The identification of cell type defining genes across human tissues and the functional study of the endothelial adhesion G protein-coupled receptor L4

Cell type specific gene expression profiles underlie differences in cell morphology, behaviour, and specialized function. Single cell RNA sequencing can be used to measure gene expression in individual cells, but challenges remain including limited read-depth, artefactual changes due to dissociation from tissue, difficulties in the analysis of fragile or morphologically complex cell types, and bias introduced from the analysis of a limited number of biological replicates. In paper I, we used an integrative correlation analysis to define cell type enriched transcripts from bulk RNAseq, generated from visceral and subcutaneous adipose tissue. We identified depot and sex-specific differences. In Paper II, we expanded our analysis to include cell types in 15 human tissue types, to create a cell type enrichment prediction atlas for all protein coding genes. A cross-tissue comparison identified shared enrichment signatures between cell types in different tissues. We also defined core identity profiles of cell types present in all or most tissue types, including endothelial cells (EC), which can vary in gene enrichment profiles across different vascular beds. The focus of paper III was the functional characterisation of one such highly EC enriched gene, adhesion G protein-coupled rector L4 (ADGRL4). EC have a major role in various biological processes, including the regulation of inflammatory responses and haemostasis. The endothelial restricted expression of ADGRL4 is indicative of an important cell type specific role in EC. We depleted ADGRL4 in EC and measured associated changes in proteome and function, under normal and cytokine stimulated conditions. Under inflammatory conditions, ADGRL4 depletion potentiated EC pro-coagulant protein expression and associated thrombin and fibrin formation. Concurrently, ADGRL4 depletion inhibited the expression of inflammation-induced interferon response genes. This indicates that ADGRL4 has a currently unappreciated role in the EC function, with a potential role in the regulation of coagulation during inflammation

Autophagy regulates neuronal excitability by controlling cAMP/Protein Kinase A signalling

Autophagy is an evolutionarily conserved process that serves to provide nutrients during starvation and to eliminate detrimental cellular components like dysfunctional organelles and damaged proteins. This role of autophagy might be critical in neurons because of their postmitotic nature. However, accumulating evidence indicates that autophagy is not merely a housekeeping process. The study shows that the crucial AuTophaGy protein ATG5 functions in neurons to regulate the cAMP-dependent protein kinase A (PKA)-mediated phosphorylation of synapse-confined proteome. This function of ATG5 is independent of bulk degradation of synaptic proteins and requires the starvation-induced targeting of PKA regulatory subunit type 1 (R1) to neuronal autophagosomes. Loss of ATG5 in either excitatory or inhibitory neurons causes a drastic accumulation of PKA R1 at synapses, which sequesters the PKA catalytic subunit and causes cAMP-dependent remodelling of the synaptic phosphoproteome. Autophagy-deficient excitatory synapses are characterized by increased thickness of the postsynaptic density and alterations in AMPA receptor GLUR1 trafficking, a phenotype that results in augmented excitatory neurotransmission and appearance of seizures in mice with glutamatergic forebrain-confined ATG5 deletion. My work has identified a previously unknown role of neuronal autophagy in regulating PKA-dependent signalling at glutamatergic synapses and suggest PKA as a target for treating autophagy-associated neurodegenerative diseases

Hippo signalling in mammalian cortical development

The cerebral cortex in humans is composed of billions of morphologically and functionally distinct neurons. Development of the neocortex requires an orchestrated succession of a series of processes, the appropriate generation, migration, and positioning of neurons, the acquisition of layer-specific transcriptional hallmarks, and the establishment of precise axonal projections. We have primarily focussed on elucidating the transcriptomic landscape of murine embryonic neural stem cells (NSCs), basal progenitors (BPs) and newborn neurons (NBNs) at the population level. I have focussed on one underexplored signalling pathway in the brain- the Hippo signalling pathway. Hippo signalling effectors are expressed dynamically during the course of development in NSCs and BPs at mRNA level. Hippo transcription factors (TFs), Tead1 and Tead3 show higher expression during gliogenesis while Tead2 is expressed at relatively higher levels during early phases of neural expansion. Known to be redundant in other biological systems, I explored different effects of three Tead TFs in NSCs using gain and loss of function. I observe reciprocal effects on neuronal migration and fate with Tead1, Tead3 and Tead2. We identified ApoE, Cyr61 and Dab2 as potential direct targets of Tead TFs in NSCs. ApoE gain of function partially recapitulates the gain of function of Tead2, reducing cell migration to the cortical plate (CP) and Dab2 gain of function recapitulates the gain of function of Tead1, an increased migration to CP. ApoE and Dab2 are involved in Reelin signalling and hence we provide the first link between Hippo and Reelin signalling pathways controlling cortical development

C-di-GMP signalling in Pseudomonas aeruginosa: connecting the dots in a multi modal network

