Alzheimer’s disease (AD) is the most common cause of dementia and quickly becoming one of the most burdening diseases of the century. Effective treatments are still missing, partially because its... Show moreAlzheimer’s disease (AD) is the most common cause of dementia and quickly becoming one of the most burdening diseases of the century. Effective treatments are still missing, partially because its pathogenesis is still incompletely understood. This thesis explores the role of iron in AD, how it interacts with the immune system to influence disease pathogenesis and whether it could serve as potential biomarker. The first part of this thesis describes the importance of translational MRI, and how it can be used to increase our understanding of neurological diseases and help identify biomarkers. Subsequently, we used translational MRI to characterize the differences in iron accumulation in the brain between patients with AD and healthy elderly. The second part of this thesis investigated how the immune cells of the brain, microglia, interact with the accumulated iron. Using a combination of advanced multispectral immunofluorescence on brain tissue from AD patients and a human stem-cell derived microglia model, we studied the activation pattern of iron-accumulating microglia in human brains and emulated microglial iron accumulation in vitro. This enabled us to study the effect of iron on the gene expression patterns and function of the brain’s immune cells. Show less
Aims: Non-invasive measures of brain iron content would be of great benefit in neurodegeneration with brain iron accumulation (NBIA) to serve as a biomarker for disease progression and evaluation... Show moreAims: Non-invasive measures of brain iron content would be of great benefit in neurodegeneration with brain iron accumulation (NBIA) to serve as a biomarker for disease progression and evaluation of iron chelation therapy. Although magnetic resonance imaging (MRI) provides several quantitative measures of brain iron content, none of these have been validated for patients with a severely increased cerebral iron burden. We aimed to validate R 2 * as a quantitative measure of brain iron content in aceruloplasminemia, the most severely iron-loaded NBIA phenotype. Methods: Tissue samples from 50 gray-and white matter regions of a postmortem aceruloplasminemia brain and control subject were scanned at 1.5 T to obtain R 2 * , and biochemically analyzed with inductively coupled plasma mass spectrometry. For gray matter samples of the aceruloplasminemia brain, sample R 2 * values were compared with postmortem in situ MRI data that had been obtained from the same subject at 3 T - in situ R 2 * . Relationships between R 2 * and tissue iron concentration were determined by linear regression analyses. Results: Median iron concentrations throughout the whole aceruloplasminemia brain were 10 to 15 times higher than in the control subject, and R 2 * was linearly associated with iron concentration. For gray matter samples of the aceruloplasminemia subject with an iron concentration up to 1000 mg/kg, 91% of variation in R 2 * could be explained by iron, and in situ R 2 * at 3 T and sample R 2 * at 1.5 T were highly correlated. For white matter regions of the aceruloplasminemia brain, 85% of variation in R 2 * could be explained by iron. Conclusions: R 2 * is highly sensitive to variations in iron concentration in the severely iron-loaded brain, and might be used as a non-invasive measure of brain iron content in aceruloplasminemia and potentially other NBIA disorders. Show less
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
Kenkhuis, B.; Somarakis, A.; Haan, L. de; Dzyubachyk, O.; IJsselsteijn, M.E.; Miranda, N.F.C.C. de; ... ; Weerd, L. van der 2021
Brain iron accumulation has been found to accelerate disease progression in amyloid-beta(A beta) positive Alzheimer patients, though the mechanism is still unknown. Microglia have been identified... Show moreBrain iron accumulation has been found to accelerate disease progression in amyloid-beta(A beta) positive Alzheimer patients, though the mechanism is still unknown. Microglia have been identified as key players in the disease pathogenesis, and are highly reactive cells responding to aberrations such as increased iron levels. Therefore, using histological methods, multispectral immunofluorescence and an automated in-house developed microglia segmentation and analysis pipeline, we studied the occurrence of iron-accumulating microglia and the effect on its activation state in human Alzheimer brains. We identified a subset of microglia with increased expression of the iron storage protein ferritin light chain (FTL), together with increased Iba1 expression, decreased TMEM119 and P2RY12 expression. This activated microglia subset represented iron-accumulating microglia and appeared morphologically dystrophic. Multispectral immunofluorescence allowed for spatial analysis of FTL(+)Iba1(+)-microglia, which were found to be the predominant A beta-plaque infiltrating microglia. Finally, an increase of FTL(+)Iba1(+)-microglia was seen in patients with high A beta load and Tau load. These findings suggest iron to be taken up by microglia and to influence the functional phenotype of these cells, especially in conjunction with A beta. Show less
