| 1. |
- Gustafsson, Gabriel
(author)
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Alpha-Synuclein Oligomers : Cellular Mechanisms and Aspects of Antibody Treatment
- 2017
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Doctoral thesis (other academic/artistic)abstract
- In Parkinson’s disease (PD) and dementia with Lewy bodies (DLB), aggregated α-synuclein deposit inside cells within the brain. Smaller soluble α-synuclein aggregates, oligomers, are present both intra- and extracellularly. The α-synuclein oligomers are known to be particularly harmful, although the underlying neurotoxic mechanisms are not fully understood. The aim of this thesis was to investigate the pathogenic roles of α-synuclein oligomers and the possibility to target such species with antibody treatment.Passive immunotherapy with α-synuclein antibodies can lead to reduced pathology and ameliorated symptoms in transgenic mice. However, it remains unknown whether the antibodies are taken up by cells or whether they act extracellularly. In Paper I, we assessed cellular internalization of various α-synuclein monoclonal antibodies. The oligomer selective mAb47 displayed the highest uptake, which was promoted by the extracellular presence of α-synuclein.Alpha-synuclein aggregates can be found in both neurons and glial cells, but the pathogenic role of glial deposits has only been sparsely investigated. In Paper II, co-cultures of neurons and glia were exposed to α-synuclein oligomers. The astrocytes in the cultures rapidly accumulated oligomers, which were only partially degraded by lysosomes. The sustained intracellular α-synuclein deposits were associated with mitochondrial stress reactions in the astrocytes. In Paper III, we sought to explore whether the astrocytic pathology induced by α-synuclein oligomers could be ameliorated by antibody treatment. Pre-incubation of oligomers with mAb47 promoted α-synuclein clearance, reduced astrocytic accumulation and rescued cells from mitochondrial stress. We could demonstrate that binding of the antibody to its antigen in the extracellular space was crucial for these effects to occur.The progressive pathology in PD is believed to be driven by cell-to-cell spreading of α-synuclein aggregates, potentially via exosomes and other extracellular vesicles (EVs). In Paper IV, we found that either fusing α-synuclein to a non-physiological protein tag or introducing the PD-causing A53T mutation directed α-synuclein towards EV secretion. Also, EV-associated α-synuclein was particularly prone to induce toxicity in recipient cells.In conclusion, this thesis sheds new light on the cellular dysfunction related to α-synuclein pathology and on how the underlying pathogenic processes may be targeted by antibody treatment.
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| 2. |
- Behere, Anish, 1993-
(author)
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Ex‘PLA’ining the progression of pathological proteins in Alzheimer’s and Parkinson’s diseases : see(d)ing is believing
- 2022
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Doctoral thesis (other academic/artistic)abstract
- Alzheimer’s disease (AD) and Parkinson’s disease (PD) are the two most common forms of neurodegenerative disorders affecting approximately 50 million people worldwide. The underlying neuropathological processes leading to AD and PD share many similarities, i.e. aberrant protein aggregation of tau and alpha-synuclein (αSyn) in the brain. Monitoring tau and αSyn aggregation is challenging, due to morphological heterogeneity of the aggregating species and problems in preserving the antigen conformation ex vivo.In paper-I, we validated the usefulness of proximity ligation assay (PLA), a technique that enabled us to visualize previously undetected early αSyn pathology in the A30P-tg mouse model of PD. We observed an age-progressive increase in the levels of phosphorylated αSyn (pSynS129) and the compactness of aggregates in the brain. Although loss of dopaminergic neurons was not found, a subtle dysregulation of other catecholamines was recorded in the older mice.In paper-II, we revealed a wide distribution of pSynS129 aggregates in alpha-synucleinopathy-patient brains. By using a PLA setup with certain antibody pair combinations on brain sections, we observed unique staining patterns, which could not be visualized using regular immunohistochemistry (IHC). In A30P-tg mice, the morphological pattern of the PLA signals indicated an intracellular shift of pSynS129 from the periphery towards the neuronal soma.In Paper-III, we demonstrated that multiplex pTauS202,T205-pTauT231, singleplex pTauT231 and singleplex pSynS129 PLAs can recognize an extensive tau and αSyn pathology compared to regular IHC. We found that using our PLA approach we could differentiate between pTauS202,T205 and pTauT231 pathology in AD brains, whereas IHC could not. Similarly, in the PD brain, singleplex pSynS129 PLA detected novel structures, i.e. apparent thick intercellular tunnelling nanotubes and early aggregates; whereas pSynS129 IHC was limited to the detection of mature pathology. Lastly, we demonstrated that our multiplex PLA approach detected co-aggregates of pSynS129-pTau.In Paper-IV, in an αSyn seeding mouse model we observed pSynS129 immunoreactivity close to the striatal injection site one day post-injection (dpi). Intriguingly, this type of staining disappeared with the concurrent formation of peri-nuclear pSynS129 inclusions in specific brain regions after 14 dpi. In parallel, astrocytic activation prior to pSynS129 inclusion formation was observed.In conclusion, we have developed several novel PLAs that detect both tau and αSyn pathology with a higher ex vivo sensitivity and specificity than currently used immunostaining methods. This thesis work provides valuable insights that potentially could be used for the development of future biomarkers for tauopathies and synucleinopathies.
