Studies on the function of PRG2/PLPPR3 in neuron morphogenesis

Neuron development follows a multifaceted sequence of cell migration, polarisation, neurite elongation, branching, tiling, and pruning. The implementation of this sequence differs between neuronal cell types and even in individual neurons between sub-compartments such as dendrites and axons. Membrane proteins are at a prime position in neurons to couple extrinsic morphogenetic signals with their intrinsic responses to orchestrate this defined morphological progression. The Phospholipid phosphatase-related / Plasticity-related gene (PLPPR/PRG)-family comprises five neuron-enriched and developmentally regulated membrane proteins with functions in cellular morphogenesis. At the start of this project, no publication had characterised the function of PLPPR3/PRG2 during neuron development. The presented work describes PLPPR3 as an axon-enriched protein localising to the plasma membrane and internal membrane compartments of neurons. Mutagenesis studies in cell lines establish the plasma membrane localisation of PLPPR3 as a regulator of its function to increase filopodia density (Chapter 2). Furthermore, the generation of a Plppr3-/- mouse line using CRISPR/Cas9 genome editing techniques (Chapter 3) enabled characterising endogenous phenotypes of PLPPR3 in neurons. In primary neuronal cultures, PLPPR3 was found to specifically control branch formation in a pathway with the phosphatase PTEN, without altering the overall growth capacity of neurons (Chapter 4). Loss of PLPPR3 specifically reduced branches forming from filopodia without affecting the stability of branches. This precise characterisation of PLPPR3 function unravelled the existence of parallel, independent programs for branching morphogenesis that are utilised and implemented differentially in developing axons and dendrites (Chapter 5). Furthermore, this thesis establishes multiple tools to study PLPPR3, the membrane lipid phosphatidylinositol-trisphosphate, and neuron morphogenesis by providing molecular tools, protocols, and semi-automated and automated image analysis pipelines (Appendix Chapter 7) and discusses experiments to test, refine and extend models of PLPPR3 function (Chapter 6). In summary, this thesis generated and utilised several tools and a Plppr3-/- mouse model to characterise PLPPR3 as a specific regulator of neuron branching morphogenesis. This precise characterisation refined and expanded the understanding of axon-specific branching morphogenesis.Nervenzellen entwickeln ihre komplexe Morphologie durch das Zusammenwirken diverser molekularer Entwicklungs-Programme der Zellkörper-Migration, der Polarisierung und der Morphogenese durch Wachstum, Verzweigung, Stabilisierung und Koordinierung ihrer Neuriten. Dabei unterscheidet sich die exakte Implementierung zwischen Nervenzell-Typen und selbst innerhalb einzelner Zellen zwischen Axonen und Dendriten. Diese unterschiedliche Morphogenese wird dabei speziell durch Membranproteine stark beeinflusst, die durch ihre Präsenz an der Plasmamembran Zell-extrinsische Signale mit den Zell-intrinsischen Morphogeneseprogrammen verbinden und beeinflussen. Die Familie der Phospholipid phosphatase-related / Plasticity-related gene (PLPPR/PRG) Proteine umfasst fünf Nervenzell-spezifische Membranproteine mit Effekten auf die Morphologie von Zellen. Zu Beginn dieses Projektes hatte noch keine Studie die Funktion des Familienmitglieds PLPPR3/PRG2 in Nervenzellen untersucht. Diese Dissertation beschreibt die Lokalisation von PLPPR3 an der Plasmamembran und in Zell-internen Membranstrukturen von Nervenzellen. Experimente in Zellkultur zeigen eine erhöhte Filopodien-Dichte nach Überexpression von PLPPR3, Mutagenese-Studien deuten eine strikte Kontrolle der Plasmamembran-Lokalisation an (Kapitel 2). Die Generierung einer Plppr3 Knockout Mauslinie mittels CRISPR/Cas9 Genom-Modifizierung (Kapitel 3) erlaubte eine Charakterisierung der endogenen Funktion von PLPPR3 in Nervenzellen. In Primärzellkultur von Nervenzellen des murinen Hippocampus zeigte sich, dass PLPPR3 im Zusammenspiel mit der Phosphatase PTEN spezifisch die Verzweigung von Nervenzellen kontrolliert, ohne deren Wachstumspotential global zu verändern (Kapitel 4). Dadurch kann PLPPR3 als ein Schalter zwischen Verzweigung und Verlängerung eines Nervenzell-Fortsatzes agieren. Der Verlust von PLPPR3 verursachte reduzierte spezifisch die Anzahl an Verzweigungen, die aus Filopodien entstanden, ohne dabei die Stabilität dieser Verzweigungen zu beeinflussen. Die präzise Charakterisierung dieser Funktion von PLPPR3 deckte auf, dass Verzweigungen von Nervenzell-Fortsätzen durch voneinander unabhängige Entwicklungsprogramme ausgebildet und stabilisiert werden können (Kapitel 5). Diese Programme werden von Axonen und Dendriten in unterschiedlicher Weise eingesetzt. Zusätzlich etabliert diese Arbeit sowohl diverse molekulare Werkzeuge und Visualisierungs-Protokolle zur Analyse von PLPPR3 und dem Membranlipid Phosphatidylinositol-Trisphosphat, als auch automatisierte Quantifizierungssoftware zur Studie der Nervenzellmorphologie (Appendix-Kapitel 7). Abschließend entwickelt und verfeinert die Dissertation mögliche Modelle zur PLPPR3-Funktion und zeigt experimentelle Strategien auf, um diese Modelle besser charakterisieren zu können (Kapitel 6). Zusammenfassend wurden in dieser Promotionsarbeit diverse Experimental- und Analyse-Strategien und eine Plppr3-/- Mauslinie entwickelt und genutzt, um PLPPR3 als einen spezifischen Regulator der Nervenzell-Morphogenese zu etablieren. Diese präzise Charakterisierung des PLPPR3 Phänotyps erlaubte zusätzlich eine Verfeinerung und Erweiterung der Erkenntnisse zur Axon-spezifischen Entwicklung von Verzweigungen

