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

Identification of genes differentially expressed in rat brain during postnatal development

During neuronal development CNS neurons extend axons over long distances. This high growth potential is lost during postnatal development resulting in very poor axonal outgrowth and regeneration in the adult CNS. This pronounced decline of axon growth potential and regenerative capability might be related to alterations in the expression level of growth-associated genes during postnatal development. The aim of the present study was the identification of candidate molecules that might be associated with axon growth, i.e. which are strongly expressed during axonal outgrowth and are downregulated as neuronal maturation proceeds. As the time periods of developmental axonal outgrowth and decrease in growth potential are well studied in rat cerebellum and entorhinal cortex, these two brain regions were chosen as model systems for analysis of gene expression patterns during axonal extension and after completion of pathway formation. In a first approach the study focused on the identification of transcription factors, because they are known to be involved in the regulation of cellular identity and differentiation and hence might also determine the intrinsic growth state of a neuron. In order to identify transcription factors from rat cerebellum and entorhinal cortex at the time of maximal axonal outgrowth, PCR with degenerate oligonucleotides, specific for the conserved DNA-binding domains of distinct transcription factor classes, was performed with cDNA from cerebellum at E18 and entorhinal cortex at P0, respectively. A limited number of PCR products could be isolated from the above brain regions by the use of primers for the POU and zinc finger family of transcription factors. Because of the small number of candidate molecules and considerable difficulties in constructing cDNA probes for further analysis this approach was not further pursued. A second approach aimed at the comparison of the transcriptional activity of young differentiating CNS neurons, which extend axons, with that of more mature neurons, which have lost growth competence. The method of suppression subtractive hybridisation (SSH) was performed in two distinct CNS tissues, rat cerebellum and entorhinal cortex, at two developmental stages (E18 and P35 for cerebellum and P0 an P10 for entorhinal cortex, respectively) in order to enrich for genes, which are downregulated during postnatal development. Several differentially expressed genes were identified, and the temporal and spatial expression pattern of some of these genes was further examined in rat brain by Northern- and in situ-hybridisation analysis at different developmental stages. One of the identified genes, rMMS2, was not known in the rat before and was characterised in this study for the first time. In addition, CRHSP-24, whose expression pattern had not previously been examined in the developing brain, was identified as a differentially expressed gene. Further analysis showed that rMMS2 and CRHSP-24 were strongly expressed in many brain regions during late embryonic and early postnatal development. Expression of both genes was significantly downregulated during the first postnatal weeks and was only weak or absent in the adult brain. As this regulated distribution correlates well with the time period of establishment of axonal connections in the developing brain, these molecules might play a role in neuronal differentiation processes. However, their function in neuronal development is not yet clear and remains to be elucidated. Because only a fraction of the enriched genes has been analysed by now the pool of subtracted genes might serve as a valuable source for the identification of further candidate genes, which might be associated with neuronal differentiation and axonal outgrowth

Identification of the role of C/EBP in neurite regeneration following microarray analysis of a L. stagnalis CNS injury model

<p>Abstract</p> <p>Background</p> <p>Neuronal regeneration in the adult mammalian central nervous system (CNS) is severely compromised due to the presence of extrinsic inhibitory signals and a reduced intrinsic regenerative capacity. In contrast, the CNS of adult <it>Lymnaea stagnalis (L. stagnalis)</it>, a freshwater pond snail, is capable of spontaneous regeneration following neuronal injury. Thus, <it>L. stagnalis </it>has served as an animal model to study the cellular mechanisms underlying neuronal regeneration. However, the usage of this model has been limited due to insufficient molecular tools. We have recently conducted a partial neuronal transcriptome sequencing project and reported over 10,000 EST sequences which allowed us to develop and perform a large-scale high throughput microarray analysis.</p> <p>Results</p> <p>To identify genes that are involved in the robust regenerative capacity observed in <it>L. stagnalis</it>, we designed the first gene chip covering ~15, 000 <it>L. stagnalis </it>CNS EST sequences. We conducted microarray analysis to compare the gene expression profiles of sham-operated (control) and crush-operated (regenerative model) central ganglia of adult <it>L. stagnalis</it>. The expression levels of 348 genes were found to be significantly altered (p < 0.05) following nerve injury. From this pool, 67 sequences showed a greater than 2-fold change: 42 of which were up-regulated and 25 down-regulated. Our qPCR analysis confirmed that CCAAT enhancer binding protein (C/EBP) was up-regulated following nerve injury in a time-dependent manner. In order to test the role of C/EBP in regeneration, C/EBP siRNA was applied following axotomy of cultured <it>Lymnaea </it>PeA neurons. Knockdown of C/EBP following axotomy prevented extension of the distal, proximal and intact neurites. <it>In vivo </it>knockdown of C/EBP postponed recovery of locomotory activity following nerve crush. Taken together, our data suggest both somatic and local effects of C/EBP are involved in neuronal regeneration.</p> <p>Conclusions</p> <p>This is the first high-throughput microarray study in <it>L. stagnalis</it>, a model of axonal regeneration following CNS injury. We reported that 348 genes were regulated following central nerve injury in adult <it>L. stagnalis </it>and provided the first evidence for the involvement of local C/EBP in neuronal regeneration. Our study demonstrates the usefulness of the large-scale gene profiling approach in this invertebrate model to study the molecular mechanisms underlying the intrinsic regenerative capacity of adult CNS neurons.</p

