Laminin-immunoreactive glia distinguish regenerative adult CNS systems from non-regenerative ones.

Most regions of the adult mammalian central nervous system (CNS) do not support axonal growth and regeneration. Laminin, expressed by cultured astrocytes and known to promote neurite outgrowth of cultured neurons, is normally present in brain basement membranes, and only transiently induced in adult brain astrocytes by injury. Here I provide three lines of evidence which suggest that the continued expression of laminin by astrocytes may be a prerequisite for axonal growth and regeneration in adult CNS. Firstly, laminin is continuously present in astrocytes of adult rat olfactory bulb apparently in close association with the olfactory nerve axons. Secondly, laminin is continuously expressed by astrocytes in adult frog brain, and sectioning of the optic tract further increases laminin immunoreactivity in astrocytes of the optic tectum during the period of axonal regeneration. Lastly, laminin appears normally in astrocytes of the frog and goldfish optic nerves which regenerate, but not in astrocytes of the rat or chick optic nerves which do not regenerate. The selective association of laminin with axons that undergo growth and regeneration in vivo is consistent with the possibility that astrocytic laminin provides these central nervous systems with their regenerative potential.     

Conditional deletion of Dicer in vascular smooth muscle cells leads to the developmental delay and embryonic mortality

Dicer is a RNAase III enzyme that cleaves double stranded RNA and generates small interfering RNA (siRNA) and microRNA (miRNA). The goal of this study is to examine the role of Dicer and miRNAs in vascular smooth muscle cells (VSMCs). We deleted Dicer in VSMCs of mice, which caused a developmental delay that manifested as early as embryonic day E12.5, leading to embryonic death between E14.5 and E15.5 due to extensive hemorrhage in the liver, brain, and skin. Dicer KO embryos showed dilated blood vessels and a disarray of vascular architecture between E14.5 and E15.5. VSMC proliferation was significantly inhibited in Dicer KOs. The expression of VSMC marker genes were significantly downregulated in Dicer cKO embryos. The vascular structure of the yolk sac and embryo in Dicer KOs was lost to an extent that no blood vessels could be identified after E15.5. Expression of most miRNAs examined was compromised in VSMCs of Dicer KO. Our results indicate that Dicer is required for vascular development and regulates vascular remodeling by modulating VSMC proliferation and differentiation.     

Sustained Down-regulation of β-Dystroglycan and Associated Dysfunctions of Astrocytic Endfeet in Epileptic Cerebral Cortex

Epilepsy is characterized by the abnormal activation of neurons in the cerebral cortex, but the molecular and cellular mechanisms contributing to the development of recurrent seizures are largely unknown. Recently, the critical involvement of astrocytes in the pathophysiology of epilepsy has been proposed. However, the nature of plastic modulations of astrocytic proteins in the epileptic cortex remains poorly understood. In this study, we utilized the zero magnesium in vitro model of epilepsy and examined the potential molecular changes of cortical astrocytes, focusing specifically on endfeet, where specialized biochemical compartments exist. We find that the continuous epileptic activation of neurons for 1 h decreases the expression level of β-dystroglycan (βDG) in acute cortical brain slices prepared from mice. This change is completely abolished by the pharmacological blockade of NMDA-type glutamate receptors as well as by matrix metalloproteinase inhibitors. Consistent with the highly specialized localization of βDG at astrocytic endfeet, where it plays a pivotal role in anchoring endfeet-enriched proteins in astrocytes, the down-regulation of βDG is accompanied by a decrease in the expression of AQP4 but not laminin. Importantly, this down-regulation of βDG persists for at least 1 h, even after the apparent recovery of neuronal activation. Finally, we show that the down-regulation of βDG is associated with the dysfunction of the endfeet at the blood-brain interface as a diffusion barrier. These results suggest that the sustained down-regulation of βDG leads to dysfunctions of astrocytic endfeet in the epileptic cerebral cortex and may contribute to the pathogenesis of epilepsy.     

