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Absence of the astrocyte-specific membrane protein MLC1 is responsible for megalencephalic leukoencephalopathy with subcortical cysts (MLC), a rare type of leukodystrophy characterized by early-onset macrocephaly and progressive white matter vacuolation that lead to ataxia, spasticity, and cognitive decline. During postnatal development (from P5 to P15 in the mouse), MLC1 forms a membrane complex with GlialCAM (another astrocytic transmembrane protein) at the junctions between perivascular astrocytic processes. Perivascular astrocytic processes along with blood vessels form the gliovascular unit. It was not previously known how MLC1 influences the physiology of the gliovascular unit. Here, using the Mlc1 knock-out mouse model of MLC, we demonstrated that MLC1 controls the postnatal development and organization of perivascular astrocytic processes, vascular smooth muscle cell contractility, neurovascular coupling, and intraparenchymal interstitial fluid clearance. Our data suggest that MLC is a developmental disorder of the gliovascular unit, and perivascular astrocytic processes and vascular smooth muscle cell maturation defects are primary events in the pathogenesis of MLC and therapeutic targets for this disease.

Intramyocellular lipid accumulation is strongly related to insulin resistance in humans, and we have shown that high glucose concentration induced de novo lipogenesis and insulin resistance in murin muscle cells. Alterations in Wnt signaling impact the balance between myogenic and adipogenic programs in myoblasts, partly due to the decrease of Wnt10b protein. As recent studies point towards a role for Wnt signaling in the pathogenesis of type 2 diabetes, we hypothesized that activation of Wnt signaling could play a crucial role in muscle insulin sensitivity. Here we demonstrate that SREBP-1c and Wnt10b display inverse expression patterns during muscle ontogenesis and regeneration, as well as during satellite cells differentiation. The Wnt/beta-catenin pathway was reactivated in contracting myotubes using siRNA mediated SREBP-1 knockdown, Wnt10b over-expression or inhibition of GSK-3beta, whereas Wnt signaling was inhibited in myoblasts through silencing of Wnt10b. SREBP-1 knockdown was sufficient to induce Wnt10b protein expression in contracting myotubes and to activate the Wnt/beta-catenin pathway. Conversely, silencing Wnt10b in myoblasts induced SREBP-1c protein expression, suggesting a reciprocal regulation. Stimulation of the Wnt/beta-catenin pathway i) drastically decreased SREBP-1c protein and intramyocellular lipid deposition in myotubes; ii) increased basal glucose transport in both insulin-sensitive and insulin-resistant myotubes through a differential activation of Akt and AMPK pathways; iii) restored insulin sensitivity in insulin-resistant myotubes. We conclude that activation of Wnt/beta-catenin signaling in skeletal muscle cells improved insulin sensitivity by i) decreasing intramyocellular lipid deposition through downregulation of SREBP-1c; ii) increasing insulin effects through a differential activation of the Akt/PKB and AMPK pathways; iii) inhibiting the MAPK pathway. A crosstalk between these pathways and Wnt/beta-catenin signaling in skeletal muscle opens the exciting possibility that organ-selective modulation of Wnt signaling might become an attractive therapeutic target in regenerative medicine and to treat obese and diabetic populations.

Aim Cerebral capillary endothelial cells (EC) form the blood–brain barrier (BBB), which regulates molecular exchange between the blood and the brain. Understanding their function during brain development is essential for optimizing treatments in neonates, children, as well as pregnant and breastfeeding women. Methods P‐glycoprotein (P‐gp/ABCB1) expression during brain development was assessed by immunohistochemistry in human cortical samples. In mice, postnatal brain microvessels were analyzed using qPCR and Western Blot, and BBB function was evaluated in vivo using [14C]sucrose to assess barrier integrity, and [3H]verapamil or [3H]rosuvastatin to assess transport activity. Results In humans, P‐gp reached mature levels in the early postnatal period. In mice, BBB integrity was established by postnatal day 5 (P5), but the expression of claudin‐5, P‐gp, and Oatp1a4 increased until P30. Brain transport of verapamil and rosuvastatin significantly decreased between P15 and P30, indicating enhanced efflux capacity. Conclusions Although BBB integrity is established at birth, BBB continues maturing throughout the postnatal period, with a predominant efflux transport. Our findings underscore the critical role of P‐gp in the acquisition of BBB gatekeeper properties. The immature BBB may result in a higher brain susceptibility to P‐gp substrates in preterm infants. P‐gp expression in the developping human cortex increases with age. In the mouse, gatekeepers of BBB integrity and transport are upregulated: tighter blood–brain barrier, increased claudin 5/Slco1a4/P‐gp expression as well as enhanced P‐gp‐mediated BBB efflux.

