KO Astrocytes Research Articles

Purinergic Signalling Mediates Aberrant Excitability of Developing Neuronal Circuits in the Fmr1 Knockout Mouse Model.

Neuronal hyperexcitability within developing cortical circuits is a common characteristic of several heritable neurodevelopmental disorders, including Fragile X Syndrome (FXS), intellectual disability and autism spectrum disorders (ASD). While this aberrant circuitry is typically studied from a neuron-centric perspective, glial cells secrete soluble factors that regulate both neurite extension and synaptogenesis during development. The nucleotide-mediated purinergic signalling system is particularly instrumental in facilitating these effects. We recently reported that within a FXS animal model, theFmr1 KO mouse, the purinergic signalling system is upregulated in cortical astrocytes leading to altered secretion of synaptogenic and plasticity-related proteins. In this study, we examined whether elevated astrocyte purinergic signalling also impacts neuronal morphology and connectivity of Fmr1 KO cortical neurons. Here, we found that conditioned media from primary Fmr1 KO astrocytes was sufficient to enhance neurite extension and complexity of both wildtype and Fmr1 KO neurons to a similar degree as UTP-mediated outgrowth. Significantly enhanced firing was also observed in Fmr1 KO neuron-astrocyte co-cultures grown on microelectrode arrays but was associated with large deficits in firing synchrony. The selective P2Y2 purinergic receptor antagonist AR-C 118925XX effectively normalized much of the aberrant Fmr1 KO activity, designating P2Y2 as a potential therapeutic target in FXS. These results not only demonstrate the importance of astrocyte soluble factors in the development of neural circuitry, but also show that P2Y purinergic receptors play a distinct role in pathological FXS neuronal activity.

IP3RPEP6, a novel peptide inhibitor of IP3 receptor channels that does not affect connexin-43 hemichannels.

Inositol 1,4,5-trisphosphate receptors (IP3 Rs) are intracellular Ca2+ -release channels with crucial roles in cell function. Current IP3 R inhibitors suffer from off-target effects and poor selectivity towards the three distinct IP3 R subtypes. We developed a novel peptide inhibitor of IP3 Rs and determined its effect on connexin-43 (Cx43) hemichannels, which are co-activated by IP3 R stimulation. IP3RPEP6 was developed by in silico molecular docking studies and characterized by on-nucleus patch-clamp experiments of IP3 R2 channels and carbachol-induced IP3 -mediated Ca2+ responses in IP3 R1, 2 or 3 expressing cells, triple IP3 R KO cells and astrocytes. Cx43 hemichannels were studied by patch-clamp and ATP-release approaches, and by inhibition with Gap19 peptide. IP3RPEP6 interactions with IP3 Rs were verified by co-immunoprecipitation and affinity pull-down assays. IP3RPEP6 concentration-dependently reduced the open probability of IP3 R2 channels and competitively inhibited IP3 Rs in an IC50 order of IP3 R2 (~3.9 μM) < IP3 R3 (~4.3 μM) < IP3 R1 (~9.0 μM), without affecting Cx43 hemichannels or ryanodine receptors. IP3RPEP6 co-immunoprecipitated with IP3 R2 but not with IP3 R1; interaction with IP3 R3 varied between cell types. The IC50 of IP3RPEP6 inhibition of carbachol-induced Ca2+ responses decreased with increasing cellular Cx43 expression. Moreover, Gap19-inhibition of Cx43 hemichannels significantly reduced the amplitude of the IP3 -Ca2+ responses and strongly increased the EC50 of these responses. Finally, we identified palmitoyl-8G-IP3RPEP6 as a membrane-permeable IP3RPEP6 version allowing extracellular application of the IP3 R-inhibiting peptide. IP3RPEP6 inhibits IP3 R2/R3 at concentrations that have limited effects on IP3 R1. IP3 R activation triggers hemichannel opening, which strongly affects the amplitude and concentration-dependence of IP3 -triggered Ca2+ responses.

