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Functional genomics approaches to study healthy and unhealthy aging in the Rhineland Study
No full textAging is a complex and heterogeneous process, characterized by a decline in physiological functions and an increased risk of various diseases, including cardiovascular, neurodegenerative, and metabolic disorders. Although the molecular hallmarks of aging have been defined, individual aging trajectories vary widely due to a combination of intrinsic and extrinsic factors. This variability complicates the development of effective treatments aimed at promoting healthy aging.
Functional genomics offers a valuable framework for investigating the molecular mechanisms underlying aging and its related traits by focusing on intricate and dynamic biological processes, such as gene regulation, epigenetic changes, and RNA activity, and their interactions. In the studies described in this thesis, I leveraged multi-omics data, including genetics, transcriptomics and epigenomics, from the population-based Rhineland Study, to explore molecular signatures associated with aging-related traits.
Specifically,
1. I investigated blood-based microRNAs associated with vascular health and cognitive function, identified the biological pathways they may regulate using gene expression data, and examined the influence of genetic variation on their expression.
2. I explored the effects of age and sex on retrotransposable elements and assessed how their dysregulation may impact downstream molecular pathways by integrating their expression profiles with those of nearby genes.
3. Finally, I used gene expression data to functionally interpret genome-wide association study (GWAS) and epigenome-wide association study (EWAS) findings, aiming to uncover biological pathways underlying associations between genetic variants and complex lipids, as well as between DNA methylation patterns and hippocampal volume and asymmetry.
I identified blood-based microRNAs associated with vascular traits, such as arterial compliance and cardiac function, and cognitive function. By integrating gene expression data, I found that these microRNAs potentially regulate key biological pathways including blood vessel development, angiogenesis, telomere maintenance, axon guidance, and synapse assembly. Additionally, genetic analyses revealed that specific SNPs influence the expression of certain microRNAs, like miR-3605-3p, highlighting the genetic regulation of microRNA expression and its impact on vascular and brain health. Moreover, I demonstrated that the expression of most retrotransposable elements increases with age, and that there are sex differences, with certain retrotransposable elements highly expressed in men. Integration with nearby gene expression data revealed co-regulation of retrotransposable elements and genes involved in immune and inflammatory pathways. Finally, using gene expression data, I functionally validated findings from GWAS on complex lipids and EWAS on hippocampal volume and asymmetry. These integrative analyses identified several genes, such as FADS1, FADS2 and ABCA7 influencing lipid metabolism, whose expression levels mediated the effects of the genetic variants on complex lipid traits, and revealed epigenetic signatures affecting hippocampal volume and asymmetry through gene expression and transcription factor regulation.
In conclusion, this work demonstrates how functional genomics approaches can identify potential biomarkers of aging-related traits and subsequently uncover the biological pathways in which they are involved. Moreover, they can provide meaningful interpretation of findings from established omics-wide association studies, providing valuable insights into the molecular mechanisms of aging
Late-life aging dynamics in <em>C. elegans</em>
No full textAging is a complex biological process marked by a progressive decline in physiological function, leading to increased vulnerability for diseases and mortality. While early and mid-life aging dynamics are well-characterized, less is known about the physiological changes that occur in late life. This thesis investigates the dynamics of aging in the model organism C. elegans, focusing on whether late life presents a deceleration in age-related changes, akin to the mortality plateau observed in many species. Our aim was to characterize proteomic and transcriptomic changes in C. elegans across the lifespan, including late life, thus contributing to a deeper understanding of the aging process.
Our methods included RNA-seq and mass spectrometry to profile age-sensitive genes (ASGs) and age-sensitive proteins (ASPs). Lifespan assays were used to identify critical periods in the aging trajectory, allowing for targeted sample collection in early, middle, and late life phases.
Our transcriptomic and proteomic analyses identified 3686 ASGs and 658 ASPs, respectively, that were used to study the kinetics of age-dependent phenotypic change across the lifespan in C. elegans. These analyses revealed two distinct phases: an initial period of age-related change, followed by a plateau phase in late life as demonstrated by a stabilization of changes in ASGs and ASPs expression. This late-life phase is also associated with a plateau in mortality rates, suggesting a biological transition where some age-sensitive pathways stabilize or even reverse.
