Phosphorylated α-synuclein in skin Schwann cells: a new biomarker for multiple system atrophy.

Multiple system atrophy (MSA) is characterized by accumulation of phosphorylated α-synuclein (p-syn) as glial cytoplasmic inclusions in the brain and a specific biomarker for this disorder is urgently needed. We aimed at investigating if p-syn can also be detected in skin Remak non-myelinating Schwann cells (RSCs) as Schwann cell cytoplasmic inclusions (SCCi) and may represent a reliable clinical biomarker for MSA. This cross-sectional diagnostic study evaluated skin p-syn in 96 patients: 46 with probable MSA (29 with parkinsonism type MSA and 17 with cerebellar type MSA), 34 with Parkinson's disease (PD) and 16 with dementia with Lewy bodies (DLB). We also included 50 healthy control subjects. Patients were recruited from five different medical centres. P-syn aggregates in skin sections were stained by immunofluorescence, followed by analyses with confocal microscopy and immuno-electron microscopy. All analyses were performed in a blinded fashion. Overall, p-syn aggregates were found in 78% of MSA patients and 100% of patients with PD/DLB, whereas they could not be detected in controls. As for neuronal aggregates 78% of MSA patients were positive for p-syn in somatic neurons, whereas all PD/DLB patients were positive in autonomic neurons. When analysing the presence of p-syn in RSCs, 74% of MSA patients were positive, whereas no such SCCi could be observed in PD/DLB patients. Analyses by immuno-electron microscopy confirmed that SCCi were only found in cases with MSA and thus absent in those with PD/DLB. In conclusion, our findings demonstrate that (i) fibrillar p-syn in RSCs is a pathological hallmark of MSA and may be used as a specific and sensitive disease biomarker; (ii) in Lewy body synucleinopathies (PD/DLB) only neurons contain p-syn deposits; and (iii) the cell-specific deposition of p-syn in the skin thus mirrors that of the brain in many aspects and suggests that non-myelinated glial cells are also involved in the MSA pathogenesis.

Proteostasis of Islet Amyloid Polypeptide: A Molecular Perspective of Risk Factors and Protective Strategies for Type II Diabetes.

The possible link between hIAPP accumulation and ß-cell death in diabetic patients has inspired numerous studies focusing on amyloid structures and aggregation pathways of this hormone. Recent studies have reported on the importance of early oligomeric intermediates, the many roles of their interactions with lipid membrane, pH, insulin, and zinc on the mechanism of aggregation of hIAPP. The challenges posed by the transient nature of amyloid oligomers, their structural heterogeneity, and the complex nature of their interaction with lipid membranes have resulted in the development of a wide range of biophysical and chemical approaches to characterize the aggregation process. While the cellular processes and factors activating hIAPP-mediated cytotoxicity are still not clear, it has recently been suggested that its impaired turnover and cellular processing by proteasome and autophagy may contribute significantly toward toxic hIAPP accumulation and, eventually, ß-cell death. Therefore, studies focusing on the restoration of hIAPP proteostasis may represent a promising arena for the design of effective therapies. In this review we discuss the current knowledge of the structures and pathology associated with hIAPP self-assembly and point out the opportunities for therapy that a detailed biochemical, biophysical, and cellular understanding of its aggregation may unveil.

Formation of amyloid in encapsulated human pancreatic and human stem cell-generated beta cell implants.

Detection of amyloid in intraportal islet implants of type 1 diabetes patients has been proposed as cause in their functional decline. The present study uses cultured adult human islets devoid of amyloid to examine conditions of its formation. After intraportal injection in patients, amyloid deposits <15 µm diameter were identified in 5%-12% of beta cell containing aggregates, 3-76 months posttransplant. Such deposits also formed in glucose-controlling islet implants in the kidney of diabetic mice but not in failing implants. Alginate-encapsulated islets formed amyloid during culture when functional, and in all intraperitoneal implants that corrected diabetes in mice, exhibiting larger sizes than in functioning nonencapsulated implants. After intraperitoneal injection in a patient, retrieved single capsules presented amyloid near living beta cells, whereas no amyloid occurred in clustered capsules with dead cells. Amyloid was also demonstrated in functional human stem cell-generated beta cell implants in subcutaneous devices of mice. Deposits up to 35 µm diameter were localized in beta cell-enriched regions and related to an elevated IAPP over insulin ratio in the newly generated beta cells. Amyloid in device-encapsulated human stem cell-generated beta cell implants marks the formation of a functional beta cell mass but also an imbalance between its activated state and its microenvironment.

