Farmaceutische, Biomedische en Dier­geneeskundige Wetenschappen

Doctoraten 2022

Ontrafelen van de effecten van verkeerd gevouwen eiwitten bij de initiatie en de progressie van de ziekte van Alzheimer - Inès Ben-Nejma (5/9/2022)

Inès Ben-Nejma

Abstract (Engels)

Alzheimer’s disease (AD) is an incurable neurodegenerative disorder, eventually leading to dementia and affecting the elderly. Due to ageing of the population, the prevalence of AD is expected to rise, with a global cost of caretaking of AD patients estimated to hundreds of billion dollars. As the aetiology of AD pathology remains elusive, the development of appropriate curative therapies requires more in-depth knowledge on the early stages of AD pathology, which is the focus of this PhD. We studied the impacts of misfolded proteins encountered in AD (Aβ, tau) in the initiation and progression of AD pathology, using highly translational in vivo MRI techniques, in combination with the Tet-Off APP and hTau.P301L mouse models, simulating Aβ and tau pathology, respectively, in a context resembling sporadic AD.

On the one hand, the Tet-Off APP mice showed early soluble Aβ-associated hyperconnectivity of brain networks prior to Aβ plaque formation, followed by a hypoconnectivity after the onset of Aβ plaque deposition which was associated with neuroinflammation surrounding the plaques. Furthermore, the effects of accumulation on the brain-wide glymphatic clearance were evaluated at a more advanced stage of AD and showed an impaired glymphatic brain fluid transport. More specifically, they demonstrated a reduced glymphatic inflow in the brain parenchyma affecting the dynamics of the glymphatic transport and thus presumably the effective clearance of brain waste, including Aβ. On the other hand, in the hTau.P301L mice, tau fibrils did not affect the functional connectivity of brain networks, despite progressive accumulation throughout the brain up to 15 weeks after seeding.

In conclusion, the results presented in this thesis indicate that degradation of the brain’s functional integrity is associated with soluble Aβ toxicity, even in a context of sporadic AD. Moreover, Aβ toxicity seems to also impact the glymphatic brain fluid circulation directly and indirectly in AD pathology, with a brain-wide spread of neuroinflammatory responses. This demonstrates as well the importance of using neuroimaging techniques to shed light on the underlying mechanisms of the early events caused by the synergy between Aβ and tau, emphasizing the clinical relevance of preclinical studies using animal models, especially for the identification of new targets, as potential therapeutic strategies for AD.

Op weg naar een beter inzicht in HINT1 neuropathie: genetische en functionele profilering - Silvia Amor Barris (6/7/2022)

Silvia Amor Barris

  • 6 juli 2022, 16u - 18u
  • Promotiezaal (CDE)
  • Online: https://eu.bbcollab.com/guest/fef44834e0c14bff9f7a5c69bfcc114c 
  • Promotor: Albena Jordanova

Abstract (Engels)

Loss-of-function mutations in the histidine triad nucleotide-binding protein 1 (HINT1) cause autosomal recessive axonal neuropathy associated with neuromyotonia (NMAN). Patients with NMAN display a progressive motor-greater-than-sensory polyneuropathy starting in the first decade of life and leading to weakness of the distal limb muscles and loss of ambulation. The clinical hallmark of the disease is neuromyotonia caused by peripheral nerve hyperexcitability that manifests as a delayed muscle relaxation after a voluntarily contraction. To date, more than 24 pathogenic variants are described to cause loss of functional HINT1 by 1) inducing protein instability followed by proteasomal degradation, 2) creating an early stop codon leading to putative non-sense mediated mRNA decay, or 3) targeting key catalytic residues rendering a non-active protein.

HINT1 is a ubiquitously expressed phosphoramidase and SUMOylase. In vitro, it can hydrolyze many AMP-substrates, yet the endogenous substrates and the biological relevance of the enzymatic activities remain unknown. In vivo, HINT1 has been linked to many different cellular functions including transcriptional regulation, modulation of G-protein couple receptor signaling and calcium signaling. Despite the plethora of known functions, its role in the maintenance of the peripheral nerve is utterly undiscovered.

