ACTAUNIVERSITATIS

UPSALIENSISUPPSALA

2015

Digital Comprehensive Summaries of Uppsala Dissertationsfrom the Faculty of Medicine 1158

Islet amyloid polypeptide (IAPP)in Type 2 diabetes and Alzheimerdisease

MARIE OSKARSSON

ISSN 1651-6206ISBN 978-91-554-9400-1urn:nbn:se:uu:diva-265501

Dissertation presented at Uppsala University to be publicly examined in B21, BMC,Husargatan 3, Uppsala, Thursday, 17 December 2015 at 13:15 for the degree of Doctor ofPhilosophy (Faculty of Medicine). The examination will be conducted in English. Facultyexaminer: Professor Mikael Oliveberg (Stockholm University, Department of Biochemistryand Biophysics).

AbstractOskarsson, M. 2015. Islet amyloid polypeptide (IAPP) in Type 2 diabetes and Alzheimerdisease. Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty ofMedicine 1158. 55 pp. Uppsala: Acta Universitatis Upsaliensis. ISBN 978-91-554-9400-1.

The misfolding and aggregation of the beta cell hormone islet amyloid polypeptide (IAPP)into amyloid fibrils is the main pathological finding in islets of Langerhans in type 2 diabetes.Pathological assemblies of IAPP are cytotoxic and believed to contribute to the loss of insulin-producing beta cells. Changes in the microenvironment that could trigger the aggregation ofIAPP are largely unknown. So is the possibility that islet amyloid can spread within or betweentissues. The present thesis have explored the roles of glycosaminoglycan heparan sulfate (HS)and the novel anti-amyloid chaperone Bri2 BRICHOS domain in the assembly of IAPP amyloidand cytotoxic IAPP aggregates. Furthermore, cross-seeding as a molecular interaction betweenthe observed connection of type 2 diabetes and Alzheimer disease has been examined.

The N-terminal region of IAPP was required for binding to HS structures and induction ofbinding promoted amyloid formation. Interference in the HS-IAPP interaction by heparanasedegradation of HS or by introducing short, soluble HS-structure fragments reduced amyloiddeposition in cultured islets. Cytotoxicity induced by extracellular, aggregating IAPP wasmediated via interactions with cell-surface HS. This suggests that HS plays an important rolein islet amyloid deposition and associated toxicity.

BRICHOS domain containing protein Bri2 was highly expressed in human beta cells andcolocalized with IAPP intracellularly and in islet amyloid deposits. The BRICHOS domaineffectively attenuated both IAPP amyloid formation and IAPP-induced cytotoxicity. Theseresults propose Bri2 BRICHOS as a novel chaperone preventing IAPP aggregation in beta cells.

The intravenous injection of IAPP, proIAPP or amyloid-β (Aβ) fibrils enhanced isletamyloidosis in transgenic human IAPP mice, demonstrating that both homologous- andheterologous seeding of islet amyloid can occur in vivo. IAPP colocalized with Aβ inbrain amyloid from AD patients, and AD patients diagnosed with T2D displayed increasedproportions of neuritic plaques, the more pathogenic plaque subtype.

In conclusion, both IAPP amyloid formation and the cytotoxic effects of IAPP is dependenton interactions with HS whereas interactions with Bri2 BRICHOS is protective. Cross-seedingbetween Aβ and IAPP can occur in vivo and the two peptides colocalize in brain amyloid inAD patients.

Keywords: amyloid, IAPP, Abeta, type 2 diabetes, Alzheimer disease, BRICHOS, heparansulfate, islet amyloid, amyloid plaques, seeding

Marie Oskarsson, Department of Medical Cell Biology, Box 571, Uppsala University,SE-75123 Uppsala, Sweden.

© Marie Oskarsson 2015

ISSN 1651-6206ISBN 978-91-554-9400-1urn:nbn:se:uu:diva-265501 (http://urn.kb.se/resolve?urn=urn:nbn:se:uu:diva-265501)

List of Papers

This thesis is based on the following papers, which are referred to in the text by their Roman numerals.

I Oskarsson, ME., Singh, K., Wang, J., Vlodavsky, I., Li, JP.,

Westermark, GT., (2015) Heparan sulfate proteoglycans are im-portant for islet amyloid formation and islet amyloid polypep-tide-induced apoptosis. Journal of Biological Chemistry, 290(24):15121–32

II Oskarsson, ME., Hermansson, E., Wang, Y., Welsh, N., Jo-hansson, J., Presto, J., Westermark, GT., (2015) The BRICHOS domain of Bri2 inhibits islet amyloid polypeptide (IAPP) fibril formation and toxicity in human beta cells. Manuscript

III Oskarsson, ME*., Paulsson, JF*., Schultz, SW., Ingelsson, M., Westermark, P., Westermark, GT., (2015) In vivo seeding and cross-seeding of localized amyloidosis: a molecular link be-tween type 2 diabetes and Alzheimer disease. American Journal of Pathology, 185(3):834–46

IV Oskarsson, ME., Kothegala, LV., Ingelsson, M., Costantino, I., William, C., Marquie, M., Frosch, MP., Hyman, BT., Westermark, GT., (2015) Levels of islet amyloid polypeptide (IAPP) and Aβ plaque subtypes in Alzheimer disease patients with type 2 diabetes. Manuscript

* These authors contributed equally to the study Reprints were made with permission from the respective publishers.

Contents

Introduction ..................................................................................................... 9 Amyloid fibrils ........................................................................................... 9 

Seeding and cross-seeding ................................................................... 11 Islet amyloid ............................................................................................. 12 

Islets of Langerhans ............................................................................. 12 Type 2 diabetes .................................................................................... 14 Islet amyloid associated with type 2 diabetes ...................................... 15 Cell death induced by aggregating IAPP ............................................. 16 

Alzheimer disease .................................................................................... 18 Type 2 diabetes - a risk factor for Alzheimer disease .......................... 19 

Heparan sulfate proteoglycans ................................................................. 19 Heparan sulfate proteoglycans in amyloid disease .............................. 20 Heparanase ........................................................................................... 21 

The BRICHOS domain ............................................................................ 21 Bri2 ...................................................................................................... 22 

Present investigations .................................................................................... 23 Aims ......................................................................................................... 23 Methodological considerations................................................................. 24 Results and discussion .............................................................................. 27 

The role of heparan sulfate in islet amyloid formation and IAPP-induced cytotoxicity (Paper I) ............................................................. 27 The Bri2 BRICHOS domain prevents IAPP amyloid formation and protects against IAPP-induced toxicity (Paper II) ............................... 28 Cross-seeding of experimental islet amyloid and IAPP-Aβ co-localization in amyloid plaques (Paper III) .......................................... 29 Levels of IAPP and plaque phenotypes in Alzheimer patients with type 2 diabetes (Paper IV) ................................................................... 30 

General discussion .................................................................................... 31 

Conclusions ................................................................................................... 35 

Sammanfattning på svenska .......................................................................... 36 

Acknowledgments......................................................................................... 38 

References ..................................................................................................... 41 

Abbreviations

Aβ Amyloid β AD Alzheimer disease AβPP Amyloid beta precursor protein CGRP Calcitonin-gene-related peptide CHO CJD

Chinese hamster ovarian cells Creutzfeldt-Jakob disease

CMV Cytomegalovirus CS Chondroitin sulfate DMSO Dimethyl sulfoxide DS Dermatan sulfate ECM Extracellular matrix ER Endoplasmatic reticulum EYFP/ECFP Enhanced yellow/cyan fluorescent protein FA Formic acid FRET Fluorescence energy transfer GAG Glycosaminoglycan GlcA Glucuronic acid GlcN Glucosamine hIAPP Human islet amyloid polypeptide Hpa Heparanase HS Heparan sulfate HSPG Heparan sulfate proteoglycans IAPP Islet amyloid polypeptide ITM2B Integral membrane protein 2B IdoA Iduronic acid IGT Impaired glucose tolerance PC Prohormone convertases PLA Proximity ligation assay SAA Serum amyloid A SDS-PAGE Sodium dodecyl sulfate polyacrylamide gel electrophoresis ThT Thioflavin T T2D Type 2 diabetes

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Introduction

Proteins are structurally complex biomolecules that are essential in all biolog-ical processes. They are synthesized on ribosomes as long linear amino acid chains and to gain functional activity, the chain must fold into its correct three-dimensional structure of alpha helices, beta sheets and turns (1). The correct fold of a protein is also called its native state. The native state represents a stable conformation where the amino acids are packed together in such a way that the free energy of the protein reaches a minimum (2). However, some proteins are metastable and more easily disrupted from this safe, native state and can partially unfold during normal ageing or disease processes due to overexpression, changes of the protein itself such as mutations or chemical modifications or changes in the microenvironment. If such misfolded proteins are not removed they can in some cases accumulate and form large, ordered aggregates called amyloid fibrils.

Amyloid fibrils Amyloid fibril is a term describing an aggregation state of proteins. The pro-teins in the fibrils are organized in a strictly ordered fashion which is generic to all amyloid fibrils, irrespective of the amino acid sequence of the protein. The definition of amyloid include 1) affinity for Congo red which should ex-hibit a green, red, yellow or orange birefringence when viewed with polarized light, 2) fibrillar appearance in ultrastructural analysis and 3) give a cross-beta diffraction pattern when analyzed by X-ray diffraction (3) .

