| 1. |
- Gestin, Maxime, 1990-
(författare)
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PepFect14, a Versatile Cell-Penetrating Peptide
- 2020
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Cell-penetrating peptides have been discovered almost three decades ago and there are, nowadays, thousands of available sequences. They offer multiple applications in the field of drug delivery as they are able to carry therapeutic macromolecules across the plasma membrane. Throughout the years, new sequences have been developed and designed to achieve new applications such as specificity for certain kinds of cargoes, intrinsic therapeutic effects and targeted delivery.In this thesis, we focused on a single most promising cell-penetrating peptide named PepFect14 and aimed at reaching a better understanding of the factors involved in the cellular uptake through paper I and paper II. Notably, in paper I we screened a library of small molecule drugs that influences signaling pathways and discovered that three drugs had an unreported influence on endocytosis. In paper II, After performing an RNA sequencing on cells treated with PepFect14, we demonstrated the involvement of autophagy in the intracellular trafficking of the cell-penetrating peptide. A second aim of this thesis, covered in paper III and paper IV, was to discover new applications for PepFect14 in order to broaden its potential. In paper III, we successfully used PepFect14 to mediate the intracellular delivery of heat shock protein 70kDa. This was the first protein delivery assisted by PepFect14. In paper IV, PepFect14 was covalently fused to mtCPP1, a cell-penetrating peptide that targets mitochondria and reduce the level of reactive oxygen species. The constructs showed the ability to keep the properties of both peptides and achieved a mitochondria-targeted antisense therapy.Overall, this thesis summarizes our effort to develop and bring to their full potential already existing cell-penetrating peptides instead of developing new sequences for each new application.
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| 2. |
- Jeppsson, Fredrik, 1975-
(författare)
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Characterization of Diagnostic Tools and Potential Treatments for Alzheimer’s Disease : PET ligands and BACE1 inhibitors
- 2016
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Alzheimer’s disease (AD) is a very complex disorder and the most common form of dementia. The two pathological hallmarks of AD are extracellular amyloid-β (Aβ) plaques in cerebral cortex, and intraneuronal neurofibrillary tangles. In the early stages of the disease it can be difficult to accurately diagnose AD, as it is difficult to distinguish from normal signs of aging. There is thus a need for sensitive non-invasive tools, able to detect pathophysiological biomarker changes. One such approach is molecular imaging of Aβ plaque load in brain, using PET (positron emission tomography) ligands.We have developed and characterized two novel Aβ plaque neuroimaging PET ligands, AZD2184 and AZD4694. The 2-pyridylbenzothiazole derivate AZD2184, is a 11C-labeled PET ligand with a higher signal-to-background ratio compared to the widely used PET ligand PIB, a 11C-labeled phenylbenzothiazole based tool. This makes it possible to detect smaller changes in Aβ plaque deposition load, and therefore theoretically, also earlier diagnosis. A drawback with 11C-labeled PET ligands is the relatively short half-life. To meet the need for PET ligands with a longer half-life, we developed the pyridylbenzofuran derivate [18F]AZD4694. Although development of fluorinated radioligands is challenging due to the lipophilic nature of aromatic fluorine, we successfully developed a 18F-labeled PET ligand with a signal-to-background ratio matching PIB, the most widely used 11C-labeled PET ligand in clinical use. 3H-labeled derivates of AZD2184, AZD4694, and PIB, showed lower binding specificity towards Aβ plaques containing ApoE. The ApoE genotype per se did not significantly affect ligand binding, instead, the amount of ApoE incorporated to the Aβ plaques appears to be of importance for the binding characteristics of these amyloid PET ligands.Beta-secretase 1 (BACE1) mediates the first step in the processing of amyloid precursor protein (APP) to Aβ peptides, making BACE1 inhibition an attractive therapeutic target in AD. We developed and characterized three novel BACE1 inhibitors, AZD3839, AZ-4217, and AZD3293. AZD3839 and AZ-4217 contains an amidine group which interacts with the catalytic aspartases Asp-32 and Asp-228 of BACE1, effectively inhibiting the enzyme. All three compounds are potent and selective inhibitors of human BACE1, with in vitro potency demonstrated in several cellular models, including primary cortical neurons. All three compound exhibited dose- and time-dependent lowering of plasma, brain, and cerebrospinal fluid Aβ levels in several species, and two of the compounds (AZD3839 and AZD3293) were progressed into clinical trials.