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| 3. |
- Scott, Pat, 1982-
(author)
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Searches for Particle Dark Matter : Dark stars, dark galaxies, dark halos and global supersymmetric fits
- 2010
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Doctoral thesis (other academic/artistic)abstract
- The identity of dark matter is one of the key outstanding problems in both particle and astrophysics. In this thesis, I describe a number of complementary searches for particle dark matter. I discuss how the impact of dark matter on stars can constrain its interaction with nuclei, focussing on main sequence stars close to the Galactic Centre, and on the first stars as seen through the upcoming James Webb Space Telescope. The mass and annihilation cross-section of dark matter particles can be probed with searches for gamma rays produced in astronomical targets. Dwarf galaxies and ultracompact, primordially-produced dark matter minihalos turn out to be especially promising in this respect. I illustrate how the results of these searches can be combined with constraints from accelerators and cosmology to produce a single global fit to all available data. Global fits in supersymmetry turn out to be quite technically demanding, even with the simplest predictive models and the addition of complementary data from a bevy of astronomical and terrestrial experiments; I show how genetic algorithms can help in overcoming these challenges.
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| 4. |
- Fagerqvist, Therese, 1983-
(author)
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Studies of α-synuclein Oligomers-with Relevance to Lewy Body Disorders
- 2013
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Doctoral thesis (other academic/artistic)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.
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| 5. |
- Almandoz Gil, Leire, 1988-
(author)
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Characterization of Physiological and Pathological Alpha-Synuclein : Implications for Parkinson’s Disease and Related Disorders
- 2018
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Doctoral thesis (other academic/artistic)abstract
- Aggregated alpha-synuclein is the main component of Lewy bodies and Lewy neurites, intraneuronal inclusions found in the brains of Parkinson’s disease (PD) and dementia with Lewy bodies (DLB) patients (synucleinopathies). Alpha-synuclein is a presynaptic protein, which is most commonly an unfolded monomer in its physiological state. However, under pathological conditions it can start to misfold and enter an aggregation pathway that will lead to the formation of oligomers of increasing size and finally insoluble fibrils. The oligomers have been hypothesized to be the most neurotoxic species, but studies of their properties have been hindered by their heterogeneity and kinetic instability. The overall aim of this thesis was to characterize and compare physiological and pathological forms of alpha-synuclein from different sources: recombinant monomers, oligomers formed in vitro through exposure to oxidative stress related reactive aldehydes, aggregates from a synucleinopathy mouse model and from synucleinopathy patients.In paper I we studied the effect of low molar excess of two lipid peroxidation products, 4-oxo-2-nonenal (ONE) and 4-hydroxy-2-nonenal (HNE), on the oligomerization of alpha-synuclein. Through biophysical methods we observed that, although both aldehydes bound to alpha-synuclein directly, ONE produced SDS-stable oligomers more rapidly than HNE. Moreover, ONE induced oligomerization at both acidic and neutral pH, while HNE only formed oligomers at neutral pH.In paper II we mapped the surface exposed epitopes of in vitro and in vivo generated alpha-synuclein species by using immunoglobulin Y antibodies raised against short linear peptides covering most of the alpha-synuclein sequence. Monomers were found to react with most antibodies, while the latter part of the N-terminus and mid-region of HNE oligomers and fibrils was found to be occluded in oligomers and fibrils. Through immunohistochemistry we compared alpha-synuclein aggregates in brain tissue from patients with synucleinopathies as well as from a mouse model expressing A30P human alpha-synuclein. Although the exposed