AAV‐mediated gene delivery in Dp71‐null mouse model with compromised barriers

Formation and maintenance of the blood–retinal barrier (BRB) is required for proper vision and breaching of this barrier contributes to the pathology in a wide variety of retinal conditions such as retinal detachment and diabetic retinopathy. Dystrophin Dp71 being a key membrane cytoskeletal protein, expressed mainly in Müller cells, its absence has been related to BRB permeability through delocalization and down‐regulation of the AQP4 and Kir4.1 channels. Dp71‐null mouse is thus an excellent model to approach the study of retinal pathologies showing blood–retinal barrier permeability. We aimed to investigate the participation of Müller cells in the BRB and in the inner limiting membrane of Dp71‐null mice compared with wild‐type mice in order to understand how these barriers work in this model of permeable BRB. To this aim, we used an Adeno‐associated virus (AAV) variant, ShH10‐GFP, engineered to target Müller cells specifically. ShH10 coding GFP was introduced by intravitreal injection and Müller cell transduction was studied in Dp71‐null mice in comparison to wild‐type animals. We show that Müller cell transduction follows a significantly different pattern in Dp71‐null mice indicating changes in viral cell‐surface receptors as well as differences in the permeability of the inner limiting membrane in this mouse line. However, the compromised BRB of the Dp71‐null mice does not lead to virus leakage into the bloodstream when the virus is injected intravitreally – an important consideration for AAV‐mediated retinal gene therapy. GLIA 2014;62:468–476     

Maintaining retinal astrocytes normalizes revascularization and prevents vascular pathology associated with oxygen‐induced retinopathy

Astrocytes are well known modulators of normal developmental retinal vascularization. However, relatively little is known about the role of glial cells during pathological retinal neovascularization (NV), a leading contributor to vision loss in industrialized nations. We demonstrate that the loss of astrocytes and microglia directly correlates with the development of pathological NV in a mouse model of oxygen‐induced retinopathy (OIR). These two distinct glial cell populations were found to have cooperative survival effects in vitro and in vivo. The intravitreal injection of myeloid progenitor cells, astrocytes, or astrocyte‐conditioned media rescued endogenous astrocytes from degeneration that normally occurs within the hypoxic, vaso‐obliterated retina following return to normoxia. Protection of the retinal astrocytes and microglia was directly correlated with accelerated revascularization of the normal retinal plexuses and reduction of pathological intravitreal NV normally associated with OIR. Using astrocyte‐conditioned media, several factors were identified that may contribute to the observed astrocytic protection and subsequent normalization of the retinal vasculature, including vascular endothelial growth factor (VEGF) and basic fibroblast growth factor (bFGF). Injection of VEGF or bFGF at specific doses rescued the retinas from developing OIR‐associated pathology, an effect that was also preceded by protection of endogenous glia from hypoxia‐induced degeneration. Together, these data suggest that vascular‐associated glia are also required for normalized revascularization of the hypoxic retina. Methods developed to target and protect glial cells may provide a novel strategy by which normalized revascularization can be promoted and the consequences of abnormal NV in retinal vascular diseases can be prevented. © 2009 Wiley‐Liss, Inc.     

