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Summary: Cells initiate fate decisions during G1 phase by converting extracellular signals into distinctive cell cycle kinetics. The DNA replication timing is determined in G1 phase; lengthened G1 and hastened S phases correlate with increased neurogenic propensity of neural progenitor cells (NPCs), although the underlying molecular control remains elusive. Here, we report that proper G1 phase completion in NPCs requires Brap, a Ras-Erk signaling modulator with ubiquitin E3 ligase activity. We identified Skp2 and Skp2-associated SCF ubiquitin ligase as a key target of Brap-mediated polyubiquitination. Loss of Brap resulted in elevated Skp2, which increased p27Kip1 destruction, leading to G1 phase truncation and premature S phase entry. The aberrantly executed G1 in Brap-mutant NPCs, followed by hindered S phase progression and increased G2 phase arrest, which together prolonged the cell cycle, impeded neuronal differentiation and culminated in microcephaly. These findings demonstrate that neuronal differentiation is potentiated during G1 phase by Brap-directed cascade of events in cell signaling and protein turnover. : Lanctot et al. show that the capacity of neural progenitors to generate cerebral cortical neurons relies on the integration of cell signaling and ubiquitin-mediated protein turnover to complete G1 phase of the cell cycle. Keywords: cell cycle, G1 phase, G1/S transition, stem/progenitor, differentiation, ubiquitination, cerebral cortical neurogenesis

Diabetic foot ulcer's impaired wound healing, which leads to the development of chronic non-healing wounds and ultimately amputation, is a major problem worldwide. Although recently endothelial progenitor cell-derived cell therapy has been used as a therapeutic intervention to treat diabetic wounds, thereby promoting neovascularization, the results, however, are not satisfactory. In this article, we have discussed the several steps that are involved in the neovascularization process, which might be impaired during diabetes. In addition, we have also discussed the reported possible interventions to correct these impairments. Thus, we have summarized neovascularization as a process with a coordinated sequence of multiple steps and thus, there is the need of a combined therapeutic approach to achieve better treatment outcomes.

SummaryRegulating cell proliferation and differentiation in CNS development requires both extraordinary complexity and precision. Neural progenitors receive graded overlapping signals from midline signaling centers, yet each makes a unique cell fate decision in a spatiotemporally restricted pattern. The Nde1-Lis1 complex regulates individualized cell fate decisions based on the geographical location with respect to the midline. While cells distant from the midline fail to self-renew in the Nde1-Lis1 double-mutant CNS, cells embedded in the signaling centers showed marked overproliferation. A direct interaction between Lis1 and Brap, a mitogen-activated protein kinase (MAPK) signaling threshold modulator, mediates this differential response to mitogenic signal gradients. Nde1-Lis1 deficiency resulted in a spatially dependent alteration of MAPK scaffold Ksr and hyperactivation of MAPK. Epistasis analyses supported synergistic Brap and Lis1 functions. These results suggest that a molecular complex composed of Nde1, Lis1, and Brap regulates the dynamic MAPK signaling threshold in a spatially dependent fashion.

Filamins are a family of actin-binding proteins responsible for diverse biological functions in the context of regulating actin dynamics and vesicle trafficking. Disruption of these proteins has been implicated in multiple human developmental disorders. To investigate the roles of different filamin isoforms, we focused on FlnA and FlnB interactions in the cartilage growth plate, since mutations in both molecules cause chondrodysplasias. Current studies show that FlnA and FlnB share a common function in stabilizing the actin cytoskeleton, they physically interact in the cytoplasm of chondrocytes, and loss of FlnA enhances FlnB expression of chondrocytes in the growth plate (and vice versa), suggesting compensation. Prolonged FlnB loss, however, promotes actin-stress fiber formation following plating onto an integrin activating substrate whereas FlnA inhibition leads to decreased actin formation. FlnA more strongly binds RhoA, although both filamins overlap with RhoA expression in the cell cytoplasm. FlnA promotes RhoA activation whereas FlnB indirectly inhibits this pathway. Moreover, FlnA loss leads to diminished expression of β1-integrin, whereas FlnB loss promotes integrin expression. Finally, fibronectin mediated integrin activation has been shown to activate RhoA and activated RhoA leads to stress fiber formation and cell spreading. Fibronectin stimulation in null FlnA cells impairs enhanced spreading whereas FlnB inhibited cells show enhanced spreading. While filamins serve a primary static function in stabilization of the actin cytoskeleton, these studies are the first to demonstrate a dynamic and antagonistic relationship between different filamin isoforms in the dynamic regulation of integrin expression, RhoGTPase activity and actin stress fiber remodeling.

