAPP oligomers to cells membrane was observed followed by insertion to cell cytoplasm and cell morphology alteration at longer incubation times.

Figure 4. hIAPP oligomers cellular localization.

Live confocal microscopy images of Rin-m cells incubated 5 μM of hIAPP-Hiytelfluor 488 oligomers for 3 hours (Green), Lysosome was stained using LysoTracker reagent (red), mitochondria was stained using MitoTracker (red). As shown, some oligomers were observed to localize in the lysosome (white) while others showed a diffusion pattern along the cell.

Figure 5. Antibodies from Type 2 Diabetes patients recognize and neutralize hIAPP oligomers.

(a) Purified antibodies from the serum of type 2 diabetes patients and healthy people (N = 3) was compared to the recognition of hIAPP oligomers (5 μg) by dot blot analysis through serial dilutions of purified antibodies. (b) Purified antibodies from the serum of type 2 diabetes (orange columns) and healthy people (grey columns, N = 10) recognition of hIAPP oligomers (5 μg) was compared by ELISPOT assay. (c) Rin-m cells treated with hIAPP oligomers (grey) with or without type 2 diabetes (orange) or healthy individuals (blue) purified antibodies in diverse concentration was examined for viability by MTT reduction assay and compared to the non-treated cells, Box-plots showing statistical distributions of cell viability after treatment (the box size represent the SD, black circles are the average, the horizontal line is the median and the upper and lower asterisks are 99^th and 1^st percentile respectively) ***P < 0.0001. (d) Type 2 diabetes antibodies recognition of different hIAPP conformers was examined by ELISPOT assay (Fibrils in blue columns, monomers in red columns and oligomers in green columns).