Young In­ves­ti­ga­tors

A highly homeostatic extracellular microenvironment is prerequisite for the proper neural activity in the central nervous system (CNS), which is ensured by the blood-brain barrier (BBB), the biointerface between the brain and the peripheral circulation. The BBB is a multicellular structure composed of endothelial cells (ECs), pericytes, and astrocytes. In addition, these cells provide the molecular substrate for migratory oligodendrocyte precursor cells (OPCs) along the vasculature during development. Failure in the recruitment of any of these cell types leads to BBB dysfunction. For instance, abnormal clustering of OPCs on the BV induces BBB breakdown, due to the contact loss of the astrocyte endfeet on the BV.

The OPC migration is mediated by cytokines as well as neurotransmitters. In vitro, using an OPC cell line, it has been shown that GABA activates GABABRs to stimulate migration. In addition, GABA derived from ECs directs interneuron migration as well, and also blocking GABA release impairs BBB integrity. Therefore, we hypothesized that EC-derived GABA guides OPC migration through GABABR signaling and such interaction promotes BBB barrier function.

To substantiate this hypothesis, we investigated OPC migration in the OPC-specific GABABR conditional knockout mice (cKO). Our preliminary data showed that in the dorsal cortex of cKO mice, the developmental expansion of OPCs at the first postnatal week was retarded while the density of these cells on the BV was increased, suggesting decelerated migration and impeded dissociation of OPCs from the BV. Furthermore, we also observed BBB leakage and sustained neuroinflammation in the cKO mouse cortex from the early postnatal week till adulthood, highly likely due to the loss of astrocyte endfeet support on the ECs. Concomitantly, a dysregulated cortical neural circuit was observed in these mice exhibiting increased excitation while suppressed inhibition.

Hence, in this proposed project, we will address the questions whether and how OPCs communicate with ECs via GABABRs during migration and how such interaction influences BBB function. For that purpose, we will investigate the GABAergic communication between ECs and OPCs employing ex vivo slice imaging. We will assess how migratory OPCs interact with ECs and astrocyte endfeet via GABABRs, using two-photon imaging in the living mouse. Finally, we will decipher the intracellular signaling pathways of GABABRs involved in the OPC-EC interaction and the BBB integrity. BBB establishment occurs during the development as well as in the repairing phase of many CNS diseases. Therefore, our study will not only bring novel insights to delineate the mechanism of BBB integrity but also guide a potential strategy for the clinical treatments against CNS diseases.

The central nervous system (CNS) plays a central role to control our body function. The CNS is normally protected against any invasions in order to avoid cell damage in the brain and in the spinal cord. However, in case of autoimmune encephalitis diseases such as multiple sclerosis (MS), autoreactive T cells infiltrate into the brain and attack myelin sheet of neurons, resulting in CNS inflammation, demyelination and neurodegeneration. The model for this disease in rodent is the experimental autoimmune encephalomyelitis (EAE), which leads to the paralysis of the animals. Interestingly, the CNS of mice and rats recover after an EAE whereas in human MS follows a chronic neurodegenerative course. This recovery from EAE in rodents rises the attention of the potential neuroprotective regulation.

Previously, we have shown the encephalitogenic T cells, rather than infiltrating the white matter, penetrate the grey matter in which they closely interact with neurons. Intriguingly, this massive T cell infiltration in the CNS is being cleaned up after animals have recovered. We therefore hypothesize that a neuroprotective mechanism takes place under EAE. Our preliminary data shows that entire cell/fragments from apoptotic encephalitogenic T cells are localized in neuronal somata in the pathological brain, indicating that neurons play a role in removing the inflammatory cells. Furthermore, we observed that during the inflammation T cell mitochondria are taken up by neurons. The neuronal mitochondria transfer was demonstrated to be part of neuroprotective mechanism by another study.

We now aim at understanding how autoreactive T cells interact with cortical neurons under inflamed conditions and decipher this interaction down to the molecular level. We will investigate the potential neuroprotective mechanism, in particular analyzing the cause of encephalitogenic T cell death and determine energy restoration in neurons through external mitochondria uptake. Finally, we will evaluate the neuroprotective outcome. We will address these aims by applying state of the art live imaging techniques combined with quantitative analysis of targeted protein and gene expression. We will observe this dynamic interaction and decipher the molecular mechanism of the potential mitochondria transfer pathway at the single cell level and in pathological brain slices. Understanding the interaction between T cells and neurons will reveal molecular cues that offer neuroprotection during autoimmune CNS diseases. We are convinced that our work will contribute to development of new strategies for the treatment of a broad spectrum of autoimmune diseases.