Involvement of the serotonin receptor 7 in synaptic plasticity in the nervous system

The serotonin receptor 7 (5-HT7R) is a G protein-coupled receptor (GPCR) involved in many physiological events of the nervous system, such as learning and memory. It is also associated with several neurological and neurodevelopmental disorders, including Autism Spectrum Disorders (ASD). Among these pathologies, Angelman Syndrome (AS) is a rare neurodevelopmental disorder with a high comorbidity with ASD, especially with regards to developmental delay and language impairment. Interestingly, several pathways altered in this disease are positively regulated by 5-HT7R. Therefore, agonists of 5-HT7R could be considered as possible candidates for the development of innovative drugs to ameliorate AS symptoms. To this aim, we identified and characterized several 5-HT7R ligands and demonstrated that their residence time is structure-dependent and related to the polarity of the aryl moiety linked to their piperazine ring. In addition, our data on neurite outgrowth in neuronal primary cultures from different brain regions suggest that the residence time of these 5-HT7R ligands correlates with their kinetics of action. Using synaptosomes an in vitro model of presynaptic terminals, we observed that the stimulation of 5-HT7R finely regulates processes involved in synaptic plasticity, such as local synthesis and secretion of selected proteins. Finally, we demonstrated that stimulation of 5-HT7R was able to rescue LTP and behavioral impairments in a mouse model of AS. Altogether, our results underline the key role played by 5-HT7R in synaptic plasticity, indicating that this receptor represents a potential innovative target for pharmacological treatments of neurodevelopmental diseases characterized by altered connectivity/plasticity such as AS

Investigating small molecule therapeutics to improve regeneration and functional recovery following peripheral nerve damage

Peripheral nerve injury (PNI) can be debilitating and results in loss of function, coupled with slow neuron regeneration. Microsurgical treatments remain the gold standard therapy, with no drug therapies currently available. Effective pharmacological treatments could potentially maintain neuronal viability, encourage axonal growth, improve axonal specificity to targets and reduce neuropathic pain. Some drugs and targets have been identified but challenges remain with clinical translation. Advancements in understanding the molecular and cellular events occurring following PNI identifies signalling pathways that could be targeted with drug therapies. The failure in drug therapies reaching PNI clinical trials may be due to the lack of effective in vitro and in vivo pre-clinical models. This study developed and applied models to be used as effective screening tools to address this need. Many compounds demonstrated positive effects on neurite growth when screened in a 3D-engineered co-culture model. NSAIDs (ibuprofen and sulindac sulfide) demonstrated beneficial effects and were studied further in two injury models demonstrating increased axonal growth and improved function. Local controlled-release drug delivery systems have become more attractive because of the drawbacks in conventional drug treatments. This study investigated drug release from various biomaterials in order to obtain an optimal material for implantation and sustained drug delivery. Suitable biomaterials were implanted in vivo to deliver ibuprofen or sulindac sulfide. Both drugs demonstrated beneficial effects on axonal regeneration and functional recovery. Embedding drugs into biocompatible and bio-degradable materials provides effective delivery systems for future translation. Studying NSAIDs revealed a previously unreported relationship between PPAR-γ affinity and regeneration. A NSAID derivative demonstrated the greatest effects on neurite growth in vitro at lower doses than other compounds tested. In summary, this work has identified therapeutic targets to aid the development of novel compounds, as well as, drug repurposing, and effective tools for the pre-clinical screening of these drugs

Computer vision profiling of neurite outgrowth mordphodynamics reveals spatio-temporal modularity of Rho GTPase signaling