Neuronal fate-restricted intermediate progenitors (IPs) are derived from the multipotent radial glia (RGs) and serve as the direct precursors for cerebral cortical neurons, but factors that control their neurogenic plasticity remain elusive. Here we report that IPs’ neuron production is enhanced by abrogating filamin function, leading to the generation of periventricular neurons independent of normal neocortical neurogenesis and neuronal migration. Loss of Flna in neural progenitor cells (NPCs) led RGs to undergo changes resembling epithelial-mesenchymal transition (EMT) along with exuberant angiogenesis that together changed the microenvironment and increased neurogenesis of IPs. We show that by collaborating with β-arrestin, Flna maintains the homeostatic signaling between the vasculature and NPCs, and loss of this function results in escalated Vegfa and Igf2 signaling, which exacerbates both EMT and angiogenesis to further potentiate IPs’ neurogenesis. These results suggest that the neurogenic potential of IPs may be boosted in vivo by manipulating Flna-mediated neurovascular communication. DOI: http://dx.doi.org/10.7554/eLife.17823.001

Human mutations in the X-linked FLNA gene are associated with a remarkably diverse phenotype, including severe arterial morphological anomalies. However, the role for filamin A (FlnA) in vascular cells remains partially understood. We used a smooth muscle (sm)-specific conditional mouse model to delete FlnA at the adult stage, thus avoiding the developmental effects of the knock-out. Inactivation of smFlnA in adult mice significantly lowered blood pressure, together with a decrease in pulse pressure. However, both the aorta and carotid arteries showed a major outward hypertrophic remodeling, resistant to losartan, and normally occurring in hypertensive conditions. Notably, arterial compliance was significantly enhanced in the absence of smFlnA. Moreover, reactivity of thoracic aorta rings to a variety of vasoconstrictors was elevated, while basal contractility in response to KCl depolarization was reduced. Enhanced reactivity to the thromboxane A2 receptor agonist U46619 was fully reversed by the ROCK inhibitor Y27632. We discuss the possibility that a reduction in arterial stiffness upon smFlnA inactivation might cause a compensatory increase in conduit artery diameter for normalization of parietal tension, independently of the ROCK pathway. In conclusion, deletion of smFlnA in adult mice recapitulates the vascular phenotype of human bilateral periventricular nodular heterotopia, culminating in aortic dilatation.

Genes disrupted in human microcephaly (meaning "small brain") define key regulators of neural progenitor proliferation and cell-fate specification. In comparison, genes mutated in human lissencephaly (lissos means smooth and cephalos means brain) highlight critical regulators of neuronal migration. Here, we report two families with extreme microcephaly and grossly simplified cortical gyral structure, a condition referred to as microlissencephaly, and show that they carry homozygous frameshift mutations in NDE1, which encodes a multidomain protein that localizes to the centrosome and mitotic spindle poles. Both human mutations in NDE1 truncate the C-terminal NDE1domains, which are essential for interactions with cytoplasmic dynein and thus for regulation of cytoskeletal dynamics in mitosis and for cell-cycle-dependent phosphorylation of NDE1 by Cdk1. We show that the patient NDE1 proteins are unstable, cannot bind cytoplasmic dynein, and do not localize properly to the centrosome. Additionally, we show that CDK1 phosphorylation at T246, which is within the C-terminal region disrupted by the mutations, is required for cell-cycle progression from the G2 to the M phase. The role of NDE1 in cell-cycle progression probably contributes to the profound neuronal proliferation defects evident in Nde1-null mice and patients with NDE1 mutations, demonstrating the essential role of NDE1 in human cerebral cortical neurogenesis.

