Alzheimer’s disease (AD) is a neurodegenerative disorder which is histopathologically characterised by amyloid plaques and neurofibrillary tangles. Amyloid plaques consist of the amyloid β-peptide (Aβ) that can form aggregates in the brain. Aβ is generated from the amyloid precursor protein (APP) through proteolytic cleavage. APP belongs to a conserved protein family that also includes the two paralogues, APP-like proteins 1 and 2 (APLP1 and APLP2). Despite the immense amount of research on APP, motivated by its implication in AD, the function of this protein family has not yet been determined. In this thesis, we have studied the expression and proteolytic processing of the APP protein family. Our results are consistent with previous findings that suggest a role for APP during neuronal development. Treatment of cells with retinoic acid (RA) resulted in increased synthesis. In addition, we observed that RA treatment shifted the processing of APP from the amyloidogenic to the non-amyloidogenic pathway. The proteins in the APP family have been hard to distinguish both with respect to function and proteolytic processing. However, for development of new drugs with APP processing enzymes as targets this is of great importance. Our studies suggest similarities, but also differences in the mechanism regulating the processing of the different paralogues. We found that brain-derived neurotrophic factor (BDNF) had different impact on the members of the APP family. Most interestingly, we also found that the mechanism behind the increased processing in response to IGF-1 was not identical between the homologous proteins. In summary, our results indicate that in terms of regulation APLP1 and APLP2 differ more from each other than from APP. Our studies open up the possibility of finding means to selectively block Aβ production without interfering with the processing and function of the paralogous proteins.

The Arctic mutation in the Amyloid-β (Aβ) domain of the Amyloid-β precursor protein (APP) causes Alzheimer’s disease (AD) and confers unique biochemical characteristics to Aβ peptides. The aims of this thesis were to evaluate a transgenic model with the Arctic mutation, and to use it to gain new insights into the mechanisms of early (pre-plaque) and late-stage Aβ pathogenesis in AD. The Arctic mutation made Aβ more prone to aggregate, to accumulate in intracellular compartments and to form extracellular plaques when the models tg-ArcSwe and tg-Swe were compared. By inhibiting APP processing genetically or pharmacologically, the intraneuronal granular immunoreactivity with antibodies binding the Aβ domain was shown to largely represent Aβ, and not APP or APP-fragments. At two months of age, the intracellularly accumulated Aβ decreased rapidly, likely because it was still accessible to intracellular clearance. Extracellular Aβ deposits emerged at 5-6 months of age and the amyloid fibril structure was more compact than in tg-Swe. Moreover, Aβ deposits in tg-ArcSwe were more resistant to chemical extraction than those of established models carrying the Swedish APP mutation only, e.g. tg-Swe mice. The stability of deposits better reflects the biochemistry of senile plaques in AD. Thus, the tg-ArcSwe model may better predict the outcome of clinical trials, particularly therapies designed to enhance clearance of Aβ aggregates and deposits. Postmortem brain of Arctic mutation carriers contained extensive parenchymal plaque pathology. Differential immunostaining patterns with C- and N-terminal Aβ antibodies revealed a subset of plaques that were unique to the brains of Arctic mutation carriers. Aβ deposits in the cerebral vessel walls were congophilic and mainly composed of full-length Aβ. In contrast, N-terminally truncated Aβ was more prominent in the parenchymal plaques, all of which essentially lacked amyloid cores. A heterogeneous assembly of mutant and wild-type Aβ was shown to favor the formation of diffuse deposits in bitransgenic mice, and such mechanisms may at least partly explain observations of plaques lacking amyloid cores in postmortem Arctic mutant brain. In the bitransgenic mice, a low level of Arctic Aβ was sufficient to facilitate aggregation of wild-type Aβ. This observation, but also our findings of differences in amyloid fibril structure in tg-ArcSwe and tg-Swe, further highlights similarities between AD and prion disorders in which PrPsc refolds PrPc and facilitates fibril formation. / (Faculty of medicine)

