| 1. |
- Ekholm Pettersson, Frida
(author)
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T-cell Differentiation and Immunological Homeostasis in Lymphopenic and Kappa Light Chain Deficient Mice
- 2002
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Doctoral thesis (other academic/artistic)abstract
- T lymphocytes are primarily involved in adaptive, cell-mediated, immune reactions. In this thesis T cells were studied regarding central and peripheral differentiation and homeostatic mechanisms for maintanance of the immune repertoire.The influence by mature T cells on thymic development was studied in C.B-17 scid/scid (SCID) mice, devoid of mature T and B cells, and whose thymocyte development is arrested at the early pro-T cell stage. When mature syngeneic T cells were injected the developmental block was overcome and there was an accumulation of CD4+CD8+ thymocytes. This event was accompanied by the maturation of medullary epithelial cells in thymus which seemed to be driven by CD8+ T cells. In the periphery there was initially a spontaneous T-cell proliferation and later, the majority of the donor T lymphocytes showed a memory phenotype with high expression of CD44 and with an early onset of proliferation and cytokine production upon stimulation. This stable pool of memory type of cells sustained for more than a year following treatment.Treating SCID mice with allogeneic T cells results in graft-versus-host disease (GVHD). Severe GVHD was dependent on the MHC-haplotype of the donor cells and was accompanied by profound alterations of the TCR-Vβ repertoire and with high production of IFN-γ.Kappa light chain (κ)-deficient mice have only half the number of B cells as their normal counterparts but normal levels of immunoglobulins. When T cells from κ-deficient mice were stimulated in vitro there was a bias towards production of B-cell stimulatory type 2 cytokines. This is proposed as a mechanism for the homeostatic control of serum immunoglobulin levels in κ-deficient mice.
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| 2. |
- Englund, Hillevi, 1980-, et al.
(author)
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Oligomerization partially explains the lowering of Aβ42 in Alzheimer's disease cerebrospinal fluid
- 2009
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In: Neuro-degenerative diseases. - : S. Karger AG. - 1660-2862 .- 1660-2854. ; 6:4, s. 139-147
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Journal article (peer-reviewed)abstract
- Background/aim: The lowering of natively analyzed Aβ42 in cerebrospinal fluid (CSF) is used as a diagnostic tool in Alzheimer’s disease (AD). Presence of Aβ oligomers can interfere with such analyses causing underestimation of Aβ levels due to epitope masking. The aim was to investigate if the lowering of CSF Aβ42 seen is caused by oligomerization. Methods: Aβ42 was analyzed under both denaturing and non-denaturing conditions. An Aβ42 oligomer ratio was calculated from these quantifications. Presence of oligomers leads to Aβ42 epitope masking during non-denaturing assays, resulting in a higher ratio. Results: The Aβ42 oligomer ratio was used for assessment of oligomerized Aβ in human CSF, after being evaluated in transgenic mouse brain homogenates. AD and mild cognitive impairment (MCI) samples displayed the expected decrease in natively measured Aβ42 compared to healthy controls and frontotemporal dementia, but not when analyzing under denaturing conditions. Accordingly, AD and MCI CSF had a higher Aβ42 oligomer ratio in CSF. Conclusion: Combining denaturing and non-denaturing quantifications of Aβ42 into an oligomer ratio enables assessment of Aβ oligomers in biological samples. The increased Aβ42 oligomer ratio for AD and MCI indicates presence of oligomers in CSF and that the lowering of natively measured Aβ42 is caused by oligomerization.
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| 3. |
- Englund, Hillevi, 1980-, et al.