Additionally, we used these datasets to identify genes that feature stability across a range of conditions and that may therefore be useful as reference genes in C. elegans. Our work identified 7 stable reference genes (pmp-3, orc-2, praf-3, aars-2, unc-16, gtf-2F1, and ZK1307.8) that maintain consistent gene expression across various conditions, including multiple age groups (days 6, 8, 12, 14, 18, 20, 24), different mutant strains (WT, age-1, daf-2, isp-1), and a range of temperatures (15°C, 20°C and 25°C), whereas previous studies typically focused on a limited set of time points.
To our knowledge, these findings represent the first study to systematically demonstrate, beyond mere lifespan data, that age-dependent changes follow a dynamic that entails a plateau phase in late life, indicating that aging-associated changes stop progressing and stabilize at advanced ages. Future studies need to address the generality of this finding by incorporating longitudinal analysis approaches and extending the assessments to additional biological systems
Vascularization of human iPSC-derived 3D retinal organoids and its impact on ganglion cell survival
No full textROs have emerged as a transformative platform for studying retinal cell development and modeling retinal diseases. These 3D structures, derived from SCs, replicate the architecture and function of the retina, offering crucial insights into retinal biology and pathology. However, a major limitation is the lack of functional vasculature, which restricts their ability to sustain nutrient and oxygen supply over time. This shortfall results in cell death and diminished functionality, particularly affecting RGCs, which are essential for transmitting visual signals from the retina to the brain. The failure of RGCs in ROs to extend axons as they do in vivo further compromise their survival and limits the organoids’ utility for studying retinal circuitry and function. The absence of a vascular network exacerbates these challenges by preventing the organoids from reaching the physiological conditions necessary for optimal cell growth and activity.
In this study, we introduce a novel approach to overcome these limitations by establishing a vascular-like system within ROs. This system incorporates functional lumens and transient pericyte-like cells, crucial for maintaining vascular integrity. The introduction of this vascular-like structure significantly enhances organoid size, reduces apoptosis in aging organoids, and preserves RGC populations. This breakthrough improves organoid viability and their potential for studying retinal diseases and therapeutic interventions. To further optimize functionality, we integrated optogene, ChRimson to label RGC axons and allow their direct recording activity on MEAs. Our results showed significantly increased RGC firing rates, with burst durations reaching up to 300 Hz, indicating robust neuronal activity. Additionally, vROs displayed synchronous activity and enhanced bursts in response to light stimulation, suggesting functional maturation of RGCs. This maturation is crucial for developing functional photoreceptor circuitry, which is essential for advancing our understanding of retinal function and devising potential therapies for retinal disorders.
In conclusion, the incorporation of a vascular-like system in ROs represents a substantial advancement, enhancing both RGC survival and functionality. This innovation addresses key limitations of traditional organoid models and opens new possibilities for using ROs as a powerful tool in biomedical research, particularly in studying retinal diseases and developing therapeutic strategies
AIM2-Inhibitory Nanobodies Reveal Differential Activation of AIM2 and NLRP3 Inflammasomes by Poxvirus DNA Genomes in Primary Human Cells
No full textPoxviruses are large double-stranded (ds)DNA viruses that replicate in the cytoplasm. While variola virus (VARV) caused millions of deaths before its eradication in 1980, monkeypox virus (MPXV) has been declared a public health emergency of international concern with over 100,000 reported cases. Inflammasomes are large macromolecular complexes that coordinate the activation of caspase-1, maturation of pro-inflammatory cytokines, and pyroptotic cell death. Poxviruses and cytosolic dsDNA have been reported to activate the AIM2 inflammasome in mice and the NLRP3 inflammasome in human monocytes. However, the inflammasome responses to poxvirus infections have not been adequately addressed in human primary cells.