The amyloid fibril core consist of a cross-beta spine (4) where beta strands in protein subunits interact via hydrogen bonds formed between the protein backbones, building up long extended beta sheets (Figure 1). Side chains pro-truding from the beta sheets interdigitate in a complementary fashion, forming a structure at the beta sheet interface called the “dry steric zipper” as it is de-void of water (5). Assembled beta sheets make up larger structures called pro-tofilaments and three to six protofilaments intertwine and form the mature amyloid fibril with a total width of ~10 nm (6). Amyloid fibrils are extremely stable mainly as a result of the numerous hydrogen bonds between the protein backbones (7), but also inter-sheet bonds between the side chains and the hy-drophobic effect of the steric zipper interface contribute to stability (5).

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Since the amyloid fibril is mainly dependent on hydrogen bonds between protein backbones, the amyloid state is considered to be a generic property of polypeptides (1). However, the propensity of proteins to form amyloid is de-termined by their amino acid sequence and to date, 31 human proteins have been found to form extracellular amyloid deposits in vivo, each connected to one disease (3). The anatomical site of deposition determines the symptoms of the disease. Some amyloid proteins form local depositions at their site of production including amyloid β (Aβ) in the brain in Alzheimer disease and IAPP in the islets of Langerhans in the pancreas in type 2 diabetes. These are classified as local amyloid diseases. In systemic amyloidosis, circulating plasma proteins form amyloid deposits in multiple organs such as the heart, kidney, spleen and liver. Systemic amyloidosis include inflammation-induced AA amyloidosis where cleaved acute phase protein serum amyloid A (SAA) form amyloid deposits throughout the body.

Although the main component in amyloid deposits is the amyloid protein, other constituents like serum amyloid P-component (SAP) (8) and proteogly-cans (9; 10) are present in all amyloid depositions.

Figure 1. General structure of the amyloid fibril. Intra- and inter beta sheet hydrogen bonds shown in red.

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Seeding and cross-seeding The formation of amyloid fibrils is a nucleation-dependent process and can be divided into three phases; lag phase, elongation phase and plateau phase (Fig-ure 2) (11). In the lag phase monomers associate into small amyloid-nuclei when at least three monomers simultaneously reaches a state of (mis)folding where segments of their peptide backbone are exposed allowing hydrogen bonds to form (12). This is called primary nucleation. These nuclei templates the misfolding of other monomers inducing rapid fibril growth by incorporat-ing monomers onto fibril ends. This is referred to as the elongation phase. The surfaces of fibrils can also catalyze the formation of new fibrils, a phenome-non called secondary nucleation (13). When equilibrium exists between mon-omers incorporated into the fibrils and monomers in solution, fibril growth levels off and a plateau phase is reached. This simplistic model for amyloid formation in vitro is assumed to also apply to the in vivo situation.

The spontaneous formation of the primary amyloid-nuclei is a rare event demonstrated by an extended lag phase in vitro. However, the addition of pre-formed fibrils to the monomers will bypass the lag phase and induce rapid fibril formation (Figure 2). This phenomenon is called seeding and represent the principle mechanism of how amyloid deposits self-propagate and prolifer-ate (11). Seeding is also the transmission mechanism behind the infectivity of prion diseases such as Creutzfeldt–Jakob disease where amyloid fibrils of prion protein (PrpSc) seed misfolding of soluble cellular PrpC (14).

Figure 2. Formation of amyloid fibrils is a nucleation-dependent process with three distinct phases; lag phase, elongation phase and plateau phase. The addition of pre-formed fibrils (seeds) will bypass the lag phase (dashed line). Seeding can be homol-ogous (green) or heterologous (red) if the seed is composed of another protein than the “seeded” protein.

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Cross-seeding, also called heterologous seeding, involves two different amy-loid proteins; the seeding protein X in fibrillar form and the seeded protein Y in solution. Amyloid formation of endogenous mouse AA-proteins was accel-erated by injections of silk from silkworm, sup35-fibrils from yeast and curli-fibrils from bacteria (15). The seeding efficacy was lower than for homolo-gous AA-fibrils but the effect was specific since non-amyloidogenic actin fi-bers did not affect amyloid formation. In vitro studies of lysozyme fibrillation demonstrated that seeding efficiency with other proteins gradually decreased with reduced sequence identity (16). Amino acid sequence homology is thought to be especially important in regions involved in the fibril core struc-ture (16).

Islet amyloid Already in 1901, Opie described lesions of “homogenous hyaline material” in the islets of Langerhans of the pancreas in a patient with diabetes (17). In 1960’s this material was recognized to be of amyloid nature (18) and its main component was long believed to be the locally produced hormone insulin. However, 85 years after its first description, islet amyloid was successfully extracted from an insulinoma rich in amyloid, solubilized in concentrated for-mic acid (FA) and sequenced (19). The main protein component turned out to be a novel peptide; Islet amyloid peptide, later renamed to Islet amyloid pol-ypeptide (IAPP). A year later, this finding was confirmed by a second group extracting the same peptide from pancreatic tissue from an individual with type 2 diabetes, naming it diabetes associated peptide (DAP) and later amylin (20). Today both IAPP and amylin appear in the literature, however this thesis will refer to the peptide with the most commonly used name IAPP.

Islets of Langerhans Islets of Langerhans are clusters of cells confined within a capsule of reticular fibers and scattered throughout the exocrine pancreas tissue (Figure 4A). The human pancreas contains about 1 million islets and they range between 50-300 µm in diameter. The islets consist of at least five different endocrine cell types of which the most prevalent are the beta cells secreting insulin and IAPP, followed by alpha cells and delta cells secreting glucagon and somatostatin respectively. Since the islets are endocrine organs, they are highly vascular-ized and innervated by autonomic nerves.

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IAPP IAPP is a 37-residue peptide hormone that belongs to the calcitonin-family together with calcitonin, two calcitonin-gene-related peptides (CGRP), inter-medin and adrenomedullin. IAPP shares more than 40 % sequence homology with the neuropeptide CGRP (21) and lower sequence homology with the other members (22). The sequence of IAPP is highly evolutionary conserved and most interspecies variations occur in the region corresponding to human IAPP (hIAPP) 20-29. Rodent IAPP have prolines in this region (residues 25, 28 and 29) (23) which makes it non-amyloidogenic due the beta sheet-break-ing property of proline side chains (Figure 3). Thus, rodents do not form islet amyloid (24) nor do they develop spontaneous diabetes. Amyloidogenic IAPP is produced by humans, non-human primates (25; 26) and in cats (27) which all can develop spontaneous type 2 diabetes with islet amyloid deposits.

Figure 3. Amino acid sequences of mouse IAPP, human IAPP and human Aβ. Pro-lines in mouse IAPP is highlighted in bold. Alignment of human IAPP and Aβ with indicated similar amino acids (thin lines) and identical amino acids (thick lines). Grey boxes indicate residues forming the beta strands involved in the beta sheets of the amyloid fibril cores (28; 29).

IAPP is predominantly expressed in the islet beta cells where it is stored and co-secreted with insulin upon glucose stimulation (30). The ratio of IAPP and insulin in the secretory granule is 1-2:50 (31) and the IAPP plasma concentra-tion during fasting conditions is around 20 pM. IAPP is produced as prepro-IAPP of 89 residues (32) and is processed into proIAPP by removal of the signal peptide upon entry to ER. A disulfide bond forms in the oxidizing en-vironment of the ER between cysteins in the N-terminal region at residues corresponding to position 2 and 7 of mature IAPP. ProIAPP is transported through the ER and Golgi apparatus and finally sorted into secretory granules where final processing takes place by sequential cleavage by prohormone con-vertase (PC) 1/3 and PC2 at dibasic residues in the C-terminal and N-terminal processing sites respectively (33; 34). The same convertases are responsible for processing of proinsulin into insulin. Mature IAPP requires trimming of the C-terminal dibasic residues and carboxyamidation of the exposed glycine (35; 36).

The physiological function of IAPP is not clear. Presence of IAPP-recep-tors on the beta cells suggest an autocrine and paracrine function within the islets (37). IAPP has concentration-dependent effects on insulin release; low

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IAPP concentrations increase insulin secretion whereas high IAPP concentra-tions reduce glucose-stimulated insulin secretion (38). In support of this, IAPP knockout mouse displayed an increased insulin secretion in response to a glu-cose challenge (39). This suggest a modulating or fine tuning role of IAPP on insulin release. Peripheral actions of IAPP include reduced food intake (40) most likely conveyed by IAPP receptors present in the brain (41).

Type 2 diabetes Diabetes mellitus is a complex metabolic disorder characterized by an insulin deficiency and disturbed glucose and lipid metabolism. Worldwide prevalence of diabetes was 382 million in 2013 and is estimated to increase to 592 million, nearly 10 % of the world’s adult population, in 2035 (42). The greatest in-crease is predicted to be seen in low- and middle-income countries. In Sweden, the greatest increase in diabetes prevalence from 2005 to 2013 was observed in the aged population, suggesting that people with diabetes in Sweden live longer perhaps due to better medication strategies or decreased mortality rates (43). There are several forms of diabetes and the most common include type 1 diabetes and type 2 diabetes. Type 1 diabetes debuts early in life and is be-lieved to be caused by an autoimmune beta cell destruction resulting in total loss of endogenous insulin production (44). Type 2 diabetes (T2D) affects nearly 90 % of the diabetes cases.

