NEURODEGENERATION
Unraveling the cellular and systemic effects of serum amyloids along the gut-brain axis in Alzheimer’s disease
Tutor: Aurélie Hofkens - Promoters: Peter Verstraelen, Winnok De Vos
Recent evidence suggests that gut inflammation and the enteric microbiome can contribute to the progression of Alzheimer's Disease (AD), a devastating neurodegenerative disorder. Our own research has identified the bacterial amyloid curli, produced by the gut microbiome, as a potent trigger of immune responses in the enteric nervous system, with serum amyloid A3 (SAA3) as a key regulator in this response. Since serum amyloids (SAAs) can penetrate the blood-brain barrier and are elevated in AD patients, they may represent a critical link in pathogenic gut-brain immune signaling. Therefore, this project aims to elucidate the mode-of-action and systemic effects of SAAs following peripheral inflammation. Using in vivo mouse models (wild type C57Bl/6, AD-relevant APPnl-g-f/MAPT, SAA knock-outs) exposed to intraperitoneal curli injections or dextran sulphate sodium (DSS)-induced colitis, we map inflammation-driven changes in the brain’s immune cell composition through transcriptomics and advanced microscopy. In parallel, we explore amyloid-induced neuroimmune signaling pathways in human and mouse cell models, including iPSC-derived microglial-cells and bone-marrow derived macrophages, by using specific compounds that modulate SAA expression. By integrating in vivo and in vitro approaches, we intend to shed light on how peripheral inflammation shapes central neuropathology, with a specific focus on the role of SAAs in mediating gut-brain communication.
Keywords: Neurodegeneration, Enteric nervous system, Gut-brain axis, Inflammation, Microscopy
Uptake and immune sensing of bacterial amyloids in the intestinal mucosa
Tutor: Dizzy De Neve - Promoters: Peter Verstraelen, Winnok De Vos
There is an intricate crosstalk between the gut and the brain in both health and disease, with gut microbiota acting as key players. Among microbial factors, bacterial amyloids such as curli act as pathogen-associated molecular patterns (PAMPs) that trigger immune responses locally in the gut and systemically, where they have been linked to the progression of neurodegenerative disorders such as Alzheimer’s disease (AD). Yet, the exact pathogenic mechanisms induced by curli remain poorly understood. This project aims to elucidate how bacterial amyloids are taken up and sensed by intestinal macrophages during inflammation, and what this means for immune communication along the gut-brain axis. The in vivo part entails the study of curli uptake in mouse models of intestinal inflammation, to identify the cells that internalize bacterial amyloids and to understand how these amyloids are processed. The in vitro part entails the stimulation of macrophage cultures with curli, to characterize the cellular pathways that are activated upon uptake, thereby focussing on lysosomal damage, inflammasome activation and interferon signaling. The project combines in vivo and in vitro work, and therefore involves a diverse set of techniques, including animal handling and tissue collection, cell culture, flow cytometry, qPCR, (immuno)fluorescent staining and confocal microscopy. These provide cellular, molecular and imaging readouts that contribute to new insights into how microbial amyloids shape gut-brain communication and the neuroinflammatory processes underlying neurodegenerative disease.
Keywords: microbiome-gut-brain axis, bacterial amyloids, immune sensing, curli uptake
Progranulin deficiency and the ageing brain: immune, glial and neuronal consequences in knockout mice
Promoter: Samir Kumar-Singh
Progranulin (PGRN) is a secreted growth factor that restrains microglial activation and supports lysosomal function. Loss-of-function mutations in the PGRN gene cause frontotemporal dementia, whereas PGRN is upregulated in Alzheimer’s disease (including in our own work on amyloid mouse models) and after brain injury such as TBI, suggesting that it is part of the brain’s damage response. How partial or complete PGRN loss shapes neuroinflammation and neurodegeneration with age is still incompletely understood. Our group has generated constitutive heterozygous and homozygous PGRN knockout mice as well as a conditional (Tie-2) knockout, and holds behavioural data for the conditional model (motor, memory and anxiety tests at 9 and 18 months) together with a large archive of paraffin-embedded and snap-frozen tissue (brain, liver, spleen, lung) and plasma from wild-type and knockout animals of different ages. No new animal experiments are needed. The student will (i) analyse the existing behavioural data; (ii) perform and quantify immunohistochemistry on paraffin-embedded brain sections for microglia (Iba1, CD68), M1/M2 polarisation, astrocytes (GFAP) and neuronal, synaptic and myelin markers, comparing genotype, gene dose (heterozygous versus homozygous) and age; (iii) measure inflammatory and growth-factor markers in snap-frozen tissue and plasma by western blotting, qPCR and multiplex immunoassays; and (iv) compare PGRN-dependent changes with the marker panel studied in TBI patients (see linked TBI project), asking whether PGRN loss produces a TBI-like inflammatory signature.
