Primary Mouse Astrocytes Research Articles

Indoxyl Sulfate Affects Glial Function Increasing Oxidative Stress and Neuroinflammation in Chronic Kidney Disease: Interaction between Astrocytes and Microglia.

Indoxyl sulfate (IS) is a protein-bound uremic toxin resulting from the metabolism of dietary tryptophan which accumulates in patients with impaired renal function, such as chronic kidney disease (CKD). IS is a well-known nephrovascular toxin but little is known about its effects on central nervous system (CNS) cells. Considering the growing interest in the field of CNS comorbidities in CKD, we studied the effect of IS on CNS cells. IS (15–60 μM) treatment in C6 astrocyte cells increased reactive oxygen species release and decreased nuclear factor (erythroid-derived 2)-like 2 (Nrf2) activation, and heme oxygenase-1 (HO-1) and NAD(P)H dehydrogenase quinone 1 expression. Moreover, IS increased Aryl hydrocarbon Receptor (AhR) and Nuclear Factor-kB (NF-kB) activation in these cells. Similiar observations were made in primary mouse astrocytes and mixed glial cells. Inducible nitric oxide synthase and cyclooxygenase-2 (COX-2) expression, tumor necrosis factor-α and interleukin-6 release and nitrotyrosine formation were increased by IS (15–60 μM) in primary mouse astrocytes and mixed glial cells. IS increased AhR and NF-kB nuclear translocation and reduced Nrf2 translocation and HO-1 expression in primary glial cells. In addition, IS induced cell death in neurons in a dose dependent fashion. Injection of IS (800 mg/kg, i.p.) into mice induced histological changes and increased COX-2 expression and nitrotyrosine formation in thebrain tissue. Taken together, our results show a significant contribution of IS in generating a neurotoxic enviroment and it could also have a potential role in neurodegeneration. IS could be considered also a potential therapeutical target for CKD-associated neurodegenerative complications.

Manganese exposure induces neuroinflammation by impairing mitochondrial dynamics in astrocytes

Chronic manganese (Mn) exposure induces neurotoxicity, which is characterized by Parkinsonian symptoms resulting from impairment in the extrapyramidal motor system of the basal ganglia. Mitochondrial dysfunction and oxidative stress are considered key pathophysiological features of Mn neurotoxicity. Recent evidence suggests astrocytes as a major target of Mn neurotoxicity since Mn accumulates predominantly in astrocytes. However, the primary mechanisms underlying Mn-induced astroglial dysfunction and its role in metal neurotoxicity are not completely understood. In this study, we examined the interrelationship between mitochondrial dysfunction and astrocytic inflammation in Mn neurotoxicity. We first evaluated whether Mn exposure alters mitochondrial bioenergetics in cultured astrocytes. Metabolic activity assessed by MTS assay revealed an IC50 of 92.68μM Mn at 24h in primary mouse astrocytes (PMAs) and 50.46μM in the human astrocytic U373 cell line. Mn treatment reduced mitochondrial mass, indicative of impaired mitochondrial function and biogenesis, which was substantiated by the significant reduction in mRNA of mitofusin-2, a protein that serves as a ubiquitination target for mitophagy. Furthermore, Mn increased mitochondrial circularity indicating augmented mitochondrial fission. Seahorse analysis of bioenergetics status in Mn-treated astrocytes revealed that Mn significantly impaired the basal mitochondrial oxygen consumption rate as well as the ATP-linked respiration rate. The effect of Mn on mitochondrial energy deficits was further supported by a reduction in ATP production. Mn-exposed primary astrocytes also exhibited a severely quiescent energy phenotype, which was substantiated by the inability of oligomycin to increase the extracellular acidification rate. Since astrocytes regulate immune functions in the CNS, we also evaluated whether Mn modulates astrocytic inflammation. Mn exposure in astrocytes not only stimulated the release of proinflammatory cytokines, but also exacerbated the inflammatory response induced by aggregated α-synuclein. The novel mitochondria-targeted antioxidant, mito-apocynin, significantly attenuated Mn-induced inflammatory gene expression, further supporting the role of mitochondria dysfunction and oxidative stress in mediating astrogliosis. Lastly, intranasal delivery of Mn in vivo elevated GFAP and depressed TH levels in the olfactory bulbs, clearly supporting the involvement of astrocytes in Mn-induced dopaminergic neurotoxicity. Collectively, our study demonstrates that Mn drives proinflammatory events in astrocytes by impairing mitochondrial bioenergetics.