T2D is a heterogeneous disease with an etiology involving a complex in-terplay between genetic and environmental factors such as age, obesity and sedentary lifestyle. The pathogenesis of T2D involves two components; low insulin sensitivity in muscle- or adipose cells referred to as peripheral insulin resistance and insufficient insulin secretion from the beta cells. Blood glucose levels remain normal or only slightly elevated during peripheral insulin re-sistance as a result of a compensatory increased insulin secretion from the beta cells. This state is referred to as impaired glucose tolerance (IGT) and may or may not progress into T2D (45). During IGT, the beta cells are under secretory stress with constant release of granule contents to compensate for the insulin resistance. When the beta cells no longer can maintain this hypersecretion of insulin due to an underlying beta cell dysfunction or reduction in beta cell mass, blood glucose levels increases and T2D is diagnosed. Dysfunctional beta cells in T2D are characterized by loss of the first phase insulin secretion (46) and are unable to maintain its normal oscillatory pattern of insulin release (47; 48). In addition, the processing of insulin from its precursor proinsulin is impaired in T2D resulting in increased secretion of proinsulin and proinsulin processing intermediates (49; 50).

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Islet amyloid associated with type 2 diabetes The main pathological hallmark of islets from T2D patients is the deposition of amyloid fibrils composed of IAPP (Figure 4B, C). Islet amyloid is present in the vast majority of patients with T2D (51-54) and in other conditions as-sociated with beta cell stress such as insulinomas (55), transplanted islets (56) and in isolated islets cultured in high glucose. Islet amyloid is also detected in the islets of aged individuals without diabetes but much less prevalent than in patients with T2D (51-54; 57). In post mortem material from patients with T2D, islet amyloid forms large extracellular deposits with adjacent remnant cords of islet cells (Figure 4B). In transgenic hIAPP rodent models and trans-planted human islets where earlier stages of amyloid deposition can be stud-ied, amyloid fibrils are found intracellular in the secretory granules (58), ex-tracellular in close proximity to the cell surface (Figure 4D, E) or at the peri-vascular basement membranes (54).

Figure 4. A) Pancreatic mouse islet with insulin reactivity, i.e. beta cells, seen in brown. B, C) An islet from a patient with type 2 diabetes with extensive islet amy-loid deposition replacing the islet cell mass. Amyloid stained with Congo red viewed with light microscopy in B and under cross-polarized light in C. D, E) Elec-tron micrograph of a human islet cultured in high glucose displaying initial amyloid depositions at the cell surface (arrows) causing cellular invaginations (dashed rec-tangle) seen magnified in E. Scale bar is 50 µm in A-C and 2 µM in D and 500 nm in E. N=nucleus.

A central question has been whether islet amyloid is a cause or merely a con-sequence of beta cell dysfunction. In a longitudinal study of non-human pri-mates which develop spontaneous diabetes, the appearance of amyloid pre-ceded fasting hyperglycemia and development of overt T2D (25). Deposition of islet amyloid during pre-diabetic stages were also demonstrated in other non-human primates (26; 59), cats (27) and transgenic hIAPP mouse models (60). Patients with severe T2D that required insulin treatment had a higher prevalence and degree of islet amyloid compared to patients without insulin

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treatment (61). Aggregation of IAPP is sporadic, but rare cases of mutations in IAPP have been discovered. A Japanese cohort of T2D patients with a mu-tation replacing serine at position 20 to a glycine in IAPP (IAPP S20G) pre-sented with early-onset and more severe T2D (62). Interestingly, IAPP S20G have an accelerated fibril formation in vitro compared to wild type IAPP (63). Several post mortem analyses have reported an association of islet amyloid with reduction of islet beta cell mass (53; 57; 64) and associations with apop-totic markers (53).

IAPP is one of the most amyloidogenic peptides known in vitro and it rap-idly forms fibrils in solution when left on the lab bench at room temperature. Still IAPP does not form amyloid in healthy individuals, despite high concen-trations both intracellularly and extracellularly in conjunction with secretion. The mechanism by which IAPP is prevented from forming fibrils during nor-mal condition and why it aggregates during type 2 diabetes is largely un-known. Overexpression and increased secretion is an important determinant but not sufficient, most evidently shown by transgenic mice overexpressing hIAPP without any spontaneous formation of amyloid (65; 66). This illustrates that other factors must be involved. Insulin is an effective inhibitor of IAPP fibril formation and it is possible that a reduced intracellular ratio of insu-lin:IAPP can trigger aggregation (31; 67). Aberrantly processed proIAPP has been suggested to form the initial amyloid fibrils which seed further amyloid growth from secreted mature IAPP (58; 68).

Cell death induced by aggregating IAPP Research during the early 1990’s by Yankner et al. were first to demonstrate amyloid-related toxicity of IAPP in cultured beta cells (69). They proposed that IAPP-induced toxicity required direct contact with the cell surface and that the mechanism of cell death was apoptosis (69). However, they also claimed that toxicity was associated to the mature fibrils, a view that came to dominate the amyloid research field for many years. Today however, the con-sensus view is that transient and soluble prefibrillar aggregates constitute the main pathological species and that different amyloid proteins form prefibrillar aggregates with a common structure and toxic mechanism (70). Based on size and structural appearance these prefibrillar species are referred to as smaller oligomers consisting of 25-500 monomers (71) or larger protofibrils with more structural resemblance to mature fibrils (72). In this thesis the term pre-fibrillar aggregates will be used. As a result of their transient nature, prefibril-lar aggregates are difficult to study and there is currently no proof of their existence in vivo (73). Indirect proof of toxicity of prefibrillar IAPP aggregates comes from observations in transgenic hIAPP rodent models where beta cell apoptosis was unrelated to the degree of islet amyloid load (74) and instead correlated with the growth of amyloid fibrils (75). In cell culture, addition of

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freshly dissolved IAPP induce toxicity whereas addition of mature fibrils have no toxic effects (71; 76).

Exposure of beta cells to exogenously added hIAPP induced cell death me-diated by caspase-3- and caspase-8 activation and upregulation of the Fas-receptor, demonstrating that beta cell death occurs via apoptosis activated by the extrinsic pathway (77-81). Apoptosis induced by endogenous hIAPP pro-duction in islets was also mediated via active caspase-3 and the Fas-receptor (79-82) in addition to upregulation of Bim-mediated intrinsic apoptotic path-way (82). The exact mechanisms that underlie islet amyloid-induced activa-tion of beta cell apoptosis are not clear. Numerous reports suggests different mechanisms, many related to events at the cell membrane. Prefibrillar IAPP aggregates have been proposed to form ion leaking pores inserted into the plasma membrane (83), form complexes with cell surface Fas-receptors (78), disrupt organelle membranes (71), cause ER stress (84), increase oxidative stress (85) and disturb autophagy (86). In addition, the amyloid fibril growth itself can impact the plasma membrane and cause local membrane leakage (87), in line with ultrastructural images from islets showing amyloid fibrils oriented perpendicular to beta cell membranes (54) (Figure 4E). In fact, forces generated by amyloid growth is in the same range as the forces observed for polymerizing actin and tubulin (88). In this context, it is also noteworthy to mention that interactions of IAPP with anionic membranes can promote fibril formation (89).

Although amyloid fibrils are not cytotoxic per se, large deposits of amyloid fibrils can affect both the function and survival of beta cells. As proposed al-ready in the initial report of islet amyloid (17), amyloid deposits surrounding the islet vasculature might limit nutrient supply and impair the ability of beta cells to monitor and respond properly to glucose changes in the blood. In ad-dition, extracellular fibrillar deposits might also disrupt the network of gap junctions between beta cells or paracrine signaling that are required for the synchronized insulin secretion.

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Alzheimer disease Alzheimer disease (AD) is a neurodegenerative disease manifested clinically by a gradual decline of memory and cognitive functions. The strongest risk factor for AD is age with an increased prevalence observed in individuals over 65 years of age. The major pathological hallmarks of AD include accumula-tions in the brain of extracellular amyloid plaques composed of the Aβ peptide and intracellular, neurofibrillary tangles composed of the hyperphosphory-lated tau protein (90; 91). Accumulations of Aβ amyloid is believed to be the underlying cause for the neuronal dysfunction and death in AD, yet, amyloid plaque load does not correlate well with the severity of cognitive symptoms (92). Aβ is released by the proteolytic cleavage of the transmembrane amyloid precursor protein (AβPP) by BACE1 and γ-secretase complex. The γ-secre-tase complex can cleave AβPP at multiple sites releasing Aβ peptides of var-ying lengths. Aβ40 is the most commonly secreted form, followed by Aβ38 and the more aggregation-prone Aβ42 (93). The vast majority (>90 %) of AD cases represent late age-of-onset sporadic cases with an unknown underlying cause, whereas a minority are caused by mutations in the AβPP gene or in the AβPP cleaving enzymes resulting in early age-of-onset familial AD (FAD).

Aβ aggregation gives rise to different types of parenchymal plaques called diffuse plaques or neuritic plaques (dense) as well as cerebral amyloid angi-opathy (CAA) in the blood vessel walls of the brain. Diffuse plaques contain mostly Aβ42 peptides (94) and are not by definition amyloid as they lack af-finity for amyloid-specific stains such as Thioflavin or Congo red. Diffuse plaques also occur frequently in non-demented aged individuals (95; 96). Neu-ritic plaques are considered more pathologically relevant. They are recognized by amyloid stains, contain mostly Aβ40 peptides and are associated with AD-related neuropathological features such as dystrophic neurites and activated immune cells (97; 98). The relationship between these two plaque types is not clear. One hypothesis suggest that the diffuse plaques are precursors of the neuritic plaques (97; 99). Patients with Downs’s syndrome (DS) have an extra copy of the AβPP gene and produce higher levels of Aβ, resulting in Aβ am-yloid deposition at ~30 years of age (100). In DS patients, Aβ42-positive dif-fuse plaques precedes the deposition of Aβ40-positive neuritic plaques sup-porting the hypothesis of diffuse plaques as precursors (101). On the other hand, patients with a rare deletion mutation in the PSEN1 gene develop only Aβ42-positive diffuse plaques (102) which evidently never “mature” into neu-ritic plaques. The same phenomenon was seen in a double transgenic mouse expressing wild type Aβ and Aβ with the arctic mutation (E22G) which de-veloped more diffuse plaques without any increase in neuritic plaques (103).