A deficit of brain dystrophin 71 impairs hypothalamic osmostat

Patients with Duchenne muscular dystrophy (DMD) and mdx mice, devoid of dystrophin proteins, show altered ionic homeostasis. To clarify dystrophin's involvement in the central control of osmotic stimuli, we investigated the effect of the disruption of Dp71, the major form of dystrophin in the brain, on the hypothalamoneurohypophysis system (HNHS) osmoregulatory response. Dp71 and Dp140 are the principal DMD gene products in the supraoptic nucleus (SON) and neurohypophysis (NH). They are present in astrocyte and pituicyte end‐feet, suggesting involvement in both intrinsic osmosensitivity of the SON and vasopressin (AVP) release from the NH. In Dp71‐null mice, the cellular distribution of Dp140 was modified, this protein being detected on the membrane of magnocellular soma. The plasma osmolality of Dp71‐null mice was lower than that of wild‐type mice under normal conditions, and this difference was maintained after salt loading, indicating a change in the set point for osmoregulation in the absence of Dp71. The increase in AVP levels detected in the SON and NH of the wild‐type was not observed in Dp71‐null mice following salt loading, and the increase in AVP mRNA levels in the SON was smaller in Dp71‐null than in wild‐type mice. This suggests that Dp71 may be involved in the functional activity of the HNHS. Its astrocyte end‐feet localization emphasizes the importance of neuronal–vascular–glial interactions for the central detection of osmolality. In the SON, Dp71 may be involved in osmosensitivity and definition of the “osmostat,” whereas, in the neurohypopohysis, it may be involved in fine‐tuning AVP release. © 2009 Wiley‐Liss, Inc.     

Generation of GFAP::GFP astrocyte reporter lines from human adult fibroblast‐derived iPS cells using zinc‐finger nuclease technology

Astrocytes are instrumental to major brain functions, including metabolic support, extracellular ion regulation, the shaping of excitatory signaling events and maintenance of synaptic glutamate homeostasis. Astrocyte dysfunction contributes to numerous developmental, psychiatric and neurodegenerative disorders. The generation of adult human fibroblast‐derived induced pluripotent stem cells (iPSCs) has provided novel opportunities to study mechanisms of astrocyte dysfunction in human‐derived cells. To overcome the difficulties of cell type heterogeneity during the differentiation process from iPSCs to astroglial cells (iPS astrocytes), we generated homogenous populations of iPS astrocytes using zinc‐finger nuclease (ZFN) technology. Enhanced green fluorescent protein (eGFP) driven by the astrocyte‐specific glial fibrillary acidic protein (GFAP) promoter was inserted into the safe harbor adeno‐associated virus integration site 1 (AAVS1) locus in disease and control‐derived iPSCs. Astrocyte populations were enriched using Fluorescence Activated Cell Sorting (FACS) and after enrichment more than 99% of iPS astrocytes expressed mature astrocyte markers including GFAP, S100β, NFIA and ALDH1L1. In addition, mature pure GFP‐iPS astrocytes exhibited a well‐described functional astrocytic activity in vitro characterized by neuron‐dependent regulation of glutamate transporters to regulate extracellular glutamate concentrations. Engraftment of GFP‐iPS astrocytes into rat spinal cord grey matter confirmed in vivo cell survival and continued astrocytic maturation. In conclusion, the generation of GFAP::GFP‐iPS astrocytes provides a powerful in vitro and in vivo tool for studying astrocyte biology and astrocyte‐driven disease pathogenesis and therapy. GLIA 2016;64:63–75     

Aquaporin 4‐Dependent expression of glial fibrillary acidic protein and tenascin‐C in activated astrocytes in stab wound mouse brain and in primary culture

We previously reported that aquaporin 4 (AQP4) has a neuroimmunological function via astrocytes and microglial cells involving osteopontin. AQP4 is a water channel localized in the endofoot of astrocytes in the brain, and its expression is upregulated after a stab wound to the mouse brain or the injection of methylmercury in common marmosets. In this study, the correlation between the expression of AQP4 and the expression of glial fibrillary acidic protein (GFAP) or tenascin‐C (TN‐C) in reactive astrocytes was examined in primary cultures and brain tissues of AQP4‐deficient mice (AQP4/KO). In the absence of a stab wound to the brain or of any stimulation of the cells, the expressions of both GFAP and TN‐C were lower in astrocytes from AQP4/KO mice than in those from wild‐type (WT) mice. High levels of GFAP and TN‐C expression were observed in activated astrocytes after a stab wound to the brain in WT mice; however, the expressions of GFAP and TN‐C were insignificant in AQP4/KO mice. Furthermore, lipopolysaccharide (LPS) stimulation activated primary culture of astrocytes and upregulated GFAP and TN‐C expression in cells from WT mice, whereas the expressions of GFAP and TN‐C were slightly upregulated in cells from AQP4/KO mice. Moreover, the stimulation of primary culture of astrocytes with LPS also upregulated inflammatory cytokines in cells from WT mice, whereas modest increases were observed in cells from AQP4/KO mice. These results suggest that AQP4 expression accelerates GFAP and TN‐C expression in activated astrocytes induced by a stab wound in the mouse brain and LPS‐stimulated primary culture of astrocytes. © 2014 Wiley Periodicals, Inc.     

