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SUMMARYAging is an intricate process that is characterized by multiple hallmarks including stem cell exhaustion, genome instability, epigenome alteration, impaired proteostasis, and cellular senescence. While each of these traits is detrimental at the cellular level, it remains unclear how they are interconnected to cause systemic organ deterioration. Here we show that abrogating Brap, a BRCA1 associated protein important for neurogenesis, results in cellular senescence with persistent DNA double-strand breaks and elevation of histone H2A mono- and poly-ubiquitination (H2Aub). The high H2Aub initiates histone proteolysis, leading to both epigenetic alteration and proteasome overflow. These defects induce neuroinflammation, impair proteostasis, accelerate neurodegeneration, and substantially shorten lifespan in mice carrying Brap deletions in the brain. We further show H2Aub is also increased in human brain tissues of Alzheimer’s disease. These data together suggest that chromatin aberrations mediated by H2Aub act as a nexus of multiple aging hallmarks and promote tissue-wide degeneration.

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.

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.

Filamins were discovered as the first family of non-muscle actin-binding protein. They are lage cytoplasmic proteins that cross-link cortical actin into a dynamic three-dimensional structure. Filamins have also been reported to interact with a large number of cellular proteins of great functional diversity, suggesting that they are unusually versatile signalling scaffolds. More recently, genetic mutations in filamin A and B have been reported to cause a wide range of human diseases, suggesting that different diseases highlight distinct filamin interactions.

Ablation of the LIS1-interacting protein Nde1 (formerly mNudE) in mouse produces a small brain (microcephaly), with the most dramatic reduction affecting the cerebral cortex. While cortical lamination is mostly preserved, the mutant cortex has fewer neurons and very thin superficial cortical layers (II to IV). BrdU birthdating revealed retarded and modestly disorganized neuronal migration; however, more dramatic defects on mitotic progression, mitotic orientation, and mitotic chromosome localization in cortical progenitors were observed in Nde1 mutant embryos. The small cerebral cortex seems to reflect both reduced progenitor cell division and altered neuronal cell fates. In vitro analysis demonstrated that Nde1 is essential for centrosome duplication and mitotic spindle assembly. Our data show that mitotic spindle function and orientation are essential for normal development of mammalian cerebral cortex.

The doublecortin-like domains (DCX), which typically occur in tandem, are novel microtubule-binding modules. DCX tandems are found in doublecortin, a 360-residue protein expressed in migrating neurons; the doublecortin-like kinase (DCLK); the product of the RP1 gene that is responsible for a form of inherited blindness; and several other proteins. Mutations in the gene encoding doublecortin cause lissencephaly in males and the 'double-cortex syndrome' in females. We here report a solution structure of the N-terminal DCX domain of human doublecortin and a 1.5 A resolution crystal structure of the equivalent domain from human DCLK. Both show a stable, ubiquitin-like tertiary fold with distinct structural similarities to GTPase-binding domains. We also show that the C-terminal DCX domains of both proteins are only partially folded. In functional assays, the N-terminal DCX domain of doublecortin binds only to assembled microtubules, whereas the C-terminal domain binds to both microtubules and unpolymerized tubulin.

Mutations in the X-linked gene Filamin A (FLNA) lead to the human neurological disorder, periventricular heterotopia (PH). Although PH is characterized by a failure in neuronal migration into the cerebral cortex with consequent formation of nodules in the ventricular and subventricular zones, many neurons appear to migrate normally, even in males, suggesting compensatory mechanisms. Here we characterize expression patterns for FlnA and a highly homologous protein Filamin B (FlnB) within the nervous system, in order to better understand their potential roles in cortical development. FlnA mRNA was widely expressed in all cortical layers while FlnB mRNA was most highly expressed in the ventricular and subventricular zones during development. In adulthood, widespread but reduced expression of FlnA and FlnB persisted throughout the cerebral cortex. FlnA and FlnB proteins were highly expressed in both the leading processes and somata of migratory neurons during corticogenesis. Postnatally, FlnA immunoreactivity was largely localized to the cell body with FlnB in the soma and neuropil during neuronal differentiation. In adulthood, diminished expression of both proteins localized to the cell soma and nucleus. Moreover, the putative FLNB homodimerization domain strongly interacted with itself or the corresponding homologous region of FLNA by yeast two-hybrid interaction, the two proteins co-localized within neuronal precursors by immunocytochemistry and the existence of FLNA-FLNB heterodimers could be detected by co-immunoprecipitation. These results suggest that FLNA and FLNB may form both homodimers and heterodimers and that their interaction could potentially compensate for the loss of FLNA function during cortical development within PH individuals.