Keywords: Progranulin, Microglia, Neuroinflammation, Ageing, Immunohistochemistry, Knockout mice
Blood platelets as biomarkers for Alzheimer’s Disease
Tutor: Bente Van den Broeck – Promoter: Frederik Denorme
Alzheimer’s disease is often diagnosed only after significant neurodegeneration, limiting treatment opportunities. We propose that blood platelets, which share molecular features with neurons, can serve as minimally invasive biomarkers for pathological processes such as amyloid-beta and tau dysregulation. We hypothesize that platelet-based biomarkers allow earlier and more accurate detection of Alzheimer’s disease, improve distinction from other dementias, and provide a cost-effective tool to monitor disease progression and therapeutic response. Establishing platelets as a reliable biomarker source could transform diagnostic practice and support the development of new therapies. In this project, we will use murine models of Alzheimer’s disease which we will follow over time. Platelets will be isolated at defined stages of disease progression and subsequently profiled for Alzheimer’s disease–associated biomarkers using transcriptomic (qPCR) and proteomic (Western blotting) analyses. In parallel, in vitro and in vivo functional assays will be performed to investigate if Alzheimer’s disease progression affects the haemostatic and thrombotic potential of platelets.
Keywords: platelets, neurodegeneration, thrombosis, bleeding, qPCR, western blotting, flow cytometry.
INFECTION AND (NEURO-)INFLAMMATION
Pseudomonas aeruginosa and Staphylococcus aureus in the human immune context: interspecies competition and host responses in humanized mouse models
Tutor: An Hotterbeekx – Promoter: Samir Kumar-Singh
Pseudomonas aeruginosa and Staphylococcus aureus are among the most frequent co-isolated pathogens in hospital-acquired and ventilator-associated pneumonia and in the cystic fibrosis lung, and co-infection is associated with a worse outcome. In an immune-humanized mouse model, we showed that P. aeruginosa causes more severe pneumonia than S. aureus, that co-infection increases the lung burden of both species, and that P. aeruginosa outcompetes S. aureus; in vitro co-culture, in contrast, is largely antagonistic, pointing to a decisive role of the human immune system in shaping the interaction. Within one of our funded projects, the student will further dissect this interaction in vitro. Reference and clinical strains will be grown alone and in co-culture (growth kinetics, CFU counts on selective media, biofilm formation and, where relevant, antibiotic susceptibility), and then exposed to human immune and epithelial cells such as monocyte-derived macrophages, neutrophils or airway epithelial cells. Host responses will be measured by phagocytosis/killing assays, multiplex cytokine and chemokine analysis, RNA extraction and qPCR, and structural changes by (immuno)fluorescence microscopy. Where useful, samples from the mouse model (lung tissue, bronchoalveolar lavage, plasma) can be used to link the in vitro findings to in vivo pathology.
Keywords: Pneumonia, Pseudomonas aeruginosa, Staphylococcus aureus, host-pathogen interaction, co-culture, Innate immunity
Targeting Platelet-Immune Interactions in Stroke
Tutor: Marie-Lore Vrancken – Promoter: Frederik Denorme
Ischemic stroke happens when a blood clot blocks blood flow to the brain, causing permanent brain damage and death. It affects about 15 million people each year, with 1 in 4 people at risk of having a stroke in their lifetime. Over the past decades, platelets have been firmly established as essential players in the pathophysiology of ischemic stroke. Despite this foundational understanding, current anti-platelet therapies remain suboptimal and are associated with a life-threatening bleeding risk. Work by our group identified platelet-immune cell cross talk as a key driver of brain injury in ischemic stroke. These platelet-immune cell interactions are unaffected by current anti-platelet agents. Our big picture goal is to find novel drugs that block these interactions and improve stroke outcomes. In this project, we will investigate how the immune receptor FCγRIIA worsens stroke outcomes by analysing brain inflammation and thrombosis as well as short and long-term stroke outcomes.
Keywords: platelets, neutrophils, thrombosis, inflammation, confocal imaging, flow cytometry, neurological behaviour phenotyping.
