Struktur-Funktionsanalyse des Transkriptionsfaktors Sox10 und Aspekte der Evolution

The invention of neural crest (NC) cells and oligodendrocytes played an important role in the emergence of the vertebrate phylum during evolution. In humans, mutations of the transcription factor SOX10 affect the development of these cell types and result in a wide range of neurocristopathies and demyelinating diseases. A direct link between SOX10 mutations and the severity of the disease has been proposed. However the exact mode of action of different mutations and more generally the importance of the different domains of the Sox10 protein on developmental processes have not been studied in detail. The first aim of this study was therefore to better characterize the functional importance of different Sox10 domains and the influence of SOX10 mutations on early NC development. Using overexpression in the chicken neural tube, I showed that Sox10 functions during early NC development rely on its DNA binding and transactivation abilities. My results moreover argue that truncated SOX10 proteins which result from nonsense mutations in exon 4 of the SOX10 gene behave as dominant negatives but that production of these proteins is prevented in human patients by nonsense mediated decay. Different mutations within the SOX10 protein can furthermore influence oligodendrocytes and NC-derived lineages differentially. This last conclusion was also confirmed in a knock-in mouse mutant where wild-type Sox10 was replaced by a mutant version without cooperative binding capabilities. In these mice, development of most NC-derived-cell types was severely impaired whereas oligodendrocyte development was not. The second aim of this study was to understand how Sox10 acquired its essential functions in oligodendrocytes and NC cells during evolution. For that purpose I analyzed the capability of Sox100B from Drosophila melanogaster to perform Sox10 specific functions in vivo. Sox100B was overexpressed in the chicken neural tube and a mutant mouse line was established where the Sox10 allele was replaced by the Sox100B open reading frame. Anaysis of these animals showed that Sox100B can take over the functions of Sox10 during early NC, Schwann cell and oligodendrocyte development arguing that an ancestral SoxE gene could have easily integrated into the gene regulatory networks that control the development of these cell types following its co-option. However, later developmental defects in several NC derived lineages in mice homozygous for the Sox100B replacement allele indicate that some degree of functional specialization and adaptation of SoxE protein properties have taken place later on.Die Entstehung von Neuralleistenzellen und Oligodendrozyten war ein wichtiger Schritt in der Evolution der Wirbeltiere. Mutationen des Transkriptionfaktors SOX10 beeinträchtigen die Entwicklung dieser Zelltypen im Menschen und verursachen Neurocristopathien und demyelinisierende Schädigungen. Es wird angenommen, dass die Art und die Position der Mutationen einen direkten Einfluß auf den Schweregrad der Krankheit haben. Die genaue Wirkungsweise der verschiedenen Mutationen, sowie die Bedeutung von verschiedenen Domänen des Sox10 Proteins für Entwicklungprozesse wurden bisher allerdings nicht genau analysiert. Im Rahmen dieser Arbeit wurden die funktionelle Bedeutung von verschiedenen SOX10 Domänen und der Einfluß von SOX10 Mutationen auf die frühe Entwicklung der Neuralleistenzellen untersucht. Die Überexpression von SOX10 im Neuralrohr von Hühner-Embryonen zeigte, dass die Funktion in der frühen Entwicklung der Neuralleistenzellen auf der DNA-Bindefähigkeit und Transaktivierungsfähigkeit des Proteins beruht. Außerdem zeigen meine Ergebnisse, dass verkürzte SOX10 Proteine, die aufgrund von Nonsense-Mutationen innerhalb des vierten Exons des SOX10 Gens produziert wurden, sich dominant negativ verhalten, dass aber die Herstellung solcher Proteine im Menschen durch „nonsense mediated decay“ vermieden wird. Verschiedene Mutationen des SOX10 Proteins hatten weiterhin unterschiedlichen Einfluß auf Oligodendrozyten und verschiedene Neuralleistenderivate. Diese Aussage wurde mittels einer knock-in Mausvariante bestätigt, in der das Wildtyp Sox10 Protein durch eine Mutante ohne kooperative DNA-Bindefähigkeit ersetzt wurde. Die Entwicklung von Neuralleistenderivaten war in diesen Mäusen stark beeinträchtigt, wohingegen die Oligodendrozytenentwicklung normal war. Im zweiten Teil dieser Arbeit sollte analysiert werden, wie Sox10 während der Evolution seine essentiellen Funktionen in Oligodendrozyten und Neuralleistenzellen erwarb. Zu diesem Zweck untersuchte ich, ob das Sox100B Protein aus Drosophila melanogaster die spezifischen Funktionen von Sox10 in vivo ausführen kann. Es wurde Sox100B im Neuralrohr von Hühner-Embryonen überexprimiert und eine Mauslinie generiert, in der das Sox10 Allel durch den offenen Leserahmen von Sox100B ersetzt wurde. Die Analyse dieser Tiere zeigte, dass Sox100B die Funktionen von Sox10 in der frühen Neuralleisten-, Schwannzellen- und Oligodendrozytenentwicklung übernehmen kann. Diese Ergebnisse beweisen, dass eine Funktionsübernahme der ursprünglichen Eigenschaften (Ko-Option) eines anzestralen SoxE Gens in das genregulatorische Netzwerk stattgefunden hat, das die Entwicklung dieser Zelltypen kontrolliert. Spätere Entwicklungsdefekte in verschiedenen Neuralleistenderivaten in Sox10Sox100B/Sox100B Mäusen zeigten aber, dass gewisse funktionelle Spezialisierungen und Adaptationen der SoxE Proteinwirkungen im Nachhinein erfolgt sind

