| 1. |
- Englund, Hillevi, 1980-
(författare)
-
Soluble amyloid-β aggregates in Alzheimer’s disease
- 2009
-
Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Soluble oligomeric aggregates of the amyloid-β (Aβ) peptide are suggested to initiate Alzheimer's disease (AD), leading to impaired synapse signalling, widespread neuronal death and loss of cognitive functions. These aggregates seem tightly linked to disease progression, and have therefore gained much attention as potential novel disease markers. In this thesis soluble oligomeric Aβ aggregates in general, and the Aβ protofibril species in particular, have been investigated with the aim to quantify and determine their role in AD pathogenesis. Sandwich-ELISAs specifically measuring Aβ42 peptides are widely used both in AD research and as complements for clinical diagnosis. Here it was demonstrated that presence of soluble Aβ aggregates disturbs such analyses, making it difficult to interpret the results. This discovery was made through analyses of samples from cell- and mouse models carrying the AD causing 'Arctic' APP mutation. When analyzed by ELISA, Aβ42 levels were reduced in Arctic samples, in contrast to levels measured by denaturing SDS-PAGE Western blot. The same divergence in Aβ42-levels between analyses was observed in CSF samples from Down syndrome infants. The discrepancy between methods was hypothesized to be due to presence of soluble Aβ aggregates leading to impaired ELISA detection caused by epitope masking. This was confirmed by developing a protofibril specific ELISA, by which samples from Arctic cell- and mouse models were demonstrated to have enhanced Aβ protofibril levels. AD patients have reduced ELISA-measured Aβ42-levels in CSF compared to healthy controls. To test if this reduction was due to oligomeric Aβ species present in AD CSF, Aβ42-levels were analyzed under both denaturing and non-denaturing conditions. These two measures were combined and an Aβ42 oligomer ratio established. Higher ratios were found in AD patients than healthy controls, implying that Aβ oligomers are present in CSF during Alzheimer pathogenesis. The observations from AD patients and young Down syndrome individuals suggest that Aβ42 oligomer formation is an early mechanism of AD pathogenesis, which potentially could be used as a biomarker to monitor disease development.
|
|
| 2. |
- Fagerqvist, Therese, 1983-
(författare)
-
Studies of α-synuclein Oligomers-with Relevance to Lewy Body Disorders
- 2013
-
Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- The protein alpha-synuclein (α-synuclein) accumulates in the brain in disorders such as Parkinson’s disease (PD) and dementia with Lewy bodies (DLB). It is believed that the monomeric form of α-synuclein can adopt a partially folded structure and start to aggregate and form intermediately sized oligomers or protofibrils. The aggregation process can continue with the formation of insoluble fibrils, which are deposited as Lewy bodies. The oligomers/protofibrils have been shown to be toxic to neurons and are therefore believed to be involved in the pathogenesis of the actual diseases. The overall aims of this thesis were to investigate the properties of α-synuclein oligomers and to generate and characterize antibodies against these species. In addition, the potential for immunotherapy of the α-synuclein oligomer-selective antibodies were evaluated in a transgenic mouse model with α-synuclein pathology.Stable, β-sheet rich α-synuclein oligomers were induced by incubation with either one of the reactive aldehydes 4-hydroxy-2-nonenal (HNE) and 4-oxo-2-nonenal (ONE). The oligomers exhibited distinct morphological properties, although both types were toxic when added to a neuroblastoma cell line. The seeding effects of ONE-induced oligomers were studied in vitro and in vivo. The oligomers induced seeding of monomeric α-synuclein in a fibrillization assay but not in a cell model or when injected intracerebrally in transgenic mice. It seemed, however, as if the oligomers affected α-synuclein turnover in the cell model.By immunizing mice with HNE-induced oligomers antibody producing hybridomas were generated. Three monoclonal antibodies were found to have strong selectivity for α-synuclein oligomers. These antibodies recognized Lewy body pathology in brains from patients with PD and DLB as well as inclusions in the brain from young α-synuclein transgenic mice, but did not bind to other amyloidogenic proteins. Finally, immunotherapy with one of the oligomer/protofibril selective antibodies resulted in lower levels of such α-synuclein species in the spinal cord of α-synuclein transgenic mice.To conclude, this thesis has focused on characterizing properties of α-synuclein oligomers. In particular, antibodies selectively targeting such neurotoxic forms were generated and evaluated for passive immunization in a transgenic mouse model. Such immunotherapy may represent a future treatment strategy against Lewy body disorders.