Reconstruction of ovine axonal cytoarchitecture enables more accurate models of brain biomechanics

There is an increased need and focus to understand how local brain microstructure affects the transport of drug molecules directly administered to the brain tissue, for example in convection-enhanced delivery procedures. This study reports a systematic attempt to characterize the cytoarchitecture of commissural, long association and projection fibres, namely the corpus callosum, the fornix and the corona radiata, with the specific aim to map different regions of the tissue and provide essential information for the development of accurate models of brain biomechanics. Ovine samples are imaged using scanning electron microscopy combined with focused ion beam milling to generate 3D volume reconstructions of the tissue at subcellular spatial resolution. Focus is placed on the characteristic cytological feature of the white matter: the axons and their alignment in the tissue. For each tract, a 3D reconstruction of relatively large volumes, including a significant number of axons, is performed and outer axonal ellipticity, outer axonal cross-sectional area and their relative perimeter are measured. The study of well-resolved microstructural features provides useful insight into the fibrous organization of the tissue, whose micromechanical behaviour is that of a composite material presenting elliptical tortuous tubular axonal structures embedded in the extra-cellular matrix. Drug flow can be captured through microstructurally-based models using 3D volumes, either reconstructed directly from images or generated in silico using parameters extracted from the database of images, leading to a workflow to enable physically-accurate simulations of drug delivery to the targeted tissue

Model and Appearance Based Analysis of Neuronal Morphology from Different Microscopy Imaging Modalities