It is suggested that Alzheimer’s disease (AD) is caused by an imbalance between production, degradation and clearance of the amyloid-β (Aβ) protein. This imbalance leads to aggregation of Aβ and tau proteins and neurodegeneration in the brain. Today there is increasing evidence that the balance between the protease cathepsin B and the protease inhibitor cystatin C affects the tendency for Aβ to aggregate. The primary aim of this thesis was to investigate Aβ, cystatin C and cathepsin B levels in blood and cerebro-spinal fluid (CSF) in relation to the risk of AD. Studies I & II were based on the re-examinations of participants, at ages 70 and 77, in the Uppsala Longitudinal Study of Adult Men (ULSAM), a community-based prospective study initiated in 1970 (participants then being 50 years of age). In ULSAM, low plasma Aβ1-40 (Study I) and low serum cystatin C levels (Study II) were associated with a higher risk of AD. Studies III & IV were based on a cross-sectional sample of people with AD, mild cognitive impairment and healthy controls, recruited at three Swedish Memory Disorder units: Uppsala University Hospital, Uppsala, Skåne University Hospital, Malmö, and Karolinska University Hospital, Huddinge, Stockholm. In Study III, CSF cystatin C levels were positively correlated with both Aβ1-42 and tau levels. In Study IV, individuals with AD had higher mean plasma cathepsin B levels than healthy controls. In conclusion, low plasma Aβ1-40 and low serum cystatin C levels may precede clinically manifest AD in elderly men, cystatin C levels are positively correlated with Aβ1-42 and tau levels in CSF, and mean plasma cathepsin B levels are higher in people with AD compared to healthy controls. In addition to Aβ1-42 and tau levels in CSF, Aβ1-40, cystatin C and cathepsin B levels in blood may reflect the risk of AD.

Alzheimer’s disease (AD) is a progressive neurodegenerative disorder, neuropathologically characterized by neurofibrillay tangles and deposition of amyloid-β (Aβ) peptides. Several mutations in the gene for amyloid precursor protein (APP) cause familial AD and affect APP processing leading to increased levels of Aβ42. However, the Arctic Alzheimer mutation (APP E693G) reduces Aβ levels. Instead, the increased tendency of Arctic Aβ peptides to form Aβ protofibrils is thought to contribute to the pathogenesis. In this thesis, the pathogenic mechanisms of the Arctic mutation were further investigated, specifically addressing if and how the mutation affects APP processing. Evidence of a shift towards β-secretase cleavage of Arctic APP was demonstrated. Arctic APP did not appear to be an inferior substrate for α-secretase, but the availability of Arctic APP for α-secretase cleavage was reduced, with diminished levels of cell surface APP in Arctic cells. Interestingly, administration of the fatty acid docosahexaenoic acid (DHA) stimulated α-secretase cleavage and partly reversed the effects of the Arctic mutation on APP processing. In contrast to previous findings, the Arctic mutation generated enhanced total Aβ levels suggesting increased Aβ production. Importantly, this thesis illustrates and explains why measures of both Arctic and wild type Aβ levels are highly dependent upon the Aβ assay used, with enzyme-linked immunosorbent assay (ELISA) and Western blot generating different results. It was shown that these differences were due to inefficient detection of Aβ oligomers by ELISA leading to an underestimation of total Aβ levels. In conclusion, the Arctic APP mutation leads to AD by multiple mechanisms. It facilitates protofibril formation, but it also alters trafficking and processing of APP which leads to increased steady state levels of total Aβ, in particular at intracellular locations. Importantly, these studies highlight mechanisms, other than enhanced production of Aβ peptide monomers, which could be implicated in sporadic AD.

The diffusible soluble oligomeric amyloid β-peptide (Aβ) has been identified as a toxic agent in Alzheimer’s disease that can cause synaptic dysfunction and memory loss, indicating its role as potential therapeutic targets for AD treatment. Recently an oligomer-specific sandwich biotin-avidin interaction based assay identified the Aβ oligomer dissociation potency of a series of dihydroxybenzoic acid (DHBA) isomers. Because the sandwich assay is an ensemble method providing limited size information, fluorescence correlation spectroscopy (FCS) was employed to provide single molecule resolution of the disassembly mechanism. Using FCS coupled with atomic force microscopy, we investigated the size distribution of fluorescein labeled synthetic Aβ oligomers at physiological concentrations, and monitored in real time the change of size and mole fraction of oligomers in the presence of dissociating agents or conditions. The higher-order dissociation process caused by DHBA isomers produced no transient oligomeric intermediates, a desirable feature for an anti-oligomer therapeutic. Urea and guanidine hydrochloride, in contrast, produced a linear dissociation with a progressive decrease of size and mole fraction of oligomers. FCS allows the facile distinction of small molecule Aβ oligomer dissociators that do not produce stable potentially toxic oligomeric Aβ intermediates.