Issue Information

Journal of NeurochemistryVolume 165, Issue 5 p. 623-624 ISSUE INFORMATIONFree Access Issue Information First published: 30 May 2023 https://doi.org/10.1111/jnc.15633 Read the full article: ‘Neuron-restrictive silencer factor/repressor element 1-silencing transcription factor (NRSF/REST) controls spatial K+ buffering in primary cortical astrocytes’ by E. Centonze, A. Marte, M. Albini, A. Rocchi, F. Cesca, M. Chiacchiaretta, T. Floss, P. Baldelli, S. Ferroni, F. Benfenati and P. Valente (J. Neurochem. vol. 165 (5), pp. 701–721) on doi:10.1111/jnc.15755 AboutPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Graphical Abstract Front cover Neuron-restrictive silencer factor/repressor element 1 (RE1)-silencing transcription factor (NRSF/REST) is a transcriptional repressor of a large cluster of neural genes containing RE1 motifs in their promoter region. NRSF/REST is ubiquitously expressed in non-neuronal cells, including astrocytes, while it is downregulated during neuronal differentiation. While neuronal NRSF/REST homeostatically regulates intrinsic excitability and synaptic transmission, the role of the high NRSF/REST expression levels in the homeostatic functions of astrocytes is poorly understood. Here, we investigated the functional consequences of NRSF/REST deletion in primary cortical astrocytes derived from NRSF/REST conditional knockout mice (KO). We found that NRSF/REST KO astrocytes displaya markedly reduced activity of inward rectifying K+ channels subtype 4.1 (Kir4.1) underlying spatial K+ buffering, associated with decreased expression and activity of the glutamate transporter-1 (GLT-1) responsible for glutamate uptake. The data indicate that astrocytic NRSF/REST directly participates in neural circuit homeostasis by regulating intrinsic excitability and excitatory transmission and that its dysfunctions may contribute to the pathogenesis of neurological disorders. Image content GFAP immunostaining (red) in Rest-KO astrocytes bearing nuclear GFP (green) staining as reporter of Cre-transduction. Read the full article ‘Neuron-restrictive silencer factor/repressor element 1-silencing transcription factor (NRSF/REST) controls spatial K+ buffering in primary cortical astrocytes’ by E. Centonze, A. Marte, M. Albini, A. Rocchi, F. Cesca, M. Chiacchiaretta, T. Floss, P. Baldelli, S. Ferroni, F. Benfenati and P. Valente (J. Neurochem. vol. 165 (5), pp. 701–721) on doi:10.1111/jnc.15755 Volume165, Issue5June 2023Pages 623-624 RelatedInformation

Astrocytes express aberrant immunoglobulins as putative gatekeeper of astrocytes to neuronal progenitor conversion

Using multi-omics analyses including RNAseq, RT-PCR, RACE-PCR, and shotgun proteomic with enrichment strategies, we demonstrated that newborn rat astrocytes produce neural immunoglobulin constant and variable heavy chains as well as light chains. However, their edification is different from the ones found in B cells and they resemble aberrant immunoglobulins observed in several cancers. Moreover, the complete enzymatic V(D)J recombination complex has also been identified in astrocytes. In addition, the constant heavy chain is also present in adult rat astrocytes, whereas in primary astrocytes from human fetus we identified constant and variable kappa chains as well as the substitution lambda chains known to be involved in pre-B cells. To gather insights into the function of these neural IgGs, CRISPR-Cas9 of IgG2B constant heavy chain encoding gene (Igh6), IgG2B overexpression, proximal labeling of rat astrocytes IgG2B and targets identification through 2D gels were performed. In Igh6 KO astrocytes, overrepresentation of factors involved in hematopoietic cells, neural stem cells, and the regulation of neuritogenesis have been identified. Moreover, overexpression of IgG2B in astrocytes induces the CRTC1-CREB-BDNF signaling pathway known to be involved in gliogenesis, whereas Igh6 KO triggers the BMP/YAP1/TEAD3 pathway activated in astrocytes dedifferentiation into neural progenitors. Proximal labeling experiments revealed that IgG2B is N-glycosylated by the OST complex, addressed to vesicle membranes containing the ATPase complex, and behaves partially like CD98hc through its association with LAT1. These experiments also suggest that proximal IgG2B-LAT1 interaction occurs concomitantly with MACO-1 and C2CD2L, at the heart of a potentially novel cell signaling platform. Finally, we demonstrated that these chains are synthesized individually and associated to recognize specific targets. Indeed, intermediate filaments Eif4a2 and Pdia6 involved in astrocyte fate constitute targets for these neural IgGs. Taken together, we hypothese that neural aberrant IgG chains may act as gatekeepers of astrocytes' fate.

Neuron-restrictive silencer factor/repressor element 1-silencing transcription factor (NRSF/REST) controls spatial K+ buffering in primary cortical astrocytes.

