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Das Heidelberger Ionenstrahl-Therapiezentrum (HIT) stellt Protonen-, Helium- und Kohlenstoff-Ionenstrahlen unterschiedlicher Energie und Intensität für die Krebsbehandlung und Sauerstoff-Ionenstrahlen für Experimente zur Verfügung. Der hierfür verwendete Beschleuniger ist darüber hinaus in der Lage auch Ionenstrahlintensitäten unterhalb der für Therapien verwendeten bereitzustellen. Allerdings ist das derzeit installierte Strahldiagnosesystems nicht in der Lage, das Strahlprofil bei solchen geringen Intensitäten (< 10^5 Ionen/s) zu messen. Dabei existieren mögliche medizinische Anwendung für diese niederintensiven Ionen-strahlen, wie beispielsweise eine neuartige und potentiell klinisch vorteilhafte Bildgebung: die Ionenradiographie. Eine essentielle Voraussetzung für diese und andere Anwendungen ist ein System zur Überwachung von Ionenstrahlen niedriger Intensität. Ein solches System wurde im Rahmen dieser Arbeit konzipiert, realisiert, getestet und optimiert.
Das Funktionsprinzip basiert auf szintillierenden Fasern, insbesondere solchen mit erhöhter Strahlungshärte für die Möglichkeit einer dauerhaften Platzierung im Therapiestrahl. Ein diese Fasern durchlaufendes Ion regt den darin enthaltenen Szintillator durch Stoßprozesse kurzzeitig an. Die dabei deponierte Energie wird anschließend in Form von Photonen wieder emittiert. Silizium-Photomultiplier sind an den Enden der Fasern montiert und wandeln die Photonensignale in verstärkte elektrische Impulse um. Diese Impulse werden von einer neuartigen und dedizierten Ausleseelektronik aufgezeichnet und verarbeitet. Ein Prototypaufbau, bestehend aus den genannten Teilen, wurde im Strahl getestet und kann das transversale Strahlprofil erfolgreich im Intensitätsbereich von 10^7 Ionen/s bis hinunter zu 10^2 Ionen/s aufzeichnen. Darüber hinaus konnte, durch die erfolgreiche Ankunftszeitmessung von einzelnen Ionen bis zu Intensitäten von 5*10^4 Ionen/s, ein Machbarkeitsnachweis für die Messung der Spur von einzelnen Teilchen erbracht werden.
Optimierung der Synthese eines neuen photolabil geschützten Nitroxid-Spin-Labels für RNA und DNA
(2023)
Im Rahmen dieser Arbeit konnte, ausgehend von den günstigen Ausgangsverbindungen Desoxyadenosin und Phthalsäureanhydrid, ein neues photolabil geschütztes Nukleotid 1 und sein Dummypartner 2 synthetisiert werden. Positiv zu bemerken ist, dass einige Schritte im Vergleich zu ähnlichen literaturbekannten Reaktionen in Einfachheit, Reinheit oder Ausbeute verbessert wurden. So konnte die Ausbeute der wichtigen Umwandlung des Amins 46 zum Iodid 47 durch den Ersatz des vorherigen DCM/DIM Gemisches durch reines DIM von 15 % auf akzeptable 50 % erhöht werden, was nicht nur Zeit, sondern auch zukünftige Chemikalienmengen einspart. Nicht nur hierbei, sondern auch bei der Nitrierung zu 61 oder auch der Oxidierung zu 63 war es von äußerster Wichtigkeit eine korrekte Temperaturkontrolle durchzuführen, da es sonst zu hohen Ausbeuteverlusten durch ungewollte Nebenreaktionen kommen konnte. Eine sehr interessante Beobachtung war die Kontrolle der Suzuki Miyaura-Kreuzkupplung durch die Anwendung verschieden starker Basen. Während schwache Basen wie KOAc nur zur Miyaura-Borilierung führten, begünstigten starke Basen wie K3PO4 die Suzuki Miyaura-Kreuzkupplung. Die Zusammenführung des Zucker Bausteins 36 und des Isoindolin-Bausteins 37 funktionierte sehr gut, sodass das Nukleotid 1 durch die Schützung der exozyklischen Amingruppe und Phosphorylierung des 3´-OH dargestellt werden konnte.
