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On the potential for GWAS with phenotypic population means and allele-frequency data (popGWAS)
(2024)
This study explores the potential of a novel genome-wide association study (GWAS) approach for identifying loci underlying quantitative polygenic traits in natural populations. Extensive population genetic forward simulations demonstrate that the approach is generally effective for oligogenic and moderately polygenic traits and relatively insensitive to low heritability, but applicability is limited for highly polygenic architectures and pronounced population structure. The required sample size is moderate with very good results being obtained already for a few dozen populations scored. The method performs well in predicting population means even with a moderate false positive rate. When combined with machine learning for feature selection, this rate can be further reduced. The data efficiency of the method, particularly when using pooled sequencing, makes GWAS studies more accessible for research in biodiversity genomics. Overall, this study highlights the promise of this popGWAS approach for dissecting the genetic basis of complex traits in natural populations.
Abstract
Seed harvesting from wild plant populations is key for ecological restoration, but may threaten the persistence of source populations. Consequently, several countries have set guidelines limiting the proportions of harvestable seeds. However, these guidelines are so far inconsistent, and they lack a solid empirical basis. Here, we use high-resolution data from 298 plant species to model the demographic consequences of seed harvesting. We find that the current guidelines do not protect populations of annuals and short-lived perennials, while they are overly restrictive for long-lived plants. We show that the maximum possible fraction of seed production – what can be harvested without compromising the long-term persistence of populations – is strongly related to the generation time of the target species. When harvesting every year, this safe seed fraction ranges from 80% in long-lived species to 2% in most annuals. Less frequent seed harvesting substantially increases the safe seed fraction: In the most vulnerable annual species, it is safe to harvest 5%, 10% or 30% of population seed production when harvesting every two, five or ten years, respectively. Our results provide a quantitative basis for seed harvesting legislations worldwide, based on species’ generation time and harvesting regime.
Significance The UN Decade on Ecosystem Restoration, 2021-2030, foresees upscaling restoration, and the demand for native seed is skyrocketing. Seeds for restoring native vegetation are often harvested in wild, but too intensive harvest can threaten the donor populations. Existing guidelines that set limits to wild seed harvest are mostly based on expert opinions, yet they commonly lack empirical basis and vary among regions in one order of magnitude. We show that the current guidelines urgently need to be reformulated, because they are overly restrictive in long-lived species, while they do not protect annual plants from extinction. Using matrix population models of nearly 300 plant species, we provide a quantitative basis for a new seed harvesting legislation world-wide.
Cyclin CLB2 mRNA localization and protein synthesis link cell cycle progression to bud growth
(2024)
Clb2 is a conserved mitotic B-type cyclin, the levels of which are finely controlled to drive progression through the cell cycle. While it is known that CLB2 transcription and Clb2 protein degradation are important for precise control of its expression, it remains unclear whether the synthesis of Clb2 is also regulated. To address whether and how Clb2 expression levels respond to cell growth changes and adapt cell cycle progression, we combined single-cell and single-molecule imaging methods to measure CLB2 mRNA and protein expression throughout the Saccharomyces cerevisiae cell cycle. We found that the CLB2 mRNA was efficiently localized to the yeast bud as soon as this compartment was formed, but strikingly the Clb2 protein accumulated in the mother nucleus. The CLB2 mRNA localization in the yeast bud by the She2-3 complex did not control protein localization but rather promoted CLB2 translation. Moreover, CLB2 mRNA bud localization and protein synthesis were coupled and dependent on a single secondary structure -a ZIP code-located in the coding sequence. In a CLB2 ZIP code mutant, mRNA localization was impaired and Clb2 protein synthesis decreased, resulting in changes in cell cycle distribution and increased size of daughter cells at birth. Finally, while in WT cells the Clb2 protein concentration followed bud growth, this relationship was impaired in the ZIP code mutant. We propose that S. cerevisiae couples the control of CLB2 mRNA bud localization and protein synthesis to coordinate cell growth and cell cycle progression. This mechanism extends our knowledge of CLB2 expression regulation, and constitutes a novel function for mRNA localization.
Research on the human and animal microbiome has become increasingly important in recent years. It is now widely accepted the gut microbiome is of crucial importance to health, as it is involved in a large number of physiological processes. The term ‘microbiome’ refers to the all living microorganisms including their genes and metabolites in a defined environment, while the specific composition of microorganisms consisting of bacteria, archaea and protozoa is referred to as the ‘microbiota’ (Lane-Petter, 1962; Lederberg and McCray, 2001).
In recent years, research has focused on various of these communities in the soil (Fierer, 2017), water (Sunagawa et al., 2015), air (Leung et al., 2014) and especially in the human gut. However, this topic is also becoming increasingly relevant for the conservation of endangered species. In the face of global mass extinctions and the listing of over 42,000 animal species as ‘critically endangered’, conservation breeding programmes are more important than ever (Díaz et al., 2019; IUCN, 2022). The responsibility for these tasks lies with zoological institutions, which are dedicated to animal conservation and the continuous monitoring of animal welfare. Microbiome research offers a non-invasive method to support species conservation. By analysing faecal samples, microbial markers can be identified that provide important information about the health status and reproductive cycle of animals (Weingrill et al., 2004; Antwis et al., 2019). Zoological facilities also provide an ideal research environment for comparing individuals from different habitats. In addition, all necessary metadata such as age, sex, kinship or medical treatment are documented and can be used for the analysis.
This is the starting point for this thesis. In order to identify such microbial markers, it is necessary to understand the microbiome of a variety of animal species. The first aim is therefore to characterise the faecal microbiota of 31 mammalian species, focusing on herbivores and carnivores. It could be shown that they differ significantly in terms of both microbial diversity and microbiota composition. Herbivorous species express a very diverse microbial composition, consisting mainly of cellulose-degrading taxa of the families Fibrobacteraceae or Spirochaetaceae. In contrast, the microbiota of carnivorous species is less diverse and is dominated by protein-degrading Fusobacteriaceae and Clostridiaceae. In addition, this thesis proves that the microbiota of herbivorous species is highly consistent, whereas the microbiota of carnivorous species is highly variable. The results of this study provide important insights for the sampling scheme of future projects. Especially when analysing carnivorous species, single samples are not sufficient to capture the full variability of the microbiome.
These results lead to the question of whether this variability can be explained by daily fluctuations in the individual microbiome and whether this can be used to distinguish between species or individuals. Using individual longitudinal data and a combined approach of clustering algorithms and dynamic time warping, it is shown that such a distinction is possible at the species and individual level. This was confirmed for both a carnivorous (Panthera tigris) and a herbivorous (Connochaetes taurinus) species. These results confirm the influence of the host individual on the faecal microbiota, in addition to the often described influence of diet (Ley et al., 2008a; Kartzinel et al., 2019).
Based on the knowledge gained from these studies, a methodology has been developed that will enable the conservation of species in the field to be supported by microbiome research in the future. The focus here lays on the identification of host-specific metadata based on the faecal microbiota. The developed regression model is able to distinguish between carnivorous, herbivorous and omnivorous hosts with up to 99% accuracy. In addition, a more accurate phylogenetic classification of the family (Canidae, Felidae, Ursidae, Herpestidae) can be made for carnivorous hosts. For herbivorous hosts, the model can predict the respective digestive system with up to 100% accuracy, distinguishing between ruminants, hindgut fermenters and a simple digestive system. The acquisition of host-specific metadata from an unknown faecal sample is an important step towards establishing microbiome research in species conservation. Field studies in particular will benefit from such new methods. Usually, costly microsatellite analysis and high-quality host DNA are required to obtain host-specific information from faecal samples. The newly developed method offers a less costly and labour-intensive alternative to conventional techniques and opens up a more accessible field for microbiome research in the field.
Aryl hydrocarbon receptor-dependent and -independent pathways mediate curcumin anti-aging effects
(2022)
The aryl hydrocarbon receptor (AhR) is a ligand-activated transcription factor whose activity can be modulated by polyphenols, such as curcumin. AhR and curcumin have evolutionarily conserved effects on aging. Here, we investigated whether and how the AhR mediates the anti-aging effects of curcumin across species. Using a combination of in vivo, in vitro, and in silico analyses, we demonstrated that curcumin has AhR-dependent or -independent effects in a context-specific manner. We found that in Caenorhabditis elegans, AhR mediates curcumin-induced lifespan extension, most likely through a ligand-independent inhibitory mechanism related to its antioxidant activity. Curcumin also showed AhR-independent anti-aging activities, such as protection against aggregation-prone proteins and oxidative stress in C. elegans and promotion of the migratory capacity of human primary endothelial cells. These AhR-independent effects are largely mediated by the Nrf2/SKN-1 pathway.
Mucormycosis is an invasive fungal infection associated with high mortality, partly due to delayed diagnosis and inadequate empiric therapy. As fungal cultures often fail to grow Mucorales, identification of respective hyphae in tissue is frequently needed for diagnosis but may be challenging. We studied fluorescence in situ hybridization (FISH) targeting specific regions of the fungal ribosomal RNA (rRNA) of Mucorales to improve diagnosis of mucormycosis from tissue samples. We generated a probe combination specifically targeting Mucorales. Probe specificity was verified in silico and using cultivated fungi. Mucorales hyphae in tissue of a mouse model demonstrated a bright cytoplasmatic hybridization signal. In tissue samples of patients with mucormycosis, a positive signal was seen in 7 of 12 (58.3%) samples. However, autofluorescence in 3 of 7 (42.9%) samples impaired the diagnostic yield. Subsequent experiments suggested that availability of nutrients and antifungal therapy may impact on the FISH signal obtained with Mucorales hyphae. Diagnosis of mucormycosis from tissue might be improved by rRNA FISH in a limited number of cases only. FISH signals may reflect different wphysiological states of fungi in tissue. Further studies are needed to define the value of FISH to diagnose mucormycosis from other clinical samples.
Chronische Entzündungen und die daraus resultierenden Morbiditäten gehören zu den häufigsten Ursachen für einen frühen Tod beim Menschen. Einer der Hauptfaktoren für die Verschlechterung des Gesundheitszustands bei Patienten mit chronischen-entzündlichen Erkrankungen ist die pathologische Infiltration von Leukozyten in gesundes Gewebe, die zu Gewebeschäden und dem Fortschreiten der Krankheit führt. Das vaskuläre Endothel, das die Innenseite der Blutgefäße auskleidet, spielt eine entscheidende Rolle bei der Entzündungsreaktion, da es als Schnittstelle für die Interaktion mit Leukozyten fungiert, um die Extravasation von Leukozyten aus dem Blutstrom in das Gewebe zu ermöglichen. Die Adhäsion von Leukozyten an die Zellen des Endothels wird dabei hauptsächlich durch die von Zytokinen ausgelösten pro-inflammatorischen NFκB- und AP-1-Signalkaskaden ermöglicht, die die Hochregulierung der wichtigsten endothelialen Adhäsionsmoleküle – ICAM-1, VCAM-1 und E-Selektin – bewirken. Eine Klasse von Wirkstoffen, die für ihre entzündungshemmenden Eigenschaften und ihren Nutzen bei der Behandlung chronischer Entzündungskrankheiten bekannt sind, sind die Mikrotubuli-bindenden-Substanzen (microtubule-targeting-agents; MTAs), die nachweislich auch den Entzündungszustand in den Zellen des Endothels und die Leukozyten-Adhäsionskaskade beeinflussen können. MTAs lassen sich in Mikrotubuli-Destabilisatoren, die eine Depolymerisation des Mikrotubuli-Zytoskeletts bewirken, und Mikrotubuli-Stabilisatoren, die die Depolymerisation der Mikrotubuli verhindern, unterteilen. Die zugrundeliegenden biomolekularen Vorgänge und Wirkungen, die die MTAs auf die Zellen des Gefäßendothels haben, und wie sie die Adhäsionskaskade der Leukozyten beeinflussen, sind jedoch weitgehend unbekannt.
