Indiana University-Purdue University Indianapolis (IUPUI) / Large-scale genome-wide association studies for Alzheimer’s disease (AD) have identified more than 20 risk loci and several pathways including lipid metabolism. Lipids are fundamental to cellular structure and organization, where they compose biological bilayer membranes surrounding the cell. In their structural role, lipids provide a scaffold for cell signaling, such as neurotransmission. There is a large body of evidence linking lipids and AD, yet the relationship between AD pathogenesis and lipid dyshomeostasis is not well understood. Here, we performed manual PubMed searches to identify the most studied lipid classes and risk genes in AD. We discussed pathological alterations of the key lipids and their potential contribution to the recent NIA-AA “A/T/N” framework. We also summarized what is known between the key lipids and etiological hypotheses of AD. Finally, we characterized relationship of the key lipids with AD genomic risk factors to identify possible downstream mechanisms of lipid dysfunction in AD. There is a large body of evidence linking lipids and AD, yet the relationship between AD pathogenesis and lipid dyshomeostasis is not well understood. In particular, we investigated the association between triglyceride (TG) species and AD. The overall goal was to test the hypothesis that TGs would associate with AD endophenotypes, based on their fatty acid composition. Diagnostic groups (cognitively normal older adults (CN), mild cognitive impairment (MCI), and AD) differed on two principal components extracted from 84 serum TG levels. Fish oil-type and olive oil-type TGs were significantly lower in MCI and AD compared to CN. Next, association analysis of TG principal components with “A/T/N/V” (amyloid-β, tau, neurodegeneration, and cerebrovascular) biomarkers for AD showed that the fish oil-type and olive oil-type TGs were also significantly associated with atrophy on MRI. Finally, a mixed model regression analysis investigated the association between baseline TGs and longitudinal changes of AD endophenotypes to show that olive oil-type TGs predicted changes in AD brain atrophy. Our results indicate that a specific subcategory of TGs is associated with an early prodromal stage of cognitive impairment and early-stage biomarkers for AD, providing the foundation for future therapeutic development related to TG metabolism. / 2023-05-05

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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 of IAPP are largely unknown. So is the possibility that islet amyloid can spread within or between tissues. 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 amyloid and cytotoxic IAPP aggregates. Furthermore, cross-seeding as a molecular interaction between the 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 of binding promoted amyloid formation. Interference in the HS-IAPP interaction by heparanase degradation of HS or by introducing short, soluble HS-structure fragments reduced amyloid deposition in cultured islets. Cytotoxicity induced by extracellular, aggregating IAPP was mediated via interactions with cell-surface HS. This suggests that HS plays an important role in islet amyloid deposition and associated toxicity. BRICHOS domain containing protein Bri2 was highly expressed in human beta cells and colocalized with IAPP intracellularly and in islet amyloid deposits. The BRICHOS domain effectively attenuated both IAPP amyloid formation and IAPP-induced cytotoxicity. These results 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 islet amyloidosis in transgenic human IAPP mice, demonstrating that both homologous- and heterologous seeding of islet amyloid can occur in vivo. IAPP colocalized with Aβ in brain amyloid from AD patients, and AD patients diagnosed with T2D displayed increased proportions of neuritic plaques, the more pathogenic plaque subtype. In conclusion, both IAPP amyloid formation and the cytotoxic effects of IAPP is dependent on interactions with HS whereas interactions with Bri2 BRICHOS is protective. Cross-seeding between Aβ and IAPP can occur in vivo and the two peptides colocalize in brain amyloid in AD patients.

Aggregation of proteins into amyloid deposits is a common feature among dozens of diseases. Two such diseases that feature amyloid deposits are Alzheimer's disease (AD) and type 2 diabetes (T2D). AD toxicity has been associated with the aggregation and accumulation of the amyloid β-peptide (Aβ); Aβ exerts its toxic effects through interactions with neuronal cell membranes. A characteristic feature of T2D is the deposition of the islet amyloid polypeptide (IAPP) in the pancreatic islets of Langerhans. It is currently unknown if IAPP aggregation is a cause or consequence of T2D, but it does lead to β-cell dysfunction and death, exacerbating the effects of diabetes. Characterizing the fundamental interactions between both Aβ and IAPP with lipid membranes and in solution will give greater insight into mechanisms of toxicity exhibited by amyloid proteins. In this work, molecular dynamics (MD) simulations were used to study the secondary, tertiary, and quatnary structure of Aβ and IAPP, in addition to peptide-membrane interactions and membrane perturbation as independently caused by both peptides. Studies were conducted to address the following questions: (1) what influence do solution conditions and oxidation state have on monomeric Aβ] (2) how and in what way does monomeric Aβ interact with model lipid membranes and what role does sequence play on these peptide-membrane interactions; (3) can MD simulations be utilized to understand Aβ tetramer formation, rearrangement, and tetramer-membrane interactions; (4) how does IAP interact with model membranes and how does that vary from non-toxic (rat) IAPP peptide-membrane interactions. These studies led to conclusions that showed variance in lipid affinity and degree of perturbation as based on peptide sequence, in addition to insight into the type of perturbation caused to membranes by these amyloid peptides. Understanding the differences in peptide-membrane interactions of amyloidogenic and non-amyloidogenic (rat) peptides gave insight into the overall mechanism of amyloidogenicity, leading to the detection of specific amino acids essential in peptide-membrane perturbation. These residues can then be targeted for novel therapeutic design to attenuate the perturbation and potential cell death as caused by these peptides. / Ph. D.