BRICHOS domain of Bri2 inhibits islet amyloid polypeptide (IAPP) fibril formation and toxicity in human beta cells.

Aggregation of islet amyloid polypeptide (IAPP) into amyloid fibrils in islets of Langerhans is associated with type 2 diabetes, and formation of toxic IAPP species is believed to contribute to the loss of insulin-producing beta cells. The BRICHOS domain of integral membrane protein 2B (Bri2), a transmembrane protein expressed in several peripheral tissues and in the brain, has recently been shown to prevent fibril formation and toxicity of Aß42, an amyloid-forming peptide in Alzheimer disease. In this study, we demonstrate expression of Bri2 in human islets and in the human beta-cell line EndoC-ßH1. Bri2 colocalizes with IAPP intracellularly and is present in amyloid deposits in patients with type 2 diabetes. The BRICHOS domain of Bri2 effectively inhibits fibril formation in vitro and instead redirects IAPP into formation of amorphous aggregates. Reduction of endogenous Bri2 in EndoC-ßH1 cells with siRNA increases sensitivity to metabolic stress leading to cell death while a concomitant overexpression of Bri2 BRICHOS is protective. Also, coexpression of IAPP and Bri2 BRICHOS in lateral ventral neurons of Drosophila melanogaster results in an increased cell survival. IAPP is considered to be the most amyloidogenic peptide known, and described findings identify Bri2, or in particular its BRICHOS domain, as an important potential endogenous inhibitor of IAPP aggregation and toxicity, with the potential to be a possible target for the treatment of type 2 diabetes.

The Amyloid Forming Peptides Islet Amyloid Polypeptide and Amyloid ß Interact at the Molecular Level.

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 ß (Aß) could constitute the link. The bimolecular fluorescence complementation (BiFC) assay was used to investigate the occurrence of heterologous interactions between IAPP and Aß and to compare the potential toxic effects of IAPP/Aß, IAPP/IAPP, and Aß/Aß 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 Aß was used to study their co-deposition and effects on longevity. We showed that the co-expression of IAPP and Aß resulted in fluorophore reconstitution to the same extent as determined for homologous IAPP/IAPP or Aß/Aß expression. The BiFC(+)/BiFC(-) ratio of lysosomal area calculations increased in transfected cells independent of the vector combinations, while only Aß/Aß expression increased mitochondrial membrane potential. Expression combinations containing Aß were necessary for the formation of a congophilic amyloid. In Drosophila melanogaster expressing IAPP/Aß, co-deposition of the amyloid-forming peptides caused reduced longevity. The BiFC results confirmed a heterologous interaction between IAPP and Aß, while co-deposits in the brain of Drosophila suggest mixed amyloid aggregates.

Electron tomography reveals the fibril structure and lipid interactions in amyloid deposits.

Electron tomography is an increasingly powerful method to study the detailed architecture of macromolecular complexes or cellular structures. Applied to amyloid deposits formed in a cell culture model of systemic amyloid A amyloidosis, we could determine the structural morphology of the fibrils directly in the deposit. The deposited fibrils are arranged in different networks, and depending on the relative fibril orientation, we can distinguish between fibril meshworks, fibril bundles, and amyloid stars. These networks are frequently infiltrated by vesicular lipid inclusions that may originate from the death of the amyloid-forming cells. Our data support the role of nonfibril components for constructing fibril deposits and provide structural views of different types of lipid-fibril interactions.

Human Astrocytes Transfer Aggregated Alpha-Synuclein via Tunneling Nanotubes.