Thanks to the analysis of an Interspecific Recombinant Congenic Strain (IRCS), we previously defined the Mafq1 quantitative trait locus as an interval on mouse Chromosome 1 associated with male hypofertility and ultrastructural abnormalities. We identified the Spermatogenesis associated protein 3 gene (Spata3 or Tsarg1) as a pertinent candidate within the Mafq1 locus and performed the CRISPR-Cas9 mediated complete deletion of the gene to investigate its function. Male mice deleted for Spata3 were normally fertile in vivo but exhibited a drastic reduction of efficiency in in vitro fertilization assays. Mobility parameters were normal but ultrastructural analyses revealed acrosome defects and an overabundance of lipids droplets in cytoplasmic remnants. The deletion of the Spata3 gene reproduces therefore partially the phenotype of the hypofertile IRCS strain.

STOX1 has been involved in genetic forms of preeclampsia. The gene encodes two major isoforms coined STOX1A and STOX1B (989 and 227 amino-acids, respectively), sharing the same DNA binding domain. The two isoforms have opposite function on major genes involved in trophoblast syncytialization and oxidative stress management. Placenta-fetal overexpression of STOX1A induces preeclampsia in mice. Here we explore the effects of STOX1B placenta-fetal overexpression. A STOX1B transgenic mouse line (expression restricted to the foeto-placental unit through ad hoc crosses) was analyzed in terms of blood pressure, proteinuria, soluble antiangiogenic factors, placental and fetal weights, maternal heart weight, and placental histology. Placental gene expression was explored by RNA-sequencing, followed by a bioinformatics analysis. Female mice carrying STOX1B placentas displayed preeclampsia features with a striking increase of genes involved in coagulation, presumably under the control of the HNF4α transcription factor. Genes specific of the spongiotrophoblast were strongly down-regulated consistently with a junctional zone reduction. This new model of preeclampsia seems connected with an enhancement of coagulation processes, similar to preeclamptic patients living at high altitude. Our model could be useful to study the features of preeclampsia in this context, and be a convenient complement to other animal models of preeclampsia.

Adrenocortical carcinomas (ACC) are aggressive and resistant to medical treatment. This study reports a single-nucleus transcriptome atlas of steroid and microenvironment cells in 38 human normal adrenals and adrenocortical tumors. We identify intermediate-state cells between glomerulosa and fasciculata, a transition state in the centripetal trans-differentiation of normal steroid cells. In tumors, steroid cells show expression programs reflecting this zonation. Although ACC microenvironment is scarce, its signatures combine with those of steroid cells into ecotypes. A first ecotype combines cancer-associated fibroblasts, tumor-associated endothelial cells, with hypoxia and mitosis signatures in steroid cells. Another ecotype combines exhausted T cells, with fasciculata steroid signature. These ecotypes are associated with poor survival. Conversely, a third ecotype combines inflammatory macrophages, with reticularis steroid signature, and better outcome. These steroid/microenvironment cells interplays improve outcome predictions and may open therapeutic options in aggressive ACC, through immune microenvironment activation by modulating glucocorticoids/androgens balance. Adrenocortical carcinomas (ACC) are aggressive and often resistant to therapy. Here, the authors provide a single-nucleus transcriptomic atlas of ACCs and normal adrenal glands, finding ecotypes in steroid and microenvironment cells that are associated with clinical outcomes.

