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Time-resolved studies define the nature of toxic IAPP intermediates, providing insight for anti-amyloidosis therapeutics.

Abedini A, Plesner A, Cao P, Ridgway Z, Zhang J, Tu LH, Middleton CT, Chao B, Sartori DJ, Meng F, Wang H, Wong AG, Zanni MT, Verchere CB, Raleigh DP, Schmidt AM - Elife (2016)

Bottom Line: These globally flexible, low order oligomers upregulate pro-inflammatory markers and induce reactive oxygen species.They do not bind 1-anilnonaphthalene-8-sulphonic acid and lack extensive β-sheet structure.Aromatic interactions modulate, but are not required for toxicity.

View Article: PubMed Central - PubMed

Affiliation: Diabetes Research Program, Division of Endocrinology, Diabetes and Metabolism, Department of Medicine, New York University School of Medicine, New York, United States.

ABSTRACT
Islet amyloidosis by IAPP contributes to pancreatic β-cell death in diabetes, but the nature of toxic IAPP species remains elusive. Using concurrent time-resolved biophysical and biological measurements, we define the toxic species produced during IAPP amyloid formation and link their properties to induction of rat INS-1 β-cell and murine islet toxicity. These globally flexible, low order oligomers upregulate pro-inflammatory markers and induce reactive oxygen species. They do not bind 1-anilnonaphthalene-8-sulphonic acid and lack extensive β-sheet structure. Aromatic interactions modulate, but are not required for toxicity. Not all IAPP oligomers are toxic; toxicity depends on their partially structured conformational states. Some anti-amyloid agents paradoxically prolong cytotoxicity by prolonging the lifetime of the toxic species. The data highlight the distinguishing properties of toxic IAPP oligomers and the common features that they share with toxic species reported for other amyloidogenic polypeptides, providing information for rational drug design to treat IAPP induced β-cell death.

No MeSH data available.


Related in: MedlinePlus

Dilution of h-IAPP by 30% into cell culture medium does not change the kinetics of amyloid formation.A stock solution of h-IAPP (20 µM) was prepared in Tris HCl buffer (20 mM, 25°C) and the reaction was monitored by thioflavin-T fluorescence. Aliquots of the stock solution were removed after 10 h of incubation at 25°C (at mid-lag phase indicated by purple and green arrows) and diluted to a final concentration of 14 µM by transferring into either warm Tris HCl buffer (20 mM, 37°C) or warm cell culture medium (supplemented RPMI containing 10% FBS, 37°C) mimicking solution conditions in the cellular assays; amyloid formation was then monitored at 37°C. The data show that while the rate of amyloid formation by oligomers modestly increases upon dilution into warm Tris HCl buffer, there is no detectable effect upon dilution into warm cell culture medium; the modest increase in rate (decrease in the lag phase length) due to the increase in temperature is offset by the effect of the medium. Data represent mean ± SD of three to six replicates per condition. Some of the error bars are the same size or smaller than the symbols in the graph.DOI:http://dx.doi.org/10.7554/eLife.12977.005
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fig2s2: Dilution of h-IAPP by 30% into cell culture medium does not change the kinetics of amyloid formation.A stock solution of h-IAPP (20 µM) was prepared in Tris HCl buffer (20 mM, 25°C) and the reaction was monitored by thioflavin-T fluorescence. Aliquots of the stock solution were removed after 10 h of incubation at 25°C (at mid-lag phase indicated by purple and green arrows) and diluted to a final concentration of 14 µM by transferring into either warm Tris HCl buffer (20 mM, 37°C) or warm cell culture medium (supplemented RPMI containing 10% FBS, 37°C) mimicking solution conditions in the cellular assays; amyloid formation was then monitored at 37°C. The data show that while the rate of amyloid formation by oligomers modestly increases upon dilution into warm Tris HCl buffer, there is no detectable effect upon dilution into warm cell culture medium; the modest increase in rate (decrease in the lag phase length) due to the increase in temperature is offset by the effect of the medium. Data represent mean ± SD of three to six replicates per condition. Some of the error bars are the same size or smaller than the symbols in the graph.DOI:http://dx.doi.org/10.7554/eLife.12977.005