ORCID

The development of new antibiotic agents is a central aspect of infection research and the rise of antibiotic resistances makes this an increasingly urgent matter. Ribosomally synthesized and post-translationally modified peptides (RiPPs) are an emerging superfamily of natural products that often exhibit promising antimicrobial activities and recent studies have demonstrated how RiPPs produced by human commensals can have host protective properties. Indeed, the rise of genome mining for the identification of novel RiPP biosynthetic gene clusters confirms that the potential for producing RiPPs is widespread amongst both beneficial human commensals as well as amongst human pathogens. Thus, it seems promising to take a closer look at the RiPPs present in the human microbiome not only in the search for novel drug leads in the fight against infectious diseases, but also for understanding their roles in and how they affect microbial communities.

The project will therefore initially focus on lead molecules able to overcome antibiotic resistances with the ultimate goal of the development of new anti-infective agents that can be translated into novel treatments. At later stages, the project will be extended to also mine metagenomic datasets to identify new RiPPs from the human microbiome and enable the study of their native functions. Thus, this project exhibits a strong synergy with project IMAGINE of the HIPS-UdS TANDEM initiative, which will provide microbiome and metagenomic datasets from clinical samples that can in turn be prospected towards identifying new, unique natural products.

The initial lead molecules investigated will be the hogocidins. These are class II lanthipeptides with a selective anti-MRSA activity and they originated from a beneficial human commensal isolated from the skin microbiota of a healthy individual. Through hogocidin secretion, the producing strain exhibits microbiome protective features and helps preventing Staphylococcus aureus colonization of the skin. Systematic structure-activity-relationship (SAR) studies will aid in lead structure optimization and will be complemented by genome mining efforts aimed at isolating closely related, naturally occurring homologs in search for superior lead structures and potential probiotics. These efforts will further establish general methodologies and work procedures that will later be utilized for heterologously producing new RiPP-based lead structures identified in the human microbiome.

The project will combine methods from biochemistry, microbiology, chemical biology, medicinal chemistry, analytical chemistry, and bioinformatics.

Research on an interaction between proteins and cell membranes is still veiled, although it is the most basic mechanism in membrane-mediated cellular processes, including antimicrobial peptide action and peptide-mediated cell membrane penetration. Recently, we studied protein-like dynamic polymers (biodynamer) of hexaethylene glycol conjugated carbazole dialdehydes, and amino acid hydrazides.

We demonstrated their strong interactions with cell membranes similar to functional peptides like cellpenetrating peptides (CPPs) or antimicrobial peptides (AMPs). Also, their superior tunability controlling their characteristics makes them a suitable research tool for investigating peptide/membrane interaction.

In this project, we aim to develop oligomeric biodynamer, oligodynamer to study various physicochemical factors affecting cell-membrane interactions, and design a new class of functional peptides more easily controllable than conventional peptides. For this purpose, we sophisticatedly control physicochemical properties of oligodynamers, such as structure, rigidity, charge, and hydrophobicity, which are expected to affect the interaction. We will investigate their effect on the oligomer/membrane interaction using microscopies, calorimetry, and the Förster resonance energy transfer (FRET) technique. We expect this project expands the applicability and potential of dynamic polymer series in biomedical fields.

For decades, neutrophils were considered as a homogeneous population of short-lived circulating and tissue patrolling immune cells that possess antimicrobial functions. However, with the emerging evidence of divers neutrophil sub-populations in health and disease with functionally different phenotypes, this long-believed traditional dogma has outdated. Surface markers, density, maturity, morphology, functional aspects and localization characterize the heterogeneity of neutrophils. However, little is known about the contribution of neutrophil subsets during viral infection-related carcinogenesis although infections with high-risk human papillomaviruses (HPV) are on the rise to the major etiologic agent for cancer at mucosal bio-interfaces.

Oncoproteins encoded by HPV have the potential to critically modulate the landscape of neutrophil plasticity and the efficacy of immunotherapeutic approaches. Notably, neutrophils are crucial for the efficacy of antibody-based immunotherapeutic approaches and thus likely affected by subset transition towards "unfavorable" neutrophils. IgA antibodies effectively engage neutrophils for antibody-dependent cellmediated cytotoxicity (ADCC) of tumor cells. We recently developed an impedance-based assay to test neutrophil activation for ADCC of adherent target cells. This assay allows an uninterrupted longterm real-time monitoring of kinetics accompanied by the detection of critical mediator release. Changes in kinetics and ADCC efficacy upon modulation of the target cells by HPV-oncoprotein expression or of the effector cells due to priming with a HPV-driven cancer cell instructed tumor microenvironment (TME) can be monitored in detail.

