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β y Supervisors: Lars Nilsson, Associate Professor Department of Public Health and Caring Sciences Uppsala University Uppsala, Sweden Lars Lannfelt, MD, Professor Department of Public Health and Caring

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β y Supervisors: Lars Nilsson, Associate Professor Department of Public Health and Caring Sciences Uppsala University Uppsala, Sweden Lars Lannfelt, MD, Professor Department of Public Health and Caring Sciences Uppsala University Uppsala, Sweden Faculty opponent: Fred van Leuven, Professor Department of Human Genetics KULeuven Leuven, Belgium Examining committee: Irina Alafuzoff, MD, Professor Department of Genetics and Pathology Uppsala University Uppsala, Sweden Karin Forsberg-Nilsson, Professor Institute for Genetics and Pathology Uppsala University Uppsala, Sweden Elisabet Londos, MD, Associate Professor Department of Clinical Sciences Malmö University Hospital/ Lund University Malmö, Sweden Chairperson: Frida Ekholm Pettersson, Assistant Professor Department of Public Health and Caring Sciences Uppsala University Uppsala, Sweden List of Papers This thesis is based on the following papers, which are referred to in the text by their roman numerals. I Philipson O, Lannfelt L, Nilsson LNG. (2009). Genetic and pharmacological evidence of intraneuronal A accumulation in APP transgenic mice. FEBS Letters, 583: II Philipson O, Hammarström P, Nilsson KPR, Portelius E, Olofsson T, Ingelsson M, Hyman BT, Blennow K, Lannfelt L, Kalimo H, Nilsson LNG. (2009). A highly insoluble state of A similar to that of Alzheimer's disease brain is found in Arctic APP transgenic mice. Neurobiology of Aging, 30: III Philipson O, Lord A, Lalowski M, Soliymani R, Thyberg J, Bogdanovic N, Tjernberg LO, Ingelsson M, Lannfelt L, Kalimo H and Nilsson LNG. Biochemical and morphological analyses of A deposits in postmortem brain of Arctic APP mutation carriers. Manuscript IV Lord A*, Philipson O*, Klingstedt T, Nilsson KPR, Hammarström P, Lannfelt L, Nilsson LNG. Arctic A selectively increases diffuse deposition of wild-type A in APP transgenic mice with the Swedish mutation. Manuscript *These authors contributed equally to the study. Reprints were made with permission from the publishers. All rights reserved. Paper I and II: Elsevier. Contents Introduction...9 Protein folding, misfolding and amyloid formation...9 Alzheimer s disease...10 Clinical symptoms, diagnosis and current treatments...10 Neuropathology...11 Genetics and risk factors...14 The amyloid cascade hypothesis...15 Processing of the Amyloid-β Precursor Protein...16 Mechanisms of A aggregation and amyloid fibril formation...19 Location of A production and intracellular aggregation...20 Formation of extracellular deposits...21 Experimental models of Alzheimer s disease...23 Transgenic models...24 Aim of the study...27 Specific aims...27 Experimental methods...28 Present investigations...31 Paper I...31 Inhibition of APP processing diminishes punctate intracellular immunostaining with A antibodies in transgenic mice...31 Paper II...36 The Arctic APP mutation alters senile plaque formation in a transgenic model of Alzheimer s disease...36 Paper III...40 Atypical A deposition in brain of Arctic APP mutation carriers...40 Paper IV...45 Arctic A modifies the aggregation pathway of wild-type A in vitro and in vivo Concluding remarks...50 Acknowledgements...53 References...55 Abbreviations APP A AD AICD Apo E APPs APPs BACE CAA CWP ELISA EM FA FAD GSIs H&E IHC MCI MS MVB mab MMSE NFT NP PBS PHF PrP PS SDS SDS-PAGE TBS tg wt Amyloid- precursor protein Amyloid- Alzheimer s disease APP intracellular domain Apolipoprotein E Soluble -secretase cleaved APP fragments Soluble -secretase cleaved APP fragments -site APP cleaving enzyme Cerebral amyloid angiopathy Cotton wool plaque Enzyme-linked immunosorbent assay Electron microscopy Formic acid Familial Alzheimer s disease -secretase inhibitors Hematoxylin & Eosin Immunohistochemistry Mild cognitive impairment Mass spectrometry Multivesicular bodies Monoclonal antibody Mini-Mental State Examination Neurofibrillary tangle Neuritic plaque Phosphate buffered saline Paired helical filament Prion protein Presenilin Sodium dodecyl sulfate SDS-Polyacrylamide Gel Electrophoresis Tris buffered saline Transgenic Wild-type Introduction Protein folding, misfolding and amyloid formation Amino acid residues linked together by peptide bonds make up the protein sequence. There are 20 naturally occurring amino acids, and the unique combination of these is referred to as the primary structure of the protein. The sequence of amino acids and the environmental conditions direct protein folding. There are different secondary structural elements, -helices and strands and loop regions. Structural rearrangements and complex assembly generates the three-dimensional conformation, the functional form of the protein. Protein folding depends on energy and it has been suggested that a protein folds correctly in the search for the lowest possible energy