To study the inflammasome response to poxviruses, we used the model poxvirus vaccinia virus (VACV) and MPXV. We employed the inflammasome reporter caspase-1-CARD-EGFP encoded by VACV to quantify inflammasome activation in primary human macrophages, CD14 monocytes, and normal human epidermal keratinocytes (NHEKs). VACV infection triggered robust inflammasome activation in IFN-γ treated primary macrophages and keratinocytes, AIM2 activation in IFN-γ treated THP-1 macrophages, and NLRP3 activation in primary CD14 monocytes. To investigate the role of AIM2 in primary cells, we generated and characterized nanobodies targeting the AIM2 pyrin domain (PYD) and identified three AIM2-inhibitory nanobodies. We solved the crystal structure of the inhibitory nanobodies in complex with AIM2 PYD and revealed that they inhibit AIM2 activation by preventing AIM2 PYD polymerization. Exploiting nanobodies with different binding epitopes, we developed bivalent nanobodies that completely block AIM2 inflammasome activation. Using VACV as both a vector and a trigger, we engineered recombinant VACV encoding AIM2-inhibitory bivalent nanobodies to express nanobodies in primary cells. We revealed that VACV infection activates the AIM2 inflammasome in primary human macrophages and keratinocytes, asserting AIM2 as a relevant sensor in human myeloid cells and primary keratinocytes. Additionally, we demonstrated that incoming VACV genomes are sufficient to trigger AIM2 inflammasome activation, while the VACV B2 poxin selectively restricts NLRP3 inflammasome activation in primary monocytes.
In conclusion, this study provides the first evidence of cell-type-specific inflammasome responses to poxvirus infection in human primary cells, uncovering potential inflammasome regulatory mechanisms that remain unknown. Additionally, we have developed the first AIM2-inhibitory nanobodies, which serve as valuable molecular tools to dissect AIM2 functions and hold potential for therapeutic application in AIM2-associated diseases
LAMTOR1 Phosphorylation Mediates Lysosomal Positioning via a Non-canonical mTORC1 Pathway
No full text(noch nicht zugänglich / not yet accessible
The role of microenvironmental cues in regulating tissue immune surveillance
No full textTissue-resident memory T (TRM) cells provide frontline immune defense against pathogens and malignancies in peripheral tissues. The tissue microenvironment plays a vital role in the development of TRM cells, shaping their abundance, phenotype and function through host-derived factors. Although tissue microenvironments are diverse in nature, they converge to instruct a common tissue-residency program in T cells that promotes local retention while inhibiting tissue-egress pathways. The cytokine transforming growth factor-beta (TGFβ) is a key regulator of TRM cell development. However, in non-epithelial sites such as the liver, TRM cells form through a TGFβ-independent tissue-residency program, which indicates that additional factors have evolved to direct tissue-residency. Beyond the effects of host-derived factors, microenvironmental perturbations such as microbial experience and chronic infections influence TRM cell dynamics, highlighting the need to understand the tissue-derived factors controlling TRM cells.
In this thesis, we examined the contribution of microenvironmental factors in shaping TRM cell development, function and persistence using genetically manipulated CD8+ T cells, multi-omics sequencing methods and a mouse model with increased microbial diversity. We found that TRM cell development across tissue sites was orchestrated by the metabolite retinoic acid (RA) through the receptor RARα. While TRM cell generation in the small intestine epithelium (SI-epithelium), liver and kidney were dependent on the RA-RARα signaling axis, the absence of RA signals induced TRM cell formation in skin and colon. Importantly, SI-epithelium TRM cells could bypass TGFβ dependency by utilizing the RA-induced differentiation pathway.
RA was also critical for the long-term maintenance of SI-epithelium TRM cells, in part by impeding retrograde migration of TRM cells to the mesenteric lymph node (mLN). In contrast, chronic infection led to SI-epithelium TRM cell erosion due to sensing of danger signals via purinergic receptor P2RX7, demonstrating that SI-epithelium TRM cell durability is dynamic.
Further, our results revealed that RA signaling induced TRM cell phenotypic and functional changes, a phenomenon reflected in mice with enhanced microbial diversity. Microbial experience also caused intestinal lengthening accompanied by increased intestinal CD8αβ+ and CD4+ T cells with altered phenotypes, suggesting that microbial diversity enhances tissue-residency and adds layers of TRM cell heterogeneity.