(author)
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Sensitive ELISA detection of amyloid-β protofibrils in biological samples
- 2007
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In: Journal of Neurochemistry. - : Wiley. - 0022-3042 .- 1471-4159. ; 103:1, s. 334-345
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Journal article (peer-reviewed)abstract
- Amyloid-β (Aβ) protofibrils are known intermediates of the in vitro Aβ aggregation process and the protofibrillogenic Arctic mutation (APPE693G) provides clinical support for a pathogenic role of Aβ protofibrils in Alzheimer's disease (AD). To verify their in vivo relevance and to establish a quantitative Aβ protofibril immunoassay, Aβ conformation dependent monoclonal antibodies were generated. One of these antibodies, mAb158 (IgG2a), was used in a sandwich ELISA to specifically detect picomolar concentrations of Aβ protofibrils without interference from Aβ monomers or the amyloid precursor protein (APP). The specificity and biological significance of this ELISA was demonstrated using cell cultures and transgenic mouse models expressing human APP containing the Swedish mutation (APPKN670/671ML), or the Swedish and Arctic mutation in combination. The mAb158 sandwich ELISA analysis revealed presence of Aβ protofibrils in both cell and animal models, proving that Aβ protofibrils are formed not only in vitro, but also in vivo. Furthermore, elevated Aβ protofibril levels in the Arctic-Swedish samples emphasize the usefulness of the Arctic mutation as a model of enhanced protofibril formation. This assay provides a novel tool for investigating the role of Aβ protofibrils in AD and has the potential of becoming an important diagnostic assay.
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| 4. |
- Englund, Hillevi, 1980-
(author)
-
Soluble amyloid-β aggregates in Alzheimer’s disease
- 2009
-
Doctoral thesis (other academic/artistic)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.
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| 5. |
- Ingelsson, Martin, et al.
(author)
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Sällsynta mutationer leder till framtidens behandling
- 2009
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In: Läkartidningen. - 0023-7205 .- 1652-7518. ; 106:20, s. 1396-1400
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Journal article (peer-reviewed)abstract
- The identification of disease-causing mutations in Alzheimer’s disease has greatly contributed to our understanding of the pathogenesis. Based on this knowledge, a number of therapeutic strategies are under development, most of which are aimed at lowering the amount of Abpeptides in the affected brain. Due to intense research efforts and massive investments at universities and in the pharmaceutical industry, the future perspectives for Alzheimer patients have never looked brighter.
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| 6. |
- Johansson, Ann-Sofi, 1978-
(author)
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Amyloid-β Protofibril Formation and Neurotoxicity : Implications for Alzheimer’s Disease
- 2007
-
Doctoral thesis (other academic/artistic)abstract
- Alzheimer’s disease (AD) is the most common cause of dementia. A characteristic feature of AD is the presence of amyloid plaques in the cortex and hippocampus of the brain. The principal component of these plaques is the amyloid-β (Aβ) peptide, a cleavage product from proteolytic processing of amyloid precursor protein (APP). A central event in AD pathogenesis is the ability of Aβ monomers to aggregate into amyloid fibrils. This process involves the formation of various Aβ intermediates, including protofibrils. Protofibrils have been implicated in familial AD, as the Arctic APP mutation is associated with enhanced rate of protofibril formation in vitro. This thesis focuses on Aβ aggregation and neurotoxicity in vitro, with special emphasis on protofibril formation. Using synthetic Aβ peptides with and without the Arctic mutation, we demonstrated that the Arctic mutation accelerated both Aβ1-42 protofibril- and fibril formation, and that these processes were affected by changes in the physiochemical environment. Oxidation of Aβ methionine delayed trimer and protofibril formation in vitro. Interestingly, these oxidized peptides did not have the neurotoxic potential of their un-oxidized counterparts, suggesting that formation of trimers and further aggregation into protofibrils is necessary for the neurotoxic actions of Aβ. In agreement, stabilization of Aβ wild type protofibrils with the omega-3 (ω3) fatty acid docosahexaenoic acid (DHA) sustained Aβ induced neurotoxicity; whereas in absence of DHA, neurotoxicity was reduced as Aβ fibrils were formed. These results suggest that the neurotoxic potential of Aβ is mainly confined to soluble aggregated forms of Aβ, not Aβ monomer/dimers or fibrillar Aβ. Stabilization of Aβ protofibrils with DHA might seem contradictory, as ω3 fatty acids generally are considered beneficial for cognition. However, we also demonstrated that DHA supplementation reduced Aβ levels in cell models of AD, providing a possible mechanism for the reported beneficial effects of DHA on cognitive measures in vivo.
