Serum response factor regulates hippocampal lamination and dendrite development and is connected with reelin signaling.
ABSTRACT: 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.
Project description:A hallmark of temporal lobe epilepsy (TLE) is hippocampal neuronal demise and aberrant mossy fiber sprouting. In addition, unrestrained neuronal activity in TLE patients induces gene expression including immediate early genes (IEGs) such as Fos and Egr1.We employed the mouse pilocarpine model to analyze the transcription factor (TF) serum response factor (SRF) in epileptogenesis, seizure induced histopathology and IEG induction. SRF is a neuronal activity regulated TF stimulating IEG expression as well as nerve fiber growth and guidance. Adult conditional SRF deficient mice (Srf CaMKCreERT2 ) were more refractory to initial status epilepticus (SE) acquisition. Further, SRF deficient mice developed more spontaneous recurrent seizures (SRS). Genome-wide transcriptomic analysis uncovered a requirement of SRF for SE and SRS induced IEG induction (e.g. Fos, Egr1, Arc, Npas4, Btg2, Atf3). SRF was required for epilepsy associated neurodegeneration, mossy fiber sprouting and inflammation. We uncovered MAP kinase signaling as SRF target during epilepsy. Upon SRF ablation, seizure evoked induction of dual specific phosphatases (Dusp5 and Dusp6) was reduced. Lower expression of these negative ERK kinase regulators correlated with altered P-ERK levels in epileptic Srf mutant animals.Overall, this study uncovered an SRF contribution to several processes of epileptogenesis in the pilocarpine model.
Project description:The Reelin ligand regulates a Dab1-dependent signaling pathway required for brain lamination and normal dendritogenesis, but the specific mechanisms underlying these actions remain unclear. We find that Stk25, a modifier of Reelin-Dab1 signaling, regulates Golgi morphology and neuronal polarization as part of an LKB1-Stk25-Golgi matrix protein 130 (GM130) signaling pathway. Overexpression of Stk25 induces Golgi condensation and multiple axons, both of which are rescued by Reelin treatment. Reelin stimulation of cultured neurons induces the extension of the Golgi into dendrites, which is suppressed by Stk25 overexpression. In vivo, Reelin and Dab1 are required for the normal extension of the Golgi apparatus into the apical dendrites of hippocampal and neocortical pyramidal neurons. This demonstrates that the balance between Reelin-Dab1 signaling and LKB1-Stk25-GM130 regulates Golgi dispersion, axon specification, and dendrite growth and provides insights into the importance of the Golgi apparatus for cell polarization.
Project description:Formation of a 6-layered cortical plate and axon tract patterning are key features of cerebral cortex development. Abnormalities of these processes may be the underlying cause for a range of functional disabilities seen in human neurodevelopmental disorders. To identify mouse mutants with defects in cortical lamination or corticofugal axon guidance, N-ethyl-N-nitrosourea (ENU) mutagenesis was performed using mice expressing LacZ reporter genes in layers II/III and V of the cortex (Rgs4-lacZ) or in corticofugal axons (TAG1-tau-lacZ). Four lines with abnormal cortical lamination have been identified. One of these was a splice site mutation in reelin (Reln) that results in a premature stop codon and the truncation of the C-terminal region (CTR) domain of reelin. Interestingly, this novel allele of Reln did not display cerebellar malformation or ataxia, and this is the first report of a Reln mutant without a cerebellar defect. Four lines with abnormal cortical axon development were also identified, one of which was found by whole-genome resequencing to carry a mutation in Lrp2. These findings demonstrated that the application of ENU mutagenesis to mice carrying transgenic reporters marking cortical anatomy is a sensitive and specific method to identify mutations that disrupt patterning of the developing brain.