Cell autonomous and noncell‐autonomous role of NF‐κB p50 in astrocyte‐mediated fate specification of adult neural progenitor cells

In previous work, we demonstrated that NF‐κB p50 acts as crucial regulator of adult hippocampal neural progenitor cells (ahNPC). Indeed, NF‐κB p50 knockout (KO) mice are characterized by remarkably reduced hippocampal neurogenesis. As a follow up to that work, herein we show that when cultured in vitro, ahNPC from wild type (WT) and p50KO mice are not significantly different in their neurogenic potential. This observation prompted us to investigate cell‐autonomous and noncell‐autonomous consequences of p50 absence on neuronal fate specification of ahNPC. In particular, we focused our attention on astrocytes, known to provide soluble proneurogenic signals, and investigated the influence of WT and p50KO astrocyte conditioned media (ACM) on WT and p50KO ahNPC differentiation. Interestingly, while WT ACM promoted both neuronal and astroglial differentiations, p50KO ACM only supported astroglial differentiation of WT ahNPC. By using a LC–MS/MS approach, we identified some proteins, which are significantly upregulated in p50KO compared with WT astrocytes. Among them, lipocalin‐2 (LCN‐2) was recognized as a novel astroglial‐derived signal regulating neuronal fate specification of ahNPC. Interestingly, LCN‐2 proneurogenic effect was greatly reduced in p50KO NPC, where LCN‐2 receptor gene expression appeared downregulated. In addition to that, we demonstrated p50KO NPC unresponsiveness to both neuronal and astroglial fate specification signals from WT and p50KO ACM, and we identified a reduced expression of α2δ1, a thrombospondin‐1 receptor, as another phenotypic change occurring in ahNPC in the absence of p50. Altogether, our data suggest that dysregulated NPC‐astrocyte communication may contribute to a reduced hippocampal neurogenesis in p50KO mice in vivo. GLIA 2016 GLIA 2017;65:169–181     

Localization of the cannabinoid type‐1 receptor in subcellular astrocyte compartments of mutant mouse hippocampus

Astroglial type‐1 cannabinoid (CB₁) receptors are involved in synaptic transmission, plasticity and behavior by interfering with the so‐called tripartite synapse formed by pre‐ and post‐synaptic neuronal elements and surrounding astrocyte processes. However, little is known concerning the subcellular distribution of astroglial CB₁ receptors. In particular, brain CB₁ receptors are mostly localized at cells' plasmalemma, but recent evidence indicates their functional presence in mitochondrial membranes. Whether CB₁ receptors are present in astroglial mitochondria has remained unknown. To investigate this issue, we included conditional knock‐out mice lacking astroglial CB₁ receptor expression specifically in glial fibrillary acidic protein (GFAP)‐containing astrocytes (GFAP‐CB₁‐KO mice) and also generated genetic rescue mice to re‐express CB₁ receptors exclusively in astrocytes (GFAP‐CB₁‐RS). To better identify astroglial structures by immunoelectron microscopy, global CB₁ knock‐out (CB₁‐KO) mice and wild‐type (CB₁‐WT) littermates were intra‐hippocampally injected with an adeno‐associated virus expressing humanized renilla green fluorescent protein (hrGFP) under the control of human GFAP promoter to generate GFAPhrGFP‐CB₁‐KO and ‐WT mice, respectively. Furthermore, double immunogold (for CB₁) and immunoperoxidase (for GFAP or hrGFP) revealed that CB₁ receptors are present in astroglial mitochondria from different hippocampal regions of CB₁‐WT, GFAP‐CB₁‐RS and GFAPhrGFP‐CB₁‐WT mice. Only non‐specific gold particles were detected in mouse hippocampi lacking CB₁ receptors. Altogether, we demonstrated the existence of a precise molecular architecture of the CB₁ receptor in astrocytes that will have to be taken into account in evaluating the functional activity of cannabinergic signaling at the tripartite synapse.     