The Drosophila Na,K-ATPase (or sodium pump) alpha-subunit gene was found to contain 10 exons and span approx. 25 kb. Two nearly adjacent transcriptional initiation sites were identified, and the 2085-nucleotide sequence upstream of the first transcriptional start was analysed for promoter activity in transfected Drosophila SL2 cells. This region was found to contain many cis-acting elements that influence promoter activity, including elements that confer 2- to 3-fold higher activity in SL2 cells cultured at 30 degrees C versus 22 degrees C. Temperature-sensitive transcriptional regulation of the Na,K-ATPase alpha-subunit in Drosophila is a plausible mechanistic candidate for the factor driving temperature-dependent up-regulation of the Na,K-ATPase alpha-subunit described here for fly strains homozygous for single P-element insertions in the alpha-subunit gene. Four new P-element insertion strains were identified in this study, each insertion site lying within the first intron of the Na,K-ATPase alpha-subunit gene. The insertion in strain 0462 resulted in cold-sensitive recessive lethality; flies homozygous for the 0462 mutation could be rescued by growth at 29-30 degrees C, a condition that partially corrected a deficiency in the level of Na,K-ATPase alpha-subunit. The high-temperature rescue of homozygous 0462 flies appeared to result primarily from improved Na,K-ATPase expression rather than an increase in the rate of ion transport per Na,K-ATPase molecule. These observations point to a role for sodium-pump activity in determining the range of temperature tolerance in Drosophila and demonstrate that relatively subtle changes in sodium-pump expression can have major consequences in whole organisms.

Aims We hypothesized that the structure and function of the mature valves is largely dependent upon how these tissues are built during development, and defects in how the valves are built can lead to the pathological progression of a disease phenotype. Thus, we sought to uncover potential developmental origins and mechanistic underpinnings causal to myxomatous mitral valve disease. We focus on how filamin-A, a cytoskeletal binding protein with strong links to human myxomatous valve disease, can function as a regulatory interface to control proper mitral valve development. Methods and results Filamin-A-deficient mice exhibit abnormally enlarged mitral valves during foetal life, which progresses to a myxomatous phenotype by 2 months of age. Through expression studies, in silico modelling, 3D morphometry, biochemical studies, and 3D matrix assays, we demonstrate that the inception of the valve disease occurs during foetal life and can be attributed, in part, to a deficiency of interstitial cells to efficiently organize the extracellular matrix (ECM). This ECM organization during foetal valve gestation is due, in part, to molecular interactions between filamin-A, serotonin, and the cross-linking enzyme, transglutaminase-2 (TG2). Pharmacological and genetic perturbations that inhibit serotonin-TG2-filamin-A interactions lead to impaired ECM remodelling and engender progression to a myxomatous valve phenotype. Conclusions These findings illustrate a molecular mechanism by which valve interstitial cells, through a serotonin, TG, and filamin-A pathway, regulate matrix organization during foetal valve development. Additionally, these data indicate that disrupting key regulatory interactions during valve development can set the stage for the generation of postnatal myxomatous valve disease.

A bang-sensitive enhancer trap line was isolated in a behavioral screen. The flies show a weak bang-sensitive paralysis, recovering after about 7 s. The P element insert is localized at 93131-2 on the salivary chromosomes, the site of the (Na + ,K + )ATPase a subunit gene. Molecular characterization demonstrates that the transposon is inserted into the first intron of this gene. This insertion leads to normal-sized transcripts, but reduced levels of expression. This change is also reflected in lower amounts of a normal-sized a subunit protein. Mutant flies show a much greater sensitivity to ouabain, likewise indicating, on a functional level, a reduction in Na + pump activity. Furthermore, the bang-sensitive behavior can also be mimicked by injecting sublethal doses of ouabain into wild-type flies. The molecular and functional evidence indicates that the insertion has produced a hypomorphic mutation of the (Na + ,K + )ATPase α subunit gene, opening the way to future studies of the regulation of the Na + pump.

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.

Cells in mechanically challenged environments must cope with high amplitude forces to maintain cell viability and tissue homeostasis. Currently, force-induced cell death and the identity of mechanoprotective factors are not defined. We examined death in cultured periodontal fibroblasts, connective tissue cells that are exposed to heavy applied forces in vivo. Static tensile forces (0.48 piconewtons/microm2 cell area) were applied through magnetite beads coated with collagen or bovine serum albumin. There was a time-dependent increase of the percentage of propidium iodide-permeable cells in force-loaded cultures incubated with collagen but not bovine serum albumin beads, indicating a role for integrins. Cells exhibited reduced mitochondrial membrane potential, increased caspase-3 activation, nuclear condensation, terminal deoxynucleotidyl transferase nick end labeling staining, and detachment from the culture dish. The caspase-3 inhibitor acetyl-Asp-Glu-Val-Asp-aldehyde reduced detachment 3-fold. There was a rapid (