Neurons in the cerebral cortex originate predominantly from asymmetrical divisions of polarized radial glial or neuroepithelial cells. Fate control of neural progenitors through regulating cell division asymmetry determines the final cortical neuronal number and organization. Haploinsufficiency of human LIS1 results in type I lissencephaly (smooth brain) with severely reduced surface area and laminar organization of the cerebral cortex. Here we show that LIS1 and its binding protein Nde1 (mNudE) regulate the fate of radial glial progenitors collaboratively. Mice with an allelic series of Lis1 and Nde1 double mutations displayed a striking dose-dependent size reduction and de-lamination of the cerebral cortex. The neocortex of the Lis1-Nde1 double mutant mice showed over 80% reduction in surface area and inverted neuronal layers. Dramatically increased neuronal differentiation at the onset of corticogenesis in the mutant led to overproduction and abnormal development of earliest-born preplate neurons and Cajal-Retzius cells at the expense of progenitors. While both Lis1 and Nde1 are known to regulate the mitotic spindle orientation, only a moderate alteration in mitotic cleavage orientation was detected in the Lis1-Nde1 double deficient progenitors. Instead, a striking change in the morphology of metaphase progenitors with reduced apical attachment to the ventricular surface and weakened lateral contacts to neighboring cells appear to hinder the accurate control of cell division asymmetry and underlie the dramatically increased neuronal differentiation. Our data suggest that maintaining the shape and cell-cell interactions of radial glial neuroepithelial progenitors by the Lis1-Nde1 complex is essential for their self renewal during the early phase of corticogenesis.

Successfully completing the S phase of each cell cycle ensures genome integrity. Impediment of DNA replication can lead to DNA damage and genomic disorders. In this study, we show a novel function for NDE1, whose mutations cause brain developmental disorders, in safeguarding the genome through S phase during early steps of neural progenitor fate restrictive differentiation. Nde1 mutant neural progenitors showed catastrophic DNA double strand breaks concurrent with the DNA replication. This evoked DNA damage responses, led to the activation of p53-dependent apoptosis, and resulted in the reduction of neurons in cortical layer II/III. We discovered a nuclear pool of Nde1, identified the interaction of Nde1 with cohesin and its associated chromatin remodeler, and showed that stalled DNA replication in Nde1 mutants specifically occurred in mid-late S phase at heterochromatin domains. These findings suggest that NDE1-mediated heterochromatin replication is indispensible for neuronal differentiation, and that the loss of NDE1 function may lead to genomic neurological disorders. DOI: http://dx.doi.org/10.7554/eLife.03297.001, eLife digest The brain is a complex organ with many different cell types that each have specialized functions. Mutations in genes that control how the brain develops can have serious consequences—and one of the most important genes involved in the development of the human brain is called NDE1. Individuals who inherit mutated copies of the NDE1 gene from both parents have brains with fewer folds than a healthy brain, and their brains are up to 90% smaller than normal. These individuals may have serious developmental disabilities, struggle with basic functions like swallowing, and die early. Moreover, having just one defective copy of the NDE1 gene has been linked to the development of cancers such as leukemia. The protein encoded by the NDE1 gene acts as a scaffold for many protein complexes and can be found throughout the cell in various cellular compartments (from the cell periphery to the nucleus). However, it was unclear where the NDE1 protein's activity was most needed in developing brain cells. Feng and Houlihan now provide a new explanation for NDE1's role in brain development. Looking for molecules that interact with the mouse version of the Nde1 protein revealed that it binds to proteins that control how DNA is packaged inside the nucleus of a cell. In doing so, Nde1 appears to protect the genome of brain stem cells, while these cells' DNA is copied and before they divide to form new cells destined to become neurons. For a cell to divide, its genetic information must be accurately copied and then segregated between the two new cells to ensure that each receives all the genetic instructions needed to develop and function properly. In brain cells from mice without functional Nde1, the DNA frequently breaks as it is copied. Brain cells that inherit damaged DNA might not function correctly, while serious breaks that affect both strands of the DNA can trigger a response that kills the mutated cell. This means, there are fewer cells that make up the outer layer of the brain, making it less wrinkled than normal. This part of the brain—called the cerebral cortex—is important for many processes including thought and memory, and it helps different areas of the brain communicate with one another. This may explain why mutations in the NDE1 gene can contribute to a variety of brain disorders. Errors during DNA replication can also cause cancer to develop. As such, the findings of Feng and Houlihan may also help to explain why some genetic mutations are associated with both cancer and brain disorders. DOI: http://dx.doi.org/10.7554/eLife.03297.002

