ABSTRACT
Intracerebral hemorrhagic stroke and vascular dementia are age- and hypertension-associated manifestations of human cerebral small vessel disease (SVD). Cerebral microvessels are formed by endothelial cells (ECs), which are connected through tight junctions, adherens junctions, and stabilizing basement membrane structures. These endothelial connections ensure both vessel stability and blood-brain barrier (BBB) functions, the latter enabling selective exchange of ions, bioactive molecules, and cells between the bloodstream and brain tissue. Srf(iECKO) mice, permitting conditional EC-specific depletion of the transcription factor Serum Response Factor (SRF), suffer from loss of BBB integrity and intracerebral hemorrhaging. Cerebral microbleeds and larger hemorrhages developed upon postnatal and adult depletion of either SRF or its cofactors Myocardin Related Transcription Factor (MRTF-A/-B), revealing essential requirements of ongoing SRF/MRTF activity for maintenance of cerebral small vessel integrity. In vivo magnetic resonance imaging allowed detection, localization, and time-resolved quantification of BBB permeability and hemorrhage formation in Srf(iECKO) brains. At the molecular level, direct and indirect SRF/MRTF target genes, encoding structural components of tight junctions (Claudins and ZO proteins), adherens junctions (VE-cadherin, α-Actinin), and the basement membrane (Collagen IV), were down-regulated upon SRF depletion. These results identify SRF and its MRTF cofactors as major transcriptional regulators of EC junctional stability, guaranteeing physiological functions of the cerebral microvasculature. We hypothesize that impairments in SRF/MRTF activity contribute to human SVD pathology.
Subject(s)
Cerebral Hemorrhage/complications , Endothelial Cells/metabolism , Serum Response Factor/metabolism , Stroke/etiology , Stroke/metabolism , Trans-Activators/metabolism , Transcription Factors/metabolism , Animals , Animals, Newborn , Astrocytes/metabolism , Astrocytes/pathology , Basement Membrane/metabolism , Basement Membrane/pathology , Blood-Brain Barrier/metabolism , Brain/blood supply , Brain/metabolism , Brain/pathology , Brain/physiopathology , Cadherins/metabolism , Cerebral Hemorrhage/metabolism , Cerebral Hemorrhage/pathology , Cerebral Hemorrhage/physiopathology , Collagen Type IV/metabolism , Down-Regulation , Evans Blue/metabolism , Exploratory Behavior , Extravasation of Diagnostic and Therapeutic Materials , Gene Deletion , Magnetic Resonance Imaging , Mice, Knockout , Microvessels/metabolism , Microvessels/pathology , Motor Activity , Permeability , Serum Response Factor/genetics , Stroke/pathology , Stroke/physiopathology , Tight Junctions/metabolism , Time FactorsABSTRACT
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 Elk3 knockout mouse line and showed that Elk3 had specific, non-redundant functions in the retinal vasculature. In Elk3(-/-) mice, post-natal retinal angiogenesis was transiently delayed until P8, after which it proceeded normally. Interestingly, tortuous arteries developed in Elk3(-/-) 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 Elk3(-/-) 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, in vitro 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 Elk3 knockout mouse is presented as a new animal model to study retinal artery tortuousity in mice and human patients.
Subject(s)
Arteries/abnormalities , Joint Instability/pathology , Neovascularization, Pathologic/pathology , Proto-Oncogene Proteins c-ets/deficiency , Proto-Oncogene Proteins c-ets/genetics , Retina/pathology , Retinal Neovascularization/pathology , Retinal Vessels/pathology , Skin Diseases, Genetic/pathology , Vascular Malformations/pathology , Angiopoietins/genetics , Angiopoietins/metabolism , Animals , Arteries/metabolism , Arteries/pathology , Disease Models, Animal , Female , Joint Instability/genetics , Joint Instability/metabolism , Male , Mice , Mice, Inbred C57BL , Mice, Knockout , Neovascularization, Pathologic/genetics , Neovascularization, Pathologic/metabolism , Receptors, TIE/genetics , Receptors, TIE/metabolism , Retina/metabolism , Retinal Neovascularization/genetics , Retinal Neovascularization/metabolism , Retinal Vessels/metabolism , Serum Response Factor/genetics , Serum Response Factor/metabolism , Signal Transduction/physiology , Skin Diseases, Genetic/genetics , Skin Diseases, Genetic/metabolism , Transcription Factors/genetics , Transcription Factors/metabolism , Vascular Endothelial Growth Factors/genetics , Vascular Endothelial Growth Factors/metabolism , Vascular Malformations/genetics , Vascular Malformations/metabolismABSTRACT
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.