|
|
| 3. |
- Lord, Anna, 1979-
(författare)
-
Targeting Early Stages of Alzheimer’s Disease in a Transgenic Model
- 2009
-
Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- The Arctic mutation causes early-onset Alzheimer’s disease (AD), and makes amyloid-β (Aβ) peptides more prone to form Aβ protofibrils. The aims of this thesis were to investigate the mechanisms of the Arctic mutation in vivo, and to use transgenic models to determine the role of early intermediates of Aβ aggregation, like protofibrils, in the pathogenesis. In addition, we aimed to evaluate protofibrils as a therapeutic target.Transgenic models with Arctic and Swedish mutations (tg-ArcSwe), and with the Swedish mutation alone (tg-Swe) were created. The Arctic mutation favored amyloidogenic processing of amyloid-β precursor protein (APP) in transgenic mice and cultured cells. The observed shift in the subcellular location and processing of APP led to increased production of intracellular Aβ in vitro, and also partly explained the early accumulation of intraneuronal Aβ in tg-ArcSwe mice. The intraneuronal Aβ in combination with enhanced levels of protofibrils appeared long before extracellular plaques emerged. Elevated protofibril levels were associated with intraneuronal Aβ and linked to spatial learning deficits in young mice, suggesting that protofibrils cause AD-related cognitive deficits. The Arctic mutation also enhanced senile plaque pathology in aged tg-ArcSwe mice, and the accelerated plaque deposition was accompanied by decreased intraneuronal Aβ. This suggests a dynamic equilibrium between the early accumulation of intraneuronal Aβ and the later senile plaque pathology.Aβ protofibrils were evaluated as a therapeutic target in tg-ArcSwe mice with passive immunization using a protofibril-selective antibody. This treatment cleared protofibrils without removing senile plaques. However, plaque formation was prevented if treatment began early, indicating that protofibrils are intermediate species of Aβ fibrillization in vivo. Targeting senile plaques with immunotherapy requires early diagnosis and intervention, whereas protofibrils can be specifically cleared from brain despite substantial AD-like deposition of insoluble Aβ. The early and persistent presence of protofibrils throughout Aβ amyloidosis makes them a promising target for future diagnostic and therapeutic strategies in AD.
|
|
| 4. |
- Philipson, Ola, 1979-
(författare)
-
Modeling Amyloid-β Pathology in Alzheimer’s Disease Using the Arctic Mutation
- 2010
-
Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- 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.
|
|
| 5. |
- Sundelöf, Johan, 1974-
(författare)
-
Amyloid β-protein, Cystatin C and Cathepsin B as Biomarkers of Alzheimer's Disease
- 2010
-
Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- 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.
|
|
| 6. |
- Cedervall, Ylva
(författare)
-
Physical Activity and Alzheimer's Disease : Measurements, Observations and Subjective Experiences
- 2014
-
Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Gait disturbances such as slow walking speed and step-to-step variability have been reported among people with mild Alzheimer’s disease (AD) and as risk factors for functional decline, dependency, and falls. Additionally, AD-related emotional reactions and decreased initiative can lead to physical inactivity. The aims of this thesis, therefore, were to explore how the ability to be physically active is affected in the early years of AD, and how people with mild AD and their cohabitants reason about physical activity as part of their everyday life.To meet the aims, an approach inspired by mixed methods research was used, covering measurements, observations and subjective experiences. Data were collected from different sources in parallel. Participants with mild AD were recruited at the Memory Clinic, Uppsala University Hospital. In Study I, a case study with two couples in which one member had AD, in-depth interviews and participating interviews were performed. Physical activity such as walking was viewed as a meaningful routine improving well-being. Participants were positive about making adjustments to enable physical activity. In Study II, the 25 participants with AD showed a significant lower walking capacity (10 m comfortable walk test, 6-minute walk test, Timed-up-and-Go test) at baseline compared to controls. The decline continued during the subsequent two years. The influence of a cognitive task on walking was distinct, despite this, participants maintained a health-promoting level of physical activity during the two-year study-period. In Study III, gait testing in the motor laboratory of 21 participants with AD showed a marked impact on gait parameters (e.g. slowed speed, decreased step length) by a cognitive task. Additionally, specific dual-task gait disturbances were frequent. In Study IV, in-depth interviews with 14 participants with AD indicated that physical activity was viewed as a meaningful activity, used as a means to maintain well-being and selfhood, and contributed to continuity in life.In conclusion, walking capacity deteriorates and declines in the early stages of AD. A simple cognitive task can have a substantially negative impact on walking already in mild AD. In contrast, people with AD can also gain “self-promoting benefits” from physical activity beyond the common health-promoting benefits.
