Synaptic Activity Regulates Mitochondrial Iron Metabolism to Enhance Neuronal Bioenergetics.

Synaptic activity is the main energy-consuming process in the central nervous system. We are beginning to understand how energy is supplied and used during synaptic activity by neurons. However, the long-term metabolic adaptations associated with a previous episode of synaptic activity are not well understood. Herein, we show that an episode of synaptic activity increases mitochondrial bioenergetics beyond the duration of the synaptic activity by transcriptionally inducing the expression of iron metabolism genes with the consequent enhancement of cellular and mitochondrial iron uptake. Iron is a necessary component of the electron transport chain complexes, and its chelation or knockdown of mitochondrial iron transporter Mfrn1 blocks the activity-mediated bioenergetics boost. We found that Mfrn1 expression is regulated by the well-known regulator of synaptic plasticity CREB, suggesting the coordinated expression of synaptic plasticity programs with those required to meet the associated increase in energetic demands.

Resolving the heterogeneity of diaphragmatic mesenchyme: a novel mouse model of congenital diaphragmatic hernia.

Congenital diaphragmatic hernia (CDH) is a relatively common developmental defect with considerable mortality and morbidity. Formation of the diaphragm is a complex process that involves several cell types, each with different developmental origins. Owing to this complexity, the aetiology of CDH is not well understood. The pleuroperitoneal folds (PPFs) and the posthepatic mesenchymal plate (PHMP) are transient structures that are essential during diaphragm development. Using several mouse models, including lineage tracing, we demonstrate the heterogeneous nature of the cells that make up the PPFs. The conditional deletion of Wilms tumor 1 homolog (Wt1) in the non-muscle mesenchyme of the PPFs results in CDH. We show that the fusion of the PPFs and the PHMP to form a continuous band of tissue involves movements of cells from both sources. The PPFs of mutant mice fail to fuse with the PHMP and exhibit increased RALDH2 (also known as ALDH1A2) expression. However, no changes in the expression of genes (including Snai1, Snai2, Cdh1 and Vim) implicated in epithelial-to-mesenchymal transition are observed. Additionally, the mutant PPFs lack migrating myoblasts and muscle connective tissue fibroblasts (TCF4+/GATA4+), suggesting possible interactions between these cell types. Our study demonstrates the importance of the non-muscle mesenchyme in development of the diaphragm.

Visceral and subcutaneous fat have different origins and evidence supports a mesothelial source.

Fuelled by the obesity epidemic, there is considerable interest in the developmental origins of white adipose tissue (WAT) and the stem and progenitor cells from which it arises. Whereas increased visceral fat mass is associated with metabolic dysfunction, increased subcutaneous WAT is protective. There are six visceral fat depots: perirenal, gonadal, epicardial, retroperitoneal, omental and mesenteric, and it is a subject of much debate whether these have a common developmental origin and whether this differs from that for subcutaneous WAT. Here we show that all six visceral WAT depots receive a significant contribution from cells expressing Wt1 late in gestation. Conversely, no subcutaneous WAT or brown adipose tissue arises from Wt1-expressing cells. Postnatally, a subset of visceral WAT continues to arise from Wt1-expressing cells, consistent with the finding that Wt1 marks a proportion of cell populations enriched in WAT progenitors. We show that all visceral fat depots have a mesothelial layer like the visceral organs with which they are associated, and provide several lines of evidence that Wt1-expressing mesothelium can produce adipocytes. These results reveal a major ontogenetic difference between visceral and subcutaneous WAT, and pinpoint the lateral plate mesoderm as a major source of visceral WAT. They also support the notion that visceral WAT progenitors are heterogeneous, and suggest that mesothelium is a source of adipocytes.

Wt1 is required for cardiovascular progenitor cell formation through transcriptional control of Snail and E-cadherin.

The epicardial epithelial-mesenchymal transition (EMT) is hypothesized to generate cardiovascular progenitor cells that differentiate into various cell types, including coronary smooth muscle and endothelial cells, perivascular and cardiac interstitial fibroblasts and cardiomyocytes. Here we show that an epicardial-specific knockout of the gene encoding Wilms' tumor-1 (Wt1) leads to a reduction in mesenchymal progenitor cells and their derivatives. We show that Wt1 is essential for repression of the epithelial phenotype in epicardial cells and during embryonic stem cell differentiation through direct transcriptional regulation of the genes encoding Snail (Snai1) and E-cadherin (Cdh1), two of the major mediators of EMT. Some mesodermal lineages do not form in Wt1-null embryoid bodies, but this effect is rescued by the expression of Snai1, underscoring the importance of EMT in generating these differentiated cells. These new insights into the molecular mechanisms regulating cardiovascular progenitor cells and EMT will shed light on the pathogenesis of heart diseases and may help the development of cell-based therapies.