Ziel dieser Studie war es, die Auswirkungen des neuartigen Mikrotubuli-Destabilisators Prätubulysin, eines Vorläufers der Tubulysine, die ursprünglich in Stämmen des Myxobakteriums Angiococcus disciformis entdeckt wurden, auf die entzündlichen Prozesse zu untersuchen, die die Leukozyten-adhäsion in TNF-aktivierten primären Endothelzellen aus der menschlichen Nabelschnurvene (HUVECs) ermöglichen. Zusätzlich wurden auch die Auswirkungen der bereits klinisch etablierten Mikrotubuli-Destabilisatoren Colchicin und Vincristin sowie des Mikrotubuli-Stabilisators Paclitaxel untersucht.
Das entzündungshemmende Potenzial von Prätubulysin wurde daher zunächst in vivo in einem Imiquimod-induzierten psoriasiformen Dermatitis-Mausmodell getestet, wobei sich zeigte, dass Prätubulysin den Entzündungszustand deutlich verringert. Um zu beweisen, dass der entzündungshemmende Effekt mit einer verringerten Interaktion von Leukozyten mit dem Endothel zusammenhängt, wurde die Wirkung von Prätubulysin in vivo mittels Intravitalmikroskopie des TNF-aktivierten Kremaster-Muskels der Maus untersucht. Dabei zeigte sich, dass die Behandlung mit Prätubulysin zu einer signifikant verringerten Adhäsion von Leukozyten an die Zellen des Gefäßendothels führte. Die verringerte Adhäsion von Leukozyten an Endothelzellen wurde auch in der in vitro Umgebung bestätigt, indem die Adhäsion von Leukozyten unter Flussbedingungen getestet wurde. Mittels Durchflusszytometrie, Western-Blot-Analyse, sowie qRT-PCR-Analyse der jeweiligen mRNA-Level konnte gezeigt werden, dass die verringerten Leukozyten-Interaktionen auf der verringerten Expression der Zelladhäsionsmoleküle ICAM-1 und VCAM-1 sowie teilweise von E-Selektin nach Behandlung mit Prätubulysin, Vincristin und Colchicin beruhen, wobei Paclitaxel keine signifikanten hemmenden Auswirkungen hatte. Weitere Untersuchungen des Einflusses von Prätubulysin auf die NFκB- und AP-1-Signalübertragung zeigten, dass diese intrazellulären Signalkaskaden durch Prätubulysin nicht behindert werden, wobei NFκB und AP-1 weitgehend in den Promotoren der Zelladhäsionsmoleküle angereichert waren, wie durch Chromatin-Immunpräzipitation nachgewiesen wurde. Darüber hinaus induzierte die Behandlung mit Prätubulysin die Aktivität der NFκB-induzierenden Kinase IKK und führte zu einem signifikanten Anstieg der Aktivität der AP-1 Upstream-Kinase JNK, wie eine Western Blot Analyse ergab. Die Prüfung der Transkriptionsaktivität von NFκB und AP-1 in Reportergen Assays zeigte, dass insbesondere die Mikrotubuli-Destabilisatoren die Promotoraktivität dieser Transkriptionsfaktoren in einer konzentrationsabhängigen Weise verringerten. Weitere Tests zur Abhängigkeit der durch Prätubulysin induzierten Hemmung der Zelladhäsionsmoleküle von der Aktivität der JNK zeigten, dass die Hemmung empfindlich auf die Aktivität dieser Kinase reagiert. Es konnte gezeigt werden, dass die Inhibition der Aktivität der JNK die Expression der Zelladhäsionsmoleküle durch die Behandlung mit Prätubulysin auf mRNA und Proteinebene wiederherstellt. Mit Hilfe der Chromatin-Immunpräzipitation konnte weiterhin gezeigt werden, dass die Behandlung mit Prätubulysin zunächst die Assoziation des Bromodomänen-enthaltenden Proteins 4 mit den Promotoren/Genen von ICAM-1 und VCAM-1 erhöhte, aber zu einem behandlungszeitabhängigen Rückgang der Anreicherung führte. Darüber hinaus wurde durch die Behandlung mit Prätubulysin auch der Abbau dieses Proteins leicht erhöht. Durch den Einsatz eines JNK Inhibitors konnte gezeigt werden, dass die Verdrängung des Bromodomänen-enthaltenden Proteins 4 von icam-1 und vcam-1, sowie der erhöhte Abbau dieses Faktors auch von der Aktivität der JNK abhängig sind. Die Verdrängung des Bromodomänen-enthaltenden Proteins 4 induzierte auch das Vorhandensein von repressiven Chromatinmarkierungen in den Genen von ICAM-1 und VCAM-1. Die Prüfung der Anreicherung der RNA-Polymerase II an den Promotoren/Genen von ICAM-1 und VCAM-1 zeigte jedoch auch eine behandlungszeitabhängige differentielle Anreicherung dieser Polymerase, wobei die Anreicherung nach kurzen Behandlungszeiten reduziert war, sich nach mittleren Behandlungszeiten erholte und nach längeren Behandlungszeiten wieder stark reduziert war. Die anschließende Prüfung der Bedeutung des Bromodomänen-enthaltenden Proteins 4 für die Expression von ICAM-1 und VCAM-1 durch Knock-down-Experimente ergab, dass das vcam-1 Gen durch Knock-down dieses Proteins unterdrückt, das icam-1 Gen jedoch induziert wird. Dies deutet auf das Vorhandensein zusätzlicher Faktoren hin, die auch auf die Aktivität der JNK reagieren und neben dem Bromodomänen-enthaltenden Proteins 4 die Transkriptionsverlängerung des icam-1 Gens bewirken.
The negative effect of fossil-based industrial processes on the environment, especially the contribution to global warming by emitting greenhouse gases such as CO2 causes a global threat to mankind. Therefore, technologies are demanded by the society for a sustainable and environmentally friendly economy. The biotechnological use of sugar-based feedstocks to produce valuable products are in conflict with, for example, food production. In order to overcome this issue, waste products such as syngas (H2, CO and CO2) or CO2 taken from the atmosphere are of increasing interest for biotechnological applications. Acetogenic bacteria are already used at industrial scale to produce sustainable and environmentally friendly biofuels from syngas. A promising candidate due to its physiological flexibility is the thermophilic acetogen Moorella thermoacetica. In contrast to most acetogens M. thermoacetica is not restricted to one energy conserving system. In addition to the Ech complex, cytochromes and quinones may be involved in energy conservation by, for example, DMSO respiration. The extra energy conserved can be used to form highly valuable but energy demanding products. In this review we give insights into the physiology of this acetogen, the current state of the art of M. thermoacetica as a platform for biotechnological applications and discuss future perspectives.
The ability of wild animals to navigate and survive in complex and dynamic environments depends on their ability to store relevant information and place it in a spatial context. Despite the centrality of spatial memory, and given our increasing ability to observe animal movements in the wild, it is perhaps surprising how difficult it is to demonstrate spatial memory empirically. We present a cognitive analysis of movements of several wolves (Canis lupus) in Finland during a summer period of intensive hunting and den-centered pup-rearing. We tracked several wolves in the field by visiting nearly all GPS locations outside the den, allowing us to identify the species, location and timing of nearly all prey killed. We then developed a model that assigns a spatially explicit value based on memory of predation success and territorial marking. The framework allows for estimation of multiple cognitive parameters, including temporal and spatial scales of memory. For most wolves, fitted memory-based models outperformed null models by 20 to 50% at predicting locations where wolves chose to forage. However, there was a high amount of individual variability among wolves in strength and even direction of responses to experiences. Some wolves tended to return to locations with recent predation success—following a strategy of foraging site fidelity—while others appeared to prefer a site switching strategy. These differences are possibly explained by variability in pack sizes, numbers of pups, and features of the territories. Our analysis points toward concrete strategies for incorporating spatial memory in the study of animal movements while providing nuanced insights into the behavioral strategies of individual predators.
Dynamic imaging of landmark organelles, such as nuclei, cell membrane, nuclear envelope, and lipid droplets enables image-based phenotyping of functional states of cells. Multispectral fluorescent imaging of landmark organelles requires labor-intensive labeling, limits throughput, and compromises cell health. Virtual staining of label-free images with deep neural networks is an emerging solution for this problem. Multiplexed imaging of cellular landmarks from scattered light and subsequent demultiplexing with virtual staining saves the light spectrum for imaging additional molecular reporters, photomanipulation, or other tasks. Published approaches for virtual staining of landmark organelles are fragile in the presence of nuisance variations in imaging, culture conditions, and cell types. This paper reports model training protocols for virtual staining of nuclei and membranes robust to cell types, cell states, and imaging parameters. We developed a flexible and scalable convolutional architecture, named UNeXt2, for supervised training and self-supervised pre-training. The strategies we report here enable robust virtual staining of nuclei and cell membranes in multiple cell types, including neuromasts of zebrafish, across a range of imaging conditions. We assess the models by comparing the intensity, segmentations, and application-specific measurements obtained from virtually stained and experimentally stained nuclei and membranes. The models rescue the missing label, non-uniform expression of labels, and photobleaching. We share three pre-trained models, named VSCyto3D, VSCyto2D, and VSNeuromast, as well as VisCy, a PyTorch-based pipeline for training, inference, and deployment that leverages the modern OME-Zarr format.
Argonaute 2 (AGO2) is an indispensable component of the RNA-induced silencing complex, operating at the translational or posttranscriptional level. It is compartmentalized into structures such as GW- and P-bodies, stress granules and adherens junctions as well as the midbody. Here we show using immunofluorescence, image and bioinformatic analysis and cytogenetics that AGO2 also resides in membrane protrusions such as open- and close-ended tubes. The latter are cytokinetic bridges where AGO2 colocalizes at the midbody arms with cytoskeletal components such as α-Τubulin and Aurora B, and various kinases. AGO2, phosphorylated on serine 387, is located together with Dicer at the midbody ring in a manner dependent on p38 MAPK activity. We further show that AGO2 is stress sensitive and important to ensure the proper chromosome segregation and cytokinetic fidelity. We suggest that AGO2 is part of a regulatory mechanism triggered by cytokinetic stress to generate the appropriate micro-environment for local transcript homeostasis.
Oncogenic transformation of lung epithelial cells is a multi-step process, frequently starting with the inactivation of tumor suppressors and subsequent activating mutations in proto-oncogenes, such as members of the PI3K or MAPK family. Cells undergoing transformation have to adjust to changes, such as metabolic requirements. This is achieved, in part, by modulating the protein abundance of transcription factors, which manifest these adjustments. Here, we report that the deubiquitylase USP28 enables oncogenic reprogramming by regulating the protein abundance of proto-oncogenes, such as c-JUN, c-MYC, NOTCH and ΔNP63, at early stages of malignant transformation. USP28 is increased in cancer compared to normal cells due to a feed-forward loop, driven by increased amounts of oncogenic transcription factors, such as c-MYC and c-JUN. Irrespective of oncogenic driver, interference with USP28 abundance or activity suppresses growth and survival of transformed lung cells. Furthermore, inhibition of USP28 via a small molecule inhibitor reset the proteome of transformed cells towards a ‘pre-malignant’ state, and its inhibition cooperated with clinically established compounds used to target EGFRL858R, BRAFV600E or PI3KH1047R driven tumor cells. Targeting USP28 protein abundance already at an early stage via inhibition of its activity therefore is a feasible strategy for the treatment of early stage lung tumours and the observed synergism with current standard of care inhibitors holds the potential for improved targeting of established tumors.