Combined HMG-COA reductase and prenylation inhibition in treatment of CCM

Cerebral cavernous malformations (CCMs) are common vascular anomalies that develop in the central nervous system and, more rarely, the retina. The lesions can cause headache, seizures, focal neurological deficits, and hemorrhagic stroke. Symptomatic lesions are treated according to their presentation; however, targeted pharmacological therapies that improve the outcome of CCM disease are currently lacking. We performed a high-throughput screen to identify Food and Drug Administration-approved drugs or other bioactive compounds that could effectively suppress hyperproliferation of mouse brain primary astrocytes deficient for CCM3. We demonstrate that fluvastatin, an inhibitor of 3-hydroxy-3-methyl-glutaryl (HMG)-CoA reductase and the N-bisphosphonate zoledronic acid monohydrate, an inhibitor of protein prenylation, act synergistically to reverse outcomes of CCM3 loss in cultured mouse primary astrocytes and in Drosophila glial cells in vivo. Further, the two drugs effectively attenuate neural and vascular deficits in chronic and acute mouse models of CCM3 loss in vivo, significantly reducing lesion burden and extending longevity. Sustained inhibition of the mevalonate pathway represents a potential pharmacological treatment option and suggests advantages of combination therapy for CCM disease.

Synthesis and Evaluation of Parenchymal Retention and Efficacy of a Metabolically Stable O-Phosphocholine-N-docosahexaenoyl-l-serine siRNA Conjugate in Mouse Brain.

Ligand-conjugated siRNAs have the potential to achieve targeted delivery and efficient silencing in neurons following local administration in the central nervous system (CNS). We recently described the activity and safety profile of a docosahexaenoic acid (DHA)-conjugated, hydrophobic siRNA (DHA-hsiRNA) targeting Huntingtin (Htt) mRNA in mouse brain. Here, we report the synthesis of an amide-modified, phosphocholine-containing DHA-hsiRNA conjugate (PC-DHA-hsiRNA), which closely resembles the endogenously esterified biological structure of DHA. We hypothesized that this modification may enhance neuronal delivery in vivo. We demonstrate that PC-DHA-hsiRNA silences Htt in mouse primary cortical neurons and astrocytes. After intrastriatal delivery, Htt-targeting PC-DHA-hsiRNA induces ∼80% mRNA silencing and 71% protein silencing after 1 week. However, PC-DHA-hsiRNA did not substantially outperform DHA-hsiRNA under the conditions tested. Moreover, at the highest locally administered dose (4 nmol, 50 μg), we observe evidence of PC-DHA-hsiRNA-mediated reactive astrogliosis. Lipophilic ligand conjugation enables siRNA delivery to neural tissues, but rational design of functional, nontoxic siRNA conjugates for CNS delivery remains challenging.

Establishment and characterization of primary astrocyte culture from adult mouse brain

As a major class of glial cells, astrocytes have been indicated to play multi-roles in physiological and pathological brain. Astrocyte cultures derived from postnatal mouse brains have been extensively used to characterize their biological properties. However, the inability to culture adult mouse primary astrocytes has long stymied studies of function in adult brain. Here, we developed a protocol to successfully establish highly enriched astrocyte cultures from the brains of adult mouse. Cortical tissues were collected to prepare cell suspension by enzymatic digestion and mechanical dissociation, and then plated onto vessels pre-coated with gelatin and matrigel and cultured in DMEM medium containing 20% fetal bovine serum (FBS). Forskolin (FSK) and glial-derived neurotrophic factor (GDNF) were use to promote astrocyte proliferation and survival respectively. These adult astrocyte cultures were identified by immunocytochemical, immunobloting and PCR analysis. Furthermore, biological and functional analysis indicated that they possess the biochemical and physiological properties of astrocytes, suggestive of a useful cell model for astroglial studies in adult brain.