DiGeorge syndrome critical region 8 (DGCR8) protein-mediated microRNA biogenesis is essential for vascular smooth muscle cell development in mice

DiGeorge Critical Region 8 (DGCR8) is a double-stranded RNA-binding protein that interacts with Drosha and facilitates microRNA (miRNA) maturation. However, the role of DGCR8 in vascular smooth muscle cells (VSMCs) is not well understood. To investigate whether DGCR8 contributes to miRNA maturation in VSMCs, we generated DGCR8 conditional knockout (cKO) mice by crossing VSMC-specific Cre mice (SM22-Cre) with DGCR8(loxp/loxp) mice. We found that loss of DGCR8 in VSMCs resulted in extensive liver hemorrhage and embryonic mortality between embryonic days (E) 12.5 and E13.5. DGCR8 cKO embryos displayed dilated blood vessels and disarrayed vascular architecture. Blood vessels were absent in the yolk sac of DGCR8 KOs after E12.5. Disruption of DGCR8 in VSMCs reduced VSMC proliferation and promoted apoptosis in vitro and in vivo. In DGCR8 cKO embryos and knockout VSMCs, differentiation marker genes, including αSMA, SM22, and CNN1, were significantly down-regulated, and the survival pathways of ERK1/2 mitogen-activated protein kinase and the phosphatidylinositol 3-kinase/AKT were attenuated. Knockout of DGCR8 in VSMCs has led to down-regulation of the miR-17/92 and miR-143/145 clusters. We further demonstrated that the miR-17/92 cluster promotes VSMC proliferation and enhances VSMC marker gene expression, which may contribute to the defects of DGCR8 cKO mutants. Our results indicate that the DGCR8 gene is required for vascular development through the regulation of VSMC proliferation, apoptosis, and differentiation.     

Astrocytes are poised for lactate trafficking and release from activated brain and for supply of glucose to neurons

Brain is a highly-oxidative organ, but during activation, glycolytic flux is preferentially up-regulated even though oxygen supply is adequate. The biochemical and cellular basis of metabolic changes during brain activation and the fate of lactate produced within brain are important, unresolved issues central to understanding brain function, brain images, and spectroscopic data. Because in vivo brain imaging studies reveal rapid efflux of labeled glucose metabolites during activation, lactate trafficking among astrocytes and between astrocytes and neurons was examined after devising specific, real-time, sensitive enzymatic fluorescent assays to measure lactate and glucose levels in single cells in adult rat brain slices. Astrocytes have a 2- to 4-fold faster and higher capacity for lactate uptake from extracellular fluid and for lactate dispersal via the astrocytic syncytium compared to neuronal lactate uptake from extracellular fluid or shuttling of lactate to neurons from neighboring astrocytes. Astrocytes can also supply glucose to neurons as well as glucose can be taken up by neurons from extracellular fluid. Astrocytic networks can provide neuronal fuel and quickly remove lactate from activated glycolytic domains, and the lactate can be dispersed widely throughout the syncytium to endfeet along the vasculature for release to blood or other brain regions via perivascular fluid flow.     

NDRG2 as a marker protein for brain astrocytes

The protein NDRG2 (N-myc downregulated gene 2) is expressed in astrocytes. We show here that NDRG2 is located in the cytosol of protoplasmic and fibrous astrocytes throughout the mammalian brain, including Bergmann glia as observed in mouse, rat, tree shrew, marmoset and human. NDRG2 immunoreactivity is detectable in the astrocytic cell bodies and excrescencies including fine distal processes. Glutamatergic and GABAergic nerve terminals are associated with NDRG2 immunopositive astrocytic processes. Müller glia in the retina displays no NDRG2 immunoreactivity. NDRG2 positive astrocytes are more abundant and more evenly distributed in the brain than GFAP (glial fibrillary acidic protein) immunoreactive cells. Some regions with very little GFAP such as the caudate nucleus show pronounced NDRG2 immunoreactivity. In white matter areas, NDRG2 is less strong than GFAP labeling. Most NDRG2 positive somata are immunoreactive for S100ß but not all S100ß cells express NDRG2. NDRG2 positive astrocytes do not express nestin and NG2 (chondroitin sulfate proteoglycan 4). The localization of NDRG2 overlaps only partially with that of aquaporin 4, the membrane-bound water channel that is concentrated in the astrocytic endfeet. Reactive astrocytes at a cortical lesion display very little NDRG2, which indicates that expression of the protein is reduced in reactive astrocytes. In conclusion, our data show that NDRG2 is a specific marker for a large population of mature, non-reactive brain astrocytes. Visualization of NDRG2 immunoreactive structures may serve as a reliable tool for quantitative studies on numbers of astrocytes in distinct brain regions and for high-resolution microscopy studies on distal astrocytic processes.     