Neuron-restrictive silencer factor/repressor element 1 (RE1)-silencing transcription factor (NRSF/REST) is a transcriptional repressor of a large cluster of neural genes containing RE1 motifs in their promoter region. NRSF/REST is ubiquitously expressed in non-neuronal cells, including astrocytes, while it is down-regulated during neuronal differentiation. While neuronal NRSF/REST homeostatically regulates intrinsic excitability and synaptic transmission, the role of the high NRSF/REST expression levels in the homeostatic functions of astrocytes is poorly understood. Here, we investigated the functional consequences of NRSF/REST deletion in primary cortical astrocytes derived from NRSF/REST conditional knockout mice (KO). We found that NRSF/REST KO astrocyte displayed a markedly reduced activity of inward rectifying K+ channels subtype 4.1 (Kir4.1) underlying spatial K+ buffering that was associated with a decreased expression and activity of the glutamate transporter-1 (GLT-1) responsible for glutamate uptake by astrocytes. The effects of the impaired astrocyte homeostatic functions on neuronal activity were investigated by co-culturing wild-type hippocampal neurons with NRSF/REST KO astrocytes. Interestingly, neurons experienced increased neuronal excitability at high firing rates associated with decrease after hyperpolarization and increased amplitude of excitatory postsynaptic currents. The data indicate that astrocytic NRSF/REST directly participates in neural circuit homeostasis by regulating intrinsic excitability and excitatory transmission and that dysfunctions of NRSF/REST expression in astrocytes may contribute to the pathogenesis of neurological disorders.

Toll-like receptor 4 activation enhances Orai1-mediated calcium signal promoting cytokine production in spinal astrocytes.

Toll-like receptor 4 (TLR4) has been implicated in pathological conditions including chronic pain. Activation of astrocytic TLRs leads to the synthesis of pro-inflammatory cytokines like interleukin 6 (IL-6) and tumor necrosis factor-ɑ (TNF-α), which can cause pathological inflammation and tissue damage in the central nervous system. However, the mechanisms of TLR4-mediated cytokine releases from astrocytes are incomplete understood. Our previous study has shown that Orai1, a key component of calcium release activated calcium channels (CRACs), mediates Ca2+ entry in astrocytes. How Orai1 contributes to TLR4 signaling remains unclear. Here we show that Orai1 deficiency drastically attenuated lipopolysaccharides (LPS)-induced TNF-α and IL-6 production in astrocytes. Acute LPS treatment did not induce Ca2+ response and had no effect on thapsigargin (Ca2+-ATPase inhibitor)-induced store-dependent Ca2+ entry. Inhibition or knockdown of Orai1 showed no reduction in LPS-induced p-ERK1/2, p-c-Jun N-terminal kinase, or p-p38 MAPK activation. Interestingly, Orai1 protein level was significantly increased after LPS exposure, which was blocked by inhibition of NF-κB activity. LPS significantly increased basal Ca2+ level and SOCE after exposure to astrocytes. Moreover, elevating extracellular Ca2+ concentration increased cytosolic Ca2+ level, which was almost eliminated in Orai1 KO astrocytes. Our study reports novel findings that Orai1 acts as a Ca2+ leak channel regulating the basal Ca2+ level and enhancing cytokine production in astrocytes under the inflammatory condition. These findings highlight an important role of Orai1 in astrocytic TRL4 function and may suggest that Orai1 could be a potential therapeutic target for neuroinflammatory disorders including chronic pain.

RGC-32 Regulates Generation of Reactive Astrocytes in Experimental Autoimmune Encephalomyelitis.

Astrocytes are increasingly recognized as critical contributors to multiple sclerosis pathogenesis. We have previously shown that lack of Response Gene to Complement 32 (RGC-32) alters astrocyte morphology in the spinal cord at the peak of experimental autoimmune encephalomyelitis (EAE), suggesting a role for RGC-32 in astrocyte differentiation. In this study, we analyzed the expression and distribution of astrocytes and astrocyte progenitors by immunohistochemistry in spinal cords of wild-type (WT) and RGC-32-knockout (KO) mice with EAE and of normal adult mice. Our analysis showed that during acute EAE, WT astrocytes had a reactive morphology and increased GFAP expression, whereas RGC-32 KO astrocytes had a morphology similar to that of radial glia and an increased expression of progenitor markers such as vimentin and fatty acid binding protein 7 (FABP7). In control mice, GFAP expression and astrocyte density were also significantly higher in the WT group, whereas the number of vimentin and FABP7-positive radial glia was significantly higher in the RGC-32 KO group. In vitro studies on cultured neonatal astrocytes from WT and RGC-32 KO mice showed that RGC-32 regulates a complex array of molecular networks pertaining to signal transduction, growth factor expression and secretion, and extracellular matrix (ECM) remodeling. Among the most differentially expressed factors were insulin-like growth factor 1 (IGF1), insulin-like growth factor binding proteins (IGFBPs), and connective tissue growth factor (CTGF); their expression was downregulated in RGC-32-depleted astrocytes. The nuclear translocation of STAT3, a transcription factor critical for astrogliogenesis and driving glial scar formation, was also impaired after RGC-32 silencing. Taken together, these data suggest that RGC-32 is an important regulator of astrocyte differentiation during EAE and that in the absence of RGC-32, astrocytes are unable to fully mature and become reactive astrocytes.