Struktur-Funktionsanalyse des Transkriptionsfaktors Sox10 und Aspekte der Evolution

The invention of neural crest (NC) cells and oligodendrocytes played an important role in the emergence of the vertebrate phylum during evolution. In humans, mutations of the transcription factor SOX10 affect the development of these cell types and result in a wide range of neurocristopathies and demyelinating diseases. A direct link between SOX10 mutations and the severity of the disease has been proposed. However the exact mode of action of different mutations and more generally the importance of the different domains of the Sox10 protein on developmental processes have not been studied in detail. The first aim of this study was therefore to better characterize the functional importance of different Sox10 domains and the influence of SOX10 mutations on early NC development. Using overexpression in the chicken neural tube, I showed that Sox10 functions during early NC development rely on its DNA binding and transactivation abilities. My results moreover argue that truncated SOX10 proteins which result from nonsense mutations in exon 4 of the SOX10 gene behave as dominant negatives but that production of these proteins is prevented in human patients by nonsense mediated decay. Different mutations within the SOX10 protein can furthermore influence oligodendrocytes and NC-derived lineages differentially. This last conclusion was also confirmed in a knock-in mouse mutant where wild-type Sox10 was replaced by a mutant version without cooperative binding capabilities. In these mice, development of most NC-derived-cell types was severely impaired whereas oligodendrocyte development was not. The second aim of this study was to understand how Sox10 acquired its essential functions in oligodendrocytes and NC cells during evolution. For that purpose I analyzed the capability of Sox100B from Drosophila melanogaster to perform Sox10 specific functions in vivo. Sox100B was overexpressed in the chicken neural tube and a mutant mouse line was established where the Sox10 allele was replaced by the Sox100B open reading frame. Anaysis of these animals showed that Sox100B can take over the functions of Sox10 during early NC, Schwann cell and oligodendrocyte development arguing that an ancestral SoxE gene could have easily integrated into the gene regulatory networks that control the development of these cell types following its co-option. However, later developmental defects in several NC derived lineages in mice homozygous for the Sox100B replacement allele indicate that some degree of functional specialization and adaptation of SoxE protein properties have taken place later on.Die Entstehung von Neuralleistenzellen und Oligodendrozyten war ein wichtiger Schritt in der Evolution der Wirbeltiere. Mutationen des Transkriptionfaktors SOX10 beeinträchtigen die Entwicklung dieser Zelltypen im Menschen und verursachen Neurocristopathien und demyelinisierende Schädigungen. Es wird angenommen, dass die Art und die Position der Mutationen einen direkten Einfluß auf den Schweregrad der Krankheit haben. Die genaue Wirkungsweise der verschiedenen Mutationen, sowie die Bedeutung von verschiedenen Domänen des Sox10 Proteins für Entwicklungprozesse wurden bisher allerdings nicht genau analysiert. Im Rahmen dieser Arbeit wurden die funktionelle Bedeutung von verschiedenen SOX10 Domänen und der Einfluß von SOX10 Mutationen auf die frühe Entwicklung der Neuralleistenzellen untersucht. Die Überexpression von SOX10 im Neuralrohr von Hühner-Embryonen zeigte, dass die Funktion in der frühen Entwicklung der Neuralleistenzellen auf der DNA-Bindefähigkeit und Transaktivierungsfähigkeit