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Type 2 diabetes - a risk factor for Alzheimer disease Numerous clinical studies have identified T2D or pre-diabetic conditions as an increased risk factor for development of AD (104-110). Leibson et al. per-formed a large scale cohort study demonstrating an increased risk for patients with T2D to develop AD compared to individuals without diabetes during an average follow-up time of 7 years (104). Also prediabetic conditions such as elevated plasma glucose levels (109; 111) and impaired acute insulin response (110) predisposed for development of dementia and AD. Some studies ob-served a stronger risk for vascular dementia rather than AD in patients with T2D (112; 113).

The underlying mechanisms behind the link between T2D and AD is cur-rently unknown. In assessments of correlations in amyloid pathology between AD- and T2D patients, the results are conflicting. One study reported that AD patients had more frequent and more extensive islet amyloid and that brain amyloid plaque burden correlated with duration of diabetes in T2D patients (108). An additive effect of diabetes and the ApoE ε4 allele on the neuritic plaque load was reported in a longitudinal study (114) whereas other studies could not find any association between increased AD pathology and T2D (115; 116). Miklossy et al. reported presence of Aβ-immunoreactivity in islet amyloid in patients with T2D (117). Also, presence of few IAPP-immunore-active deposits adjacent to Aβ-deposits in the parenchyma and vessel walls in AD brain have recently been described (118).

Heparan sulfate proteoglycans Proteoglycans are complex macromolecules composed of long unbranched polysaccharide chains of 50-200 monosaccharide units (119) covalently at-tached to a core protein. These polysaccharide chains called glycosaminogly-cans (GAGs) can be of different types, the most common are heparan sulfate (HS), chondroitin sulfate (CS) and dermatan sulfate (DS). HS and the closely related heparin are built up from repeating disaccharides of glucuronic acid (GlcA) or iduronic acid (IdoA) combined with N-acetylated glucosamine (GlcNAc) or N-sulfated glucosamine (GlcNS) (Figure 5A). Whereas heparin production is restricted to the mast cells, HS is ubiquitously expressed in all tissues and found at the cell surface attached to core proteins syndecan or glypican or in the extracellular matrix attached to core proteins perlecan, agrin or collagen XVIII (Figure 5B) (120). HS and heparin are highly negatively charged due the presence of multiple sulfate groups, either arranged in small tracts as in HS or more uniformly spread as in heparin. HS interact with a vast number of bioactive molecules and are thus vital for a wide range of essential biological processes including receptor signaling, cellular adhesion and bar-rier formation in basement membranes, reviewed in (120). The interaction of

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HS with their target proteins occur through electrostatic binding between the negatively charged sulfate groups and positively charged amino acid residues (121; 122). Sulfation patterns on the chains are dynamic and can vary with cell type or external stimuli, thereby altering HS interaction with ligands and ultimately their function (123).

Figure 5. Structure of heparan sulfate proteoglycan (HSPG). A) HS is attached to a core protein and can be cleaved by heparanase. B) HSPG can be associated to the cell surface attached to syndecans and glypicans, or found in the extracellular space attached to perlecan.

Heparan sulfate proteoglycans in amyloid disease Proteoglycans, in particular heparan sulfate proteoglycans (HSPGs) are con-sistently found in all types of amyloid depositions irrespective of amyloid pre-cursor protein or the tissue affected, including senile plaques and islet amyloid (124; 125). The source of HS in islet amyloid is likely the beta cells themselves as they contain high levels of HS (126; 127) associated to cell surface syndecans and glypicans as well as secreted associated to perlecan (127-129). HS and heparin are potent inducers of in vitro fibril formation of many amy-loid proteins, including IAPP (130), proIAPP (131) and Aβ (132). Other GAGs such as CS and DS also promote fibril formation but with less potency than HS (133; 134). HS is believed to accelerate amyloid formation through a scaffolding mechanism where binding to multiple amyloid proteins increases the local concentration or aligns the proteins to facilitate interactions (135). HS could also stabilize the beta sheet structure in the initial seeds (134) or prevent mature fibrils from dissociation (136) or proteolytic degradation (137). HS amyloid-promoting effect is strongly dependent on HS chain length

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(122; 138) and overexpression of heparanase in mice models reduce deposi-tion of AA-amyloid (139) and Aβ (140). These results suggests that HS has a direct role in amyloid formation through interactions with the amyloid pro-teins that promotes amyloid fibril formation or stability.

Heparanase Heparanase is the only known mammalian enzyme that can fragment HS chains (141). It is an endo-β-glucuronidase that specifically cleave the bond between GlcA and 6-O-sulfated GlcNS saccharides (142) generating a range of differently sized HS fragments (Figure 5A) (143). Heparanase is activated by proteolytic cleavage of inactive proheparanase of 65 kDa by cathepsin-L to generate a non-covalently linked heterodimer of 8 kDa and 50 kDa subunits (144). The cleavage of HSPG in the extracellular matrix by heparanase is im-portant in processes such as angiogenesis and inflammation (145). Secreted heparanase can also bind cell surface HSPG, promote their internalization and degrade HS in acidic endosomes or lysosomes (146; 147). Human heparanase is mainly located intracellularly whereas the chicken heparanase contains a longer and more hydrophobic signal peptide than the human heparanase, pro-moting its secretion and localization to the cell membrane (148).

The BRICHOS domain In 2002, a novel protein domain of ~100 residues was discovered by Sanchez-Pulido et al (149) given the name BRICHOS based on the three protein fami-lies in which the domain was identified in; Bri (see below), chondromodulin-I and surfactant protein C (SP-C). Since the discovery, the list of BRICHOS domain containing proteins have grown, to date including more than 300 pro-teins divided into 12 different protein families (150). The crystal structure of proSP-C BRICHOS revealed that the domain folds into two alpha-helices sur-rounding five central beta–strands, and the surface of the strands facing the first alpha helix, called Face A, is proposed to contain its ligand-interaction site (151). So far, only the crystal structure of proSP-C BRICHOS is available, but it is likely to be similar to other BRICHOS domains, including the Bri2 BRICHOS (152). Although the exact target motif for the binding of BRICHOS’ Face A in ligands is unknown, it appears to involve stretches of at least 5-7 amino acids (153), with specific properties (charge, hydrophobi-city etc.) in a beta-hairpin secondary structure (151), rather than just recogni-tion of a specific primary amino acid sequence.

BRICHOS was early predicted to possess a chaperone activity (149). This was supported by the finding that mutations in the proSP-C BRICHOS domain was associated with amyloid formation of the transmembrane region SP-C leading to the respiratory disorder interstitial lung disease (ILD) (151). It is

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believed that each BRICHOS-containing protein also encompasses another region rich in beta-sheets and prone to form amyloid, for example SP-C in proSP-C or Bri2-23 in Bri2 (see below), and that the BRICHOS domain pre-vent amyloid formation of the other region during synthesis, either through intra- or intermolecular interactions (149). Interestingly, this anti-amyloid chaperone activity of the BRICHOS domain on endogenous regions appear to extend also to amyloid-prone regions in other proteins, such as Aβ (152; 154-156).

Bri2 Bri2, also known as integral membrane protein 2B (ITM2B), is a type II trans-membrane protein which belongs to an evolutionary conserved protein family composed of at least two other members, Bri1 and Bri3 (157). While Bri1 and Bri3 show more restricted expression patterns limited mainly to osteo- and chondrogenic tissues or brain respectively (157; 158), Bri2 mRNA is ubiqui-tously expressed in many if not all human tissues with especially high levels in the heart, placenta with lower levels detected in kidney and whole pancreas (159). Full length Bri2 contains 266 amino acids with a calculated molecular weight of 30 kDa. Bri2, like most BRICHOS containing proteins, comprises five regions; a short N-terminal region facing the cytosol (position 1-51), a transmembrane region (position 52-75), a linker region (positions 76-137) fol-lowed by the BRICHOS domain (position 137-231) and the C-terminal pep-tide Bri2-23 (position 244-266) (Figure 6). It contains one N-glycosylation site (Asp-170) and can be enzymatically processed at three positions; in the transmembrane region by SPPL2a/2b (160), in the linker region by ADAM10 (160) and at position 243-244 by furin-like proteases releasing a 23 residue peptide Bri2-23 (161). Mutations in the Bri2 gene (ITM2B) results in exten-sion of the Bri2-23 peptide to 34-residue peptides referred to as ABri and ADan which deposits as brain amyloid in familial British dementia (FBD) (162) and familial Danish dementia (FDD) (163).

Figure 6. Schematic structure of Bri2. The BRICHOS domain between residues 137-231 is indicated.

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Present investigations

Aims The overall aim of this thesis was to investigate potential molecules and mech-anisms involved in the formation of islet amyloid and investigate a molecular interaction between type 2 diabetes and Alzheimer disease. The specific aims for each paper were:

I Investigate the effects of heparanase overexpression on islet amyloid formation and the role of heparan sulfate in IAPP-in-duced apoptosis.

II Investigate the expression of Bri2 in human beta cells and de-

termine the potential role of its BRICHOS domain as an inhib-itor of IAPP fibril formation and IAPP-induced apoptosis.

III Investigate seeding and cross-seeding of experimental islet am-

yloid and investigate a molecular interaction between IAPP and Aβ in amyloid deposits from patients with type 2 diabetes and Alzheimer disease.

