The Co-oligomers of Aß42 and Human Islet Amyloid Polypeptide Exacerbate Neurotoxicity and Alzheimer-like Pathology at Cellular Level.

The Aß hypothesis has long been central to Alzheimer's disease (AD) theory, with a recent surge in attention following drug approvals targeting Aß plaque clearance. Aß42 oligomers (AßO) are key neurotoxins. While ß-amyloid (Aß) buildup is a hallmark of AD, postmortem brain analyses have unveiled human islet amyloid polypeptide (hIAPP) deposition in AD patients, suggesting a potential role in Alzheimer's pathology. This study investigates the neurotoxic effects of co-aggregates of Aß42 and hIAPP, specifically focusing on their impact on cell survival, apoptosis, and AD-like pathology. We analyzed and compared the impact of AßO and Aß42-hIAPP on cell survival in SH-SY5Y cells, apoptosis and inducing AD-like pathology in glutamatergic neurons. Aß42-hIAPP co-oligomers exhibited significantly greater toxicity, causing 2.3-3.5 times higher cell death compared to AßO alone. Furthermore, apoptosis rates were significantly exacerbated in glutamatergic neurons when exposed to Aß42-hIAPP co-oligomers. The study also revealed that Aß42-hIAPP co-oligomers induced typical AD-like pathology in glutamatergic neurons, including the presence of Aß deposits (detected by 6E10 and 4G8 immunofluorescence) and alterations in tau protein (changes in total tau HT7, phosphorylated tau AT8, AT180). Notably, Aß42-hIAPP co-oligomers induced a more severe AD pathology compared to AßO alone. These findings provide compelling evidence for the heightened toxicity of Aß42-hIAPP co-oligomers on neurons and their role in exacerbating AD pathology. The study contributes novel insights into the pathogenesis of Alzheimer's disease, highlighting the potential involvement of hIAPP in AD pathology. Together, these findings offer novel insights into AD pathogenesis and routes for constructing animal models.

Oxidant-modified amylin fibrils and aggregates alter the inflammatory profile of multiple myeloid cell types, but are non-toxic to islet ß cells.

Diabetes mellitus currently affects ∼10% of the population worldwide, with Type 2 predominating, and this incidence is increasing steadily. Both Type 1 and 2 are complex diseases, involving ß-cell death and chronic inflammation, but the pathways involved are unresolved. Chronic inflammation is characterized by increased oxidant formation, with this inducing protein modification, altered function and immunogenicity. Amylin, a peptide hormone co-secreted with insulin by ß-cells, has attracted considerable interest for its amyloidogenic properties, however, the effects that oxidants have on amylin aggregation and function are poorly understood. Amylin was exposed in vitro to hypochlorous acid, hydrogen peroxide and peroxynitrous acid/peroxynitrite to investigate the formation of post-translational oxidative modifications (oxPTMs, via mass spectrometry) and fibril formation (via transmission electron microscopy). Amylin free acid (AFA) was also examined to investigate the role of the C-terminal amide in amylin. Oxidant exposure led to changes in aggregate morphology and abundance of oxPTMs in a concentration-dependent manner. The toxicity and immunogenic potential of oxidant-modified amylin or AFA on pancreatic islet cells (INS-1E), human monocyte cell line (THP-1) and monocyte-derived dendritic cells (moDCs) were examined using metabolic activity and cytokine assays, and flow cytometry. No significant changes in vitality or viability were detected, but exposure to oxidant-modified amylin or AFA resulted in altered immunogenicity when compared to the native proteins. THP-1 and moDCs show altered expression of activation markers and changes in cytokine secretion. Furthermore, oxidant-treated amylin and AFA promoted maturation of THP-1 and pre-mature moDCs, as determined by changes in size, and maturation markers.

Cyanidin-3-O-Glucoside improves the viability of human islet cells treated with amylin or Aß1-42 in vitro.