Manganese Exposure Induces Neuroinflammation by Impairing Mitochondrial Dynamics in Astrocytes

Chronic manganese (Mn) exposure induces neurotoxicity, which is characterized by Parkinsonian symptoms resulting from impairment in the extrapyramidal motor system of the basal ganglia. Mitochondrial dysfunction and oxidative stress are considered key pathophysiological features of Mn neurotoxicity. Recent evidence suggests astrocytes as a major target of Mn neurotoxicity since Mn accumulates predominantly in astrocytes. However, the primary mechanisms underlying Mn‐induced astroglial dysfunction and its role in metal neurotoxicity are not completely understood. In this study, we examined the interrelationship between mitochondrial dysfunction and astrocytic inflammation in Mn neurotoxicity. We first evaluated whether Mn exposure alters mitochondrial bioenergetics in cultured astrocytes. Metabolic activity assessed by MTS assay revealed an IC50 of ~ 100 μM Mn at 24 h in both primary mouse astrocytes (PMAs) and the human astrocytic cell line U373s. Furthermore, Mn treatment reduced mitochondrial mass, indicative of impaired mitochondrial function and biogenesis, which is substantiated by the significant reduction in mRNA of mitofusin‐2, a protein that serves as a ubiquitination target for mitophagy. Seahorse analysis of bioenergetics status in Mn‐treated astrocytes revealed that Mn significantly impaired the basal mitochondrial oxygen consumption rate as well as the ATP‐linked respiration rate. The effect of Mn on mitochondrial energy deficits was further supported by a reduction in ATP production. Mn‐exposed PMAs also exhibited a severe quiescent energy phenotype, which was substantiated by the inability of oligomycin to increase the extracellular acidification rate. Since astrocytes regulate immune functions in CNS, we also evaluated whether Mn modulates astrocytic inflammation. Mn exposure in astrocytes not only stimulated the release of proinflammatory cytokines, but also exacerbated the inflammatory response induced by aggregated α‐synuclein. Lastly, the novel mitochondria‐targeted antioxidant, mito‐apocynin, significantly attenuated Mn‐induced inflammatory gene expression in U373s, further supporting the role of mitochondria dysfunction and oxidative stress in mediating astrogliosis. Collectively, we demonstrate for the first time that Mn drives proinflammatory events in astrocytes by impairing mitochondrial bioenergetics.Support or Funding InformationNIH grants ES026892 and NS088206

Chronic infusion of SOD1G93A astrocyte-secreted factors induces spinal motoneuron degeneration and neuromuscular dysfunction in healthy rats.

Amyotrophic lateral sclerosis is a fatal neurodegenerative disease and studies in vitro show that motoneuron degeneration is triggered by non-cell-autonomous mechanisms. However, whether soluble toxic factor(s) released by mutant superoxide dismutase 1 (SOD1) expressing astrocytes induces death of motoneurons and leads to motor dysfunction in vivo is not known. To directly test this, healthy adult rats were treated with conditioned media derived from primary mouse astrocytes (ACM) that express human (h) SOD1G93A (ACM-hG93A) via chronic osmotic pump infusion in the lumbar spinal cord. Controls included ACM derived from transgenic mice expressing hSOD1WT (ACM-hWT) or non-transgenic mouse SOD1WT (ACM-WT) astrocytes. Rats chronically infused with ACM-hG93A started to develop motor dysfunction at 8 days, as measured by rotarod performance. Additionally, immunohistochemical analyses at day 16 revealed reactive astrogliosis and significant loss of motoneurons in the ventral horn of the infused region. Controls did not show significant motor behavior alterations or neuronal damage. Thus, we demonstrate that factors released in vitro from astrocytes derived from ALS mice cause spinal motoneuron death and consequent neuromuscular dysfunction in vivo.

Oxidized and degraded mitochondrial polynucleotides (DeMPs), especially RNA, are potent immunogenic regulators in primary mouse macrophages

Certain mitochondrial components can act as damage-associated molecular patterns (DAMPs) or danger signals, triggering a proinflammatory response in target (usually immune) cells. We previously reported the selective degradation of mitochondrial DNA and RNA in response to cellular oxidative stress, and the immunogenic effect of this DNA in primary mouse astrocytes. Here, we extend these studies to assess the immunogenic role of both mitochondrial DNA and RNA isolated from hydrogen peroxide (HP) treated HA1 cells (designated “DeMPs” for degraded mitochondrial polynucleotides) using mouse bone marrow derived macrophages (BMDMs), a conventional immune cell type. DeMPs and control mitochondrial DNA (cont mtDNA) and RNA (cont mtRNA) were transfected into BMDMs and cell-free media analyzed for the presence of proinflammatory cytokines (IL-6, MCP-1, and TNFα) and Type I interferon (IFN-α and IFN-β). Cont mtDNA induced IL-6 and MCP-1 production, and this effect was even greater with DeMP DNA. A similar response was observed for Type I interferons. An even stronger induction of proinflammatory cytokine and type 1 interferons was observed for cont mtRNA. However, contrary to DeMP DNA, DeMP RNA attenuated rather than potentiated the cont mtRNA cytokine inductions. This attenuation effect was not accompanied by an IL-10 or TGFβ anti-inflammatory response. All DeMP effects were observed at multiple oxidant concentrations. Finally, DeMP production and immunogenicity overlaps with cellular adaptive response and so may contribute to cellular oxidant protection. These results provide new insight into the immunogenicity of mitochondrial polynucleotides, and identify new roles and selective consequences of cellular oxidation.