Neurite outgrowth is essential to build the neuronal processes that produce axons and dendrites that connect the adult brain. In cultured cells, the neurite outgrowth process is highly dynamic, and consists of a series of repetitive morphogenetic sub-processes (MSPs), such as neurite initiation, elongation, branching, growth cone motility and collapse (da Silva and Dotti 2002). Neurons also actively migrate, which might in part reflect neuronal migration during brain development. Each of the different MSPs inherent to neurite outgrowth and cell migration is likely to be regulated by precise spatio-temporal signaling networks that control cytoskeletal dynamics, trafficking and adhesion events. These MSPs can occur on a range of time and length scales. For example, microtubule bundling in the neurite shaft can be maintained during hours, while growth cone filopodia dynamically explore their surrounding on time scales of seconds and length scales of single microns. This implies that a correct understanding of these processes will require analysis with an adequate spatio-temporal resolution. The Rho family of GTPases are signaling switches that regulate a wide variety of cellular processes, such as actin and adhesion dynamics, gene transcription, and neuronal differentiation (Boguski and McCormick 1993). Rho GTPases are activated by guanine nucleotide exchange factors (GEFs), and are switched off by GTPase activating proteins (GAPs). Upon activation, Rho GTPases can associate with effectors to initiate a downstream response. Current models propose that Rac1 and Cdc42 regulate neurite extension, while RhoA controls growth cone collapse and neurite retraction (da Silva and Dotti 2002). However, until now the effects of Rho GTPases on neurite outgrowth have mostly been assessed using protein mutants in steady-state experiments, most often at late differentiation stages, which do not provide any insight about the different MSPs during neurite outgrowth. However, our proteomic analysis of biochemically-purified neurites from N1E-115 neuronal-like cells (Pertz et al. 2008), has suggested the existence of an unexpectedly complex 220 proteins signaling network consisting of multiple GEFs, GAPs, Rho GTPases, effectors and additional interactors. This is inconsistent with the simplistic view that classical experiments have provided before. In order to gain insight into the complexity of this Rho GTPase signaling network, we performed a siRNA screen that targets each of these 220 proteins individually. We hypothesized that specific spatio-temporal Rho GTPase signaling networks control different MSPs occurring during neurite outgrowth, and therefore designed an integrated approach to capture the whole morphodynamic continuum of this process. Perturbations of candidates that lead to a similar phenotype might be part of a given spatio-temporal signaling network. This approach consisted of: 1) A high content microscopy platform that allowed us to produce 8000 timelapse movies of 660 siRNA perturbations; 2) A custom built, computer vision approach that allowed us to automatically segment and track neurite and soma morphodynamics in the timelapse movies (collaboration with the group of Pascal Fua, EPFL, Lausanne); 3) A sophisticated statistical analysis pipeline that allowed the extraction of morphological and morphodynamic signatures (MDSs) relevant to each siRNA perturbation (collaboration with the group of Francois Fleuret, IDIAP). Analysis of our dataset revealed that each siRNA perturbation led to a quantifiable phenotype, emphasizing the quality of our proteomic dataset. Hierarchical clustering of the MDSs revealed the existence of 24 phenoclusters that provide information about neurite length, branching, number of neurites, soma migration speed, and a panel of additional morphological and morphodynamic features that are more difficult to grasp using visual inspection. This complex phenotypic space can more easily be understood when classified according to the first 4 features. Our screen then suggests the existence of 4 major morphodynamic phenotypes that define distinct stages of the neurite outgrowth process. These consist of phenotypes with short neurites, multiple short neurites, long neurites, and long and branched neurites. Further subdivision using the other features provides more information, with cell migration features being very often affected. This implies a high overlap between the signaling machinery that regulates the neurite outgrowth and cell migration processes. The high phenotypical redundancy (24 clusters for 220 candidate genes) provides only limited information to deduce unambiguous signaling networks regulating distinct MSPs. Further knowledge acquired from other approaches we used to study Rho GTPase signaling (FRET biosensors, and other live cell imaging techniques), made us realize that some morphodynamic phenotypes can only be understood when growth cone dynamics are inspected at a much higher resolution. For this purpose, we decided to further investigate a defined subset of genes using high resolution live cell imaging and a custom built growth cone segmentation and tracking pipeline for accurate quantification (collaboration with the group of Gaudenz Danuser, Harvard Medical School, Boston). These results shed light into how distinct cytoskeletal networks enabling growth cone advance can globally impact the neurite outgrowth process. A clear understanding of spatio-temporal Rho GTPase signaling will therefore require multi-scale approaches. Our results provide the first insight into the complexity of spatio-temporal Rho GTPase signaling during neurite outgrowth. The technologies we devised and our initial results, pave the way for a systems biology understanding of these complex signaling systems

Identification and characterization of long-range SOX9 enhancers in limb development

The transcription factor Sox9 is a master regulator of skeletogenesis. Heterozygous mutations of human SOX9 result in Campomelic Dysplasia (CD), in which affected individuals display distinct abnormalities in limbs and other skeletal assemblies. Recently, chromosomal translocations and deletions at >1Mb from SOX9 have been detected in some CD patients, suggesting the requirement of long‐range regulatory elements in mediating both spatiotemporal and dosage of Sox9 during limb development. To this end, we exploited several published ChIP‐Seq data, and identified nine, evolutionarily conserved, putative limb enhancers of SOX9, namely E1Sox9 to E9Sox9. Transgenic mouse embryos carrying E1Sox9‐driven LacZ reporter showed discrete transgene expression at the pre‐scapular domain where endogenous Sox9 is also expressed. Bioinformatic analyses on our candidate enhancers result in the identification of several signaling effector binding motifs, and indeed, we revealed that BMP‐Smad and Shh‐Gli pathways are possible upstream regulatory networks that govern the spatiotemporal and dosage of limb Sox9 expression via our predicted enhancers, respectively. Our results unveil the underlying molecular control in governing the complex patterning of Sox9 expression in the developing limb, and provide new molecular insight to the etiology of CD syndrome.postprin