Molecular networks and gene identification studies in frontotemporal dementia - Cemile Koçoğlu (23/02/2022)
- 23 February 2022, 4pm - 6pm
- Online: https://eu.bbcollab.com/guest/6e49ada3d64c4a2b986750a92513d53e
- Promoters: Julie van der Zee, Christine Van Broeckhoven
Genetic and functional evidence linking noncoding variants with neurodegenerative brain diseases - Alexandros Frydas (25/01/2022)
- 25 January 2022, 4pm - 6pm
- Online defence
- Promoters: Christine Van Broeckhoven, Julie van der Zee
Genetic variants across the genome can lead to phenotypic variability for different traits and are a major cause for disease. A large portion of these alterations reside in the part of the genome that does not encode proteins, termed “noncoding”. After the first successful effort to sequence the entire human genome, we now reckon the largest portion of the noncoding genome to encompass regulatory sequences with distinct functions in diverse cellular responses. Variation within these elements has been linked with dysregulation of molecular processes and implicated with complex disorders, including neurodegenerative brain diseases (NBDs). These are caused by progressive deterioration and neuronal cell death in diverse brain regions, resulting in varied clinical symptoms, ranging from loss of cognitive function to movement impairments. Genetic heritability of NBDs is only partially explained by known mutations in the coding genome. Hundreds of loci of the noncoding genome have been associated with increased risk for NBDs by genome wide association studies (GWAS), although functional characterization is often lacking.
The aim of my PhD is to investigate the impact of noncoding genetic variants in NBDs and how they could account for the missing genetic heritability or the different aspects of heterogeneity characterizing each disorder. First, I investigated the impact of noncoding variants in the granulin gene (GRN) in patients with frontotemporal dementia (FTD). FTD is the second most common form of dementia following Alzheimer’s disease (AD), for people below 65 years old. The described mechanism for GRN-associated FTD is haploinsufficiency, with heterozygous loss-of-function mutations leading to partial loss of progranulin (PGRN), following degradation of the mutant transcript. I functionally characterized noncoding elements, termed upstream open reading frames (uORFs), which are harbored in the main and an alternative 5’ untranslated region (UTRs) of the GRN gene. I initially estimated the presence of transcripts containing the alternative long 5’ UTR in disease-affected brain regions after analyzing differential splicing events in brain RNA-seq datasets. Reporter assays revealed significant repressive effects for both uORFs of the main 5’ UTR as well as for a short uORF of the longer alternative 5’ UTR. Genetic screening of a well-characterized Flanders-Belgian FTD patient cohort, identified one variant residing in the uORFs of the main 5’ UTR which significantly mitigated their repressive effect. These findings propose novel regulatory elements that could mediate PGRN levels and warrant further investigation into their function in disease-relevant cell types. Next, I studied the genetic variability in microRNA (miRNAs) in AD and FTD patients. Whole exome sequencing (WES) data was available for a subset of FTD patients from the Flanders-Belgian cohort are used to search for variants in brain-expressed miRNAs. Four rare seed variants were identified in four miRNAs. Significant association of rare variants in the MIR885 gene with FTD were found. Also, the association was significant after expanding the screening for MIR885 to the Flanders-Belgian AD cohort. The variants are predicted to dysregulate expression of the miRNA precursor or alter the target pools of the mature miRNA sequences, suggesting different routes of functional investigation to delineate a connection between this miRNA and neurodegenerative processes.
These findings are placed in a broader context regarding functional investigation of noncoding variation in Chapter General Discussion. I discuss different state-of-the-art approaches to unravel functional properties of noncoding regions and how they could be used as a go-to-way to dissect the hundreds of susceptibility loci emerging by NBD GWAS. Further, I propose ways to improve the discovery curve of such studies, which seem to be reaching a plateau. Taken together, results obtained in this PhD expand knowledge about understudied parts of the genome involved in disease pathogenesis and propose novel targets that might be valuable for diagnosis and therapeutic strategies.
New perspectives on genetically-associated molecular and clinical processes in Alzheimer’s disease - Fahri Küçükali (24/01/2022)
- 24 January 2022, 4pm - 6pm
- Online defence
- Promoter: Kristel Sleegers
Increased life expectancy is in line with the increasing prevalence of dementia, leading to an increasing substantial burden on public health and society. The most common cause of dementia is Alzheimer’s disease (AD), a neurodegenerative disease whose clinical manifestations are progressive memory loss and cognitive impairment. As the in vivo biomarker studies indicate that AD pathophysiology in brain typically start decades earlier than the onset of these symptoms, AD is defined as a continuum. Moreover, AD is a complex disease with a high heritability. Over the past three decades genetic studies have revealed the genetic associations and significantly improved our understanding of AD pathophysiology. However, there are still barriers to progress in these studies, such as the difficulty of identifying novel genetic variants associated with clinical and molecular processes in Alzheimer’s continuum and the complexity of functionally interpreting these genetic signals to reveal high likely prioritized genes and mechanisms of actions. By using a distinct set of modern molecular genetic techniques, we aimed to address these barriers in three parts. First, we sequenced three candidate genes within ZCWPW1 complex risk locus in over 2,000 Belgian AD patients and controls. We identified that the ultra-rare predicted loss-of-function (pLoF) mutations in ZCWPW1 were enriched in patients. Second, we sequenced the exome of 450 EMIF-AD subjects and accessed the whole-genome data of 808 ADNI subjects, both paired with AD-relevant traits such as the clinical, cognitive, cerebrospinal fluid, and MRI phenotypes. Using these two cohorts, we conducted the first comprehensive gene-based exome-wide association studies of these traits. One highlighted association was between pLoF mutations in RBKS and cognitive impairment, where substantial number AD patients could benefit from a potential therapeutic for slower cognitive decline. Third, in one of the largest genome-wide association study (GWAS) of 789,000 subjects, we identified an unprecedented number of 42 new genetic risk loci for AD. We designed a systematic gene prioritization approach that included molecular QTL-GWAS integration analyses of AD risk. This revealed highly likely candidate risk genes in 75% of these novel loci. These new genes include SHARPIN and RBCK1, members of LUBAC complex, that directly participate in regulation of TNF-α signaling pathway, together with several other prioritized genes. Taken together, these novel perspectives on the genetically-associated molecular and clinical processes can be insightful for better understanding the mechanisms underlying the AD pathophysiology, and consequently may lead to development of better preventive and therapeutic opportunities for AD.
