Activity In Primary Astrocytes Research Articles

Elevated calcineurin activity in primary astrocytes leads to the dephosphorylation of connexin 43 in conjunction with increased membrane permeability.

Hyperactivation of the Ca2+/calmodulin-dependent phosphatase calcineurin (CN) is observed in reactive astrocytes associated with neuroinflammation and progressive degenerative diseases, like Alzheimer's disease. Apart from key transcription factors (e.g. nuclear factor of activated t cells and nuclear factor-κB) very few other CN-dependent pathways have been studied in astrocytes. The hemichannel protein, connexin 43 (Cx43) is found at high levels in astrocytes and contains a CN-sensitive Ser residue near its carboxy terminus. CN-dependent dephosphorylation of Cx43 has been reported in primary astrocytes treated with injurious stimuli, but much remains unknown about CN/Cx43 interactions in the context of neuroinflammation and disease. Western blots were used to assess total Cx43 and dephosphorylated Cx43 subtypes in rat embryonic primary astrocytes treated with a hyperactive CN fragment (ΔCN, via adenovirus), or with a proinflammatory cytokine cocktail. Under similar treatment conditions, an ethidium bromide (EtBr) uptake assay was used to assess membrane permeability. Effects of ΔCN and cytokines were tested in the presence or absence of the CN inhibitor, cyclosporin A. A connexin inhibitor, carbenoxolone was also used in EtBr assays to assess the involvement of connexins in membrane permeability. Treatment with ΔCN or cytokines increased dephosphorylated Cx43 levels in conjunction with increased membrane permeability (elevated EtBr uptake). Effects of ΔCN or cytokine treatment were blocked by cyclosporine A. Treatment-induced changes in EtBr uptake were also inhibited by carbenoxolone. The results suggest that Cx43 hemichannels could be an important mechanism through which astrocytic CN disrupts neurologic function associated with neurodegenerative disease.

Anti-inflammatory potency of novel ecto-5′-nucleotidase/CD73 inhibitors in astrocyte culture model of neuroinflammation

Three novel cytosine-derived α,β-methylene diphosphonates designated MRS4598, MRS4552, and MRS4602 were tested in the range of 1 × 10−9 to 1 × 10−3 M for their efficacy and potency in inhibiting membrane-bound ecto-5′-nucleotidase/CD73 activity in primary astrocytes in vitro. The compounds were also tested for their ability to attenuate the reactive astrocyte phenotype induced by proinflammatory cytokines. The main findings are as follows: A) The tested compounds induced concentration-dependent inhibition of CD73 activity, with maximal inhibition achieved at ∼1 × 10−3M; B) All compounds showed high inhibitory potency, as reflected by IC50 values in the submicromolar range; C) All compounds showed high binding capacity, as reflected by Ki values in the low nanomolar range; D) Among the tested compounds, MRS4598 showed the highest inhibitory efficacy and potency, as reflected by IC50 and Ki values of 0.11 μM and 18.2 nM; E) Neither compound affected astrocyte proliferation and cell metabolic activity at concentrations near to IC50; E) MRS4598 was able to inhibit CD73 activity in reactive astrocytes stimulated with TNF-α and to induce concentration-dependent inhibition of CD73 in reactive astrocytes stimulated with IL-1β, with an order of magnitude higher IC50 value; F) MRS4598 was the only compound tested that was able to induce shedding of the CD73 from astrocyte membranes and to enhance astrocyte migration in the scratch wound migration assay, albeit at concentration well above its IC50 value. Given the role of CD73 in neurodegenerative diseases, MRS4598, MRS4552, and MRS4602 are promising pharmacological tools for the treatment of neurodegeneration and neuroinflammation.

298-OR: Variant-to-Gene Mapping at the Childhood Obesity Locus on chr12q13 and Subsequent Luciferase Assay Analyses Implicate rs71329as a Causal Variant within the 3’ UTR of FAIM2