des Proteins beruht. Außerdem zeigen meine Ergebnisse, dass verkürzte SOX10 Proteine, die aufgrund von Nonsense-Mutationen innerhalb des vierten Exons des SOX10 Gens produziert wurden, sich dominant negativ verhalten, dass aber die Herstellung solcher Proteine im Menschen durch „nonsense mediated decay“ vermieden wird. Verschiedene Mutationen des SOX10 Proteins hatten weiterhin unterschiedlichen Einfluß auf Oligodendrozyten und verschiedene Neuralleistenderivate. Diese Aussage wurde mittels einer knock-in Mausvariante bestätigt, in der das Wildtyp Sox10 Protein durch eine Mutante ohne kooperative DNA-Bindefähigkeit ersetzt wurde. Die Entwicklung von Neuralleistenderivaten war in diesen Mäusen stark beeinträchtigt, wohingegen die Oligodendrozytenentwicklung normal war. Im zweiten Teil dieser Arbeit sollte analysiert werden, wie Sox10 während der Evolution seine essentiellen Funktionen in Oligodendrozyten und Neuralleistenzellen erwarb. Zu diesem Zweck untersuchte ich, ob das Sox100B Protein aus Drosophila melanogaster die spezifischen Funktionen von Sox10 in vivo ausführen kann. Es wurde Sox100B im Neuralrohr von Hühner-Embryonen überexprimiert und eine Mauslinie generiert, in der das Sox10 Allel durch den offenen Leserahmen von Sox100B ersetzt wurde. Die Analyse dieser Tiere zeigte, dass Sox100B die Funktionen von Sox10 in der frühen Neuralleisten-, Schwannzellen- und Oligodendrozytenentwicklung übernehmen kann. Diese Ergebnisse beweisen, dass eine Funktionsübernahme der ursprünglichen Eigenschaften (Ko-Option) eines anzestralen SoxE Gens in das genregulatorische Netzwerk stattgefunden hat, das die Entwicklung dieser Zelltypen kontrolliert. Spätere Entwicklungsdefekte in verschiedenen Neuralleistenderivaten in Sox10Sox100B/Sox100B Mäusen zeigten aber, dass gewisse funktionelle Spezialisierungen und Adaptationen der SoxE Proteinwirkungen im Nachhinein erfolgt sind

The enteric nervous system is a potential autoimmune target in multiple sclerosis

Multiple sclerosis (MS) is a chronic autoimmune disease of the central nervous system (CNS) in young adults that has serious negative socioeconomic effects. In addition to symptoms caused by CNS pathology, the majority of MS patients frequently exhibit gastrointestinal dysfunction, which was previously either explained by the presence of spinal cord lesions or not directly linked to the autoimmune etiology of the disease. Here, we studied the enteric nervous system (ENS) in a B cell-and antibody-dependent mouse model of MS by immunohistochemistry and electron microscopy at different stages of the disease. ENS degeneration was evident prior to the development of CNS lesions and the onset of neurological deficits in mice. The pathology was antibody mediated and caused a significant decrease in gastrointestinal motility, which was associated with ENS gliosis and neuronal loss. We identified autoantibodies against four potential target antigens derived from enteric glia and/or neurons by immunoprecipitation and mass spectrometry. Antibodies against three of the target antigens were also present in the plasma of MS patients as confirmed by ELISA. The analysis of human colon resectates provided evidence of gliosis and ENS degeneration in MS patients compared to non-MS controls. For the first time, this study establishes a pathomechanistic link between the well-established autoimmune attack on the CNS and ENS pathology in MS, which might provide a paradigm shift in our current understanding of the immunopathogenesis of the disease with broad diagnostic and therapeutic implications

Modulation of lipopolysaccharide-induced neuronal response by activation of the enteric nervous system.