Abstract 97: Analysis of Pten-null mouse astrocytes revealed overlapping transcribed genes with human glioblastoma transcriptome

Abstract Glioblastoma is an aggressive form of brain cancer with universally poor prognosis. Even with optimal therapy, the cancer recurs frequently. Phosphatase and tensin homolog (PTEN) is a tumor suppressor gene whose mutations are often found in multiple human cancers. PTEN encodes a lipid phosphatase and serves as a negative regulator of the phosphoinositide 3-kinase (PI3K) pathway. Cumulative evidence suggests that PTEN mutations play significant roles in tumorigenesis of glioblastoma as 60% of glioblastoma patients acquired mutations in this gene. Based on our preliminary data, transcriptomic profiling of primary astrocytes derived from Pten knockout mice revealed an overlapping enrichment of mitotic regulators and cell cycle related pathways with human GBM. 4 genes (RCC1, RCC2, NEK2, and CHK2) were validated to be significantly upregulated in PTEN KO astrocytes by qRT-PCR. Regulator of chromatin condensation 2 (RCC2) has been reported to be guanine-nucleotide exchange factor (GEF) for Rac1 and RalA GTPase and act as a regulator of cell cycle progression. Accumulating evidence revealed a pro-tumorigenic role of RCC2 in multiple cancers such as tumor growth and tumor metastasis. Via siRNA inhibition of RCC2, we demonstrated that knocking down of RCC2 in glioblastoma cell lines suppresses cell growth and RCC2 is essential for both G1-S and G2-M progression. Additionally, RCC2 knocking down and a PI3K inhibitor LY294002 inhibit glioblastoma cell viability in a synergistic manner. In summary, PTEN deficiency leads to the upregulation of RCC2, which may promote tumorigenesis of glioblastoma by dysregulating cell cycle progression.To further explore the RCC2 roles, we will find the potential downstream target of RCC2 in dysregulating cell cycle and its potential therapeutic effect in vivo. Funding: General Research Fund Grant #14113519 from The Research Grants Council of Hong Kong Citation Format: Tian Liu, Penelope Mei-Yu Or, Chi-Wai Wong, Stanley Kwok-Kuen Cheung, Yubing Wang, Yiwei Wang, Andrew M. Chan. Analysis of Pten-null mouse astrocytes revealed overlapping transcribed genes with human glioblastoma transcriptome [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 97.

Astrocyte morphogenesis is dependent on BDNF signaling via astrocytic TrkB.T1.

Brain-derived neurotrophic factor (BDNF) is a critical growth factor involved in the maturation of the CNS, including neuronal morphology and synapse refinement. Herein, we demonstrate astrocytes express high levels of BDNF's receptor, TrkB (in the top 20 of protein-coding transcripts), with nearly exclusive expression of the truncated isoform, TrkB.T1, which peaks in expression during astrocyte morphological maturation. Using a novel culture paradigm, we show that astrocyte morphological complexity is increased in the presence of BDNF and is dependent upon BDNF/TrkB.T1 signaling. Deletion of TrkB.T1, globally and astrocyte-specifically, in mice revealed morphologically immature astrocytes with significantly reduced volume, as well as dysregulated expression of perisynaptic genes associated with mature astrocyte function. Indicating a role for functional astrocyte maturation via BDNF/TrkB.T1 signaling, TrkB.T1 KO astrocytes do not support normal excitatory synaptogenesis or function. These data suggest a significant role for BDNF/TrkB.T1 signaling in astrocyte morphological maturation, a critical process for CNS development.

Membralin deficiency dysregulates astrocytic glutamate homeostasis leading to ALS-like impairment.