Bacteria rely on complex regulatory networks to control their cellular programme in order to respond to extracellular factors in an environment or host setting. Bis-(3′-5′)-cyclic dimeric guanosine monophosphate (c-di-GMP) is a signalling molecule used by several bacterial species to facilitate some of those important lifestyle decisions. One of the most complex and elaborated c-di-GMP signalling networks is found in the human opportunistic pathogen Pseudomonas aeruginosa, which is a major cause of chronic infection in cystic fibrosis patients. The genome of P. aeruginosa alone encodes 41 proteins harbouring the potential to make or break c-di-GMP. Using a methodical deletion approach, single and multiple c-di-GMP metabolizing genes were knocked out and their role in attachment, biofilm, motility, virulence and antimicrobial resistance systematically assessed, providing a comprehensive pool of data for the c-di-GMP research community. Interestingly, a very large number of c-di-GMP deletion mutants showed an altered biofilm/attachment profile, indicating that the network is of great importance for motile to sessile transition. We further uncovered that among all c-di-GMP regulating enzymes, PA0285 is one of the most potent phosphodiesterases regulating intracellular c-di-GMP levels leading to the most pronounced biofilm attachment phenotype. It is uniquely controlled by its dual domain structure containing both an EAL domain responsible for breaking c-di-GMP as well as a regulatory GGDEF domain characteristic of c-di-GMP synthesis. Both domains have been shown to be essential for the PA0285 mediated phenotypes in biofilm attachment and intracellular c-di-GMP. Further, targeted protein-protein interaction analysis revealed the molecular mechanisms of DgcP (PA5487), a c-di-GMP synthesising cyclase. involved in the convergence of c-di-GMP and cyclic adenosine monophosphate (cAMP) signalling pathways facilitating touch-down and attachment behaviour of P. aeruginosa.Open Acces

Mechanism of actin polymerization by adhesion and degranulation-promoting adaptor protein (ADAP)

