Identification of new functions of SRF-mediated transcription during the development of neuronal networks

Im Rahmen der vorliegenden Arbeit wurden neue Funktionen des Serum Response Factors (SRF) beim Aufbau neuronaler Netzwerke entdeckt: Im Manuskript 1 (Stritt et al., 2009) wurde die Transkription von Genen auf genomweiter Ebene im Gehirn von wildtypischen und SRF-defizienten Mäusen verglichen. Durch die Analyse der GeneChip-Daten ging hervor, dass myelin- und oligodendrocytenspezifische Gene in den Srf Mutanten drastisch herunterreguliert waren. Hieraus vermutete Defekte in der axonalen Myelinisierung und in der Oligodendrocytendifferenzierung konnten in vivo und in vitro in den Srf Mutanten bzw. in SRF-defizienten Zellkulturen bestätigt werden. Damit wurde eine völlig neue Rolle von SRF als Regulator der Myelinisierung und Oligodendrocytendifferenzierung aufgedeckt. Durch weitere Untersuchungen wurde das insulin growth factor binding protein CTGF (connective tissue growth factor) im Gehirn als SRF-reguliertes Signalmolekül identifiziert, das von Neuronen sekretiert wird. Unsere Ergebnisse ergaben zusammenfassend, dass neuronales SRF über CTGF auf parakrine Weise die Myelinisierung und Oligodendrocytendifferenzierung beeinflussen kann, indem CTGF auf die Insulinsignalgebung wirkt und die fördernde Wirkung von insulin growth factors (IGFs) auf die Myelinisierung antagonisiert. Im Manuskript 2 (Stern et al., 2009) der vorliegenden Arbeit konnte gezeigt werden, dass Aktin nicht nur die Funktion als Bestandteil des zytoplasmatischen Zellgerüsts ausübt, sondern dass Aktin-Signalgebung in Neuronen aktiv an SRF-vermittelter Genexpression beteiligt ist, wodurch neuronale Motilität moduliert wird. Im Manuskript 3 (Stritt and Knöll, 2009) dieser Arbeit wurden neue Funktionen von SRF in der hippocampalen Entwicklung identifiziert. Histologische Untersuchungen ergaben, dass SRF im Hippocampus Aufbau von Zell- und Faserschichten, Dendritenwachstum und die Bildung dendritischer Dornfortsätze reguliert. Da die in den Srf Mutanten beobachteten Defekte in der hippocampalen Entwicklung stark an Mutanten des Reelin-Signalweges erinnerten, wurde außerdem eine mögliche Interaktion zwischen SRF und Reelin analysiert. Hierbei konnte gezeigt werden, dass SRF den Ort der Reelin-Signalgebung beeinflusst, da in Srf Mutanten Reelin-exprimierende Zellen ektopisch lokalisiert waren. Des Weiteren konnte eine durch Reelin induzierte Signalkaskade in SRF-vermittelter Transkription resultieren. Zusätzlich konnte festgestellt werden, dass Reelin und SRF gemeinsam Neuritenwachstum regulieren, wobei SRF eventuell als Mediator von Reelin-induziertem Neuritenwachstum fungiert.In the present study we could identify new functions of the Serum Response Factor (SRF) during the development of neuronal networks: Within manuscript 1 (Stritt et al., 2009) we compare gene expression on a genome-wide level between brains of wildtype and SRF-deficient mice. Analysis of the GeneChips revealed that genes, associated with myelination and differentiation of oligodendrocytes, were strongly downregulated in Srf mutants. We therefore concluded that axonal myelination and oligodendrocyte differentiation might be affected in Srf mutants. Indeed, we could in vivo and in vitro observe severe defects in myelination and oligodendrocyte differentiation, both, in Srf mutants, as well as in SRF-deficient cultures. Thus, we uncovered a new role of SRF in regulating myelination and oligodendrocyte differentiation during brain development. We identified CTGF (connective tissue growth factor) - an insulin growth factor binding protein, that can be secreted by neurons - to be regulated by SRF. Altogether, our results showed, that neuronal SRF can regulate myelination and oligodendrocyte differentiation in a paracrine manner, by transcriptionally regulating Ctgf, whose gene product can affect Insulin signaling and can counteract IGF1 (insulin growth factor1)-stimulated oligodendrocyte differentiation. In manuscript 2 (Stern et al., 2009) of the present work we could show, that actin not only functions in neurons as a part of the cytosceleton, but is also actively functioning as a signal mediator, involved in SRF-dependent gene expression, that leads to neuronal motility. In manuscript 3 (Stritt and Knöll, 2009) of this work we identified new functions of SRF in hippocampal development. Immunohistological studies revealed that SRF regulates hippocampal lamination, dendritic branching and dendritic spine number. As these phenotypes observed in Srf mutants resemble mice lacking components of the Reelin signaling cascade, we analysed a potential interaction between SRF and Reelin signaling. In support of such an interaction, our data indicate that SRF influences location of Reelin-expressing cells. We also observed that Reelin can activate SRF-dependent gene transcription. Additionally, we could show that Reelin and SRF act together in regulating neurite outgrowth, with SRF being a mediator of Reelin-induced neurite outgrowth