Islet transplantation is being considered as an alternative treatment for type 1 diabetes. Despite recent progress, transplant recipients continue to experience progressive loss of insulin independence. Cyanidin-3-O-Glucoside (C3G) has shown to be protective against damage that may lead to post-transplant islet loss. In this study, human islets cultured with or without C3G were treated with human amylin, Aß1-42, H2O2, or rapamycin to mimic stresses encountered in the post-transplant environment. Samples of these islets were collected and assayed to determine C3G's effect on cell viability and function, reactive oxygen species (ROS), oxidative stress, amyloid formation, and the presence of inflammatory as well as autophagic markers. C3G treatment of human islets exposed to either amylin or Aß1-42 increased cell viability (p<0.01) and inhibited amyloid formation (p<0.01). A reduction in ROS and an increase in HO-1 gene expression as well as in vitro islet function were also observed in C3G-treated islets exposed to amylin or Aß1-42, although not significantly. Additionally, treatment with C3G resulted in a significant reduction in the protein expression of inflammatory markers IL-1ß and NLRP3 (p<0.01) as well as an increase in LC3 autophagic marker (p<0.05) in human islets treated with amylin, Aß1-42, rapamycin, or H2O2. Thus, C3G appears to have a multi-faceted protective effect on human islets in vitro, possibly through its anti-oxidant property and alteration of inflammatory as well as autophagic pathways.

Distinct Modes of Action of IAPP Oligomers on Membranes.

Islet amyloid polypeptide (IAPP, also known as amylin) is a peptide hormone that is co-secreted with insulin by pancreatic ß-cells and forms amyloid aggregates in type II diabetes. Various lines of evidence indicate that oligomers of this peptide may induce toxicity by disrupting or forming pores in cell membranes, but the structure of these pores is unknown. Here, we create models of pores for both helical and ß-structured peptides using implicit membrane modeling and test their stability using multimicrosecond all-atom simulations. We find that the helical peptides behave similarly to antimicrobial peptides; they remain stably inserted in a highly tilted or partially unfolded configuration creating a narrow water channel. Parallel helix orientation creates a somewhat larger pore. An octameric ß barrel of parallel ß-hairpins is highly stable in the membrane, whereas the corresponding barrel made of antiparallel hairpins is not. We propose that certain experiments probe the helical pore state while others probe the ß-structured pore state; this provides a possible explanation for lack of correlation that is sometimes observed between in vivo toxicity and in vitro liposome permeabilization experiments.

The role of mitochondrial apoptotic pathway in islet amyloid-induced ß-cell death.

Islet amyloid, formed by aggregation of human islet amyloid polypeptide (hIAPP), contributes to ß-cell death in type 2 diabetes. We previously showed that extracellular hIAPP aggregates promote Fas-mediated ß-cell apoptosis. Here, we tested if hIAPP aggregates can trigger the mitochondrial apoptotic pathway (MAP). hIAPP aggregation in Ad-hIAPP transduced INS-1 and human islet ß-cells promoted cytochrome c release, caspase-9 activation and apoptosis, which were reduced by Bax inhibitor. Amyloid formation in hIAPP-expressing mouse islets during culture increased caspase-9 activation in ß-cells. Ad-hIAPP transduced islets from CytcKA/KA and BaxBak ßDKO mice (models of blocked MAP), had lower caspase-9-positive and apoptotic ß-cells than transduced wild-type islets, despite comparable amyloid formation. Blocking Fas (markedly) and Bax or caspase-9 (modestly) reduced ß-cell death induced by extracellular hIAPP aggregates. These findings suggest a role for MAP in amyloid-induced ß-cell death and a potential strategy to reduce intracellular amyloid ß-cell toxicity by blocking cytochrome c apoptotic function.

Tryptophan-galactosylamine conjugates inhibit and disaggregate amyloid fibrils of Aß42 and hIAPP peptides while reducing their toxicity.

