This thesis aimed to gain more insight into the role of iron in neurodegenerative diseases using high-field MRI. I investigated the pathological correlates of susceptibility-based contrasts on MRI,... Show moreThis thesis aimed to gain more insight into the role of iron in neurodegenerative diseases using high-field MRI. I investigated the pathological correlates of susceptibility-based contrasts on MRI, and how iron accumulation is associated with disease progression both ex vivo and in vivo. Show less
The presence of protein aggregates is commonly believed to be an important risk factor for immunogenicity of therapeutic proteins. Among all types of aggregates, dimers are relatively abundant in... Show moreThe presence of protein aggregates is commonly believed to be an important risk factor for immunogenicity of therapeutic proteins. Among all types of aggregates, dimers are relatively abundant in most commercialized monoclonal antibody (mAb) products. The aim of this study was to investigate the immunogenicity of artificially created mAb dimers relative to that of unstressed and stressed mAb monomers. A monoclonal murine IgG1 (mIgG1) antibody was exposed to low pH, elevated temperature, or UV irradiation to induce dimerization. Dimers and monomers were purified via size-exclusion chromatography. Physicochemical analysis revealed that upon all stress conditions, new deamidation or oxidation or both of amino acids occurred. Nevertheless, the secondary and tertiary structures of all obtained dimers were similar to those of unstressed mIgG1. Isolated dimers were administered subcutaneously in Balb/c mice, and development of antidrug antibodies and accumulation of follicular T helper cells in draining lymph nodes and spleens were determined. None of the tested dimers or stressed monomers were found to be more immunogenic than the unstressed control in our mouse model. In conclusion, both dimers and monomers generated by using 3 different stress factors have a low immunogenicity similar to that of the unstressed monomers. Show less
Parkinson’s disease is a neurodegenerative disease characterized by the presence of abnormal deposits of aggregated proteins in the brain tissue, known as Lewy bodies. The major components of Lewy... Show moreParkinson’s disease is a neurodegenerative disease characterized by the presence of abnormal deposits of aggregated proteins in the brain tissue, known as Lewy bodies. The major components of Lewy bodies are aggregated forms of a small presynaptic protein known as α-synuclein (α-syn). In this thesis we report on the intricacies of α-syn aggregation. Using an array of biophysical techniques we were able to observe the formation of the earliest α-syn oligomeric species – relatively stable dimers and tetramers – which are more easily formed than commonly assumed. Fluorescent labelling was shown to significantly affect the morphology of α-syn aggregates, which limits the applicability of this technique. From the growth kinetics of α-syn fibrillar seeds we conclude that the elongation of fibrils proceeds by a different mechanism than primary nucleation. Further, we studied the effect of solution conditions and surface effects on the growth of the α-syn aggregates. Using total internal reflection microscopy and confocal fluorescence imaging we observed the elongation of individual fibrils in real time, showing that this process proceeds by leaps and bounds. Show less
Dissolving microneedles are an attractive approach for non-invasive delivery of drugs via the skin, particularly when the doses are in the microgram or low-milligram range. The aim of the study was... Show moreDissolving microneedles are an attractive approach for non-invasive delivery of drugs via the skin, particularly when the doses are in the microgram or low-milligram range. The aim of the study was to develop hyaluronan-based, monoclonal IgG-loaded microneedles for intradermal delivery enabling efficient penetration and rapid dissolution in the skin while preserving protein stability. Microscopic analysis showed successful preparation of sharp microneedles with the tip length of ~ 280 μm and with up to 10% (w/w) of IgG content. The water content of the microneedles was ~ 12% and was not affected by the protein content. The protein distribution was uniform within microneedle tips and individual arrays but some array-to-array variation of IgG level within a single preparation batch was detected. After dissolution of microneedle arrays in PBS, > 80% of protein was recovered and no conformational changes were detected by fluorescence spectroscopy. At submicron level, only weak and reversible interaction between HA and IgG was found by asymmetric flow field flow fractionation analysis after the dissolution of prepared microneedles. Although, the formation of insoluble micron-size particles was detected by flow imaging microscopy the IgG amount incorporated into these particles was negligible (< 5%). Finally, microneedles were able to penetrate into the epidermis of ex vivo human skin followed by the rapid dissolution of the microneedle tips in the skin. After 10 min of application, the majority of the original tip length was dissolved and IgG and hyaluronan were co-deposited until a depth of 150–200 μm in the skin. In conclusion, developed hyaluronan-based dissolving microneedles allow rapid non-invasive intradermal protein delivery. Show less
This dissertation mainly focuses on interdisciplinary approaches for biomedical knowledge discovery. This required special efforts in developing systematic strategies to integrate various data... Show moreThis dissertation mainly focuses on interdisciplinary approaches for biomedical knowledge discovery. This required special efforts in developing systematic strategies to integrate various data sources and techniques, leading to improved discovery of mechanistic insights on human diseases. Chapter one looks at the possibility in which combining various bioinformatics-based strategies can significantly improve the characterization of the OPMD mouse model. We discuss that this approach in knowledge discovery, on the basis of our extensive analysis, helped us to shed some light on how this model system relates to OPMD pathophysiology in human. In Chapter two, we expand on this combinatory approach by conducting a cross-species data analysis. In this study, we have looked for common patterns that emerge by assessing the