Overall, our findings reveal additional microenvironmental cues that are critical for balancing TRM cell abundance and function across tissues. This research provides a rationale for targeting environmental regulators to modulate TRM cells in a tissue-specific manner for improved immune defenses
The role of cysteines and S-palmitoylation in NLRP3 inflammasome activation
No full textNucleotide oligomerization domain (NOD)-like receptor protein 3 (NLRP3) is a cytosolic pattern recognition receptor with a critical role in numerous autoinflammatory disorders and non-communicable diseases characterized by chronic inflammation. In innate immune cells, such as macrophages, NLRP3 assembles into a multimolecular complex known as inflammasome, in response to intracellular homeostatic disturbances caused by tissue damage or infection. Activation of the NLRP3 inflammasome triggers a pro-inflammatory signaling cascade, culminating in pyroptosis – a lytic, inflammatory form of cell death – and the release of the cytokine interleukin-1β. Despite extensive research, the molecular mechanisms governing NLRP3 activation remain incompletely understood. In this study, I identified a pivotal role for cysteine residues in NLRP3 activation. Mechanistically, we demonstrated that a few residues undergo S-palmitoylation, a reversible lipid modification involving the attachment of a long-chain fatty acid. This modification enhances protein membrane association and stability, thereby facilitating NLRP3 trafficking to organelles, promoting inflammasome assembly and sustaining cytokine release. Moreover, I identified the palmitoyltransferase ZDHHC7 as a critical enzyme required for activation.
These findings establish S-palmitoylation as a key post-translational regulatory mechanism of NLRP3, providing new insights into inflammasome activation and uncovering potential therapeutic targets for inflammation-associated diseases
Deep brain imaging of neuronal activity in sensory thalamus during innate and learned avoidance behavior
No full textAvoidance behaviors are critical for survival, enabling organisms to respond effectively to potential threats in their environment. While much is known about how cortical and limbic brain areas contribute to avoidance behavior, the role of the auditory thalamus (medial geniculate body - MGB) in mediating these active defensive responses has not been addressed to this date. Here I investigate how MGB neurons encode both innate and learned avoidance. Using a miniature microscope imaging approach, I monitor the activity of MGB neurons in freely moving mice across multiple days of exploratory tests and active avoidance task. I found that MGB neurons display a variety of plasticity profiles upon danger-predicting auditory stimuli. Furthermore, the population level encoding of auditory stimuli underwent prominent changes as learning progresses, resulting in a coherent neural representation in MGB. Besides the learned avoidance, the role of MGB in encoding innate avoidance behavior is explored. Notably, MGB exhibited distinctly opposing patterns of activity, dynamically encoding innate safety and danger during exploration of different environments. Moreover, the data shows MGB neurons modify their responses after avoidance training which has not been manifested in behavioral read-outs post-training. This research extends our understanding of sensory processing in the medial geniculate body and provides new insights into the neural mechanisms underlying avoidance behavior
The Phenotypic Correlates and Genetic Basis of the Central Neuroendocrine System
No full textThe hypothalamus is considered the body’s principal center for maintaining homeostasis and orchestrating the secretion of multiple hormones. The pineal gland closely interacts with the hypothalamus and forms an essential component of circadian regulation via melatonin secretion. Assessing their structural characteristics and phenotypic correlates across the lifespan could therefore provide novel insights into the neurobiological basis of age-associated dysregulation of a range of bodily functions. However, comprehensive population-based studies were lacking.
Using cross-sectional data from the Rhineland Study and the UK Biobank Study, two large-scale population-based cohort studies, we conducted the projects to address this gap. In both cohorts, volumes of hypothalamic structures and pineal gland were obtained from 3T structural MRI through an automatic parcellation (FastSurfer-HypVINN). Standardized cognitive domain scores were derived from extensive neuropsychological test batteries. Genotype data underwent rigorous quality control and were imputed using high-quality reference panels.