Following up blood biomarkers of traumatic brain injury in the brain
Promoter: Samir Kumar-Singh
Traumatic brain injury (TBI) triggers an acute inflammatory and neurodegenerative response, but it remains unclear which circulating proteins truly reflect processes in the injured brain. Together with the CENTER-TBI consortium, our group has profiled a broad panel of cytokines, chemokines, growth factors and neurodegeneration-related proteins by Olink in approximately 1,800 blood samples from TBI patients across the injury spectrum and from controls within Europe. In this project the student will take the most informative candidates emerging from this analysis (e.g. microglial/myeloid, astroglial, axonal and synaptic markers) and test whether they are also regulated in brain tissue and cells. Validation will rely on complementary systems: (i) human brain tissue (TBI and control, where accessible through the biobank and ethical approval) and archived mouse brain tissue with established neuroinflammation, used to validate antibodies and assays; (ii) injury-relevant in vitro models, in which primary or iPSC-derived microglia, astrocytes and neurons are exposed to inflammatory or mechanical stress and the candidate markers are measured in cells and conditioned medium; and (iii) public TBI brain transcriptomic and proteomic datasets, for in silico comparison with the blood signature. The student will use western blotting, ELISA/multiplex immunoassays, qPCR, (multiplex) immunohistochemistry with digital image analysis, cell culture and basic bioinformatics/statistics (R), and will learn how to interpret biomarker data across blood, tissue, and cells.
Keywords: Traumatic brain injury, Neuroinflammation, Biomarkers, Olink, Western blotting, Microglia
Unravelling the etiopathogenesis of post-infection syndromes: following up circulating markers of Long COVID and post-mpox condition
Tutor: An Hotterbeekx, Isaac Barhishindi – Promoter: Samir Kumar-Singh
A substantial minority of patients develop persistent symptoms after infections such as COVID-19 (Long COVID) or mpox, yet the underlying mechanisms are unknown and no biomarkers or treatments exist. In our Long COVID cohort constituting >400 individuals followed for up to 24 months, multiplex profiling on multiparametric assays (MSD and Olink) revealed an IL-17F-centred Th17/Th2 module, altered vascular-repair factors and phenotype-specific trajectories, and similar MSD data are available from mpox patients. Analogous to the TBI project, the student will follow up the most informative circulating markers beyond plasma. Using the existing datasets, the student will (i) compare MSD and Olink measurements of overlapping analytes and test whether the Long COVID signature is also found after mpox, using R and mixed-effects/latent-factor models; and (ii) test the biological meaning of the top markers (e.g. IL-17F, vascular-repair factors) in human in vitro models, in which primary airway or skin epithelial and endothelial cells are exposed to recombinant cytokines or patient-derived plasma and barrier integrity, repair and inflammatory responses are measured by ELISA/multiplex immunoassays, qPCR, western blotting and (immuno)fluorescence microscopy. Where available, the cellular source of the markers is explored by flow cytometry of stored PBMC or by immunohistochemistry on archived tissue.
Keywords: Post-infection syndrome, Long COVID, Mpox, etiopathogenesis
CANCER
Characterization of the interplay between glioma-associated macrophages and tumor cells in human model systems
Tutor: Mariken Lathouwers – Promotor: Winnok De Vos
Glioblastoma (GBM) is a highly aggressive brain tumor characterized by infiltrative cancer cells that display significant phenotypic plasticity and an immunosuppressive microenvironment. Key players of this microenvironment are brain-resident microglia and bone marrow-derived macrophages, together known as the tumor associated macrophages (TAMs), which are recruited and reprogrammed by the tumor to support its growth and survival. To better understand the interactions between cancer cells and TAMs, we are developing a model that recapitulates this interplay in a fully human 3D context. To this end, we generate both types of TAMs from human induced pluripotent stem cells and co-culture them with patient-derived glioblastoma stem-like cells (GSC) in 2D and 3D systems. We are currently mapping the molecular identity and cell state of the microglial TAM and GSC populations, using cyclic immunofluorescence and molecular assays. Thereafter, we intend to document the impact of co-culture on their phenotype using confocal and lightsheet microscopy complemented by ELISA. Additionally, we will study their migration and functional behavior using live cell imaging. Once optimized we will expand the model with the macrophage-derived TAM population and evaluate the potential of pharmacological interventions aimed at re-educating TAM. This way we hope to unveil novel therapeutic entry points for a disease that still has no cure.