Many lines of evidence suggest that the Parkinson's disease (PD)-related protein α-synuclein (α-SYN) can propagate from cell to cell in a prion-like manner. However, the cellular mechanisms behind the spreading remain elusive. Here, we show that human astrocytes derived from embryonic stem cells actively transfer aggregated α-SYN to nearby astrocytes via direct contact and tunneling nanotubes (TNTs). Failure in the astrocytes' lysosomal digestion of excess α-SYN oligomers results in α-SYN deposits in the trans-Golgi network followed by endoplasmic reticulum swelling and mitochondrial disturbances. The stressed astrocytes respond by conspicuously sending out TNTs, enabling intercellular transfer of α-SYN to healthy astrocytes, which in return deliver mitochondria, indicating a TNT-mediated rescue mechanism. Using a pharmacological approach to inhibit TNT formation, we abolished the transfer of both α-SYN and mitochondria. Together, our results highlight the role of astrocytes in α-SYN cell-to-cell transfer, identifying possible pathophysiological events in the PD brain that could be of therapeutic relevance.SIGNIFICANCE STATEMENT Astrocytes are the major cell type in the brain, yet their role in Parkinson's disease progression remains elusive. Here, we show that human astrocytes actively transfer aggregated α-synuclein (α-SYN) to healthy astrocytes via direct contact and tunneling nanotubes (TNTs), rather than degrade it. The astrocytes engulf large amounts of oligomeric α-SYN that are subsequently stored in the trans-Golgi network region. The accumulation of α-SYN in the astrocytes affects their lysosomal machinery and induces mitochondrial damage. The stressed astrocytes respond by sending out TNTs, enabling intercellular transfer of α-SYN to healthy astrocytes. Our findings highlight an unexpected role of astrocytes in the propagation of α-SYN pathology via TNTs, revealing astrocytes as a potential target for therapeutic intervention.

Islet amyloid polypeptide, islet amyloid, and diabetes mellitus.

Islet amyloid polypeptide (IAPP, or amylin) is one of the major secretory products of ß-cells of the pancreatic islets of Langerhans. It is a regulatory peptide with putative function both locally in the islets, where it inhibits insulin and glucagon secretion, and at distant targets. It has binding sites in the brain, possibly contributing also to satiety regulation and inhibits gastric emptying. Effects on several other organs have also been described. IAPP was discovered through its ability to aggregate into pancreatic islet amyloid deposits, which are seen particularly in association with type 2 diabetes in humans and with diabetes in a few other mammalian species, especially monkeys and cats. Aggregated IAPP has cytotoxic properties and is believed to be of critical importance for the loss of ß-cells in type 2 diabetes and also in pancreatic islets transplanted into individuals with type 1 diabetes. This review deals both with physiological aspects of IAPP and with the pathophysiological role of aggregated forms of IAPP, including mechanisms whereby human IAPP forms toxic aggregates and amyloid fibrils.

Transplantation of macroencapsulated human islets within the bioartificial pancreas ßAir to patients with type 1 diabetes mellitus.

Macroencapsulation devices provide the dual possibility of immunoprotecting transplanted cells while also being retrievable, the latter bearing importance for safety in future trials with stem cell-derived cells. However, macroencapsulation entails a problem with oxygen supply to the encapsulated cells. The ßAir device solves this with an incorporated refillable oxygen tank. This phase 1 study evaluated the safety and efficacy of implanting the ßAir device containing allogeneic human pancreatic islets into patients with type 1 diabetes. Four patients were transplanted with 1-2 ßAir devices, each containing 155 000-180 000 islet equivalents (ie, 1800-4600 islet equivalents per kg body weight), and monitored for 3-6 months, followed by the recovery of devices. Implantation of the ßAir device was safe and successfully prevented immunization and rejection of the transplanted tissue. However, although beta cells survived in the device, only minute levels of circulating C-peptide were observed with no impact on metabolic control. Fibrotic tissue with immune cells was formed in capsule surroundings. Recovered devices displayed a blunted glucose-stimulated insulin response, and amyloid formation in the endocrine tissue. We conclude that the ßAir device is safe and can support survival of allogeneic islets for several months, although the function of the transplanted cells was limited (Clinicaltrials.gov: NCT02064309).

The human serum protein C4b-binding protein inhibits pancreatic IAPP-induced inflammasome activation.

