scholarly journals The Amyloid Forming Peptides Islet Amyloid Polypeptide and Amyloid β Interact at the Molecular Level

2021 ◽  
Vol 22 (20) ◽  
pp. 11153
Author(s):  
Ye Wang ◽  
Gunilla T. Westermark
Keyword(s):  
Drosophila Melanogaster ◽  
Membrane Potential ◽  
Mitochondrial Membrane Potential ◽  
Mitochondrial Membrane ◽  
Islet Amyloid Polypeptide ◽  
Amyloid Β ◽  
Epidemiological Studies ◽  
Bimolecular Fluorescence Complementation ◽  
Amyloid Formation ◽  
Islet Amyloid

Epidemiological studies support a connection between the two common disorders, type-2 diabetes and Alzheimer’s disease. Both conditions have local amyloid formation in their pathogenesis, and cross-seeding between islet amyloid polypeptide (IAPP) and amyloid β (Aβ) could constitute the link. The bimolecular fluorescence complementation (BiFC) assay was used to investigate the occurrence of heterologous interactions between IAPP and Aβ and to compare the potential toxic effects of IAPP/Aβ, IAPP/IAPP, and Aβ/Aβ expression in living cells. Microscopy was used to confirm the fluorescence and determine the lysosomal, mitochondrial areas and mitochondrial membrane potential , and a FACS analysis was used to determine ROS production and the role for autophagy. Drosophila melanogaster expressing IAPP and Aβ was used to study their co-deposition and effects on longevity. We showed that the co-expression of IAPP and Aβ resulted in fluorophore reconstitution to the same extent as determined for homologous IAPP/IAPP or Aβ/Aβ expression. The BiFC(+)/BiFC(-) ratio of lysosomal area calculations increased in transfected cells independent of the vector combinations, while only Aβ/Aβ expression increased mitochondrial membrane potential. Expression combinations containing Aβ were necessary for the formation of a congophilic amyloid. In Drosophila melanogaster expressing IAPP/Aβ, co-deposition of the amyloid-forming peptides caused reduced longevity. The BiFC results confirmed a heterologous interaction between IAPP and Aβ, while co-deposits in the brain of Drosophila suggest mixed amyloid aggregates.

scholarly journals Stabilization and structural analysis of a membrane-associated hIAPP aggregation intermediate

eLife ◽  
2017 ◽  
Vol 6 ◽  
Author(s):  
Diana C Rodriguez Camargo ◽  
Kyle J Korshavn ◽  
Alexander Jussupow ◽  
Kolio Raltchev ◽  
David Goricanec ◽  
...  
Keyword(s):  
Type 2 Diabetes ◽  
Cell Death ◽  
Islet Amyloid Polypeptide ◽  
Amyloid Β ◽  
Md Simulations ◽  
Human Islet ◽  
Amyloid Formation ◽  
Islet Amyloid ◽  
Induce Cell Death

Membrane-assisted amyloid formation is implicated in human diseases, and many of the aggregating species accelerate amyloid formation and induce cell death. While structures of membrane-associated intermediates would provide tremendous insights into the pathology and aid in the design of compounds to potentially treat the diseases, it has not been feasible to overcome the challenges posed by the cell membrane. Here, we use NMR experimental constraints to solve the structure of a type-2 diabetes related human islet amyloid polypeptide intermediate stabilized in nanodiscs. ROSETTA and MD simulations resulted in a unique β-strand structure distinct from the conventional amyloid β-hairpin and revealed that the nucleating NFGAIL region remains flexible and accessible within this isolated intermediate, suggesting a mechanism by which membrane-associated aggregation may be propagated. The ability of nanodiscs to trap amyloid intermediates as demonstrated could become one of the most powerful approaches to dissect the complicated misfolding pathways of protein aggregation.

scholarly journals Development of a No-Wash Assay for Mitochondrial Membrane Potential Using the Styryl Dye DASPEI

2010 ◽  
Vol 15 (9) ◽  
pp. 1071-1081 ◽  
Author(s):  
Kristian H. R. Jensen ◽  
Jens C. Rekling
Keyword(s):  
Membrane Potential ◽  
Mitochondrial Dysfunction ◽  
Mitochondrial Membrane Potential ◽  
Cho Cells ◽  
Mitochondrial Membrane ◽  
Large Scale ◽  
Chinese Hamster ◽  
Amyloid Β ◽  
Drug Target Discovery ◽  
Cellular Models