Putative endothelial progenitor cells are associated with flow-mediated dilation in refractory hypertensives.

BACKGROUND: Hypertension has been related to endothelial dysfunction. Patients with refractory hypertension (RH) have a reduced number of endothelial progenitor cells (EPCs). AIM: To evaluate if blood EPC levels relate to endothelium-dependent vasodilation (ED-VD) in RH. METHODS: We analyzed 29 RH confirmed by 24-h ambulatory blood pressure monitoring and assessed complete clinical and laboratory evaluation. EPCs were isolated from peripheral mononuclear cells (MNC) by flow cytometry. ED-VD was determined measuring flow-mediated dilation (FMD) by venous occlusion plethysmography. Results. Circulating EPCs/10(5) MNC (median [Q1-Q3]): 23.0 [4.5-53.8]. FMD (median [Q1-Q3]): 211.7 [79.5-365.8]%. Significant correlations with log-FMD: EPCs (r = 0.469; p = 0.018) and homocysteine (r = -0.414; p = 0.045). There was no collinearity between EPCs and homocysteine. FMD did not correlate with age, gender, office BP, 24-h systolic blood pressure or 24-h diastolic blood pressure, laboratory parameters, C-reactive-protein, left ventricular-mass index, dyslipidaemia, smoking habit and statin or angiotensin system blockers treatment. Multiple linear regression analysis showed that after age-adjustment, EPC (p = 0.027) and homocysteine (p = 0.004) were the only variables that predicted FMD (R = 0.740). After dividing patients according to EPC number, patients in the lower tertile showed a significantly reduced FMD compared with those in the group of the two upper tertiles of EPC: log-FMD (mean+/-SD): 4.7+/-0.9 vs 5.6+/-0.8, respectively (p = 0.031). CONCLUSIONS: ED-VD independently correlates with circulating EPCs in RH. Homocysteine is also an independent predictor of lower FMD in such patients.

The transcription factors Slug and Snail act as repressors of Claudin-1 expression in epithelial cells.

Claudin-1 is an integral membrane protein component of tight junctions. The Snail family of transcription factors are repressors that play a central role in the epithelial-mesenchymal transition, a process that occurs during cancer progression. Snail and Slug members are direct repressors of E-cadherin and act by binding to the specific E-boxes of its proximal promoter. In the present study, we demonstrate that overexpression of Slug or Snail causes a decrease in transepithelial electrical resistance. Overexpression of Slug and Snail in MDCK (Madin-Darby canine kidney) cells down-regulated Claudin-1 at protein and mRNA levels. In addition, Snail and Slug are able to effectively repress human Claudin-1-driven reporter gene constructs containing the wild-type promoter sequence, but not those with mutations in two proximal E-box elements. We also demonstrate by band-shift assay that Snail and Slug bind to the E-box motifs present in the human Claudin-1 promoter. Moreover, an inverse correlation in the levels of Claudin-1 and Slug transcripts were observed in breast cancer cell lines. E-box elements in the Claudin-1 promoter were found to play a critical negative regulatory role in breast cancer cell lines that expressed low levels of Claudin-1 transcript. Significantly, in invasive human breast tumours, high levels of Snail and Slug correlated with low levels of Claudin-1 expression. Taken together, these results support the hypothesis that Claudin-1 is a direct downstream target gene of Snail family factors in epithelial cells.

Opposite effects of tumor necrosis factor and soluble fibronectin on junctional adhesion molecule-A in endothelial cells.

Junctional adhesion molecule-A (JAM-A) regulates key inflammatory responses, such as edema formation and leukocyte transmigration. Although it has been reported that the inflammatory cytokine tumor necrosis factor (TNF) causes the disassembly of JAM-A from the intercellular junctions, the mechanism has not been elucidated fully. Here, we report that TNF enhances the solubility of JAM-A in Triton X-100 and increases the amount of Triton-soluble JAM-A dimers at the cell surface but does not change the total levels of cellular JAM-A. Thus we hypothesized that TNF causes the redistribution of JAM-A from the junctions to the cell surface and that junction disassembly is sufficient to account for JAM-A redistribution. Intriguingly, however, even after complete disassembly of the junctions (with EDTA and trypsin), higher levels of JAM-A are detectable at the cell surface (by FACS analysis) in cells that had been previously incubated in the presence of TNF than in its absence. Thus we propose that TNF causes not only the disassembly of JAM-A from the junctions and its subsequent redistribution to the cell surface but also its dispersal in such a way that JAM-A becomes more easily accessible to the antibodies used for FACS analysis. Finally, we evaluated whether soluble fibronectin might attenuate the effects of TNF on JAM-A, as some inflammatory conditions are associated with the depletion of plasma fibronectin. We found that fibronectin reduces the effect of TNF on the disassembly of JAM-A, but not on its dispersal, thus further stressing that disassembly and dispersal can be functionally dissociated.