Subject(s)
Retina/metabolism , Retinal Diseases/metabolism , Serum Response Factor/metabolism , Vascular Diseases/metabolism , Animals , Cell Nucleus/metabolism , Cytoplasm/metabolism , Gene Deletion , Gene Expression Regulation , Mice , Mice, Knockout , Neovascularization, Pathologic , Neovascularization, Physiologic , Phenotype , RNA, Messenger/metabolism , Retina/pathology , Tamoxifen/pharmacology , Trans-Activators/metabolism , Transcription Factors/metabolism , Vascular Endothelial Growth Factor A/metabolismABSTRACT
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.
Subject(s)
Cell Adhesion Molecules, Neuronal/metabolism , Dendrites/physiology , Extracellular Matrix Proteins/metabolism , Hippocampus/growth & development , Nerve Tissue Proteins/metabolism , Serine Endopeptidases/metabolism , Serum Response Factor/metabolism , Signal Transduction/physiology , Animals , Cell Adhesion Molecules, Neuronal/genetics , Cells, Cultured , Dendrites/ultrastructure , Extracellular Matrix Proteins/genetics , Hippocampus/anatomy & histology , Hippocampus/metabolism , Mice , Mice, Transgenic , Mutation , Nerve Tissue Proteins/genetics , Neurons/cytology , Neurons/physiology , Phenotype , Reelin Protein , Serine Endopeptidases/genetics , Serum Response Factor/genetics , Transcription, GeneticABSTRACT
Neuronal motility relies on actin treadmilling. In addition to regulating cytoskeletal dynamics in the cytoplasm, actin modulates nuclear gene expression. We present a hitherto unappreciated cross talk of actin signaling with gene expression governing neuronal motility. Toward this end, we used a novel approach using mutant actins either favoring (G15S) or inhibiting (R62D) F-actin assembly. Overexpressing these mutant actins in mouse hippocampal neurons not only modulated growth-cone function but also neurite elongation, which was ambiguous by traditional pharmacological interference. G15S actin enhanced neurite outgrowth and filopodia number. In contrast, R62D reduced neurite length and impaired growth-cone filopodia formation. Growth-cone collapse induced by ephrin-As, a family of repulsive axon guidance molecules, is impaired upon R62D expression, resulting in perseverance of ring-shaped F-actin filaments. R62D-induced phenotypes strongly resemble neurons lacking SRF (Serum Response Factor). SRF controls gene transcription of various actin isoforms (e.g., Actb, Acta1) and actin-binding proteins (e.g., Gsn) and is the archetypical transcription factor to study actin interplay with transcription. We show that neuronal motility evoked by these actin mutants requires SRF activity. Further, constitutively active SRF partially rescues R62D-induced phenotypes. Conversely, actin signaling regulates neuronal SRF-mediated gene expression. Notably, a nucleus-resident actin (R62D(NLS)) also regulates SRF's transcriptional activity. Moreover, R62D(NLS) decreases neuronal motility similar to the cytoplasmic R62D actin mutant although R62D(NLS) has no access to cytoplasmic actin dynamics. Thus, herein we provide first evidence that neuronal motility not only depends on cytoplasmic actin dynamics but also on the availability of actin to modulate nuclear functions such as gene transcription.
Subject(s)
Actins/physiology , Cell Movement/physiology , Cell Nucleus/physiology , Neurons/physiology , Serum Response Factor/physiology , Transcription, Genetic/physiology , Actins/ultrastructure , Animals , Cell Nucleus/ultrastructure , Growth Cones/physiology , Growth Cones/ultrastructure , Mice , Mutation , Neurons/ultrastructure , Serum Response Factor/deficiency , Serum Response Factor/geneticsABSTRACT
In neurons, serum response factor (SRF)-directed transcription regulates migration, axon pathfinding and synapse function. We found that forebrain-specific, neuron-restricted SRF ablation in mice elevated oligodendrocyte precursors while inhibiting terminal oligodendrocyte differentiation. Myelin gene and protein expression were downregulated and we observed a lack of oligodendrocytes in mixed neuron/glia and oligodendrocyte-enriched cultures derived from Srf(-/-) mutants. Ultrastructural inspection revealed myelination defects and axonal degeneration in Srf(-/-) mutants. Consistent with our finding that neuronal SRF depletion impaired oligodendrocyte fate in a non-cell autonomous manner, neuron-restricted expression of constitutively active SRF-VP16 affected neighboring oligodendrocyte maturation. Genome-wide transcriptomics identified candidate genes for paracrine regulation of oligodendrocyte development, including connective tissue growth factor (CTGF), whose expression is repressed by SRF. Adenovirus-mediated CTGF expression in vivo revealed that CTGF blocks excessive oligodendrocyte differentiation. In vitro, CTGF-mediated inhibition of oligodendrocyte maturation involved sequestration and thereby counteraction of insulin growth factor 1-stimulated oligodendrocyte differentiation.