Amyloid is defined as extracellular protein aggregates with a characteristic fibrillar ultra-structure, Congo red affinity and a unique x-ray diffraction pattern. At present, 25 different human amyloid fibril proteins have been identified, and amyloid aggregation is associated with pathological manifestations such as Alzheimer’s disease, spongiform encephalopathy and type 2 diabetes. Amyloid aggregation triggers apoptosis by incorporation of early oligomers in cellular membranes, causing influx of ions. Amyloid is the only visible pathological islet alteration in subjects with type 2 diabetes, and islet amyloid polypeptide (IAPP) is the major islet amyloid fibril component. IAPP is produced by beta-cells and co-localized with insulin in the secretory granules. Both peptides are synthesised as pro-molecules and undergo proteolytic cleavage by the prohormone convertase 1/3 and 2. Although IAPP is the main amyloid constituent, both proIAPP and proIAPP processing intermediates have been identified in islet amyloid. The aim of this thesis was to study the role of impaired processing of human proIAPP in early islet amyloidogenesis. Five cell lines with individual processing properties were transfected with human proIAPP and expression, aggregation and viability were studied. Cells unable to process proIAPP into IAPP or to process proIAPP at the N-terminal processing site accumulated intracellular amyloid-like aggregates and underwent apoptosis. Further, proIAPP immunoreactivity was detected in intracellular amyloid-like aggregates in betacells from transgenic mice expressing human IAPP and in transplanted human beta-cells. ProIAPP was hypothesized to act as a nidus for further islet amyloid deposition, and to investigate this theory, amyloid-like fibrils produced from recombinant IAPP, proIAPP and insulin C-peptide/A-chain were injected in the tail vein of transgenic mice expressing the gene for human IAPP. Pancreata were recovered after 10 months and analysed for the presence of amyloid. Both IAPP and proIAPP fibrils but not des-31,32 proinsulin fibrils, caused an increase in affected islets and also an increase of the amyloid amount. This finding demonstrates a seeding capacity of proIAPP on IAPP fibrillogenesis. IAPP has been known for some time to trigger apoptosis in cultured cells, and a novel method for real time detection of apoptosis in beta-cells was developed. Aggregation of recombinant proIAPP and proIAPP processing intermediates were concluded to be inducers of apoptosis as potent as IAPP fibril formation. From the results of this study, a scenario for initial islet amyloidogenesis is proposed. Initial amyloid formation occurs intracellularly as a result of alterations in beta-cell processing capacity. When the host cell undergoes apoptosis intracellular proIAPP amyloid becomes extracellular and can act as seed for further islet amyloid deposition.

Diss. (sammanfattning) Linköping : Univ., 2001. / Härtill 5 uppsatser.

Plaques of amyloid β peptide (Aβ) are a hallmark trait of Alzheimer’s disease (AD). However, the precise role of Aβ aggregates is not well understood. Recent studies have identified that naturally occurring N-terminal truncation and pyroglutamylation of Aβ significantly increases its neurotoxicity by an unknown mechanism. Content of pyroglutamylated Aβ (pE-Aβ) in AD brains has been shown to reach up to 50% of total Aβ. Modified pE-Aβ co-aggregates with Aβ by a seeding mechanism and forms structurally distinct and highly toxic oligomers. We studied structural transitions of the full-length Aβ1-42, its pyroglutamylated form AβpE3-42, their 9:1 (Aβ1-42/AβpE3-42) and 1:1 molar combinations. Transmission electron microscopy was used to directly visualize the fibrils of the samples in a buffer mimicking physiological environment. Atomic force microscopy measurements were done to determine rate of second nucleation events in fibrils. Thioflavin-T fluorescence indicated that low ionic strength suppressed the aggregation of AβpE3-42 but promoted that of Aβ1-42, suggesting different paths of fibrillogenesis of unmodified Aβ and pE- Aβ. Interestingly, AβpE3-42 at only 10% significantly facilitated the fibrillization of Aβ1-42 at near-physiological ionic strength but had little effect at low salt. Circular dichroism and Fourier transform infrared (FTIR) spectroscopy were used to characterize the structural transitions during fibrillogenesis. In aqueous buffer, both unmodified Aβ and pE-Aβ peptides adopted parallel intermolecular β-structure. Interestingly, AβpE3-42 contained lower β-sheet content than 13C-Aβ1-42, while retaining significantly larger fractions of α-helical and turn structures. Structural details of Aβ and pE-Aβ combinations were unveiled by isotope-edited FTIR spectroscopy, using 13C-labeled Aβ1-42 and unlabeled AβpE3-42. When exposed to environmental humidity, AβpE3-42 not only maintained an increased fraction of α-helix but also was able to reverse 13C-Aβ1-42 β-sheet structure. These data provide a novel structural mechanism for pE-Aβ hypertoxicity; pE-Aβ undergoes faster nucleation due to its increased hydrophobicity, thus promoting formation of smaller, hypertoxic oligomers of partial α-helical structure.