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| 7. |
- Johansson, Ann-Sofi, et al.
(author)
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Docosahexaenoic acid stabilizes soluble amyloid-β protofibrils and sustains amyloid-β induced neurotoxicity in vitro
- 2007
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In: The FEBS Journal. - : Wiley. - 1742-464X .- 1742-4658. ; 274:4, s. 990-1000
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Journal article (peer-reviewed)abstract
- Enrichment of diet and culture media with the polyunsaturated fatty acid docosahexaenoic acid has been found to reduce the amyloid burden in mice and lower amyloid-β (Aβ) levels in both mice and cultured cells. However, the direct interaction of polyunsaturated fatty acids, such as docosahexaenoic acid, with Aβ, and their effect on Aβ aggregation has not been explored in detail. Therefore, we have investigated the effect of docosahexaenoic acid, arachidonic acid and the saturated fatty acid arachidic acid on monomer oligomerization into protofibrils and protofibril fibrillization into fibrils in vitro, using size exclusion chromatography. The polyunsaturated fatty acids docosahexaenoic acid and arachidonic acid at micellar concentrations stabilized soluble Aβ42 wild-type protofibrils, thereby hindering their conversion to insoluble fibrils. As a consequence, docosahexaenoic acid sustained amyloid-β-induced toxicity in PC12 cells over time, whereas Aβ without docosahexaenoic acid stabilization resulted in reduced toxicity, as Aβ formed fibrils. Arachidic acid had no effect on Aβ aggregation, and neither of the fatty acids had any protofibril-stabilizing effect on Aβ42 harboring the Arctic mutation (AβE22G). Consequently, AβArctic-induced toxicity could not be sustained using docosahexaenoic acid. These results provide new insights into the toxicity of different Aβ aggregates and how endogenous lipids can affect Aβ aggregation.
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| 8. |
- Kamali-Moghaddam, Masood, et al.
(author)
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Sensitive detection of A beta protofibrils by proximity ligation : relevance for Alzheimer's disease
- 2010
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In: BMC Neuroscience. - : Springer Science and Business Media LLC. - 1471-2202. ; 11, s. 124-
-
Journal article (peer-reviewed)abstract
- Background: Protein aggregation plays important roles in several neurodegenerative disorders. For instance, insoluble aggregates of phosphorylated tau and of A beta peptides are cornerstones in the pathology of Alzheimer's disease. Soluble protein aggregates are therefore potential diagnostic and prognostic biomarkers for their cognate disorders. Detection of the aggregated species requires sensitive tools that efficiently discriminate them from monomers of the same proteins. Here we have established a proximity ligation assay (PLA) for specific and sensitive detection of A beta protofibrils via simultaneous recognition of three identical determinants present in the aggregates. PLA is a versatile technology in which the requirement for multiple target recognitions is combined with the ability to translate signals from detected target molecules to amplifiable DNA strands, providing very high specificity and sensitivity. Results: For specific detection of A beta protofibrils we have used a monoclonal antibody, mAb158, selective for A beta protofibrils in a modified PLA, where the same monoclonal antibody was used for the three classes of affinity reagents required in the assay. These reagents were used for detection of soluble Ab aggregates in solid- phase reactions, allowing detection of just 0.1 pg/ml A beta protofibrils, and with a dynamic range greater than six orders of magnitude. Compared to a sandwich ELISA setup of the same antibody the PLA increases the sensitivity of the Ab protofibril detection by up to 25- fold. The assay was used to measure soluble Ab aggregates in brain homogenates from mice transgenic for a human allele predisposing to A beta aggregation. Conclusions: The proximity ligation assay is a versatile analytical technology for proteins, which can provide highly sensitive and specific detection of A beta aggregates - and by implication other protein aggregates of relevance in Alzheimer's disease and other neurodegenerative disorders.
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| 9. |
- Lannfelt, Lars, et al.