We dissected the genetic and molecular aspects of HINT1 neuropathy and described protein structure - molecular (dys)function correlations. We identified two novel causal variants expanding the genetic, phenotypic, and geographic landscape of NMAN. Furthermore, we established an experimental pipeline to systematically model all reported missense mutations in HINT1, providing an important tool for assessing the pathogenicity of newly identified variants of unknow significance. The functional characterization of NMAN- associated genetic defects allowed us to define three mutational clusters in the protein structure, paving the way for patient stratification strategies in the development of new therapies. Finally, we used transcriptome profiling to identify cellular processes affected by HINT1 deficiency and suggest actin cytoskeleton and calcium dynamics to play a prominent role in the disease mechanisms. Taken together, our results contribute to a better understanding of HINT1 function and its role in inherited peripheral neuropathies.

Moleculaire epidemiologie en populatiestructuur van Mycobacterium tuberculosis-complex stammen bij tuberculosepatiënten: implicaties voor diagnostische benaderingen - Mebrat Ejo Kitata (16/06/2022)

Mebrat Ejo Kitata

Abstract (Engels)

Tuberculosis (TB) is the 2nd most deadly infectious disease, and its implication for health and economy is increasingly recognized in different countries. In this PhD study, we characterized Mycobacterium tuberculosis complex (MTBc) isolates collected from TB patients in Ethiopia and Niger, two countries in East and West Africa that carry a high burden of TB. In addition, we tested the efficiency of current diagnostic practices for differentiation of MTBc lineages (Ls) and exclusion of drug-resistant TB. In Ethiopia, five MTBc lineages (L1–L4 and L7) were identified from newly recruited pulmonary and lymph node TB patients, with predominance of East-African-Indian (L3) and Euro-American (L4) strains, but a lower than expected prevalence of Ethiopia L7 isolates. The genotypes were similarly distributed between pulmonary and lymph node TB, and all lineages were equally isolated on culture medium and recognized as MTBc by the rapid MPT64 assay. Additionally, analysis of rifampicin-/multidrug-resistant (RR/MDR) TB isolates in Northwest Ethiopia showed four MTBc lineages (L1, L3, L4, and L7), with an expected high rate of the most common rpoB_S450L and katG_S315T mutations causing MDR, but a low proportion of gyrA and rrs mutations causing resistance to second-line drugs. Characterization of RR/MDR-TB isolates from Niger revealed the six major MTBc lineages, L1-L6, with a predominance of the L4 “Ghana” and “Cameroon” families. We noticed a significantly faster conversion to smear-negative microscopy results during treatment and a more likely favorable outcome for patients infected by “Cameroon” isolates (log-rank p < 0.01). Other risk factors such as gender, age, HIV status, body mass index, and treatment regimen were not associated with sputum smear- and culture- conversion. As a supplemental study, we incorporated retrospective rapid molecular testing (MTBDRsl)  results from the STREAM stage 1 central database to assess its negative predictive value (NPV) directly from sputum samples compared to phenotypic drug-susceptibility testing. We found 9 (2.3%) fluoroquinolones (FQ)- and 5 (1.3%) second-line injectables (SLI)- discordant phenotypic/genotypic drug-susceptibility testing results. The NPV of MTBDRsl for exclusion of FQ resistance was 99.2% and for SLI resistance 100%. In conclusion, our studies provided great insights into the population structure of the MTBc in Ethiopia and Niger, demonstrating that treatment response varied by lineage. Finally, MTBDRsl is suitable for excluding resistance to second-line agents from clinical samples at the start of RR/MDR-TB treatment, including testing of paucibacillary specimens.