Project description:One pathway regulating the migration of neurons during development of the mammalian cortex involves the extracellular matrix protein Reelin. Reelin and components of its signaling cascade, the lipoprotein receptors ApoER2 and Vldlr and the intracellular adapter protein Dab1 are pivotal for a correct layer formation during corticogenesis. The olfactory bulb (OB) as a phylogenetically old cortical region is known to be a prominent site of Reelin expression. Although some aspects of Reelin function in the OB have been described, the influence of Reelin on OB layer formation has so far been poorly analyzed. Here we studied animals deficient for either Reelin, Vldlr, ApoER2 or Dab1 as well as double-null mutants. We performed organotypic migration assays, immunohistochemical marker analysis and BrdU incorporation studies to elucidate roles for the different components of the Reelin signaling cascade in OB neuroblast migration and layer formation. We identified ApoER2 as being the main receptor responsible for Reelin mediated detachment of neuroblasts and correct migration of early generated interneurons within the OB, a prerequisite for correct OB lamination.
Project description:<h4>Unlabelled</h4>The mechanisms controlling cortical dendrite initiation and targeting are poorly understood. Multiphoton imaging of developing mouse cortex reveals that apical dendrites emerge by direct transformation of the neuron's leading process during the terminal phase of neuronal migration. During this ?110 min period, the dendritic arbor increases ?2.5-fold in size and migration arrest occurs below the first stable branch point in the developing arbor. This dendritic outgrowth is triggered at the time of leading process contact with the marginal zone (MZ) and occurs primarily by neurite extension into the extracellular matrix of the MZ. In reeler cortices that lack the secreted glycoprotein Reelin, a subset of neurons completed migration but then retracted and reorganized their arbor in a tangential direction away from the MZ soon after migration arrest. For these reeler neurons, the tangential oriented primary neurites were longer lived than the radially oriented primary neurites, whereas the opposite was true of wild-type (WT) neurons. Application of Reelin protein to reeler cortices destabilized tangential neurites while stabilizing radial neurites and stimulating dendritic growth in the MZ. Therefore, Reelin functions as part of a polarity signaling system that links dendritogenesis in the MZ with cellular positioning and cortical lamination.<h4>Significance statement</h4>Whether the apical dendrite emerges by transformation of the leading process of the migrating neuron or emerges de novo after migration is completed is unclear. Similarly, it is not clear whether the secreted glycoprotein Reelin controls migration and dendritic growth as related or separate processes. Here, multiphoton microscopy reveals the direct transformation of the leading process into the apical dendrite. This transformation is coupled to the successful completion of migration and neuronal soma arrest occurs below the first stable branch point of the nascent dendrite. Deficiency in Reelin causes the forming dendrite to avoid its normal target area and branch aberrantly, leading to improper cellular positioning. Therefore, this study links Reelin-dependent dendritogenesis with migration arrest and cortical lamination.
Project description:During brain development, growth cones respond to attractive and repulsive axon guidance cues. How growth cones integrate guidance instructions is poorly understood. Here, we demonstrate a link between BDNF (brain derived neurotrophic factor), promoting axonal branching and ephrin-A5, mediating axonal repulsion via Eph receptor tyrosine kinase activation. BDNF enhanced growth cone filopodial dynamics and neurite branching of primary neurons. We show that ephrin-A5 antagonized this BDNF-evoked neuronal motility. BDNF increased ERK phosphorylation (P-ERK) and nuclear ERK entry. Ephrin-A5 suppressed BDNF-induced ERK activity and might sequester P-ERK in the cytoplasm. Neurotrophins are well established stimulators of a neuronal immediate early gene (IEG) response. This is confirmed in this study by e.g. c-fos, Egr1 and Arc upregulation upon BDNF application. This BDNF-evoked IEG response required the transcription factor SRF (serum response factor). Notably, ephrin-A5 suppressed a BDNF-evoked neuronal IEG response, suggesting a role of Eph receptors in modulating gene expression. In opposite to IEGs, long-term ephrin-A5 application induced cytoskeletal gene expression of tropomyosin and actinin. To uncover specific Eph receptors mediating ephrin-As impact on neurotrophin signaling, EphA7 deficient mice were analyzed. In EphA7 deficient neurons alterations in growth cone morphology were observed. However, ephrin-A5 still counteracted neurotrophin signaling suggesting that EphA7 is not required for ephrin and BDNF crosstalk. In sum, our data suggest an interaction of ephrin-As and neurotrophin signaling pathways converging at ERK signaling and nuclear gene activity. As ephrins are involved in development and function of many organs, such modulation of receptor tyrosine kinase signaling and gene expression by Ephs might not be limited to the nervous system.