A key limitation of genome-wide association studies (GWAS) is that they do not specify either the underlying causal variant (s) or the effector gene (s) at a given locus. The widely reproduced obesity FAIM2 locus, named arbitrarily after the nearest gene, is relatively understudied given it is more strongly associated in children than in adults. Our previously reported trans-ancestral GWAS meta-analysis of childhood obesity enabled fine-mapping of the FAIM2 locus; in the ‘credible set’ of 6 SNPs, rs71329 had the highest probability of being causal. This non-coding variant resides in the 3’ untranslated region (UTR) of FAIM2. Given that obesity-related GWAS signals have been reported to be enriched in genes relevant to neuronal function, we generated ATAC-seq and high-resolution promoter-focused Capture C data in both primary astrocytes and ESC-derived hypothalamic neurons to carry out variant-to-gene mapping at this locus. We observed that rs71329 occupied a region of open chromatin, therefore a putatively functional element, and made physical contact with the FAIM2 promoter. These findings were also supported by both GTEx testes eQTL data and a transcriptome-wide association study in brain tissue. We sought to determine in vitro if the rs71329alleles influence enhancer activity in primary astrocytes. Using technical replicates repeated 8 times, the non-risk allele region increased luciferase reporter expression relative to the FAIM2 promoter alone by a fold change of 1.87, while the risk allele decreased expression by a fold change of 0.68 (non-risk allele+FAIM2 promoter vs. risk allele+FAIM2 promoter P<0.0001) . Our results show that rs71329resides in an enhancer element and suggest that the risk allele within the 3’ UTR of FAIM2 decreases the expression of this putative effector gene, which then confers risk of childhood obesity and in turn type 2 diabetes later in life. Disclosure S.H.Littleton: None. J.Bradfield: None. J.A.Pippin: None. C.Su: None. A.Chesi: None. A.D.Wells: Stock/Shareholder; Johnson & Johnson. R.I.Berkowitz: Research Support; Eisai Inc., Novo Nordisk. M.C.Pahl: None. S.F.Grant: None. Funding National Institutes of Health (HD056465)

Microglia-Specific Promoter Activities of HEXB Gene.

Adeno-associated virus (AAV)-mediated genetic targeting of microglia remains a challenge. Overcoming this hurdle is essential for gene editing in the central nervous system (CNS). Here, we characterized the minimal/native promoter of the HEXB gene, which is known to be specifically and stably expressed in the microglia during homeostatic and pathological conditions. Dual reporter and serial deletion assays identified the critical role of the natural 5’ untranslated region (−97 bp related to the first ATG) in driving transcriptional activity of the mouse Hexb gene. The native promoter region of mouse, human, and monkey HEXB are located at −135, −134, and −170 bp to the first ATG, respectively. These promoters were highly active and specific in microglia with strong cross-species transcriptional activities, but did not exhibit activity in primary astrocytes. In addition, we identified a 135 bp promoter of CD68 gene that was highly active in microglia but not in astrocytes. Considering that HEXB is specifically expressed in microglia, these data suggest that the newly characterized microglia-specific HEXB minimal/native promoter can be an ideal candidate for microglia-targeting AAV gene therapy in the CNS.

GPR120 Signaling Controls Amyloid-β Degrading Activity of Matrix Metalloproteinases.

Alzheimer's disease (AD) is characterized by the extensive deposition of amyloid-β peptide (Aβ) in the brain. Brain Aβ level is regulated by a balance between Aβ production and clearance. The clearance rate of Aβ is decreased in the brains of sporadic AD patients, indicating that the dysregulation of Aβ clearance mechanisms affects the pathologic process of AD. Astrocytes are among the most abundant cells in the brain and are implicated in the clearance of brain Aβ via their regulation of the blood-brain barrier, glymphatic system, and proteolytic degradation. The cellular morphology and activity of astrocytes are modulated by several molecules, including ω3 polyunsaturated fatty acids, such as docosahexaenoic acid, which is one of the most abundant lipids in the brain, via the G protein-coupled receptor GPR120/FFAR4. In this study, we analyzed the role of GPR120 signaling in the Aβ-degrading activity of astrocytes. Treatment with the selective antagonist upregulated the matrix metalloproteinase (MMP) inhibitor-sensitive Aβ-degrading activity in primary astrocytes. Moreover, the inhibition of GPR120 signaling increased the levels of Mmp2 and Mmp14 mRNAs, and decreased the expression levels of tissue inhibitor of metalloproteinases 3 (Timp3) and Timp4, suggesting that GPR120 negatively regulates the astrocyte-derived MMP network. Finally, the intracerebral injection of GPR120-specific antagonist substantially decreased the levels of TBS-soluble Aβ in male AD model mice, and this effect was canceled by the coinjection of an MMP inhibitor. These data indicate that astrocytic GPR120 signaling negatively regulates the Aβ-degrading activity of MMPs.SIGNIFICANCE STATEMENT The level of amyloid β (Aβ) in the brain is a crucial determinant of the development of Alzheimer's disease. Here we found that astrocytes, which are the most abundant cell type in the CNS, harbor degrading activity against Aβ, which is regulated by GPR120 signaling. GPR120 is involved in the inflammatory response and obesity in peripheral organs. However, the pathophysiological role of GPR120 in Alzheimer's disease remains unknown. We found that selective inhibition of GPR120 signaling in astrocytes increased the Aβ-degrading activity of matrix metalloproteases. Our results suggest that GPR120 in astrocytes is a novel therapeutic target for the development of anti-Aβ therapeutics.