Mechanism of 2,3,4',5-Tetrahydroxystilbene 2-O-β-D-Glucoside-Induced Upregulation of Glutamate Transporter 1 Protein Expression in Mouse Primary Astrocytes

Glutamate transporter-1 (GLT-1), a major glutamate transporter expressed in astrocytes, takes up excess glutamate from the micro-environment in order to prevent excitotoxicity. Drugs that increase GLT-1 expression may have therapeutic effects in disorders associated with neuronal excitotoxicity. 2,3,4',5-tetrahydroxystilbene 2-O-β-<smlcap>D</smlcap>-glucoside (TSG), a monomer of stilbene from polygonummultiflorum, exerts neuroprotection in a range of experimental models such as Alzheimer's disease and brain ischemia. In this study, we evaluated the effect of TSG on GLT-1 protein expression in mouse primary-cultured astrocytes. Results showed that TSG markedly increased the GLT-1 protein expression level in mouse primary-cultured astrocytes in a dose- and time-dependent manner, and this increase was mediated by the activation of protein kinase B (Akt) but not by the activation of extracellular signal-regulated protein kinase 1/2. Furthermore, inhibition of cAMP response element-binding protein, but not nuclear factor kappa B, abolished the TSG-mediated increase in GLT-1 protein expression in cultured astrocytes. Collectively, these findings may provide novel insights into the mechanism for TSG in neuroprotection, and would help search new agents targeting neurodegenerative disorders associated with impaired astrocytic glutamate transporters.

LRRK2 kinase inhibition, a potential Parkinson’s disease therapy, induces LRRK2 protein destabilization and proteasomal degradation