Antivirale immuunrespons-dynamiek tijdens varicella-zoster-virus infectie in een humaan iPSC-afgeleid neuronaal model en bij herpes zoster patiënten - Marlies Boeren (8/6/2022)

Marlies Boeren

Abstract (Engels)

Varicella-zoster virus (VZV) naturally infects more than 95% of the population which results in varicella (i.e., chickenpox). Upon infection, VZV particles may access sensory nerve endings in the skin and can be transported to neural ganglia, via retrograde axonal transport, where VZV latency is established. In more than one out of four people, VZV will eventually reactivate from this latent state causing herpes zoster (HZ, i.e. shingles). In addition, a substantial part of HZ patients suffers from long-lasting pain after the rash has disappeared, adding to the burden of disease. Although effective vaccines against HZ are now available, vaccination coverage worldwide is relatively low. Hence, VZV and HZ remain a major burden for the foreseeable future.

The humanotropic and neurotropic nature of VZV is a major hurdle in the field which hampers the advancement of our understanding of VZV infection dynamics and pathogenesis. Due to VZV’s strictly humanotropic character, no small animal model can fully recapitulate VZV disease. To overcome this, we aimed to develop a human iPSC-derived neuronal model in a chambered system that separates cell bodies and axon termini, which allows mimicking the natural route of VZV infection via axon termini. We showed that, following VZV infection, activation of interferon-stimulated genes (ISGs) depends on exogenous interferon-α (IFNα). Indeed, VZV infection of hiPSC-neurons via their axon termini, resulted in VZV spread throughout the neuronal cultures, without the production of IFNα or ISGs which have direct antiviral effector functions. In contrast, exogenous treatment of hiPSC-neurons with IFNα resulted in a reduction of VZV spread and in the upregulation of ISGs. Taken together, we suggest that whilst hiPSC-neurons are good IFNα-responders, they seem poor IFNα-producers thereby being unable to limit VZV spread in hiPSC-neuronal cultures. Importantly, this may imply that other cell types within the nervous system are essential as IFNα-producers. Hence, we believe that future studies investigating innate immune responses to VZV infection of neuronal cells should be carried out by including additional immune cell types in the cultures.

In the second part of this thesis, we investigated the adaptive immune response dynamics during VZV reactivation in HZ patients. We found that VZV-specific antibody titers were still significantly higher one year after the HZ episode as compared to controls, raising the possibility that continuous subclinical VZV reactivation may contribute to high VZV IgG titers in HZ patients.

In the last part of this thesis, transcriptomic analyses on whole blood from HZ patients and controls were carried out. Our data showed activation of several host immune pathways during VZV reactivation, especially related to the type I IFN response, but also related to adaptive immune responses. In addition, we found that upregulation of complement component 4 binding protein alpha (C4BPA), a major inhibitor of the complement system, may be a potential risk factor for the development of HZ but this needs further investigation. Indeed, a genome-wide association study could reveal if genetic variants are implicated in HZ disease.

Het beoordelen van antilichaam responsen tegen het dengue virus in het kader van de opkomende arbovirussen – van basis immunologie naar nieuwe toepassingen voor diagnostiek - Francesca Falconi (2/6/2022)

Francesca Falconi

  • 2 juni 2022, 16u - 18u
  • Aula Janssens (ITG)
  • Promotoren: Kevin Ariën, Peter Delputte

Abstract (Engels)

Dengue virus (DENV) is the causative agent of dengue fever (DF), the most prevalent arthropod-borne viral disease worldwide. DF presents with non-specific symptoms similar to other febrile diseases that overlap geographically. Therefore, diagnosis cannot be made on clinical and epidemiological data alone and thus require laboratory techniques for case confirmation.

Antibody-based assays are simple, affordable, applicable at the point of care and offer a wide diagnostic window. However, the high genetic similarity of DENV serotypes and also with other flaviviruses such as Zika, results in cross-reactive (CR) antibodies complicating serological diagnostics.

​Consequently, the search for and selection of appropriate biomaterials that enable capturing of highly specific anti-DENV antibodies is crucial to improve dengue diagnosis. The main objective of this PhD thesis was to (1) delineate the dynamics of the antibody responses against DENV at epitope level, (2) to identify regions able to capture DENV-specific Abs, and (3) investigate the value of these biomaterials for diagnostic purposes.