Mechanisms underlying motor neuron degeneration in amyotrophic lateral sclerosis (ALS) are yet unclear. Specific deletion of the ER-component membralin in astrocytes manifested postnatal motor defects and lethality in mice, causing the accumulation of extracellular glutamate through reducing the glutamate transporter EAAT2. Restoring EAAT2 levels in membralin KO astrocytes limited astrocyte-dependent excitotoxicity in motor neurons. Transcriptomic profiles from mouse astrocytic membralin KO motor cortex indicated significant perturbation in KEGG pathway components related to ALS, including downregulation of Eaat2 and upregulation of Tnfrsf1a. Changes in gene expression with membralin deletion also overlapped with mouse ALS models and reactive astrocytes. Our results shown that activation of TNF receptor (TNFR1)-NFκB pathway known to suppress Eaat2 transcription was upregulated with membralin deletion. Further, reduced membralin and EAAT2 levels correlated with disease progression in spinal cord from SOD1-mutant mouse models, and reductions in membralin/EAAT2 were observed in human ALS spinal cord. Importantly, overexpression of membralin in SOD1G93A astrocytes decreased TNFR1 levels and increased EAAT2 expression, and improved motor neuron survival. Importantly, upregulation of membralin in SOD1G93A mice significantly prolonged mouse survival. Together, our study provided a mechanism for ALS pathogenesis where membralin limited glutamatergic neurotoxicity, suggesting that modulating membralin had potentials in ALS therapy.

A Parkinson's disease gene, DJ-1, repairs brain injury through Sox9 stabilization and astrogliosis.

Defects in repair of damaged brain accumulate injury and contribute to slow-developing neurodegeneration. Here, we report that a deficiency of DJ-1, a Parkinson's disease (PD) gene, delays repair of brain injury due to destabilization of Sox9, a positive regulator of astrogliosis. Stereotaxic injection of ATP into the brain striatum produces similar size of acute injury in wild-type and DJ-1-knockout (KO) mice. However, recovery of the injury is delayed in KO mice, which is confirmed by 9.4T magnetic resonance imaging and tyrosine hydroxylase immunostaining. DJ-1 regulates neurite outgrowth from damaged neurons in a non-cell autonomous manner. In DJ-1 KO brains and astrocytes, Sox9 protein levels are decreased due to enhanced ubiquitination, resulting in defects in astrogliosis and glial cell-derived neurotrophic factor/ brain-derived neurotrophic factor expression in injured brain and astrocytes. These results indicate that DJ-1 deficiency causes defects in astrocyte-mediated repair of brain damage, which may contribute to the development of PD.

Truncated TrkB.T1-Mediated Astrocyte Dysfunction Contributes to Impaired Motor Function and Neuropathic Pain after Spinal Cord Injury

Following spinal cord injury (SCI), astrocytes demonstrate long-lasting reactive changes, which are associated with the persistence of neuropathic pain and motor dysfunction. We previously demonstrated that upregulation of trkB.T1, a truncated isoform of the brain-derived neurotrophic factor receptor (BDNF), contributes to gliosis after SCI, but little is known about the effects of trkB.T1 on the function of astrocytes. As trkB.T1 is the sole isoform of trkB receptors expressed on astrocytes, we examined the function of trkB.T1-driven astrocytes in vitro and in vivo Immunohistochemistry showed that trkB.T1+ cells were significantly upregulated 7 d after injury, with sustained elevation in white matter through 8 weeks. The latter increase was predominantly found in astrocytes. TrkB.T1 was also highly expressed by neurons and microglia/macrophages at 7 d after injury and declined by 8 weeks. RNA sequencing of cultured astrocytes derived from trkB.T1+/+ (WT) and trkB.T1-/- (KO) mice revealed downregulation of migration and proliferation pathways in KO astrocytes. KO astrocytes also exhibited slower migration/proliferation in vitro in response to FBS or BDNF compared with WT astrocytes. Reduced proliferation of astrocytes was also confirmed after SCI in astrocyte-specific trkB.T1 KO mice; using mechanical allodynia and pain-related measurements on the CatWalk, these animals also showed reduced hyperpathic responses, along with improved motor coordination. Together, our data indicate that trkB.T1 in astrocytes contributes to neuropathic pain and neurological dysfunction following SCI, suggesting that trkB.T1 may provide a novel therapeutic target for SCI.SIGNIFICANCE STATEMENT Neuropathic pain after spinal cord injury (SCI) may in part be caused by upregulation of the brain-derived neurotrophic factor (BDNF) receptor trkB.T1, a truncated isoform of BDNF. TrkB.T1 is the only isoform of tropomyosin-related receptor kinase type B (trkB) receptors expressed on astrocytes. Here, we showed that trkB.T1 is significantly increased in the injured mouse spinal cord, where it is predominantly found in astrocytes. RNA sequencing of cultured astrocytes demonstrated downregulation of migration and proliferation pathways in trkB.T1 KO astrocytes. This was validated in vivo, where deletion of trkB.T1 in astrocytes reduced cell proliferation and migration. After SCI, astrocyte-specific trkB.T1 KO mice showed reduced hyperpathic responses and improved motor coordination. Therefore, the trkB.T1 receptor plays a significant pathophysiological role after SCI, and may provide a novel therapeutic target for SCI.