京都大学 / 0048 / 新制・課程博士 / 博士(医学) / 甲第22636号 / 医博第4619号 / 新制||医||1044(附属図書館) / 京都大学大学院医学研究科医学専攻 / (主査)教授 高橋 淳, 教授 古川 壽亮, 教授 村井 俊哉 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

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.

Copper is one of the essential metal ions for aerobic organisms. Two well known functions of copper in the biological systems are electron transfer and molecular oxygen interaction. Thus, this metal can be found in haemocyanin, an oxygen carrier protein, and superoxide dismutase, an enzyme that involves in electron transfer. In addition, having a positive redox potential allows copper to be involved in redox chemistry. It is the redox properties of copper that are responsible for many important biochemical processes. Although the copper-containing oxidases have been well studied over the years, certain mechanistic details such as reaction intermediates remain to be elucidated. Several research groups have been trying to study this by trying to mimic the native systems, synthesizing bulky organic molecules with copper-binding and oxidative capabilities. However, these model systems are only applicable in organic solvents at low temperatures. In this study, three naturally occurring peptides, amyloid-ß, bacitracin, and histatin 5, have been shown to display the oxidative chemistry when complexed with CuII. A combination of spectroscopic (UV-Vis and NMR) and reactivity was used in studying their metal-binding properties as well as in elucidating their catalytic mechanism.

Studies presented in this dissertation were conducted to gain more insight into the role of phospholipid asymmetry and amyloid-β (Aβ)-induced oxidative stress in brain of subjects with amnestic mild cognitive impairment (aMCI) and Alzheimer disease (AD). AD is a largely sporadic, age-associated neurodegenerative disorder clinically characterized by the vast, progressive loss of memory and cognition commonly in populations over the age of ~65 years, with the exception of those with familial AD, which develop AD symptoms as early as ~30 years-old. Neuropathologically, both AD and FAD can be characterized by synapse and neuronal cell loss in conjunction with accumulation of neurofibrillary tangles and senile plaques. Elevated levels of oxidative stress and damage to brain proteins, lipids, and nucleic acids are observed, as well. Likewise, aMCI, arguably the earliest form of AD, displays many of these same clinical and pathological characteristics, with a few exceptions (e.g., no dementia) and to a lesser extent. Studies in this dissertation focused on the contributions of oxidative stress to the exposure of phosphatidylserine (PtdSer) to the outer-leaflet of the lipid membrane, how and when PtdSer asymmetric collapse contributes to the progression of aMCI, AD, and FAD, and the role played by methionine-35 (Met-35) of Aβ in oxidative stress and damage, as measured in a transgenic mouse model of Aβ pathology. Normally, the PtdSer is sequestered to the cytosolic, inner-leaflet of the bilayer by the adenosine triphosphate (ATP)-dependent, membrane-bound translocase, flippase, which unidirectionally transports PtdSer inward against its concentration gradient. Oxidative stress-induced modification of flippase and/or PtdSer, however, leads to prolonged extracellular exposure of PtdSer on the outer membrane leaflet, a known signal for both early apoptosis and selective recognition and mononuclear phagocytosis of dying cells. Within the inferior parietal lobule (IPL) of subjects with aMCI and AD, a significant collapse in PtdSer asymmetry was found in association with increased levels of both pro- and anti-apoptotic proteins, Bax, caspase-3, and Bcl-2. Moreover, a significant collapse in PtdSer asymmetry was also found in whole brain of human double-mutant knock-in mouse models of Aβ pathology, together with significantly reduced Mg2+ATPase activity, representing flippase activity, and increased levels of pro-apoptotic caspase-3. Significant PtdSer externalization corresponded to the age at which significant soluble Aβ(1-42) deposition occurs in this particular mouse model (9 months), and not of plaque deposition (12 months), suggesting that elevated levels of Aβ(1-42), together with increasing oxidative stress and apoptosis, may contribute to altered PtdSer membrane localization. Also in this dissertation, transgenic mice carrying Swedish and Indiana mutations on the human amyloid precursor protein (APPSw,In) and APPSw,In mice carrying a Met35Leu mutation on Aβ were derived to investigate the role of Met-35 in Aβ(1-42)-induced oxidative stress in vivo. Oxidative stress analyses revealed that Aβ-induced oxidative stress requires the presence of Met-35, as all indices of oxidative damage (i.e., protein carbonylation, nitration, and protein-bound 4-hydroxy-2-trans-nonenal [HNE]) in brain of Met35Leu mice were completely prevented. Moreover, immunohistochemical analyses indicated that the Met35Leu mutation influences plaque formation, as a clear reduction in Aβ-immunoreactive plaques in Met35Leu mice was found in conjunction with a significant increase in microglial activation. In contrast, behavioral analyses suggested that spatial learning and memory was independent of Met-35 of Aβ, as Met35Leu mice demonstrated inferior water-maze performance compared to non-transgenic mice. Differential expression and redox proteomic analyses to pinpoint proteins significantly altered by the APPSw,In and Met35Leu mutations was performed, as well. Expression proteomics showed significant increases and decreases in APPSw,In and Met35Leu mouse brain, respectively, in proteins involved in cell signaling, detoxification, structure, metabolism, molecular chaperoning, protein degradation, mitochondrial function, etc. Redox proteomics found many of these same proteins to be oxidatively modified (i.e., protein carbonylation and nitration) in both APPSw,In and Met35Leu mouse brain, providing additional insights into the critical nature of Met-35 of Aβ for in vivo oxidative stress in a mammalian species brain, and strongly suggesting similar importance of Met-35 of Aβ(1-42) in brain of subjects with aMCI and AD. Taken together, studies presented in this dissertation demonstrate the role of oxidative stress-induced alteration of PtdSer asymmetry and Met-35 in Aβ-induced oxidative stress in aMCI, AD, and FAD brain.