Self-assembly of proteins into amyloid fibrils is a hallmark of various diseases, including Alzheimer's disease (AD) and Type-2 diabetes Mellitus (T2DM). Aggregation of specific peptides, like Aß42 in AD and hIAPP in T2DM, causes cellular dysfunction resulting in the respective pathology. While these amyloidogenic proteins lack sequence homology, they all contain aromatic amino acids in their hydrophobic core that play a major role in their self-assembly. Targeting these aromatic residues by small molecules may be an attractive approach for inhibiting amyloid aggregation. Here, various biochemical and biophysical techniques revealed that a panel of tryptophan-galactosylamine conjugates significantly inhibit fibril formation of Aß42 and hIAPP, and disassemble their pre-formed fibrils in a dose-dependent manner. They are also not toxic to mammalian cells and can reduce the cytotoxicity induced by Aß42 and hIAPP aggregates. These tryptophan-galactosylamine conjugates can therefore serve as a scaffold for the development of therapeutics towards AD and T2DM.

Protein-conformational diseases in childhood: Naturally-occurring hIAPP amyloid-oligomers and early ß-cell damage in obesity and diabetes.

BACKGROUND AND AIMS: This is the first time that obesity and diabetes mellitus (DM) as protein conformational diseases (PCD) are reported in children and they are typically diagnosed too late, when ß-cell damage is evident. Here we wanted to investigate the level of naturally-ocurring or real (not synthetic) oligomeric aggregates of the human islet amyloid polypeptide (hIAPP) that we called RIAO in sera of pediatric patients with obesity and diabetes. We aimed to reduce the gap between basic biomedical research, clinical practice-health decision making and to explore whether RIAO work as a potential biomarker of early ß-cell damage. MATERIALS AND METHODS: We performed a multicentric collaborative, cross-sectional, analytical, ambispective and blinded study; the RIAO from pretreated samples (PTS) of sera of 146 pediatric patients with obesity or DM and 16 healthy children, were isolated, measured by sound indirect ELISA with novel anti-hIAPP cytotoxic oligomers polyclonal antibody (MEX1). We carried out morphological and functional studied and cluster-clinical data driven analysis. RESULTS: We demonstrated by western blot, Transmission Electron Microscopy and cell viability experiments that RIAO circulate in the blood and can be measured by ELISA; are elevated in serum of childhood obesity and diabetes; are neurotoxics and works as biomarkers of early ß-cell failure. We explored the range of evidence-based medicine clusters that included the RIAO level, which allowed us to classify and stratify the obesity patients with high cardiometabolic risk. CONCLUSIONS: RIAO level increases as the number of complications rises; RIAOs > 3.35 µg/ml is a predictor of changes in the current indicators of ß-cell damage. We proposed a novel physio-pathological pathway and shows that PCD affect not only elderly patients but also children. Here we reduced the gap between basic biomedical research, clinical practice and health decision making.

Inhibition of human islet amyloid polypeptide aggregation and cellular toxicity by oleuropein and derivatives from olive oil.

Loss of ß-cell function and ß-cell death is the key feature of type 2 diabetes mellitus (T2DM). One hypothesis for the mechanism of this feature is amyloid formation by the human islet amyloid polypeptide (hIAPP). Despite the global prevalence of T2DM, there are no therapeutic strategies for the treatment of or prevention of amylin amyloidosis. Clinical trials and population studies indicate the healthy virtues of the Mediterranean diet, especially the extra virgin olive oil (EVOO) found in this diet. This oil is enriched in phenolic compounds shown to be effective against several aging and lifestyle diseases. Oleuropein (Ole), one of the most abundant polyphenols in EVOO, has been reported to be anti-diabetic. Some of Ole's main derivative have attracted our interest due to their multi-targetted effects, including interference with amyloid aggregation path. However, the structure-function relationship of Ole and its metabolites in T2DM are not yet clear. We report here a broad biophysical approach and cell biology techniques that enabled us to characterize the different molecular mechanisms by which tyrosol (TYR), hydroxytyrosol (HT), oleuropein (Ole) and oleuropein aglycone (OleA) modulate the hIAPP fibrillation in vitro and their effects on cell cytotoxicity. The OleA formed by enolic acid and hydroxytyrosol moiety was found to be more active than the Ole and HT at low micromolar concentrations. We further demonstrated that OleA inhibit the cytotoxicity induced by hIAPP aggregates by protecting more the cell membrane from permeabilization and then from death. These findings highlight the benefits of consuming EVOO and the great potential of its polyphenols, mainly OleA. Moreover, they support the possibility to validate and optimize the possible pharmacological use of EVOO polyphenols for T2DM prevention and therapy and also for many other amyloid related diseases.

