Dataset Information

Drosophila melanogaster as a model system for studies of islet amyloid polypeptide aggregation.

ABSTRACT:

Background

Recent research supports that aggregation of islet amyloid polypeptide (IAPP) leads to cell death and this makes islet amyloid a plausible cause for the reduction of beta cell mass, demonstrated in patients with type 2 diabetes. IAPP is produced by the beta cells as a prohormone, and proIAPP is processed into IAPP by the prohormone convertases PC1/3 and PC2 in the secretory granules. Little is known about the pathogenesis for islet amyloid and which intracellular mechanisms are involved in amyloidogenesis and induction of cell death.

Methodology/principal findings

We have established expression of human proIAPP (hproIAPP), human IAPP (hIAPP) and the non-amyloidogenic mouse IAPP (mIAPP) in Drosophila melanogaster, and compared survival of flies with the expression driven to different cell populations. Only flies expressing hproIAPP in neurons driven by the Gal4 driver elav(C155,Gal4) showed a reduction in lifespan whereas neither expression of hIAPP or mIAPP influenced survival. Both hIAPP and hproIAPP expression caused formation of aggregates in CNS and fat body region, and these aggregates were both stained by the dyes Congo red and pFTAA, both known to detect amyloid. Also, the morphology of the highly organized protein granules that developed in the fat body of the head in hIAPP and hproIAPP expressing flies was characterized, and determined to consist of 15.8 nm thick pentagonal rod-like structures.

Conclusions/significance

These findings point to a potential for Drosophila melanogaster to serve as a model system for studies of hproIAPP and hIAPP expression with subsequent aggregation and developed pathology.

SUBMITTER: Schultz SW

PROVIDER: S-EPMC3114789 | biostudies-literature | 2011

REPOSITORIES: biostudies-literature

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Publications

Drosophila melanogaster as a model system for studies of islet amyloid polypeptide aggregation.

Schultz Sebastian Wolfgang SW Nilsson K Peter R KP Westermark Gunilla Torstensdotter GT

PloS one 20110614 6

<h4>Background</h4>Recent research supports that aggregation of islet amyloid polypeptide (IAPP) leads to cell death and this makes islet amyloid a plausible cause for the reduction of beta cell mass, demonstrated in patients with type 2 diabetes. IAPP is produced by the beta cells as a prohormone, and proIAPP is processed into IAPP by the prohormone convertases PC1/3 and PC2 in the secretory granules. Little is known about the pathogenesis for islet amyloid and which intracellular mechanisms ar ...[more]

PMID: 21695120

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Project description:Amyloid diseases are degenerative pathologies, highly prevalent today because they are closely related to aging, that have in common the erroneous folding of intrinsically disordered proteins (IDPs) which aggregate and lead to cell death. Type 2 Diabetes involves a peptide called human islet amyloid polypeptide (hIAPP), which undergoes a conformational change, triggering the aggregation process leading to amyloid aggregates and fibers rich in β-sheets mainly found in the pancreas of all diabetic patients. Inhibiting the aggregation of amyloid proteins has emerged as a relevant therapeutic approach and we have recently developed the design of acyclic flexible hairpins based on peptidic recognition sequences of the amyloid β peptide (Aβ1-42) as a successful strategy to inhibit its aggregation involved in Alzheimer's disease. The present work reports the extension of our strategy to hIAPP aggregation inhibitors. The design, synthesis, conformational analyses, and biophysical evaluations of dynamic β-hairpin like structures built on a piperidine-pyrrolidine β-turn inducer are described. By linking to this β-turn inducer three different arms (i) pentapeptide, (ii) tripeptide, and (iii) α/aza/aza/pseudotripeptide, we demonstrate that the careful selection of the peptide-based arms from the sequence of hIAPP allowed to selectively modulate its aggregation, while the peptide character can be decreased. Biophysical assays combining, Thioflavin-T fluorescence, transmission electronic microscopy, capillary electrophoresis, and mass spectrometry showed that the designed compounds inhibit both the oligomerization and the fibrillization of hIAPP. They are also capable to decrease the aggregation process in the presence of membrane models and to strongly delay the membrane-leakage induced by hIAPP. More generally, this work provides the proof of concept that our rational design is a versatile and relevant strategy for developing efficient and selective inhibitors of aggregation of amyloidogenic proteins.

Project description:Aims/hypothesisThe islet vasculature, including its constituent islet endothelial cells, is a key contributor to the microenvironment necessary for normal beta cell health and function. In type 2 diabetes, islet amyloid polypeptide (IAPP) aggregates, forming amyloid deposits that accumulate between beta cells and islet capillaries. This process is known to be toxic to beta cells but its impact on the islet vasculature has not previously been studied. Here, we report the first characterisation of the effects of IAPP aggregation on islet endothelial cells/capillaries using cell-based and animal models.MethodsPrimary and immortalised islet endothelial cells were treated with amyloidogenic human IAPP (hIAPP) alone or in the presence of the amyloid blocker Congo Red or the Toll-like receptor (TLR) 2/4 antagonist OxPAPc. Cell viability was determined0 along with mRNA and protein levels of inflammatory markers. Islet capillary abundance, morphology and pericyte coverage were determined in pancreases from transgenic mice with beta cell expression of hIAPP using conventional and confocal microscopy.ResultsAggregated hIAPP decreased endothelial cell viability in immortalised and primary islet endothelial cells (by 78% and 60%, respectively) and significantly increased expression of inflammatory markers Il6, Vcam1 and Edn1 mRNA relative to vehicle treatment in both cell types (p<0.05; n=4). Both cytotoxicity and the proinflammatory response were ameliorated by Congo Red (p<0.05; n=4); whereas TLR2/4-inhibition blocked inflammatory gene expression (p<0.05; n=6) without improving viability. Islets from high-fat-diet-fed amyloid-laden hIAPP transgenic mice also exhibited significantly increased expression of most markers of endothelial inflammation (p<0.05; n=5) along with decreased capillary density compared with non-transgenic littermates fed the same diet (p<0.01). Moreover, a 16% increase in capillary diameter was observed in amyloid-adjacent capillaries (p<0.01), accompanied by a doubling in pericyte structures positive for neuron-glial antigen 2 (p<0.001).Conclusions/interpretationIslet endothelial cells are susceptible to hIAPP-induced cytotoxicity and exhibit a TLR2/4-dependent proinflammatory response to aggregated hIAPP. Additionally, we observed amyloid-selective effects that decreased islet capillary density, accompanied by increased capillary diameter and increased pericyte number. Together, these data demonstrate that the islet vasculature is a target of the cytotoxic and proinflammatory effects of aggregated hIAPP that likely contribute to the detrimental effects of hIAPP aggregation on beta cell function and survival in type 2 diabetes.

Dataset Information

Drosophila melanogaster as a model system for studies of islet amyloid polypeptide aggregation.

Background

Methodology/principal findings

Conclusions/significance

Publications

Drosophila melanogaster as a model system for studies of islet amyloid polypeptide aggregation.

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