Dynamic inositol trisphosphate-mediated calcium signals within astrocytic endfeet underlie vasodilation of cerebral arterioles

Active neurons communicate to intracerebral arterioles in part through an elevation of cytosolic Ca(2+) concentration ([Ca(2+)](i)) in astrocytes, leading to the generation of vasoactive signals involved in neurovascular coupling. In particular, [Ca(2+)](i) increases in astrocytic processes ("endfeet"), which encase cerebral arterioles, have been shown to result in vasodilation of arterioles in vivo. However, the spatial and temporal properties of endfoot [Ca(2+)](i) signals have not been characterized, and information regarding the mechanism by which these signals arise is lacking. [Ca(2+)](i) signaling in astrocytic endfeet was measured with high spatiotemporal resolution in cortical brain slices, using a fluorescent Ca(2+) indicator and confocal microscopy. Increases in endfoot [Ca(2+)](i) preceded vasodilation of arterioles within cortical slices, as detected by simultaneous measurement of endfoot [Ca(2+)](i) and vascular diameter. Neuronal activity-evoked elevation of endfoot [Ca(2+)](i) was reduced by inhibition of inositol 1,4,5-trisphosphate (InsP(3)) receptor Ca(2+) release channels and almost completely abolished by inhibition of endoplasmic reticulum Ca(2+) uptake. To probe the Ca(2+) release mechanisms present within endfeet, spatially restricted flash photolysis of caged InsP(3) was utilized to liberate InsP(3) directly within endfeet. This maneuver generated large amplitude [Ca(2+)](i) increases within endfeet that were spatially restricted to this region of the astrocyte. These InsP(3)-induced [Ca(2+)](i) increases were sensitive to depletion of the intracellular Ca(2+) store, but not to ryanodine, suggesting that Ca(2+)-induced Ca(2+) release from ryanodine receptors does not contribute to the generation of endfoot [Ca(2+)](i) signals. Neuronally evoked increases in astrocytic [Ca(2+)](i) propagated through perivascular astrocytic processes and endfeet as multiple, distinct [Ca(2+)](i) waves and exhibited a high degree of spatial heterogeneity. Regenerative Ca(2+) release processes within the endfeet were evident, as were localized regions of Ca(2+) release, and treatment of slices with the vasoactive neuropeptides somatostatin and vasoactive intestinal peptide was capable of inducing endfoot [Ca(2+)](i) increases, suggesting the potential for signaling between local interneurons and astrocytic endfeet in the cortex. Furthermore, photorelease of InsP(3) within individual endfeet resulted in a local vasodilation of adjacent arterioles, supporting the concept that astrocytic endfeet function as local "vasoregulatory units" by translating information from active neurons into complex InsP(3)-mediated Ca(2+) release signals that modulate arteriolar diameter.     

The beta-amyloid peptide of Alzheimer's disease decreases adhesion of vascular smooth muscle cells to the basement membrane

Cerebral amyloid angiopathy (CAA) is a major feature of Alzheimer's disease pathology. In CAA, degeneration of vascular smooth muscle cells (VSMCs) occurs close to regions of the basement membrane where the amyloid protein (Abeta) builds up. In this study, the possibility that Abeta disrupts adhesive interactions between VSMCs and the basement membrane was examined. VSMCs were cultured on a commercial basement membrane substrate (Matrigel). The presence of Abeta in the Matrigel decreased cell-substrate adhesion and cell viability. Full-length oligomeric Abeta was required for the effect, as N- and C-terminally truncated peptide analogues did not inhibit adhesion. Abeta that was fluorescently labelled at the N-terminus (fluo-Abeta) bound to Matrigel as well as to the basement membrane heparan sulfate proteoglycan (HSPG) perlecan and laminin. Adhesion of VSMCs to perlecan or laminin was decreased by Abeta. As perlecan influences VSMC viability through the extracellular signal-regulated kinase (ERK)1/2 signalling pathway, the effect of Abeta1-40 on ERK1/2 phosphorylation was examined. The level of phospho-ERK1/2 was decreased in cells following Abeta treatment. An inhibitor of ERK1/2 phosphorylation enhanced the effect of Abeta on cell adhesion. The studies suggest that Abeta can decrease VSMC viability by disrupting VSMC-extracellular matrix (ECM) adhesion.     