state [1]. It is thought that this is achieved by using a local energy minima or funnel along the route from the random coil to the fully folded protein [2]. Chaperones, a diverse group of proteins assist in and prevent erroneous folding by keeping newly synthesised proteins in a favourable conformation. This process prevents unwanted protein aggregation. If the protein fails to fold correctly, there are cellular mechanisms detecting and removing unfolded or misfolded proteins, i.e. the ubiquitinproteasome system [3]. Alternatively, aggregates can undergo microtubilimediated transport to a cytoplasmic site near the centrosome, where the misfolded proteins form an aggresome. The cell can also sequester and dispose parts of the cytoplasm in a membrane structure, the autophagosome, in a process called macroautophagy [4, 5]. If these mechanisms fail, proteins may undergo further conformational changes and self-associate into assemblies of misfolded proteins. Proteins can then form stable aggregates with a -sheet conformation, a structure called amyloid. The -sheet with the constituent -strands is arranged parallel or anti-parallel running perpendicular to the axis of the fibril. These insoluble structures are generally highly protease-resistant and there are limited cellular pathways for their degradation. Amyloid fibrils are 7-12 nm in diameter [6] and normally composed of 2-6 fibrillar subunits arranged in parallel, the protofilaments. A number of factors determine the propensity of a protein for amyloid fibril formation e.g. molecular charge, hydrophobicity, conformational stability, solubility of the folded conformation and secondary structure propensity [7]. Amyloid is a generic term, which is defined by certain criteria. It is formed in vivo (in vitro derivates should be referred to as 9 amyloid-like fibrils), X-ray diffraction reveals a cross beta sheet structure and Congo red stained fibrils display birefringence under polarized light [8]. The amyloid fibril is typically unbranched when viewed in electron microscope. In humans, there are almost 30 known amyloid forming peptides, and yet others form amyloid-like fibrils under suitable conditions [9, 10]. A large number of diseases, described as protein misfolding disorders are associated with amyloidosis. Misfolded aggregated proteins deposited in tissues is a common feature of several neurodegenerative disorders, including amyloid- (A ) in Alzheimer s disease (AD), superoxide dismutase-1 in familial amyotrophic lateral sclerosis and the prion protein (PrP Sc ) in Creutzfeldt-Jakob s disease. Alzheimer s disease Clinical symptoms, diagnosis and current treatments AD, the most common age-related neurodegenerative disorder, is believed to affect as many as million people worldwide [11]. With increasing life expectancy, the worldwide prevalence is expected to triple within the next 50 years [12]. The clinical symptoms and neuropathology characterizing the disease was first described by the German physican, Alois Alzheimer, at a scientific meeting in Tübingen The patient, a woman who died in her fifties, suffered from severe memory loss, disorientation and hallucinations. AD has an insidious onset and a slow but irreversible progress where the end stage is often characterized by a complete loss of independence. Death is usually caused by secondary infection and typically occurs 5-15 years after onset of disease symptoms [11]. Early symptoms of AD are impaired acquisition and retention of memories, visuospatial deficits, difficulty handling complex tasks and a loss of language skills. Increased memory loss in combination with personality changes often contributes to a descending performance in social life, sometimes accompanied by depression. For the clinical diagnosis of AD, one needs to take into account the medical history and to exclude alternative causes of cognitive decline and dementia. The DSM-IV [13] and ICD-10 criteria [14], proclaim a trustworthy AD diagnosis of patients presenting with persistent ( 6 months) and progressive memory loss and other cognitive deficits resulting in impaired social and functional activities of daily life. A common test to evaluate cognitive status is the Mini-Mental State Examination (MMSE). In addition, functional and structural brain imaging techniques, such as computed tomography (CT), magnetic resonance imaging (MRI), position emission tomography (PET) [15] and single photon emission computed tomography are valuable in the differential diagnosis of AD e.g. to distinguish AD from dementia caused by stroke, tumor and subdural hematoma. 