IV Investigate IAPP levels and Aβ plaque profiles in Alzheimer

disease patients with and without type 2 diabetes

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Methodological considerations This section contains some considerations regarding the methods used in pa-per I-IV. For complete descriptions of all material and methods used, see in-dividual papers.

Thioflavin T assay (paper I and II) Amyloid fibril formation was monitored with Thioflavin T (ThT) which binds to structures rich in beta sheets resulting in a dramatic increase in fluorescence intensity at 480 nm under 440 nm excitation.

IAPP is a highly amyloidogenic peptide and prevention of its aggregation prior to experiments was important. A stock solution of 5 mM synthetic IAPP (Keck Biotechnology, Yale) in DMSO were aliquoted to avoid repeated freeze-thaw cycles and stored at -80°C. All surfaces were treated with Sigma-cote (Sigma) to avoid surface-induced aggregation and IAPP solutions were prepared immediately prior to the assay from the IAPP stock. Fibrillation ex-periments were carried out as described with short 2 sec bursts of shaking at 100 rpm before each reading (15 min intervals).

The increase in ThT fluorescence intensity is explained by a rotational re-striction of ThT imposed by rigidity of the binding site on the beta sheet sur-faces (164). These binding sites can differ in both number or in individual affinity for ThT and give different fluorescent intensities which are unrelated to the amount of fibrils (63; 165). We therefore only interpreted ThT curves based on time until the increase in fluorescence was seen (the lag phase) rather than draw any conclusions on amount of fibrils formed based on height of the curves.

FRET assay (paper I and II) We developed a FRET-based cell assay to determine real-time apoptotic re-sponses to aggregating IAPP. CHO cells were stably transfected with a vector (166) coding for CMV-promotor-based expression of the FRET pair ECFP-EYFP linked via the amino acid sequence Asp-Glu-Val-Asp (DEVD) targeted by activated caspase-3. Caspase-3 cleavage of the DEVD linker separates the fluorophores and reduce FRET which can be measured in a plate reader (Fig-ure 7).

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Figure 7. Schematic drawing illustrating the loss of FRET between ECFP and EYFP after caspase-3 cleavage of the DEVD-containing linker.

Cells were detached mechanically with a scraper and seeded into a multiwell plate and grown over night. Mechanical detachment was used over enzymatic based methods in order to preserve cell surface proteins. Plated cells were cul-tured for 48 h in 4 % DMSO to increase vector expression by activating the silenced CMV promotor, a phenomenon often observed in stably transfected cells over time (167). DMSO has toxic effects on cells and could potentially interfere in the subsequent apoptosis assay. We determined 4 % DMSO as the lowest concentration yielding sufficient ECFP-DEVD-EYFP levels to meas-ure changes in FRET in response to apoptotic inducer staurosporine. Culture in 4 % DMSO in itself did not induce apoptosis, however it reduced prolifer-ation rates of the cells. Prior to the assay, the cells were incubated 1-2 h in DMSO-free medium followed by 30 min in 120 mM NaCl, 4.7 mM KCl, 2.5 mM CaCl2, 1.2 mM MgSO4, 0.5 mM KH2PO4, pH 7.4 with 20 mM HEPES and 2 mM glucose (KRGH buffer) to remove all DMSO and phenol red from the medium. To reduce inter-assay differences in both fibrillation kinetics and fibril morphologies, all IAPP-solutions were seeded with sonicated preformed IAPP fibrils (125 nM peptide) from one single stock. Seeds were added also to the negative control groups.

An advantage with this real-time approach is that it can follow cytotoxicity over time in the same cell population. This gives a more comprehensive view of the apoptotic responses in contrast to end-point measurements such as 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay, TUNEL staining or apoptosis immunoreactivity in cellular extracts. This is especially important in studies of apoptotic responses to such dynamic pro-cesses as amyloid fibril aggregation. A disadvantage with the assay is the use of a non-beta cell, immortalized cell line, therefore the physiological relevance of their apoptotic responses for human beta cells in islets can be questioned. Previously, ECFP-DEVD-EYFP have been expressed in mouse beta cell line βTC-6 where IAPP-induced apoptosis was demonstrated (76). In addition, our assay uses exogenously added IAPP which only provide information on ex-tracellular IAPP-induced toxicity while in the islets IAPP amyloid occur both inside the cell (58) as well as in the extracellular space.

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Proximity ligation assay (paper II, III and IV) In situ proximity ligation assay (PLA) is an antibody-based method to visual-ize co-localization of antibody targets (168). The two target proteins are first recognized by their respective primary antibody made from different species (Figure 8). Subsequent incubation with species-specific oligonucleotide-con-jugated secondary antibodies (PLA probes) will only generate an amplifiable circular DNA strand if both target proteins are in close proximity (<40 nm distance) (168). The circular DNA strand is amplified up to 1300-fold with polymerase (169) and the generated DNA tangle, still attached to the target proteins via the antibodies, is detected after hybridization with fluorescence-labeled complementary oligonucleotides. A PLA signal is seen as a small sub-micrometer red fluorescent spot appearing above the focus of the section since the long, amplified DNA strand extends out. This makes the PLA signals easy to distinguish from autofluorescent structures such as lipofuscin granules which are abundant in the brain tissue.

PLA shows superior specificity and sensitivity compared to conventional double-immunofluorescence labeling often used to determine co-localization. The high specificity of PLA derives from the requirement of dual antibody binding and the amplification of the DNA strand enables visualization of events that would be below the limit-of-detection with conventional immuno-fluorescence. The high resolution of co-localization (<40 nm) determined with PLA also makes it easier to discriminate from mere adjacency on the µm-scale as a result of the compact, crowded milieu of an organelle and in particular an amyloid deposit.

Figure 8. Principles of proximity ligation assay (PLA). A) Two primary antibodies from different species binds its target proteins (black and grey spheres) and is recog-nized by the species-specific secondary antibodies conjugated with short oligonucle-otides (PLA probes). Two soluble oligonucleotides are added and will only form a circular DNA after a ligation step if the secondary antibodies are in close proximity. B) The circular DNA is amplified and C) detected with fluorescently labelled com-plementary oligonucleotides (stars). Illustration inspired by Trifilieff P. et al. (170).

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Results and discussion The role of heparan sulfate in islet amyloid formation and IAPP-induced cytotoxicity (Paper I) The effect of reduced length of HS chains on islet amyloid formation was in-vestigated by generating mice that express human heparanase and human IAPP (hpa-hIAPP). Human heparanase was expressed as a chimeric form with the signal peptide from chicken heparanase, enhancing its secretion and local-ization to the cell membrane (148). High levels of active heparanase were de-tected in isolated hpa-hIAPP islets and a significant reduction of HS chain length was observed from whole pancreata. Based on the pronounced expres-sion of heparanase and HS in islets but not in exocrine pancreas tissue (126), reduced HS length should reflect islet-associated HS. Isolated islets from hpa-hIAPP mice cultured in high glucose medium, which stimulates IAPP secre-tion, for 19 days developed significantly less amyloid compared to hIAPP is-lets. HS and other GAGs are important for beta cell survival and hormone release (126; 171; 172) which has complicated interpretations of studies re-porting reduced amyloid in islets when GAG synthesis have been completely blocked (172). In addition, HS can affect Aβ secretion by directly interfering in BACE1 cleavage of AβPP (173). To exclude altered IAPP secretion as a cause for the reduced amyloid load, we verified that overexpression of hepa-ranase did not impair beta cell function evident by the unaltered glucose-in-duced secretion of IAPP from hpa-hIAPP islets compared to hIAPP islets. In-stead, we hypothesize that the reduction in islet amyloid deposition by over-expression of heparanase results from the digestion of HS on cell surfaces and in the extracellular matrix, reducing the availability of HS which otherwise would act as scaffolds for amyloid formation. A similar hypothesis have been suggested for other amyloid proteins (122; 139; 140). This hypothesis is in accordance with our observation that both the binding to IAPP and the IAPP amyloid-promoting effect of heparin (an analogue to HS) decreased with re-duced chain length. In line with this, Jha et al. demonstrated that shorter hep-arin chains display both longer lag phase and slower growth rates of IAPP fibril formation compared to full length heparin (138).

Transgenic hIAPP mouse islets cultured in high glucose medium supple-mented with 12mer heparin fragments developed less amyloid compared to islets cultured without any heparin fragments, a finding which was repeated with cultured human islets. This suggest that in addition to the reduced HS chain length also soluble HS fragments generated from the heparanase cleav-age in hpa-hIAPP islets are capable of reducing islet amyloid deposition. The small, soluble HS fragments likely interfere in the binding of IAPP to cell surface- and extracellular matrix-associated HS, thereby neutralizing IAPP and preventing amyloid formation in the islets. Short heparin fragments or

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heparin-like fragments have previously been shown to reduce amyloid for-mation of AA-proteins (174; 175).

Effects of cellular HS on IAPP-induced apoptosis was determined in a cell model expressing a reporter molecule for caspase-3 activation. HS-containing cells (CHOwt) showed a time-dependent increase in apoptosis when exposed to extracellular IAPP, whereas HS-deficient cells remained unaffected when exposed to the same IAPP concentration. In addition, presence of soluble hep-arin/heparin fragments reduced the total apoptotic response induced by IAPP in HS-containing cells. The soluble heparin/heparin fragments likely compete with IAPP binding to cell surface-associated HS thereby redirecting IAPP fi-brillation away from the vicinity of the cell surface where it is likely to exert its toxic effects.

