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The synaptonemal complex (SC) is a highly conserved structure in sexually reproducing organism. It has a tripartite, ladder-like organization and mediates the stable pairing, called synapsis, of the homologous chromosomes during prophase of meiosis I. Failure in homolog synapsis result in aneuploidy and/or apoptosis of the developing germ cells.
Since 1956, the SC is subject of intense research and its presence was described in various species from yeast to human. Its structure was maintained during millions of years of evolution consist-ing of two parallel lateral elements (LEs), joined by numerous transverse filaments (TFs) which run perpendicular to the LEs and an electron dense central element (CE) in the middle of the SC. Individual protein components, however, were characterized only in few available model organ-isms, as for example Saccharomyces cerevisiae, Arabidopsis thaliana, Drosophila melanogaster, Ceanorhabditis elegans and Mus musculus. Rather unexpectedly, these characterizations failed to detect an evolutionary homology between the protein components of the different SCs. This fact challenged the general idea of a single origin of the SC in the evolution of meiosis and sexual reproduction.
This thesis now addressed itself to the task to unravel the discrepancy between the high conser-vation of the SC structure and its diverse and apparently non-homologous protein composition, focusing on the animal kingdom. It is the first study dealing with the evolution of the SC in Meta-zoa and demonstrates the monophyly of the mammalian SC components in metazoan species. The thesis demonstrates that at least four out of seven murine SC proteins emerged in Eumeta-zoa at the latest and have been likewise part of an ancient SC as it can be found in the present-day cnidarian species Hydra. This SC displays the common organization and already possesses the minimal protein kit corresponding to the three different structural domains: LEs, TFs and the CE. Additionally, the individual phylogenies of the murine SC proteins revealed the dynamic evolu-tionary history of the ancient SC. Further components were added during the diversification of Bilateria and vertebrates while ancestral proteins likely duplicated in the vertebrate lineage and diversified or got lost in the branch leading to ecdysozoan species. It is hypothesized that the apparently non-homologous SC proteins in D. melanogaster and C. elegans actually do derive from the ancient SC proteins but diversified beyond recognition during the fast evolution of Ar-thropoda and Nematoda.
The study proposes Hydra as an alternative invertebrate model system for meiosis and SC re-search to the standard organisms D. melanogaster and C. elegans. Recent results about the cni-darian SC as well as the possible application of standard methods is discussed and summarized in the concluding section.
Based on genetic association and functional imaging studies, reduced function of tryptophan hydroxylase-2 (TPH2) has been shown to be critically involved in the pathophysiology of anxiety-disorders and depression. In order to elucidate the impact of a complete neuronal 5-HT deficiency, mice with a targeted inactivation of the gene encoding Tph2 were generated. Interestingly, survival of Tph2-/- mice, the formation of serotonergic neurons and the pathfinding of their projections was not impaired. Within this thesis, I investigated the influence of 5-HT deficiency on the γ-amino butyric acid (GABA) system. The GABAergic system is implicated in the pathophysiology of anxiety disorders. Therefore, measurement of GABA concentrations in different limbic brain regions was carried out. These measurements were combined with immunohistochemical estimation of GABAergic cell subpopulations in the dorsal hippocampus and amygdala. In Tph2-/- mice GABA concentrations were increased exclusively in the dorsal hippocampus. In heterozygous Tph2+/- mice concentrations of GABA were increased in the amygdala compared to Tph2-/- and wt control mice, while the reverse was found in the prefrontal cortex. The changes in GABA concentrations were accompanied by altered cell density of GABAergic neurons within the basolateral complex of the amygdala and parvalbumin (PV) neurons of the dorsal hippocampus and by adaptational changes of 5-HT receptors. Thus, adaptive changes during the development on the GABA system may reflect altered anxiety-like and depressive-like behavior in adulthood. Moreover, chronic mild stress (CMS) rescues the depressive-like effects induced by 5-HT deficiency. In contrast, 5-HT is important in mediating an increased innate anxiety-like behavior under CMS conditions. This is in line with a proposed dual role of 5-HT acting through different mechanisms on anxiety and depressive-like behavior, which is influenced by gene-environment interaction effects. Further research is needed to disentangle these complex networks in the future.