International audienceBackground:Evidence continues to mount concerning the importance of the enteric nervous system (ENS) incontrolling numerous intestinal functions in addition to motility and epithelial functions. Nevertheless, little isknown concerning the direct participation of the ENS in the inflammatory response of the gut during infectious orinflammatory insults. In the present study we analyzed the ENS response to bacterial lipopolysaccharide, inparticular the production of a major proinflammatory cytokine, tumor necrosis factor-alpha (TNF-α).Methods:TNF-αexpression (measured by qPCR, quantitative Polymerase Chain Reaction) and production(measured by ELISA) were measured in human longitudinal muscle-myenteric plexus (LMMP) and rat ENS primarycultures (rENSpc). They were either treated or not treated with lipopolysaccharide (LPS) in the presence or not ofelectrical field stimulation (EFS). Activation of extracellular signal-regulated kinase (ERK) and 5?-adenosinemonophosphate-activated protein kinase (AMPK) pathways was analyzed by immunocytochemistry and Westernblot analysis. Their implications were studied using specific inhibitors (U0126, mitogen-activated protein kinasekinase, MEK, inhibitor and C compound, AMPK inhibitor). We also analyzed toll-like receptor 2 (TLR2) expression andinterleukin-6 (IL-6) production after LPS treatment simultaneously with EFS or TNF-α-neutralizing antibody.Results:Treatment of human LMMP or rENSpc with LPS induced an increase in TNF-αproduction. Activation of theENS by EFS significantly inhibited TNF-αproduction. This regulation occurred at the transcriptional level. Signalinganalyses showed that LPS induced activation of ERK but not AMPK, which was constitutively activated in rENSpcneurons. Both U0126 and C compound almost completely prevented LPS-induced TNF-αproduction. In the presenceof LPS, EFS inhibited the ERK and AMPK pathways. In addition, we demonstrated using TNF-α-neutralizing antibody thatLPS-induced TNF-αproduction increased TLR2 expression and reduced IL-6 production.Conclusions:Our results show that LPS induced TNF-αproduction by enteric neurons through activation of thecanonical ERK pathway and also in an AMPK-dependent manner. ENS activation through the inhibition of thesepathways decreased TNF-αproduction, thereby modulating the inflammatory response induced by endotoxin

Genome-wide analysis of 944 133 individuals provides insights into the etiology of haemorrhoidal disease

Objective Haemorrhoidal disease (HEM) affects a large and silently suffering fraction of the population but its aetiology, including suspected genetic predisposition, is poorly understood. We report the first genome-wide association study (GWAS) meta-analysis to identify genetic risk factors for HEM to date. Design We conducted a GWAS meta-analysis of 218 920 patients with HEM and 725 213 controls of European ancestry. Using GWAS summary statistics, we performed multiple genetic correlation analyses between HEM and other traits as well as calculated HEM polygenic risk scores (PRS) and evaluated their translational potential in independent datasets. Using functional annotation of GWAS results, we identified HEM candidate genes, which differential expression and coexpression in HEM tissues were evaluated employing RNA-seq analyses. The localisation of expressed proteins at selected loci was investigated by immunohistochemistry. Results We demonstrate modest heritability and genetic correlation of HEM with several other diseases from the GI, neuroaffective and cardiovascular domains. HEM PRS validated in 180 435 individuals from independent datasets allowed the identification of those at risk and correlated with younger age of onset and recurrent surgery. We identified 102 independent HEM risk loci harbouring genes whose expression is enriched in blood vessels and GI tissues, and in pathways associated with smooth muscles, epithelial and endothelial development and morphogenesis. Network transcriptomic analyses highlighted HEM gene coexpression modules that are relevant to the development and integrity of the musculoskeletal and epidermal systems, and the organisation of the extracellular matrix. Conclusion HEM has a genetic component that predisposes to smooth muscle, epithelial and connective tissue dysfunction