​The characterization of the temporal evolution of the IgG and IgM response following dengue infection was done using a high-density peptide microarray spanning the entire proteomes and diversity of DENV1-4, Zika (ZIKV) and yellow fever virus (YFV). Sera from a cohort of dengue infected individuals from Peru, as representatives for secondary infections, and overseas travelers, as representatives for primary infections, were used. We found that reactive linear epitopes were located across the proteome both in structural proteins and in non-structural proteins and were targeted by either IgM and/or IgG antibodies. Subsequent analysis of the antibody response against the peptide libraries from DENV, ZIKV and YFV and the further alignment of the highly immunoreactive regions onto the DENV proteome allowed the identification of 15 and 12 DENV-type-specific epitopes and 13 and 12 flavivirus-CR epitopes, targeted by IgM and IgG antibodies, respectively.

The diagnostic potential of 20 peptides was further assessed using a bead-based multiplex peptide immunoassay on a large panel of carefully selected human serum samples. Individual peptides offered satisfactory diagnostic performance, but the combination of multiple peptides and using a machine learning algorithm substantially improved the sensitivity and specificity of the assay. In summary, this work has mapped in great detail the immunodominant linear epitopes across the DENV polyprotein and provided proof-of-concept that this approach offers clear potential for the development of highly accurate next-generation dengue serological tests.

Richting nieuwe behandelingsopties voor trypanosomiasis bij mens en dier: van mechanistische inzichten in antiparasitaire geneesmiddelen tot de immunologische aspecten van vroege infectie - Dorien Mabille (23/05/2022)

Dorien Mabille

  • 23 mei 2022, 16u - 18u
  • Auditorium O1 (CDE)
  • Promotoren: Guy Caljon, Louis Maes

Abstract (Engels)

Human African trypanosomiasis is a neglected tropical disease caused by the tsetse fly-transmitted unicellular protozoa Trypanosoma brucei gambiense and T. b. rhodesiense. Inoculation of the parasite into the skin results in infection of the host. The first stage of the disease is characterized by non-specific flu-like symptoms due to parasite multiplication in the lymphatics and the bloodstream. Parasite invasion of the central nervous system causes severe neurological symptoms. A wide range of other trypanosome species are responsible for disease in wild and domestic animals causing a widespread socioeconomic problem. The therapies that are currently available are limited in number and face disadvantages of toxicity and drug resistance. Despite decades of research, effective vaccines to protect humans and animals from infection are still lacking. In addition, asymptomatic individuals who remain undiagnosed and hence untreated can still contribute to disease transmission. The development of novel treatment strategies will therefore be crucial to achieve disease elimination.

 The first aim of this thesis was to evaluate and characterize novel anti-trypanosomal compounds. The medicinal chemistry group of the UGent developed a series of nucleoside analogues based on structural elements of the natural nucleoside antibiotics tubercidin and cordycepin, which were previously shown to have high antiparasitic potency but accompanied by extreme host toxicity. We identified 2 promising nucleoside analogues with high activity against Stage-II disease. Mechanistic evaluation using a whole-genome RNAi library identified TbAT1, ADKIN and 4EIP as involved in their mode-of-action. Evaluation of the adenosine analogues for the treatment of animal trypanosomiasis was able to identify compounds showing a broad activity profile across multiple parasite species/strains.

The second aim was to characterize the early immunological events that occur upon the bite of an infected tsetse fly to gather the scientific knowledge necessary to develop novel intervention strategies based on the host-parasite interplay. Neutrophils were previously identified as being rapidly recruited to the site of infection, exhibiting a surprising pro-parasitic response. Detailed in vitro and in vivo analysis of the role of the neutrophil showed that neutrophils become activated upon parasitic stimulation but without a negative impact om parasite replication. In naturally infected mouse models, neutrophils play a role in parasite retention at the site of infection and impact on systemic organ colonization. Analysis of parasite burdens in various tissues identified the lungs as a site of strong parasite proliferation pointing to a potential role as parasitic tissue reservoir. Parasite presence resulted in a large influx of immune cells and change in the transcriptional profile, however, without detrimental effect on lung function. Depletion of specific immune cell subsets including eosinophils, NK cells and IgM+ B cells may underly the increased susceptibility of patients to opportunistic infections.