Mitochondrial dysfunction is a hallmark of several diseases and may also result from drugs with unwanted side effects on mitochondrial biochemistry. The mitochondrial membrane potential is a good indicator of mitochondrial function. Here, the authors have developed a no-wash mitochondrial membrane potential assay using 2-(4-(dimethylamino)styryl)-N-ethylpyridinium iodide (DASPEI), a rarely used mitochondrial potentiometric probe, in a 96-well format using a fluorescent plate reader. The assay was validated using 2 protonophores (CCCP, DNP), which are known uncouplers, and the neuroleptic thioridazine, which is a suspected mitochondrial toxicant. CCCP and DNP have short-term depolarizing effects, and thioridazine has long-term hyperpolarizing effects on the mitochondrial membrane potential of Chinese hamster ovary (CHO) cells. The assay also detected changes of the mitochondrial membrane potential in CHO cells exposed to cobalt (mimicking hypoxia) and in PC12 cells exposed to amyloid β, demonstrating that the assay can be used in cellular models of hypoxia and Alzheimer’s disease. The assay needs no washing steps, has a Z′ value >0.5, can be used on standard fluorometers, has good post liquid-handling stability, and thus is suitable for large-scale screening efforts. In summary, the DASPEI assay is simple and rapid and may be of use in toxicological testing, drug target discovery, and mechanistic models of diseases involving mitochondrial dysfunction.

scholarly journals Inhibition of Alzheimer's amyloid-β peptide-induced reduction of mitochondrial membrane potential and neurotoxicity by gelsolin

2005 ◽  
Vol 26 (6) ◽  
pp. 849-855 ◽  
Author(s):  
Hongjiang Qiao ◽  
Richard C. Koya ◽  
Koji Nakagawa ◽  
Hiroki Tanaka ◽  
Hisakazu Fujita ◽  
...  
Keyword(s):  
Membrane Potential ◽  
Mitochondrial Membrane Potential ◽  
Mitochondrial Membrane ◽  
Amyloid Β ◽  
Amyloid Β Peptide

Polymorphic cross-seeding amyloid assemblies of amyloid-β and human islet amyloid polypeptide

2015 ◽  
Vol 17 (35) ◽  
pp. 23245-23256 ◽  
Author(s):  
Mingzhen Zhang ◽  
Rundong Hu ◽  
Hong Chen ◽  
Yung Chang ◽  
Jie Ma ◽  
...  
Keyword(s):  
Alzheimer’S Disease ◽  
Type 2 Diabetes ◽  
Alzheimer's Disease ◽  
Islet Amyloid Polypeptide ◽  
Amyloid Β ◽  
Epidemiological Studies ◽  
Human Islet ◽  
Islet Amyloid ◽  
Human Islet Amyloid Polypeptide

Epidemiological studies have shown that the development of Alzheimer's disease (AD) is associated with type 2 diabetes (T2D), but it still remains unclear how AD and T2D are connected.

scholarly journals Amyloid fibril structure of islet amyloid polypeptide by cryo-electron microscopy reveals similarities with amyloid beta

2020 ◽  
Author(s):  
Christine Röder ◽  
Tatsiana Kupreichyk ◽  
Lothar Gremer ◽  
Luisa U. Schäfer ◽  
Karunakar R. Pothula ◽  
...  
Keyword(s):  
Electron Microscopy ◽  
Early Onset ◽  
Critical Role ◽  
Islet Amyloid Polypeptide ◽  
Amyloid Β ◽  
Amyloid Β Peptide ◽  
Amyloid Formation ◽  
Islet Amyloid ◽  
Cryo Electron Microscopy

AbstractA critical role of the hormone islet amyloid polypeptide (IAPP) is vividly discussed for Type 2 Diabetes (T2D), where amyloid deposits in pancreatic islets consisting of fibrillar IAPP have been associated with beta cell loss. Here, we applied cryo-electron microscopy to elucidate the structure of IAPP fibrils prepared at physiological pH and reconstructed densities of three dominant polymorphs. An atomic model of the main polymorph comprising residues 13 – 37 in a density map of 4.2 Å resolution reveals two S-shaped, intertwined protofilaments. The segment 21-NNFGAIL-27, which is essential for IAPP amyloidogenicity, forms the protofilament interface together with tyrosine 37 and the amidated C-terminus. The main IAPP fibril polymorph resembles polymorphs of the Alzheimer disease (AD)-associated amyloid-β peptide (Aβ), which is striking in light of the epidemiological link between T2D and AD and reports on IAPP-Aβ cross-seeding in vivo. The results structurally link the early-onset T2D IAPP genetic polymorphism S20G with the early-onset AD Arctic mutation E22G of Aβ, rationalize previous data on IAPP fibrils, help to elucidate mechanisms of amyloid formation and toxicity, and support the design of fibril growth inhibitors as well as imaging probes for early detection of IAPP fibrils.