Regression of retinal capillaries following N‐methyl‐D‐aspartate‐induced neurotoxicity in the neonatal rat retina

Degeneration of retinal capillaries occurs following N‐methyl‐D ‐aspartate (NMDA)‐induced retinal neurotoxicity, and the degree of capillary degeneration decreases in an age‐dependent manner. To determine the role of vascular endothelial growth factor (VEGF) in the high susceptibility of capillaries to neuronal damage during the early postnatal stage, this study compares the vascular regression patterns between NMDA‐treated retinas and retinas treated with N‐[2‐chloro‐4‐{(6,7‐dimethoxy‐4‐quinazolinyl)oxy}phenyl]‐N′‐propylurea (KRN633), a VEGF receptor tyrosine kinase inhibitor, in neonatal rats. Two days after a single intravitreal injection of NMDA (200 nmol/eye) on postnatal day (P) 7, substantial retinal neuron loss and delayed expansion of the retinal vascular bed were observed. The reduction in the capillary density in the central retina reached statistical significance 4 days after NMDA treatment. In retinas of rats injected subcutaneously with KRN633 (10 mg/kg) on P7 and P8, simplified vasculature attributable to capillary regression and prevention of endothelial cell growth were seen on P9, whereas no visible changes in the morphology of the retinal layers were observed. The degree of capillary degeneration in NMDA‐treated retinas was less than that in KRN633‐treated retinas. No apparent changes in immunoreactivities for VEGF were found 2 days after NMDA treatment. These results indicate that neuronal cell loss in the retina precedes retinal capillary degeneration following NMDA treatment, and VEGF‐dependent immature capillaries might be more susceptible to NMDA‐induced neuronal damage. © 2014 Wiley Periodicals, Inc.     

Astrocyte‐targeted production of interleukin‐6 reduces astroglial and microglial activation in the cuprizone demyelination model: Implications for myelin clearance and oligodendrocyte maturation

Multiple sclerosis (MS) is a chronic demyelinating disease of the central nervous system. Interleukin (IL)−6 is a pleiotropic cytokine with a potential role in MS. Here we used transgenic mice with astrocyte‐targeted production of IL‐6 (GFAP‐IL6Tg) to study the effect of IL‐6 in the cuprizone‐induced demyelination paradigm, which is an experimental model of de‐ and re‐myelination, both hallmarks of MS. Our results demonstrated that cuprizone‐treated GFAP‐IL6Tg mice showed a significant reduction in astroglial and especially microglial activation/accumulation in the corpus callosum in comparison with the corresponding cuprizone‐treated wild type (WT). Production of a key microglial attracting chemokine CXCL10, as well as CXCL1 and CCL4 was lower in cuprizone‐treated GFAP‐IL6Tg mice compared with cuprizone‐treated WT. Reduced microglial cell accumulation was associated with inefficient removal of degraded myelin and axonal protection in cuprizone‐treated GFAP‐IL6Tg mice, compared with WT mice at the peak of demyelination. In addition, transgenic production of IL‐6 did not alter initial oligodendrocyte (OL) apoptosis and oligodendrocyte precursor cell recruitment to the lesion site, but it impaired early OL differentiation, possibly due to impaired removal of degraded myelin. Indeed, a microglial receptor involved in myelin phagocytosis, TREM2, as well as the phagolysosomal protein CD68 were lower in cuprizone‐treated GFAP‐IL6Tg compared with WT mice. Our results show for the first time that astrocyte‐targeted production of IL‐6 may play a role in modulating experimental demyelination induced by cuprizone. Further understanding of the IL‐6‐mediated molecular mechanisms involved in the regulation of demyelination is needed, and may have implications for the development of future therapeutic strategies for the treatment of MS. GLIA 2016;64:2104–2119     