In cultured motoneurons of a mouse model for the motoneuron disease spinal muscular atrophy (SMA), reduced levels of the protein SMN (survival of motoneurons) cause defects in axonal growth. This correlates with reduced β-actin mRNA and protein in growth cones, indicating that anterograde transport and local translation of β-actin mRNA are crucial for motoneuron function. However, direct evidence that indeed local translation is a physiological phenomenon in growth cones of motoneurons was missing. Here, a lentiviral GFP-based reporter construct was established to monitor local protein synthesis of β-actin mRNA. Time-lapse imaging of fluorescence recovery after photobleaching (FRAP) in living motoneurons revealed that β-actin is locally translated in the growth cones of embryonic motoneurons. Interestingly, local translation of the β-actin reporter construct was differentially regulated by different laminin isoforms, indicating that laminins provide extracellular cues for the regulation of local translation in growth cones. Notably, local translation of β-actin mRNA was deregulated when motoneurons of a mouse model for type I SMA (Smn-/-; SMN2) were analyzed. In situ hybridization revealed reduced levels of β-actin mRNA in the axons of Smn-/-; SMN2 motoneurons. The distribution of the β-actin mRNA was not modified by different laminin isoforms as revealed by in situ hybridization against the mRNA of the eGFP encoding element of the β-actin reporter. In case of the mRNA of α-actin and γ-actin isoforms, the endogenous mRNA did not localize to the axons and the localization pattern was not affected by the SMN levels expressed in the cell. Taken together our findings suggest that regulation of local translation of β-actin in growth cones of motoneurons critically depends on laminin signaling and the amount of SMN protein. Embryonic stem cell (ESC)-derived motoneurons are an excellent in vitro system to sort out biochemical and cellular pathways which are defective in neurodegenerative diseases like SMA. Here, a protocol for the differentiation and antibody-mediated enrichment of ESC-derived motoneurons is presented, which was optimized during the course of this study. Notably, this study contributes the production and purification of highly active recombinant sonic hedgehog (Shh), which was needed for the efficient differentiation of mouse ESCs to motoneurons. ESC-derived motoneurons will now offer high amounts of cellular material to allow the biochemical identification of disease-relevant molecular components involved in regulated local protein synthesis in axons and growth cones of motoneurons.
Platelet activation and aggregation at sites of vascular injury involves massive cytoskeletal re-organization, which is required for proper platelet function. Moreover, the cytoskeleton plays central roles in megakaryo- and thrombopoiesis. Thus, cytoskeletal protein aberrations can be the underlying reason for many pathological phenotypes. Although intensive research is carried out to identify the key players involved in cytoskeletal reorganization, the signaling cascades orchestrating these complex processes are still poorly understood. This thesis investigates the role of three actin-binding proteins, Coactosin-like (Cotl) 1, Profilin (Pfn) 1 and Thymosin (T) β4, in platelet formation and function using genetically modified mice.
ADF-H-containing proteins such as Twinfilin or Cofilin are well characterized as regulators of thrombopoesis and cytoskeletal reorganization. Although Cotl1 belongs to the ADF-H protein family, lack of Cotl1 did not affect platelet count or cytoskeletal dynamics. However, Cotl1-deficiency resulted in significant protection from arterial thrombus formation and ischemic stroke in vivo. Defective GPIb-vWF interactions and altered second wave mediator release present potential reasons for the beneficial effect of Cotl1-deficiency. These results reveal an unexpected function of Cotl1 as a regulator of thrombosis and hemostasis, establishing it as a potential target for a safe therapeutic therapy to prevent arterial thrombosis or ischemic stroke.
Recent studies showed that the organization of the circumferential actin cytoskeleton modulates calpain-mediated αIIbβ3 integrin closure, thereby also controlling αIIbβ3 integrin localization. The second part of this thesis identified the actin-sequestering protein Pfn1 as a central regulator of platelet integrin function as Pfn1-deficient platelets displayed almost abolished αIIbβ3 integrin signaling. This translated into a profound protection from arterial thrombus formation and prolonged tail bleeding times in vivo which was caused by enhanced calpain-dependent integrin closure. These findings further emphasize the importance of a functional actin cytoskeleton for intact platelet function in vitro and in vivo.