miRNA Biogenesis Enzyme Drosha Is Required for Vascular Smooth Muscle Cell Survival

miRNA biogenesis enzyme Drosha cleaves double-stranded primary miRNA by interacting with double-stranded RNA binding protein DGCR8 and processes primary miRNA into precursor miRNA to participate in the miRNA biogenesis pathway. The role of Drosha in vascular smooth muscle cells (VSMCs) has not been well addressed. We generated Drosha conditional knockout (cKO) mice by crossing VSMC-specific Cre mice, SM22-Cre, with Drosha ^loxp/loxp mice. Disruption of Drosha in VSMCs resulted in embryonic lethality at E14.5 with severe liver hemorrhage in mutant embryos. No obvious developmental delay was observed in Drosha cKO embryos. The vascular structure was absent in the yolk sac of Drosha homozygotes at E14.5. Loss of Drosha reduced VSMC proliferation in vitro and in vivo. The VSMC differentiation marker genes, including αSMA, SM22, and CNN1, and endothelial cell marker CD31 were significantly downregulated in Drosha cKO mice compared to controls. ERK1/2 mitogen-activated protein kinase and the phosphatidylinositol 3-kinase/AKT were attenuated in VSMCs in vitro and in vivo. Disruption of Drosha in VSMCs of mice leads to the dysregulation of miRNA expression. Using bioinformatics approach, the interactions between dysregulated miRNAs and their target genes were analyzed. Our data demonstrated that Drosha is required for VSMC survival by targeting multiple signaling pathways.     

Phenotypic and gene expression modification with normal brain aging in GFAP-positive astrocytes and neural stem cells

Astrocytes secrete growth factors that are both neuroprotective and supportive for the local environment. Identified by glial fibrillary acidic protein (GFAP) expression, astrocytes exhibit heterogeneity in morphology and in the expression of phenotypic markers and growth factors throughout different adult brain regions. In adult neurogenic niches, astrocytes secrete vascular endothelial growth factor (VEGF) and fibroblast growth factor-2 (FGF-2) within the neurogenic niche and are also a source of special GFAP-positive multipotent neural stem cells (NSCs). Normal aging is accompanied by a decline in CNS function and reduced neurogenesis. We asked whether a decreased availability of astrocyte-derived factors may contribute to the age-related decline in neurogenesis. Determining alterations of astrocytic activity in the aging brain is crucial for understanding CNS homeostasis in aging and for assessing appropriate therapeutic targets for an aging population. We found region-specific alterations in the gene expression of GFAP, VEGF, and FGF-2 and their receptors in the aged brain corresponding to changes in astrocytic reactivity, supporting astrocytic heterogeneity and demonstrating a differential aging effect. We found that GFAP-positive NSCs uniquely coexpress both VEGF and its key mitotic receptor Flk-1 in both young and aged hippocampus, indicating a possible autocrine/paracrine signaling mechanism. VEGF expression is lost once NSCs commit to a neuronal fate, but Flk-1-mediated sensitivity to VEGF signaling is maintained. We propose that age-related astrocytic changes result in reduced VEGF and FGF-2 signaling, which in turn limits NSC and progenitor cell maintenance and contributes to decreased neurogenesis.     

Vascular remodeling in hypertensive transgenic mice.

We physiologically and histopathologically analyzed vascular damage due to hypertension and vascular remodeling in hypertensive transgenic mice (Tsukuba hypertensive mice; THM). Pubertal (6-week-old) THM already had hypertension similar to blood pressure in adult THM due to an enhanced renin angiotensin system (RAS). They progressively developed remarkable vascular hypertrophy composed of dedifferentiation of vascular smooth muscle cells (VSMCs) and extracellular matrix accumulation in the thoracic aorta, and VSMC hyperplasia was predominant in the abdominal aorta. THM are therefore a useful animal model for studying vascular remodeling mediated by enhanced RAS.     