Neurodevelopmental psychiatric disorders (NPDs) like attention deficit hyperactivity disorder (ADHD), autism spectrum disorder (ASD), and schizophrenia, affect millions of people worldwide. Despite recent progress in NPD research, much remains to be discovered about their underpinnings, therapeutic targets, effects of biological sex and age. Risk factors influencing brain development and signalling include prenatal inflammation and genetic variation. This dissertation aimed to build upon these findings by combining behavioural, molecular, and neuromorphological investigations in mouse models of such risk factors, i.e. maternal immune activation (MIA), neuron-specific overexpression (OE) of the cytoplasmatic isoforms of the RNA-binding protein RBFOX1, and neuronal deletion of the small Ras GTPase DIRAS2.
Maternal infections during pregnancy pose an increased risk for NPDs in the offspring. While viral-like MIA has been previously established elsewhere, this study was the first in our institution to implement the model. I validated NPD-relevant deficits in anxiety- and depression-like behaviours, as well as dose- and sex-specific social deficits in mouse offspring following MIA in early gestation. Proteomic analyses in embryonic and adult hippocampal (HPC) synaptoneurosomes highlighted novel and known targets affected by MIA. Analysis of the embryonic dataset implicated neurodevelopmental disruptions of the lipid, polysaccharide, and glycoprotein metabolism, important for proper membrane function, signalling, and myelination, for NPD-pertinent sequelae. In adulthood, the observed changes encompassed transmembrane trafficking and intracellular signalling, apoptosis, and cytoskeletal organisation pathways. Importantly, 50 proteins altered by MIA in embryonic and adult HPC were enriched in the NPD-relevant synaptic vesicle cycle. A persistently upregulated protein cluster formed a functional network involved in presynaptic signalling and proteins downregulated in embryos but upregulated in adults by MIA were correlated with observed social deficits. 49/50 genes encoding these proteins were significantly associated with NPD- and comorbidity-relevant traits in human phenome-wise association study data for psychiatric phenotypes. These findings highlight NPD-relevant targets for future study and early intervention in at-risk individuals. MIA-evoked changes in the neuroarchitecture of the NPD-relevant HPC and prefrontal cortex (PFC) of male and female mice highlighted sex- and region-specific alterations in dendritic and spine morphology, possibly underlining behavioural phenotypes.
To further investigate genetic risk factors of NPDs, I performed a study based on the implications of RBFOX1’s pleiotropic role in neuropsychiatric disorders and previous preclinical findings. Cytoplasmatic OE of RBFOX1, which affects the stability and translation of thousands of targets, was used to disseminate its role in morphology and behaviour. RBFOX1 OE affected dendritic length and branching in the male PFC and led to spine alterations in both PFC and HPC. Due to previously observed ASD-like endophenotypes in our Rbfox1 KO mice and the importance of gene × environment effects on NPD susceptibility, I probed the interaction of cytoplasmatic OE and a low-dose MIA on offspring. Both RBFOX1 OE alone and with MIA led to increased offspring loss during the perinatal period. Preliminary data suggested that RBFOX1 OE × MIA might increase anxiety- and anhedonia-like behaviours. Morphological changes in the adult male OE HPC and PFC suggested increased spine density and reduced dendritic complexity. A small post-mortem study in human dorsolateral PFC of older adults did not reveal significant effects of a common risk variant on RBFOX1 abundance.
To expand upon NPD genetic risks, I evaluated the effects of a homo- (KO) or heterozygous (HET) Diras2 deletion in a novel, neuron-specific mouse model. DIRAS2’s function is largely unknown, but it has been associated with ADHD in humans and neurodevelopment in vitro. In adult mice, there were subtle sex-specific effects on behaviour, i.e. more pronounced NPD-relevant deficits in males, in keeping with human data. KO mice had subtly improved cognitive performance, while HET mice exhibited behaviours in line with core ADHD symptoms, e.g. earning difficulties (females), response inhibition deficits and hyperactivity (males), suggesting Diras2 dose-sensitivity and sex-specificity. The morphological findings revealed multiple aberrations in dendritic and spine morphology in the adult PFC, HPC, and amygdala of HET males. KOs changes in spine and dendritic morphology were exclusively in the PFC and largely opposite to those in HETs and NPD-like phenotypes. Region- and genotype-specific expression changes in Diras2 and Diras1 were observed in six relevant brain regions of adult HET and KO females, also revealing differences in the survival and morphology regulator mTOR, which might underlie observed differences.
In conclusion, the effects of MIA and partial Diras2 knockdown resembled each other in core, NPD-associated behavioural and morphological phenotypes, while cytoplasmatic RBFOX1 OE and full Diras2 KO differed from those. My findings suggest complex dose- and sex-dependent relationships between these prenatal and genetic interventions, whose NPD-relevant influences might converge onto neurodevelopmental molecular pathways. An assessment of such putative overlap, based on available data from the MIA proteomic analyses of embryonic and adult HPC, suggested the three models might be linked via downstream targets, interactions, and upstream regulators. Future studies should disseminate both distinct and shared aspects of MIA, RBFOX1, and DIRAS2 relevant to NPDs and build upon these findings.
Mitochondria are dynamic organelles exhibiting diverse shapes. While the variation of shapes, ranging from spheres to elongated tubules, and the transition between them, are clearly seen in many cell types, the molecular mechanisms governing this morphological variability remain poorly understood. Here, we propose a novel shaping mechanism based on the interplay between the inner and outer mitochondrial membranes. Our biophysical model suggests that the difference in surface area, arising from the pulling of the inner membrane into cristae, correlates with mitochondrial elongation. Analysis of live cell super-resolution microscopy data supports this correlation, linking elongated shapes to the extent of cristae in the inner membrane. Knocking down cristae shaping proteins further confirms the impact on mitochondrial shape, demonstrating that defects in cristae formation correlate with mitochondrial sphericity. Our results suggest that the dynamics of the inner mitochondrial membrane are important not only for simply creating surface area required for respiratory capacity, but go beyond that to affect the whole organelle morphology. This work explores the biophysical foundations of individual mitochondrial shape, suggesting potential links between mitochondrial structure and function. This should be of profound significance, particularly in the context of disrupted cristae shaping proteins and their implications in mitochondrial diseases.
It is widely acknowledged that biodiversity change is affecting human well-being by altering the supply of Nature's Contributions to People (NCP). Nevertheless, the role of individual species in this relationship remains obscure. In this article, we present a framework that combines the cascade model from ecosystem services research with network theory from community ecology. This allows us to quantitatively link NCP demanded by people to the networks of interacting species that underpin them. We show that this “network cascade” framework can reveal the number, identity and importance of the individual species that drive NCP and of the environmental conditions that support them. This information is highly valuable in demonstrating the importance of biodiversity in supporting human well-being and can help inform the management of biodiversity in social-ecological systems.
Tree-related microhabitats (TReMs) have been proposed as important indicators of biodiversity to guide forest management. However, their application has been limited mostly to temperate ecosystems, and it is largely unknown how the diversity of TReMs varies along environmental gradients. In this study, we assessed the diversity of TReMs on 180 individual trees and 44 plots alongside a large environmental gradient on Kilimanjaro, Tanzania. We used a typology adjusted to tropical ecosystems and a tree-climbing protocol to obtain quantitative information on TreMs on large trees and dense canopies. We computed the diversity of TReMs for each individual tree and plot and tested how TReM diversity was associated with properties of individual trees and environmental conditions in terms of climate and human impact. We further used non-metric multidimensional scaling (NMDS) to investigate the composition of TReM assemblages alongside the environmental gradients. We found that diameter at breast height (DBH) and height of the first branch were the most important determinants of TReM diversity on individual trees, with higher DBH and lower first branch height promoting TReM diversity. At the plot level, we found that TReM diversity increased with mean annual temperature and decreased with human impact. The composition of TReMs showed high turnover across ecosystem types, with a stark difference between forest and non-forest ecosystems. Climate and the intensity of human impact were associated with TReM composition. Our study is a first test of how TReM diversity and composition vary along environmental gradients in tropical ecosystems. The importance of tree size and architecture in fostering microhabitat diversity underlines the importance of large veteran trees in tropical ecosystems. Because diversity and composition of TReMs are sensitive to climate and land-use effects, our study suggests that TReMs can be used to efficiently monitor consequences of global change for tropical biodiversity.
Tree-related microhabitats (TReMs) have been proposed as important indicators of biodiversity to guide forest management. However, their application has been limited mostly to temperate ecosystems, and it is largely unknown how the diversity of TReMs varies along environmental gradients. In this study, we assessed the diversity of TReMs on 180 individual trees and 46 plots alongside a large environmental gradient on Kilimanjaro, Tanzania. We used a typology adjusted to tropical ecosystems and a tree-climbing protocol to obtain quantitative information on TreMs on large trees and dense canopies. We computed the diversity of TReMs for each individual tree and plot and tested how TReM diversity was associated with properties of individual trees and environmental conditions in terms of climate and human impact. We further used non-metric multidimensional scaling (NMDS) to investigate the composition of TReM assemblages alongside the environmental gradients. We found that diameter at breast height (DBH) and height of the first branch were the most important determinants of TReM diversity on individual trees, with higher DBH and lower first branch height promoting TReM diversity. At the plot level, we found that TReM diversity increased with mean annual temperature and decreased with human impact. The composition of TReMs showed high turnover across ecosystem types, with a stark difference between forest and non-forest ecosystems. Climate and the intensity of human impact were associated with TReM composition. Our study is a first test of how TReM diversity and composition vary along environmental gradients in tropical ecosystems. The importance of tree size and architecture in fostering microhabitat diversity underlines the importance of large veteran trees in tropical ecosystems. Because diversity and composition of TReMs are sensitive to climate and land-use effects, our study suggests that TReMs can be used to efficiently monitor consequences of global change for tropical biodiversity.
This thesis investigates the structure of the translocase of the outer membrane (TOM) complex in mitochondria, focusing on the TOM holo complex through single-particle electron cryo-microscopy (cryoEM) complemented by mass spectrometry and computational structure prediction. Mitochondria, crucial for energy production in eukaryotic cells, import most of their proteins from the cytoplasm. These proteins enter through the TOM complex, which in its core form consists of a membrane-embedded homodimer of Tom40 pores, two Tom22 cytoplasmic receptors, and six small TOM stabilizing subunits (Tom7, Tom6, and Tom5). The holo complex includes two additional subunits, Tom70 and Tom20, whose stoichiometry and positioning are less understood due to their easy dissociation during isolation of the complex. CryoEM analysis revealed the high-resolution structure of the Neurospora crassa TOM core complex at 3.3 Å, containing all core subunits, and the presence of a central phospholipid causing the Tom40 dimer to tilt to 20°. Furthermore, a 4 Å resolution map indicated the binding of a precursor protein as it transitions through the translocation barrel. Finally, at 6-7 Å resolution, the structure of the TOM holo complex highlighted Tom20's flexibility as it interacts with the core complex, emphasizing its role in protein translocation. This work provides significant insights into the architecture and functioning of the TOM complex, contributing to the understanding of mitochondrial protein import mechanisms.
This work focused on the biosynthesis and characterization of esterified lipid mediators. Lipid mediators were generally thought to exert their effects as free molecules, and their esterification was regarded as a storage mechanism. However, more recent studies indicate that esterified lipid mediators are a distinct class of mediators. When this thesis started back in 2017, the idea of esterified lipids as a new class of mediators was relatively new so that respective compounds were either quite expensive or not commercially available at all. Therefore, a biosynthetic approach had to be established first to enable the study of the new lipid mediator class. Within the cell, esterified lipids are produced by activation and subsequent incorporation of polyunsaturated fatty acids. These steps are enzymatically catalyzed by members of the acyl-CoA synthetase family and the lysophosphatidylcholine acyltransferase family, respectively. Therefore, the enzymes acyl-CoA synthetase long-chain family member 4 (ACSL4) and lysophosphatidylcholine acyltransferase 2 (LPCAT2) were selected for a biosynthetic approach due to their broad substrate acceptance.