Mentions: Amyloid formation by h-IAPP, like that of other amyloidogenic proteins, comprises three distinct phenomenological phases: a lag, growth and saturation phase (Figure 1B). Little or no amyloid is formed in the lag phase and little is known about the nature of the species that populate this phase. Secondary nucleation leads to production of new fibrils, either by breakage of the small number of fibrils present or by templating new aggregates off the surface of existing fibrils. We developed time-resolved assays that allow concurrent biophysical, biochemical and biological characterization of the ensemble of species produced during IAPP amyloid formation (Figure 2A). Physiologically relevant solution conditions were found such that assembly of IAPP occurs on long time scales. The time scale is sufficiently long enough that the presence of toxic species can be detected indirectly by removing aliquots and applying them to cultured rat INS-1 β-cells or murine pancreatic islets. Stock solutions of h-IAPP, h-IAPP mutants and non-toxic, non-amyloidogenic rat IAPP (r-IAPP) were prepared by dissolving the peptides in buffer (time-zero) and incubating them at 25°C (pH 7.4). Aliquots were removed at various time points over the course of aggregation and characterized by the amyloid sensitive dye thioflavin-T and by transmission electron microscopy (TEM); aliquots were also applied to cultured β-cells at the same time points. Addition of aliquots to the cells involves only a 30% dilution of the peptide stock solutions. Control experiments using photochemical induced cross-linking and thioflavin-T kinetic assays of amyloid formation in buffer at 25°C reveal that this modest dilution does not significantly alter the distribution of oligomers, nor does it significantly alter the time course of amyloid formation. The same dilution into cell culture medium at 37°C has no significant effect on the time course (Figure 2—figure supplements 1 and 2). Toxicity was assessed by measuring loss in cellular metabolic function, detected by Alamar Blue reduction assays; production of reactive oxygen species (ROS); upregulation of inflammatory markers; production of cleaved caspase-3; and by observed changes in cellular morphology by light microscopy. These real-time experiments probe kinetic species produced during the course of h-IAPP amyloid formation, and are fundamentally different from the common approach in which peptide is added to cells upon dissolution in cell culture medium and toxicity monitored after subsequent incubation times on cells. This experimental design also differs from studies that attempt to trap non-amyloidogenic oligomers using surfaces such as gold particles, detergents or micelles. It is not known how surface-trapping techniques affect the conformational properties of oligomers (Bram et al., 2014; Kayed, 2003). The experiments reported here provide critical information about toxic, amyloidogenic IAPP oligomers in solution.10.7554/eLife.12977.003Figure 2.Toxic h-IAPP species are transiently populated lag phase intermediates.


Time-resolved studies define the nature of toxic IAPP intermediates, providing insight for anti-amyloidosis therapeutics.

Abedini A, Plesner A, Cao P, Ridgway Z, Zhang J, Tu LH, Middleton CT, Chao B, Sartori DJ, Meng F, Wang H, Wong AG, Zanni MT, Verchere CB, Raleigh DP, Schmidt AM - Elife (2016)