Lis1-Nde1 integrates cerebral cortical neurogenesis with neuronal migration by stabilizing the basal-lateral surface of radial glial cells., Radial glial cells (RGCs) are distinctive neural stem cells with an extraordinary slender bipolar morphology and dual functions as precursors and migration scaffolds for cortical neurons. Here we show a novel mechanism by which the Lis1-Nde1 complex maintains RGC functions through stabilizing the dystrophin/dystroglycan glycoprotein complex (DGC). A direct interaction between Nde1 and utrophin/dystrophin allows for the assembly of a multi-protein complex that links the cytoskeleton to the extracellular matrix of RGCs to stabilize their lateral membrane, cell-cell adhesion, and radial morphology. Lis1-Nde1 mutations destabilized the DGC and resulted in deformed, disjointed RGCs and disrupted basal lamina. Besides impaired RGC self-renewal and neuronal migration arrests, Lis1-Nde1 deficiencies also led to neuronal over-migration. Additional to phenotypic resemblances of Lis1-Nde1 with DGC, strong synergistic interactions were found between Nde1 and dystroglycan in RGCs. As functional insufficiencies of LIS1, NDE1, and dystroglycan all cause lissencephaly syndromes, our data demonstrated that a three-dimensional regulation of RGC's cytoarchitecture by the Lis1-Nde1-DGC complex determines the number and spatial organization of cortical neurons as well as the size and shape of the cerebral cortex., Author Summary The processes of neurogenesis and neuronal migration within the developing cerebral cortex must be tightly orchestrated to enable ordered generation and transportation of neurons to designated cortical layers. The mechanism by which these two processes are integrated remains elusive. Radial glial cells, the major neural stem cells in the developing brain, serve both as progenitors and migration scaffolds for cortical neurons as they migrate. The cortical developmental disease lissencephaly (smooth brain) is a result of defects in neurogenesis and neuronal migration, and is associated with the protein LIS1 and its binding partner NDE1. In this study, we show that several key players in human cerebral cortical development, including LIS1, NDE1, dystrophin, and dystroglycan, form a molecular complex to regulate cortical neurogenesis and neuronal migration in a mouse model. This multi-protein complex is active on the basal-lateral surface of radial glial cells, which is known to provide guidance to migrating neurons. When we depleted NDE1 in mice, dystrophin and dystroglycan were lost from the membrane and radial glial cells were deformed, indicating the importance of the multi-protein complex for proper cell morphology. This effect on morphology resulted in a loss of normal migration and cortical phenotypes similar to lissencephaly. Our findings suggest that genes that regulate the structure and function of the basal-lateral membrane of radial glial cells may integrate the dual functions of these cells and determine the size, shape, and function of the cerebral cortex.

LIS1, a microtubule-associated protein, is required for neuronal migration, but the precise mechanism of LIS1 function is unknown. We identified a LIS1 interacting protein encoded by a mouse homolog of NUDE, a nuclear distribution gene in A. nidulans and a multicopy suppressor of the LIS1 homolog, NUDF. mNudE is located in the centrosome or microtubule organizing center (MTOC), and interacts with six different centrosomal proteins. Overexpression of mNudE dissociates γ-tubulin from the centrosome and disrupts microtubule organization. Missense mutations that disrupt LIS1 function block LIS1-mNudE binding. Moreover, misexpression of the LIS1 binding domain of mNudE in Xenopus embryos disrupts the architecture and lamination of the CNS. Thus, LIS1-mNudE interactions may regulate neuronal migration through dynamic reorganization of the MTOC.

International audience; Mutations in the filamin A (FlnA) gene are frequently associated with severe arterial abnormalities, although the physiological role for this cytoskeletal element remains poorly understood in vascular cells. We used a conditional mouse model to selectively delete FlnA in smooth muscle (sm) cells at the adult stage, thus avoiding the developmental effects of the knockout. Basal blood pressure was significantly reduced in conscious smFlnA knockout mice. Remarkably, pressure-dependent tone of the resistance caudal artery was lost, whereas reactivity to vasoconstrictors was preserved. Impairment of the myogenic behavior was correlated with a lack of calcium influx in arterial myocytes upon an increase in intraluminal pressure. Notably, the stretch activation of CaV1.2 was blunted in the absence of smFlnA. In conclusion, FlnA is a critical upstream element of the signaling cascade underlying the myogenic tone. These findings allow a better understanding of the molecular basis of arterial autoregulation and associated disease states.