In a first attempt, recombinant protein expression in E. coli was studied. While the expression and purification of C-terminally His6x-tagged ACSL4 resulted in a pure and active protein, the expression of LPCAT2 turned out quite troublesome. Although several expression and purification parameters were varied, including purification tags, buffer compositions, and chromatography strategies, successful purification of LPCAT2 was not achieved.
Instead, a second approach was studied. This time, stably transfected cells overexpressing ACSL4 and/or LPCAT2 were generated from the human embryonal kidney (HEK) 293T cell line. Stably transfected cell lines were characterized on protein level and regarding their oxylipin profile. After confirming the overexpression and functionality of the enzymes, lipoxygenases (LOs) were co-expressed in a doxycycline-inducible manner to prevent premature cell death due to increased oxidative stress. As a result, LO product formation was enhanced and enabled the investigation of specific oxylipins. Since increased lipid peroxidation is also a key component of the ferroptosis cell death mechanisms, cell lines were investigated towards their cell viability. Indeed, expression of ACSL4 and/or LPCAT2 promoted cell death when treated with the ferroptosis inducers erastin or RSL3, even in the absence of LO expression. Furthermore, analysis by laser scanning confocal microscopy revealed that the localization of 15-LO1 was altered in the presence of LPCAT2, similar to treatment with RSL3 in vector control cells.
In conclusion, a stable overexpression system of ACSL4 and/or LPCAT2 was successfully established in HEK293T cells, which enabled the synthesis and characterization of esterified oxylipins. Interestingly, characterization of the cell lines revealed a correlation with the cell death mechanism ferroptosis. Although the expression of ACSL4 has already been reported as a biomarker for ferroptosis, this is the first time that a potential connection of LPCAT2 with ferroptosis was demonstrated. As a result, this may provide new therapeutic options for ferroptosis-related pathologies such as neurodegeneration, autoimmune diseases, or tumorigenesis.
The ligand-sensing transcription factor Nurr1 emerges as a promising therapeutic target for neurodegenerative pathologies but Nurr1 ligands for functional studies and therapeutic validation are lacking. Here pronounced Nurr1 modulation by statins for which clinically relevant neuroprotective effects are demonstrated, is reported. Several statins directly affect Nurr1 activity in cellular and cell-free settings with low micromolar to sub-micromolar potencies. Simvastatin as example exhibits anti-inflammatory effects in astrocytes, which are abrogated by Nurr1 knockdown. Differential gene expression analysis in native and Nurr1-silenced cells reveals strong proinflammatory effects of Nurr1 knockdown while simvastatin treatment induces several neuroprotective mechanisms via Nurr1 involving changes in inflammatory, metabolic and cell cycle gene expression. Further in vitro evaluation confirms reduced inflammatory response, improved glucose metabolism, and cell cycle inhibition of simvastatin-treated neuronal cells. These findings suggest Nurr1 involvement in the well-documented but mechanistically elusive neuroprotection by statins.
Human feline leukaemia virus subgroup C receptor-related proteins 1 and 2 (FLVCR1 and FLVCR2) are members of the major facilitator superfamily1. Their dysfunction is linked to several clinical disorders, including PCARP, HSAN and Fowler syndrome2,3,4,5,6,7. Earlier studies concluded that FLVCR1 may function as a haem exporter8,9,10,11,12, whereas FLVCR2 was suggested to act as a haem importer13, yet conclusive biochemical and detailed molecular evidence remained elusive for the function of both transporters14,15,16. Here, we show that FLVCR1 and FLVCR2 facilitate the transport of choline and ethanolamine across the plasma membrane, using a concentration-driven substrate translocation process. Through structural and computational analyses, we have identified distinct conformational states of FLVCRs and unravelled the coordination chemistry underlying their substrate interactions. Fully conserved tryptophan and tyrosine residues form the binding pocket of both transporters and confer selectivity for choline and ethanolamine through cation–π interactions. Our findings clarify the mechanisms of choline and ethanolamine transport by FLVCR1 and FLVCR2, enhance our comprehension of disease-associated mutations that interfere with these vital processes and shed light on the conformational dynamics of these major facilitator superfamily proteins during the transport cycle.
Background: Despite advances in treatment of patients with non-small cell lung cancer, carriers of certain genetic alterations are prone to failure. One such factor frequently mutated, is the tumor suppressor PTEN. These tumors are supposed to be more resistant to radiation, chemo- and immunotherapy.
Results: We demonstrate that loss of PTEN led to altered expression of transcriptional programs which directly regulate therapy resistance, resulting in establishment of radiation resistance. While PTEN-deficient tumor cells were not dependent on DNA-PK for IR resistance nor activated ATR during IR, they showed a significant dependence for the DNA damage kinase ATM. Pharmacologic inhibition of ATM, via KU-60019 and AZD1390 at non-toxic doses, restored and even synergized with IR in PTEN-deficient human and murine NSCLC cells as well in a multicellular organotypic ex vivo tumor model.
Conclusion: PTEN tumors are addicted to ATM to detect and repair radiation induced DNA damage. This creates an exploitable bottleneck. At least in cellulo and ex vivo we show that low concentration of ATM inhibitor is able to synergise with IR to treat PTEN-deficient tumors in genetically well-defined IR resistant lung cancer models.
Human feline leukemia virus subgroup C receptor-related proteins 1 and 2 (FLVCR1 and 2) are members of the major facilitator superfamily1. Their dysfunction is linked to several clinical disorders, including PCARP, HSAN, and Fowler syndrome2–7. Earlier studies concluded that FLVCR1 may function as a putative heme exporter8–12, while FLVCR2 was suggested to act as a heme importer13, yet conclusive biochemical and detailed molecular evidence remained elusive for the function of both transporters14–17. Here, we show that FLVCR1 and FLVCR2 facilitate the transport of choline and ethanolamine across human plasma membranes, utilizing a concentration-driven substrate translocation process. Through structural and computational analyses, we have identified distinct conformational states of FLVCRs and unraveled the coordination chemistry underlying their substrate interactions. Within the binding pocket of both transporters, we identify fully conserved tryptophan and tyrosine residues holding a central role in the formation of cation-π interactions, essential for choline and ethanolamine selectivity. Our findings not only clarify the mechanisms of choline and ethanolamine transport by FLVCR1 and FLVCR2, enhancing our comprehension of disease-associated mutations that interfere with these vital processes, but also shed light on the conformational dynamics of these MFS-type proteins during the transport cycle.
The hydrothermal vent tubeworm Riftia pachyptila hosts a single 16S rRNA phylotype of intracellular sulfur-oxidizing symbionts, which vary considerably in cell morphology and exhibit a remarkable degree of physiological diversity and redundancy, even in the same host. To elucidate whether multiple metabolic routes are employed in the same cells or rather in distinct symbiont subpopulations, we enriched symbionts according to cell size by density gradient centrifugation. Metaproteomic analysis, microscopy, and flow cytometry strongly suggest that Riftia symbiont cells of different sizes represent metabolically dissimilar stages of a physiological differentiation process: While small symbionts actively divide and may establish cellular symbiont-host interaction, large symbionts apparently do not divide, but still replicate DNA, leading to DNA endoreduplication. Moreover, in large symbionts, carbon fixation and biomass production seem to be metabolic priorities. We propose that this division of labor between smaller and larger symbionts benefits the productivity of the symbiosis as a whole.
How the brain evolved remains a mystery. The goal of this thesis is to understand the fundamental processes that are behind the evolutionary history of the brain. Amniotes appeared 320 million years ago with the transition from water to land. This early group bifurcated into sauropsids (reptiles and birds) and synapsids (mammals). Amniote brains evolved separately and display obvious structural and functional differences. Although those differences reflect brain diversification, all amniote brains share a common ancestor and their brains show multiple derived similarities: equivalent structures, networks, circuits and cell types have been preserved during millions of years. Finding these differences and similarities will help us understand brain historical evolution and function. Studying brain evolution can be approached from various levels, including brain structure, circuits, cell types, and genes. We propose a focus on cell types for a more comprehensive understanding of brain evolution. Neurons are the basic building blocks and the most diverse cell types in the brain. Their evolution reflects changes in the developmental processes that produce them, which in turn may shape the neural circuits they belong to. However, there is currently a lack of a unified criteria for studying the homology of connectivity and development between neurons. A neuron’s transcriptome is a molecular representation of its identity, connectivity, and developmental/evolutionary history. Hence the comparison of neuronal transcriptomes within and across species is a new and transformative development in the study of brain evolution. As an alternative, comparing neuronal transcriptomes across different species can provide insights into the evolution of the brain. We propose that comparing transcriptomes can be a way to fill this gap and unify these criteria. In previous studies, published in Science (Tosches et al., 2018) and Nature (Norimoto et al., 2020), we leveraged scRNAseq in reptiles to re-evaluate the origins and evolution of the mammalian cerebral cortex and claustrum. Motivated by the success of this approach, in this thesis we have now expanded single-cell profiling to the entire brain of a lizard species, the Australian dragon Pogona vitticeps, with a special focus in thalamus and prethalamus of. This approach allowed us to study the evolution of neuron types in amniotes. Therefore, we aimed to build a multilevel atlas of the lizard brain based on histology and transcriptomic and compare it to an equal mouse dataset (Zeisel et al., 2018).
Our atlas reveals a general structure that is consistent with that for other amniote brains, allowing us to make a direct comparison between lizard and mouse, despite their evolutionary divergence 320 million years ago. Through our analysis of the transcriptomes present in various neuron types, we have uncovered a core of conserved classes and discovered a fascinating dichotomy of new and conserved neuron types throughout the brain. This research challenges the traditional notion that certain brain regions are more conserved than others.
Our research also has uncovered the evolutionary history of the lizard thalamus and prethalamus by comparing them to homologous brain regions of the mouse. This pioneering research sheds new light on our understanding of the evolutionary history of the lizard brain. We propose a new classification of the lizard thalamic nuclei based on
transcriptomics. Our research revealed that the thalamic neuron types in lizards can be grouped into two large, conserved categories from the medial to lateral thalamus. These categories are encoded by a common set of effector genes, linking theories based on connectivity and molecular studies of these areas. In our data we have seen that there is a conservation of the medial-lateral transcriptomic axis in mouse and lizard, this conservation was most likely already present in the common ancestor. Although there is a shared medial-lateral axis, a deeper study of the thalamic cell types has allowed us to see the existence of a partial diversification of the thalamic population, specifically in the sensory-related lateral thalamus; in opposition, the medial thalamic nuclei neuron-types have been preserved.
On the other hand, the comparison with the mammalian prethalamus allowed us to confirm that the lizard ventromedial thalamic neuron types are homologous to mouse reticular thalamic neuron types (Díaz et al., 1994), even if they do not express the classical Reticular thalamic nucleus (RTn) marker PV/pvalb. We also discovered that there has been a simplification in the mammalian prethalamic neuron types in favor of an increase in the number of Interneurons (IN) types within their thalamus. We suggest that the loss of GABAergic neuronal types in the mammalian prethalamus is linked to the need for a more efficient control of the thalamo-pallial communication in mammals, while in lizards, where thalamo-pallial communication is probably simpler, the diversity prethalamus presents a higher diversity.
Biological membranes serve as physical barriers in cells and organelles, enabling the maintenance of chemical or ionic gradients that are essential for triggering various integral, peripheral, or lipid-anchored membrane proteins, necessary for their life-essential functions. The study of membrane proteins has unique challenges due to their hydrophobic nature, limited expression levels, and inherent flexibility. Single-particle analysis (SPA) enables the determination of high-resolution three-dimensional structures using minimal amounts of specimen without the need for crystallization. Additionally, cryogenic electron tomography (cryo-ET) and subtomogram averaging (StA) offer the ability to study membrane protein complexes, cellular architecture, and molecular interactions while preserving close-to-life conditions. With ongoing improvements in cryo-EM technologies, obtaining high-resolution structures of membrane proteins in vitro can allow people to understand their mechanisms and functions, and to facilitate the design and optimization of new therapeutic agents. Furthermore, there has been significant growth in the structural characterization of membrane proteins in situ, as studying biomolecules within their physiological context is an ultimate goal in structural biology for a comprehensive understanding of molecular networks in cells.