Perlecan maintains the integrity of cartilage and some basement membranes

Perlecan is a heparan sulfate proteoglycan that is expressed in all basement membranes (BMs), in cartilage, and several other mesenchymal tissues during development. Perlecan binds growth factors and interacts with various extracellular matrix proteins and cell adhesion molecules. Homozygous mice with a null mutation in the perlecan gene exhibit normal formation of BMs. However, BMs deteriorate in regions with increased mechanical stress such as the contracting myocardium and the expanding brain vesicles showing that perlecan is crucial for maintaining BM integrity. As a consequence, small clefts are formed in the cardiac muscle leading to blood leakage into the pericardial cavity and an arrest of heart function. The defects in the BM separating the brain from the adjacent mesenchyme caused invasion of brain tissue into the overlaying ectoderm leading to abnormal expansion of neuroepithelium, neuronal ectopias, and exencephaly. Finally, homozygotes developed a severe defect in cartilage, a tissue that lacks BMs. The chondrodysplasia is characterized by a reduction of the fibrillar collagen network, shortened collagen fibers, and elevated expression of cartilage extracellular matrix genes, suggesting that perlecan protects cartilage extracellular matrix from degradation.     

Carotid artery ligation induced intimal thickening and proliferation is unaffected by ageing

Following interventions to treat atherosclerosis, such as coronary artery bypass graft surgery, restenosis occurs in approximately 40% of patients. Identification of proteins regulating intimal thickening could represent targets to prevent restenosis. Our group previously demonstrated that in a murine model of vascular occlusion, Wnt4 protein expression and β-catenin signalling was upregulated which promoted vascular smooth muscle cell (VSMC) proliferation and intimal thickening. In this study, the effect of age on VSMC proliferation, intimal hyperplasia and Wnt4 expression was investigated. In vitro proliferation of VSMCs isolated from young (2 month) or old (18–20 month) C57BL6/J mice was assessed by immunocytochemistry for EdU incorporation. As previously reported, 400 ng/mL recombinant Wnt4 protein increased proliferation of VSMCs from young mice. However, this response was absent in VSMCs from old mice. As our group previously reported reduced intimal hyperplasia in Wnt4^+/? mice compared to wildtype controls, we hypothesised that impaired Wnt4 signalling with age may result in reduced neointimal formation. To investigate this, carotid artery ligation was performed in young and old mice and neointimal area was assessed 21 days later. Surprisingly, neointimal area and percentage lumen occlusion were not significantly affected by age. Furthermore, neointimal cell density and proliferation were also unchanged. These data suggest that although Wnt4-mediated proliferation was impaired with age in primary VSMCs, carotid artery ligation induced neointimal formation and proliferation were unchanged in old mice. These results imply that Wnt4-mediated proliferation is unaffected by age in vivo, suggesting that therapeutic Wnt4 inhibition could inhibit restenosis in patients of all ages.     

Yap1 protein regulates vascular smooth muscle cell phenotypic switch by interaction with myocardin

The Hippo-Yap (Yes-associated protein) signaling pathway has emerged as one of the critical pathways regulating cell proliferation, differentiation, and apoptosis in response to environmental and developmental cues. However, Yap1 roles in vascular smooth muscle cell (VSMC) biology have not been investigated. VSMCs undergo phenotypic switch, a process characterized by decreased gene expression of VSMC contractile markers and increased proliferation, migration, and matrix synthesis. The goals of the present studies were to investigate the relationship between Yap1 and VSMC phenotypic switch and to determine the molecular mechanisms by which Yap1 affects this essential process in VSMC biology. Results demonstrated that the expression of Yap1 was rapidly up-regulated by stimulation with PDGF-BB (a known inducer of phenotypic switch in VSMCs) and in the injured vessel wall. Knockdown of Yap1 impaired VSMC proliferation in vitro and enhanced the expression of VSMC contractile genes as well by increasing serum response factor binding to CArG-containing regions of VSMC-specific contractile genes within intact chromatin. Conversely, the interaction between serum response factor and its co-activator myocardin was reduced by overexpression of Yap1 in a dose-dependent manner. Taken together, these results indicate that down-regulation of Yap1 promotes VSMC contractile phenotype by both up-regulating myocardin expression and promoting the association of the serum response factor-myocardin complex with VSMC contractile gene promoters and suggest that the Yap1 signaling pathway is a central regulator of phenotypic switch of VSMCs.     