Die Synthese der 14mer DNA bzw. RNA Sequenzen mit den neuen Nukleotiden 1 und 2 funktionierten mit zufriedenstellenden Ausbeuten, nur die Abspaltung der Pac-Gruppe benötigte etwas harschere Bedingungen von 50 °C in 32 % Ammoniak über Nacht. Die photolabile Schutzgruppe in Strang (V) mDNA-Tetramethyl konnte nun abgespalten und das Nitroxid an Luft reoxidiert werden. Anhand von EPR-Spektren und einer HPLC Analyse ergab sich jedoch eine Abspalteffizienz von nur 70 %. Dies bedeutet, dass für künftige PELDOR Messungen eine Aufreinigung des Spaltgemisches zur Isolierung des Radikal-Strangs von Nöten ist.
Anhand des Schmelzpunkts der verschiedenen Duplexe wurde anschließend die mögliche Anwendung des Nukleotids weiter analysiert. Hierbei stellte sich heraus, dass der Benzolring sowohl in 2 als auch in 1 eine erhebliche Destabilisierung des Duplex erzeugte. Somit ist das neue photolabil geschützte Nukleotid als EPR Sonde in der Mitte von Sequenzen nur bedingt geeignet. Zukünftige Experimente könnten das neue Spin Label nicht in der Mitte, sondern an den Enden der Sequenzen ähnlich anderer Arbeiten[92,94,164] einbauen, wo die Destabilisierung eine geringere Auswirkung hat, oder die Sequenz für eine bessere Stabilisierung verlängern.[164] Bei ausreichenden Duplexstabilitäten könnten hiermit dann PELDOR Messungen durchgeführt werden. Ähnlich starre, sterisch anspruchsvolle Nukleotide zeigten auch ähnliche Schmelzpunkte für ihre Duplexe[120], dennoch wurden sie für weitere Markierungsexperimente verwendet. Hierbei handelte es sich jedoch nicht um EPR Sonden, sondern um Fluoreszenzmarker. Da auch das neue Spin-Label 1 ein großes π-System besitzt, könnte eine komplett neue Herangehensweise die Anwendung als Fluoreszenzmarker sein. Genaue Absorptionsmessungen müssten noch durchgeführt werden, jedoch zeigte das Spin Label sehr stark fluoreszierende Eigenschaften unter der UV-Lampe während der Säulenchromatographie. Hierbei würde die Synthese um einiges kürzer ausfallen, da das EPR-aktive Nitroxid nicht mehr benötigt wird und geschützt werden muss, was Zeit und Chemikalien spart.
Zusammenfassend wurde über eine 22-stufige Synthese ein neues photolabil geschütztes Spin-Label synthetisiert, in ein 14mer integriert, erfolgreich entschützt und mittels EPR-Spektroskopie vermessen. Schmelzpunktmessungen zeigten jedoch eine große Destabilisierung und deuten darauf hin, dass 1 und Nukleotide mit ähnlich Benzolringen nur eingeschränkt als EPR-aktive Nukleotide geeignet sind.
Diese Arbeit hatte das Ziel, die Größe einer DVT-Aufnahme (FOV) mit der Größe der durch die Indikationsstellung definierten Region (ROI) zu vergleichen. Durch eine speziell dafür entwickelte Software sollten Messungen in den Datensätzen ermöglicht werden. Die dazu verwendeten 332 Datensätze wurden zufallsverteilt aus den mit einem Orthophos SL-3D DVT-Gerät der Firma Dentsply Sirona in der Poliklinik für zahnärztliche Chirurgie und Implantologie des ZZMK (Carolinum) in Frankfurt angefertigten Röntgenaufnahmen selektiert.
Es wurde die Auswertungssoftware ExRoi entwickelt, mit der die Werte des axialen Durchmessers, die Höhe (vertikale Dimension) sowie die Distanz der Mittelpunkte von FOV und ROI direkt in den Datensätzen bestimmt werden konnten. Zusätzlich wurde festgehalten, welche rechtfertigenden Indikationen gestellt und welche Auflösungsmodi verwendet wurden.