Aims: Aceruloplasminemia is an ultra-rare neurodegenerative disorder associated with massive brain iron deposits, of which the molecular composition is unknown. We aimed to quantitatively determine... Show moreAims: Aceruloplasminemia is an ultra-rare neurodegenerative disorder associated with massive brain iron deposits, of which the molecular composition is unknown. We aimed to quantitatively determine the molecular iron forms in the aceruloplasminemia brain, and to illustrate their influence on iron-sensitive MRI metrics.Methods: The inhomogeneous transverse relaxation rate (R2*) and magnetic susceptibility obtained from 7 T MRI were combined with Electron Paramagnetic Resonance (EPR) and Superconducting Quantum Interference Device (SQUID) magnetometry. The basal ganglia, thalamus, red nucleus, dentate nucleus, superior- and middle temporal gyrus and white matter of a post-mortem aceruloplasminemia brain were studied. MRI, EPR and SQUID results that had been previously obtained from the temporal cortex of healthy controls were included for comparison.Results: The brain iron pool in aceruloplasminemia detected in this study consisted of EPR-detectable Fe3+ ions, magnetic Fe3+ embedded in the core of ferritin and hemosiderin (ferrihydrite-iron), and magnetic Fe3+ embedded in oxidized magnetite/maghemite minerals (maghemite-iron). Ferrihydrite-iron represented above 90% of all iron and was the main driver of iron-sensitive MRI contrast. Although deep gray matter structures were three times richer in ferrihydrite-iron than the temporal cortex, ferrihydrite-iron was already six times more abundant in the temporal cortex of the patient with aceruloplasminemia compared to the healthy situation (162 & micro;g/g vs. 27 & micro;g/g), on average. The concentrations of Fe3+ ions and maghemite-iron in the temporal cortex in aceruloplasminemia were within the range of those in the control subjects.Conclusions: Iron-related neurodegeneration in aceruloplasminemia is primarily associated with an increase in ferrihydrite-iron, with ferrihydrite-iron being the major determinant of iron-sensitive MRI contrast. Show less
Bulk, M.; Hegeman-Kleinn, I.; Kenkhuis, B.; Suidgeest, E.; Roon-Mom, W. van; Lewerenz, J.; ... ; Weerd, L. van der 2020
Previous MRI studies consistently reported iron accumulation within the striatum of patients with Huntington's disease (HD). However, the pattern and origin of iron accumulation is poorly... Show morePrevious MRI studies consistently reported iron accumulation within the striatum of patients with Huntington's disease (HD). However, the pattern and origin of iron accumulation is poorly understood. This study aimed to characterize the histopathological correlates of iron-sensitive ex vivo MRI contrast change in HD brains. To this end, T2*-weighted 7T MRI was performed on postmortem tissue of the striatum of three control subjects and 10 HD patients followed by histological examination. In addition, formalin-fixed paraffin-embedded material of three control subjects and 14 HD patients was selected for only histology to identify the cellular localization of iron using stainings for iron, myelin, microglia and astrocytes. As expected HD striata showed prominent atrophy. Compared to controls, the striatum of HD patients was in general more hypointense on T2*-weighted highfield MRI and showed a more intense histopathological staining for iron. In addition, T2*-weighted MRI identified large focal hypointensities within the striatum of HD patients. Upon histological examination, these large focal hypointensities frequently colocalized with enlarged perivascular spaces and iron was found within the vessel wall and reactive astrocytes. In conclusion, we show that the striatum of HD patients has a distinctive phenotype on T2*-weighted MRI compared to control subjects. On ex vivo MRI, these contrast changes are heavily biased by enlarged perivascular spaces from which it is currently unknown whether this is a fixation artefact or a disease specific observation. Clinically, the observation of iron within reactive astrocytes is of importance for the interpretation and understanding of the potential underlying mechanisms of T2*-weighted MRI results in HD patients. Show less
Kenkhuis, B.; Jonkman, L.E.; Bulk, M.; Buijs, M.; Boon, B.D.C.; Bouwman, F.H.; ... ; Weerd, L. van der 2019