Alzheimer s (DA) is a neurodegenerative disease evidenced by cognitive disorders and attention deficit and learning, and is the main cause of dementia in the elderly. The amyloid hypothesis posits that extracellular amyloid-β (Aβ) deposits are the fundamental etiological factor of the disease. However, the AD etiology has yet to be fully understood and common treatments remain largely non-efficacious. Caernorhabditis elegans transgenic strains expressing toxic Aβ has been employed as AD in vivo model in order to elucidate mechanisms and verifying the effectiveness of pharmacological compounds. The organoselenium compound tested in this study, Diphenyl-diselenide (PhSe)2, has shown efficacy in ameliorate several parametres in neurodegenerative disease models. In the present study, we analyzed the effects of (PhSe)2 chronic treatment on Aβ peptide-induced toxicity in C. elegans. This data shows that chronic exposure to (PhSe)2 attenuated oxidative stress induced by Aβ with concomitant recovery of associative learning memory in worms. In addition, (PhSe)2 decreased Aβ transgene expression, suppressing the Aβ peptide and down-regulating hsp-16.2 by reducing the need of this chaperone under Aβ toxicity. This observations suggest that (PhSe)2 plays an important role in protection against oxidative stress-induced toxicity, this representing a promising potential pharmaceutical modality by attenuating Aβ expression. / A Doença de Alzheimer (DA) é uma doença neurodegenerativa evidenciada por distúrbios cognitivos e déficit de atenção e aprendizagem, sendo a principal causa de demência em idosos. A Hipótese Amilóide postula o acúmulo de depósitos extracelulares do peptídio β-amilóide (Aβ) no cérebro como o principal fator da doença. Entretanto, sua etiologia ainda não está completamente elucidada e seu tratamento visa apenas a melhora dos sintomas. Cepas transgênicas do nematódeo Caernorhabditis elegans que expressam as espécies tóxicas Aβ, têm sido utilizadas como modelos in vivo de DA para elucidar mecanismos e verificar a eficácia de novas moleculas. O disseleneto de difenila ((PhSe)2), composto orgânico de selênio utilizado nesse estudo, tem demonstrado eficácia em melhorar diversos parâmetros em modelos de doenças neurodegenerativas. No presente estudo foram analisados os efeitos do tratamento crônico com (PhSe)2 na toxicidade induzida pela Aβ em C. elegans. Os resultados mostraram que a exposição crônica ao (PhSe)2 atenuou o estresse oxidativo induzido pela Aβ, além de recuperar a memória associativa no nematódeo. Além disso, o (PhSe)2 diminuiu a expressão do gene Aβ, levando a supressão do peptídio Aβ e reduzindo a expressão do gene hsp-16.2, por diminuir a necessidade desta chaperona frente a toxicidade Aβ. Estes dados sugerem que o (PhSe)2 desempenha um importante papel na proteção contra a toxicidade induzida por estresse oxidativo, além de representar um promissor agente farmacológico por atenuar a expressão do Aβ.