Fig 2: IAPP immunoreactivity is present both intra- and extracellularly in hproIAPP and hIAPP expressing flies.The nls-GFP (green) expression was driven by elavC155,Gal4 and included to help to identify the cell nuclei in expressing cells. In control flies (A), IAPP-immunoreactivity is absent as expected. In hproIAPP expressing flies, the IAPP-reactivity (red) is in (B) primarily present extracellularly, separated from the nls-GFP signal, while in (C) the reactivity is intracellular and present adjacent to a nls-GFP labelled nucleus. In hIAPP expressing flies (D), IAPP-reactivity (red) is seen both extra- and intracellularly. The study was performed on dissected whole mount fly brains. To enhance the intensity of the GFP-signal the brains were also labelled with antibodies specific for GFP. The cell nuclei were labelled with TO-PRO-3 and visualized as blue. Flies were 30 days old.

Fig 3: One letter code for human and mouse IAPP residues 1–37.Amino acid identity is shown in green and residue substitutions are shown in black, except for the three proline residues substitutions present in mouse IAPP that are shown in red (A). Proline residues are known beta-breakers and it is believed that these residues hinder IAPP-aggregation. ProIAPP processing. In man, proIAPP is synthesised as a 67 residue long polypeptide that undergoes posttranslational cleavage at dibasic residues (red) performed by the prohormone convertases PC2 and PC1/3. The two basic residues that remain at the C-terminus after PC1/3 processing are removed by carboxypeptidase E. Thereafter, the remaining C-terminal glycine residue is used for C-terminal amidation. A disulphide-bond is formed between residues 2 and 7 (B). Survival curve for flies expressing hproIAPP shows a reduction in life span. Expression of proIAPP was directed to neurons by the elavC155,Gal4 driver (C). This expression resulted in a significant reduction of lifespan (green), when compared to control flies (elavC155,Gal4, black) or flies containing UAS-proIAPP only (grey). These latter flies contain the inserted DNA, but do not express the corresponding protein due to absence of Gal4. (D) Comparison of the survival of hIAPP (brown) and elav C155,Gal4, (black) expressing flies shows that hIAPP expression does not reduce the survival (p>0.05). The hproIAPP (green) expressing flies showed a significant shorter lifespan than hIAPP (brown, p<0.001). (E) Comparison of the survival of mIAPP (blue) with control flies elavC155, Gal4, (black) showed that mIAPP expression does not reduce the survival of the flies (p>0.05). Flies expressing mIAPP (blue) lived longer than hproIAPP expressing flies (green, p<0.001). (F) The median survival for control flies (black), hproIAPP (green), hIAPP (brown) and mIAPP (blue) are 49, 37, 48 and 42 days, respectively, and the survival of hproIAPP flies is significantly reduced when compared to flies from the control, hIAPP and mIAPP strains (p<0.001).The survival for hIAPP flies is not reduced when compared to mIAPP or control flies. The survival studies were performed in an incubator with 70% humidity, at 26°C.

Fig 4: Expressed hproIAPP and hIAPP driven by elavC155,Gal4 are secreted from the neurons.(A) The green section highlights the studied area and the carton in (B) is an overview of the most important histological features present in this region. (C, D, E) Inside the humoral-brain-barrier, elav positive neurons are shown (green-light blue) and hIAPP (red) is localized to this area (C, D). (F, G, H) cover an area including both sides of the blood-brain-barrier where the neurons (green-light blue) are present at the bottom part and the fat body in the top part. In (F, G) are the cells in the fat body recognized by IAPP antibodies (red). Immunolabeling of IAPP in the fat body cells demonstrates that hproIAPP (F) and hIAPP (G) are secreted from the neurons, transferred over the humoral-brain-barrier and taken up by cells in the fat body. Cell nuclei are labelled with TOPRO-3 and visualized as blue. Flies were 40 days old.

Fig 5: Transgene expression is increased in young flies.The hIAPP, hproIAPP and mIAPP expression driven by the elavC155, Gal4 driver was analysed in 5 days (A, B) and 15 days (C–J) old flies. Immunolabeling of whole brain mounts from these younger flies revealed more neuronal immunoreactivity than detected in 30 and 40 days old flies. The depicted immunoreactivity is within the humoral-brain-barrier, the expected site of expression. Immunolabeling was performed with a monoclonal antibody that reacts with human and mouse IAPP. Arrows indicates reactivity.