Glio-vascular modifications caused by Aquaporin-4 deletion in the mouse retina

Aquaporin-4 (AQP4) is the Central Nervous System water channel highly expressed at the perivascular glial domain. In the retina, two types of AQP4 expressing glial cells take part in the blood-retinal barrier (BRB), astrocytes and Müller cells. The aim of the present study is to investigate the effect of AQP4 deletion on the retinal vasculature by looking at typical pathological hallmark such as BRB dysfunction and gliotic condition.AQP4 dependent BRB properties were evaluated by measuring the number of extravasations in WT and AQP4 KO retinas by Evans blue injection assay. AQP4 deletion did not affect the retinal vasculature, as assessed by Isolectin B4 staining, but caused BRB impairment to the deep plexus capillaries while the superficial and intermediate capillaries were not compromised. To investigate for gliotic responses caused by AQP4 deletion, Müller cells and astrocytes were analysed by immunofluorescence and western blot, using the Müller cell marker Glutamine Synthetase (GS) and the astrocyte marker GFAP. While GS expression was not altered in AQP4 KO retinas, a strong GFAP upregulation was found at the level of AQP4 KO astrocytes at the superficial plexus and not at Müller cells at the intermediate and deep plexi. These data, together with the upregulation of inflammatory markers (TNF-α, IL-6, IL-1β and ICAM-1) in AQP4 KO retinas indicated AQP4 deletion as responsible for a gliotic phenotype. Interestingly, no GFAP altered expression was found in AQP4 siRNA treated astrocyte primary cultures. All together these results indicate that AQP4 deletion is directly responsible for BRB dysfunction and gliotic condition in the mouse retina. The selective activation of glial cells at the primary plexus suggests that different regulatory elements control the reaction of astrocytes and Müller cells. Finally, GFAP upregulation is strictly linked to gliovascular crosstalk, as it is absent in astrocytes in culture. This study is useful to understand the role of AQP4 in the perivascular domain in the retina and its possible implications in the pathogenesis of retinal vascular diseases and of Neuromyelitis Optica, a human disease characterized by anti-AQP4 auto-antibodies.

ID: 122: The gp130 cytokines mediate differential signal pathway activation and transcriptional responses in astrocytes versus microglia

The gp130 cytokines include IL-6, IL-11, LIF and OSM. These cytokines have important roles in neuroinflammation and neurodevelopment. However, the nature of any cell-specific responses of astrocytes and microglia to this cytokine family and their molecular basis is not well understood. Here, the gp130 family cytokine signal pathway activation and global transcriptional responses were examined in murine astrocytes and microglia in vitro. In astrocytes, OSM-induced STAT1 phosphorylation was significantly greater than for hyperIL-6 (IL-6 linked to the soluble IL-6 receptor) or LIF. However, STAT3 phosphorylation was induced to similar levels for all three cytokines. Conversely, in microglia which lacked the OSMR, hyperIL-6 and LIF but not OSM, induced phosphorylation of STAT3 and only minimal phosphorylation of STAT1. In STAT1 KO astrocytes, OSM conferred a stronger activation of STAT3 compared with WT astrocytes. Transcriptomic analysis of WT and STAT1 KO astrocyte response to OSM, hyperIL-6 or LIF revealed up-regulation and downregulation of many transcripts with OSM > IL-6 > LIF. A dominant IFN-like response to OSM was largely abolished in STAT1 KO astrocytes, suggesting that a large portion of OSM-regulated genes is STAT1-dependent. The gene expression profile of OSM- and hyperIL-6 treated astrocytes was somewhat similar while that for LIF differed the most. Despite signalling via common gp130 pathways, our findings show that specificity is achieved through multiple mechanisms including cell-restricted expression of some receptors, varying strength of signal pathway activation and differential activation of STAT1 and STAT3, which mediate differential gene expression and potential functions by the gp130 cytokines in astrocytes and microglia.

Astrocyte-expressed FABP7 regulates dendritic morphology and excitatory synaptic function of cortical neurons.