The Bri2 BRICHOS domain prevents IAPP amyloid formation and protects against IAPP-induced toxicity (Paper II) Expression of the BRICHOS containing protein Bri2 have so far been demon-strated in the brain and the anti-amyloidogenic effects of its BRICHOS do-main have mainly been studied in the context of neurodegenerative diseases like AD (152; 156; 176-178). In paper II we demonstrated Bri2 protein ex-pression in human islets and in the human beta cell line EndoC-βH1. Bio-chemical analysis of islet- and cell extracts with Bri2 antibodies revealed a protein with molecular weight corresponding to full length Bri2 in addition to lower molecular weight proteins matching predicted fragments generated by enzymatic processing of Bri2. In particular, a small protein likely correspond-ing to the released BRICHOS domain (predicted molecular weight of ~13 kDa) was detected. Furthermore, double immunolabelling revealed Bri2-reac-tivity in insulin-positive cells and its accumulation in subcellular compart-ments in, or close to, trans-Golgi. Moreover, we detected co-localization (<40 nm proximity) between Bri2 and IAPP using PLA technique both intracellu-larly in beta cells and in extracellular islet amyloid deposits in patients with T2D.

The Bri2 BRICHOS domain (residues 113-231 of Bri2) effectively inhib-ited amyloid fibril formation of IAPP in vitro. The inhibiting effect was also achieved when added during the elongation phase of fibrillation, however only at low IAPP concentrations. Analyses with transmission electron microscopy (TEM) verified the absence of fibrils in IAPP samples incubated in the pres-ence of Bri2 BRICHOS and instead large amorphous aggregates were de-tected, positive for both IAPP- and Bri2-reactivity. However, no IAPP mono-mers could be detected. This is in contrast to the anti-amyloidogenic effects of Bri2 BRICHOS observed for Aβ in vitro, where Bri2 BRICHOS kept Aβ as a monomer during the extended lag phase (152).

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Preformed IAPP fibrils incubated with Bri2 BRICHOS for 3 h and ana-lyzed with immuno-electron microscopy displayed substantial Bri2-reactivity decorating the surfaces of the fibrils, in line with the observed binding of proSP-C BRICHOS to Aβ42 fibrils (13). In a seeding experiment, these Bri2 BRICHOS-decorated IAPP fibrils exhibited a reduced seeding capacity com-pared to uncoated fibrils.

To bring the finding of Bri2 BRICHOS inhibition of IAPP fibril formation from the test tube into a more biological relevant situation we used a cellular assay to measure the apoptotic response to externally added IAPP in the pres-ence of Bri2 BRICHOS. Exposure of cells (CHOwt) to increasing concentra-tions of monomeric IAPP (5-50 µM) resulted in a concentration dependent increase in apoptosis. Presence of Bri2 BRICHOS at a constant concentration of 5 µM effectively reduced IAPP-induced cellular apoptosis at lower IAPP concentrations. However when the ratio exceeded 4:1 (20 µM IAPP: 5 µM Bri2 BRICHOS) the rescue effect was lost.

The role of Bri2 under conditions of beta cell stress when the risk of IAPP aggregation is high was studied by siRNA-induced knock down of Bri2 in EndoC-βH1 cells. Cells with reduced Bri2 expression were exposed to high glucose (28 mM), high fat (1.5 mM palmitate) or high glucose with high fat for two days. Results showed that cells with reduced Bri2 expression were more susceptible to cell death when exposed to high glucose with high fat compared to control cells. In Drosophila melanogaster, the transgenic expres-sion of IAPP in a subpopulation of neurons resulted in cell loss. This toxic effect of IAPP expression was alleviated by co-expressing Bri2 BRICHOS in the same cells.

Cross-seeding of experimental islet amyloid and IAPP-Aβ co-localization in amyloid plaques (Paper III) There are several reports of interactions between IAPP and Aβ in vitro. O’Nu-allain et al. demonstrated that Aβ fibrils were efficient seeds of IAPP fibrilla-tion and that IAPP fibrils could seed Aβ, yet much less potent than homolo-gous seeding between Aβ and Aβ fibrils (179). Monomers of IAPP and Aβ mixed together formed hetero-assemblies which delayed fibril formation and suppressed cytotoxicity (180; 181). Modified IAPP with N-methylations at position 24 and 26 is non-amyloidogenic and can inhibit fibril formation of IAPP as well as Aβ (181; 182). This prompted us to investigate whether cross-seeding could occur between these peptides in vivo.

Transgenic mice expressing human IAPP develop islet amyloid after 12 months when fed a diet high in fat (183). Transgenic hIAPP mice, that re-ceived a single intravenous injection at <6 weeks of age with preformed IAPP- or proIAPP fibrils, developed more extensive islet amyloid deposition com-pared to control mice after 10 months on high fat diet. Similarly, mice injected

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with Aβ fibrils also developed extensive islet amyloid although less compared to mice injected with IAPP fibrils. Both the number of mice with islet amyloid and the number of islets with amyloid per mouse was significantly increased in all injected groups. The amyloid in the injected mice was located mainly perivascular in the islets and consisted only of IAPP. No reactivity of the in-jected fibril proteins (proIAPP or Aβ) could be detected. This demonstrates in vivo evidence that islet amyloid formation can be induced by blood-borne am-yloid seeds and that cross-seeding between Aβ and IAPP can occur. Further-more, the data demonstrates that degree of sequence or structural similarity is an important factor for the seeding potency, since homologous seeding with IAPP fibrils was more potent than heterologous seeding with proIAPP- or Aβ fibrils.

To analyze if the two peptides also co-localized in amyloid deposits in hu-mans, we first analyzed islet amyloid from patients with T2D for Aβ-immu-noreactivity. However, no Aβ-reactivity could be detected in islet amyloid us-ing two sensitive techniques (western blot and PLA), despite previous reports of Aβ in islet amyloid (117). We next investigated IAPP-reactivity in brain temporal cortex tissue, and indeed IAPP immunoreactivity was detected with western blot in brain extracts from both AD patients and non-AD patients. However, the FA-fractions from the brains of AD patients contained more IAPP-reactivity compared to the FA-fractions from the brains of non-AD pa-tients. One of the AD patients was diagnosed with T2D and interestingly also showed the lowest levels of IAPP amongst the AD patients. Since the FA frac-tion contain the insoluble protein pool, including proteins from amyloid de-posits, we analyzed whether the increased IAPP reactivity in AD brains could result from IAPP deposition in the Aβ plaques. Using PLA technique we de-tected co-localization between IAPP and Aβ in both neuritic plaques, diffuse plaques and in blood vessel-associated amyloid. Generation of PLA signals requires the proteins to be within 40 nm from each other which suggests that deposition of IAPP and Aβ are mixed, rather than forming separate deposits adjacent to each other as was claimed in a previous study (118).

Levels of IAPP and plaque phenotypes in Alzheimer patients with type 2 diabetes (Paper IV) To pursue and extend our findings from paper III, we analyzed brain IAPP levels and Aβ-amyloid pathology in an AD cohort with or without concomi-tant T2D. The analyzed material consisted of postmortem brain tissue from the frontal and temporal cortex of AD patients with clinically diagnosed T2D (n=9, AD/T2D+) or without T2D (n=15, AD/T2D-). IAPP was detected in FA fractions from the brains of both AD/T2D+ and AD/T2D- and IAPP co-local-ized with Aβ in both diffuse- and neuritic plaques, thereby confirming the findings from paper III in this larger AD cohort. Although Aβ levels were

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similar in both groups, IAPP levels were generally lower in AD/T2D+ com-pared to AD/T2D-, in line with the single observation from the AD patient with T2D in paper III. A yet unanswered question is whether the IAPP in the brain is locally produced or if it originates from production in the pancreas and then translocates into the brain. The low IAPP levels in the FA fraction might reflect a reduced IAPP secretion from dysfunctional beta cells during end-stage T2D (184; 185), suggesting that the majority of IAPP deposited in plaques originated from peripheral production in the pancreas. IAPP has been reported to readily cross the blood brain barrier, even more efficiently than insulin (186; 187).

AD/T2D+ patients contained a higher proportion of neuritic plaques over diffuse plaques compared to AD/T2D-. Neuritic plaques are more associated with morphological transformed glial cells and dystrophic neurites (98) and progression from early AD to end-stage AD is characterized by an increase of neuritic plaques and a decrease of diffuse plaques (188). In light of this, our results suggest that AD patients with concurrent T2D have a plaque phenotype linked to a more severe and advanced form of AD pathology.

General discussion Results from paper I provide further proof to the current view holding aggre-gation events at the plasma membrane responsible for cell death induced by extracellular aggregating IAPP (54; 69; 78; 83; 87). Based on data from paper I we suggest that beta cell surface-associated HS acts as a potential recruiter of IAPP to the plasma membrane. Once at the plasma membrane, IAPP’s bind-ing to HS itself, and potentially also interactions with adjacent phospholipids (89), catalyzes IAPP fibril formation initiating the toxic response in the cell, perhaps by complexing with the Fas-receptor (78). This implies that anything interfering in the binding between IAPP and HS would alleviate IAPP-induced toxicity. In agreement with that, we demonstrated that the addition of soluble short HS structures reduced the total apoptotic response induced by IAPP. A recent report demonstrated that truncated IAPP 16-37 lacking the N-terminal residues important for HS binding, showed reduced cytotoxicity despite its high propensity to form amyloid fibrils in (189). On the contrary, events pro-moting IAPP-HS binding would accordingly increase amyloid formation and amyloid-induced toxicity. Aberrant prohormone processing in the beta cells is associated with T2D (190) and unprocessed proIAPP with intact N- and C-terminal processing sites of Lys-Arg is present in islet amyloid (58; 191). Early aggregates of proIAPP have been suggested to serve as seeds for further islet amyloid deposition (58). It is conceivable that an increased release of the more positively charged proIAPP enhance binding to the negatively charged HS (131) thereby promoting fibril formation. In addition, altered HS structures (i.e. length, sulfation pattern) in islets or released from infiltrating cells (192)

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induced by a general ageing process or a diabetic milieu could affect HS in-teractions with IAPP (193-195).