AIMS/HYPOTHESIS: Inflammasome activation and subsequent IL-1ß production is a driver of islet pathology in type 2 diabetes. Oligomers, but not mature amyloid fibrils, of human islet amyloid polypeptide (IAPP), which is co-secreted with insulin, trigger NOD-like receptor pyrin domain containing-3 (NLRP3) inflammasome activation. C4b-binding protein (C4BP), present in serum, binds to IAPP and affects transition of IAPP monomers and oligomers to amyloid fibrils. We therefore hypothesised that C4BP inhibits IAPP-mediated inflammasome activation and IL-1ß production. METHODS: Macrophages were exposed to IAPP in the presence or absence of plasma-purified human C4BP, and inflammasome activation was assessed by IL-1ß secretion as detected by ELISA and reporter cell lines. IAPP fibrillation was assessed by thioflavin T assay. Uptake of IAPP-C4BP complexes and their effects on phagolysosomal stability were assessed by flow cytometry and confocal microscopy. The effect of C4BP regulation of IAPP-mediated inflammasome activation on beta cell function was assessed using a clonal rat beta cell line. Immunohistochemistry was used to examine the association of IAPP amyloid deposits and macrophage infiltration in isolated human and mouse pancreatic islets, and expression of C4BP from isolated human pancreatic islets was assessed by quantitative PCR, immunohistochemistry and western blot. RESULTS: C4BP significantly inhibited IAPP-mediated IL-1ß secretion from primed macrophages at physiological concentrations in a dose-dependent manner. C4BP bound to and was internalised together with IAPP. C4BP did not affect IAPP uptake into phagolysosomal compartments, although it did inhibit its formation into amyloid fibrils. The loss of macrophage phagolysosomal integrity induced by IAPP incubation was inhibited by co-incubation with C4BP. Supernatant fractions from macrophages activated with IAPP inhibited both insulin secretion and viability of clonal beta cells in an IL-1ß-dependent manner but the presence of C4BP during macrophage IAPP incubation rescued beta cell function and viability. In human and mouse islets, the presence of amyloid deposits correlated with higher numbers of infiltrating macrophages. Isolated human islets expressed and secreted C4BP, which increased with addition of IL-1ß. CONCLUSIONS/INTERPRETATION: IAPP deposition is associated with inflammatory cell infiltrates in pancreatic islets. C4BP blocks IAPP-induced inflammasome activation by preventing the loss of macrophage phagolysosomal integrity required for NLRP3 activation. The consequence of this is the preservation of beta cell function and viability. C4BP is secreted directly from human pancreatic islets and this increases in response to inflammatory cytokines. We therefore propose that C4BP acts as an extracellular chaperone protein that limits the proinflammatory effects of IAPP.

C4b-binding Protein Protects ß-Cells from Islet Amyloid Polypeptide-induced Cytotoxicity.

C4BP (C4b-binding protein) is a polymer of seven identical α chains and one unique ß chain synthesized in liver and pancreas. We showed previously that C4BP enhances islet amyloid polypeptide (IAPP) fibril formation in vitro Now we report that polymeric C4BP strongly inhibited lysis of human erythrocytes incubated with monomeric IAPP, whereas no lysis was observed after incubation with preformed IAPP fibrils. In contrast, incubation with the monomeric α-chain of C4BP was less effective. These data indicate that polymeric C4BP with multiple binding sites for IAPP neutralizes lytic activity of IAPP. Furthermore, addition of monomeric IAPP to a rat insulinoma cell line (INS-1) resulted in decreased cell viability, which was restored in the presence of physiological concentrations of C4BP. Treatment of INS-1 cells and primary rat islets with IAPP also diminished their ability to secrete insulin upon stimulation with glucose, which was reversed in the presence of C4BP. Further, C4BP was internalized together with IAPP into INS-1 cells. Pathway analyses of mRNA expression microarray data indicated that cells exposed to C4BP and IAPP in comparison with IAPP alone increased expression of genes involved in cholesterol synthesis. Depletion of cholesterol through methyl-ß-cyclodextrin or cholesterol oxidase abolished the protective effect of C4BP on IAPP cytotoxicity of INS-1 cells. Also, inhibition of phosphoinositide 3-kinase but not NF-κB had a similar effect. Taken together, C4BP protects ß-cells from IAPP cytotoxicity by modulating IAPP fibril formation extracellularly and also, after uptake by the cells, by enhancing cholesterol synthesis.

Heparan Sulfate Proteoglycans Are Important for Islet Amyloid Formation and Islet Amyloid Polypeptide-induced Apoptosis.