Fatty acid binding protein 7 (FABP7) expressed by astrocytes in developing and mature brains is involved in uptake and transportation of fatty acids, signal transduction, and gene transcription. Fabp7 knockout (Fabp7 KO) mice show behavioral phenotypes reminiscent of human neuropsychiatric disorders such as schizophrenia. However, direct evidence showing how FABP7 deficiency in astrocytes leads to altered brain function is lacking. Here, we examined neuronal dendritic morphology and synaptic plasticity in medial prefrontal cortex (mPFC) of Fabp7 KO mice and in primary cortical neuronal cultures. Golgi staining of cortical pyramidal neurons in Fabp7 KO mice revealed aberrant dendritic morphology and decreased spine density compared with those in wild-type (WT) mice. Aberrant dendritic morphology was also observed in primary cortical neurons co-cultured with FABP7-deficient astrocytes and neurons cultured in Fabp7 KO astrocyte-conditioned medium. Excitatory synapse number was decreased in mPFC of Fabp7 KO mice and in neurons co-cultured with Fabp7 KO astrocytes. Accordingly, whole-cell voltage-clamp recording in brain slices from pyramidal cells in the mPFC showed that both amplitude and frequency of action potential-independent miniature excitatory postsynaptic currents (mEPSCs) were decreased in Fabp7 KO mice. Moreover, transplantation of WT astrocytes into the mPFC of Fabp7 KO mice partially attenuated behavioral impairments. Collectively, these results suggest that astrocytic FABP7 is important for dendritic arbor growth, neuronal excitatory synapse formation, and synaptic transmission, and provide new insights linking FABP7, lipid homeostasis, and neuropsychiatric disorders, leading to novel therapeutic interventions.

Connexin43 hemichannels mediate secondary cellular damage spread from the trauma zone to distal zones in astrocyte monolayers.

The mechanism of secondary damage spread after brain trauma remains unsolved. In this work, we redirected the attention to astrocytic communication pathways. Using an in vitro trauma model that consists of a scratch injury applied to an astrocyte monolayer, we found a significant and transient induction of connexin43 (Cx43) hemichannel activity in regions distal from the injury, which was maximal ∼1 h after scratch. Two connexin hemichannel blockers, La(3+) and the peptide Gap26, abolished the increased activity, which was also absent in Cx43 KO astrocytes. In addition, the scratch-induced increase of hemichannel activity was prevented by inhibition of P2 purinergic receptors. Changes in hemichannel activity took place with a particular spatial distribution, with cells located at ∼17 mm away from the scratch presenting the highest activity (dye uptake). In contrast, the functional state of gap junction channels (dye coupling) was not significantly affected. Cx43 hemichannel activity was also enhanced by the acute extracellular application of 60 mM K(+) . The increase in hemichannel activity was associated with an increment in apoptotic cells at 24 h after scratch that was totally prevented by Gap26 peptide. These findings suggest that Cx43 hemichannels could be a new approach to prevent or reduce the secondary cell damage of brain trauma.

Proteomic Differential Display Analysis Reveals Decreased Expression of PEA-15 and Vimentin in FABP7-Deficient Astrocytes

Fatty acid binding proteins (FABPs) are intracellular lipid chaperones which mediate the uptake, transportation and signaling roles of fatty acids. Former studies suggest that FABP7 regulates proliferation and differentiation of radial glia and astrocytes and is associated with various central nervous system diseases including glioma and neuropsychiatric diseases. However, the underlying mechanism is poorly understood. To further explore the mechanistic roles of FABP7 on astrocyte function, the proteome of astrocytes cultured from Fabp7 KO mice was compared with wild-type counterparts by two-dimensional gel electrophoresis (2-DE). We found that 16 protein spots showed differential expression intensity on 2-DE gels. Among them, seven spots appeared elevated, and nine spots appeared decreased in Fabp7 KO astrocytes. Selected spots were analyzed using liquid chromatography tandem mass spectrometry (LC-MS/MS) analysis and their protein identity was subsequently revealed using protein databases. Four spots including phosphoprotein enriched in astrocytes 15 (PEA-15), GFAP, vimentin and FABP7 were selected for further confirmation by Western blotting. Consistently, there was a significant decrease in expression of PEA-15 and vimentin, both of which play significant roles in proliferation, differentiation, maturation, and survival of astrocytes. These results suggest the molecular mechanism how FABP7 controls astrocyte function and provide new insight to explain the role of FABP7 in glioma and reactive gliosis.