(author)
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Translating research on brain aging into public health : a new type of immunotherapy for Alzheimer's disease
- 2010
-
In: Nutrition reviews. - : Oxford University Press (OUP). - 0029-6643 .- 1753-4887. ; 68:12, s. S128-S134
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Research review (peer-reviewed)abstract
- The identification of disease-causing mutations in Alzheimer's disease has contributed greatly to the understanding of the pathogenesis of this disease. The amyloid-beta (A beta) peptide has come into focus and is believed to be central to the pathogenesis of Alzheimer's disease. With only symptomatic treatment available, efforts to develop new therapeutics aimed at lowering the amount of A beta peptides in the affected brain have intensified. In particular, immunotherapy against A beta peptides has attracted considerable interest, as it offers the possibility to generate highly specific molecules targeting highly specific moieties. Due to intense research efforts and massive investments at universities and in the pharmaceutical industry, the outlook for patients and their relatives has never been brighter.
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| 10. |
- Lord, Anna, 1979-, et al.
(author)
-
Amyloid-β protofibril levels correlate with spatial learning in Arctic Alzheimer’s disease transgenic mice
- 2009
-
In: The FEBS Journal. - : Wiley. - 1742-464X .- 1742-4658. ; 276:4, s. 995-1006
-
Journal article (peer-reviewed)abstract
- Oligomeric assemblies of amyloid-β (Aβ) are suggested to be central in the pathogenesis of Alzheimer's disease because levels of soluble Aβ correlate much better with the extent of cognitive dysfunctions than do senile plaque counts. Moreover, such Aβ species have been shown to be neurotoxic, to interfere with learned behavior and to inhibit the maintenance of hippocampal long-term potentiation. The tg-ArcSwe model (i.e. transgenic mice with the Arctic and Swedish Alzheimer mutations) expresses elevated levels of Aβ protofibrils in the brain, making tg-ArcSwe a highly suitable model for investigating the pathogenic role of these Aβ assemblies. In the present study, we estimated Aβ protofibril levels in the brain and cerebrospinal fluid of tg-ArcSwe mice, and also assessed their role with respect to cognitive functions. Protofibril levels, specifically measured with a sandwich ELISA, were found to be elevated in young tg-ArcSwe mice compared to several transgenic models lacking the Arctic mutation. In aged tg-ArcSwe mice with considerable plaque deposition, Aβ protofibrils were approximately 50% higher than in younger mice, whereas levels of total Aβ were exponentially increased. Young tg-ArcSwe mice showed deficits in spatial learning, and individual performances in the Morris water maze were correlated inversely with levels of Aβ protofibrils, but not with total Aβ levels. We conclude that Aβ protofibrils accumulate in an age-dependent manner in tg-ArcSwe mice, although to a far lesser extent than total Aβ. Our findings suggest that increased levels of Aβ protofibrils could result in spatial learning impairment.
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| 11. |
- Lord, Anna, et al.
(author)
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An amyloid-beta protofibril-selective antibody prevents amyloid formation in a mouse model of Alzheimer's disease
- 2009
-
In: Neurobiology of Disease. - : Elsevier BV. - 0969-9961 .- 1095-953X. ; 36:3, s. 425-434
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Journal article (peer-reviewed)abstract
- Human genetics link Alzheimer's disease pathogenesis to excessive accumulation of amyloid-beta (Abeta) in brain, but the symptoms do not correlate with senile plaque burden. Since soluble Abeta aggregates can cause synaptic dysfunctions and memory deficits, these species could contribute to neuronal dysfunction and dementia. Here we explored selective targeting of large soluble aggregates, Abeta protofibrils, as a new immunotherapeutic strategy. The highly protofibril-selective monoclonal antibody mAb158 inhibited in vitro fibril formation and protected cells from Abeta protofibril-induced toxicity. When the mAb158 antibody was administered for 4 months to plaque-bearing transgenic mice with both the Arctic and Swedish mutations (tg-ArcSwe), Abeta protofibril levels were lowered while measures of insoluble Abeta were unaffected. In contrast, when treatment began before the appearance of senile plaques, amyloid deposition was prevented and Abeta protofibril levels diminished. Therapeutic intervention with mAb158 was however not proven functionally beneficial, since place learning depended neither on treatment nor transgenicity. Our findings suggest that Abeta protofibrils can be selectively cleared with immunotherapy in an animal model that display highly insoluble Abeta deposits, similar to those of Alzheimer's disease brain.