transcriptome data from three OPMD model systems and patients. This strategy led to unravelling the most prominent molecular pathway involved in OPMD pathology. The third chapter achieves a similar goal to identify similar molecular and pathophysiological features between OPMD and the common process of skeletal muscle ageing. Engaging in a study in which the focus was made on the universality of biological processes, in the light of evolutionary mechanisms and common functional features, led to novel discoveries. This work helped us uncover remarkable insights on molecular mechanisms of ageing muscles and protein aggregation. Chapters four and five take a different route by tackling the field of computational biology. These chapters aim to extend network inference by providing novel strategies for the exploitation and integration of multiple data sources. We show that these developments allow us to infer more robust regulatory mechanisms to be identified while translations and predictions are made across very different datasets, platforms, and organisms. Finally, the dissertation is concluded by providing an outlook on ways the field of systems biology can evolve in order to offer enhanced, diversified and robust strategies for knowledge discovery. Show less
Huntington disease (HD) is the best know of the polyglutamine disorders which are caused by the excessive expansion of a CAG repeat in a transcribed gene. It is estimated that there are 1300 HD... Show moreHuntington disease (HD) is the best know of the polyglutamine disorders which are caused by the excessive expansion of a CAG repeat in a transcribed gene. It is estimated that there are 1300 HD patients in the Netherlands and even more people that are currently at risk. Translation of the CAG repeat results in proteins with an expanded polyglutamine repeat. Expansion of this repeat above a threshold of 36 results in an aggregation prone protein leading to disease onset around mid-life. In all polyglutamine diseases an inverse correlation is found between repeat expansion and age of disease onset. Neuronal intranuclear inclusions (NII) which contain at least part of the expanded protein are identified as hallmarks of the polyglutamine diseases. Inclusions have been suggested to function as protective storage sites implying that soluble misfolded proteins are the primary cause of toxicity. The aggregation prone properties of these proteins are causing the pathological gain of function due to interference with normal cellular function. The ubiquitin proteasome system (UPS) is responsible for the main protein degradation within the cell. The UPS contributes to cellular homeostasis by regulating the expression of essential proteins and degradation of excessive proteins. In addition, the UPS is involved in cellular protein quality control and responsible for the degradation of aberrant proteins that accumulate in neurodegeneration. Also expanded polyglutamine proteins can be degraded by the UPS in order to protect the cell from this aggregation prone protein. However, the expanded repeat is also difficult to degrade causing impairment of the proteasome and consequential accumulation. The general introduction in Chapter 1 gives an overview of several of the important aspects leading to neurodegeneration in HD and specifically the contribution of the UPS. Chapter 2 describes the discovery of molecular misreading which occurs on GAGAG motifs in different genes. The frameshift mutant of ubiquitin B (UBB+1) is formed by a dinucleotide deletion (_GU) in the mRNA. The mutant ubiquitin protein that is formed can no longer ubiquitinate substrate proteins but is a target for ubiquitination and subsequent proteasomal degradation. UBB+1 accumulates in several neurodegenerative diseases and is an in vivo marker for proteasomal inhibition. In Chapter 3, we investigated the accumulation of UBB+1 in HD and Spinocerebellar ataxia-3 (SCA3) and potential contribution to neurodegeneration. UBB+1 was found to accumulate in the cytoplasm and NII in the affected brain regions of HD and SCA3 demonstrating in vivo proteasome inhibition in these disorders. In a cellular model of HD we observed that UBB+1 results in inhibition of the proteasome which causes increased aggregate formation. In addition, a synergistic increase in polyglutamine induced cell death was found upon expression of UBB+1. These findings implicate UBB+1 as an aggravating factor in polyglutamine induced neurodegeneration and stresses the importance of the UPS for degradation of aberrant polyglutamine and UBB+1 proteins. UBB+1 transgenic mice show a mild inhibition of the proteasome. In Chapter 4 we tested the influence of this UPS inhibition by UBB+1 on Htt aggregation in vivo. Expression of expanded polyglutamine protein in the striatum of the UBB+1 transgenic mice showed a strong increase in NII formation compared to wildtype littermates. These results demonstrate in vivo that minor differences in UPS capacity can have major detrimental effects on the neuropathology of HD. The ubiquitin conjugating enzyme E2-25K has been shown to interact directly with Htt independent of polyglutamine repeat length. In Chapter 5, we investigated the localization of E2-25K in HD as well as the contribution to neurodegeneration. E2-25K co-localizes with a subset of NII in HD brain as well as with aggregates in apoptotic cells in vitro. Dominant negative E2-25K __ lacking the catalytic tail domain __ as well as an antisense construct decreased aggregate formation of expanded Htt. Additionally, mutant and antisense E2-25K reduced polyglutamine-induced cell death. These findings show that ubiquitination of E2-25K-targets contributes to aggregate formation as well as neuronal cell death in HD. Finally, in Chapter 6 the different findings of this thesis are discussed as well as further research and perspectives. In conclusion, the findings of this thesis illustrate the importance of the UPS for the cellular clearance of toxic proteins involved in neurodegeneration. The precise mechanism of specific neuronal dysfunction in HD is still unclear but is triggered by the gain of function of the polyglutamine repeat. Impairment of the UPS results in the further accumulation of aberrant proteins and subsequent neuronal dysfunction. Since HD is caused by protein expression from a mutant allele, more efficient degradation could protect the neurons from harmful polyglutamine proteins. Show less