We employed multivariable linear regression to assess associations of volumes of hypothalamic structures and pineal gland with age, sex as well as cognitive performance. We observed that, in older individuals, volumes of total hypothalamus, anterior hypothalamus, posterior hypothalamus and mammillary bodies were smaller, while those of medial hypothalamus and tuberal region were larger. Volumes of all hypothalamic structures were larger in men compared to women. In both sexes, larger volumes of total hypothalamus, anterior hypothalamus, posterior hypothalamus and mammillary bodies were associated with better domain-specific cognitive performance, whereas larger medial hypothalamus and tuberal region were associated with worse domain-specific cognitive performance. Additionally, we found that the pineal gland volume was larger in women and exhibited an accelerated decrease with age. Additionally, pineal gland volume was associated with larger volumes of total brain, grey matter, hippocampus, cerebellum and hypothalamus, as well as higher mean cortical thickness. Importantly, after adjustment for generalized brain atrophy, larger pineal gland volume was associated with better global cognitive performance as well as with a faster processing speed in both datasets. We further conducted the first GWAS of pineal gland volume, demonstrating 19% heritability and identifying 34 genome-wide significant loci, with highly concordant results between the two independent cohorts. Gene mapping and functional annotation highlighted multiple genes and pathways involved in numerous traits including sleep, metabolism, neurodevelopment and neurodegeneration. Mendelian randomization indicated a causal relationship between pineal gland volume and daytime napping.
In summary, this thesis investigated age- and sex-related changes in volumes of hypothalamic structures (including its subregions) and pineal gland across adult lifespan, as well as their association with cognitive function. Additionally, the genetic architecture of pineal gland volume was investigated. The findings described in this thesis advance our understanding of the neurobiological mechanisms underlying age-associated disruptions in bodily homeostatic functions, particularly in the context of age-related neurodegenerative disorders
Zusammensetzung und Lokalisation von Harnsteinen in Abhängigkeit von Alter und Geschlecht der Steinpatienten im Zeitraum von 2007 bis 2020
No full textDas Harnsteinleiden unterliegt einer komplexen Pathogenese, die sich in Abhängigkeit der Steinzusammensetzung voneinander unterscheidet und bis heute noch nicht in vollem Umfang geklärt ist. Es handelt sich um ein multifaktorielles Krankheitsbild, das sich in der Prävalenz und der Erscheinungsform mit der Entwicklung einer Bevölkerung verändern kann. Mithilfe dieser Studie wurde in einer großen Stichprobe die jüngste Entwicklung hinsichtlich des Vorkommens unterschiedlicher Harnsteinbestandteile in Deutschland im Zeitraum von 2007 bis 2020 analysiert. Alle Harnsteine wurden mittels Infrarotspektroskopie untersucht. Die häufigsten Harnsteinhauptkomponenten waren hierbei Kalziumoxalate, Karbonatapatit und Harnsäure-Anhydrat. Es bestanden geschlechtsspezifische Unterschiede. Männer waren, mit Ausnahme der Bildung von Karbonatapatit, Struvit und seltenen Phosphaten, deutlich öfter von der Erkrankung betroffen als Frauen sowie im Durchschnitt älter. Mit fortschreitenden Studienjahren konnte ein steigender Anteil an Frauen sowie hochbetagter Harnsteinpatienten erfasst werden. Die Inzidenz bei Patienten von 40 bis 59 Jahren überwog unabhängig vom Geschlecht der aller anderer Altersgruppen. Lediglich 1,3 Prozent aller Erkrankten waren jünger als 20 Jahre alt. Einzelne Substanzen hatten davon abweichende Inzidenzspitzen. Während das Auftreten von Cystinsteinen mit einem jungen Erkrankungsalter assoziiert war, traten harnsäurehaltige Steine und Struvitsteine in vergleichsweise höherem Lebensalter auf und waren häufiger in der Blase und der Harnröhre lokalisiert. Geschlechtsunabhängig lagen Whewellit- und Harnsäuresteine zudem häufiger vor als Weddellit- und Harnsäure-Dihydratsteine. Auch das Patientenalter unterschied sich zwischen den Hydratformen einer Harnsteinsubstanz. Patienten mit Whewellit- oder Harnsäure-Anhydratsteinen wiesen ein höheres Durchschnittsalter sowie höhere prozentuale Anteile in älteren Altersgruppen auf als Patienten mit führendem Anteil der jeweiligen Dihydratform. Die vorliegende Studie bekräftigt Unterschiede in dem Vorkommen einzelner Harnsteinbildner hinsichtlich des Alters und des Geschlechts der Betroffenen sowie der Lokalisation