Tβ4 is a moonlighting protein, acting as one of the major actin-sequestering proteins in cells of higher eukaryotes and exerting various paracrine functions including anti-inflammatory, immunomodulatory and pro-angiogenic effects. Although excessively studied, its role for cytoskeletal dynamics, the distinction between endo- and exogenous protein function and its uptake and release mechanisms are still poorly understood. Constitutive Tβ4-deficiency resulted in thrombocytopenia accompanied by a largely diminished G-actin pool in platelets and divergent effects on platelet reactivity. Pre-incubation of platelets with recombinant Tβ4 will help to understand the function of endo- and exogenous protein, which is under current investigation.
Maintenance of tumor vasculature integrity is indispensable for tumor growth and thus affects tumor progression. Previous studies have identified platelets as major regulators of tumor vascular integrity, as their depletion selectively renders tumor vessels highly permeable, causing massive intratumoral hemorrhage. While these results establish platelets as potential targets for anti-tumor therapy, depletion is not a treatment option due to the essential role of platelets for hemostasis. This thesis demonstrates for the first time that functional inhibition of glycoprotein (GP) VI on the platelet surface rapidly induces tumor hemorrhage and diminishes tumor growth similar to complete platelet depletion but without inducing systemic bleeding complications. Both, the intratumoral bleeding and tumor growth arrest could be reverted by depletion of Ly6G+ cells confirming them to be responsible for the induction of bleeding and necrosis within the tumor. In addition, GPVI inhibition increased intra-tumoral accumulation of co-administered chemotherapeutic agents, thereby resulting in a profound anti-tumor effect. In summary, this thesis manifests platelet GPVI as a key regulator of vascular integrity specifically in growing tumors, serving as a potential basis for the development of anti-tumor strategies.
In the second part of this thesis, light is shed on the modulating role of bridging integrator 2 (BIN2) in platelet Ca2+ signaling. Stromal interaction molecule 1 (STIM1) mediated store-operated calcium entry (SOCE) is the major route of Ca2+ influx in platelets, triggered by inositol trisphosphate receptor (IP3R)-dependent Ca2+ store release. In this thesis, the BAR domain superfamily member BIN2 was identified as the first Ca2+ signaling modulator, interacting with both, STIM1 and IP3R in platelets. Deletion of BIN2 resulted in reduced Ca2+ store release and Ca2+ influx in response to all tested platelet agonists. These defects were a consequence of impaired IP3R function in combination with defective STIM1-mediated SOC channel activation, while Ca2+ store content and agonist-induced IP3 production were unaltered. These results establish BIN2 as a central regulator of platelet Ca2+ signaling.
The third part of this thesis focuses on the effect of the soluble neuronal guidance protein Sema7A on platelet function. Rosenberger et al. discovered that Sema7A cleavage from red blood cells increases the formation of platelet-neutrophil complexes, thereby reinforcing thrombo-inflammation in myocardial ischemia-reperfusion injury (MIRI). This thesis establishes soluble Sema7A as a stimulator of platelet thrombus formation via its interaction with platelet GPIbα, thereby reinforcing PNC formation. Thus, interfering with the GPIb-Sema7A interaction during MIRI represents a potential strategy to reduce cardiac damage and improve clinical outcome following MI.