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| 12. |
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| 13. |
- Lord, Anna, 1979-
(author)
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Targeting Early Stages of Alzheimer’s Disease in a Transgenic Model
- 2009
-
Doctoral thesis (other academic/artistic)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.
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| 14. |
- Sahlin, Charlotte, et al.
(author)
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Docosahexaenoic acid stimulates non-amyloidogenic APP processing resulting in reduced Aβ levels in cellular models of Alzheimer's disease
- 2007
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In: European Journal of Neuroscience. - : Wiley. - 0953-816X .- 1460-9568. ; 26:4, s. 882-889
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Journal article (peer-reviewed)abstract
- Epidemiological studies suggest that a high intake of polyunsaturated fatty acids, such as docosahexaenoic acid (DHA), is associated with a reduced risk of Alzheimer's disease. Here, we examined the effects of DHA on amyloid precursor protein (APP) processing in cellular models of Alzheimer's disease by analysing levels of different APP fragments, including amyloid-β (Aβ). DHA administration stimulated non-amyloidogenic APP processing and reduced levels of Aβ, providing a mechanism for the reported beneficial effects of DHA in vivo. However, an increased level of APP intracellular domain was also observed, highlighting the need to increase our knowledge about the relevance of this fragment in Alzheimer's disease pathogenesis. In conclusion, our results suggest that the proposed protective role of DHA in Alzheimer's disease pathogenesis might be mediated by altered APP processing and Aβ production.
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| 15. |
- Sandberg, Anders, et al.
(author)
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Stabilization of neurotoxic Alzheimer amyloid-beta oligomers by protein engineering
- 2010
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In: Proceedings of the National Academy of Sciences of the United States of America. - : Proceedings of the National Academy of Sciences. - 0027-8424 .- 1091-6490. ; 107:35, s. 15595-15600
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Journal article (peer-reviewed)abstract
- Soluble oligomeric aggregates of the amyloid-beta peptide (Abeta) have been implicated in the pathogenesis of Alzheimer's disease (AD). Although the conformation adopted by Abeta within these aggregates is not known, a beta-hairpin conformation is known to be accessible to monomeric Abeta. Here we show that this beta-hairpin is a building block of toxic Abeta oligomers by engineering a double-cysteine mutant (called Abetacc) in which the beta-hairpin is stabilized by an intramolecular disulfide bond. Abeta(40)cc and Abeta(42)cc both spontaneously form stable oligomeric species with distinct molecular weights and secondary-structure content, but both are unable to convert into amyloid fibrils. Biochemical and biophysical experiments and assays with conformation-specific antibodies used to detect Abeta aggregates in vivo indicate that the wild-type oligomer structure is preserved and stabilized in Abetacc oligomers. Stable oligomers are expected to become highly toxic and, accordingly, we find that beta-sheet-containing Abeta(42)cc oligomers or protofibrillar species formed by these oligomers are 50 times more potent inducers of neuronal apoptosis than amyloid fibrils or samples of monomeric wild-type Abeta(42), in which toxic aggregates are only transiently formed. The possibility of obtaining completely stable and physiologically relevant neurotoxic Abeta oligomer preparations will facilitate studies of their structure and role in the pathogenesis of AD. For example, here we show how kinetic partitioning into different aggregation pathways can explain why Abeta(42) is more toxic than the shorter Abeta(40), and why certain inherited mutations are linked to protofibril formation and early-onset AD.