Due to the amphipathic nature of membrane proteins, their production, purification, and isolation pose significant challenges compared to soluble proteins. To maintain the membrane protein fold in an aqueous buffer after disrupting lipid membranes, the use of detergents, amphipols, lipid nanodiscs, saposin-lipoprotein (salipro), styrene-maleic acid co-polymer lipid particles (SMALPS) is common and often essential. A limitation of the membrane-mimetic systems is the absence of an actual lipid bilayer environment. To address this issue, membrane proteins can be reconstituted into liposomes, and this closed membrane environment closely mimics the physiological conditions of the proteins. The use of liposomes for structure determination is expected to significantly expand in the in vitro study of membrane proteins and membrane-associated proteins, particularly for capturing transient complexes in specific functional states.
Resolving the structures of membrane proteins in their native cellular context is considered the ideal approach for understanding their functions and associated molecular networks. While single-particle cryo-EM can achieve higher resolution than subtomogram averaging, it often requires at least partial purification of the target molecules from their native environment inside cells and tissues. By combining averaging tools on subvolumes obtained through cryo-ET, structures can currently be determined at resolutions of 10-30 Å. With ongoing advancements and refinements in cryo-ET methodologies, routine high-resolution structure determination in situ is poised to become a valuable tool for both structural and cell biologists in the long run, and the field holds great promise for further expanding our understanding of cellular structures and processes at the molecular level.
The main aim of this thesis is to further our knowledge of the structure and function of a small prokaryotic voltage-gated sodium ion channel, NaChBac in liposomes, and a large knob complex found on the surface of Plasmodium falciparum-infected human erythrocyte by cryo-ET and StA.
Chapter 2 presents the first StA map of the 120-kDa NaChBac embedded in liposomes under a resting membrane potential at a modest resolution of 16 Å. The approach presented in this study, which can be widely applied to cryo-EM analysis of membrane proteins, with a specific focus on membrane proteins with small soluble domains, lays the foundation for cryo-ET and StA of integral or peripheral membrane proteins whose functions are affected by transmembrane electrochemical gradients and/or membrane curvatures. Chapter 3 shows the first cryo-EM structure of the supramolecular knob complex in P. falciparum-infected human erythrocyte. While a previous study provided an overall architectural view of knobs using negative stain tomography, the in situ structure bridges this gap, guiding future investigations into the molecular composition and the role of these native knobs in Plasmodium infection and immunity.
This thesis opens up several promising lines for future studies of membrane proteins in vitro and in situ, where other membrane proteins can be studied in physiologically relevant environments. Already with the present generation of cryo-EM hardware and software, this thesis represents pioneering research in the field of membrane protein structural biology.
An important goal is to identify the direct activation domain (AD)-interacting components of the transcriptional machinery within the context of native complexes. Toward this end, we first demonstrate that the multisubunit TFIID, SAGA, mediator, and Swi/Snf coactivator complexes from transcriptionally competent whole-cell yeast extracts were all capable of specifically interacting with the prototypic acidic ADs of Gal4 and VP16. We then used hexahistidine tags as genetically introduced activation domain-localized cross-linking receptors. In combination with immunological reagents against all subunits of TFIID and SAGA, we systematically identified the direct AD-interacting subunits within the AD-TFIID and AD-SAGA coactivator complexes enriched from whole-cell extracts and confirmed these results using purified TFIID and partially purified SAGA. Both ADs directly cross-linked to TBP and to a subset of TFIID and SAGA subunits that carry histone-fold motifs.
Owing to their morphological complexity and dense network connections, neurons modify their proteomes locally, using mRNAs and ribosomes present in the neuropil (tissue enriched for dendrites and axons). Although ribosome biogenesis largely takes place in the nucleus and perinuclear region, neuronal ribosomal protein (RP) mRNAs have been frequently detected remotely, in dendrites and axons. Here, using imaging and ribosome profiling, we directly detected the RP mRNAs and their translation in the neuropil. Combining brief metabolic labeling with mass spectrometry, we found that a group of RPs rapidly associated with translating ribosomes in the cytoplasm and that this incorporation was independent of canonical ribosome biogenesis. Moreover, the incorporation probability of some RPs was regulated by location (neurites vs. cell bodies) and changes in the cellular environment (following oxidative stress). Our results suggest new mechanisms for the local activation, repair and/or specialization of the translational machinery within neuronal processes, potentially allowing neuronal synapses a rapid means to regulate local protein synthesis.
Owing to their morphological complexity and dense network connections, neurons modify their proteomes locally, using mRNAs and ribosomes present in the neuropil (tissue enriched for dendrites and axons). Although ribosome biogenesis largely takes place in the nucleus and perinuclear region, neuronal ribosomal protein (RP) mRNAs have been frequently detected remotely, in dendrites and axons. Here, using imaging and ribosome profiling, we directly detected the RP mRNAs and their translation in the neuropil. Combining brief metabolic labeling with mass spectrometry, we found that a group of RPs quickly associated with translating ribosomes in the cytoplasm and that this incorporation is independent of canonical ribosome biogenesis. Moreover, the incorporation probability of some RPs was regulated by location (neurites vs. cell bodies) and changes in the cellular environment (in response to oxidative stress). Our results suggest new mechanisms for the local activation, repair and/or specialization of the translational machinery within neuronal processes, potentially allowing remote neuronal synapses a rapid solution to the relatively slow and energy-demanding requirement of nuclear ribosome biogenesis.
Protein turnover, the net result of protein synthesis and degradation, enables cells to remodel their proteomes in response to internal and external cues. Previously, we analyzed protein turnover rates in cultured brain cells under basal neuronal activity and found that protein turnover is influenced by subcellular localization, protein function, complex association, cell type of origin, and by the cellular environment (Dörrbaum et al., 2018). Here, we advanced our experimental approach to quantify changes in protein synthesis and degradation, as well as the resulting changes in protein turnover or abundance in rat primary hippocampal cultures during homeostatic scaling. Our data demonstrate that a large fraction of the neuronal proteome shows changes in protein synthesis and/or degradation during homeostatic up- and down-scaling. More than half of the quantified synaptic proteins were regulated, including pre- as well as postsynaptic proteins with diverse molecular functions.
Mitochondrial matrix peptidase CLPP is crucial during cell stress. Its loss causes Perrault syndrome type 3 (PRLTS3) with infertility, neurodegeneration and growth deficit. Its target proteins are disaggregated by CLPX, which also regulates heme biosynthesis via unfolding ALAS enzyme, providing access of pyridoxal-5’-phosphate (PLP). Despite efforts in diverse organisms with multiple techniques, CLPXP substrates remain controversial. Here, avoiding recombinant overexpression, we employed complexomics in mitochondria from three mouse tissues to identify endogenous targets. CLPP absence caused accumulation and dispersion of CLPX-VWA8 as AAA+ unfoldases, and of PLPBP. Similar changes and CLPX-VWA8 comigration were evident for mitoribosomal central protuberance clusters, translation factors like GFM1-HARS2, RNA granule components LRPPRC-SLIRP, and enzymes OAT-ALDH18A1. Mitochondrially translated proteins in testis showed reductions to <30% for MTCO1-3, misassembly of complex-IV supercomplex, and accumulated metal-binding assembly factors COX15-SFXN4. Indeed, heavy metal levels were increased for iron, molybdenum, cobalt and manganese. RT-qPCR showed compensatory downregulation only for Clpx mRNA, most accumulated proteins appeared transcriptionally upregulated. Immunoblots validated VWA8, MRPL38, MRPL18, GFM1 and OAT accumulation. Coimmunoprecipitation confirmed CLPX binding to MRPL38, GFM1 and OAT, so excess CLPX and PLP may affect their activity. Our data elucidate mechanistically the mitochondrial translation fidelity deficits, which underlie progressive hearing impairment in PRLTS3.
Viruses that carry a positive-sense, single-stranded (+ssRNA) RNA translate their genomes soon after entering the host cell to produce viral proteins, with the exception of retroviruses. A distinguishing feature of retroviruses is reverse transcription, where the +ssRNA genome serves as a template to synthesize a double-stranded DNA copy that subsequently integrates into the host genome. As retroviral RNAs are produced by the host cell transcriptional machinery and are largely indistinguishable from cellular mRNAs, we investigated the potential of incoming retroviral genomes to directly express proteins. Here we show through multiple, complementary methods that retroviral genomes are translated after entry. Our findings challenge the notion that retroviruses require reverse transcription to produce viral proteins. Synthesis of retroviral proteins in the absence of productive infection has significant implications for basic retrovirology, immune responses and gene therapy applications.
Viruses that carry a positive-sense, single-stranded RNA translate their genomes after entering the host cell to produce viral proteins, with the exception of retroviruses. A distinguishing feature of retroviruses is reverse transcription, where the ssRNA genome serves as a template to synthesize a double-stranded DNA copy that subsequently integrates into the host genome. As retroviral RNAs are produced by the host transcriptional machinery and are largely indistinguishable from cellular mRNAs, we investigated the potential of incoming retroviral genomes to express proteins. Here we show through various biochemical methods that HIV-1 genomes are translated after entry, in case of minimal or full-length genomes, envelopes using different cellular entry pathways and in diverse cell types. Our findings challenge the dogma that retroviruses require reverse transcription to produce viral proteins. Synthesis of retroviral proteins in the absence of productive infection has significant implications for basic retrovirology, immune responses and gene therapy applications.
We examined the feedback between the major protein degradation pathway, the ubiquitin-proteasome system (UPS), and protein synthesis in rat and mouse neurons. When protein degradation was inhibited, we observed a coordinate dramatic reduction in nascent protein synthesis in neuronal cell bodies and dendrites. The mechanism for translation inhibition involved the phosphorylation of eIF2α, surprisingly mediated by eIF2α kinase 1, or heme-regulated kinase inhibitor (HRI). Under basal conditions, neuronal expression of HRI is barely detectable. Following proteasome inhibition, HRI protein levels increase owing to stabilization of HRI and enhanced translation, likely via the increased availability of tRNAs for its rare codons. Once expressed, HRI is constitutively active in neurons because endogenous heme levels are so low; HRI activity results in eIF2α phosphorylation and the resulting inhibition of translation. These data demonstrate a novel role for neuronal HRI that senses and responds to compromised function of the proteasome to restore proteostasis.
Impact of pectin dietary supplementation on experimental food allergy via gut microbiota modulation
(2023)
In recent years, dietary fibers gained focus in regard of their immune-modulatory effects and the potentially beneficial effect on allergies. The dietary fiber and prebiotic pectin is able to promote growth and activity of beneficial bacteria and thereby induce modulation of different immune responses. However, structurally different types of pectin might promote different immune-modulatory responses and to date the optimal pectin type for induction of beneficial health effects is not identified. Furthermore, it is still unclear, whether pectins provide a beneficial effect on certain allergies, such as food allergy.
Having this in consideration, this study examined the immune-modulatory effects of structurally different pectins on naive as well as peach allergic mice. Furhtermore, the impact of dietary pectin supplementation on composition and diversity of the murine gut microbiota was determined.
This study showed that dietary pectin intervention was able to suppress allergy-related Th2 responses considering humoral and cellular immune responses. Only apple-derived high-methoxyl pectin revealed an impact on total IgA levels and affected the microbial richness. Furthermore, it is not known whether the effects observed with the two pectins are caused by modulations of the bacterial composition or induced at least partly by direct interaction with the immune cells. Further studies are required to fully understand the mechanisms underlying the immune-modulatory capacities of different pectins.