Although great efforts to characterize the embryonic phase of brain microvascular system development have been made, its postnatal maturation has barely been described. Here, we compared the molecular and functional properties of brain vascular cells on postnatal day (P)5 vs. P15, via a transcriptomic analysis of purified mouse cortical microvessels (MVs) and the identification of vascular-cell-type-specific or -preferentially expressed transcripts. We found that endothelial cells (EC), vascular smooth muscle cells (VSMC) and fibroblasts (FB) follow specific molecular maturation programs over this time period. Focusing on VSMCs, we showed that the arteriolar VSMC network expands and becomes contractile resulting in a greater cerebral blood flow (CBF), with heterogenous developmental trajectories within cortical regions. Samples of the human brain cortex showed the same postnatal maturation process. Thus, the postnatal phase is a critical period during which arteriolar VSMC contractility required for vessel tone and brain perfusion is acquired and mature.

Intramyocellular lipid accumulation is strongly related to insulin resistance in humans, and we have shown that high glucose concentration induced de novo lipogenesis and insulin resistance in murin muscle cells. Alterations in Wnt signaling impact the balance between myogenic and adipogenic programs in myoblasts, partly due to the decrease of Wnt10b protein. As recent studies point towards a role for Wnt signaling in the pathogenesis of type 2 diabetes, we hypothesized that activation of Wnt signaling could play a crucial role in muscle insulin sensitivity. Here we demonstrate that SREBP-1c and Wnt10b display inverse expression patterns during muscle ontogenesis and regeneration, as well as during satellite cells differentiation. The Wnt/[beta]-catenin pathway was reactivated in contracting myotubes using siRNA mediated SREBP-1 knockdown, Wnt10b over-expression or inhibition of GSK-3[beta], whereas Wnt signaling was inhibited in myoblasts through silencing of Wnt10b. SREBP-1 knockdown was sufficient to induce Wnt10b protein expression in contracting myotubes and to activate the Wnt/[beta]-catenin pathway. Conversely, silencing Wnt10b in myoblasts induced SREBP-1c protein expression, suggesting a reciprocal regulation. Stimulation of the Wnt/[beta]-catenin pathway i) drastically decreased SREBP-1c protein and intramyocellular lipid deposition in myotubes; ii) increased basal glucose transport in both insulin-sensitive and insulin-resistant myotubes through a differential activation of Akt and AMPK pathways; iii) restored insulin sensitivity in insulin-resistant myotubes. We conclude that activation of Wnt/[beta]-catenin signaling in skeletal muscle cells improved insulin sensitivity by i) decreasing intramyocellular lipid deposition through downregulation of SREBP-1c; ii) increasing insulin effects through a differential activation of the Akt/PKB and AMPK pathways; iii) inhibiting the MAPK pathway. A crosstalk between these pathways and Wnt/[beta]-catenin signaling in skeletal muscle opens the exciting possibility that organ-selective modulation of Wnt signaling might become an attractive therapeutic target in regenerative medicine and to treat obese and diabetic populations.

The brain dense vascular network is essential for distributing oxygen and nutrients to neural cells. The network develops during embryogenesis and leads to the formation of the endothelial blood-brain barrier (BBB). This barrier is surrounded by mural cells (pericytes and vascular smooth muscle cells (VSMCs)) and fibroblasts. Here, we compared the molecular and functional properties of brain vascular cells on postnatal day (P)5 vs. P15, via a transcriptomic analysis of purified mouse cortical microvessels (MVs) and the identification of vascular-cell-type-specific or -preferentially expressed transcripts. We found that endothelial cells (ECs), VSMCs and fibroblasts follow specific molecular maturation programs over this time period. In particular, ECs acquire P-glycoprotein (P-gP)-mediated efflux capacities. The arterial VSMC network expands, acquires contractile proteins (such as smooth muscle actin (SMA) and myosin heavy chain 11 (Myh11)) and becomes contractile. We also analyzed samples of human brain cortex from the early prenatal stage through to adulthood: the expression of endothelial P-gP increased at birth and Myh11 in VSMCs acts as a developmental switch (as in the mouse) at birth and up to the age of 2 of 5 years. Thus, in both mice and humans, the early postnatal phase is a critical period during which the essential properties of cerebral blood vessels (i.e. the endothelial efflux of xenobiotics and other molecules, and the VSMC contractility required for vessel tone and brain perfusion) are acquired and mature.