The aim of the suggested project is to analyze how natural occurring, and tumor virus-infection TME-induced neutrophil subsets affect antibodybased immunotherapeutic approaches and how unfavorable alterations could be reverted by novel pharmacological compounds. Compounds capable to revert TME-related neutrophil subset transition could support immunotherapeutic approaches even in a broader perspective for malignant and inflammatory diseases. In summary, investigating how viral infection-related cancer instructed TME affects neutrophil subset transition at a single cell level combined with protein activity and functional read outs will help on the one side to identify promising markers for patient’s risk and therapy stratification and on the other side to identify promising novel small molecules for potential clinical development.

ORCID

Epigenetic signatures regulate how the genetic code inscribed in DNA sequence is interpreted and thus govern the state and function of each individual cell, ultimately giving rise to hundreds of different, highly specialized cell types. Understanding gene regulation is therefore paramount for understanding normal cell development as well as aberrant cellular phenotypes in disease.

In this project, we will develop a framework of computational methods to define epigenetic cell states associated with changes in the cell environment and to dissect the regulatory dynamics involved in the immune response to pathogen exposure. Our collaboration partners are generating single-cell, multiomics profiles of human immune cell states in naïve samples and samples challenged with exposure to different pathogens, including HIV, flu, MRSA/MSSA, Anthrax and Organophosphates.

We will develop and apply methods for the integrative analysis of epigenomic data by modeling the interplay between DNA sequence, DNA methylation, chromatin accessibility and gene expression. We will identify and characterize genomic elements involved in the epigenetic regulation of cell state and we will derive celltype-specific, multi-omics biomarkers of pathogen exposure. Integrating DNA sequence, epigenetic profiles and regulator expression, we will quantify and compare the activity of transcription factors (TFs). The methods developed in this project will be made available in user-friendly and scalable software tools.

The project directly synergizes with ongoing efforts at UdS in the NanoBioMed area that emphasize single-cell approaches and contribute to enhanced understanding of gene regulation. Our computational methods are directly transferable to other systems and tissues involving dynamic regulation of cell state, including changes induced by metabolism, drug-based perturbations, microbiome and cellular interfaces

Understanding the physical processes at the membrane of cells is of great scientific and technological importance. Examples include transport of sub-nanometer particles across the membrane channels, conformational deformations of the cell membrane to internalize larger particles, and formation of microtubule- or actin-based protrusions.

In this proposed project, we aim at understanding the mechanisms of two distinct processes:

1. formation and growth of microtentacles (McTNs) and
2. wrapping of nanoparticles by lipid membranes.

McTNs are one-dimensional microtubule-based protrusions that are generated by circulating tumor cells (CTCs) and contribute to cell adhesion and aggregation. CTCs play a crucial role in the spread of tumors during metastasis. There are anti-cancer drugs that stabilize microtubules or depolymerize f-actin to block the progression of mitosis and prevent cell division of cancerous tumor cells. However, the main drawback of this type of drugs is that they promote the formation of McTNs (by weakening the actin cortex against growing microtubules), thus, increase the chance of metastasis.

In a preliminary study in collaboration with the groups of Profs. Lautenschläger and Santen, we have managed to generate McTNs in non-cancerous cells using specific drugs that influence the actin cortex. We will further develop and automate our image processing package for 3D reconstruction of cells, which enables us to systematically characterize the structure and dynamics of McTNs for various types and concentrations of drugs. We will also develop a model for microtubule bundle growth and stabilization against barriers and study the mechanisms of McTN formation under the influence of drugs. The long-term aim is to better understand the conditions initiating metastasis and to design more efficient drugs and strategies for cancer treatment.

Our second major goal is to elucidate the mechanism of wrapping of nanoparticles by lipid membranes. In a related study, we previously clarified the role of endo/exocytosis rates and microtubule-assisted transport on receptor clustering at the mammalian cell surface with the group of Prof. Schmitt. Here, we plan to solely focus on phagocytosis and numerically investigate how the interplay between particle properties (such as its size, shape, and softness), elasticity of the membrane and actin cortex, particle-membrane adhesion, and coupling between actin cortex and plasma membrane determines the success rate of wrapping. We also expect that the contribution of actin polymerization at the leading edge of the membrane is influential. The simulation results will be validated by comparing them to two types of experiments: imaging single phagocytosis events at the cell membrane, or statistically evaluating the internalization efficiency of cells upon varying the key parameters in an environment containing many target particles. Phagocytosis is of vital importance for fighting infection by neutrophils. Thus, in a broader perspective, investigation of their wrapping mechanism is in line with other extensive studies at Saarland University on the mechanisms of search, response, and clearance in the immune system. The results may also help to design novel drug delivery strategies.

Our proposed study potentially suggests new experiments on cell membranes and can trigger new collaborations with other research groups of the university.