Event Abstract Back to Event LRRK2 kinase inhibition, a potential Parkinson’s disease therapy, induces LRRK2 protein destabilization and proteasomal degradation Evy Lobbestael1*, Laura Civiero2, Tina De Wit1, Jean-Marc Taymans3, Elisa Greggio2 and Veerle Baekelandt1 1 KU Leuven, Neurosciences, Belgium 2 University of Padova, Italy 3 INSERM-CHRU de Lille-Université de Lille, France Leucine-rich repeat kinase 2 (LRRK2) kinase activity is increased in several pathogenic mutations, including the most common mutation, G2019S, and plays a role in Parkinson’s disease (PD) pathobiology. LRRK2 kinase inhibitors are currently one of the most prevailing disease-modifying therapeutic PD strategies and although several lines of evidence support beneficial effects, many questions need to be answered before clinical applications can be envisaged. We aimed to gain insight in the LRRK2 kinase inhibitor-induced reduction in LRRK2 protein levels. We used six different LRRK2 kinase inhibitors administered to mice, primary cultures and cell lines and monitored LRRK2 (phosphorylation) levels via immunoblotting at different time points. LRRK2 kinase inhibition induces LRRK2 dephosphorylation and can reduce LRRK2 protein levels of overexpressed wild-type and G2019S, but not A2016T or K1906M, LRRK2 as well as endogenous LRRK2 in mouse brain and primary astrocytes. This effect could be reversed by proteasomal inhibition, but not by lysosomal inhibition, while mRNA levels remained unaffected. Using LRRK2 S910A and S935A phosphorylation mutants, we show that dephosphorylation of these sites is not required for LRRK2 degradation. Interestingly, a truncated form of LRRK2, detected in mouse kidney, is dephosphorylated upon LRRK2 kinase inhibition, but not destabilized. In conclusion, we show that LRRK2 kinase inhibitor-induced LRRK2 destabilization is mediated by proteasomal degradation. This effect appears to be cell type specific and is not (only) dependent on dephosphorylation at LRRK2 S910 and S935. Increasing our insight in the molecular consequences of LRRK2 kinase inhibition will be crucial in the development of LRRK2-based PD therapies. Keywords: LRRK2, Kinase inhibition, Parkinson's disease, therapy, proteasomal degradation Conference: 12th National Congress of the Belgian Society for Neuroscience, Gent, Belgium, 22 May - 22 May, 2017. Presentation Type: Oral Presentation Topic: Disorders of the Nervous System Citation: Lobbestael E, Civiero L, De Wit T, Taymans J, Greggio E and Baekelandt V (2019). LRRK2 kinase inhibition, a potential Parkinson’s disease therapy, induces LRRK2 protein destabilization and proteasomal degradation. Front. Neurosci. Conference Abstract: 12th National Congress of the Belgian Society for Neuroscience. doi: 10.3389/conf.fnins.2017.94.00100 Copyright: The abstracts in this collection have not been subject to any Frontiers peer review or checks, and are not endorsed by Frontiers. They are made available through the Frontiers publishing platform as a service to conference organizers and presenters. The copyright in the individual abstracts is owned by the author of each abstract or his/her employer unless otherwise stated. Each abstract, as well as the collection of abstracts, are published under a Creative Commons CC-BY 4.0 (attribution) licence (https://creativecommons.org/licenses/by/4.0/) and may thus be reproduced, translated, adapted and be the subject of derivative works provided the authors and Frontiers are attributed. For Frontiers’ terms and conditions please see https://www.frontiersin.org/legal/terms-and-conditions. Received: 21 Apr 2017; Published Online: 25 Jan 2019. * Correspondence: Dr. Evy Lobbestael, KU Leuven, Neurosciences, Leuven, Belgium, evy.lobbestael@kuleuven.be Login Required This action requires you to be registered with Frontiers and logged in. To register or login click here. Abstract Info Abstract The Authors in Frontiers Evy Lobbestael Laura Civiero Tina De Wit Jean-Marc Taymans Elisa Greggio Veerle Baekelandt Google Evy Lobbestael Laura Civiero Tina De Wit Jean-Marc Taymans Elisa Greggio Veerle Baekelandt Google Scholar Evy Lobbestael Laura Civiero Tina De Wit Jean-Marc Taymans Elisa Greggio Veerle Baekelandt PubMed Evy Lobbestael Laura Civiero Tina De Wit Jean-Marc Taymans Elisa Greggio Veerle Baekelandt Related Article in Frontiers Google Scholar PubMed Abstract Close Back to top Javascript is disabled. Please enable Javascript in your browser settings in order to see all the content on this page.

Hyperhomocysteinemia causes ER stress and impaired autophagy that is reversed by Vitamin B supplementation.

Hyperhomocysteinemia (HHcy) is a well-known risk factor for stroke; however, its underlying molecular mechanism remains unclear. Using both mouse and cell culture models, we have provided evidence that impairment of autophagy has a central role in HHcy-induced cellular injury in the mouse brain. We observed accumulation of LC3B-II and p62 that was associated with increased MTOR signaling in human and mouse primary astrocyte cell cultures as well as a diet-induced mouse model of HHcy, HHcy decreased lysosomal membrane protein LAMP2, vacuolar ATPase (ATP6V0A2), and protease cathepsin D, suggesting that lysosomal dysfunction also contributed to the autophagic defect. Moreover, HHcy increased unfolded protein response. Interestingly, Vitamin B supplementation restored autophagic flux, alleviated ER stress, and reversed lysosomal dysfunction due to HHCy. Furthermore, the autophagy inducer, rapamycin was able to relieve ER stress and reverse lysosomal dysfunction caused by HHcy in vitro. Inhibition of autophagy by HHcy exacerbated cellular injury during oxygen and glucose deprivation and reperfusion (OGD/R), and oxidative stress. These effects were prevented by Vitamin B co-treatment, suggesting that it may be helpful in relieving detrimental effects of HHcy in ischemia/reperfusion or oxidative stress. Collectively, these findings show that Vitamin B therapy can reverse defects in cellular autophagy and ER stress due to HHcy; and thus may be a potential treatment to reduce ischemic damage caused by stroke in patients with HHcy.

(-)-Epicatechin, a Natural Flavonoid Compound, Protects Astrocytes Against Hemoglobin Toxicity via Nrf2 and AP-1 Signaling Pathways.