Serum Response Factor Regulates Hippocampal Lamination and Dendrite Development and Is Connected with Reelin Signaling ▿ †

During brain development, neurons and their nerve fibers are often segregated in specific layers. The hippocampus is a well-suited model system to study lamination in health and aberrant cell/fiber lamination associated with neurological disorders. SRF (serum response factor), a transcription factor, regulates synaptic-activity-induced immediate-early gene (IEG) induction and cytoskeleton-based neuronal motility. Using early postnatal conditional SRF ablation, we uncovered distorted hippocampal lamination, including malpositioning of granule cell neurons and disruption of layer-restricted termination of commissural-associational and mossy fiber axons. Besides axons, dendrite branching and spine morphogenesis in Srf mutants were impaired, offering a first morphological basis for SRF's reported role in learning and memory. Srf mutants resemble mice lacking components of the reelin signaling cascade, a fundamental signaling entity in brain lamination. Our data indicate that reelin signaling and SRF-mediated gene transcription might be connected: reelin induces IEG and cytoskeletal genes in an SRF-dependent manner. Further, reelin-induced neurite motility is blocked in Srf mutants and constitutively active SRF rescues impaired neurite extension in reeler mouse mutants in vitro. In sum, data provided in this report show that SRF contributes to hippocampal layer and nerve fiber organization and point at a link between Srf gene transcription and reelin signaling

Endothelial SRF/MRTF ablation causes vascular disease phenotypes in murine retinae

Retinal vessel homeostasis ensures normal ocular functions. Consequently, retinal hypovascularization and neovascularization, causing a lack and an excess of vessels, respectively, are hallmarks of human retinal pathology. We provide evidence that EC-specific genetic ablation of either the transcription factor SRF or its cofactors MRTF-A and MRTF-B, but not the SRF cofactors ELK1 or ELK4, cause retinal hypovascularization in the postnatal mouse eye. Inducible, EC-specific deficiency of SRF or MRTF-A/MRTF-B during postnatal angiogenesis impaired endothelial tip cell filopodia protrusion, resulting in incomplete formation of the retinal primary vascular plexus, absence of the deep plexi, and persistence of hyaloid vessels. All of these features are typical of human hypovascularization-related vitreoretinopathies, such as familial exudative vitreoretinopathies including Norrie disease. In contrast, conditional EC deletion of Srf in adult murine vessels elicited intraretinal neovascularization that was reminiscent of the age-related human pathologies retinal angiomatous proliferation and macular telangiectasia. These results indicate that angiogenic homeostasis is ensured by differential stage-specific functions of SRF target gene products in the developing versus the mature retinal vasculature and suggest that the actin-directed MRTF-SRF signaling axis could serve as a therapeutic target in the treatment of human vascular retinal diseases

<i>Elk3</i> Deficiency Causes Transient Impairment in Post-Natal Retinal Vascular Development and Formation of Tortuous Arteries in Adult Murine Retinae

<div><p>Serum Response Factor (SRF) fulfills essential roles in post-natal retinal angiogenesis and adult neovascularization. These functions have been attributed to the recruitment by SRF of the cofactors Myocardin-Related Transcription Factors MRTF-A and -B, but not the Ternary Complex Factors (TCFs) Elk1 and Elk4. The role of the third TCF, Elk3, remained unknown. We generated a new <i>Elk3</i> knockout mouse line and showed that Elk3 had specific, non-redundant functions in the retinal vasculature. In <i>Elk3(−/−)</i> mice, post-natal retinal angiogenesis was transiently delayed until P8, after which it proceeded normally. Interestingly, tortuous arteries developed in <i>Elk3(−/−)</i> mice from the age of four weeks, and persisted into late adulthood. Tortuous vessels have been observed in human pathologies, e.g. in ROP and FEVR. These human disorders were linked to altered activities of vascular endothelial growth factor (VEGF) in the affected eyes. However, in <i>Elk3(−/−)</i> mice, we did not observe any changes in VEGF or several other potential confounding factors, including mural cell coverage and blood pressure. Instead, concurrent with the post-natal transient delay of radial outgrowth and the formation of adult tortuous arteries, Elk3-dependent effects on the expression of Angiopoietin/Tie-signalling components were observed. Moreover, <i>in vitro</i> microvessel sprouting and microtube formation from P10 and adult aortic ring explants were reduced. Collectively, these results indicate that Elk3 has distinct roles in maintaining retinal artery integrity. The <i>Elk3</i> knockout mouse is presented as a new animal model to study retinal artery tortuousity in mice and human patients.</p></div