Amylin and beta amyloid proteins interact to form amorphous heterocomplexes with enhanced toxicity in neuronal cells.

Human pancreatic islet amyloid polypeptide (hIAPP) and beta amyloid (Aß) can accumulate in Type 2 diabetes (T2D) and Alzheimer's disease (AD) brains and evidence suggests that interaction between the two amyloidogenic proteins can lead to the formation of heterocomplex aggregates. However, the structure and consequences of the formation of these complexes remains to be determined. The main objective of this study was to characterise the different types and morphology of Aß-hIAPP heterocomplexes and determine if formation of such complexes exacerbate neurotoxicity. We demonstrate that hIAPP promotes Aß oligomerization and formation of small oligomer and large aggregate heterocomplexes. Co-oligomerized Aß42-hIAPP mixtures displayed distinct amorphous structures and a 3-fold increase in neuronal cell death as compared to Aß and hIAPP alone. However, in contrast to hIAPP, non-amyloidogenic rat amylin (rIAPP) reduced oligomer Aß-mediated neuronal cell death. rIAPP exhibited reductions in Aß induced neuronal cell death that was independent of its ability to interact with Aß and form heterocomplexes; suggesting mediation by other pathways. Our findings reveal distinct effects of IAPP peptides in modulating Aß aggregation and toxicity and provide new insight into the potential pathogenic effects of Aß-IAPP hetero-oligomerization and development of IAPP based therapies for AD and T2D.

Analysis of Baboon IAPP Provides Insight into Amyloidogenicity and Cytotoxicity of Human IAPP.

The polypeptide hormone islet amyloid polypeptide (IAPP) forms islet amyloid in type 2 diabetes, a process which contributes to pancreatic ß-cell dysfunction and death. Not all species form islet amyloid, and the ability to do so correlates with the primary sequence. Humans form islet amyloid, but baboon IAPP has not been studied. The baboon peptide differs from human IAPP at three positions containing K1I, H18R, and A25T substitutions. The K1I substitution is a rare example of a replacement in the N-terminal region of amylin. The effect of this mutation on amyloid formation has not been studied, but it reduces the net charge, and amyloid prediction programs suggest that it should increase amyloidogenicity. The A25T replacement involves a nonconservative substitution in a region of IAPP that is believed to be important for aggregation, but the effects of this replacement have not been examined. The H18R point mutant has been previously shown to reduce aggregation in vitro. Baboon amylin forms amyloid on the same timescale as human amylin in vitro and exhibits similar toxicity toward cultured ß-cells. The K1I replacement in human amylin slightly reduces toxicity, whereas the A25T substitution accelerates amyloid formation and enhances toxicity. Photochemical cross-linking reveals that the baboon amylin, like human amylin, forms low-order oligomers in the lag phase of amyloid formation. Ion-mobility mass spectrometry reveals broadly similar gas phase collisional cross sections for human and baboon amylin monomers and dimers, with some differences in the arrival time distributions. Preamyloid oligomers formed by baboon amylin, but not baboon amylin fibers, are toxic to cultured ß-cells. The toxicity of baboon oligomers and lack of significantly detectable toxicity with exogenously added amyloid fibers is consistent with the hypothesis that preamyloid oligomers are the most toxic species produced during IAPP amyloid formation.