Formation of islet amyloid have been proposed to be a contributing factor in the poor survival of transplanted islets (56). Heparin-immobilization onto islet surfaces is currently in trials to minimize blood clotting and inflammatory reactions during islet transplantation (196). Based on our findings in paper I, it is possible that such immobilized heparin molecules could serve as scaffolds for amyloid formation, potentially aggravating islet survival post-transplanta-tion.

One argument against that HS would be an amyloid-promoting molecule is the fact that IAPP is frequently exposed to such ubiquitously present mole-cules like HS and still, islet amyloid is a rare finding in individuals without diabetes (51-53). Even in the absence of interactions with HS, IAPP is one of the most fibrillogenic peptides in vitro. This suggests that components in the beta cells that limit IAPP fibril formation must exist in vivo. Data in paper II proposes that Bri2 and in particular its BRICHOS domain, previously only studied in the context of neurodegenerative diseases, could be a molecular chaperone inhibiting amyloid formation of IAPP in beta cells. Paper II pre-sents the first description of Bri2 protein expression in islets of Langerhans and beta cells. Genetic and transcriptional data have been available supporting beta cell expression of Bri2; the identification of binding sites for multiple islet-specific transcription factors in flanking regions of the human ITM2B gene (197) and high levels of Bri2 mRNA in human islets and a human beta cell line (198). We demonstrated the co-localization of Bri2 and IAPP intra-cellularly in beta cells, likely in the ER or Golgi. Their co-localization in the secretory granules cannot be discerned from our results, but should not be ruled out. In addition to full length Bri2, also shorter Bri2 fragments were detected including a possible liberated BRICHOS domain suggesting that beta cells contain enzymes capable of processing Bri2. Indeed, expression of furin and ADAM10 have been reported in islets (199; 200), however at low levels and Bri2 processing by other enzymes present in beta cells remains to be in-vestigated. The secretion of a released BRICHOS domain from beta cells is supported by the co-localization of Bri2 BRICHOS and IAPP in extracellular amyloid deposits from patients with T2D. Based on these findings, we con-clude that Bri2 BRICHOS inhibition of IAPP amyloid formation could take place both intracellularly as well as in the extracellular space.

So, what species of IAPP does Bri2 BRICHOS recognize and interact with? The requirement of only substoichiometric amounts seen by us and in other studies rule out the possibility that BRICHOS acts by simple monomer deple-tion (152). We identified an interaction of Bri2 BRICHOS with the surface of mature IAPP fibrils, which reduced the fibrils’ seeding capacity. In addition to fibril surfaces, Bri2 BRICHOS may also interact with monomers adopting an abnormal conformation prone to aggregate into fibrils. Based on available

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information of general binding motifs of BRICHOS domains, the binding mo-tif should be 5-7 residues (153), contain charged residues (at least in the case of Bri2-BRICHOS) (201) and the protein should be in a β-hairpin confor-mation (152). Dupuis et al. demonstrated two main structural species popu-lated by IAPP monomers in solution; an helix-coil or a beta-hairpin and that the latter represented the more aggregation-prone variant (202). Residues 18-23 of IAPP (His-Ser-Ser-Asn-Asn-Phe) is exposed in the turn of the beta-hairpin but is incorporated into an helix in the helix-coil variant of IAPP mon-omers (202). Interestingly this region shows similarities with residue 11-16 of Bri2-23 (His-Phe-Glu-Asn-Lys-Phe) and 3 out of 4 of the strictly conserved residues predicted for BRICHOS binding in this region (underlined in bold) (201) is present in IAPP 18-23. Based on this, it is possible that Bri2 BRICHOS recognizes IAPP 18-23 exposed when monomers acquire the more aggregation-prone beta-hairpin conformation. Bri2 BRICHOS interaction may result in refolding IAPP back into the more native and less aggregation prone helix-coil structure, thus preventing progression into prefibrillar toxic aggregates and amyloid fibrils. This hypothesis is similar to observations from proSP-C BRICHOS which binds to SP-C, the amyloid-prone transmembrane region of proSP-C, only when it is unfolded and not when it is in an alpha helical structure (203).

Combined results from paper I and paper II also supports that rapid amyloid formation is less cytotoxic, and inversely that slow amyloid formation can po-tentiate cytotoxicity. Presence of soluble heparin promotes rapid formation of inert amyloid fibrils, remote from the cell surfaces and thereby reduces the time during which toxic prefibrillar species exist and can act on the cell. In contrast, high IAPP concentrations in the presence of Bri2 BRICHOS extend the fibril formation process and thus the time under which toxic prefibrillar species are formed which lead to increased cytotoxicity. This supports the concept that prefibrillar species formed during the fibril formation process al-ternatively the growth of fibrils itself confer the initial IAPP-induced toxicity (204). However, although the above may be true in the cell assay, the build-up of islet amyloid mass in vivo (See Figure 4B) will eventually have detri-mental effects on the beta cells and destroy the architecture of the islet.

In paper III and IV we explored the interaction between amyloid peptides IAPP and Aβ. Specifically we demonstrated that local IAPP amyloid deposi-tion in the pancreatic islets of transgenic hIAPP mice could be enhanced by blood-borne IAPP fibrils as well as by proIAPP- and Aβ fibrils. Furthermore, we demonstrated the co-localization of IAPP with Aβ in brain-derived amy-loid in AD patients and a higher proportion of neuritic plaques, the more path-ogenic plaque subtype, in AD patients with concomitant T2D. These observa-tions from an experimental mouse model and patient material implies that a molecular interaction between IAPP and Aβ could affect disease events and be a possible factor in the clinical link between AD and T2D.

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Reports on seeding as a cause for disease development comes from studies on transmissible prion diseases, but also from animal models of AA-amyloi-dosis (15; 205). The potency of seeding is quite remarkable and as little as <1ng of protein can seed efficiently in a mouse model for AA-amyloidosis (205). Seeds can remain in the body for a long time, up to 40-50 years in hu-mans (206; 207) and when conditions for amyloid growth are met i.e. in-creased levels of amyloid precursor protein (205), the seeds accelerate aggre-gation which otherwise would occur much later or not at all.

Patients with familial polyneuropathy (FAP) develop systemic amyloidosis of mutated transthyretin (TTR), however the liver which is the main site of TTR production remains functionally normal without visible amyloid deposi-tions. Due to shortage of livers for donation, these livers from FAP patients can be used in so-called domino transplantations into patients with hepatic malignancies or end-stage liver disease. Several studies have reported that such recipients of FAP livers develop ATTR amyloidosis already a few years after the transplantation (208-210). Also, treatment with human cadaver-de-rived growth hormone from undiagnosed cases of Creutzfeldt-Jakob disease (CJD) can induce iatrogenic CJD characterized by brain amyloid deposition of not only prions (211) but also Aβ (212) in the recipients. Work by Juckers group have experimentally demonstrated that two intraperitoneal injections one weak apart with Aβ-rich brain extracts induced brain Aβ amyloid deposi-tion in APP transgenic mice 7 months later (213). The seeds appeared to enter the brain at multiple sites and interestingly, seeds could be detected in periph-eral blood leucocytes (214).

The concept of a prion-like mechanisms behind the enhancement and spreading of amyloid proteins have been discussed mostly in terms of homo-logue seeding and in neurodegenerative diseases (215). However, the concept could be extended to also include endogenous intrapatient cross-seeding events of two different amyloid proteins. However, such seeding events should be rare owing to the strong dependency of sequence similarities for efficient seeding (16). For IAPP and Aβ these requirements are met by their sequence- and length similarities and they indeed show an ability to both bind (180; 181) and cross-seed each other in vitro (179) and in vivo as observed in paper III.

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Conclusions

I The N-terminal region of IAPP is required for binding to HS structures and the binding promotes formation of amyloid fi-brils. Overexpression of heparanase reduce islet amyloid for-mation and cell surface-associated heparan sulfate mediate IAPP-induced apoptosis.

II Bri2 protein is highly expressed in human beta cells and co-lo-

calizes with IAPP both intracellularly and in islet amyloid de-posits. The BRICHOS domain of Bri2 is a potent inhibitor of IAPP fibril formation and IAPP-induced apoptosis, both in vitro and in vivo.

III Intravenous injections of IAPP, proIAPP and Aβ fibrils enhance

islet amyloidosis in transgenic human IAPP mice. IAPP co-lo-calize with Aβ in brain amyloid plaques in patients with Alz-heimer disease, but no Aβ could be detected in islet amyloid in patients with type 2 diabetes.

IV Alzheimer disease patients also diagnosed with type 2 diabetes

have a higher proportion of neuritic plaques, the more patho-genic plaque subtype compared to patients only diagnosed with Alzheimer disease.