MicroRNA‐365 modulates astrocyte conversion into neuron in adult rat brain after stroke by targeting Pax6

Reactive astrocytes induced by ischemia can transdifferentiate into mature neurons. This neurogenic potential of astrocytes may have therapeutic value for brain injury. Epigenetic modifications are widely known to involve in developmental and adult neurogenesis. PAX6, a neurogenic fate determinant, contributes to the astrocyte‐to‐neuron conversion. However, it is unclear whether microRNAs (miRs) modulate PAX6‐mediated astrocyte‐to‐neuron conversion. In the present study we used bioinformatic approaches to predict miRs potentially targeting Pax6, and transient middle cerebral artery occlusion (MCAO) to model cerebral ischemic injury in adult rats. These rats were given striatal injection of glial fibrillary acidic protein targeted enhanced green fluorescence protein lentiviral vectors (Lv‐GFAP‐EGFP) to permit cell fate mapping for tracing astrocytes‐derived neurons. We verified that miR‐365 directly targets to the 3′‐UTR of Pax6 by luciferase assay. We found that miR‐365 expression was significantly increased in the ischemic brain. Intraventricular injection of miR‐365 antagomir effectively increased astrocytic PAX6 expression and the number of new mature neurons derived from astrocytes in the ischemic striatum, and reduced neurological deficits as well as cerebral infarct volume. Conversely, miR‐365 agomir reduced PAX6 expression and neurogenesis, and worsened brain injury. Moreover, exogenous overexpression of PAX6 enhanced the astrocyte‐to‐neuron conversion and abolished the effects of miR‐365. Our results demonstrate that increase of miR‐365 in the ischemic brain inhibits astrocyte‐to‐neuron conversion by targeting Pax6, whereas knockdown of miR‐365 enhances PAX6‐mediated neurogenesis from astrocytes and attenuates neuronal injury in the brain after ischemic stroke. Our findings provide a foundation for developing novel therapeutic strategies for brain injury.     

Intermediate filaments attenuate stimulation‐dependent mobility of endosomes/lysosomes in astrocytes

Intermediate filament (IF) proteins upregulation is a hallmark of astrocyte activation and reactive gliosis, but its pathophysiological implications remain incompletely understood. A recently reported association between IFs and directional mobility of peptidergic vesicles allows us to hypothesize that IFs affect vesicle dynamics and exocytosis‐mediated astrocyte communication with neighboring cells. Here, we ask whether the trafficking of recycling vesicles (i.e., those fused to and then retrieved from the plasma membrane) and endosomes/lysosomes depends on IFs. Recycling vesicles were labeled by antibodies against vesicle glutamate transporter 1 (VGLUT1) and atrial natriuretic peptide (ANP), respectively, and by lysotracker, which labels endosomes/lysosomes. Quantitative fluorescence microscopy was used to monitor the mobility of labeled vesicles in astrocytes, derived from either wild‐type (WT) mice or mice deficient in glial fibrillary acidic protein and vimentin (GFAP^−/−Vim^−/−), the latter lacking astrocyte IFs. Stimulation with ionomycin or ATP enhanced the mobility of VGLUT1‐positive vesicles and reduced the mobility of ANP‐positive vesicles in WT astrocytes. In GFAP^−/−Vim^−/− astrocytes, both vesicle types responded to stimulation, but the relative increase in mobility of VGLUT1‐positive vesicles was more prominent compared with nonstimulated cells, whereas the stimulation‐dependent attenuation of ANP‐positive vesicles mobility was reduced compared with nonstimulated cells. The mobility of endosomes/lysosomes decreased following stimulation in WT astrocytes. However, in GFAP^−/−Vim^−/− astrocytes, a small increase in the mobility of endosomes/lysosomes was observed. These findings show that astrocyte IFs differentially affect the stimulation‐dependent mobility of vesicles. We propose that upregulation of IFs in pathologic states may alter the function of astrocytes by deregulating vesicle trafficking. © 2010 Wiley‐Liss, Inc.     