Analysis of Prairie Vole Amylin Reveals the Importance of the N-Terminus and Residue 22 in Amyloidogenicity and Cytotoxicity.

Amyloid formation by amylin contributes to ß-cell dysfunction in type 2 diabetes. The features that control the amyloidogenicity and toxicity of amylin are not understood. Not all species form islet amyloid, and its presence or absence correlates with the in vitro behavior of the polypeptide. Rats do not develop type 2 diabetes or islet amyloid, and rat amylin is non-amyloidogenic, except at very high concentrations. This has led to the notion that rodent amylins are non-amyloidogenic. Prairie vole amylin has an unusual sequence compared to those of human and rat amylin, including nonconservative Lys-1 to Glu and Asn-22 to Gly substitutions. The first reduces the net charge on the peptide, while the second disrupts a potential network of side chain hydrogen bonds in the amyloid fiber, a so-called Asn ladder. The prairie vole polypeptide forms amyloid more slowly than human amylin and is considerably less cytotoxic. An Asn-22 to Gly substitution in human amylin significantly reduces toxicity, increasing the effective concentration of amylin required to reach 50% toxicity by >7-fold, but has modest effects on the time to form amyloid. A Lys-1 to Glu replacement has a weaker effect but does reduce toxicity relative to that of human amylin, without having a significant impact on the time to form amyloid. The effect of the Lys-1 to Glu substitution on amyloid kinetics is more significant in Tris buffer than in phosphate-buffered saline. This work demonstrates that the N-terminus of amylin plays a role in modulating toxicity and highlights the key role of position 22.

The small molecule inhibitor anle145c thermodynamically traps human islet amyloid peptide in the form of non-cytotoxic oligomers.

Type 2 diabetes (T2DM) is associated with aggregation of the human islet amyloid polypeptide (hIAPP) into cytotoxic amyloid species. Here we tested the effect of a diphenylpyrazole (DPP)-derived small molecule inhibitor, anle145c, on cytotoxicity and on aggregation properties of hIAPP. We demonstrate that incubation of hIAPP with the inhibitor yields ~10 nm-sized non-toxic oligomers, independent of the initial aggregation state of hIAPP. This suggests that anle145c has a special mode of action in which anle145c-stabilized oligomers act as a thermodynamic sink for the preferred aggregation state of hIAPP and anle145c. We also demonstrate that the inhibitor acts in a very efficient manner, with sub-stoichiometric concentrations of anle145c being sufficient to (i) inhibit hIAPP-induced death of INS-1E cells, (ii) prevent hIAPP fibril formation in solution, and (iii) convert preformed hIAPP fibrils into non-toxic oligomers. Together, these results indicate that anle145c is a promising candidate for inhibition of amyloid formation in T2DM.

Amyloidogenicity and cytotoxicity of des-Lys-1 human amylin provides insight into amylin self-assembly and highlights the difficulties of defining amyloidogenicity.

The polypeptide amylin is responsible for islet amyloid in type 2 diabetes, a process which contributes to ß-cell death in the disease. The role of the N-terminal region of amylin in amyloid formation is relatively unexplored, although removal of the disulfide bridged loop between Cys-2 and Cys-7 accelerates amyloid formation. We examine the des Lys-1 variant of human amylin (h-amylin), a variant which is likely produced in vivo. Lys-1 is a region of high charge density in the h-amylin amyloid fiber. The des Lys-1 polypeptide forms amyloid on the same time scale as wild-type amylin in phosphate buffered saline, but does so more rapidly in Tris. The des Lys-1 variant is somewhat less toxic to cultured INS cells than wild type. The implications for the in vitro mechanism of amyloid formation and for comparative analysis of amyloidogenicity are discussed.

Human islet amyloid polypeptide (hIAPP) aggregation in type 2 diabetes: Correlation between intrinsic physicochemical properties of hIAPP aggregates and their cytotoxicity.