Deposition of ß cell toxic islet amyloid is a cardinal finding in type 2 diabetes. In addition to the main amyloid component islet amyloid polypeptide (IAPP), heparan sulfate proteoglycan is constantly present in the amyloid deposit. Heparan sulfate (HS) side chains bind to IAPP, inducing conformational changes of the IAPP structure and an acceleration of fibril formation. We generated a double-transgenic mouse strain (hpa-hIAPP) that overexpresses human heparanase and human IAPP but is deficient of endogenous mouse IAPP. Culture of hpa-hIAPP islets in 20 mm glucose resulted in less amyloid formation compared with the amyloid load developed in cultured islets isolated from littermates expressing human IAPP only. A similar reduction of amyloid was achieved when human islets were cultured in the presence of heparin fragments. Furthermore, we used CHO cells and the mutant CHO pgsD-677 cell line (deficient in HS synthesis) to explore the effect of cellular HS on IAPP-induced cytotoxicity. Seeding of IAPP aggregation on CHO cells resulted in caspase-3 activation and apoptosis that could be prevented by inhibition of caspase-8. No IAPP-induced apoptosis was seen in HS-deficient CHO pgsD-677 cells. These results suggest that ß cell death caused by extracellular IAPP requires membrane-bound HS. The interaction between HS and IAPP or the subsequent effects represent a possible therapeutic target whose blockage can lead to a prolonged survival of ß cells.

In vivo seeding and cross-seeding of localized amyloidosis: a molecular link between type 2 diabetes and Alzheimer disease.

Several proteins have been identified as amyloid forming in humans, and independent of protein origin, the fibrils are morphologically similar. Therefore, there is a potential for structures with amyloid seeding ability to induce both homologous and heterologous fibril growth; thus, molecular interaction can constitute a link between different amyloid forms. Intravenous injection with preformed fibrils from islet amyloid polypeptide (IAPP), proIAPP, or amyloid-beta (Aß) into human IAPP transgenic mice triggered IAPP amyloid formation in pancreas in 5 of 7 mice in each group, demonstrating that IAPP amyloid could be enhanced through homologous and heterologous seeding with higher efficiency for the former mechanism. Proximity ligation assay was used for colocalization studies of IAPP and Aß in islet amyloid in type 2 diabetic patients and Aß deposits in brains of patients with Alzheimer disease. Aß reactivity was not detected in islet amyloid although islet ß cells express AßPP and convertases necessary for Aß production. By contrast, IAPP and proIAPP were detected in cerebral and vascular Aß deposits, and presence of proximity ligation signal at both locations showed that the peptides were <40 nm apart. It is not clear whether IAPP present in brain originates from pancreas or is locally produced. Heterologous seeding between IAPP and Aß shown here may represent a molecular link between type 2 diabetes and Alzheimer disease.

Efficient amyloid A clearance in the absence of immunoglobulins and complement factors.

Amyloid A amyloidosis is a protein misfolding disease characterized by deposition of extracellular aggregates derived from the acute-phase reactant serum amyloid A protein. If untreated, amyloid A amyloidosis leads to irreversible damage of various organs, including the kidneys, liver, and heart. Amyloid A deposits regress upon reduction of serum amyloid A concentration, indicating that the amyloid can be efficiently cleared by natural mechanisms. Clearance was proposed to be mediated by humoral immune responses to amyloid. Here, we report that amyloid clearance in mice lacking complement factors 3 and 4 (C3C4(-/-)) was equally efficient as in wild-type mice (C57BL/6), and was only slightly delayed in agammaglobulinemic mice (J(H-/-)). Hence, antibodies or complement factors are not necessary for natural amyloid clearance, implying the existence of alternative physiological pathways for amyloid removal.

Insulin release from isolated cat islets of Langerhans.