Dilution of h-IAPP by 30% into cell culture medium does not change the kinetics of amyloid formation.A stock solution of h-IAPP (20 µM) was prepared in Tris HCl buffer (20 mM, 25°C) and the reaction was monitored by thioflavin-T fluorescence. Aliquots of the stock solution were removed after 10 h of incubation at 25°C (at mid-lag phase indicated by purple and green arrows) and diluted to a final concentration of 14 µM by transferring into either warm Tris HCl buffer (20 mM, 37°C) or warm cell culture medium (supplemented RPMI containing 10% FBS, 37°C) mimicking solution conditions in the cellular assays; amyloid formation was then monitored at 37°C. The data show that while the rate of amyloid formation by oligomers modestly increases upon dilution into warm Tris HCl buffer, there is no detectable effect upon dilution into warm cell culture medium; the modest increase in rate (decrease in the lag phase length) due to the increase in temperature is offset by the effect of the medium. Data represent mean ± SD of three to six replicates per condition. Some of the error bars are the same size or smaller than the symbols in the graph.DOI:http://dx.doi.org/10.7554/eLife.12977.005
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fig2s2: Dilution of h-IAPP by 30% into cell culture medium does not change the kinetics of amyloid formation.A stock solution of h-IAPP (20 µM) was prepared in Tris HCl buffer (20 mM, 25°C) and the reaction was monitored by thioflavin-T fluorescence. Aliquots of the stock solution were removed after 10 h of incubation at 25°C (at mid-lag phase indicated by purple and green arrows) and diluted to a final concentration of 14 µM by transferring into either warm Tris HCl buffer (20 mM, 37°C) or warm cell culture medium (supplemented RPMI containing 10% FBS, 37°C) mimicking solution conditions in the cellular assays; amyloid formation was then monitored at 37°C. The data show that while the rate of amyloid formation by oligomers modestly increases upon dilution into warm Tris HCl buffer, there is no detectable effect upon dilution into warm cell culture medium; the modest increase in rate (decrease in the lag phase length) due to the increase in temperature is offset by the effect of the medium. Data represent mean ± SD of three to six replicates per condition. Some of the error bars are the same size or smaller than the symbols in the graph.DOI:http://dx.doi.org/10.7554/eLife.12977.005
Mentions: Amyloid formation by h-IAPP, like that of other amyloidogenic proteins, comprises three distinct phenomenological phases: a lag, growth and saturation phase (Figure 1B). Little or no amyloid is formed in the lag phase and little is known about the nature of the species that populate this phase. Secondary nucleation leads to production of new fibrils, either by breakage of the small number of fibrils present or by templating new aggregates off the surface of existing fibrils. We developed time-resolved assays that allow concurrent biophysical, biochemical and biological characterization of the ensemble of species produced during IAPP amyloid formation (Figure 2A). Physiologically relevant solution conditions were found such that assembly of IAPP occurs on long time scales. The time scale is sufficiently long enough that the presence of toxic species can be detected indirectly by removing aliquots and applying them to cultured rat INS-1 β-cells or murine pancreatic islets. Stock solutions of h-IAPP, h-IAPP mutants and non-toxic, non-amyloidogenic rat IAPP (r-IAPP) were prepared by dissolving the peptides in buffer (time-zero) and incubating them at 25°C (pH 7.4). Aliquots were removed at various time points over the course of aggregation and characterized by the amyloid sensitive dye thioflavin-T and by transmission electron microscopy (TEM); aliquots were also applied to cultured β-cells at the same time points. Addition of aliquots to the cells involves only a 30% dilution of the peptide stock solutions. Control experiments using photochemical induced cross-linking and thioflavin-T kinetic assays of amyloid formation in buffer at 25°C reveal that this modest dilution does not significantly alter the distribution of oligomers, nor does it significantly alter the time course of amyloid formation. The same dilution into cell culture medium at 37°C has no significant effect on the time course (Figure 2—figure supplements 1 and 2). Toxicity was assessed by measuring loss in cellular metabolic function, detected by Alamar Blue reduction assays; production of reactive oxygen species (ROS); upregulation of inflammatory markers; production of cleaved caspase-3; and by observed changes in cellular morphology by light microscopy. These real-time experiments probe kinetic species produced during the course of h-IAPP amyloid formation, and are fundamentally different from the common approach in which peptide is added to cells upon dissolution in cell culture medium and toxicity monitored after subsequent incubation times on cells. This experimental design also differs from studies that attempt to trap non-amyloidogenic oligomers using surfaces such as gold particles, detergents or micelles. It is not known how surface-trapping techniques affect the conformational properties of oligomers (Bram et al., 2014; Kayed, 2003). The experiments reported here provide critical information about toxic, amyloidogenic IAPP oligomers in solution.10.7554/eLife.12977.003Figure 2.Toxic h-IAPP species are transiently populated lag phase intermediates.

Bottom Line: These globally flexible, low order oligomers upregulate pro-inflammatory markers and induce reactive oxygen species.They do not bind 1-anilnonaphthalene-8-sulphonic acid and lack extensive β-sheet structure.Aromatic interactions modulate, but are not required for toxicity.

View Article: PubMed Central - PubMed

Affiliation: Diabetes Research Program, Division of Endocrinology, Diabetes and Metabolism, Department of Medicine, New York University School of Medicine, New York, United States.

ABSTRACT
Islet amyloidosis by IAPP contributes to pancreatic β-cell death in diabetes, but the nature of toxic IAPP species remains elusive. Using concurrent time-resolved biophysical and biological measurements, we define the toxic species produced during IAPP amyloid formation and link their properties to induction of rat INS-1 β-cell and murine islet toxicity. These globally flexible, low order oligomers upregulate pro-inflammatory markers and induce reactive oxygen species. They do not bind 1-anilnonaphthalene-8-sulphonic acid and lack extensive β-sheet structure. Aromatic interactions modulate, but are not required for toxicity. Not all IAPP oligomers are toxic; toxicity depends on their partially structured conformational states. Some anti-amyloid agents paradoxically prolong cytotoxicity by prolonging the lifetime of the toxic species. The data highlight the distinguishing properties of toxic IAPP oligomers and the common features that they share with toxic species reported for other amyloidogenic polypeptides, providing information for rational drug design to treat IAPP induced β-cell death.

No MeSH data available.


Related in: MedlinePlus