Platelets apoptosis in rats after global brain ischemia

2018 ◽  
pp. 27-35
Author(s):  
А.А. Соколовская ◽  
Э.Д. Вирюс ◽  
В.В. Александрин ◽  
А.С. Роткина ◽  
К.А. Никифорова ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
Ischemic Stroke ◽  
Membrane Potential ◽  
Mitochondrial Membrane Potential ◽  
Brain Ischemia ◽  
Mitochondrial Membrane ◽  
Male Rats ◽  
Global Brain Ischemia ◽  
Platelet Apoptosis ◽  
Global Brain

Цель исследования. Ишемические повреждения головного мозга, являются одной из наиболее частой причин инвалидности и смертности во всем мире. Недавно была установлена роль апоптоза тромбоцитов в патофизиологии инсульта, однако его механизмы до сих пор остаются невыясненными. Несмотря на различные экспериментальные модели, направленные на мониторинг апоптоза тромбоцитов, результаты, относительно изучения и выявления апоптоза тромбоцитов при ишемии головного мозга у крыс, весьма немногочисленны. Цель исследования - анализ апоптоза тромбоцитов с помощью метода проточной цитофлуориметрии на модели глобальной ишемии мозга у крыс. Методика. В экспериментах использовано 6 крыс-самцов Вистар в возрасте от 5 до 6 мес., разделенных на 2 группы: интактный контроль (К) и глобальная ишемия головного мозга. Модель глобальной ишемии головного мозга у крыс воспроизводилась путём билатеральной окклюзии общих сонных артерий на фоне гипотензии. Уровень системного артериального давления снижали посредством кровопотери до 40-45 мм рт. ст. Суспензию тромбоцитов крыс получали методом гельфильтрации с использованием сефарозы 2B. Для анализа экстернализации фосфатидилсерина (ФС) тромбоциты крыс инкубировали с Аннексином V-PE в связывающем буфере. Для оценки митохондриального мембранного потенциала (ММП) тромбоциты инкубировали с катионным красителем JC-1. После инкубации образцы немедленно анализировали на проточном цитофлуориметре FACSCalibur (Becton Dickinson, США). Результаты. Согласно полученным данным, экстернализация ФС на тромбоцитах крыс, перенесших инсульт, была значительно выше (53,45 ± 4,21%), чем в контрольной группе крыс (5,27 ± 2,40%). Данный эффект подтверждается выраженной деполяризацией митохондриальных мембран (DYm). После экспериментальной ишемии мозга почти 40% тромбоцитов было деполяризовано. Заключение. Использованный в работе подбор методов и маркеров обеспечивает понимание механизмов апоптоза тромбоцитов как в экспериментальных, так и в клинических условиях. Полученные данные позволяют сделать заключение, что апоптоз тромбоцитов является одним из факторов развития глобальной ишемии головного мозга у крыс. Результаты могут быть использованы для понимания механизмов, участвующих в развитии ишемического повреждения, что, в свою очередь, может быть использовано при разработке новых терапевтических стратегий. Aim. Stroke is one of the most common causes of disability and mortality worldwide. Multiple experimental models of stroke have focused on monitoring of platelet apoptosis. However, studies on and detection of platelet apoptosis in rats with ischemic stroke are very scarce. We investigated platelet apoptosis in rats with global brain ischemia using flow cytometry. Methods. Experiments were carried out on healthy, adult Wistar male rats weighing 300-350 g. The rats were divided into the following 2 groups: intact rats and rats with global brain ischemia. Global brain ischemia was induced by two-vessel (2-VO) carotid occlusion in combination with hypotension. Systemic blood pressure was reduced by 40-45 mm Hg by inducing haemorrhage. Platelets were isolated by gel filtration on Sepharose 2B. For evaluation of phosphatidylserine (PS) externalization, platelets were incubated with Annexin V-PE and analyzed on FACSCalibur (BD Biosciences). Mitochondrial membrane potential (DY) was measured during platelets apoptosis using JC-1, a mitochondrial membrane potential indicator. Platelets were analyzed by flow cytometry immediately after the incubation. Results. PS externalization on platelets was significantly greater after global brain ischemia (53.45 ± 4.21%) than in the control group (5.27 ± 2.40%). Pronounced depolarization of mitochondrial membrane potential (DYm) confirmed this finding. In the rat group with experimental brain ischemia, almost 40% (35.24 ± 5.21%) of platelets were depolarized. Conclusion. Our results provide insight into mechanisms involved in platelet apoptosis during ischemic stroke and can be used in further development of new therapeutic strategies.

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