Feline diabetes mellitus is a common endocrine disease with increasing prevalence. It shows similarities with human type 2 diabetes and is characterized by insulin resistance and deficient insulin secretion. Moreover, cats and humans belong to the very few species that form amyloid depositions in the pancreatic islets. However, little is known about cat islet function and no studies have addressed insulin secretion from isolated islets ex vivo. The aim of this study was to establish a protocol for isolation of islets of Langerhans from pancreata of cats euthanized due to disease, and to evaluate insulin secretion responses to various physiological and pharmacological stimuli. Collagenase digestion of pancreatic tissue from 13 non-diabetic cats and two cats with diabetic ketoacidosis yielded individual islets surrounded by a layer of exocrine tissue that was reduced after two days in culture. Histological examination showed islet amyloid in pancreatic biopsies from most non-diabetic and in one diabetic cat. Islets from non-diabetic cats cultured at 5.5 mM glucose responded with increased insulin secretion to 16.7 mM glucose, 30 mM K+ and 20 µM of the sulfonylurea glipizide (2-3 times basal secretion at 3 mM glucose). The glucagon-like peptide-1 receptor agonist exendin-4 (100 nM) had no effect under basal conditions but potentiated glucose-triggered insulin release. Only one of nine islet batches from diabetic cats released detectable amounts of insulin, which was enhanced by exendin-4. Culture of islets from non-diabetic cats at 25 mM glucose impaired secretion both in response to glucose and K+ depolarization. In conclusion, we describe a procedure for isolation of islets from cat pancreas biopsies and demonstrate that isolated cat islets secrete insulin in response to glucose and antidiabetic drugs. The study provides a basis for future ex vivo studies of islet function relevant to the understanding of the pathophysiology and treatment of feline diabetes.

Efficient Vascular and Neural Engraftment of Stem Cell-Derived Islets.

Pluripotent stem cell-derived islets (SC-islets) have emerged as a new source for ß-cell replacement therapy. The function of human islet transplants is hampered by excessive cell death posttransplantation; contributing factors include inflammatory reactions, insufficient revascularization, and islet amyloid formation. However, there is a gap in knowledge of the engraftment process of SC-islets. In this experimental study, we investigated the engraftment capability of SC-islets at 3 months posttransplantation and observed that cell apoptosis rates were lower but vascular density was similar in SC-islets compared with human islets. Whereas the human islet transplant vascular structures were a mixture of remnant donor endothelium and ingrowing blood vessels, the SC-islets contained ingrowing blood vessels only. Oxygenation in the SC-islet grafts was twice as high as that in the corresponding grafts of human islets, suggesting better vascular functionality. Similar to the blood vessel ingrowth, reinnervation of the SC-islets was four- to fivefold higher than that of the human islets. Both SC-islets and human islets contained amyloid at 1 and 3 months posttransplantation. We conclude that the vascular and neural engraftment of SC-islets are superior to those of human islets, but grafts of both origins develop amyloid, with potential long-term consequences.

Islet amyloid polypeptide triggers limited complement activation and binds complement inhibitor C4b-binding protein, which enhances fibril formation.

Islet amyloid polypeptide (IAPP) is synthesized in pancreatic ß-cells and co-secreted with insulin. Aggregation and formation of IAPP-amyloid play a critical role in ß-cell death in type 2 diabetic patients. Because Aß-fibrils in Alzheimer disease activate the complement system, we have here investigated specific interactions between IAPP and complement factors. IAPP fibrils triggered limited activation of complement in vitro, involving both the classical and the alternative pathways. Direct binding assays confirmed that IAPP fibrils interact with globular head domains of complement initiator C1q. Furthermore, IAPP also bound complement inhibitors factor H and C4b-binding protein (C4BP). Recombinant C4BP mutants were used to show that complement control protein (CCP) domains 8 and 2 of the α-chain were responsible for the strong, hydrophobic binding of C4BP to IAPP. Immunostaining of pancreatic sections from type 2 diabetic patients revealed the presence of complement factors in the islets and varying degree of co-localization between IAPP fibrils and C1q, C3d, as well as C4BP and factor H but not membrane attack complex. Furthermore, C4BP enhanced formation of IAPP fibrils in vitro. We conclude that C4BP binds to IAPP thereby limiting complement activation and may be enhancing formation of IAPP fibrils from cytotoxic oligomers.

Studies on alpha-synuclein and islet amyloid polypeptide interaction.