(-)-Epicatechin is a brain-permeable, natural product found at high concentrations in green tea and cocoa. Our previous research has shown that (-)-epicatechin treatment reduces hemorrhagic stroke injury via nuclear factor erythroid 2-related factor 2 (Nrf2) signaling pathway in vivo. However, the mechanism of action of this compound in modulation of oxidant stress and in protection against hemoglobin-induced astrocyte injury is unclear. Therefore, we explored the cellular and molecular mechanisms that underlie these protective effects in vitro. Mouse primary astrocytes isolated from wild-type mice and Nrf2 knockout (KO) mice were preconditioned with hemoglobin to simulate intracerebral hemorrhage (ICH) in vitro. Effects of (-)-epicatechin were measured by Western blotting, immunostaining, MTT assay, and reactive oxidant stress (ROS) assay. (-)-Epicatechin increased Nrf2 nuclear accumulation and cytoplasmic levels of superoxide dismutase 1 (SOD1) in wild-type astrocytes but did not increase SOD1 expression in Nrf2 knockout (KO) astrocytes. Furthermore, (-)-epicatechin treatment did not alter heme oxygenase 1 (HO1) expression in wild-type astrocytes after hemoglobin exposure, but it did decrease HO1 expression in similarly treated Nrf2 KO astrocytes. In both wild-type and Nrf2 KO astrocytes, (-)-epicatechin suppressed phosphorylated JNK and nuclear expression of JNK, c-jun, and c-fos, indicating that inhibition of activator protein-1 (AP-1) activity by (-)-epicatechin is Nrf2-independent. These novel findings indicate that (-)-epicatechin protects astrocytes against hemoglobin toxicity through upregulation of Nrf2 and inhibition of AP-1 activity. These cellular and molecular effects may partially explain the cerebroprotection as we previously observed for (-)-epicatechin in animal models of ICH.

Systemic and Brain Pharmacokinetics of Perforin Inhibitor Prodrugs.

We have recently reported that by converting a perforin inhibitor into an l-type amino acid transporter 1 (LAT1)-utilizing prodrug its cellular uptake can be greatly increased. The aim of the present study was to determine the in vivo and brain pharmacokinetics of two perforin inhibitors and their LAT1-utilizing prodrugs 1 and 2. In addition, the brain uptake mechanism and entry into primary mouse cortical neurons and astrocytes were evaluated. After 23 μmol/kg i.p. bolus injection, the prodrugs' unbound area under the concentration curve in brain was 0.3 nmol/g × min, whereas the parent drugs could not reach the brain. The unbound brain concentrations of the prodrugs after 100 μM in situ mouse brain perfusion were 521.4 ± 46.9 and 126.9 ± 19.9 pmol/g for prodrugs 1 and 2, respectively. The combination of competing transporter substrates for LAT1, l-tryptophan, and for organic anion transporting polypeptides, probenecid, decreased the brain concentrations to 352.4 ± 44.5 and 70.9 ± 7.0 pmol/g, respectively. In addition, in vitro uptake studies showed that at 100 μM prodrug 1 had 3.4-fold and 4.5-fold higher uptake rate into neurons and astrocytes, respectively, compared to its parent drug. Thus, the prodrugs enhance significantly the therapeutic potential of the parent drugs for the treatment of disorders of central nervous system in which neuroinflammation is involved.

Tibolone protects astrocytic cells from glucose deprivation through a mechanism involving estrogen receptor beta and the upregulation of neuroglobin expression

Tibolone, a synthetic steroid used for the prevention of osteoporosis and the treatment of climacteric symptoms in post-menopausal women, may exert tissue selective estrogenic actions acting on estrogen receptors (ERs). We previously showed that tibolone protects human T98G astroglial cells against glucose deprivation (GD). In this study we have explored whether the protective effect of tibolone on these cells is mediated by ERs. Experimental studies showed that both ERα and ERβ were involved in the protection by tibolone on GD cells, being ERβ preferentially involved on these actions over ERα. Tibolone increased viability of GD cells by a mechanism fully blocked by an ERβ antagonist and partially blocked by an ERα antagonist. Furthermore, ERβ inhibition prevented the effect of tibolone on nuclear fragmentation, ROS and mitochondrial membrane potential in GD cells. The protective effect of tibolone was mediated by neuroglobin. Tibolone upregulated neuroglobin in T98G cells and primary mouse astrocytes by a mechanism involving ERβ and neuroglobin silencing prevented the protective action of tibolone on GD cells. In summary, tibolone protects T98G cells by a mechanism involving ERβ and the upregulation of neuroglobin.

Laquinimod prevents cuprizone-induced demyelination independent of Toll-like receptor signaling.