In the last decade, the redox interconversion between metal thiolate and disulfide compounds has been extensively investigated for copper, but not for other transition metal ions. In this... Show moreIn the last decade, the redox interconversion between metal thiolate and disulfide compounds has been extensively investigated for copper, but not for other transition metal ions. In this thesis, our investigations are described of the possibility to extend the metal thiolate/disulfide redox interconversion reactions to cobalt or iron compounds. A number of cobalt(II) disulfide and cobalt(III) thiolate compounds of different ligands and different anions are reported in this thesis. It was revealed that the anion of cobalt(II) salts, the structure of disulfide ligands, and the type of solvent influence the formation of either cobalt(II) disulfide or cobalt(III) thiolate compounds. However, a consistent trend cannot be provided to predict which of the species is generated. An important conclusion of this work is that the cobalt(II) disulfide to cobalt(III) thiolate interconversion reaction might be related to the ligand field strength of the ligand, and the binding strength and ligand field strength of the anions and solvent used. Apart from the cobalt compounds, two iron(II) disulfide compounds were reported in this thesis as well. However, so far we were not able to trigger the conversion of these compounds to their respective iron(III) thiolate compounds. Show less
In this dissertation, the synthesis and characterization of a series of iron complexes based on different ligand platforms are described. The complexes are subsequently studied for their... Show moreIn this dissertation, the synthesis and characterization of a series of iron complexes based on different ligand platforms are described. The complexes are subsequently studied for their activity in catalytic water oxidation with the help of a variety of electroanalytical techniques. The results show that the catalytic activity of structurally related iron complexes correlates strongly with the electronics of the iron centre. Another potentially very important aspect in the field of homogeneous electrocatalysis which has so far received only very little attention in published literature is the influence of the nature of the electrode material on the resulting electrochemistry. The results discussed in thesis show that interactions between the working electrode and the catalyst in solution can exhibit a strong influence on the resulting electrochemistry. Overall, the results of this work demonstrate that iron-based complexes can indeed be made to work as electrocatalysts for the water oxidation reaction. Furthermore, the results show that the electronic structure of the iron centre is a promising target for the design of new and improved catalysts. Finally, the results also highlight the importance of trying out different electrode materials as part of routine tests of new potential electrocatalysts. Show less
Cellulose makes up one of the most abundant renewable materials, present in all kinds of plant biomass (Pauly and Keegstra 2010). However, to be able to utilize the cellulose as feedstock, it needs... Show moreCellulose makes up one of the most abundant renewable materials, present in all kinds of plant biomass (Pauly and Keegstra 2010). However, to be able to utilize the cellulose as feedstock, it needs to be separated from lignin which cements the cellulose and hemi-cellulose fibers. Lignolytic peroxidases can be produced by Aspergillus niger, and its production was found to be improved by heme supplementation, suggesting a limiting effect of this co-factor during heterologous expression. The research described in this thesis explores fungal heme biosynthesis and its regulation by means of heme deficient mutant strains and overexpression strains of heme biosynthesis genes or corroborated iron metabolism with the final aim to increase the available intracellular heme for peroxidase production. Using heme deficient strains, we demonstrated that A. niger is capable of heme uptake and utilzation and that siroheme synthesis derives from the first half of the heme biosynthesis pathway as well. The tight regulation on heme biosynthesis on transcription and (post)translational level prohibits large changes in heme content, and indicated a bottleneck on the level of ferrochelatase and possible uroporphyrinogen III decarboxylase and coproporphyrinogen III oxidase and questions whether A. niger would be the most suitable host for heterologous peroxidase production. Show less
The research presented in this thesis provides several novel insights regarding the _-thalassemia intermedia phenotype. Earlier studies observed that patients with _-thalassemia intermedia... Show moreThe research presented in this thesis provides several novel insights regarding the _-thalassemia intermedia phenotype. Earlier studies observed that patients with _-thalassemia intermedia experience a clinical complications profile that is different from that in patients with _-thalassemia major; which was primarily attributed to their transfusion-independence. In this work, a variety of clinical morbidities were explored and their associations with the underlying disease pathophysiology and risk factors were examined. The morbidities evaluated throughout the studies involved several organs and organ systems including the vasculature (venous thrombosis, pulmonary artery hypertension, cerebrovascular disease, and leg ulcers), heart, liver, kidney, endocrine glands (diabetes mellitus, hypothyroidism, and hypogonadism), bone (osteoporosis), and the hematopoietic system (extramedullary hematopoietic tumors). Findi ngs confirm that _-thalassemia intermedia should no longer be regarded as a mild form of thalassemia as patients experience serious manifestations involving almost every organ system. Show less