The role of sclerostin in vascular calcification and the calcification paradox - Annelies De Maré (18/01/2022)
Annelies De Maré
- 18 January 2022, 4pm - 6pm
- Online defence
- Promoters: Patrick D'Haese, Anja Verhulst
Ectopic vascular calcification involves the deposition of calcium-phosphate crystals in the medial layer of the arterial wall. Although vascular calcification is part of the normal aging process, it is accelerated in patients with chronic kidney disease (CKD), diabetes and other metabolic disorders. In these patient populations, vascular calcification importantly contributes to increased morbidity and mortality. An imbalance in osteochondrogenic signaling and anticalcific events, allows the development of arterial calcification. Since vascular media calcification highly resembles bone development and metabolism, the (canonical) Wnt/b-catenin signaling, known to be a crucial regulator of bone turnover, might be involved. Furthermore, ectopic vascular calcification is also frequently accompanied by a reduced bone mineral density and disturbed bone turnover, a phenomenon that is referred to as the calcification paradox. In this thesis, the possible involvement of the Wnt/b-catenin signaling was investigated by elucidating the role of sclerostin, a negative regulator of this pathway, during vascular calcification.
In CKD patients, both serum and urinary sclerostin concentrations are elevated, which is indicative of increased sclerostin production. In this regard, we found that skeletal sclerostin expression was only moderately correlated with circulating sclerostin levels. Therefore it is possible that extra-osseous-produced sclerostin may contribute to the total serum sclerostin concentration and that sclerostin produced in the vasculature, can spill over into the circulation. This is in line with our observations in warfarin-exposed rats. In these rats, we found increased serum levels of sclerostin along with the local production of sclerostin in the calcified vessels. Since osteocytic sclerostin expression remained unchanged, these increased levels are likely to originate from local production in the calcified vessels. Remarkably, despite normal renal function, a mild decrease in bone and mineralized area was observed in this model. Based on these observations, vascular-derived sclerostin might be involved in the calcification paradox by exerting endocrine effects on the bone compartment.
To help elucidate the role of sclerostin during vascular calcification, we investigated calcification development in mice with genetic depletion of sclerostin (Sost-/- mice) and in mice treated with an anti-sclerostin antibody. In both models, absence of (functional) sclerostin led to a significantly higher vascular calcium content, indicating a protective role for sclerostin during vascular calcification development.
To conclude, this project not only demonstrated that sclerostin is being produced and secreted by transdifferentiated VSMCs, likely in an attempt to slow down vascular calcification but also that sclerostin seems to function as a messenger in the calcification paradox.
From heme protein to biosensor: investigation of Caenorhabditis elegans Globin-3 and heme protein immobilization on porous materials - Zainab Hafideddine (17/01/2022)
- 17 January 2022, 4pm - 6pm
- Online defence
- Promoters: Luc Moens, Sabine Van Doorslaer
Biosensors are nowadays ubiquitous in medical research as well as in environmental monitoring, food control, drug discovery and military applications. Protein-based electrochemical biosensors are based on the detection of an electrical signal produced after a biochemical reaction between the target analyte and the adsorbed proteins on the biosensor. The coupling of biosensors to heme proteins allows a highly sensitive and selective detection of a range of small molecules such as H2O2, H2S, NO and phenolic compounds. These small molecules are typical byproducts of many industrial processes and are extremely toxic at high concentration. The heme iron of the heme protein changes its oxidation state upon binding with the target molecule which can be detected by the electrode of the biosensor. However, proteins are not stable outside their physiological environment, and therefore immobilization of the heme proteins on the electrode surface is necessary. Ordered mesoporous materials, such as mesoporous titania (TiO2) and silica (SiO2), have been widely considered for the immobilization of proteins because of their advantageous properties that can be fine-tuned for interaction with specific proteins to enhance protein activity.
The characterization of new heme proteins is essential and is therefore the focus of the first part of the thesis. The 34 heme proteins of the nematode Caenorhabditis elegans are found to possess widely divergent functions, several participating in redox and signaling reactions which are interesting for future biosensing applications. Since knockout of glb-3 resulted in a severe reduction in fertility and motility of the nematode, a biophysical characterization of GLB-3 is performed for the first time in this work.
In the second part, the correlation between the structural changes and the activity of the heme proteins upon adsorption is unraveled by means of several techniques. Horse heart myoglobin, human neuroglobin and horseradish peroxidase are well-characterized heme proteins and were immobilized in different mesoporous materials. Electron paramagnetic resonance spectroscopy (EPR) was the main technique used in this part to study the active site, i.e. the heme group, of the heme proteins before and after immobilization. Moreover, a first immobilization test was performed on GLB-3 in TiO2 to illustrate the importance of optimizing each adsorption experiment when working with a different protein. Additionally, to obtain more insight in the structural changes of neuroglobin after immobilization, site-directed spin labelling in combination with EPR was performed. Finally, the generation of reactive oxygen species on the titania surface was analyzed via EPR.