Finally, the obtained results generated evidence that dietary pectin intervention can beneficially modulate the immune response in healthy mice and – at least partially – suppress allergy-related immune responses in a model of food allergy, depending on the structural characteristics of the used pectin.
Two new species of Fomitopsidaceae, Pseudofomitopsis fusca R.Saha, A.K.Dutta & K.Acharya sp. nov. and Fomitopsis benghalensis R.Saha, A.K.Dutta & K.Acharya sp. nov., are described from West Bengal, India, based on morphological and molecular phylogenetic analyses (nuclear ITS sequence). Pseudofomitopsis fusca sp. nov. possesses perennial, triquetrous to ungulate, sessile basidiocarps with a shiny, glabrous, azonate, dark brown upper surface, a yellowish grey pore surface with angular pores (3–5 per mm), a dimitic type of hyphal system with clamped generative hyphae, fusoid cystidioles; ellipsoid, cotton blue positive, and basidiospores 3–5 × 1.5–3.5 µm. Fomitopsis benghalensis sp. nov. is characterized by its annual, resupinate basidiocarp with pilose, bluish white to orange-grey, warty, woody upper surface, bluish-white pore surface, circular to angular pores (5–7 per mm), a trimitic type of hyphal system with clamped generative hyphae, fusoid cystidioles, and cylindrical to elongate basidiospores (5.5–8 × 2.5–3.5 µm). The new taxa are compared to closely related taxa. Photomicrographs of the basidiocarps, along with detailed morphological descriptions and a molecular sequence-based phylogenetic tree, are provided.
The prefrontal cortex (PFC) is considered the cognitive center of the mammalian brain. It is involved in a variety of cognitive functions such as decision making, working memory, goal-directed behavior, processing of emotions, flexible action selection, attention, and others (Fuster, 2015). In rodents, these functions are associated with the medial prefrontal cortex (mPFC). Experiments in mice and rats have shown that neurons in the mPFC are necessary for successful performance of many cognitive tasks. Moreover, measurements of neural activity in the mPFC show excitation or inhibition in different cells in relation to specific aspects of the tasks to be solved. To date, however, it is largely unknown whether prefrontal neurons are stably activated during the same behaviors within a task and whether similar aspects are represented by the same neurons in different tasks. In addition, it is unclear how specifically neurons are activated, for example, whether cells that are activated in response to reward are activated in a different task without reward in a different situation or remain inactive. To address these questions, we recorded the same neurons in the mPFC of mice over the course of several weeks while the animals performed various behaviors.
To do this, we expressed GCaMP6 in pyramidal neurons in the mPFC of mice. A small lens was implanted in the same location and a miniature microscope ("miniscope") was used to record neural activity. Later the extracted neurons got aligned based on their shape and position across multiple days and sessions. The mice performed five different behavioral tests while neural activity was measured: A spatial working memory test in a T-maze, exploration of the elevated plus maze (EPM), a novel object recognition (NO) test including free open field (OF) exploration, a social interaction (SI) test and discriminatory auditory fear conditioning (FC). Each task was repeated at least twice to check for stable task encoding across sessions. Behavioral performance and neural correlates to specific task events were similar to earlier studies across all tasks. We utilized generalized linear models (GLM) to determine which behavioral variables most strongly influence neural activity in the mPFC. The position of the mouse in the environment was found to explain most of the variance in neural activity, together with movement speed they were the strongest predictors of neural activity across all tasks. Reward time points in the working memory test, the conditioned stimulus after fear conditioning, or head direction in general were also strongly encoded in the mPFC.
Many of the recorded neurons showed a stable spatial activity profile across multiple sessions of the same task. Similarly, cells that coded for position in one task tended to code for position in other tasks. Not only did the same cells code for position across multiple tasks, but cells also coded for movement speed and head direction. This indicates that at least these general behavioral variables are each represented by the same neurons in the mPFC. Interestingly, the stability of position or speed coding did not depend on the time between two sessions, but only on whether it was within the same or across different tasks. Within the same task, stability was slightly higher than across different tasks.
To find out whether task-specific behavioral aspects were also stably encoded in the mPFC, difference scores as the difference in neural activity between two task aspects like left- and right-choice trials or exposed and enclosed locations were calculated. Many cells encoded these aspects stably across different sessions of each task. Both the left-right differences in the different phases of the working memory test, the open-closed-arm differences in the elevated plus maze, the different activity between center and corners in the open field, the social target-object differences in the social interaction test, and the differences between the two tones during fear conditioning were all stably encoded across the population of mPFC cells. Only the distinction between the novel and the familiar object during object recognition was not stably encoded, but also the preference for the novel object was not present in the second session of novel object exploration.
There was also an overlap in coding for different aspects within a task across multiple sessions. For example, cells stably encoded left-right differences in the T-maze between different sessions as a function of walking direction across different phases of working memory, an aspect that we could already show within one session (Vogel, Hahn et al., 2022). During fear conditioning, the same cells showed a discrimination between CS+ and CS- that also responded to the start of CS+.
Consistency in the neurons activity across different tasks was also found, but only between tasks with similar demands, the elevated plus-maze and free exploration of the open field. Cells that were more active in the open arms also showed more activity in the center of the open field and vice versa. This could be an indicator that the cells were coding for anxiety or exposure across those tasks, indicating that neurons in the mPFC also stably encode general task aspects independent of the specific environment. However, it remains unclear what exactly these neurons encode; in the case of a general fear signal, one would also expect activation during fear conditioning which could not be found.
Overall, we found that neurons in the mPFC of mice encoded multiple general behavioral variables across multiple tasks and task-specific variables were encoded stably within each of the tested tasks. However, we found little task-specific variables that were systematically encoded by the same neurons with the exception being the elevated plus-maze and open field exploration, two tasks with similar features.
In (eco-)toxicological studies the light/dark transition (LDT) test is one of the most frequently used behaviour assays with zebrafish eleutheroembryos. However, study results vary regarding data presentation and analysis and mostly focus on a limited amount of the recorded data. In this study, we investigated whether monitoring two behavioural outcomes (time and distance moved) together with analysing multiple parameters can improve test sensitivity and data interpretation. As a proof of principle 5-day old zebrafish (Danio rerio) eleutheroembryos exposed to either endocrine disruptors (EDs) or acetylcholine esterase (AChE) inhibitors were investigated. We analysed conventional parameters such as mean and sum and implemented additional endpoints such as minimum or maximum distance moved and new parameters assessing the bursting response of eleutheroembryos. Furthermore, changes in eleutheroembryonic behaviour during the moment of the light to dark transition were added. To improve data presentation control-normalised results were displayed in radar charts, enabling the simultaneous presentation of different parameters in relation to each other. This enabled us to identify parameters most relevant to a certain behavioural response. A cut off threshold using control data was applied to identify parameters that were altered in a biological relevant manner. Our approach was able to detect effects on different parameters that remained undetected when analysis was done using conventional bar graphs on - in most cases analysed - averaged, mean distance moved values. By combining the radar charts with additional parameters and by using control-based thresholds, we were able to increase the test sensitivity and promote a deeper understanding of the behaviour response of zebrafish eleutheroembryos in the LDT test and thereby increased its usability for behavioural toxicity studies.
Life and biological resilience rely on the execution of precise gene expression profiles. A key mechanism to ensure cellular homeostasis is the regulation of protein synthesis. Recent studies have unveiled an intrinsic regulatory capacity of ribosomes, previously considered mere executors of mRNA translation. Neurons in particular finely regulate protein synthesis, at both global and local levels. This sustains their complex morphology and allows them to rapidly transmit, integrate, and respond to external stimuli. In this thesis, I investigated the neuronal ribosome and how subcellular environments and physiological perturbations shape it, by profiling its molecular composition, functional interconnections, and cellular distribution.
First, I used genetic engineering, biochemical purification, and mass spectrometry, to characterize in an unbiased manner the translation machinery specifically from excitatory and inhibitory neurons of the mouse cortex. I found that neuronal ribosomes commonly interact with RNA-binding proteins, components of the cytoskeleton, and proteins associated with the endoplasmic reticulum and vesicles. In line with the requirement for local protein synthesis in the distal parts of neurons, we observed that neuronal ribosomes preferentially interact with proteins involved in cellular transport. Remarkably, I observed a strong association between ribosomes and pre-synaptic vesicles, which suggests a potential regulatory interaction between local translation and neuronal activity.
Intriguingly, I and others have observed mRNAs encoding for core ribosomal proteins (RPs) among the genes most enriched in neuronal processes. This observation challenges two historical assumptions of ribosome biology: (1) new RPs are incorporated only into newly forming ribosomes, and (2) this incorporation occurs only in the nucleus and perinuclear region. In my PhD, I aimed to directly test these two assumptions and if proven wrong ask whether and why neurons would localize RP mRNAs far from their known assembly site.
Employing a combination of metabolic labeling and highly sensitive mass spectrometry techniques, I discovered that a subset of RPs rapidly and dynamically binds on and off mature ribosomes. Strikingly, this incorporation does not depend on the supply of new ribosomes from the nucleus. Therefore, my data refuted the assumption that ribosomes are built and degraded as a unit and revealed a more dynamic view of these machines, which can actively exchange core components. In particular, I found that the association of certain exchanging RPs is influenced by location (e.g., cell body versus neurites) and cellular state (e.g., post-oxidative stress). Neurons may use this mechanism to repair and/or specialize their protein synthesis machinery in a rapid and context-dependent manner.
Finally, I asked whether some steps of ribosome biogenesis could also take place in distal processes. Although most steps of ribosome assembly occur within the nucleus, the final stages of maturation are known to occur in the cytosol. By combining several imaging and biochemical approaches, I found that cytosolic (but not nuclear) pre-ribosomal particles are present in neuronal processes. Through the incorporation of new RPs into these immature particles, neurons may be able to locally “turn on” previously incompetent ribosomes. This may enable regions near synapses to enhance and customize their translational capacity, independently of the central pool of ribosomes from the cell body. Indeed, I observed that synaptic plasticity induces a maturation of cytosolic pre-ribosomes.
In summary, this thesis shows how neuronal ribosomes can sense cellular states, respond by adjusting their core composition, and in doing so influence the local capacity for protein synthesis. By overturning long-held assumptions in ribosome biology, this work highlights new molecular mechanisms of gene expression and enriches our understanding of the rapid and dynamic strategies cells employ to operate, thrive, and adaptively respond to environmental changes.
Long non-coding RNAs are a very versatile class of molecules that can have important roles in regulating a cells function, including regulating other genes on the transcriptional level. One of these mechanisms is that RNA can directly interact with DNA thereby recruiting additional components such as proteins to these sites via an RNA:dsDNA triplex formation. We genetically deleted the triplex forming sequence (FendrrBox) from the lncRNA Fendrr in mice and found that this FendrrBox is partially required for Fendrr function in vivo. We found that the loss of the triplex forming site in developing lungs causes a dysregulation of gene programs associated with lung fibrosis. A set of these genes contain a triplex site directly at their promoter and are expressed in lung fibroblasts. We biophysically confirmed the formation of an RNA:dsDNA triplex with target promoters in vitro. We found that Fendrr with the Wnt signalling pathway regulates these genes, implicating that Fendrr synergizes with Wnt signalling in lung fibrosis.