Project description:Our goal was to identify phosphorylation sites that regulate serum response factor (SRF) activity to gain a better understanding of the signaling mechanisms that regulate SRF's involvement in smooth muscle cell (SMC)-specific and early response gene expression.By screening phosphorylation-deficient and mimetic mutations in SRF(-/-) embryonic stem cells, we identified T159 as a phosphorylation site that significantly inhibits SMC-specific gene expression in an embryonic stem cell model of SMC differentiation. This residue conforms to a highly conserved consensus cAMP-dependent protein kinase (PKA) site, and in vitro and in vivo labeling studies demonstrated that it was phosphorylated by PKA. Results from gel shift and chromatin immunoprecipitation assays demonstrated that T159 phosphorylation inhibited SRF binding to SMC-specific CArG elements. Interestingly, the myocardin factors could at least partially rescue the effects of the T159D mutation under some conditions, but this response was promoter specific. Finally, PKA signaling had much less of an effect on c-fos promoter activity and SRF binding to the c-fos CArG.Our results indicate that phosphorylation of SRF by PKA inhibits SMC-specific transcription suggesting a novel signaling mechanism for the control of SMC phenotype.
Project description:It is widely accepted that amyloid precursor protein (APP) plays a central role in the pathogenesis of Alzheimer's disease. In addition, APP has been proposed to have functions in numerous biological processes including neuronal proliferation, differentiation, migration, axon guidance, and neurite outgrowth, as well as in synapse formation and function. However, germline knockout of APP yields relatively subtle phenotypes, and brain development appears grossly normal. This is thought to be due in part to functional compensation by APP family members and other type I transmembrane proteins. Here, we have generated a conditional mouse knockout for APP that is controlled temporally using CreER and tamoxifen administration. We show that total cortical expression of APP is reduced following tamoxifen administration during embryonic time points critical for cortical lamination, and that this results in displacement of Reelin-positive cells below the cortical plate with a concurrent elevation in Reelin protein levels. These results support a role for APP in cortical lamination and demonstrate the utility of a conditional knockout approach in which APP can be deleted with temporal control in vivo. This new tool should be useful for many different applications in the study of APP function across the mammalian life span.
Project description:Serum response factor (SRF) is an essential transcription factor that influences many cellular processes including cell proliferation, migration, and differentiation. SRF directly regulates and is required for immediate early gene (IEG) and actin cytoskeleton-related gene expression. SRF coordinates these competing transcription programs through discrete sets of cofactors, the ternary complex factors (TCFs) and myocardin-related transcription factors (MRTFs). The relative contribution of these two programs to in vivo SRF activity and mutant phenotypes is not fully understood. To study how SRF utilizes its cofactors during development, we generated a knock-in <i>Srf<sup>aI</sup></i> allele in mice harboring point mutations that disrupt SRF-MRTF-DNA complex formation but leave SRF-TCF activity unaffected. Homozygous <i>Srf<sup>aI/aI</sup></i> mutants die at E10.5 with notable cardiovascular phenotypes, and neural crest conditional mutants succumb at birth to defects of the cardiac outflow tract but display none of the craniofacial phenotypes associated with complete loss of SRF in that lineage. Our studies further support an important role for MRTF mediating SRF function in cardiac neural crest and suggest new mechanisms by which SRF regulates transcription during development.
Project description:Neuronal migration is a fundamental component of brain development whose failure is associated with various neurological and psychiatric disorders. Reelin is essential for the stereotypical inside-out sequential lamination of the neocortex, but the molecular mechanisms of its action still remain unclear. Here we show that regulation of Notch activity plays an important part in Reelin-signal-dependent neuronal migration. We found that Reelin-deficient mice have reduced levels of the cleaved form of Notch intracellular domain (Notch ICD) and that loss of Notch signaling in migrating neurons results in migration and morphology defects. Further, overexpression of Notch ICD mitigates the laminar and morphological abnormalities of migrating neurons in Reeler. Finally, our in vitro biochemical studies show that Reelin signaling inhibits Notch ICD degradation via Dab1. Together, our results indicate that neuronal migration in the developing cerebral cortex requires a Reelin-Notch interaction.