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| 16. |
- Sehlin, Dag
(author)
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Aβ Conformation Dependent Antibodies and Alzheimer's Disease
- 2010
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Doctoral thesis (other academic/artistic)abstract
- Soluble intermediates of the amyloid-β (Aβ) aggregation process are suggested to play a central role in the pathogenesis of Alzheimer’s disease (AD) by causing synaptic dysfunction and neuronal loss. In this thesis, soluble Aβ aggregates have been studied with a particular focus on the Aβ protofibril, which has served as the antigen for developing conformation dependent monoclonal antibodies. Antibodies generated from mice immunized with Aβ protofibrils were characterized regarding Aβ binding properties and the amino acid sequences of their antigen binding sites. A conformation dependent IgG antibody, mAb158, was further characterized and found to bind to Aβ protofibrils with a 200-fold higher affinity than to monomeric Aβ without affinity for soluble amyloid-β precursor protein (AβPP) or other amyloidogenic proteins. A sandwich enzyme-linked immunosorbent assay (ELISA) based on mAb158 was used to measure soluble Aβ protofibrils in brain extracts from AβPP-transgenic mice. Low levels of protofibrils could also be detected in human AD brain. However, positive signals generated from measurements in AD and control CSF samples were attributed to interference from heterophilic antibodies (HA), generating false positive signals by cross-binding the assay antibodies; consequently, a study on HA interference in Aβ oligomer ELISAs was initiated. A large set of plasma and CSF samples from AD and non-AD subjects were analyzed with and without measures taken to block HA interference, revealing that virtually all signals above the assay limit of detection were false and generated by HA interference. Many types of soluble Aβ aggregates have been described and suggested to impair neuron and synapse function. To investigate the soluble Aβ pool, synthetic Aβ and brain extracts from AβPP-transgenic mice and AD patients were ultracentrifuged on a density gradient to separate Aβ by size under native conditions. Four distinct gradient fractions were defined based on the appearance of synthetic Aβ in atomic force microscopy (AFM) and immunoreactivity in our protofibril specific sandwich ELISA. Interestingly, most Aβ from AD patients and AβPP-transgenic mice separated in the same fraction as toxic synthetic protofibrils.
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| 17. |
- Sehlin, Dag, et al.
(author)
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Heavy-Chain Complementarity-Determining Regions Determine Conformation Selectivity of Anti-A beta Antibodies
- 2011
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In: Neurodegenerative Diseases. - : S. Karger AG. - 1660-2854 .- 1660-2862. ; 8:3, s. 117-123
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Journal article (peer-reviewed)abstract
- Background/Aims: Amyloid-beta (Abeta) protofibrils are neurotoxic soluble intermediates in the Abeta aggregation process eventually forming senile plaques in Alzheimer's disease. This Abeta species is a potential biomarker for Alzheimer's disease and also a promising target for immunotherapy. In this study, we investigated the characteristics of conformation-dependent Abeta antibodies specific for Abeta protofibrils. Methods: Mice were immunized with Abeta protofibrils to generate hybridomas producing Abeta-specific monoclonal antibodies. Binding of antibodies to different Abeta conformations was investigated with inhibition ELISA. The antibodies' complementarity-determining region (CDR) sequences were determined and compared. Results: A majority of the antibodies were of the IgM class, all selectively binding to aggregated Abeta. Two IgG antibodies were generated: one with selective affinity for Abeta protofibrils and the other bound Abeta in all conformations. A high degree of similarity between the heavy-chain CDRs of the conformation-dependent antibodies was found, and all high-affinity Abeta antibodies displayed a high degree of sequence similarity in the light-chain CDRs. Conclusion: Sequence similarity in the heavy-chain CDRs is associated with conformation selectivity of the antibodies, while sequence similarity in the light-chain CDRs correlates with the affinity for Abeta.
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| 18. |
- Sehlin, Dag, et al.
(author)
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Interference from Heterophilic Antibodies in Amyloid-beta Oligomer ELISAs
- 2010
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In: Journal of Alzheimer's Disease. - : IOS Press. - 1387-2877 .- 1875-8908. ; 21:4, s. 1295-1301
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Journal article (peer-reviewed)abstract
- Amyloid-beta (Abeta) oligomers of different sizes and forms have recently been the focus for many Alzheimer's disease (AD) researchers. Various immunoassays have been used to detect low concentrations of these elusive Abeta species in different forms of human samples using little or no sample dilutions. However, the possibility that positive results may be caused by interference from heterophilic antibodies (HA) is often overlooked. HA, which recognize immunoglobulins from other species, are present in human plasma and cerebrospinal fluid (CSF) and may cause interference in sandwich immunoassays like enzyme-linked immunosorbent assays (ELISAs) by cross-binding the capture and detection antibodies of the assay. They thus may generate a false positive signal. Here we show that when assessing the Abeta oligomer content in plasma samples from 44 individuals with a sandwich ELISA, none of the 21 positive signals remained when the assay was repeated in the presence of factors blocking HA. Similarly, in CSF samples from 104 individuals, the signals from the 22 positive samples were strongly reduced when analyzed after anti-HA treatment. Taken together, HA interference is a problem that needs to be addressed when measuring low levels of an antigen in human plasma and CSF samples.