Precise regulation of gene expression networks is required to develop and maintain a healthy organism before and after birth and throughout adulthood. Such networks are mostly comprised of regulatory proteins, but meanwhile many long non-coding transcripts (lncRNAs) are shown to participate in these regulatory processes. The functions and mechanisms of these lncRNAs vary greatly, however they are often associated with transcriptional regulation. Three lncRNAs, namely Sweetheart RNA (Swhtr), Fetal-lethal noncoding developmental regulatory RNA / Foxf1 adjacent non-Coding developmental regulatory RNA (Fendrr) and lncFsd2, were studied in this work to demonstrate the variety of cellular and biological processes that require lncRNA-mediated fine-tuning, in regard to the cardiopulmonary system.
Swhtr was found to be expressed exclusively in cardiomyocytes and became critical for regeneration after myocardial injury. Mice lacking Swhtr did not show issues under normal conditions, but failed to undergo compensatory hypertrophic remodeling after injury, leading to increased mortality. This effect was rescued by re-expressing Swhtr, demonstrating importance of the RNA. Genes dependent on Swhtr during cardiac stress were found to likely be regulated by NKX2-5 through physical interaction with Swhtr. Fendrr was found to be expressed in lung and interacted with target promoters through its RNA:dsDNA binding domain, the FendrrBox, which was partially required for Fendrr function. Fendrr, together with activated WNT signaling, regulated fibrosis related target genes via the FendrrBox in fibroblasts. LncFsd2, an ubiquitously expressed lncRNA, showed possible interaction with the striated muscle specific Fsd2, but its exact function and regulatory role remain unclear in muscle physiology. Immunoprecipitation and subcellular fractionation experiments suggest that lncFsd2 might be involved in nuclear retention of Fsd2 mRNA, thus fine-tuning FSD2 protein expression. These investigations have shed light on the roles of these lncRNAs in stress responses, fibrosis-related gene regulation, and localization processes, advancing our understanding of cardiovascular and pulmonary maintenance, reaction to injury, and diseases. The diverse and intricate roles of these three lncRNAs highlight how they influence various cellular processes and disease states, offering avenues for exploring lncRNA functions in different biological contexts.
All-optical closed-loop voltage clamp for precise control of muscles and neurons in live animals
(2023)
Excitable cells can be stimulated or inhibited by optogenetics. Since optogenetic actuation regimes are often static, neurons and circuits can quickly adapt, allowing perturbation, but not true control. Hence, we established an optogenetic voltage-clamp (OVC). The voltage-indicator QuasAr2 provides information for fast, closed-loop optical feedback to the bidirectional optogenetic actuator BiPOLES. Voltage-dependent fluorescence is held within tight margins, thus clamping the cell to distinct potentials. We established the OVC in muscles and neurons of Caenorhabditis elegans, and transferred it to rat hippocampal neurons in slice culture. Fluorescence signals were calibrated to electrically measured potentials, and wavelengths to currents, enabling to determine optical I/V-relationships. The OVC reports on homeostatically altered cellular physiology in mutants and on Ca2+-channel properties, and can dynamically clamp spiking in C. elegans. Combining non-invasive imaging with control capabilities of electrophysiology, the OVC facilitates high-throughput, contact-less electrophysiology in individual cells and paves the way for true optogenetic control in behaving animals.
Mitochondria perform essential energetic, metabolic and signalling functions within the cell. To fulfil these, the integrity of the mitochondrial proteome has to be preserved. Therefore, each mitochondrial subcompartment harbours its own system for protein quality control. However, if the capacity of mitochondrial chaperones and proteases is overloaded, mitochondrial misfolding stress (MMS) occurs. Upon this stress condition, mitochondria communicate with the nucleus to increase the transcription of nuclear encoded mitochondrial chaperones and proteases. This proteotoxic stress pathway was termed the mitochondrial unfolded protein response (UPRmt) aiming at restoring protein homeostasis. Despite being discovered over 25 years ago, the signalling molecules released by stressed mitochondria as well as the corresponding receptor and transcription factor remain poorly understood. With this study, we aimed at characterising the underlying signalling events and mechanisms of how mitochondria react to misfolded proteins. First, we aimed to establish different methods to induce MMS that triggers the transcriptional induction of mitochondrial chaperones and proteases detected by quantitative polymerase chain reaction. We were able to induce UPRmt signalling by overexpression of an aggregation-prone protein and by knock-down or inhibition of mitochondrial protein quality control components. To study the signalling in a time-resolved manner, we focused on the usage of the mitochondrial HSP90 inhibitor GTPP and the mitochondrial LONP1 protease inhibitor CDDO.
Early time point RNA sequencing analysis of cells stressed with GTPP or CDDO revealed upregulated genes in response to oxidative stress. Indeed, measurements of mitochondrial superoxide with the fluorescent dye MitoSOX showed increased levels of reactive oxygen species (ROS) upon MMS induction. In contrast, there was no induction of mitochondrial chaperones and proteases when combining MMS with antioxidants. Compartment-specific targeting of the hydrogen peroxide sensor HyPer7 revealed increased ROS levels in the intermembrane space and matrix of mitochondria, followed by elevated ROS levels in the cytosol at later time points. The importance of cytosolic ROS for the signalling was supported by preventing UPRmt induction with an inhibitor blocking the outer mitochondrial membrane pore. Thus, ROS were identified as an essential UPRmt signal.
To understand which cytosolic factor is modified by ROS, redox proteomics was performed. Here, reversible changes on cysteine residues of the HSP40 co-chaperone DNAJA1 were observed upon MMS. Consequently, transcriptional induction of UPRmt genes was abolished by DNAJA1 knock-down. To understand the function of DNAJA1 during UPRmt signalling, quantitative interaction proteomics upon MMS revealed an increased binding to mitochondrial proteins and its interaction partner HSP70. Immunoprecipitation confirmed a ROS-dependent interaction between HSP40 and HSP70. Increased binding to mitochondrial proteins represented a cytosolic interaction of DNAJA1 with mitochondrial precursor proteins, whose accumulation was confirmed by western blot. Moreover, a fluorescent protein targeted to mitochondria accumulated in the cytosol during GTPP treatment, confirming a reduced import efficiency upon MMS. Preventing the accumulation of precursors by a translation inhibitor or depletion of a general mitochondrial transcription factor resulted in reduced UPRmt activation. Thus, DNAJA1 is essential for UPRmt signalling, since its oxidation by mitochondrial ROS and its enhanced recruitment to mitochondrial precursors allows the integration of both MMS-induced signals.
To link these findings to an increased transcription of mitochondrial chaperones and proteases, we screened for transcription factors accumulating in the nucleus upon MMS by cellular fractionation mass spectrometry. We demonstrated that specifically HSF1 accumulates in nuclei of cells stressed with GTPP or CDDO. Depletion of HSF1 by knock-down or knock-out resulted in the abrogation of the UPRmt-specific transcriptional response. HSF1 activation was visualised by nuclear accumulation on western blot, a process inhibited by ROS and precursor suppression. Moreover, DNAJA1 depletion prevented HSF1 activation. Ultimately, we proved by immunoprecipitation that the inhibitory interaction between HSF1 and HSP70 is reduced upon MMS.
Thus, we conclude that MMS increases mitochondrial ROS that are released into the cytosol. In addition, the import efficiency is reduced upon MMS, resulting in the accumulation of non-imported mitochondrial precursor proteins in the cytosol. Both signals are recognised via DNAJA1 oxidation and substrate binding. The concurrent recruitment of HSP70 to DNAJA1 results in the loss of the inhibitory HSP70-HSF1 interaction. Thus, active HSF1 can migrate to the nucleus to initiate transcription of mitochondrial chaperones and proteases. These findings are in accordance with observations in yeast, where mistargeted mitochondrial proteins activate cellular stress responses. Our results highlight a surprising interconnection and dependence of the mitochondrial and the cytosolic proteostasis network, in which the UPRmt is activated by a combination of two mitochondria-specific proteotoxic stress signals.
This dissertation constitutes a series of successive research papers, starting with the characterization of various optogenetic tools up to the establishment of purely optical electrophysiology in living animals.
Optogenetics has revolutionized neurobiology as it allows stimulation of excitable cells with exceptionally high spatiotemporal resolution. To cope with the increasing complexity of research issues and accompanying demands on experimental design, the broadening of the optogenetic toolbox is indispensable. Therefore, one goal was to establish a wide variety of novel rhodopsin-based actuators and characterize them, among others, with respect to their spectral properties, kinetics, and efficacy using behavioral experiments in Caenorhabditis elegans. During these studies, the applicability of highly potent de- and hyperpolarizers with adapted spectral properties, altered ion specificity, strongly slowed off-kinetics, and inverted functionality was successfully demonstrated. Inhibitory anion channelrhodopsins (ACRs) stood out, filling the gap of long-sought equivalent hyperpolarizing tools, and could be convincingly applied in a tandem configuration combined with the red-shifted depolarizer Chrimson for bidirectional stimulation (Bidirectional Pair of Opsins for Light-induced Excitation and Silencing, BiPOLES). A parallel study aimed to compare various rhodopsin-based genetically encoded voltage indicators (GEVIs) in the worm: In addition to electrochromic FRET-based GEVIs that use lower excitation intensity, QuasAr2 was particularly convincing in terms of voltage sensitivity and photostability in C. elegans. However, classical optogenetic approaches are quite static and only allow perturbation of neural activity. Therefore, QuasAr2 and BiPOLES were combined in a closed-loop feedback control system to implement the first proof-of-concept all-optical voltage clamp to date, termed the optogenetic voltage clamp (OVC). Here, an I-controller generates feedback of light wavelengths to bidirectionally stimulate BiPOLES and keep QuasAr’s fluorescence at a desired level. The OVC was established in body wall muscles and various types of neurons in C. elegans and transferred to rat hippocampal slice culture. In the worm, it allowed to assess altered cellular physiology of mutants and Ca2+-channel characteristics as well as dynamical clamping of distinct action potentials and associated behavior.
Ultimately, the optogenetic actuators and sensors implemented in the course of this cumulative work enabled to synergistically combine the advantages of imaging- and electrode-based techniques, thus providing the basis for noninvasive, optical electrophysiology in behaving animals.
Rafts: Rafts sind spezialisierte Domänen biologischer Membranen, die sich durch ihre spezifische Lipid- und Proteinzusammensetzung auszeichnen (zur Übersicht siehe Simons und Toomre, 2000). Die am besten beschriebenen Rafts sind die Caveolae, doch es gibt noch weitere weniger gut charakterisierte Rafttypen. Rafts werden verschiedene zelluläre Funktionen zugeschrieben wie z.B. gerichteter Transport von Membranproteinen, Endozytose und Signaltransduktion. Diese Funktionen erfüllen sie vornehmlich, indem sie verschiedene Proteine und Lipide bedingt durch ihre biophysikalischen Eigenschaften selektiv aufnehmen oder ausschließen. Viele Raftproteine sind über gesättigte Acylketten, wie Myristat oder Palmitat, oder einen GPIAnker mit der Membran assoziiert. Transmembranproteine, wie z.B. der EGFRezeptor, können jedoch auch in Rafts angereichert sein. Besonders an der Plasmamembran dienen Rafts als Signaltransduktionszentren, indem sie beteiligte Rezeptoren und Signalmoleküle konzentrieren.
Reggie-Proteine: Bei der Suche nach Proteinen, die bei der Regeneration von verletzten Sehnerven von Fischen hochreguliert werden, wurden Reggie-1 und Reggie-2 entdeckt (Schulte et al., 1997). Gleichzeitig wurden diese Proteine bei der Suche nach neuen Raftproteinen gefunden und als Flotillin-1 (=Reggie-2) und Flotillin-2 (=Reggie-1) bezeichnet (Bickel et al., 1997). Reggie-1 und -2 haben ein Molekulargewicht von 47 kDa und sind auf Aminosäuren-Basis zu 44% identisch. Homologe zu Reggie-1 wurden bislang in Mensch, Maus, Ratte und Fisch, wie auch in D. melanogaster gefunden. Die evolutionäre Konservierung der Reggies ist, mit beispielsweise 80% zwischen Ratte und Goldfisch, sehr hoch und weist auf eine wichtige Funktion hin, die Sequenzkonservierung verlangt. Reggie-1 wird ubiquitär exprimiert, wogegen Reggie-2 ein weniger verbreitetes Expressionsmuster aufweist. Reggie-1 ist vornehmlich an der Plasmamembran und an Endosomen lokalisiert. Die subzelluläre Lokalisation von Reggie-2 hängt vom Zelltyp ab...