The neuronal morphology analysis is key for understanding how a brain works. This process requires the neuron imaging system with single-cell resolution; however, there is no feasible system for the human brain. Fortunately, the knowledge can be inferred from the model organism, Drosophila melanogaster, to the human system. This dissertation explores the morphology analysis of Drosophila larvae at single-cell resolution in static images and image sequences, as well as multiple microscopy imaging modalities. Our contributions are on both computational methods for morphology quantification and analysis of the influence of the anatomical aspect. We develop novel model-and-appearance-based methods for morphology quantification and illustrate their significance in three neuroscience studies. Modeling of the structure and dynamics of neuronal circuits creates understanding about how connectivity patterns are formed within a motor circuit and determining whether the connectivity map of neurons can be deduced by estimations of neuronal morphology. To address this problem, we study both boundary-based and centerline-based approaches for neuron reconstruction in static volumes. Neuronal mechanisms are related to the morphology dynamics; so the patterns of neuronal morphology changes are analyzed along with other aspects. In this case, the relationship between neuronal activity and morphology dynamics is explored to analyze locomotion procedures. Our tracking method models the morphology dynamics in the calcium image sequence designed for detecting neuronal activity. It follows the local-to-global design to handle calcium imaging issues and neuronal movement characteristics. Lastly, modeling the link between structural and functional development depicts the correlation between neuron growth and protein interactions. This requires the morphology analysis of different imaging modalities. It can be solved using the part-wise volume segmentation with artificial templates, the standardized representation of neurons. Our method follows the global-to-local approach to solve both part-wise segmentation and registration across modalities. Our methods address common issues in automated morphology analysis from extracting morphological features to tracking neurons, as well as mapping neurons across imaging modalities. The quantitative analysis delivered by our techniques enables a number of new applications and visualizations for advancing the investigation of phenomena in the nervous system

Advances in Neural Signal Processing

Neural signal processing is a specialized area of signal processing aimed at extracting information or decoding intent from neural signals recorded from the central or peripheral nervous system. This has significant applications in the areas of neuroscience and neural engineering. These applications are famously known in the area of brain–machine interfaces. This book presents recent advances in this flourishing field of neural signal processing with demonstrative applications

Advances in Neural Signal Processing

Neural signal processing is a specialized area of signal processing aimed at extracting information or decoding intent from neural signals recorded from the central or peripheral nervous system. This has significant applications in the areas of neuroscience and neural engineering. These applications are famously known in the area of brain–machine interfaces. This book presents recent advances in this flourishing field of neural signal processing with demonstrative applications

Caracterização do papel da Gαo na neuritogénese: um destaque para o complexo Gαo-Proteina percursora de amilóide