Virus-gastheer interacties in de replicatiecyclus van chikungunya virus - Lien De Caluwé (1/4/2022)

Lien De Caluwé

  • 1 april 2022, 16u - 18u
  • Aula Janssens (Instituut voor Tropische Geneeskunde Antwerpen)
  • Promotoren: Kevin Ariën, Xaveer Van Ostade

Abstract (Engels)

The prevalence of arthropod-borne viruses (arboviruses) dramatically increased in the last decade in diseased-endemic countries and the viruses have and are arriving in previously unaffected countries. The dispersal of these arboviruses is progressively being facilitated by climate change and increased transcontinental movements. Arboviruses are capable of efficiently replicating in both their vertebrate host and invertebrate vectors. Chikungunya virus (CHIKV), member of the Alphavirus genus, is such a re-emerging arbovirus which has increasingly affected public health on all inhabited continents.

Generally, viruses are dependent on their host cell to complete all steps in the viral replication cycle. In order to do so, many virus-host protein-protein interactions are necessary. To date the virus-host and virus-vector interactome for CHIKV is poorly understood. So far, multiple attachment factors used for CHIKV entry have been identified but the whole picture remains incomplete. We applied an affinity-based purification approach coupled to mass spectrometry analysis (AP-MS) to identify protein-protein interactions that facilitate the entry of CHIKV in both human and mosquito cells. We were able to identify and validate the involvement of the CD147 protein complex in the entry of CHIKV in human but not mosquito cells. Our AP-MS strategy further identified seven attachment candidates for CHIKV in mosquito cells whereof two proteins (ATPB and RPSA) were previously described as attachment factors for CHIKV and/or related alphaviruses in mosquito cells. Our results add to current knowledge on alphaviral entry and indicate that this process is a versatile multi-route step where various attachment factors and endocytic pathways are used.

CHIKV has a broad cellular tropism, however one marked exception is the resistance of the A549 cell line to CHIKV infection. This resistance could indicate the presence of a restrictive mechanism for CHIKV in A549 cells and understanding the viral and host proteins involved would be of great interest. We used the high genomic plasticity of CHIKV to adapt the virus to efficient growth in this restrictive cell line. We identified four adaptive mutations after serially passaging CHIKV, two in the structural and two in the non-structural proteins and assessed their contribution to the increased viral replication in A549 cells. While the adaptive mutations in the non-structural proteins also stimulate viral replication in Vero cells, the mutations in the structural proteins negatively impact viral replication in Vero cells. Further identification of this natural block to CHIKV in A549 cells is subject of future research. In conclusion, the work presented in this thesis adds to a better general understanding of the CHIKV replication cycle but many unknowns remain.

De bijdrage van zeldzame varianten in ABCA7 tot de genetische etiologie van de ziekte van Alzheimer - Liene Bossaerts (22/03/2022)

Liene Bossaerts

  • 22 maart 2022, 16u - 18u
  • Auditorium O1 (CDE) 
  • Online: https://eu.bbcollab.com/guest/4b377e04d966415cbc3ceb2f65887080
  • Promotoren: Christine Van Broeckhoven, Sebastiaan Engelborghs

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Abstract (Engels)

Despite decades of research, no treatment is available to halt or slow down Alzheimer’s disease (AD), the leading cause of dementia. Unravelling the molecular mechanisms underlying the pathogenesis of AD is imperative to find novel therapeutic targets. Genome-wide association studies in large AD and control cohorts identified ABCA7 as a risk gene for AD. ABCA7, or the ATP-binding cassette subfamily A member 7 gene, encodes a transmembrane protein involved in lipid metabolism and phagocytosis, and was found to regulate the generation and clearance of amyloid β, an important neuropathological hallmark of AD. Post-GWAS studies identified an enrichment of rare heterozygous premature termination codon (PTC) mutations in AD patients versus healthy control individuals. In fact, our research group was one of the first to report this association in a Belgian cohort of 772 AD patients and 757 control individuals.