Platelet activation and aggregation at sites of vascular injury are essential processes to limit blood loss but they also contribute to arterial thrombosis, which can lead to myocardial infarction and stroke. Stable thrombus formation requires a series of events involving platelet receptors which contribute to adhesion, activation and aggregation of platelets. Regulation of receptor expression by (metallo-)proteinases has been described for several platelet receptors, but the molecular mechanisms are ill-defined. The signaling lymphocyte activation molecule (SLAM) family member CD84 is expressed in immune cells and platelets, however its role in platelet physiology was unclear. In this thesis, CD84 deficient mice were generated and analyzed. In well established in vitro and in vivo assays testing platelet function and thrombus formation, CD84 deficient mice displayed phenotypes indistinguishable from wild-type controls. It was concluded that CD84 in platelets does not function as modulator of thrombus formation, but rather has other functions. In line with this, in the second part of this thesis, a novel regulation mechanism for platelet CD84 was discovered and elucidated. Upon platelet activation, the N-terminus of CD84 was found to be cleaved exclusively by the a disintegrin and metalloproteinase 10 (ADAM10), whereas the intracellular part was cleaved by calpain. In addition, regulation of the platelet activating collagen receptor glycoprotein VI (GPVI) was studied and it was shown that GPVI is in contrast to CD84 differentially regulated by ADAM10 and ADAM17. A novel role of CD84 under pathophysiological conditions was revealed as CD84 deficient mice were protected from ischemic stroke in the model of transient middle cerebral artery occlusion and this protection was based on the lack of CD84 in T cells. Ca2+ is an essential second messenger that facilitates activation of platelets and diverse functions in different eukaryotic cell types. Store-operated Ca2+ entry (SOCE) represents the major mechanism leading to rise in intracellular Ca2+ concentration in non-excitable cells. The Ca2+ sensor STIM1 (stromal interaction molecule 1) and the SOC channel subunit protein Orai1 are established mediators of SOCE in platelets. STIM2 is the major STIM isoform in neurons, but the role of the SOC channel subunit protein Orai2 in platelets and neurons has remained elusive. In the third part of this thesis, Orai2 deficient mice were generated and analyzed. Orai2 was dispensable for platelet function, however, Orai2 deficient mice were protected from ischemic neurodegeneration and this phenotype was attributed to defective SOCE in neurons.
Activated platelets and coagulation jointly contribute to physiological hemostasis. However, pathological conditions can also trigger unwanted platelet activation and initiation of coagulation resulting in thrombosis and precipitation of ischemic damage of vital organs such as the heart or brain. The specific contribution of procoagulant platelets, positioned at the interface of the processes of platelet activation and coagulation, in ischemic stroke had remained uninvestigated. The first section of the thesis addresses this aspect through experiments conducted in novel megakaryocyte- and platelet-specific TMEM16F conditional KO mice (cKO). cKO platelets phenocopied defects in platelets from Scott Syndrome patients and had severely impaired procoagulant characteristics. This led to decelerated platelet-driven thrombin generation and delayed fibrin formation. cKO mice displayed prolonged bleeding times and impaired arterial thrombosis. However, infarct volumes in cKO mice were comparable to wildtype (WT) mice in an experimental model of ischemic stroke. Therefore, while TMEM16F-regulated platelet procoagulant activity is critical for hemostasis and thrombosis, it is dispensable for cerebral thrombo-inflammation in mice.
The second section describes the generation and initial characterization of a novel knockin mouse strain that expresses human coagulation factor XII (FXII) instead of endogenous murine FXII. These knockin mice had normal occlusion times in an experimental model of arterial thrombosis demonstrating that human FXII is functional in mice. Therefore, these mice constitute a valuable tool for testing novel pharmacological agents against human FXII – an attractive potential target for antithrombotic therapy.
Glycoprotein (GP)VI and C-type lectin-like receptor 2 (CLEC-2)-mediated (hem)immunoreceptor tyrosine-based activation motif (ITAM) signaling represent a major pathway for platelet activation. The last section of the thesis provides experimental evidence for redundant functions between the two members of the Grb2 family of adapter proteins - Grb2 and Gads that lie downstream of GPVI and CLEC-2 stimulation. In vitro and in vivo studies in mice deficient in both Grb2 and Gads (DKO) revealed that DKO platelets had defects in (hem)ITAM-stimulation-specific activation, aggregation and signal transduction that were more severe than the defects observed in single Grb2 KO or Gads KO mice. Furthermore, the specific role of these adapters downstream of (hem)ITAM signaling was essential for maintenance of hemostasis but dispensable for the known CLEC-2 dependent regulation of blood-lymphatic vessel separation.