A large number of pathological diseases are known now to be associated with the misfolding and the aberrant oligomerization and deposition of peptides and proteins into various aggregates. One of these peptides is islet amyloid polypeptide (IAPP), which is responsible for amyloid formation in type 2 diabetes. The mechanism of IAPP amyloid formation in vivo and in vitro is not well understood and the factors behind the peptide aggregates toxicity are not fully defined. Therefore, the precise nature of toxic agents still remains to be elucidated. In this context, first we used a complementary biophysical approach to undertake a systematic study of the hIAPP aggregation process with focus on the lag phase, followed by the study of their degrees of toxicity when added to the extracellular medium of pancreatic cells. The structural properties of hIAPP aggregates are characterized by evaluating their size with DLS, their surface hydrophobicity with ANS, and the interactions between monomers through the intrinsic fluorescence of aromatic residues or by the quenching of these residues mainly the tyrosine in position 37. Our results indicate that despite the method used to study hIAPP aggregation, the obtained curve is easily well fitted in a sigmoidal curve but with some differences. In fact, the analysis of the kinetic parameters gives different information about the hIAPP aggregation process such as lag time and growth rate. Moreover, a high surface hydrophobicity and small size of the aggregates, mainly for the species formed during the lag time, shows strong correlation with the cytotoxicity. These findings provide new insights into the structural changes during hIAPP aggregation and are consistent with a model in which the exposure of hydrophobic surfaces and the small size of aggregates formed during the early stage of the process are crucial for their cytotoxicity.

Nitration of hIAPP promotes its toxic oligomer formation and exacerbates its toxicity towards INS-1 cells.

Amyloid formation of human islet amyloid polypeptide (hIAPP) is one of the most common pathological features of type 2 diabetes (T2D). Increasing evidences have shown that the overproduction of reactive oxygen species (ROS) and reactive nitrogen species (RNS) play an important role in the development of the T2D. Interestingly, our previous studies indicated that heme could bind to hIAPP, and the complex might induce the nitration of tyrosine residue (Y37) of hIAPP in the presence of hydrogen peroxide and nitrite. However, it remains unclear about effect of the nitration on the implicated function of hIAPP in the development of T2D. In this study, fluorescent assays, transmission electron microscopy (TEM), atomic force microscope (AFM) were used to demonstrate that nitration of hIAPP significantly decreased its fibril formation. But the decreased fibril formation was not through the diminished aggregation of hIAPP monomer as suggested by the results of circular dichroism spectroscopy (CD) and gel electrophoresis assay. Surface-enhanced raman spectroscopy (SERS) indicated that nitration of hIAPP impaired the intermolecular hydrogen bonding. On the basis of these results, we hypothesize that nitration of hIAPP may block the intermolecular hydrogen bonding, leading to the inhibition of its fibril formation. In addition, cytotoxicity study of native and modified hIAPP was also performed on INS-1 cells, which revealed exacerbated toxicity of hIAPP by its nitration. The findings in this study that nitration of hIAPP promotes its oligomer formation and thus exacerbates its cytotoxicity suggests a possible link between the nitrite (or the sum of nitrite and nitrate) levels and T2D, and ameliorated nitration of hIAPP by diminishing nitrative stress might be a promising therapeutic strategy for T2D.

Silibinin protects rat pancreatic ß-cell through up-regulation of estrogen receptors' signaling against amylin- or Aß1-42 -induced reactive oxygen species/reactive nitrogen species generation.