Hyperoxia causes reduced density of retinal astrocytes in the central avascular zone in the mouse model of oxygen-induced retinopathy

The mouse model of oxygen-induced retinopathy (OIR) is commonly used to investigate various aspects of the pathogenesis of the retinopathy of prematurity (ROP) as well as angiogenesis in general. Retinal astrocytes were suggested to be involved in retinal angiogenesis. This study aimed to describe their localization and cell density during the course of physiological vascularization and pathological revascularization.Mice expressing H2B-GFP (green fluorescent protein fused to histone 2B) from the endogenous Pdgfra promoter were kept in 75% oxygen from P7 (post natal day 7) to P12 (mouse model of OIR). Retinal flatmounts or cryosections were immunostained for glial fibrillary acidic protein (Gfap), glutamine synthetase (Glul), collagen IV (Col IV), desmin (Des), caspase 3 (Casp3), paired box 2 (Pax2), or Ki67. Astrocytic nuclei were counted with the ImageJ macro AuTOCellQuant. The hypoxic state of the retina was investigated by Hypoxyprobe.The GFP signal of the Pdgfra reporter mice co-localized with Pax2, a nuclear marker for retinal astrocytes. This bright label was much easier to quantify than Gfap or Pax2 staining. Quantification of the cell density of astrocytes during physiological development specified the spreading of astrocytes in a concentrical wave from the optic nerve head towards the periphery. Astrocyte density was reduced during the remodelling of the primary vascular plexus into a hierarchical vascular tree (maximal astrocyte density at P1: 2800 astrocytes/mm2, final astrocyte density: 800 astrocytes/mm2). In the OIR model, cell density of astrocytes was elevated in the peripheral vascularized zone. In contrast, astrocyte density dropped to a half (400 astrocytes/mm2) of the normal value in the central avascular zone during the hyperoxic phase between P8 and P10 by apoptosis and rose only after P17 as the retinal network normalized. An additional drop of astrocyte density was observed within the angles between the large vessels of the central avascular zone during hypoxia between P12 and P17. Astrocyte density was not altered at vascular tufts.The hyperoxia effect on astrocytes including the reduced astrocyte density is not the reason for vascular tuft formation. Hypoxia-affected astrocytes in combination with a reduced astrocytic network in the central avascular zone during the hypoxic phase are important determinants in the formation of pathological features during retinal revascularization.     

Developmental and post‐injury cortical gliogenesis: A Genetic fate‐mapping study with Nestin‐CreER mice

The primary sources of cortical gliogenesis, either during development or after adult brain injury, remain uncertain. We previously generated Nestin‐CreER mice to fate‐map the progeny of radial glial cells (RG), a source of astrocytes and oligodendrocytes in the nervous system. Here, we show that Nestin‐CreER mice label another population of glial progenitors, namely the perinatal subventricular zone (SVZ) glioblasts, if they are crossed with stop‐floxed EGFP mice and receive tamoxifen in late embryogenesis (E16–E18). Quantification showed E18 tamoxifen‐induction labeled more perinatal SVZ glioblasts than RG and transitional RG combined in the newborn brain (54% vs. 22%). Time‐lapse microscopy showed SVZ‐glioblasts underwent complex metamorphosis and often‐reciprocal transformation into transitional RG. Surprisingly, the E10‐dosed RG progenitors produced astrocytes, but no oligodendrocytes, whereas E18‐induction fate‐mapped both astrocytes and NG2+ oligodendrocyte precursors in the postnatal brain. These results suggest that cortical oligodendrocytes mostly derive from perinatal SVZ glioblast progenitors. Further, by combining genetic fate‐mapping and BrdU‐labeling, we showed that cortical astrocytes cease proliferation soon after birth (<P10) and only undergo nonproliferative gliosis (i.e., increased GFAP expression without cell‐division) after stab‐wound injury in adult brains. By contrast, 9.7% of cortical NG2+ progenitors remained mitotic at P29, and the ratio rose to 13.8% after stab‐wound injury. Together, these results suggest NG2+ progenitors, rather than GFAP+ astrocytes, are the primary source of proliferative gliosis after adult brain injury. © 2008 Wiley‐Liss, Inc.     