Keystone mutualisms, such as corals, lichens or mycorrhizae, sustain fundamental ecosystem functions. Range dynamics of these symbioses are, however, inherently difficult to predict because host species may switch between different symbiont partners in different environments, thereby altering the range of the mutualism as a functional unit. Biogeographic models of mutualisms thus have to consider both the ecological amplitudes of various symbiont partners and the abiotic conditions that trigger symbiont replacement. To address this challenge, we here investigate 'symbiont turnover zones'--defined as demarcated regions where symbiont replacement is most likely to occur, as indicated by overlapping abundances of symbiont ecotypes. Mapping the distribution of algal symbionts from two species of lichen-forming fungi along four independent altitudinal gradients, we detected an abrupt and consistent β-diversity turnover suggesting parallel niche partitioning. Modelling contrasting environmental response functions obtained from latitudinal distributions of algal ecotypes consistently predicted a confined altitudinal turnover zone. In all gradients this symbiont turnover zone is characterized by approximately 12°C average annual temperature and approximately 5°C mean temperature of the coldest quarter, marking the transition from Mediterranean to cool temperate bioregions. Integrating the conditions of symbiont turnover into biogeographic models of mutualisms is an important step towards a comprehensive understanding of biodiversity dynamics under ongoing environmental change.
Bioactive small molecules are used in many research areas as important tools to uncover biological pathways, interpret phenotypic changes, deconvolute protein functions and explore new therapeutic strategies in disease relevant cellular model systems. To unlock the full potential of these small molecules and to ensure reliability of results obtained in cellular assays, it is crucial to understand the properties of these small molecules. These properties encompass their activity and potency on their designated target(s), their selectivity towards unintended off-targets and their phenotypic effects in a cellular system. Approved drugs often engage with multiple targets, which can be beneficial for some applications such as treatment of cancer where several pathways need to be inhibited for treatment efficacy. However, targeting multiple key proteins in diverse pathways also increases the possibility for unspecific or unwanted side effects. For many drugs the entire target space that they modulate is not known. This makes it difficult to use these drugs for target deconvolution or functional assays with the aim to understand the underlying biological processes. In contrast to drugs, for mechanistic studies, a good alternative are chemical tool compounds so called chemical probes that are usually exclusively selective as well as chemogenomic compounds, that inhibit several targets but have narrow selectivity profiles. Because they are mechanistic tools, chemical tool compounds must meet stringent quality criteria and they are therefore well characterized in terms of their potency, selectivity and cellular on-target activity. To ensure that an observed phenotypic effect caused by a compound can be attributed to the described target(s), it is essential to study also properties of chemical tools leading to unspecific cellular effects. There are a variety of unspecific effects that can be caused by physiochemical compound properties that can interfere with phenotypic assays as well as functional compound evaluations. One of these effects is low solubility causing toxicity or intrinsic fluorescence potentially interfering with assay readouts. But unanticipated cellular responses can also arise from unspecific binding, accumulation in cellular compartments or damage caused to organelles such as mitochondria or the cytoskeleton that can result in the induction of diverse forms of cell death.
In this study, we investigated the influence of a variety of small molecules on distinct cell states, by establishing and validating high-content imaging assays, which we called Multiplex assay. This assay portfolio enabled us to detect different cellular responses using diverse fluorescent reporters, such as the influence of a compound on cell viability, induction of cell death programs and modulation of the cell cycle. Additionally, general compound properties such as precipitation and intrinsic fluorescence were simultaneously detected. The assay is adaptable to assess other cellular properties of interest, such as mitochondrial health, changes in cytoskeletal morphology or phospholipidosis. A significant advantage of the assay is that we are using live cells, so we can capture dynamic cellular changes and fluctuations that can be crucial for the understanding of cellular responses.
Der Hirntumor Glioblastom (GBM) ist aufgrund seines infiltrativen Wachstums, der hohen intra- und intertumoralen Heterogenität, der hohen Therapieresistenz als auch aufgrund der sogenannten gliomartigen Stammzellen sehr schwer zu behandeln und führt fast immer zu Rezidiven. Da es in den letzten Jahrzehnten kaum Fortschritte in der Behandlung des GBMs gab, bis auf die Therapie mit Tumortherapiefeldern, wird weiterhin nach alternativen Zelltodtherapien geforscht, wie zum Beispiel dem Autophagie-abhängigen Zelltod. Der Autophagie-abhängige Zelltod ist durch einen erhöhten autophagischen Flux gekennzeichnet und obwohl die Autophagie, als auch selektive Formen wie die Lysophagie und Mitophagie, normalerweise als überlebensfördernde Mechanismen gelten, konnten viele Studien eine duale Rolle in der Tumorentstehung, -progression und -behandlung aufzeigen, die vor allem vom Tumortyp und stadium abhängt. Um die zugrunde liegenden Mechanismen des durch Medikamente induzierten Autophagie-abhängigen Zelltods im GBM weiter zu entschlüsseln, habe ich in meiner Dissertation verschiedene Substanzen untersucht, die einen Autophagie-abhängigen Zelltod induzieren.
In einer zuvor in unserem Labor durchgeführten Studie konnte gezeigt werden, dass das Antipsychotikum Pimozid (PIMO) und der Opioidrezeptor-Antagonist Loperamid (LOP) einen Autophagie-abhängigen Zelltod in GBM Zellen induzieren können. Darauf aufbauend habe ich die Fähigkeit zur Induktion des Autophagie-abhängigen Zelltods in weiteren Zellmodellen validiert. Dies bestätigte einen erhöhten autophagischen Flux nach PIMO und LOP Behandlung, während der Zelltod als auch der autophagische Flux in Autophagie-defizienten Zellen reduziert war. In weiteren Versuchen konnte ich die Involvierung der LC3-assoziierten Phagozytose (LAP), ein Signalweg der auf die Funktion einiger autophagischer Proteine angewiesen ist, ausschließen. Weiterhin konnte ich eine massive Störung des Cholesterin- und Lipidstoffwechsels beobachten. Unter anderem akkumulierte Cholesterin in den Lysosomen gefolgt von massiven Schäden des lysosomalen Kompartiments und der Permeabiliserung der lysosomalen Membran. Dies trug einerseits zur Aktivierung überlebensfördernder Lysophagie als auch der Zell-schädigenden „Bulk“-Autophagie bei. Letztendlich konnte aber die erhöhte Lysophagie die Zellen nicht vor dem Zelltod retten und die Zellen starben einen Autophagie-abhängigen lysosomalen Zelltod. Da die Eignung von LOP als Therapie für das GBM aufgrund der fehlenden Blut-Hirn-Schranken Permeabilität und von dem Antipsychotikum PIMO aufgrund teils schwerer Nebenwirkungen eingeschränkt ist, habe ich mich im weiteren Verlauf meiner Dissertation mit einer Substanz mit einem anderen Wirkmechanismus beschäftigt.
Der Eisenchelator und oxidative Phosphorylierungs (OXPHOS) Inhibitor VLX600 wurde zuvor berichtet mitochondriale Dysfunktion und Zelltod in Kolonkarzinomzellen zu induzieren. Allerdings hat meines Wissens nach bisher noch keine Studie die therapeutische Eignung von VLX600 für das GBM untersucht. Hier zeige ich eine neuartige Autophagie-abhängige Zelltod-induzierende Fähigkeit von VLX600 für GBM Zellen, da der Zelltod signifikant in Autophagie-defizienten Zellen aber nicht durch Caspase-Inhibitoren gehemmt wurde und der autophagische Flux erhöht war. Darüber hinaus konnte ich die Hemmung der OXPHOS und die Induktion von mitochondrialem Stress in GBM Zellen bestätigen und weiterhin aufzeigen, dass VLX600 nicht nur die mitochondriale Homöostase stört, sondern auch zu einer BNIP3-BNIP3L-abhängigen Mitophagie führt, die wahrscheinlich durch HIF1A reguliert wird aber keinen erkennbaren Nettoeffekt auf den von VLX600 induzierten Zelltod hat. Demnach induziert VLX600 letale „Bulk“-Autophagie in den hier verwendeten Zellmodellen. Darüber hinaus konnte ich zeigen, dass die Eisenchelatierung durch VLX600 eine große Rolle für den von VLX600-induzierten Zelltod spielt aber auch für die Mitophagie Induktion, Histon Lysin Methylierung und den ribosomalen Stress. Letztendlich ist es wahrscheinlich ein Zusammenspiel all dieser Faktoren, die zur Zelltodinduktion durch VLX600 führen und interessanterweise werden Eisenchelatoren bereits in präklinischen und klinischen Studien für Krebstherapien untersucht. Dabei könnten gewisse metabolische Eigenschaften verschiedener Tumorzellen die Sensitivität von Wirkstoffen, die auf den Metabolismus wirken wie VLX600, beeinflussen was in zukünftigen Studien beachtet werden sollte um den bestmöglichsten Therapieerfolg zu erzielen. Zusammenfassend unterstützt meine Dissertation die duale Rolle der Autophagie, die stark vom jeweiligen Kontext abhängt und befürwortet die weitere Forschung von Substanzen, die einen Autophagie-abhängigen Zelltod induzieren, für das GBM.
EphrinB2 and GRIP1 stabilize mushroom spines during denervation-induced homeostatic plasticity
(2021)
Highlights
• Denervation induces mushroom spine loss and AMPAR redistribution to the surface
• GRIP1 and ephrinB2 mediate homeostatic mechanisms after lesion
• Stimulation with the ephrinB2 receptor EphB4 promotes a surface shift of AMPARs
• AMPARs surface shift restores impaired spine recovery after lesion in GRIP1 mutants
Summary
Despite decades of work, much remains elusive about molecular events at the interplay between physiological and structural changes underlying neuronal plasticity. Here, we combined repetitive live imaging and expansion microscopy in organotypic brain slice cultures to quantitatively characterize the dynamic changes of the intracellular versus surface pools of GluA2-containing α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptors (AMPARs) across the different dendritic spine types and the shaft during hippocampal homeostatic plasticity. Mechanistically, we identify ephrinB2 and glutamate receptor interacting protein (GRIP) 1 as mediating AMPAR relocation to the mushroom spine surface following lesion-induced denervation. Moreover, stimulation with the ephrinB2 specific receptor EphB4 not only prevents the lesion-induced disappearance of mushroom spines but is also sufficient to shift AMPARs to the surface and rescue spine recovery in a GRIP1 dominant-negative background. Thus, our results unravel a crucial role for ephrinB2 during homeostatic plasticity and identify a potential pharmacological target to improve dendritic spine plasticity upon injury.
Highlights
• Enables immunostaining and visualization of epitopes deep within brain slices
• Utilizes expansion microscopy to increase imaging resolution
• Optimized for brain organotypic slice cultures and tested in acute brain slices
• Analysis workflow for protein distribution (surface vs. intracellular pool) using Imaris
Publisher’s note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics.
Summary
Assessing protein distribution with super-resolution in tissue is often complicated and restrictive. Here, we describe a protocol for immunostaining and expansion microscopy imaging of mouse brain organotypic slice cultures. We detail an Imaris analysis workflow to analyze the surface vs intracellular distribution of AMPA receptors at super-resolution during homeostatic plasticity. We have optimized the protocol for brain organotypic slice culture and tested in acute brain slices. This protocol is suitable to study protein distribution under multiple plasticity paradigms.