Amylin and amyloid-ß (Aß) were found to induce reactive oxygen species (ROS) and reactive nitrogen species (RNS) in rat pancreatic ß-cell line, INS-1 cells, leading to cell death. In this study, we report on reciprocal relationship between the expression of estrogen receptors (ERs) α and ß (ERα and ERß) and generation of ROS/RNS in amylin/Aß1-42 -treated INS-1 cells. That is, pharmacological activation of ERs in INS-1 cells significantly decreases ROS/RNS generation, but blockage of ERs increases ROS/RNS generation. Silibinin is a natural polyphenolic flavonoid isolated from milk thistle with phytoestrogen activities, also known as silybin. Treatment with silibinin down-regulated ROS/RNS production induced by treatment with amylin/Aß1-42 in the cells. Silencing ERs expression with siRNAs targeting ERs showed that the protective effect of silibinin was markedly weakened, indicating that silibinin protection is largely attributed to activation of ERs' signaling. The binding of silibinin to ERs implies that the protective effect of silibinin on amylin/Aß1-42 -treated INS-1 cells owes to down-regulation of ROS/RNS through the activation of ERs phosphorylation. Amylin and Aß1-42 cotreatment enhanced furthermore ROS/RNS generation and cytotoxicity through further down-regulation of ERs phosphorylation, and this was reversed by silibinin. Silibinin also protects INS-1 cells from amylin and Aß1-42 cotreatment. These results indicate that protective effect of silibinin is mediated by enhancement of ERs phosphorylation that depresses ROS/RNS generation in amylin/Aß1-42 -treated INS-1 cells.

Sub-Toxic Human Amylin Fragment Concentrations Promote the Survival and Proliferation of SH-SY5Y Cells via the Release of VEGF and HspB5 from Endothelial RBE4 Cells.

Human amylin is a 37-residue peptide hormone (hA1-37) secreted by ß-cells of the pancreas and, along with insulin, is directly associated with type 2 diabetes mellitus (T2DM). Amyloid deposits within the islets of the pancreas represent a hallmark of T2DM. Additionally, amylin aggregates have been found in blood vessels and/or brain of patients with Alzheimer's disease, alone or co-deposited with ß-amyloid. The purpose of this study was to investigate the neuroprotective potential of human amylin in the context of endothelial-neuronal "cross-talk". We initially performed dose-response experiments to examine cellular toxicity (quantified by the [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] MTT assay) of different hA17⁻29 concentrations in endothelial cells (RBE4). In the culture medium of these cells, we also measured heat shock protein B5 (HspB5) levels by ELISA, finding that even a sub-toxic concentration of hA17⁻29 (3 µM) produced an increase of HspB5. Using a cell medium of untreated and RBE4 challenged for 48 h with a sub-toxic concentration of hA17⁻29, we determined the potential beneficial effect of their addition to the medium of neuroblastoma SH-SY5Y cells. These cells were subsequently incubated for 48 h with a toxic concentration of hA17⁻29 (20 µM). We found a complete inhibition of hA17⁻29 toxicity, potentially related to the presence in the conditioned medium not only of HspB5, but also of vascular endothelial growth factor (VEGF). Pre-treating SH-SY5Y cells with the anti-Flk1 antibody, blocking the VEGF receptor 2 (VEGFR2), significantly decreased the protective effects of the conditioned RBE4 medium. These data, obtained by indirectly measuring VEGF activity, were strongly corroborated by the direct measurement of VEGF levels in conditioned RBE4 media as detected by ELISA. Altogether, these findings highlighted a novel role of sub-toxic concentrations of human amylin in promoting the secretion of proteic factors by endothelial cells (HspB5 and VEGF) that support the survival and proliferation of neuron-like cells.

Graphene quantum dots against human IAPP aggregation and toxicity in vivo.

The development of biocompatible nanomaterials has become a new frontier in the detection, treatment and prevention of human amyloid diseases. Here we demonstrated the use of graphene quantum dots (GQDs) as a potent inhibitor against the in vivo aggregation and toxicity of human islet amyloid polypeptide (IAPP), a hallmark of type 2 diabetes. GQDs initiated contact with IAPP through electrostatic and hydrophobic interactions as well as hydrogen bonding, which subsequently drove the peptide fibrillization off-pathway to eliminate the toxic intermediates. Such interactions, probed in vitro by a thioflavin T kinetic assay, fluorescence quenching, circular dichroism spectroscopy, a cell viability assay and in silico by discrete molecular dynamics simulations, translated to a significant recovery of embryonic zebrafish from the damage elicited by IAPP in vivo, as indicated by improved hatching as well as alleviated reactive oxygen species production, abnormality and mortality of the organism. This study points to the potential of using zero-dimensional nanomaterials for in vivo mitigation of a range of amyloidosis.