Objective:To test whether Toll-like receptor (TLR) signaling plays a key role for reduced nuclear factor B (NF-κB) activation after laquinimod treatment in the model of cuprizone-induced demyelination, oligodendrocyte apoptosis, inflammation, and axonal damage.Methods:Ten-week-old C57BL/6J, TLR4−/−, and MyD88−/− mice received 0.25% cuprizone for 6 weeks and were treated daily with 25 mg/kg laquinimod or vehicle. After 6 weeks of demyelination, extent of demyelination, oligodendrocyte density, microglia infiltration, and axonal damage were analyzed in the corpus callosum. Additionally, we analyzed primary mouse astrocytes from C57BL/6J, TLR4−/−, MyD88−/−, and TRIF−/− mice for alteration in NF-κB signaling.Results:Vehicle-treated controls from C57BL/6J, TLR4−/−, and MyD88−/− mice displayed extensive callosal demyelination as well as microglial activation. In contrast, mice treated with 25 mg/kg laquinimod showed mainly intact callosal myelin. The demyelination score was significantly higher in all untreated mice compared to mice treated with laquinimod. There were significantly fewer APP-positive axonal spheroids, Mac3-positive macrophages/microglia, and less oligodendrocyte apoptosis in the corpus callosum of laquinimod-treated mice in comparison to untreated controls. Stimulated primary mouse astrocytes from laquinimod-treated groups show reduced NF-κB activation compared to vehicle-treated controls.Conclusions:Our results confirm that laquinimod prevents demyelination in the cuprizone mouse model for multiple sclerosis via downregulation of NF-κB activation. This laquinimod effect, however, does not involve upstream Toll-like receptor signaling.

Identification of RNA-Binding Protein LARP4B as a Tumor Suppressor in Glioma.

Transposon-based insertional mutagenesis is a valuable method for conducting unbiased forward genetic screens to identify cancer genes in mice. We used this system to elucidate factors involved in the malignant transformation of neural stem cells into glioma-initiating cells. We identified an RNA-binding protein, La-related protein 4b (LARP4B), as a candidate tumor-suppressor gene in glioma. LARP4B expression was consistently decreased in human glioma stem cells and cell lines compared with normal neural stem cells. Moreover, heterozygous deletion of LARP4B was detected in nearly 80% of glioblastomas in The Cancer Genome Atlas database. LARP4B loss was also associated with low expression and poor patient survival. Overexpression of LARP4B in glioma cell lines strongly inhibited proliferation by inducing mitotic arrest and apoptosis in four of six lines as well as in two patient-derived glioma stem cell populations. The expression levels of CDKN1A and BAX were also upregulated upon LARP4B overexpression, and the growth-inhibitory effects were partially dependent on p53 (TP53) activity in cells expressing wild-type, but not mutant, p53. We further found that the La module, which is responsible for the RNA chaperone activity of LARP4B, was important for the growth-suppressive effect and was associated with BAX mRNA. Finally, LARP4B depletion in p53 and Nf1-deficient mouse primary astrocytes promoted cell proliferation and led to increased tumor size and invasiveness in xenograft and orthotopic models. These data provide strong evidence that LARP4B serves as a tumor-suppressor gene in glioma, encouraging further exploration of the RNA targets potentially involved in LARP4B-mediatd growth inhibition. Cancer Res; 76(8); 2254-64. ©2016 AACR.

Structural Basis for Nucleotide Hydrolysis by the Acid Sphingomyelinase-like Phosphodiesterase SMPDL3A

Sphingomyelin phosphodiesterase, acid-like 3A (SMPDL3A) is a member of a small family of proteins founded by the well characterized lysosomal enzyme, acid sphingomyelinase (ASMase). ASMase converts sphingomyelin into the signaling lipid, ceramide. It was recently discovered that, in contrast to ASMase, SMPDL3A is inactive against sphingomyelin and, surprisingly, can instead hydrolyze nucleoside diphosphates and triphosphates, which may play a role in purinergic signaling. As none of the ASMase-like proteins has been structurally characterized to date, the molecular basis for their substrate preferences is unknown. Here we report crystal structures of murine SMPDL3A, which represent the first structures of an ASMase-like protein. The catalytic domain consists of a central mixed β-sandwich surrounded by α-helices. Additionally, SMPDL3A possesses a unique C-terminal domain formed from a cluster of four α-helices that appears to distinguish this protein family from other phosphoesterases. We show that SMDPL3A is a di-zinc-dependent enzyme with an active site configuration that suggests a mechanism of phosphodiester hydrolysis by a metal-activated water molecule and protonation of the leaving group by a histidine residue. Co-crystal structures of SMPDL3A with AMP and α,β-methylene ADP (AMPCP) reveal that the substrate binding site accommodates nucleotides by establishing interactions with their base, sugar, and phosphate moieties, with the latter the major contributor to binding affinity. Our study provides the structural basis for SMPDL3A substrate specificity and sheds new light on the function of ASMase-like proteins.