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| 3. |
- Koistinen, Niina, 1972-
(författare)
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The amyloid-β precursor protein (APP)-binding protein Fe65 and APP processing
- 2018
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Alzheimer’s disease (AD) is a neurodegenerative disorder characterized by abnormal deposition of neurotoxic amyloid-β (Aβ) peptide. Aβ is generated by sequential cleavage of the amyloid-β precursor protein (APP) by β- and then γ-secretase. However, APP can also be processed by α- and γ-secretase, instead resulting in generation of neuroprotective sAPPα. Increased APP phosphorylation and altered expression levels of the brain enriched Fe65 protein have been observed in the brains of AD patients. Fe65 can not only interact with membrane tethered APP, but can also localized into the nucleus and act as a transcriptional regulator together with the APP intracellular domain (AICD), generated after γ-secretase processing. How APP processing, APP/Fe65 interaction, and the nuclear AICD/Fe65 complex is regulated has not yet been fully understood. The aim of this thesis was therefore to further elucidate how Fe65 is regulated and how APP Ser675 phosphorylation affects APP processing.We could identify several factors regulating Fe65. First, we identified that neuronal differentiation induces Fe65 phosphorylation (paper I), and that phosphorylated forms of Fe65 were preferentially localized outside the nucleus (paper II). Second, we found that the APP binding PTB2 domain of Fe65, rather than the previously proposed N-terminal WW domain, is important for the nuclear localization of Fe65 (paper II). In addition, we surprisingly found that mutation of S228 in the Fe65 N-terminus could increase the APP/Fe65 interaction (paper III). Third, both α- and γ-secretase inhibitors decreased Fe65 nuclear localization similarly, indicating an important role of α-secretase in regulating Fe65 nuclear localization (papers II and III). Lastly, we could in paper IV for the first time show that phosphorylation of APP at Ser675 regulates APP processing at the plasma membrane, resulting in reduced levels of sAPPα. These results, together with the observation that APP Ser675 phosphorylation occur in AD brains, suggest that Ser675 phosphorylation could contribute to AD pathology by decreasing α-secretase processing and instead increasing the levels of Aβ.In summary these studies have contributed to understanding of APP processing and the interplay between Fe65 and APP, two suggested key players in AD.
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| 4. |
- Menon, Preeti, 1988-
(författare)
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The amyloid-β precursor protein (APP) and its adaptor protein Fe65 : Two key players in Alzheimer’s disease
- 2020
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Alzheimer’s disease (AD) is a neurodegenerative disease characterized by the abnormal accumulation and aggregation of amyloid beta (Aβ) peptides within the brain. Generation of Aβ occur when the amyloid-beta precursor protein (APP) is proteolytically processed by β- and then γ-secretase in the amyloidogenic pathway. However, if APP instead is cleaved by α- and γ-secretase in the non-amyloidogenic pathway, Aβ formation is prevented and neuroprotective sAPPα is generated. In addition to these canonical processing pathways, APP can also be cleaved along non-canonical pathways by Δ, η, caspase or Meprinβ, resulting in numerous fragments that have different functional properties. The trafficking and processing of APP is a complex process and can be regulated by the adaptor protein Fe65. Following γ-secretase mediated cleavage of APP, the intracellular domain of APP and Fe65 can together translocate into the nucleus and regulate nuclear signaling. However, the exact mechanisms of how APP processing and APP/Fe65 nuclear signaling are regulated is still unclear. The aim of this thesis was to study different factors that may influence the regulation of APP processing and Fe65 nuclear localization. We found that phosphorylation of APP at Ser675 alters APP processing resulting in reduced levels of sAPPα and total sAPP, without affecting the plasma membrane level of APP. We could further observe an increased level of a slower migrating C99 like CTF, which was not generated by β-secretase cleavage of APP as there was no expression of BACE1 in the cell model used. Instead, generation of this CTF was blocked upon Meprinβ siRNA knockdown. Taken together these findings suggest that APP-Ser675 phosphorylation promotes Meprinβ processing of APP. In another study, we found that mutation of Ser228 at the Fe65 N-terminal