10 Laboratory tests are also valuable in the differential diagnosis. A high level of total tau and/or phosphorylated tau (ptau) and a low level of the 42 amino acid long A (A 42) in the cerebrospinal fluid (CSF) is suggestive of AD [16, 17]. However, a neuropathological examination has to be carried out for a definite diagnosis of AD. The validity of the clinical assessments of patients having probable or possible AD can reach 85-90% when the brain is neuropathologically examined [18]. Above 65 years of age, the prevalence and incidence of dementia increases twofold every succeeding five years, with an estimated prevalence of ~20% of AD among the population over the age of 85 [19]. Today only symptomatic treatment, which helps some patients to temporarily maintain their cognitive abilities, is available. However it does not affect the underlying disease process, synaptic loss and neuronal atrophy leading to neurotransmitter loss and cognitive deficits. The cholinergic neurons projecting from the basal forebrain to the hippocampus and the cerebral cortex degenerate early in the disease process. These pathways are important for memory functions; consequently cholinergic substitution therapy for AD, which can alleviate cognitive symptoms of mild-tomoderate AD, was developed. These drugs (e.g. Aricept ) inhibit acetylcholinesterase, which degrades acetylcholine at the synaptic cleft [20]. The release and uptake of the neurotransmitter glutamate is also deemed to be dysfunctional in AD. Memantine, an uncompetitive N-methyl D-aspartate (NMDA) receptor antagonist, is thought to prevent glutamate-induced neurotoxicity by inhibiting overactive NMDA receptors and thereby reducing excessive calcium influx. It is approved for treatment of moderateto-severe AD [21]. Since current treatments do not target the underlying neurodegenerative processes it is of great importance to develop therapeutic approaches that target molecular pathways involved in AD pathogenesis [22]. There is also a need for better differential diagnosis in the early stages of the disease and objective disease monitoring with biomarkers. It would lead to more valuable clinical trials and in the future, it could help to enable therapeutic prevention. Neuropathology The brain of an AD patient typically shows gross atrophy of the hippocampus, the parietal and temporal lobes of the cerebral cortex and enlarged ventricles [23] as illustrated in Figure 1A-B. AD is also characterized by reduction in synaptic density and loss of neurons e.g. in the hippocampal formation [24]. The neuritic plaque (NP) of Aβ and the neurofibrillary tangle (NFT) are the two neuropathological hallmarks of AD. In the 1960s, with the advent of electron microscopy, Michael Kidd and Robert Terry described the ultrastructure of filaments in NFT in AD brain 11 [25], and later the microtubule protein tau was shown to be the main constituent [26]. In the mid 1980s, amyloid- (A ) was purified and partially sequenced from angiopathic meningeal vessels of AD brains and identified as the main component of the vascular [27] and soon thereafter, parenchymal amyloid [28] Figure 1C-F. NFTs mainly consist of paired helical filaments with hyperphosphorylated forms of the microtubule-associated protein tau, as their main constituent. Tau protein regulates the assembly and stability of microtubules, an essential part of the cytoskeleton, which is vital to vesicle transport. In AD brain, the tau protein in intracellular tangles is abnormally phosphorylated and dissociated from the microtubules. An increased soluble pool of tau undergoing conformational changes is likely an important early step in the assembly of tau filaments [29]. A definite postmortem diagnosis of AD is dependent on the presence of NP and NFT, their frequency and location in the brain and the age of the patient. From the early guidelines regarding the hallmarks of AD pathology [30], more refined criteria for scoring NP [31] and staging NFT/neuropil thread pathology [32] were established and became widely accepted. Later, these critera were combined in NIA-RI recommendation [33], which took into account the distribution and extent of both hallmarks (NP and NFT/ neuropil threads). Based on the presence of the two patholological hallmarks, the likelihood of dementia being a result of AD is staged as low, intermediate or high. Instead of chemical staining procedures (Thioflavin S and Silver stains), current recommendations emphasize the need for molecular classification with immunohistochemistry and assessment of regional distribution of the pathologies. The AD related A pathology phases 1-5 relate to the spread of A -pathology throughout the brain [34] with deposits first being found in the neocortex and only in the latest phase in the cerebellum. In general, the severity of hyperphosphorylated tau pathology (Braak stages I to VI) is based on density and regional distribution of AT8- positive neurofibrillary threads in hippocampus (Braak I-III), temporal cortex (Braak IV) and occipital cortex (Braak V-VI) [35, 36]. 12 Figure 1. Neuropathological changes in a sporadic AD brain. Macroscopic changes of AD brain (A) as compared to a normal brain (B). The AD brain is severely degenerated with enlarged ventricles, widened sulci and an atrophied hippocampus. The two neuropatholological hallmarks of AD, neuritic plaques (NPs) (open arrow) and neurofibrillary tangles (NFTs, closed arrow) are visualized by a silver staining technique in the entorhinal cortex (C). Parenchymal, extracellular deposits (closed arrows) and cerebral amyloid angiopathy (open arrow) are stained for amyloid- (D). Hyperphosphorylated tau in neurofibrillary threads and NFT are stained with the AT8-antibody (E) and dystrophic neurites clustered within and around amyloid plaques (F). Images are kindly provided by Professor Hannu Kalimo. 