Keywords: Glioblastoma, glioma-associated macrophages, iPSC-derived organoids
Super-resolved analysis of DNA damage repair in glioblastoma
Tutor: Mirthe Vandenputte - Promoter: Winnok De Vos
Glioblastoma is among the most aggressive and treatment-resistant cancers. Despite multimodal treatment with radio- and chemotherapy, tumor recurrence is almost universal, leaving patients with a prognosis of less than 2 years. Glioma stem-like cells (GSCs) are key drivers of therapy resistance and recurrence, because these cells can engage adaptive transcriptional programs that allow them to cope with high levels of genotoxic stress. It is becoming clear that this adaptability does not necessarily rely on upregulation of individual DNA damage repair (DDR) proteins, but rather on a dynamic usage of different repair pathways. In line with this so-called DNA repair plasticity, we have identified DDR pathway dependencies in glioblastoma cell lines, which we coupled to specific pharmacological targets. Building on this work we now aim to validate whether similar dependencies are present in patient-derived GSCs, whether they can be connected to specific cell states and whether they can be therapeutically exploited. To characterize the dynamics of DDR pathways (i.e., repair factor recruitment and DNA lesion resolution), we will exploit live cell imaging and superresolution microscopy. These analyses will be complemented by western blotting and qPCR to quantify changes in the protein levels, resp. gene expression of selected DDR markers. By integrating imaging and molecular analyses, this project aims to gain deeper insight into the adaptive mechanisms that allow GSCs to survive therapeutic DNA damage and to identify molecular vulnerabilities that could potentially be exploited to improve treatment efficacy and limit GBM recurrence.
Keywords: DNA damage, glioma stem-like cells, Glioblastoma, qPCR, western blotting, confocal microscopy
Nuclear envelope stress in glioblastoma
Tutor: Isa Decuypere - Promoter: Winnok De Vos
Glioblastoma (GBM) is the most common and aggressive primary brain tumor, with a median survival of only 15 months after diagnosis and no cure. A key challenge in treating GBM is its heterogeneity, as glioma stem-like cells (GSCs) can self-renew, change phenotypes, and interact with the surrounding tumor microenvironment. Although extensive research has identified numerous molecular alterations underlying GBM progression and treatment resistance, the contribution of altered cellular biophysical properties remains less well understood. Both within the tumor bulk and during invasion, GBM cells are exposed to mechanical forces from the surrounding environment. These forces can deform the nucleus and place the nuclear envelope under mechanical stress. Such nuclear envelope (NE) stress can perturb cellular homeostasis and promote DNA damage, genome instability, and inflammatory signaling, with potential consequences for tumor cell survival, plasticity, and disease progression. To understand the impact of NE stress on GBM aggressiveness, we are now investigating how GSCs respond to mechanically induced NE stress and to what extent their vulnerability to NE stress correlates with cell state plasticity. To recapitulate the mechanical stress of the tumor microenvironment, we will culture the GSCs on polyacrylamide hydrogels from low stiffness mimicking healthy brain tissue (100 Pa) to the high stiffness characteristic of the GBM tumor mass (10 kPa). We will systematically evaluate the degree of NE stress as a function of the cell state in the GSC panel using cyclic immunostaining. Furthermore, to study invasion in a physiologically relevant context, we will exploit an assembloid model, which consists of growing GSCs as tumoroids and combining them with separately grown iPSC-derived cerebral organoids. We will evaluate the degree of NE stress as a function of cell state and infiltration depth using section-based and in toto imaging approaches. This way we will gain a cell-state-resolved view on NE stress in the GSCs and enable us to assess its relationship with their infiltration into the surrounding tissue.
Keywords: glioblastoma, glioma stem-like cells, cell biology, nuclear envelope, live cell imaging, assembloids
Identifying drivers of cellular plasticity in glioblastoma using live state reporters
Tutor: Ritik Mehta – Promoter: Winnok De Vos
Glioblastoma (GBM) is a lethal brain cancer with no cure. Central to the pathology is a profound cell state heterogeneity and plasticity, which drives therapy resistance and recurrence. Increasing evidence indicates that these GBM states depend on the cues present in the local tumor microenvironment, such as non-neoplastic interactions and the mechanical landscape. However, classical in vitro cultures fail to recapitulate this complexity, and molecular analyses only allow for end-point assessment, making it a daunting task to identify the causal drivers of state plasticity and their vulnerabilities. In order to study GBM plasticity in a systematic and physiologically relevant manner, we are using a panel of patient-derived glioma stem-like cells (GSCs), which we have transduced to obtain fluorescent cell state reporters, enabling spatio-temporal evaluation with live microscopy. Initially, placing these transgenic lines in controlled biochemical and mechanical assays allows us to isolate the effects of individual cues on plasticity. By then co-culturing them as 3D tumoroids with cerebral organoids, we can evaluate the cellular plasticity in physiological models. This way, we intend to shed light on the drivers of cell state plasticity and open avenues for the development of new targeted treatments.
Keywords: Glioblastoma, organoids, mechanobiology, live microscopy