Deficiency of peroxisome proliferator-activated receptor α attenuates apoptosis and promotes migration of vascular smooth muscle cells

Peroxisome proliferator-activated receptor (PPAR) α is widely expressed in the vasculature and has pleiotropic and lipid-lowering independent effects, but its role in the growth and function of vascular smooth muscle cells (VSMCs) during vascular pathophysiology is still unclear. Herein, VSMC-specific PPARα-deficient mice (Ppara^ΔSMC) were generated by Cre-LoxP site-specific recombinase technology and VSMCs were isolated from mice aorta. PPARα deficiency attenuated VSMC apoptosis induced by angiotensin (Ang) II and hydrogen peroxide, and increased the migration of Ang II-challenged cells.     

Smooth muscle cell-driven vascular diseases and molecular mechanisms of VSMC plasticity

Vascular smooth muscle cells (VSMCs) are the major cell type in blood vessels. Unlike many other mature cell types in the adult body, VSMC do not terminally differentiate but retain a remarkable plasticity. Fully differentiated medial VSMCs of mature vessels maintain quiescence and express a range of genes and proteins important for contraction/dilation, which allows them to control systemic and local pressure through the regulation of vascular tone. In response to vascular injury or alterations in local environmental cues, differentiated/contractile VSMCs are capable of switching to a dedifferentiated phenotype characterized by increased proliferation, migration and extracellular matrix synthesis in concert with decreased expression of contractile markers. Imbalanced VSMC plasticity results in maladaptive phenotype alterations that ultimately lead to progression of a variety of VSMC-driven vascular diseases. The nature, extent and consequences of dysregulated VSMC phenotype alterations are diverse, reflecting the numerous environmental cues (e.g. biochemical factors, extracellular matrix components, physical) that prompt VSMC phenotype switching. In spite of decades of efforts to understand cues and processes that normally control VSMC differentiation and their disruption in VSMC-driven disease states, the crucial molecular mechanisms and signalling pathways that shape the VSMC phenotype programme have still not yet been precisely elucidated. In this article we introduce the physiological functions of vascular smooth muscle/VSMCs, outline VSMC-driven cardiovascular diseases and the concept of VSMC phenotype switching, and review molecular mechanisms that play crucial roles in the regulation of VSMC phenotypic plasticity.     

Coupling of neural activity to blood flow in olfactory glomeruli is mediated by astrocytic pathways

Functional neuroimaging uses activity-dependent changes in cerebral blood flow to map brain activity, but the contributions of presynaptic and postsynaptic activity are incompletely understood, as are the underlying cellular pathways. Using intravital multiphoton microscopy, we measured presynaptic activity, postsynaptic neuronal and astrocytic calcium responses, and erythrocyte velocity and flux in olfactory glomeruli during odor stimulation in mice. Odor-evoked functional hyperemia in glomerular capillaries was highly correlated with glutamate release, but did not require local postsynaptic activity. Odor stimulation induced calcium transients in astrocyte endfeet and an associated dilation of upstream arterioles. Calcium elevations in astrocytes and functional hyperemia depended on astrocytic metabotropic glutamate receptor 5 and cyclooxygenase activation. Astrocytic glutamate transporters also contributed to functional hyperemia through mechanisms independent of calcium rises and cyclooxygenase activation. These local pathways initiated by glutamate account for a large part of the coupling between synaptic activity and functional hyperemia in the olfactory bulb.     

Do neurons in the vertebrate CNS migrate on laminin?

In adult rat brain the extracellular matrix glycoprotein, laminin, is found only in basement membranes, but is transiently expressed by astrocytes after brain injury. Here, I show that laminin also appears in immature brain cells during CNS development, and that its presence coincides with phases of neuronal migration. In early embryos, laminin is seen throughout the whole thickness of the forming brain, and is apparently synthesized by the cells, as judged by its intracytoplasmic localization. As development proceeds, intracellular laminin becomes restricted to the periventricular regions while punctate deposits of laminin follow the course of vimentin-positive radial glial fibers. In most brain regions, the adult pattern of laminin expression is achieved by birth. In the post-natal rat cerebellum, however, laminin is detected in external granule cells, in Purkinje cells, and in punctate deposits along the radial Bergmann glial fibers. By day 24 after birth, when the migration of external granule cells is complete, all laminin immunoreactivity disappears from these structures. The transient expression of laminin in regions where neurons are migrating raises the possibility that laminin plays a role in neuronal migration during CNS development.