Methylglyoxal Induces Changes in the Glyoxalase System and Impairs Glutamate Uptake Activity in Primary Astrocytes.

The impairment of astrocyte functions is associated with diabetes mellitus and other neurodegenerative diseases. Astrocytes have been proposed to be essential cells for neuroprotection against elevated levels of methylglyoxal (MG), a highly reactive aldehyde derived from the glycolytic pathway. MG exposure impairs primary astrocyte viability, as evaluated by different assays, and these cells respond to MG elevation by increasing glyoxalase 1 activity and glutathione levels, which improve cell viability and survival. However, C6 glioma cells have shown strong signs of resistance against MG, without significant changes in the glyoxalase system. Results for aminoguanidine coincubation support the idea that MG toxicity is mediated by glycation. We found a significant decrease in glutamate uptake by astrocytes, without changes in the expression of the major transporters. Carbenoxolone, a nonspecific inhibitor of gap junctions, prevented the cytotoxicity induced by MG in astrocyte cultures. Thus, our data reinforce the idea that astrocyte viability depends on gap junctions and that the impairment induced by MG involves glutamate excitotoxicity. The astrocyte susceptibility to MG emphasizes the importance of this compound in neurodegenerative diseases, where the neuronal damage induced by MG may be aggravated by the commitment of the cells charged with MG clearance.

Beneficial Function of Cell Division Cycle 2 Activity in Astrocytes on Axonal Regeneration after Spinal Cord Injury

Migrating activity of reactive astrocytes induced after spinal cord injury (SCI) controls glial scar formation by limiting inflammatory responses around the injury area, and, therefore, can be beneficial for regenerative responses of spinal axons. Recently, we found that cell division cycle 2 (cdc2) activity in primary astrocytes facilitated neurite outgrowth of co-cultured neurons. Here, we investigated the effects of cdc2 activity on regenerative processes in vivo after SCI. Administration of the cdc2 inhibitor purvalanol A restricted compaction of the injury cavity and astrocyte infiltration into the cavity. After SCI, regenerative responses of anterogradely labeled corticospinal tract (CST) axons were attenuated by purvalanol A treatment. Using the polymeric tube that was implanted into the spinal cord as a nerve guide conduit, we found that purvalanol A treatments reduced astrocyte migration into the tube graft and, in parallel, retarded the extension of spinal axons into the tube. These results suggest that astrocytes with cdc2 activity may play a permissive role in mediating regrowth of spinal axons after lesion.

Antidepressants inhibit DNA methyltransferase 1 through reducing G9a levels

The discovery of epigenetic processes as possible pivotal regulatory mechanisms in psychiatric diseases raised the question of how psychoactive drugs may impact the epigenetic machinery. In the present study we set out to explore the specificity and the mode of action of the reported inhibitory effect of the TCA (tricyclic antidepressant) amitriptyline on DNMT (DNA methyltransferase) activity in primary astrocytes from the rat cortex. We found that the impact on DNMT was shared by another TCA, imipramine, and by paroxetine, but not by venlafaxine or the mood stabilizers carbamazepine and valproic acid. DNMT activity in subventricular neural stem cells was refractory to the action of ADs (antidepressants). Among the established DNMTs, ADs primarily targeted DNMT1. The reduction of enzymatic DNMT1 activity was neither due to reduced DNMT1 expression nor due to direct drug interference. We tested putative DNMT1-inhibitory mechanisms and discovered that a known stimulator of DNMT1, the histone methyltransferase G9a, exhibited decreased protein levels and interactions with DNMT1 upon AD exposure. Adding recombinant G9a completely reversed the AD repressive effect on DNMT1 function. In conclusion, the present study presents a model where distinct ADs affect DNMT1 activity via G9a with important repercussions for possible novel treatment regimes.