Adhesion and degranulation-promoting adaptor protein (ADAP) is known to play an important role in indirectly regulating integrins upon T-cell receptor or chemokine receptor stimulation and as a result is crucial for T cell migration and adhesion. ADAP has also been reported to bind to the known actin regulators Ena/VASP and Nck. Here, in this work we show that ADAP interacts with actin directly and induces actin polymerization in vitro. The region responsible for the polymerization activity was narrowed down to the disordered N-terminal 381 residues of ADAP. ADAP also induces bundling of actin filaments in an in vitro co-sedimentation assay. The fragments of ADAP that showed activity in polymerization assays were also active on bundling actin filaments. The formation of filaments and actin fibers by ADAP and various ADAP fragments were confirmed by negative stain electron microscopy showing bundles of distinct sizes and patterns when comparing ADAP_1-381, ADAP-full length and the ADAP/SKAP55 complex. The involvement of the N-terminus of ADAP in actin remodeling was further confirmed in Jurkat T cells where adhesion and migration was seen to be impaired and total F-actin content was significantly reduced upon stimulation in cells expressing ADAP missing its N-terminal region. NMR investigations also confirmed the binding of ADAP to monomeric as well as filamentous actin and provided information about multiple epitopes in the N-terminal 200 residues of ADAP involved in direct interaction with monomeric actin. Further confirmation of these epitopes was obtained from crosslinking mass spectrometry where short ADAP motifs, often enriched in lysine-proline dipeptides were found to bind to all four sub-domains on monomeric actin suggesting a multivalent interaction mode. In contrast, the same ADAP motifs were seen to converge to the actin dimer interface along the axis of the F-actin filament. The enhanced actin polymerization activity of ADAP in the presence of actin binding proteins like profilin (a barbed end actin elongator) and CapZ (a barbed end capping protein) indicate that ADAP polymerizes through the pointed end of the actin filament. However, the addition of cofilin initially leads to enhanced polymerization activity by ADAP followed by strong depolymerization. This suggests that cofilin has an overlapping binding site with ADAP on the filament as also suggested by our docking models. Overall, these results direct towards a novel mechanism of actin polymerization by the intrinsically disordered region of ADAP, which enhances the local concentration of actin by a sponge-like, multivalent mode of interaction that leads to instant polymerization, formation of filaments and actin bundles.Das Adhäsions- und degranulationsfördernde Adaptorprotein (ADAP) spielt bekanntermaßen eine wichtige Rolle bei der indirekten Regulierung von Integrinen nach Stimulation durch T-Zell-Rezeptoren oder Chemokinrezeptoren und ist daher für die Migration und Adhäsion von T-Zellen entscheidend. Es wurde auch berichtet, dass ADAP an die bekannten Aktinregulatoren Ena/VASP und Nck bindet. In dieser Arbeit zeigen wir, dass ADAP direkt mit Aktin interagiert und in vitro zur Polymerisation von Aktin führt. Die Region, die für die Polymerisationsaktivität verantwortlich ist, wurde auf die ungeordneten N-terminalen 381 Aminosäuren von ADAP eingegrenzt. ADAP induziert auch die Bündelung von Aktinfilamenten in einem in vitro Co-Sedimentationstest. Die Fragmente von ADAP, die in Polymerisationstests Aktivität zeigten, waren auch bei der Bündelung von Aktinfilamenten aktiv. Die Bildung von Filamenten und Aktinfasern durch ADAP und verschiedene ADAP-Fragmente wurde durch Negativ-Stain-Elektronenmikroskopie bestätigt, die beim Vergleich von ADAP_1-381, ADAP in voller Länge und dem ADAP/SKAP55-Komplex Bündel unterschiedlicher Größe und Muster zeigte. Die Beteiligung des N-Terminus von ADAP am Aktin-Remodeling wurde ferner in Jurkat-T-Zellen bestätigt, bei denen eine Beeinträchtigung der Adhäsion und Migration festgestellt wurde und der Gesamt-F-Aktin-Gehalt nach Stimulation in Zellen, die ADAP ohne N-Terminus exprimieren, signifikant reduziert war. NMR-Untersuchungen bestätigten auch die Bindung von ADAP an monomeres und filamentöses Aktin und lieferten Informationen über mehrere Epitope in den N-terminalen 200 Aminosäureresten von ADAP, die an dieser direkten Interaktion beteiligt sind. Eine weitere Bestätigung dieser Epitope lieferte die Crosslinking-Massenspektrometrie, bei der festgestellt wurde, dass kurze ADAP-Motive, die häufig mit Lysin-Prolin-Dipeptiden angereichert sind, an allen vier Subdomänen von monomerem Aktin binden, was auf einen multivalenten Interaktionsmodus schließen lässt. Im Gegensatz dazu wurde festgestellt, dass dieselben ADAP-Motive entlang der Achse des F-Aktinfilaments an der Schnittstelle der Aktin-Dimere konvergieren. Die verstärkte Aktinpolymerisationsaktivität von ADAP in Gegenwart von Aktinbindungsproteinen wie Profilin (ein Aktinverlängerer) und CapZ (ein Kappenprotein) deutet darauf hin, dass ADAP über das minus-Ende des Aktinfilaments polymerisiert. Die Zugabe von Cofilin führt jedoch zunächst zu einer verstärkten Polymerisationsaktivität von ADAP, gefolgt von einer starken Depolymerisation. Dies deutet darauf hin, dass Cofilin eine überlappende Bindungsstelle mit ADAP auf dem Filament hat, wie auch unsere Docking-Modelle nahelegen. Insgesamt deuten diese Ergebnisse auf einen neuartigen Mechanismus der Aktinpolymerisation durch den intrinsisch ungeordneten Bereich von ADAP hin, der die lokale Aktinkonzentration durch einen schwammartigen, multivalenten Interaktionsmodus erhöht, der zu sofortiger Polymerisation, Bildung von Filamenten und Aktinbündeln führt

Mechanisms of Ewing sarcoma metastasis : biochemistry and biophysics

Ewing sarcoma (ES) is a special type of bone cancer, first described by Dr. James Ewing in his paper __Diffusive endothelioma of bone__. Today Ewing sarcoma represents the second most common bone cancer among adolescents and young adults. Contrary to the positive achievement in treatment of localized tumors, the long-term (5-years) survival for Ewing sarcoma patients with metastasis, however, remain below the 30% mark. In this thesis a report on experimental work aiming for a better understanding of the mechanisms underlying Ewing sarcoma metastasis is presented. Two distinct mechanisms are investigated: (1) a biochemical approach in which the initial steps in the CXCR4 signaling cascade are followed, and (2) a biophysical approach in which the guidance of Ewing sarcoma metastasis by the stiffness of their microenvironment is demonstrated. The results presented in this thesis provide deeper insights into the mechanisms controlling signaling of the chemokine receptor CXCR4 and into the role of the micro-environment in Ewing sarcoma cells behavior.Through various experimental approaches it was shown that both biochemical and biophysical guidance control how Ewing sarcoma develops into its distinct metastatic phenotype.Biological and Soft Matter Physic

NMDAR-PSD95-nNOS Axis-Mediated Molecular Mechanisms in the Basolateral Amygdala Underlying Fear Consolidation