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Sammanfattning på svenska

Vissa sjukdomstillstånd medför att aggregat av felveckade proteiner som kal-las amyloidfibriller ansamlas i vävnader. I amyloidfibrillerna ordnar sig pro-teinerna med vätebindningar i ett väldigt regelbundet mönster och fibrillerna ser nästan exakt likadana ut oavsett vilket protein som utgör byggstenarna i dem. Typ 2 diabetes drabbar framförallt äldre personer och orsakas av att be-tacellerna i de Langerhanska öarna i pankreas inte kan utsöndra tillräckligt med insulin vilket leder till förhöjt blodglukos. Hos patienter med typ 2 dia-betes bildas amyloid i öarna som byggs upp av ett betacellshormon som kallas islet amyloid polypeptide (IAPP). IAPP-amyloid, eller små förstadier till amyloidfibrillen s.k. prefibrillära aggregat, är toxiska för betacellerna och tros bidra till betacellsdöd och därmed också utvecklingen av typ 2 diabetes. Trots att IAPP snabbt bildar amyloidfibriller i provröret så är det till stor del okänt varför IAPP-amyloid ansamlas i öarna vid typ 2 diabetes. Syftet med en del av denna avhandling har varit att studera potentiella molekyler i cellen som skulle kunna påverka både bildningen av IAPP-amyloid men även den celldöd som induceras av aggregerande IAPP.

Vi har kunnat visa att en kolhydratstrukturer som kallas heparansulfat är viktig för bildningen av IAPP-amyloid. Heparansulfat finns överallt i kroppen, både på cellytor och i cellernas omgivningar. I isolerade öar från genmodi-ferade möss som bildar humant IAPP med ett samtidigt överuttryck av det enzym (heparanase) som bryter ner heparansulfat kunde vi uppmäta mindre IAPP-amyloid efter odling i hög glukos under lång tid. Även öar från humana IAPP möss som odlats i närvaro av korta heparansulfat-liknande fragment uppvisade mindre mängd deponerad amyloid. Vidare kunde vi visa att celler som helt saknade heparansulfat på cellytan inte var mottagliga för den celldöd som orsakas av aggregerande IAPP. Sammantaget visar dessa resultat att IAPP’s interaktioner med heparansulfat påskyndar amyloidbildningen och att interaktionen med heparansulfat på cellytan är viktig för att IAPP ska kunna inducera celldöd.

Som nämnts så bildar IAPP obehindrat amyloidfibriller i ett provrör på bara några timmar. Ändå bildas inte IAPP-amyloid hos friska människor trots att IAPP finns i höga koncentrationer både i betacellen och utanför när IAPP pre-cis utsöndrats. Det borde alltså finnas naturliga hämmare av amyloidbildning i betacellerna. Vi har kunnat visa att en sådan hämmare skulle kunna vara ett protein som heter Bri2 som innehåller en domän som kallas BRICHOS. För

37

första gången har vi visat att Bri2 uttrycks i betacellerna och att det samloka-liserar med IAPP både inne i cellen och i IAPP-amyloiden. BRICHOS-domä-nen från Bri2 kunde effektivt hämma IAPP-amyloidbildning och dess förmåga att inducera celldöd. Baserat på dessa resultat föreslår vi att Bri2 och i synner-het dess BRICHOS-domän skulle kunna vara en molekyl i betacellen som skyddar mot uppkomst av IAPP-amyloid.

Amyloidbildning liknar på många sätt bildningen av iskristaller där en initial iskärna fungerar som en katalysator och får omgivande vattenmolekyler att genast rätta sig efter iskärnan och forma is. På samma sätt kan ett litet frö av amyloid, kallad seed, få omgivande korrekt veckade proteiner att snabbt felveckas och byggas in i amyloidfibrillen. Detta fenomen kallas seeding och är den mekanism som gör prion-sjukdomar smittbara. I den andra delen av avhandlingen visade vi att en injektion av amyloidfibriller av IAPP in i blod-banan hos unga, humana IAPP möss gav upphov till en ökad ö-amyloidmängd vid 10 månaders ålder. Intressant nog gav även injektioner av amyloidfibriller bestående av ett helt annat protein, amyloid-β (Aβ), en ökad ö-amyloidmängd. Detta kallas cross-seeding eftersom det involverar två olika amyloid proteiner, ett i amyloidform som ”seedar” och ett annat som blir ”seedat”. Aβ bildar amyloidinlagringarna, kallade amyloida plack, i hjärnan hos patienter med Alzheimers sjukdom. Risken för att insjukna i Alzheimers har visats sig vara förhöjd hos patienter med typ 2 diabetes varför vi frågade oss om en interakt-ion, och i förlängningen en möjlig cross-seeding mekanism, mellan IAPP och Aβ kunde utgöra en koppling mellan de två sjukdomarna. Och mycket riktigt fann vi samlokalisation av IAPP med Aβ i amyloidinlagringarna i hjärnan hos Alzheimer patienter. Däremot kunde vi inte finna Aβ i ö-amyloid hos typ 2 diabetes patienter. Vidare fann vi en förhöjd inlagring av neuritiska amyloid-plack, en mer patologiskt kopplad plack-typ, i hjärnan hos Alzheimer patienter med typ 2 diabetes jämfört med Alzheimer patienter utan diabetes. Baserat på dessa resultat finns det en möjlighet att interaktioner mellan IAPP och Aβ skulle kunna utgöra en av kopplingarna mellan typ 2 diabetes och Alzheimers sjukdom.

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Acknowledgments

There are many people that I would like to thank which have contributed in many different ways to the work behind this thesis:

First of all my supervisor Gunilla Westermark for your phenomenal ability to evoke curiosity and enthusiasm for research. Your supervision has from the start focused on learning and development, encouraging me to always try new ideas and methods. I greatly appreciate the time you’ve taken to discuss re-search and shared your vast knowledge on amyloid but also on laboratory techniques and protocols. Lastly, thanks for all the good food, interesting books and movies about various wars, terrorists or just simple criminals and for dressing up Frank “preppy style”. Per Westermark for teaching me a lot about amyloid and for always reflect-ing on nomenclatures. Plaques should be spheres. Jin-Ping Li, my co-supervisor for your expertise in heparan sulfate and all the help during work with paper I. All my co-authors in the papers. Martin Ingelsson for the collaboration in the Alzheimer projects. The BRICHOS-group from KI; Jan Johansson, Jenny Presto, Erik Her-mansson and co-workers for nice collaboration. Marianne Ljungkvist for all the excellent technical assistance, help in keep-ing the lab in order and for being such a good roommate. I’m glad you did not leave before me. Ye Wang and Xiaohong Gu for all the support, nice comments and for fun travel-company to conferences around the world. And Lakshmi Kothegala for nice collaboration in paper IV. Former members of the group; Sebastian Schultz for making the time in Linköping so much fun and for teaching me a lot about research – like dis-

39

secting out fly brains. Sofia Nyström for letting me be your esthetical advi-sor, Johan Paulsson for laying the first stone one paper III, Jana Spona-rova and Aida Vahdat Shariatpanahi. All the senior researchers at the department of Medical Cell Biology for the tough questions at brain pubs and for increasing my knowledge on beta cells, islets, and diabetes, especially Michael Welsh, Leif Jansson, Nils Welsh, Stellan Sandler and Arne Andersson. All present and former PhD-students at Medical Cell Biology for interesting brain pub presentations, PhD-student days, all the well-organized “julgrans-plundringar” and for making the days so much fun, especially Sara U (friluftsmänniskan med ett finger över till vattenläckor), Evelina (entreprenö-ren), Gustaf (för Upptågs-eran), Liza (är du adlig?), Karin, Sara M (lycka till!), Nikhil (christmas-party fellow organizer), Kailash (christmas-party fel-low organizer), Daniel, Ebba, Parham and David. Joey Lau, My Quach and Sara Bohman for all the support, sushi-lunches, dinners and much more. The fika-crew on B3 level 1; Lisbeth Sagulin, Monica Sandberg, Mats Hjortberg and Stellan Sandler. Björn Åkerblom, Per Holmfeldt, Camilla Sävmarker and Shumin Pan for invaluable help with teaching matters and administrative issues. Leif Ljung for fixing the electron microscope whenever needed and Göran Ståhl for fix-ing everything else. Former fellow-PhD students from the amyloid field; Paul O’Callaghan and Fredrik Noborn – for all discussions on HS role in amyloidosis and Paul for coffee-consumption-retreats. Elisabet Ihse for managing to arrange at least one “amyloid-beer”. Ellahe for all the laughs at conferences and dinners. Everyone attending the JoLaWe amyloid-seminars for interesting presenta-tions and feedback. All the students that have done their projects in the group. Veronica Langenes och Emma Söderberg för åren i Linköping då vi lärde oss om citronsyracykeln och Cri du chat tillsammans. Nu slår vi rekord i hög-skolepoäng!

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Plan 12 på Cellbiologen i Linköping, Åsa, Cecilia, Siri, Tobias och Ia. Sak-nar er än idag. Mina vänner, framförallt Marie-Louise, Hanna, Daniel, Alexandra, Jenny, Monique och Mathieu, för att ni är så fina, smarta och roliga. Hela familjen Östberg och min familj; pappa, mamma, Andreas och Mi-chaela för att ni visat intresse för innehållet i denna bok och för allt stöd, på alla möjliga sätt. Sist men inte minst, Frank och Gustav - Tack för precis allt annat i mitt liv än det som ryms i denna lilla bok! ___________________________________ Tack även till Bergmarks resestipendium och Anna-Maria Lundins stipendie-fond (Smålands nation) för finansiering av konferensresor.

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Acta Universitatis UpsaliensisDigital Comprehensive Summaries of Uppsala Dissertationsfrom the Faculty of Medicine 1158

Editor: The Dean of the Faculty of Medicine

A doctoral dissertation from the Faculty of Medicine, UppsalaUniversity, is usually a summary of a number of papers. A fewcopies of the complete dissertation are kept at major Swedishresearch libraries, while the summary alone is distributedinternationally through the series Digital ComprehensiveSummaries of Uppsala Dissertations from the Faculty ofMedicine. (Prior to January, 2005, the series was publishedunder the title “Comprehensive Summaries of UppsalaDissertations from the Faculty of Medicine”.)

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