Inhibitory effect of the antidepressant imipramine on NF-κB-dependent CXCL1 expression in TNFα-exposed astrocytes

Neuroinflammation is associated with the pathophysiology of various neurodegenerative diseases. Emerging evidence indicates that imipramine, a tricyclic antidepressant commonly used in depressive disorders, exhibits neuroprotective activity partly through anti-inflammatory effects. However, the molecular mechanisms underlying imipramine-mediated anti-inflammatory response are poorly understood. In this study, rat primary cultured astrocytes were used to elucidate the effect of the imipramine on TNFα-induced inflammatory responses. The results clearly demonstrated that imipramine reduced TNFα-induced CXCL1 expression through suppression of NF-κB-dependent CXCL1 promoter activity in primary astrocytes. In addition, we found that imipramine suppressed TNFα-induced phosphorylation of inhibitor of κBα (IκBα) and p65/RelA nuclear factor-κB (NF-κB), as well as the nuclear translocation of p65/RelA in primary cultured astrocytes. Chemotaxis assay demonstrated that astrocyte-derived CXCL1 contributed to migration of BV2 microglial cells toward astrocytes. This response was significantly blocked by treatment of astrocytes with imipramine or NF-κB inhibitor BAY11-7082. This study indicates that the antidepressant imipramine inhibits TNFα-induced CXCL1 expression via down-regulation of NF-κB signaling pathway in astrocytes and suggests that imipramine has a potential as an anti-inflammatory drug.

Apolipoprotein E-specific innate immune response in astrocytes from targeted replacement mice

BackgroundInheritance of the three different alleles of the human apolipoprotein (apo) E gene (APOE) are associated with varying risk or clinical outcome from a variety of neurologic diseases. ApoE isoform-specific modulation of several pathogenic processes, in addition to amyloid β metabolism in Alzheimer's disease, have been proposed: one of these is innate immune response by glia. Previously we have shown that primary microglia cultures from targeted replacement (TR) APOE mice have apoE isoform-dependent innate immune activation and paracrine damage to neurons that is greatest with TR by the ε4 allele (TR APOE4) and that derives from p38 mitogen-activated protein kinase (p38MAPK) activity.MethodsPrimary cultures of TR APOE2, TR APOE3 and TR APOE4 astrocytes were stimulated with lipopolysaccharide (LPS). ApoE secretion, cytokine production, and nuclear factor-kappa B (NF-κB) subunit activity were measured and compared.ResultsHere we showed that activation of primary astrocytes from TR APOE mice with LPS led to TR APOE-dependent differences in cytokine secretion that were greatest in TR APOE2 and that were associated with differences in NF-κB subunit activity.ConclusionOur results suggest that LPS activation of innate immune response in TR APOE glia results in opposing outcomes from microglia and astrocytes as a result of TR APOE-dependent activation of p38MAPK or NF-κB signaling in these two cell types.

Transcriptional regulation of human excitatory amino acid transporter 1 (EAAT1): cloning of the EAAT1 promoter and characterization of its basal and inducible activity in human astrocytes

Excitatory amino acid transporter 1 (EAAT1) is one of the two glial glutamate transporters that clear the extracellular glutamate generated during neuronal signal transmission. Here, we cloned and characterized a 2.1-kb promoter region of human EAAT1 and investigated its function in the transcriptional regulation of the EAAT1 gene in human primary astrocytes. The full-length promoter region lacked TATA and CCAAT boxes and an initiator element, it contained several potential transcription factor-binding sites and it exhibited promoter activity in primary astrocytes and in several types of transformed cells. Consecutive 5'-deletion analysis of the EAAT1 promoter indicated the presence of negative and positive regulatory regions and a putative core promoter between -57 bp and +20 bp relative to the transcription start site (TSS). The core promoter contained a single GC-box in position -52/-39 and one E-box near the TSS and the GC-box site that was responsible for 90% of the basal promoter activity as determined by mutational analysis. Electrophoretic mobility shift, supershift and competition assays demonstrated binding of stimulating proteins (Sp) 1 and 3 to the GC-box and upstream stimulating factor (USF) 1 to the E-box. Treatment of primary human astrocytes with cellular modulators 8-bromo cyclic AMP and epidermal growth factor increased EAAT1 promoter activity in transient transfection assays and increased cellular EAAT1 mRNA expression and glutamate uptake by astrocytes. Conversely, tumor necrosis factor-alpha reduced both EAAT promoter activity and cellular EAAT1 mRNA expression. These results enable studies of transcriptional regulation of EAAT1 gene at the promoter level.