ROS­mediated autophagy through the AMPK signaling pathway protects INS­1 cells from human islet amyloid polypeptide­induced cytotoxicity.

Oligomerization of human islet amyloid polypeptide (hIAPP) is toxic and contributes to progressive reduction of ß cell mass in patients with type 2 diabetes mellitus. Autophagy is a highly conserved homeostatic mechanism in eukaryotes. Previous studies have confirmed that hIAPP can promote autophagy in ß cells, but the underlying molecular mechanism and cellular regulatory pathway of hIAPP­induced autophagy remains not fully elucidated. Accumulation of reactive oxygen species (ROS) causes hIAPP induced­ß cell death. At present, little is known about the association between hIAPP­induced oxidative stress and autophagy in ß cells. Therefore, the present study investigated the underlying molecular mechanism and regulatory pathway of hIAPP­induced autophagy. Transmission electron microscopy was used to observe the number of autophagosome in cells. Cell viability was determined by an MTT test. A 2',7'­dichlorofluorescin diacetate assay was used to measure the relative levels of reactive ROS. Western blotting was used to detect expression of adenosine monophosphate­activated protein kinase (AMPK) and autophagic markers p62 and microtubule associated protein 1 light chain 3. The results demonstrated that hIAPP induces autophagy through ROS­mediated AMPK signaling pathway in INS­1 cells. Upregulation of autophagy by AMPK activator 5­aminoimidazole­4­carboxamide1­ß­D­ribofuranoside decreased ROS and malondialdehyde generation, whereas inhibition of autophagy by 3­methyladenine and AMPK inhibitor compound C aggravated hIAPP­induced oxidative stress and toxicity in INS­1 cells. Taken together, the present study suggested that hIAPP induces autophagy via a ROS­mediated AMPK signaling pathway. Furthermore, autophagy serves as a cell­protective mechanism against hIAPP­induced toxicity and chemical promotion of autophagy through AMPK signaling pathway attenuates hIAPP induced cytotoxicity and oxidative stress in INS­1 cells.

The receptor for advanced glycation endproducts is a mediator of toxicity by IAPP and other proteotoxic aggregates: Establishing and exploiting common ground for novel amyloidosis therapies.

Proteotoxicity plays a key role in many devastating human disorders, including Alzheimer's, Huntington's and Parkinson's diseases; type 2 diabetes; systemic amyloidosis; and cardiac dysfunction, to name a few. The cellular mechanisms of proteotoxicity in these disorders have been the focus of considerable research, but their role in prevalent and morbid disorders, such as diabetes, is less appreciated. There is a large body of literature on the impact of glucotoxicity and lipotoxicity on insulin-producing pancreatic ß-cells, and there is increasing recognition that proteotoxicty plays a key role. Pancreatic islet amyloidosis by the hormone IAPP, the production of advanced glycation endproducts (AGE), and insulin misprocessing into cytotoxic aggregates are all sources of ß-cell proteotoxicity in diabetes. AGE, produced by the reaction of reducing sugars with proteins and lipids are ligands for the receptor for AGE (RAGE), as are the toxic pre-fibrillar aggregates of IAPP produced during amyloid formation. The mechanisms of amyloid formation by IAPP in vivo or in vitro are not well understood, and the cellular mechanisms of IAPP-induced ß-cell death are not fully defined. Here, we review recent findings that illuminate the factors and mechanisms involved in ß-cell proteotoxicity in diabetes. Together, these new insights have far-reaching implications for the establishment of unifying mechanisms by which pathological amyloidoses imbue their injurious effects in vivo.