Introduction: Parkinson's disease and type 2 diabetes have both elements of local amyloid depositions in their pathogenesis. In Parkinson's disease, alpha-synuclein (aSyn) forms insoluble Lewy bodies and Lewy neurites in brain neurons, and in type 2 diabetes, islet amyloid polypeptide (IAPP) comprises the amyloid in the islets of Langerhans. In this study, we assessed the interaction between aSyn and IAPP in human pancreatic tissues, both ex vivo and in vitro. Material and Methods: The antibody-based detection techniques, proximity ligation assay (PLA), and immuno-TEM were used for co-localization studies. Bifluorescence complementation (BiFC) was used for interaction studies between IAPP and aSyn in HEK 293 cells. The Thioflavin T assay was used for studies of cross-seeding between IAPP and aSyn. ASyn was downregulated with siRNA, and insulin secretion was monitored using TIRF microscopy. Results: We demonstrate intracellular co-localization of aSyn with IAPP, while aSyn is absent in the extracellular amyloid deposits. ASyn reactivity is present in the secretory granules of ß-cells and some α-cells in human islets. The BiFC-expression of aSyn/aSyn and IAPP/IAPP in HEK293 cells resulted in 29.3% and 19.7% fluorescent cells, respectively, while aSyn/IAPP co-expression resulted in ∼10% fluorescent cells. Preformed aSyn fibrils seeded IAPP fibril formation in vitro, but adding preformed IAPP seeds to aSyn did not change aSyn fibrillation. In addition, mixing monomeric aSyn with monomeric IAPP did not affect IAPP fibril formation. Finally, the knockdown of endogenous aSyn did not affect ß cell function or viability, nor did overexpression of aSyn affect ß cell viability. Discussion: Despite the proximity of aSyn and IAPP in ß-cells and the detected capacity of preformed aSyn fibrils to seed IAPP in vitro, it is still an open question if an interaction between the two molecules is of pathogenic significance for type 2 diabetes.

CLEC11A improves insulin secretion and promotes cell proliferation in human beta-cells.

Beta-cell dysfunction is a hallmark of disease progression in patients with diabetes. Research has been focused on maintaining and restoring beta-cell function during diabetes development. The aims of this study were to explore the expression of C-type lectin domain containing 11A (CLEC11A), a secreted sulphated glycoprotein, in human islets and to evaluate the effects of CLEC11A on beta-cell function and proliferation in vitro. To test these hypotheses, human islets and human EndoC-ßH1 cell line were used in this study. We identified that CLEC11A was expressed in beta-cells and alpha-cells in human islets but not in EndoC-ßH1 cells, whereas the receptor of CLEC11A called integrin subunit alpha 11 was found in both human islets and EndoC-ßH1 cells. Long-term treatment with exogenous recombinant human CLEC11A (rhCLEC11A) accentuated glucose-stimulated insulin secretion, insulin content, and proliferation from human islets and EndoC-ßH1 cells, which was partially due to the accentuated expression levels of transcription factors MAFA and PDX1. However, the impaired beta-cell function and reduced mRNA expression of INS and MAFA in EndoC-ßH1 cells that were caused by chronic palmitate exposure could only be partially improved by the introduction of rhCLEC11A. Based on these results, we conclude that rhCLEC11A promotes insulin secretion, insulin content, and proliferation in human beta-cells, which are associated with the accentuated expression levels of transcription factors MAFA and PDX1. CLEC11A, therefore, may provide a novel therapeutic target for maintaining beta-cell function in patients with diabetes.

ß-cell loss and ß-cell apoptosis in human type 2 diabetes are related to islet amyloid deposition.

Amyloid deposition and reduced ß-cell mass are pathological hallmarks of the pancreatic islet in type 2 diabetes; however, whether the extent of amyloid deposition is associated with decreased ß-cell mass is debated. We investigated the possible relationship and, for the first time, determined whether increased islet amyloid and/or decreased ß-cell area quantified on histological sections is correlated with increased ß-cell apoptosis. Formalin-fixed, paraffin-embedded human pancreas sections from subjects with (n = 29) and without (n = 39) diabetes were obtained at autopsy (64 ± 2 and 70 ± 4 islets/subject, respectively). Amyloid and ß cells were visualized by thioflavin S and insulin immunolabeling. Apoptotic ß cells were detected by colabeling for insulin and by TUNEL. Diabetes was associated with increased amyloid deposition, decreased ß-cell area, and increased ß-cell apoptosis, as expected. There was a strong inverse correlation between ß-cell area and amyloid deposition (r = -0.42, P < 0.001). ß-Cell area was selectively reduced in individual amyloid-containing islets from diabetic subjects, compared with control subjects, but amyloid-free islets had ß-cell area equivalent to islets from control subjects. Increased amyloid deposition was associated with ß-cell apoptosis (r = 0.56, P < 0.01). Thus, islet amyloid is associated with decreased ß-cell area and increased ß-cell apoptosis, suggesting that islet amyloid deposition contributes to the decreased ß-cell mass that characterizes type 2 diabetes.