13 Genetics and risk factors Early-onset Alzheimer s disease In some families, AD has been described with an autosomal dominant pattern of inheritance, often with an early age of onset ( 65 years) [37]. Despite the difference in age of onset, the clinical picture and the neuropathology is generally similar to sporadic AD. This is important since it implies that studies of FAD can help us understand the molecular mechanisms of sporadic AD. Several missense mutations in APP and the presenilin genes (PS1 and PS2) that are inherited in an autosomal dominant mode have been identified. Almost all of these mutations have a complete penetrance. Most APP mutations are located in or around the A domain and alter proteolytic processing of APP [38]. Missense mutations close to the C- terminus of the A domain increase the production of A 42 or the ratio of A 42/A 40, while a mutation close to the N-terminus, the Swedish (Swe) mutation, increases the levels of both A 42 and A 40 [39]. The Swedish mutation (APP K670N, M671L) is a double mutation at codons 670 and 671 caused by two base pair substitutions [40]. The APP gene is located on chromosome 21 and Down s syndrome patients with trisomy chromosome 21, almost invariably develop dementia. This is most likely due to the gene dosage effect with enhanced A production, since the neuropathology is highly similar to AD. APP gene duplications have also been found in families with a variant of AD [41]. Mutations within the A -domain of APP result in different clinical and pathologic phenotypes. Several pathogenic intra-a mutations have been identified, all positioned near the central hydrophobic cluster in position in the A sequence. The Arctic (Arc) mutation (APP E693G) identified in a family from northern Sweden, is located at position 22 in the A sequence, where glutamic acid is substituted for a glycine. It drastically alters the kinetics of A aggregation and promotes A protofibril and fibril formation in vitro. Individuals in the family with the Arctic mutation have a disease onset ranging from 52 to 62 years and at postmortem examination, the brains show severe parenchymal A deposition and cerebral angiopathy in the absence of hemorrhages [42]. Despite the importance of tau and the formation of NFTs in AD, mutations in the tau gene have not been associated with AD. Instead such genetic lesions causes frontotemporal lobar degeneration [43], indicating that altered tau metabolism (and likely tangle formation) is sufficient to generate neurodegeneration and dementia. 14 Late-onset Alzheimer s disease Late-onset AD (representing more than 95% of total AD cases) affects individuals above 65 years of age and is commonly perceived as sporadic in its origin. Some of the late onset cases have a family history of AD [44], and based on twin studies an almost 80% heritability of AD has been estimated [45]. Age is the main risk factor for AD. Females are overrepresented among AD patients, more than would be anticipated by increased life expectancy among women [46]. The only well established genetic risk factor for developing late onset AD is apolipoprotein E (ApoE). Three variants of ApoE exist ( 2, 3 and 4). The 4 allele is associated with increased risk for developing AD [47] and the 2 allele confers some protection. The ApoE gene is located on chromosome 19q and generates a 35 kda plasma protein with important functions in cholesterol transport, metabolism and storage [48]. Some studies show that ApoE affects the process of A fibrillization [49, 50]. Other non-a components, e.g. heparan sulfate (a glycosaminoglycan) has been found codeposited in plaques of AD [51] and likely play a role in the A fibrillogenesis, as shown in vitro [52]. Recently three new risk factor genes, clusterin (Apo J), PICALM and complement receptor 1 (CR1) [53, 54] were identified. Their impact on disease susceptibility is much less than that of the ApoE 4 allele. The amyloid cascade hypothesis The amyloid cascade hypothesis states that the accumulation of A is the primary pathogenic process which instigates all other abnormal processes in AD brain. Formation of NFT, cell loss, inflammation and neurotransmission deficiencies are considered downstream events contributing to neuronal dysfunction and dementia [20, 55]. However, the severity of dementia equates better to the number and location of NFTs than to the extent of senile plaque deposition [56], a finding that is inconsistent with the initial amyloid cascade hypothesis [57]. The relationship between NP and the NFT remains poorly understood. Transgenic mice expressing mutant APP and tau suffer from more severe tau pathology than mice expressing only mutant tau. In contrast, t
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