The general aim of this PhD work was to explore the genetic contribution of rare ABCA7 variants to AD. To get more insights in the mutational spectrum and prevalence of PTC mutations, as well as to better understand the variability in onset age of PTC carriers, we first expanded the ABCA7 screening to a larger Belgian patient (n = 1376) and control (n = 976) cohort. This screening revealed 67 PTC mutations in the patient cohort and 18 in the control group.

We also investigated the frequency and pathogenicity of missense, indel and splice variants in the Belgian AD and control cohort. We explored the effect of missense mutations, selected from the Belgian patient cohort, on the subcellular localization of ABCA7 in HeLa cells and show for the first time that these mutations can induce mislocalization and impaired trafficking to the plasma membrane, resulting in a loss-of-functional ABCA7. In addition, we describe co-segregation of a mislocalizing missense mutation with AD in a pedigree showing an autosomal dominant inheritance pattern.

Neuropathological examination in ABCA7 PTC and missense mutation carriers revealed prominent levels of cerebral amyloid angiopathy (CAA) additional to AD pathological hallmarks. Therefore, we investigated the contribution of rare ABCA7 variants to CAA in a cohort of 83 Belgian CAA patients. This pilot study reveals a significant role for ABCA7 in the pathogenesis of CAA.

In conclusion, this work contributes to the establishment of ABCA7 as a strong genetic risk factor for AD and CAA. In addition, we highlight new pathogenic mechanisms through which rare variants in ABCA7 contribute to AD and CAA.


Cellulaire profilering onthult moleculaire kenmerken en kwetsbaarheden van neuroblastoom - Sofia Zanotti (16/03/2022)

Sofia Zanotti

  • 16 maart 2022, 13u - 15u
  • Online: https://eu.bbcollab.com/guest/46c488635d7645dcb3276df7da67aba5
  • Promotoren: Winnok De Vos, Frank Speleman

Abstract (Engels)

Neuroblastoma (NB) is a pediatric tumor of the developing sympathetic nervous system. Roughly half of all high risk NBs show MYCN oncogene amplification and elevated levels of replicative stress (RS). To avoid toxic DNA damage levels, NB cells strongly activate DNA damage repair pathways. We reasoned that this feature may represent a synthetic lethal target offering an exploitable therapeutic vulnerability. To interrogate the levels of RS, we established a microscopy-based assay that provides a comprehensive readout on RS- associated DNA damage at the single cell level. It is based on immunofluorescence labeling for markers of single and double stranded breaks (RPA32, resp. yH2AX) and cell cycle status (DNA-binding dye and EdU labeling). Exhaustive extraction and integration of cellular features (e.g., by including morphological and texture descriptors) from the obtained images allowed moving from classical, one-dimensional endpoints (such as signal intensity) to multiparametric signatures that more accurately describe the impact of pharmacological perturbations and reveal off-target effects. We benchmarked the assay using DNA damaging agents with known mechanism of action (RRM2 inhibitor triapine, hydroxyurea and etoposide) and subsequently evaluated inhibitors for CHK1 and WEE1 cell cycle checkpoint kinases, revealing a synergetic cytotoxic effect. After having established this valuable imaging pipeline, we turned our attention to interrogating the early mechanisms involved in NB development upon MYCN-activation. We explored the early changes in the transcriptional landscape of an inducible RPE1–MYCN human cell model. Following attenuation of the initially induced MYCN gene expression signature, we found an unexpected growth reduction that was accompanied by upregulation of pivotal cell cycle regulators such as p53 and CDKN1A and their respective protein products. In-depth analysis revealed transcriptional and phenotypic evidence of nucleolar stress, suggesting translational overload. Supported by bulk and single-cell transcriptomics, we propose that MYCN-induced nucleolar stress drives cells into a pre-senescent state that is not fully committed to, possibly due to an inadequate checkpoint response. With this work, we demonstrated that RS can indeed be pharmacologically exploited as a vulnerability of NB cells and gain insight into the complex interaction between MYCN ectopic expression and senescence-like cellular responses. In conclusion, this PhD provides a biological and technological basis for further unveiling critical dependencies that might serve as novel drug targets for MYCN driven malignancies.