|
|
| 7. |
- Johansson, Hans-Erik, 1960-
(författare)
-
The Impact of Bariatric Surgery on Obesity related Metabolic Traits with Specific Emphasis on Glucose, Insulin and Proinsulin
- 2010
-
Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Hyperproinsulinemia is associated with type 2 diabetes (T2DM) and obesity and is a predictor for future coronary heart disease. This thesis examines the effect of bariatric surgery on glucometabolic status including insulin and proinsulin responses after meal. Further we explored longitudinally the effects of bariatric surgery on glucose, insulin and proinsulin secretion as well as lipids, liver enzymes and magnesium concentrations. We explored by a standardised meal test the postprandial dynamics of proinsulin and insulin and effects on glucose and lipids in patients treated with gastric bypass (RYGBP) surgery and in patients treated with bileopancreatic diversion with duodenal switch surgery (BPD-DS). Comparisons were made to morbidly obese patients and normal weight controls (NW). RYGBP surgery markedly lowers fasting and postprandial proinsulin concentrations although BMI was higher compared to NW-controls. BPD-DS surgery induces a large weight loss and normalises postprandial responses of glucose, proinsulin and insulin and markedly lowers triglycerides. We evaluated non-diabetic morbidly obese patients who underwent bariatric surgery followed-up for up to four years after surgery. Long-term follow-up showed that RYGBP surgery is not only characterized by markedly and sustained lowered BMI but also lowered concentrations of proinsulin, insulin, ALT and increased HDL-C possibly via reduced hepatic insulin resistance. We also examined how magnesium status is affected by bariatric surgery as magnesium has been shown to be inversely related to glucose and to insulin resistance. The serum magnesium concentrations increased by 6% after RYGBP and 10% after BPD-DS. In summary, RYGBP and BPD-DS surgery results in marked weight loss, alterations in insulin and proinsulin dynamics, lowered fasting and postprandial proinsulin concentrations and improved glucometabolic and magnesium status.
|
|
| 8. |
- Keskin, Isil, 1987-
(författare)
-
SOD, ORF and ALS: On the role of SOD1 and C9ORF72 in the pathogenesis of ALS
- 2016
-
Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Amyotrophic lateral sclerosis (ALS) is characterized by adult-onset degeneration of upper and lower motor neurons. Symptoms begin focally in one muscle and then spread contiguously, resulting in progressive paralysis and death from respiratory failure. Hexanucleotide repeat expansion in C9ORF72 is the most common genetic cause, however, mutations in SOD1 were the first identified and are found in 1-9% of patients. Misfolded SOD1 aggregates in the CNS are hallmarks of ALS associated with SOD1 mutations. However, accumulation of misfolded or aggregated SOD1 protein has also been reported in sporadic and familial ALS without SOD1 mutations, suggesting that wild-type SOD1 could play a role in ALS pathology in general.The aims of this thesis are: 1) To describe the resulting disease phenotype and specific characteristics of the SOD1 protein carrying the stable disease- associated mutation L117V. 2) To set up cell-based in vitro models to study the mechanisms of SOD1 misfolding and aggregation under physiologically relevant expression levels. 3) To compare SOD1 activity in patient-derived samples and screen for underlying causes of deviant SOD1 activities in individuals lacking SOD1 mutations.1) We identified a novel L117V SOD1 mutant in two families of Syrian origin that co-segregated with the disease. This mutation was associated with slow disease progression, reduced penetrance and a uniform phenotype. The L117V mutant protein was indistinguishable from wild-type SOD1 in terms of stability, dismutation activity and misfolding in patient-derived cell lines.2) We established patient-derived fibroblast and iPSC-MN lines expressing mutant SOD1 at physiological levels as in vitro models to study misfolding and aggregation of SOD1. We investigated the effects of several cellular pathway disturbances on SOD1 misfolding. Misfolded SOD1 was increased by inhibition of the ubiquitin-proteasome pathway in fibroblasts derived from both patients and controls. An