Die Stichprobe bestand aus Aufnahmen mit einem axialen Durchmesser von 8 cm [VOL 1] (n= 76, entsprechend 46,39%), 5 cm Durchmesser [VOL 2] (n = 102, entsprechend 30,72%) und 11 cm Durchmesser [VOL 3] (n= 154, entsprechend 22,89%). 95,18% der Aufnahmen wurden im HD-Modus mit laut Herstellerangaben vier Mal so vielen Aufnahmen im Vergleich zum SD-Modus angefertigt. Hauptindikationen waren Implantat Planung (45,1%) und Planungen komplizierter Zahn-Extraktionen (25,5%).
Die Messungen zum Vergleich des axialen Durchmessers zeigten, dass bei Verwendung des VOL 2 die ROI im Mittel den größten Anteil der FOV nutzt (78,52 %), den kleinsten Anteil nutzt durchschnittlich VOL 1 (56,04 %). Dazwischen liegt VOL 3 (69,12 %). In der Vertikalen nutzt die ROI von VOL 3 mittelwertig den größten Anteil der FOV (81,87 %), den kleinsten Mittelwert hat VOL 1 (58,76 %). VOL 2 liegt zwischen diesen Werten (64,47 %).
In allen Fällen war das FOV größer als die ROI und die ROI lag im Bereich des gewählten FOV.
Die Mittelpunkte von FOV und ROI lagen im Mittel in der axialen Ebene in Abhängigkeit vom gewählten Volumen um rund 9-13 mm auseinander, in der coronalen und sagittalen Ebene um rund 5-6mm.
Aus diesen Ergebnissen kann für das verwendete Gerät eine gute Trefferquote für die ROI abgeleitet werden. Höhe und Durchmesser des FOV hätten in den meisten Fällen kleiner gewählt werden können, liegen aber angesichts der vorhandenen Auswahl-Optionen des Röntgengeräts zur Dimensionierung der Volumina in einem akzeptablen Bereich.
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.
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.
Das Ziel in der vorliegenden systematischen Literaturrecherche ist es, den aktuellen Stand des Wissens über die Erfolgsfaktoren von Einzelzahnimplantaten, bei denen eine Sofortbelastung auf den provisorischen Implantatkronen zum Antagonisten erfolgt, wiederzugeben. In diesem Rahmen wurden Zusammenhänge zwischen der Implantatüberlebensrate, der Primärstabilität, der Implantatregion, des Implantatdesigns und des Implantationszeitpunktes getrennt nach okklusalem Belastungsprotokoll (mit versus ohne antagonistische Okklusionskontakte auf den provisorischen Implantatkronen) und der postoperativen Verhaltensweisen der Patienten analysiert. Der Prozess der systematischen Literaturrecherche wurde nach den PRISMA-Guidelines für den Suchzeitraum von 2012 bis 2022 durchgeführt.
Um die Forschungsfrage zu beantworten, wurde eine qualitative Analyse in deskriptiver Form durchgeführt.
In den analysierten Studien, wurde am häufigsten ein Eindrehmoment von ≥ 35 Ncm als Kriterium für eine Sofortbelastung gewählt. Die meisten Implantate wurden in der Oberkieferregion 15 bis 25 inseriert. Bei den Implantatkronen mit antagonistischen Okklusionskontakten fielen die Überlebensraten insgesamt niedriger aus als in den Studien, in denen die Implantatkronen ohne antagonistische Okklusionskontakte eingegliedert wurden. Die Überlebensraten waren weitestgehend vergleichbar mit den Ergebnissen konventioneller Belastungsprotokolle. Die Datenlage anhand dieser Recherche spricht zurzeit für die Realisierung der Sofortbelastung von provisorischen Implantatkronen ohne antagonistische Okklusionskontakte in Verbindung mit postoperativen Verhaltensmaßnahmen.
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.
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.
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.