Indiana University-Purdue University Indianapolis (IUPUI)Fear is an evolutionarily conserved response that can facilitate avoidance learning and promote survival, but excessive and persistent fear responses lead to development of phobias, generalized fear, and post-traumatic stress disorder. The primary goal of experiments in this dissertation is to determine the molecular mechanisms underlying formation of fear memories. The acquisition and consolidation of fear is dependent upon activation of N-methyl-D-aspartic acid receptors (NMDARs). Stimulation of NMDARs recruits neuronal nitric oxide synthase (nNOS) to the synaptic scaffolding protein, postsynaptic density protein 95 (PSD95), to produce nitric oxide (NO). Our laboratory has previously shown that disruption of the PSD95-nNOS interaction attenuates fear consolidation and impairs long-term potentiation of basolateral amygdala (BLA) neurons in a rodent model of auditory fear conditioning. However, the molecular mechanisms by which disrupting the PSD95-nNOS interaction attenuates fear consolidation are not well understood. Here, we used pharmacological and genetic approaches to study the effects underlying nNOS activity in the BLA during fear consolidation. During the early stage of fear memory consolidation (4-6 hours after fear acquisition), we observed increased α- Amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor (AMPAR)-mediated current and synaptosomal AMPAR GluR1 subunit trafficking in the BLA; while during the late stage (24h after fear acquisition), we detected a combination of enhanced AMPAR- and NMDAR-mediated currents, increased synaptosomal NMDAR NR2B subunit expression, and phosphorylation of synaptosomal AMPAR GluR1 and NMDAR NR2B subunits in the BLA. Importantly, we showed that pharmacological and genetic blockade of nNOS activity inhibits all of these glutamatergic synaptic plasticity changes in the BLA. Additionally, we discovered whole transcriptome changes in the BLA following fear consolidation. In the group with pharmacological inhibition of nNOS activity, however, gene expression levels resembled control-like levels. We also observed altered expression of multiple genes and identified the insulin-like growth factor system, D3/D4 dopamine receptor binding, and cGMP effects as key pathways underlying nNOSmediated consolidation of fear. Our results reveal nNOS-mediated, sequentially orchestrated synaptic plasticity changes facilitated by AMPA and NMDA receptors in the BLA during early and late stages of fear memory consolidation. We also report novel genetic targets and pathways in the BLA underlying NMDAR-PSD95-nNOS axis-mediated formation of fear memories

Characterization and Pharmacological Modulation of Ion Transport Mechanisms Involved in the Innate Defence of Human Airway Epithelium

The airway epithelium plays a protective role against microbial pathogens delivered by inhaled air. We used cultured bronchial epithelia, differentiated in vitro under air-liquid interface condition, to study the defence mechanisms of the airway epithelium against pathogens, with a particular focus on those regulated by intracellular Ca2+. As a first step, we investigated the response to inflammatory stimuli, in particular IL-4 and IL-17/TNF-α,which both elicited profound changes in transepithelial ion transport mechanisms but with some important differences. IL-4 strongly increased the expression and function of the Ca2+-activated Cl- channel TMEM16A, and decreased the Na+ absorption mediated by ENaC. In contrast, the IL-17/TNF-α treatment had no effect on TMEM16A and markedly increased ENaC function. This condition could promote dehydration of the airway surface, and impairment of mucociliary clearance, particularly in patients affected by cystic fibrosis (CF) which suffer from defective function of CFTR, a second type of Cl- channel. We thought that potentiators of TMEM16A-mediated Cl- secretion could be useful to improve airway surface hydration and hence mucociliary clearance, in particular under inflammatory conditions associated with IL-17/TNF-α and in CF patients. To find TMEM16A potentiators, we screened a large (11,300 compounds) library with a functional assay. We found three active molecules, ARN7149, ARN4550, and ARN11391. Analysis of mechanism of action of these compounds indicate they act upstream TMEM16A. In particular, ARN7149 is a potentiator of the purinergic P2RY2 receptor, whereas ARN11391 is a potentiator of the inositol triphosphate receptor ITPR1. Interestingly, ARN7149 and ARN4550 also caused inhibition of ENaC, probably by causing breakdown of PIP2, a known regulator of ENaC function. Combined effects on TMEM16A (potentiation) and ENaC (inhibition) could have beneficial effects on mucociliary clearance.We also evaluated the specificity of known pharmacological inhibitors of TMEM16A, in particular niclosamide, which has been used to assign physiological roles to TMEM16A. Surprisingly, most compounds, except Ani9, showed an indirect mechanism of action involving alteration of mechanisms that control intracellular Ca2+ mobilization. Therefore, conclusions based on many TMEM16A inhibitors need to be reconsidered. Finally, we investigated the role of TRPV4, a Ca2+ channel that acts as a sensor of mechanical and chemical stimuli. It has been shown that TRPV4 activation enhances the beating of cilia in the airway epithelium. We found that TRPV4 has an additional role in innate defence mechanisms. Indeed, stimulation of TRPV4-dependent Ca2+ influx resulted in H2O2 release, a potential bactericidal molecule