APOLD1 loss causes endothelial dysfunction involving cell junctions, cytoskeletal architecture, and Weibel-Palade bodies, while disrupting hemostasis

International audienceVascular homeostasis is impaired in various diseases thereby contributing to the progression of their underlying pathologies. The endothelial immediate early gene Apolipoprotein L domain-containing 1 (APOLD1) helps regulate endothelial function. However, its precise role in endothelial cell (EC) biology remains unclear. We have immuno-localized APOLD1 to EC cell contacts and to Weibel- Palade bodies (WPB) where it associates with von Willebrand factor (VWF) tubules. Silencing of APOLD1 in primary human ECs disrupted the cell junction-cytoskeletal interface, thereby altering endothelial permeability accompanied by spontaneous release of WPB contents. This resulted in an increased presence of WPB cargoes, notably VWF and ANGPT2 in the extracellular medium. Autophagy flux, previously recognized as an essential mechanism for the regulated release of WPBs, was impaired in the absence of APOLD1. In addition, we report APOLD1 as a candidate gene for a novel inherited bleeding disorder (IBD) across three generations of a large family associating an atypical bleeding diathesis with episodic impaired microcirculation. A dominant heterozygous nonsense APOLD1:p.R49* variant segregated to affected family members. Compromised vascular integrity resulting from an excess of plasma ANGPT2, and locally impaired availability of VWF may explain the unusual clinical profile of APOLD1:p.R49* patients. In summary, our findings identify APOLD1 as an important regulator of vascular homeostasis and raise the need to consider testing of EC function in patients with IBDs without apparent platelet or coagulation defects

Incidence (%) of tortuous arteries.

<p>(* p<0.05 ** p<0.01 *** p<0.001, na = not analysed, ns = not significant, n = number of retinae analysed).</p><p>Incidence (%) of tortuous arteries.</p

Microvessel sprouting and microtube formation is impaired in P10 and adult <i>Elk3(−/−)</i> knockout aortic ring explants.

<p>(A left) Aortic rings from <i>Elk3(+/+)</i> control animals show robust microvessel sprouting on day 5 of culture. In contrast, aortic rings from <i>Elk3(−/−)</i> knockout animals show drastically reduced microvessel sprouting. (A middle) Control <i>Elk3(+/+)</i>, but not <i>Elk3(−/−)</i> knockout aortic ring endothelial cells form microtubes in Matrigel after three weeks of culture. (A right) Phalloidin staining of aortic ring endothelial cells cultivated in Matrigel shows interconnected ECs and tip cells with filopodia in control explants, whereas <i>Elk3(−/−)</i> knockout aortic ring endothelial cells lack connections and instead have degenerated bulbs. (B, C) Kinetics of aortic ring microvessel sprouting for four pairs of adult (B) and four pairs of P10 (C) <i>Elk3(+/+)</i> control and <i>Elk3(−/−)</i> knockout animals observed during <i>in vitro</i> growth (for each time point mean +/<i>−</i> s.e.m. presented). Scale bar in (A left and middle) 500 µm, in (A right) 50 µm.</p

Deletion of <i>Elk3</i> leads to delayed retinal angiogenesis during early post-natal stages in <i>Elk3(−/−)</i> knockout mice.

<p>(A) IsolectinB4 staining of flat-mounts of retinae from P4 <i>Elk3(+/+)</i> control and <i>Elk3(−/−)</i> knockout mice. The red lines outline the whole retinal area, the yellow lines the areas covered by blood vessels. (B) Quantitation of retinal area covered by blood vessels (% vascularization) at P4. (C) Higher magnification of the angiogenic front in P4 control and <i>Elk3(−/−)</i> knockout retinae. (D) IsolectinB4 staining of retinal flat-mounts of a P6 <i>Elk3(+/+)</i> control and <i>Elk3(−/−)</i> knockout mouse. The red arrow points towards delayed angiogenic front in the <i>Elk3(−/−)</i> retina. (E) Quantitation of retinal area covered by blood vessels (% vascularization) at P6. (F) Higher magnification of the angiogenic front in P6 control and <i>Elk3(−/−)</i> knockout retinae. (G) IsolectinB4 staining of retinal flat-mounts from P8 <i>Elk3(+/+)</i> control and <i>Elk3(−/−)</i> knockout mice. The red arrow points towards the delayed angiogenic front in the <i>Elk3(−/−)</i> retina. (H) Quantitation of retinal area covered by blood vessels (% vascularization) at P8. (I) Higher magnification of the angiogenic front in P8 control and <i>Elk3</i> knockout retinae. All quantitation data are normalized to the control  =  100%. The data shown are means +/− s.e.m. Statistical significance using Student's t-test is indicated with * p<0.05 ** p<0.01 *** p<0.001. Scale bar in (A, D, G) 1000 µm, in (C, F, I) 100 µm.</p