Amyloidogenic diseases, such as Alzheimer's disease (AD) and Type II Diabetes (T2D), are characterized by the accumulation of amyloid aggregates. Despite having very different amino-acid sequences, the underlying amyloidogenic proteins form similar supramolecular fibril structures that are highly stable and resistant to physical and chemical denaturation. AD is characterized by two toxic lesions: extracellular amyloid β-peptide (Aβ) plaques and intracellular neurofibrillary tangles composed of microtubule-associated protein tau. Similarly, a feature of T2D is the deposition of islet amyloid polypeptide (IAPP) aggregates in and around the pancreas. The mechanisms by which Aβ, tau, and IAPP aggregate, and cause cell death is unknown; thus, gaining greater insight into the stabilizing forces and initial unfolding events is crucial to our understanding of these amyloidogenic diseases. This work uses molecular dynamics (MD) simulations to study the secondary, tertiary, and quaternary structure of Aβ, tau, and IAPP. Specifically, this work used the Drude polarizable force field (FF), which explicitly represents electronic polarization allowing charge distributions to change in response to perturbations in local electric fields. This model allows us to describe the role charge plays on protein folding and stability and how perturbations to the charge state drive pathology. Studies were conducted to address the following questions: 1) What are the stabilizing forces of fibril and oligomeric structures? 2) How do charge-altering mutations modulate the conformational ensemble and thermodynamic properties of Aβ? 3) How do charge-altering post-translational modifications of Aβ and tau modulate changes in the conformational ensembles? These studies establish that shifts in local microenvironments play a role in fibril and oligomer stability. Furthermore, these studies found that changes in protein sequence and charge are sufficient to disrupt and change the secondary and tertiary structure of these amyloidogenic proteins. Overall, this dissertation describes how charge modulates protein unfolding and characterizes the mechanism of those changes. In the long term, this work will help in the development of therapeutics that can target these changes to prevent protein aggregation that leads to cell death. / Doctor of Philosophy / Protein aggregation is the hallmark of many chronic diseases, such as Alzheimer's disease (AD) and Type II Diabetes (T2D). The formation of two toxic aggregates: amyloid β-peptide (Aβ) plaques and neurofibrillary tangles composed of microtubule-associated protein tau are some of the key characteristics of AD. In addition, the formation of islet amyloid polypeptide (IAPP) aggregates in the pancreas is thought to play a role in the development of T2D. The pathways by which the proteins Aβ, tau, and IAPP aggregate are unknown; thus, gaining a greater insight into the properties that may cause these diseases is necessary to develop treatments. By studying these proteins at the atomistic level, we can understand how small changes to these proteins alter how they misfold in a way that promotes toxicity. Herein, we used a computational technique called molecular dynamics (MD) simulations to gain new insights into how protein structure changes. We explored the dynamics of these proteins and investigated the role that charge plays in protein folding and described how charge modulates protein folding and characterized the mechanism of those changes. This work serves as a characterization of protein folding and sets the ground for future structural studies and drug development.