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| 19. |
- Sehlin, Dag, et al.
(author)
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Large Aggregates Are the Major Soluble Ab Species in AD Brain Fractionated with Density Gradient Ultracentrifugation
- 2012
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In: PLOS ONE. - : Public Library of Science (PLoS). - 1932-6203. ; 7:2
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Journal article (peer-reviewed)abstract
- Soluble amyloid-β (Aβ) aggregates of various sizes, ranging from dimers to large protofibrils, have been associated with neurotoxicity and synaptic dysfunction in Alzheimer's Disease (AD). To investigate the properties of biologically relevant Aβ species, brain extracts from amyloid β protein precursor (AβPP) transgenic mice and AD patients as well as synthetic Aβ preparations were separated by size under native conditions with density gradient ultracentrifugation. The fractionated samples were then analyzed with atomic force microscopy (AFM), ELISA, and MTT cell viability assay. Based on AFM appearance and immunoreactivity to our protofibril selective antibody mAb158, synthetic Aβ42 was divided in four fractions, with large aggregates in fraction 1 and the smallest species in fraction 4. Synthetic Aβ aggregates from fractions 2 and 3 proved to be most toxic in an MTT assay. In AβPP transgenic mouse brain, the most abundant soluble Aβ species were found in fraction 2 and consisted mainly of Aβ40. Also in AD brains, Aβ was mainly found in fraction 2 but primarily as Aβ42. All biologically derived Aβ from fraction 2 was immunologically discriminated from smaller species with mAb158. Thus, the predominant species of biologically derived soluble Aβ, natively separated by density gradient ultracentrifugation, were found to match the size of the neurotoxic, 80–500 kDa synthetic Aβ protofibrils and were equally detected with mAb158.
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| 20. |
- Söllvander, Sofia, 1983-
(author)
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Amyloid-β Protofibrils in Alzheimer´s Disease : Focus on Antibodies, Inflammation and Astrocytes
- 2015
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Doctoral thesis (other academic/artistic)abstract
- Soluble amyloid-beta (Aβ) aggregates, including Aβ protofibrils, play a central role in Alzheimer’s disease (AD) and constitute a potential diagnostic biomarker and a therapeutic target. Aβ protofibrils promote synapse dysfunction and neurodegeneration, but the mechanisms behind these effects remain unclear. The aim of this thesis was to increase the knowledge of Aβ protofibrils in AD pathology.When measuring low abundant antigens, such as soluble Aβ aggregates, in plasma and CSF by immunoassays, there is a possibility of interference by heterophilic antibodies (HA). In paper I, we show that HA generate false positive signals, by cross-binding the assay antibodies, when plasma and CSF from AD patients and healthy controls were analyzed for soluble Aβ aggregates, using sandwich ELISAs.Natural anti-Aβ antibodies exist in AD patients and healthy individuals. Circulating Aβ and anti-Aβ antibodies may form immune complexes, masking epitopes on the anti-Aβ antibody, which makes the anti-Aβ antibody concentration difficult to measure. In paper II, the ELISpot technique enabled us to successfully measure B cell production of anti-Aβ antibodies. Our results show that anti-Aβ protofibril antibody production is present in both AD patients and healthy individuals, but is significantly higher in AD patients, indicating that the immune system attempt to eliminate the toxic Aβ species.Insufficient lysosomal degradation is proposed to cause sporadic AD. In paper III, we used a co-culture system of astrocytes, neurons and oligodendrocytes, to clarify the role of astrocytes in Aβ protofibril clearance. Astrocytes are the most prominent glial cell type in the brain, but their role in AD remains elusive. We found that astrocytes effectively engulf, but inefficiently degrade Aβprotofibrils. This result in a high intracellular load of toxic, partly N-terminally truncated Aβ and lysosomal dysfunction. Moreover, we found that secretion of microvesicles, containing N-terminally truncated Aβ, induce neuronal apoptosis. In paper IV, we show that treatment with the protofibril selective antibody mAb158 lead to enhanced Aβ clearance and thereby prevent Aβ neurotoxicity.Taken together, this thesis contributes with important knowledge on the role of Aβ protofibrils in AD pathogenesis and technical aspects that should be considered when measuring Aβ in human tissues.