epitopes were found to be similar overall, subtle differences were detected in the C-terminus.An additional aim of this thesis was to characterize synaptic aggregates of alpha-synuclein. In paper III we obtained synaptosomal preparations of the A30P mouse model and found that a subset of the alpha-synuclein present in the synaptosomes was proteinase K resistant and therefore aggregated. Further biochemical analyses showed that the aggregated alpha-synuclein mainly was of human, i.e. transgenic, origin and that Ser 129 was not phosphorylated, which otherwise is a common post translational modification of alpha-synuclein in Lewy bodies.It has been suggested that alpha-synuclein plays a role in neurotransmitter release by binding to the SNARE protein VAMP-2 and thereby chaperoning the SNARE complex assembly. In paper IV we used proximity ligation assay to visualize the co-localization of alpha-synuclein and the SNARE proteins in primary neurons from non-transgenic and A30P transgenic mice.In conclusion, in this thesis we have characterized a variety of alpha-synuclein species and shed light on the diversity of alpha-synuclein aggregates. Additionally, we have characterized synaptic species of alpha-synuclein and analyzed the co-localization between alpha-synuclein and SNARE proteins in neurons.
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| 6. |
- Brolin, Emma
(author)
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Pathophysiological effects of alpha-synuclein on SNARE complex proteins in models of alpha-synucleinopathies
- 2022
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Doctoral thesis (other academic/artistic)abstract
- Accumulation and spread of alpha-synuclein (α-syn) aggregates are central to the disease pathogenesis of Parkinson’s disease and dementia with Lewy bodies, collectively known as α-synucleinopathies. Native α-syn is a monomeric presynaptic protein that can act as a molecular chaperone for the soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) complex assembly. The aim of this thesis was to investigate the pathophysiological effect of different α-syn species on SNARE protein distribution and to study the involvement of extracellular vesicles (EVs) in the propagation of α-syn pathology.In paper I, the co-localization between α-syn and the SNARE proteins VAMP-2, SNAP-25 and syntaxin-1, was analyzed in primary cortical neurons from transgenic (tg) human A30P α-syn and wild type (wt) mice using proximity ligation assay (PLA). The results demonstrated that SNARE proteins co-localized with total α-syn mainly in neuronal processes, and with A30P α-syn predominantly in the cell soma. In paper II, we investigated how altered molecular properties of α-syn could affect its cellular processing. Different α-syn constructs were expressed in SH-SY5Y cells and the culture medium was analyzed for free-floating α-syn, as well as for α-syn within the EV fraction. Modifications in the N-terminal increased the EV secretion and enhanced the cell-to-cell transfer of α-syn. In paper III, the synaptic α-syn species of the A30P tg mouse brain were biochemically characterized and their effect on SNARE protein distribution was analyzed with western blot and PLA. We found that synaptosomal α-syn aggregates were mainly composed of non-phosphorylated human A30P α-syn. A decrease of intact SNARE complexes was observed in the tg A30P synaptosomes and in the prefrontal cortex, even though the total levels of SNARE proteins were unchanged in A30P compared to wt mice.In paper IV, we studied the effect of α-syn monomers and α-syn preformed fibrils (PFFs) on SNARE protein distribution in wt primary neurons, using PLA. Both short- and long-term exposure to α-syn monomers or PFFs altered the co-localization of SNARE proteins. Promoting the long-term uptake of α-syn by using a protein delivery reagent, further increased SNARE protein redistribution. In contrast, a PFF-induced SNARE protein redistribution was not observed when lysosomal degradation was inhibited. Interestingly, addition of EVs from monomer- and PFF-treated astrocytes also affected SNARE protein distribution in recipient neurons. Taken together, the results from this thesis indicate that synaptic α-syn aggregates and EV-associated α-syn could be promising therapeutic targets in the α-synucleinopathies.
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