Late-onset Alzheimer’s disease (LOAD) is the most common form of dementia, with an incidence of up to 50% in western populations over the age of 85 and a high heritability (up to 80%). The identification of risk factors for the development of LOAD is imperative for improving our understanding of this disease and for identifying therapeutic targets for treatment or prevention. Currently, the major known risk factors for the development of LOAD are age and the ApoE ε4 genotype. Previous studies have implicated plasma levels of the amyloid beta (Aß) peptide as a LOAD-associated quantitative trait and identification of loci influencing this trait could provide new insights into LOAD. In this thesis, plasma levels of the Aß peptides Aß40 and Aß42 have been measured in two isolated populations and genome-wide linkage and association analyses were performed. The genome-wide association analyses identified a number of promising quantitative trait loci; highlighting both novel and previously reported LOAD genes for further study, whilst also providing an excellent resource for genetic convergence studies with other LOAD related traits. Several studies have reported an association between levels of oxidative stress and levels of Aß such that increasing levels of Aß appear to increase markers for oxidative stress and vice versa. The role of oxidative stress in LOAD and aging was therefore also investigated through analysis of mitochondrial mutational burden and DNA damage respectively, using DNA isolated from both blood and the brain and by carrying out a candidate gene association study of loci involved in mitochondrial function in LOAD cases and controls. Approaches to the investigation of mitochondrial genetics for the study of LOAD are comprehensively reviewed, adapted and tested and the results indicate a need for additional research in this aspect of the disease. This thesis therefore presents a focus on two aspects of genetic research into LOAD, a complex disease with multiple environmental and genetic influences which aims to advance our understanding of the disease and bring us closer to treatment and prevention strategies.

The self-assembly of proteins into filamentous structures underpins many aspects of biology, from dynamic cell scaffolding proteins such as actin, to the amyloid plaques responsible for a number of degenerative diseases. Typically, these self-assembly processes have been treated as nucleated, reversible polymerisation reactions, where dynamic fluctuations in a population of monomers eventually overcome an energy barrier, forming a stable aggregate that can then grow and shrink by the addition and loss of more protein from its ends. The nucleated, reversible polymerisation framework is very successful in describing a variety of protein systems such as the cell scaffolds actin and tubulin, and the aggregation of haemoglobin. Historically, amyloid fibrils were also thought to be described by this model, but measurements of their aggregation kinetics failed to match the model's predictions. Instead, recent work indicates that autocatalytic polymerisation - a process by which the number of growth competent species is increased through secondary nucleation, in proportion to the amount already present - is better at describing their formation. In this thesis, I will extend the predictions made in this mean-field, autocatalytic polymerisation model through use of kinetic Monte Carlo simulations. The ubiquitous sigmoid-like growth curve of amyloid fibril formation often possesses a notable quiescent lag phase which has been variously attributed to primary and secondary nucleation processes. Substantial variability in the length of this lag phase is often seen in replicate experimental growth curves, and naively may be attributed to fluctuations in one or both of these nucleation processes. By comparing analytic waiting-time distributions, to those produced by kinetic Monte Carlo simulation of the processes thought to be involved, I will demonstrate that this cannot be the case in sample volumes comparable with typical laboratory experiments. Experimentally, the length of the lag phase, or "lag time", is often found to scale with the total protein concentration, according to a power law with exponent γ. The models of nucleated polymerisation and autocatalytic polymerisation predict different values for this scaling exponent, and these are sometimes used to identify which of the models best describes a given protein system. I show that this approach is likely to result in a misidentification of the dominant mechanisms under conditions where the lag phase is dominated by a different process to the rest of the growth curve. Furthermore, I demonstrate that a change of the dominant mechanism associated with total protein concentration will produce "kinks" in the scaling of lag time with total protein concentration, and that these may be used to greater effect in identifying the dominant mechanisms from experimental kinetic data. Experimental data for bovine insulin aggregation, which is well described by the autocatalytic polymerisation model for low total protein concentrations, displays an intriguing departure from the predicted behaviour at higher protein concentrations. Additionally, the protein concentration at which the transition occurs, appears to be affected by the presence of salt. Coincident with this, an apparent change in the fibril structure indicates that different aggregation mechanisms may operate at different total protein concentrations. I demonstrate that a transition whereby the self-assembly mechanisms change once a critical concentration of fibrils or fibrillar protein is reached, can explain the observed behaviour and that this predicts a substantially higher abundance of shorter laments - which are thought to be pathogenic - at lower total protein concentrations than if self-assembly were consistently autocatalytic at all protein concentration. Amyloid-like loops have been observed in electron and atomic-force microscographs, together with non-looped fibrils, for a number of different proteins including ovalbumin. This implies that fibrils formed of these proteins are able to grow by fibrillar end-joining, and not only monomer addition as is more commonly assumed. I develop a simple analytic expression for polymerisation by monomer addition and fibrillar end-joining, (without autocatalysis) and show that this is not sufficient to explain the growth curves obtained experimentally for ovalbumin. I then demonstrate that the same data can be explained by combining fibrillar end-joining and fragmentation. Through the use of an analytic expression, I estimate the kinetic rates from the experimental growth curves and, via simulation, investigate the distribution of lament and loop lengths. Together, my findings demonstrate the relative importance of different molecular mechanisms in amyloid fibril formation, how these might be affected by various environmental parameters, and characteristic behaviour by which their involvement might be detected experimentally.