Phospholipase C‐epsilon links G‐protein coupled receptor activation to inflammatory responses in astrocytes (1066.5)

Neuroinflammation underlies many diseases of the central nervous system (CNS) and the role of astrocytes in this process is increasingly recognized. Thrombin and the lysophospholipids, LPA and S1P are generated during injury and can activate G‐protein coupled receptors (GPCRs) on astrocytes. We hypothesized that GPCRs that couple to RhoA induce inflammatory gene expression in astrocytes through phospholipase C‐epsilon (PLCε). Using wild‐type (WT) and PLCε (KO) astrocytes, we recently demonstrated that thrombin, LPA, and S1P signal through PLCε to mediate sustained PKD activation and subsequently activation of NFκB to induce expression of inflammatory genes including COX‐2, IL‐1β, and IL‐6 (Dusaban, SS et al. PNAS, 2013). We are currently investigating the molecular mechanism by which PLCε mediates sustained PKD activation and downstream inflammatory responses. Preliminary data using Golgi targeted PKD FRET reporters suggests that PLCε mediates sustained activation of PKD at the Golgi. We also find that disrupting the Golgi with brefeldin A inhibits both PKD activation and COX‐2 expression. We further determined that COX‐2 gene expression was induced through PLCε following in vitro wounding and in vivo cortical stab wound injury. In light of growing evidence for a role of glial cells and S1P receptors in neuroinflammation, we investigated the S1P receptor subtypes coupled to PLCε and its downstream targets. Taking advantage of receptor KO astrocytes and receptor knockdown, we determined that coupling occurs primarily through the S1P3 receptor. Future work is aimed at identifying other GPCRs that signal through PLCε to mediate neuroinflammatory responses.Grant Funding Source: GM 36927

β−Amyloid exacerbates inflammation in astrocytes lacking fatty acid amide hydrolase through a mechanism involving PPAR‐α, PPAR‐γ and TRPV1, but not CB1 or CB2 receptors

The endocannabinoid system may regulate glial cell functions and their responses to pathological stimuli, specifically, Alzheimer's disease. One experimental approach is the enhancement of endocannabinoid tone by blocking the activity of degradative enzymes, such as fatty acid amide hydrolase (FAAH). We examined the role of FAAH in the response of astrocytes to the pathologic form of β-amyloid (Aβ). Astrocytes from wild-type mice (WT) and from mice lacking FAAH (FAAH-KO) were incubated with Aβ for 8, 24 and 48 h, and their inflammatory responses were quantified by elisa, western-blotting and real-time quantitative-PCR. FAAH-KO astrocytes were significantly more responsive to Aβ than WT astrocytes, as shown by the higher production of pro-inflammatory cytokines. Expression of COX-2, inducible NOS and TNF-α was also increased in Aβ-exposed KO astrocytes compared with that in WTs. These effects were accompanied by a differential pattern of activation of signalling cascades involved in mediating inflammatory responses, such as ERK1/2, p38MAPK and NFκB. PPAR-α and PPAR-γ as well as transient receptor potential vanilloid-1 (TRPV1), but not cannabinoid CB₁ or CB₂ receptors, mediate some of the differential changes observed in Aβ-exposed FAAH-KO astrocytes. The pharmacological blockade of FAAH did not render astrocytes more sensitive to Aβ. In contrast, exogenous addition of several acylethanolamides (anandamide, palmitoylethanolamide and oleoylethanolamide) induced an antiinflammatory response. The genetic deletion of FAAH in astrocytes exacerbated their inflammatory phenotype against Aβ in a process involving PPAR-α, PPAR-γ and TRPV1 receptors.

The role of phospholipase c‐epsilon in COX‐2 expression and inflammation

Brain injury triggers a response characterized by astrocyte proliferation, inflammatory gene expression, and migration. During injury thrombin is generated and can activate the G‐protein coupled protease‐activated receptor 1 (PAR1). Using primary mouse astrocytes, we demonstrated that PAR1 activation triggers phosphoinositide hydrolysis through the novel phospholipase C epsilon (PLCε). Here we asked whether PLCε signaling mediates inflammatory gene expression in astrocytes in response to PAR1 activation. Thrombin stimulation induces 8 and 2.5‐fold increases in COX‐2 mRNA and protein expression, respectively, and these are significantly inhibited in PLCε KO astrocytes. Protein kinase D (PKD) is also activated in response to thrombin and lost in PLCε KO astrocytes, and studies using siRNA or inhibitors show PKD activation is important for COX‐2 expression. Furthermore, we found that thrombin induces NFκB translocation, which in turn regulates COX‐2 expression and that this is dependent on PLCε and PKD. Finally, using scratch assays and a stab wound model to mimic astrogliosis in vitro and in vivo, respectively, we demonstrate PLCε dependent COX‐2 expression. Our studies show that PLCε is the link between activation of PAR1 and COX‐2 expression and this is mediated through PKD and NFκB activation. In vitro and in vivo data indicate that this pathway has important implications in astrogliosis.