age-related decline in proteasome activity could contribute to the late onset of ALS.Next, we studied the effects of low oxygen tension on misfolding and aggregation of SOD1 in patient-derived cells. Low O2 tensions were found to markedly increase C57-C146 disulphide reduction, misfolding and aggregation of SOD1. Importantly, the largest effects were detected in iPSC-MNs. This suggests that motor neurons are specifically vulnerable to misfolding and aggregation of SOD1 under low O2 tension.3) We compared the enzymatic activity of SOD1 in blood samples from a large number of ALS patients and controls. We screened for potential underlying causes of deviant SOD1 activities in individuals lacking SOD1 mutations. No aberrations in copy number, other large structural changes in introns and exons or intronic mutations in the 30-50 bp flanking the exons were found in the 142 outliers, with either very low or very high SOD1 dismutation activities. However, hemoglobinopathies, including thalassemias and iron deficiency anemia, were associated with high SOD1/mg Hb ratios. Erythrocytes from patients with destabilizing SOD1 mutations showed half the normal activity. There were no significant differences in SOD1 activity between control individuals and ALS patients without a coding SOD1 mutation, or carriers of TBK1 mutations or the hexanucleotide repeat expansion in C9ORF72. Our result suggests that SOD1 enzymatic activity is not associated with the disease in non-SOD1 mutation ALS.
|
|
| 9. |
- O'Callaghan, Paul
(författare)
-
Heparan Sulfate in the Amyloidosis and Inflammation of Alzheimer’s Disease
- 2011
-
Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Alzheimer’s disease (AD) is a neurodegenerative disorder, with extensive evidence implicating the misfolding, aggregation and deposition of the amyloid-β (Aβ) peptide as central to the pathogenesis. Heparan sulfate (HS) is an interactive glycosaminoglycan, attached to core proteins as HS proteoglycans (HSPGs). HSPGs are present on cell surfaces and in the extracellular matrix where they facilitate multiple signaling functions, but HS is also consistently present in all amyloid deposits, including those of AD. In amyloidosis HS has been studied as an aggregation template, promoting fibril formation and serving a scaffold function in the resulting deposits. The objective of this thesis was to assess how cell surface HS is potentially implicated in Aβ amyloidosis and the associated neuroinflammation of AD. In AD brain we determined that HS predominantly accumulated in Aβ deposits with dense cores and found glial-expressed HSPGs within these deposits. Aβ elevated HSPG levels in primary glial cultures, implicating activated glia as one source of the Aβ-associated HS. Next, we determined that microglial HSPGs are critical for the upregulation of interleukin-1β and tumor necrosis factor-α following exposure to lipopolysaccharide, an established inflammatory insult. Together these results raise the possibility that Aβ-induced expression of microglial HSPGs may promote neuroinflammation. Multiple mechanisms of Aβ toxicity have been proposed and different Aβ assemblies exert their toxicity through alternative routes. We found that three different preparations of Aβ aggregates all exhibited HS-dependent cytotoxicity, which in part correlated with Aβ internalization. Furthermore, heparin treatment attenuated Aβ cytotoxicity and uptake. In Aβ-positive AD microvasculature, HS deposited with Apolipoprotein E (ApoE) and its receptor, the low density lipoprotein receptor-related protein 1 (LRP1). In cell culture, HS and LRP1 co-operated in Aβ interactions and the addition of ApoE increased the levels of cell-associated Aβ in a HS- and LRP1-dependent manner. This ApoE-mediated increase in cell-associated Aβ may promote toxicity and vascular degeneration, but equally HS-mediated internalization of Aβ could represent a clearance route across the blood-brain-barrier.The findings presented here illustrate multiple roles for cell-surface HSPGs in interactions relevant to the pathogenesis of AD.
|
|
| 10. |
- Sahlin, Charlotte, 1977-
(författare)
-
Pathogenic Mechanisms of the Arctic Alzheimer Mutation
- 2007
-
Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- 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.
|
|