Osteopontin is indispensable for activation of astrocytes in injured mouse brain and primary culture

ABSTRACT

Objectives: Osteopontin (OPN) is an inflammatory cytokine inducer involved in cell proliferation and migration in inflammatory diseases or tumors. To investigate the function of OPN in astrocyte activation during brain injury, we compared OPN-deficient (OPN/KO) with wild-type (WT) mouse brains after stab wound injury and primary culture of astrocytes.

Methods: Primary cultures of astrocytes were prepared from either WT or OPN/KO postnatal mouse brains. Activation efficiency of astrocytes in primary culture was accessed using Western blotting by examining the protein levels of glial fibrillary acidic protein (GFAP) and tenascin-C (TN-C), which are markers for reactive astrocytes, following lipopolysaccharide (LPS) stimulation. Furthermore, the stab wound injury on the cerebral cortex as a brain traumatic injury model was used, and activation of astrocytes and microglial cells was investigated using immunofluorescent analysis on fixed brain sections.

Results: Primary cultures of astrocytes prepared from WT or OPN/KO postnatal mouse brains showed that only 25% of normal shaped astrocytes in a flask were produced in OPN/KO mice. The expression levels of both GFAP and TN-C were downregulated in the primary culture of astrocytes from OPN/KO mice compared with that from WT mice. By the immunofluorescent analysis on the injured brain sections, glial activation was attenuated in OPN/KO mice compared with WT mice.

Discussion: Our data suggest that OPN is essential for proper astrocytic generation in vitro culture prepared from mouse cerebral cortex. OPN is indispensable for astrocyte activation in the mouse brain injury model and in LPS stimulated primary culture.

Abbreviations: AQP4: aquaporin 4; BBB: blood brain barrier; BrdU: bromo-deoxy uridine; CNS: central nervous system; GFAP: glial fibllirary acidic protein; IgG: immunoglobulin G; LPS: lipopolysaccharide; OPN: osteopontin; OPN/KO: osteopontin-deficient; TN-C: tenascin-C     

Prion protein and its ligand stress inducible protein 1 regulate astrocyte development

Prion protein (PrP^C) interaction with stress inducible protein 1 (STI1) mediates neuronal survival and differentiation. However, the function of PrP^C in astrocytes has not been approached. In this study, we show that STI1 prevents cell death in wild‐type astrocytes in a protein kinase A‐dependent manner, whereas PrP^C‐null astrocytes were not affected by STI1 treatment. At embryonic day 17, cultured astrocytes and brain extracts derived from PrP^C‐null mice showed a reduced expression of glial fibrillary acidic protein (GFAP) and increased vimentin and nestin expression when compared with wild‐type, suggesting a slower rate of astrocyte maturation in PrP^C‐null animals. Furthermore, PrP^C‐null astrocytes treated with STI1 did not differentiate from a flat to a process‐bearing morphology, as did wild‐type astrocytes. Remarkably, STI1 inhibited proliferation of both wild‐type and PrP^C‐null astrocytes in a protein kinase C‐dependent manner. Taken together, our data show that PrP^C and STI1 are essential to astrocyte development and act through distinct signaling pathways. © 2009 Wiley‐Liss, Inc.