dramatically increased the interaction between Fe65 and full-length APP. Moreover, this enhanced interaction resulted in decreased levels of non-amyloidogenic processing of APP and thus neuroprotective sAPPα. This suggest that the level of Fe65-APP interaction is important in regulating APP processing. Therefore, we also wanted to elucidate more about how the adaptor protein Fe65 is regulated. We found that Fe65 is likely phosphorylated on several residues in the N-terminus and that these phosphorylated forms preferentially localized in the cytoplasm. In addition, we could show that the nuclear level and nuclear/cytoplasmic ratio of Fe65 was decreased upon mutation of Fe65-Ser228 to glutamic acid, mimicking phosphorylation. Taken together this suggest that phosphorylation of Ser228 together with other residues in the N-terminus of Fe65 negatively regulate the Fe65 nuclear localization. In a third study, we could also show that the Fe65 PTB2 domain, rather than the WW domain, plays an important role in localizing Fe65 to the nucleus. Lastly, using different inhibitors, we found that blocking α-secretase processing decrease the Fe65 nuclear localization to the same extent as γ-secretase inhibition in both undifferentiated and RA or PMA differentiated cells. This suggest that α-secretase processing of APP or other Fe65 interacting transmembrane proteins play a more important role in regulation of Fe65 nuclear localization than previously thought. Interestingly, while ADAM10 was the most important α-secretase mediating this effect in undifferentiated cells, other α-secretases, likely ADAM17, played a more important role in RA or PMA differentiated neuroblastoma cells.In summary, the results obtained in this thesis have increased the understanding of APP processing and how the adaptor protein Fe65 may act as a molecular switch altering APP cleavage.
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| 5. |
- Niss, Frida
(författare)
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RNA binding proteins and epigenetics in SCA7
- 2019
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Licentiatavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Polyglutamine diseases are a group of nine disorders that includes, among others SCA7. The common denominator is an expanded glutamine tract in the respective disease protein caused by unstable replication during meiosis. Most research within this field points to a combination of gain-of-function and loss-of-function mechanisms causing all polyglutamine diseases. Using a SCA7 model we are thus attempting to study both of these mechanisms. The glutamine tract expansion responsible for SCA7 is located in the protein Ataxin-7, which like the other polyglutamine proteins aggregates into large inclusions in patient cells. In a gain-of-function mechanism, the aggregates are suggested to cause stress to the cell by e.g. sequestering vital proteins into the aggregates, which could disrupt their function. RNA-binding proteins such as FUS and TDP-43 are often found in aggregates in neurodegenerative diseases, and have been observed in SCA7 aggregates as well. However, if disruption of FUS and TDP-43 function occurs, or if it plays a role in SCA7 pathology is unclear. We found a high rate of co-aggregation of FUS with Ataxin-7 using immunofluorescence and filter trap assays. Furthermore, we found that both the localization and function of FUS was altered in a SCA7 cell model using cell fractionations and RT-PCR. Additionally, we found that TDP-43 also co-aggregated with Ataxin-7 and phosphorylation of TDP-43 was increased during the disease phenotype.Wild-type Ataxin-7 normally functions within chromatin regulation processes, and loss-of-function pathology in SCA7 could therefore involve a disruption of these processes. We have developed a method, FRIC, that enables us to study chromatin organization in live cells using confocal microscopy and fluorescently tagged histones. Using inhibitors of HATs and HDACs, as well as a previously known protein that regulates chromatin structure, we were able to observe changes in chromatin structure in the nuclear periphery, confirming the usefulness of FRIC. Additionally, we investigated the involvement of an inner nuclear membrane protein, Samp1, in chromatin organization and found Samp1 to be instrumental in organizing peripheral chromatin.Taken together, the results from these two studies indicate that SCA7 pathology disturbs RNA-binding protein mediated transcriptional regulation in a gain-of-function mechanism, and that FRIC is a powerful new tool for examining chromatin regulation in diseases with disrupted transcription, like SCA7.
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