Regulatory sequences for the transcription of the laminin B2 gene in astrocytes

Astrocytes synthesize only the B2 chain of laminin and that this chain is sufficient to stimulate neurite outgrowth. In this study, we have examined laminin B1 and B2 promoter constructs in various cell types in order to understand the transcriptional regulation of laminin B2 gene in astrocytes. Comparison of nuclear factor binding by Southwestern analysis with the highly active B2 promoter fragment revealed different patterns of nuclear factor binding. In HepG2 cells, two proteins of 105 and 98 kDa were identified while, in primary astrocytes, human U251 and rat C6 glioma cells, a greater number of nuclear proteins ranging from 43 to 212 kDa were detected. The laminin B1 promoter construct was inactive in transient transfection experiments in astrocytes yet active in the HepG2 hepatoma cells which synthesize both the B1 and B2 chains. In contrast, the laminin B2 promoter construct was active in both astrocytes and HepG2 cells. These results are consistent with the lack of laminin B1 mRNA expression in astrocytes and suggest that the differential regulation of the laminin B1 and B2 gene is controlled at the transcriptional level. Delineation of the 5′-flanking regions responsible for basal levels of B2 laminin promoter activity revealed a silencer-like segment between −830 and −224 which reduced promoter activity. Deletion analysis further revealed that B2 laminin promoter possesses a highly active short promoter (−94 to +106) and basal transcriptional activity resides within −61 to +106. DNase 1 footprinting, gel-shift competition assays and site-directed mutagenesis of a highly active short promoter revealed that this region contained binding sites for cell-type nuclear factors. The shortest construct containing only residues −21 to +106 was inactive in HepG2 and U251 glioma cells. In primary astrocytes, however, this construct showed a high level of transcriptional activity. Deletion of 47 bp (+59 to +106) in 5′-UTR completely blocked promoter activity in astrocytes confirming that this downstream region is important for transcriptional activity in primary astrocytes. Together, these results suggest that astrocytes may utilize mutually exclusive transcription factors and regulatory sequences, in addition to common factors in the control of the laminin B2 promoter.

TGF-beta suppression of IFN-gamma-induced class II MHC gene expression does not involve inhibition of phosphorylation of JAK1, JAK2, or signal transducers and activators of transcription, or modification of IFN-gamma enhanced factor X expression.

TGF-beta is a widely expressed immunoregulatory protein that exerts a diverse range of effects on many types of cells. One of the effects of TGF-beta is the inhibition of both constitutive and cytokine-inducible class II MHC gene expression. In this study, we demonstrate that TGF-beta inhibits expression of class II MHC surface protein, mRNA, and promoter activity in primary astrocytes, and that this inhibition is both dose and time dependent. TGF-beta does not act to inhibit IFN-gamma-induced gene expression in a global fashion, as induction of ICAM-1 and IRF-1 gene expression by IFN-gamma is unaffected by treatment with TGF-beta. Furthermore, TGF-beta does not affect events that are involved in IFN-gamma-induced intracellular signaling such as tyrosine phosphorylation of JAK1, JAK2, and STAT1 alpha, nor does it affect IFN-gamma induction of the class II X2 box binding protein IFN-gamma enhanced factor X. We speculate that TGF-beta may be exerting its effects by modulating the expression or function of constitutively expressed factors responsible for regulation of class II MHC gene expression in astrocytes.

Multinuclear NMR Studies on the Energy Metabolism of Glial and Neuronal Cells

In this multinuclear NMR study myo-inositol is identified as a glia-specific marker for in vivo NMR studies. The unusually high inositol concentration may participate in the osmoregulatory system in astrocytes. Primary astrocytes also synthesize and export high amounts of hypotaurine, an intermediate of taurine synthesis. Taurine--another osmolyte--is synthesized from cysteine by astrocytes but not by primary neurons. Taurine as well as hypotaurine is accumulated by neurons from the extracellular medium. 13C NMR labelling results with 2-13C pyruvate indicate a considerable contribution of the anaplerotic pathway in primary neurons from rat. The activity is only half of the activity in primary astrocytes. The ratio of pyruvate carboxylase/malic enzyme activity versus pyruvate dehydrogenase activity reflects the degree of maturation. The 13C isotopomer ratio of glutamate and glutamine is different for pure astrocyte cultures. Therefore, the different isotopomer ratios of glutamate to glutamine obtained from intact brain studies alone do not prove TCA cycle compartimentation in the brain. Finally, the PCr/ATP ratio in primary astrocytes is 3 times higher than in primary neurons. This has to be considered in case of recovery from ischemic insults.