Several lines of evidence implicate a dysregulation of tryptophan hydroxylase (TPH)-dependent serotonin (5-HT) synthesis in emotions and stress and point to their potential relevance to the etiology and pathogenesis of various neuropsychiatric disorders. However, the differential expression pattern of the two isoforms TPH1 and TPH2 which encode two forms of the rate-limiting enzyme of 5-HT synthesis is controversial. Here, a comprehensive spatio-temporal analysis clarifies TPH1 and TPH2 expression during pre- and postnatal development of the mouse brain and in adult human brain as well as in peripheral organs including the pineal gland. Four different methods (real time PCR, in situ hybridization, immunohistochemistry and Western blot analysis) were performed to systematically control for tissue-, species- and isoform-specific expression on both the pre- and posttranslational level. TPH2 expression was consistently detected in the raphe nuclei, as well as in fibres in the deep pineal gland and in the gastrointestinal tract. Although TPH1 expression was found in these peripheral tissues, no significant TPH1 expression was detected in the brain, neither during murine development, nor in mouse and human adult brain. Also under conditions like stress and clearing the tissue from blood cells, no changes in expression levels were detectable. Furthermore, the reuptake of 5-HT into the presynaptic neuron by the serotonin transporter (SERT) is the major mechanism terminating the neurotransmitter signal. Thus, mice with a deletion in the Sert gene (Sert KO mice) provide an adequate model for human affective disorders to study lifelong modified 5-HT homeostasis in interaction with stressful life events. To further explore the role of TPH isoforms, Tph1 and Tph2 expression was studied in the raphe nuclei of Sert deficient mice under normal conditions as well as following exposure to acute immobilization stress. Interestingly, no statistically significant changes in expression were detected. Moreover, in comparison to Tph2, no relevant Tph1 expression was detected in the brain independent from genotype, gender and treatment confirming expression in data from native animals. Raphe neurons of a brain-specific Tph2 conditional knockout (cKO) model were completely devoid of Tph2-positive neurons and consequently 5-HT in the brain, with no compensatory activation of Tph1 expression. In addition, a time-specific Tph2 inducible (i) KO mouse provides a brain-specific knockdown model during adult life, resulting in a highly reduced number of Tph2-positive cells and 5-HT in the brain. Intriguingly, expression studies detected no obvious alteration in expression of 5-HT system-associated genes in these brain-specific Tph2 knockout and knockdown models. The findings on the one hand confirm the specificity of Tph2 in brain 5-HT synthesis across the lifespan and on the other hand indicate that neither developmental nor adult Tph2-dependent 5-HT synthesis is required for normal formation of the serotonergic system, although Tph1 does not compensate for the lack of 5-HT in the brain of Tph2 KO models. A further aim of this thesis was to investigate the expression of the neuropeptide oxytocin, which is primarily produced in the hypothalamus and released for instance in response to stimulation of 5-HT and selective serotonin reuptake inhibitors (SSRIs). Oxytocin acts as a neuromodulator within the central nervous system (CNS) and is critically involved in mediating pain modulation, anxiolytic-like effects and decrease of stress response, thereby reducing the risk for emotional disorders. In this study, the expression levels of oxytocin in different brain regions of interest (cortex, hippocampus, amygdala, hypothalamus and raphe nuclei) from female and male wildtype (WT) and Sert KO mice with or without exposure to acute immobilization stress were investigated. Results showed significantly higher expression levels of oxytocin in brain regions which are involved in the regulation of emotional stimuli (amygdala and hippocampus) of stressed male WT mice, whereas male Sert KO as well as female WT and Sert KO mice lack these stress-induced changes. These findings are in accordance with the hypothesis of oxytocin being necessary for protection against stress, depressive mood and anxiety but suggest gender-dependent differences. The lack of altered oxytocin expression in Sert KO mice also indicates a modulation of the oxytocin response by the serotonergic system and provides novel research perspectives with respect to altered response of Sert KO mice to stress and anxiety inducing stimuli.