Neuronal migration, like the migration of many cell types, requires an extensive rearrangement of cell shape, mediated by changes in the cytoskeleton. The genetic analysis of human brain malformations has identified several biochemical players in this process, including doublecortin (DCX) and LIS1, mutations of which cause a profound migratory disturbance known as lissencephaly ('smooth brain') in humans. Studies in mice have identified additional molecules such as Cdk5, P35, Reelin, Disabled and members of the LDL superfamily of receptors. Understanding the cell biology of these molecules has been a challenge, and it is not known whether they function in related biochemical pathways or in very distinct processes. The last year has seen rapid advances in the biochemical analysis of several such molecules. This analysis has revealed roles for some of these molecules in cytoskeletal regulation and has shown an unexpected conservation of the genetic pathways that regulate neuronal migration in humans and nuclear movement in simple eukaryotic organisms.

How mitotic cell fate is regulated in the developing mammalian CNS is an important but largely unanswered question. Recently reporting in Neuron, Godin et al. showed that Huntingtin, the protein mutated in Huntington's disease, is required for both cerebral cortical neurogenesis and mitotic spindle function in neural progenitors.

Long-range, directed migration is particularly dramatic in the cerebral cortex, where postmitotic neurons generated deep in the brain migrate to form layers with distinct form and function. In the X-linked dominant human disorder periventricular heterotopia (PH), many neurons fail to migrate and persist as nodules lining the ventricular surface. Females with PH present with epilepsy and other signs, including patent ductus arteriosus and coagulopathy, while hemizygous males die embryonically. We have identified the PH gene as filamin 1 ( FLN1 ), which encodes an actin-cross-linking phosphoprotein that transduces ligand–receptor binding into actin reorganization, and which is required for locomotion of many cell types. FLN1 shows previously unrecognized, high-level expression in the developing cortex, is required for neuronal migration to the cortex, and is essential for embryogenesis.

The inv(16) chromosomal aberration is found in 10% of the cases with AML. The resultant CBFB-MYH11 fusion gene that plays a key-role in the leukemia development, but for full transformation, additional genetic hits are necessary. We report that the MYH11 gene overlaps with the NDE1 gene. At their 3 prime ends the genes overlap at the genomic, transcript as well as coding level and as a consequence, the inv(16) disrupts NDE1 in addition to CBFB and MYH11 in 90% of CBFB-MYH11 positive cases. To determine the relevance of Nde1 function in hematopoiesis leukemia development, hematopoiesis was studied in Nde1-/− and Nde1+/− mutant mice. A significant percentage of the Nde1 null mutant mice developed a myeloproliferative (MPD) phenotype at 6–12 months of age. This phenotype was observed in 6 out of 15 (40%) Nde1−/− mice, 2 out of 15 (13%) Nde1+/− mice and none of 4 wild type mice. All mice with the MPD phenotype exhibited a profound splenomegaly with spleen sizes ranging from 5 to 20-fold larger than normal wild type controls. Histopathological analysis demonstrated hypercellular bone marrow, effacement of normal splenic architecture with expansion of a myeloid cells, and infiltration of proliferating myeloid cells in various organs . A 10-fold increase in Gr1+/Mac1+ double positive mature myeloid cells in the spleen was confirmed by flow-cytometric analysis. Peripheral blood analyses showed that the white blood cell counts were slightly elevated in Nde1 mutant mice that had not yet developed a MPD phenotype compared to wild type mice. A somewhat higher white blood cell count was observed in Nde1 mutant mice with MPD phenotype. In the Nde1 mutant mice with or without splenomegaly we did not observe an increase in the number of B- and T-cells as measured by flowcytometer using various lymphoid cell surface markers. Based on these data we conclude that loss of Nde1 function and Nde1 haploinsufficiency results in the onset of a MPD phenotype. Earlier studies showed that Nde1 is a centrosomal protein. To test whether Nde1 is involved in spindle pole formation and chromosome segregation, we analyzed the mitotic behaviour of mouse embryo fibroblasts (MEFs). From passage 3 to passage 10, aneuploid metaphases increased much more rapidly in Nde1−/− MEFs than in the wild type controls. In addition, a broader spectrum of abnormal chromosome numbers was seen in Nde1−/− MEFs. Thus, Nde1 deficiency results in chromosomal instability. To study whether the numerical chromosome abnormalities were associated with aberrant spindle formation we stained Nde1−/− MEFs with a monoclonal antibody against tubulin. Multipolar spindles and misassembled spindles were more frequently observed in Nde1−/− MEFs compared to wild type cells. We conclude that the abnormal chromosomal segregation of Nde1−/− MEFs is associated with defective mitotic spindle structure. Since Nde1 is disrupted in the majority of inv(16) positive AML patients and the disruption in mice resulted in genetic instability and the onset of myeloproliferative disease, we suggest that the disruption of Nde1 in inv(16)+ AML may serve as additional genetic defect that contributes to leukemogenesis.

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.