IL-17 induces MIP-1α expression in primary mouse astrocytes via TRPC channel.

Our previous study demonstrated IL-17-mediated induction of MIP-1α through its binding to the cognate IL-17RA and MIP-1α was involved in astrocyte activation. Transient receptor potential canonical (TRPC) channel was involved in astrocyte activation, however, whether TRPC channel regulates MIP-1α expression in the context of multiple sclerosis (MS) remains largely unknown. In this study we identify the essential role of TRPC channel in IL-17-mediated MIP-1α expression and astrocyte activation. Moreover, treatment of astrocytes with IL-17 activated MAPKs and PI3K/Akt signaling pathways with downstream NF-κB pathways. Interestingly, the TRPC blocker-SKF96365 (10μM) and Norgestimate (10μM) significantly inhibited the increased expression of MIP-1α via suppression of IL-17-mediated ERK, p38 and JNK MAPKs and PI3K/Akt pathway activation, thereby underscoring the role of TRPC channel in this process. Together these data underpin the role of TRPC channel as a novel target that regulates MIP-1α expression and cell activation-mediated by IL-17 with implications for therapeutic intervention for reversal of neuroinflammation inflicted by IL-17. Understanding the regulation of MIP-1α expression may provide insights into the development of potential therapeutic targets for neuroinflammation associated with MS.

A chemical proteomic atlas of brain serine hydrolases identifies cell type-specific pathways regulating neuroinflammation.

Metabolic specialization among major brain cell types is central to nervous system function and determined in large part by the cellular distribution of enzymes. Serine hydrolases are a diverse enzyme class that plays fundamental roles in CNS metabolism and signaling. Here, we perform an activity-based proteomic analysis of primary mouse neurons, astrocytes, and microglia to furnish a global portrait of the cellular anatomy of serine hydrolases in the brain. We uncover compelling evidence for the cellular compartmentalization of key chemical transmission pathways, including the functional segregation of endocannabinoid (eCB) biosynthetic enzymes diacylglycerol lipase-alpha (DAGLα) and -beta (DAGLβ) to neurons and microglia, respectively. Disruption of DAGLβ perturbed eCB-eicosanoid crosstalk specifically in microglia and suppressed neuroinflammatory events in vivo independently of broader effects on eCB content. Mapping the cellular distribution of metabolic enzymes thus identifies pathways for regulating specialized inflammatory responses in the brain while avoiding global alterations in CNS function.

Interleukin 1β Regulation of the System xc− Substrate-specific Subunit, xCT, in Primary Mouse Astrocytes Involves the RNA-binding Protein HuR

System xc(-) is a heteromeric amino acid cystine/glutamate antiporter that is constitutively expressed by cells of the CNS, where it functions in the maintenance of intracellular glutathione and extracellular glutamate levels. We recently determined that the cytokine, IL-1β, increases the activity of system xc(-) in CNS astrocytes secondary to an up-regulation of its substrate-specific light chain, xCT, and that this occurs, in part, at the level of transcription. However, an in silico analysis of the murine xCT 3'-UTR identified numerous copies of adenine- and uridine-rich elements, raising the possibility that undefined trans-acting factors governing mRNA stability and translation may also contribute to xCT expression. Here we show that IL-1β increases the level of mRNA encoding xCT in primary cultures of astrocytes isolated from mouse cortex in association with an increase in xCT mRNA half-life. Additionally, IL-1β induces HuR translocation from the nucleus to the cytoplasm. RNA immunoprecipitation analysis reveals that HuR binds directly to the 3'-UTR of xCT in an IL-1β-dependent manner. Knockdown of endogenous HuR protein abrogates the IL-1β-mediated increase in xCT mRNA half-life, whereas overexpression of HuR in unstimulated primary mouse astrocytes doubles the half-life of constitutive xCT mRNA. This latter effect is accompanied by an increase in xCT protein levels, as well as a functional increase in system xc(-) activity. Altogether, these data support a critical role for HuR in mediating the IL-1β-induced stabilization of astrocyte xCT mRNA.