The DREAM complex plays an important role in regulation of gene expression during the cell cycle. It was previously shown that the DREAM subunits LIN9 and B-MYB are required for early embryonic development and for the maintenance of the inner cell mass in vitro. In this work the effect of LIN9 or B-MYB depletion on embryonic stem cells (ESC) was examined. It demonstrates that LIN9 and B-MYB knock down changes the cell cycle distribution of ESCs and results in an accumulation of cells in G2 and M and in an increase of polyploid cells. By using genome-wide expression studies it was revealed that the depletion of LIN9 leads to downregulation of mitotic genes and to upregulation of differentiation-specific genes. ChIP-on chip experiments determined that mitotic genes are direct targets of LIN9 while lineage specific markers are regulated indirectly. Importantly, depletion of LIN9 does not alter the expression of the pluripotency markers Sox2 and Oct4 and LIN9 depleted ESCs retain alkaline phosphatase activity. I conclude that LIN9 is essential for proliferation and genome stability of ESCs by activating genes with important functions in mitosis and cytokinesis. The exact molecular mechanisms behind this gene activation are still unclear as no DREAM subunit features a catalytically active domain. It is assumed that DREAM interacts with other proteins or co-factors for transcriptional activation. This study discovered potential binding proteins by combining in vivo isotope labeling of proteins with mass spectrometry
(MS) and further analysed the identified interaction of the tight junction protein ZO-2 with DREAM which is cell cycle dependent and strongest in S-phase. ZO-2 depletion results in reduced cell proliferation and decreased G1 gene expression. As no G2/M genes, typical DREAM targets, are affected upon ZO-2 knock down, it is unlikely that ZO-2 binding is needed for a functional DREAM complex. However, this work demonstrates that with (MS)-based quantitative proteomics, DREAM interacting proteins can be identified which might help to elucidate the mechanisms underlying DREAM mediated gene activation.
Cellular proliferation, differentiation and survival in response to extracellular signals are controlled by the signal transduction pathway of Ras, Raf and MAP kinase. The Raf proteins are serine/threonine kinases with essential function in growth/differentiation/survival - related signal transduction events. In mammals, three functional (A-, B-, and C-Raf) genes were described. Biochemical studies suggest overlapping and differential utilization of Raf isozymes. However, the frequent co-expression of Raf isozymes and their multiple activators and effectors impedes the full understanding of their specific roles. The elucidation of these roles is important due to the involvement of the Ras/Raf/MEK/MAP kinase cascade in human disorders especially in tumor development and progression. B-Raf was shown to posses the strongest kinase activity among Raf kinases and display antiapoptotic properties. Mice deficient in B-Raf show overall growth retardation and die between E10.5 and E12.5 of vascular defects caused by excessive death of differentiated endothelial cells. To elucidate the redundancy of Raf isozymes during embryonic development and to rescue B-Raf-/- (KO) phenotype, B-Raf alleles were disrupted by introducing A-Raf cDNA under the control of endogenous B-Raf promoter. The resulting BRaf A-Raf/A-Raf (KIN) phenotype depends on genetic background. The living embryos displaying normal development but size reduction were found with low incidence at E12.5d-16.5d. All of them displayed the rescue of vascular system. One adult p20 mouse without any visible defects in development and behavior was obtained. On the other hand, the processes of neurogenesis and neural precursors migration in survived embryos were disturbed which led in some cases to underdevelopment of different brain compartments. TUNEL and cell proliferation (PCNA staining) assays revealed more apoptotic (E13.5d) and less proliferating(E12.5d cells within ventricular and sub-ventricular zones of brain ventricles and in striatum of KIN embryos. In addition, more apoptotic cells were detected in many other tissues of E13.5d and in lung of E16.5d KIN embryos but not in adult KIN mouse. p20 KIN mouse demonstrated reduced fraction of neural precursor cells in sub-granular zone of hippocampus and mature neurons in olfactory bulb. The other processes of neurogenesis were not disturbed in adult KIN animal. Fibroblasts obtained from KIN embryos demonstrated less proliferative ability and were more susceptible to apoptotic stimuli compared to WT. This was accompanied by the reduction of active ERK and Akt required for survival, and with decrease of inactive phosphorylated BAD. The kinetic of both ERK and Akt phosphorylation upon serum stimulation was delayed. All these data indicate that moderate A-Raf kinase activity can prevent the endothelial apoptosis but is not enough to completely rescue the other developmental consequences.