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| 21. |
- Söllvander, Sofia, et al.
(author)
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Increased Number of Plasma B Cells Producing Autoantibodies Against A beta(42) Protofibrils in Alzheimer's Disease
- 2015
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In: Journal of Alzheimer's Disease. - 1387-2877 .- 1875-8908. ; 48:1, s. 63-72
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Journal article (peer-reviewed)abstract
- The Alzheimer's disease (AD)-related peptide amyloid-beta (A beta) has a propensity to aggregate into various assemblies including toxic soluble A beta protofibrils. Several studies have reported the existence of anti-A beta antibodies in humans. However, it is still debated whether levels of anti-A beta antibodies are altered in AD patients compared to healthy individuals. Formation of immune complexes with plasma A beta makes it difficult to reliably measure the concentration of circulating anti-A beta antibodies with certain immunoassays, potentially leading to an underestimation. Here we have investigated anti-A beta antibody production on a cellular level by measuring the amount of anti-A beta antibody producing cells instead of the plasma level of anti-A beta antibodies. To our knowledge, this is the first time the anti-A beta antibody response in plasma has been compared in AD patients and age-matched healthy individuals using the enzyme-linked immunospot (ELISpot) technique. Both AD patients and healthy individuals had low levels of B cells producing antibodies binding A beta(40) monomers, whereas the number of cells producing antibodies toward A beta(42) protofibrils was higher overall and significantly higher in AD compared to healthy controls. This study shows, by an alternative and reliable method, that there is a specific immune response to the toxic A beta protofibrils, which is significantly increased in AD patients.
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| 22. |
- Wiberg, Henning, et al.
(author)
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Separation and characterization of aggregated species of amyloid-beta peptides
- 2010
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In: Analytical and Bioanalytical Chemistry. - : Springer Science and Business Media LLC. - 1618-2642 .- 1618-2650. ; 397:6, s. 2357-2366
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Journal article (peer-reviewed)abstract
- We have investigated the use of isoelectric focusing and immunodetection for the separation of low molecular weight species of amyloid-beta (A beta) peptides from their aggregates. From solutions of A beta(1-40) or A beta(1-42) monomeric peptides, low molecular weight material appeared at a pI value of ca. 5, while the presence of aggregates was detected as bands, observed at a pI of 6-6.5. The formation of A beta aggregates (protofibrils) was verified by a sandwich ELISA, employing the protofibril conformation-selective antibody mAb158. In order to study the aggregation behavior when using a mixture of the monomers, we utilized the IEF separation combined with Western blot using two polyclonal antisera, selective for A beta(1-40) and A beta(1-42), respectively. We conclude that both monomers were incorporated in the aggregates. In a further study of the mixed aggregates, we used the protofibril conformation-selective antibody mAb158 for immunoprecipitation, followed by nanoelectrospray mass spectrometry (IP-MS). This showed that the A beta(1-42) peptide is incorporated in the aggregate in a significantly larger proportion than its relative presence in the original monomer composition. IP-MS with mAb158 was also performed, and compared to IP-MS with the A beta-selective antibody mAb1C3, where a monomeric A beta(1-16) peptide was added to the protofibril preparation. A beta(1-16) is known for its poor aggregation propensity, and acted therefore as a selectivity marker. The results obtained confirmed the protofibril conformation selectivity of mAb158.
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