Doutoramento em BiomedicinaGαo is the most abundant Gα subunit present in the brain, however, its specific functions are still far from clear. Studies of the signaling pathways modulated by Gαo have uncovered potential roles for Gαo in the development of the nervous system, especially in neuritogenesis. The characterization of Gαo interactome has also been crucial for the better understanding of this protein’s functions. One of the Gαo interacting proteins is the amyloid precursor protein (APP), a protein that is involved in several physiological functions, such as cell survival, neuronal migration, and neuronal differentiation. APP is also best known for its involvement in Alzheimer’s Disease (AD). APP binds and activates Gαo, an interplay that was associated with neuronal migration and AD. However, so far, no published study has investigated the effects of the APP-Gαo interaction on neuritogenesis. The main goal of this work was thus to characterize Gαo role on neuritogenesis by focusing the research on the neuritogenic effects of the Gαo-APP complex. First, by using SH-SY5Y neuroblastoma cells, we studied the impact of APP serine 655 (S655) phosphorylation on the APP-Gαo interaction. Through the use of two APP mutants mimicking the phosphorylated and dephosphorylated state of S655, SE and SA APP respectively, we have demonstrated that S655 phosphorylation increases APP efficiency to bind and activate Gαo. Moreover, we present evidence that APP modulates Gαo neuritogenic effects in a phosphodependent mechanism. STAT3 and ERK1/2 signaling displayed a sequential activation on this neuritogenic mechanism, with STAT3 being mainly involved in the formation of new processes, while ERK1/2 was more involved in neuritic elongation. We also present data supporting a role for the APP-Gαo complex on dendritogenesis in rat primary neuronal cultures. The second part of this work focused on unraveling the mechanisms involved in the control of APP and Gαo cellular protein levels. We identified the lysosome as a new pathway by which Gαo is degraded, as an effect of SA APP overexpression. We also provide evidence that this degradation mechanism might be part of chaperone-mediated autophagy, through which APP-Gαo signaling might be regulated. Finally, due to our interest in studying neuronal differentiation and a lack of reliable tools to analyze phase contrast images, we developed NeuronRead, an ImageJ macro capable of semi-automated analysis of both phase contrast and fluorescence neuronal images. NeuronRead was extensively validated and used to monitor SH-SY5Y differentiation upon modulation of Gαo activity. With this work, we delivered new data that advances knowledge on the function and regulation of the Gαo-APP complex in a neuronal context, and provided the scientific community with a new tool for the study of neuronal differentiation.Gαo é a subunidade Gα mais abundante no cérebro, no entanto, as suas funções especificas ainda estão longe de serem claras. Estudos das vias de sinalização moduladas pela Gαo têm exposto potenciais papéis para a Gαo no desenvolvimento do sistema nervoso, especialmente em neuritogénese. A caracterização do interactoma da Gαo também tem sido crucial para uma melhor compreensão das funções desta proteína. Uma das proteínas interatoras da Gαo é a proteina precursora de amiloide (APP), uma proteina que se encontra envolvida em várias funções fisiológicas, como sobrevivência celular, migração neuronal, e diferenciação neuronal. APP também é mais conhecida pelo seu envolvimento da Doença de Alzheimer (AD). APP liga-se e ativa a Gαo, uma interação que tem sido associada com migração neuronal e AD. No entanto, até agora, não existem estudos publicados que investiguem a interação APP-Gαo na neuritogénese. O principal objetivo deste trabalho foi então caracterizar o papel da Gαo na neuritogénese através do foco na investigação dos efeitos neuritogénico do complexo Gαo-APP. Primeiro, através do uso de células de neuroblastoma SH-SY5Y, estudámos o impacto da fosforilação da serina 655 (S655) da APP na interação APP-Gαo. Através do uso de dois mutantes da APP que mimetizam o estado fosforilado e desfosforilado da S655, SE e SA APP respetivamente, demonstrámos que a fosforilação da S655 aumenta a eficiência da APP em ligar e ativar a Gαo. Além disso, apresentamos provas de que a APP modula os efeitos neuritogénicos da Gαo num mecanismo fosfo-dependente. Neste mecanismo neuritogénico, a sinalização da STAT3 e ERK1/2 exibiram uma ativação sequencial, com a STAT3 participando na formação de novos processos e a ERK1/2 na elongação dos mesmos. Apresentamos ainda dados que suportam um papel da APP-Gαo na dendritogénese em culturas neuronais primárias. A segunda parte deste trabalho focou-se na investigação de mecanismos envolvidos no controlo dos níveis proteicos celulares da APP e Gαo. Identificámos o lisossoma como um novo processo pelo qual a Gαo é degradada em consequência da sobre expressão da SA APP. Também mostramos provas de que este mecanismo pode fazer parte de autofagia mediada por chaperonas, através do qual a sinalização da APP-Gαo poderá estar a ser regulada. Finalmente, devido ao nosso interesse em estudar diferenciação neuronal e à falta de ferramentas para este estudo em imagens de contraste de fase, criámos o NeuronRead, uma macro do ImageJ capaz de analisar de forma semiautomática imagens neuronais de contraste de fase e fluorescência. NeuronRead foi extensivamente validado, e usado para monitorizar a diferenciação de células SH-SY5Y após modulação da atividade da Gαo. Com este trabalho contribuímos com novos dados que ajudam na compreensão da função e regulação do complexo Gαo-APP, e disponibilizamos para a comunidade cientifica uma nova ferramenta para o estudo da diferenciação neurona