Moleculaire netwerken en gen identificatie studies in frontotemporale dementie - Cemile Koçoğlu (23/02/2022)

Cemile Koçoğlu

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Abstract (Engels)

Frontotemporal dementia (FTD) is an early-onset dementia belonging to a group of disorders caused by progressive neurodegeneration in the frontal and temporal brain lobes. Genetics play a strong role in FTD, and to date, causal mutations were discovered in several genes explaining approximately 20% of all FTD patients. Characterization of the role of these genes revealed physical and functional interactions between several FTD-associated proteins. Therefore, we hypothesized that the interactors of established FTD proteins can provide insights into the missing heritability of FTD. We prioritized candidates from: i) overrepresented Gene Ontology biological processes in the protein interaction network of the known FTD genes (FTD-PIN), and ii) the endolysosomal pathway which was previously demonstrated to be pivotal in FTD and other dementias. We analyzed association in the prioritized genes with disease of rare (minor allele frequency < 1% in gnomAD Non-Finnish European exomes cohort) protein-altering variants identified in patient and control cohorts. We constructed an FTD-PIN with the physical interactors of 14 established FTD genes. Subsequently we performed functional enrichment analysis and identified enriched biological processes in FTD development linked to waste disposal, cell death, immune system, and response to stimulus. SKAT-O analysis in the genes belonging to these processes (n=440) suggested TNFAIP3 as the top gene (Praw = 0.7x10-3). Functional enrichment on the TNFAIP3-subnetwork from the FTD-PIN pointed to the involvement of immune signaling networks. In parallel, using a pathway-centric approach, we prioritized 50 genes involved in the endolysosomal pathway based on their tolerance to variation and expression levels in brain. In search of shared genetic factors across dementias, we analyzed these genes in a cohort comprising FTD, Alzheimer’s disease and Lewy body dementia patients and controls. Our analysis showed suggestive enrichment of missense variants in the AP2A2 gene (Praw = 0.048) in dementia patients. We confirmed this observation via burden testing against the gnomAD non-Finnish European exome controls (Fisher’s exact test, two-sided P = 4.54x10-4). AP2A2 is a member of AP2-complex in clathrin-mediated endocytosis. Characterization of the impact of these variants in the endocytosis machinery is needed to provide insights into the role of AP2A2 in neurodegeneration. In conclusion, our findings demonstrate the added value of integrating network and pathway analysis to increase the power of rare variant association analyses. Such approaches will provide a deeper resolution in the biological processes implicated in FTD and hold the promise to highlight pathways to target for diagnosis and therapeutic strategy development. 

Genetisch en functioneel bewijs linkt niet-coderende variaties met neurodegeneratieve hersenziekten - Alexandros Frydas (25/01/2022)

Alexandros Frydas

  • 25 januari 2022, 16u - 18u
  • Online verdediging
  • Promotoren: Christine Van Broeckhoven, Julie van der Zee

Abstract (Engels)

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.

Nieuwe Perspectieven op de Genetische Basis van Moleculaire en Klinische Processen in de Ziekte van Alzheimer - Fahri Küçükali (24/01/2022)

Fahri Küçükali

  • 24 januari 2022, 16u - 18u
  • Online verdediging
  • Promotor: Kristel Sleegers

Abstract (Engels)

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.

De rol van sclerostine in vasculaire calcificatie en de calcificatieparadox - Annelies De Maré (18/01/2022)

Annelies De Maré

  • 18 januari 2022, 16u - 18u
  • Online verdediging
  • Promotoren: Patrick D'Haese, Anja Verhulst

Abstract (Engels)

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.

Van heemeiwit tot biosensor: onderzoek van Caenorhabditis elegans Globine-3 en heemeiwitimmobilizatie in poreuze materialen - Zainab Hafideddine (17/01/2022)

Zainab Hafideddine

  • 17 januari 2022, 16u - 18u
  • Online verdediging
  • Promotoren: Luc Moens, Sabine Van Doorslaer

Abstract (Engels)

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.