Background
The glutamate/glutamine cycle is an integral component of brain homeostasis. Neurons release glutamate, which is the most abundant neurotransmitter in the brain involved in learning, memory, and cognition. In excess, glutamate is neurotoxic and is linked to a number of neurodegenerative diseases such as Alzheimer’s disease [
1], Parkinson’s disease [
2], and neuroAIDS [
3,
4]. Both astrocytes and microglia have specific transporters to clear extracellular glutamate and convert it into glutamine in a regulated process. Upon glutamate uptake, glutamine synthetase (GS) catalyzes the ATP-dependent reaction of glutamate and ammonia into glutamine. Glutamine is released and in turn is taken up by neurons for conversion back to glutamate by glutaminase [
5].
Astrocytes have a dominant role in the glutamate/glutamine cycle. They clear >90 % of excess glutamate [
5,
6] through excitatory amino acid transporters (EAATs) 1 and 2. Although EAAT1 and EAAT2 are primarily expressed on astrocytes, EAAT2 (or GLT-1 in rodents) is the major transporter of glutamate on astrocytes [
7,
8]. The mechanism by which astrocytes regulate the expression of two key proteins in the glutamate/glutamine cycle, EAAT2 and GS, are not clearly understood. Studies have identified a role of nuclear factor kappa B (NF-kB) in the regulation of astrocytic EAAT2; however, NF-kB activation appears to have a bidirectional role. In mouse and human astrocytes, epidermal growth factor (EGF) stimulates NF-kB to induce EAAT2 expression [
9‐
12]. However, in human astrocytes, TNF-α stimulation of NF-kB reduces EAAT2 protein expression [
13]. Both scenarios require external stimulation of NF-kB activation suggesting a non-NF-kB-dependent regulatory pathway for baseline conditions of EAAT2 protein expression. Of note, although basal (e.g., without an inducing signal) expression of EAAT2 in rodent-derived astrocytes is little to undetectable [
9,
12], in human-derived astrocytes and astroglioma cell lines, EAAT2 expression is measureable making these cells ideal to study agents capable of down-modulating baseline EAAT2 [
7,
8,
14]. Regulation of astrocytic GS remains less defined although studies have indicated GS regulation by transient molecules including extracellular signals, inflammatory mediators, and hormones [
13,
15‐
17]. Studies of rat liver epithelial cell line RL-ET-14, mouse hepatocellular carcinoma, and human pancreatic tumors have revealed β-catenin regulation of EAAT2/GLT-1 and GS [
15,
18,
19]. Collectively, these studies reveal the complex and highly regulated nature of glutamate cycling protein regulation under basal and inducible conditions.
Using microarray analysis, we previously showed that β-catenin knockdown in human progenitor-derived astrocyte (PDAs) leads to modulation of genes relevant in regulating aspects of glutamate neurotransmission [
20]. This finding prompted further analysis of the direct role of β-catenin on key players in glutamate/glutamine cycling (EAAT2 and GS). β-Catenin is a central mediator of the Wnt/β-catenin pathway. In association with members of the T cell factor/lymphoid enhancer factor (TCF/LEF) family of transcription factors (TCF-1, TCF-3, TCF-4, LEF1), β-catenin functions as a transcriptional regulator of gene expression. β-Catenin also binds to cadherins to facilitate cell adhesion and cell-to-cell communication [
21]. The Wnt/β-catenin pathway is engaged by Wnt ligands, small secreted glycoproteins, which bind to members of the seven-transmembrane G protein-coupled Frizzled receptors and co-receptor lipoprotein receptor-related protein 5 or 6 (LRP5/6). This induces a complex signaling cascade resulting in glycogen synthetase kinase 3β (GSK3β) destabilization to hypophosphorylate β-catenin and prevention of proteosomal degradation. Relocation of β-catenin to the nucleus allows either association with TCF/LEF for gene regulation or relocation to the cell membrane for cadherin binding.
We evaluated here the role of β-catenin in regulating the expression of EAAT2 and GS in human astrocytes and mouse medial prefrontal cortex (mPFC). We show that β-catenin positively regulates EAAT2 and GS expression but through association with different TCF/LEF members. In regulating EAAT2 and GS expression, β-catenin interacts with TCF-1 and TCF-3, respectively. These findings demonstrate that the β-catenin pathway is a critical regulator of EAAT2 and GS expression, which can be harnessed to remove excess glutamate in neurodegenerative diseases.
Methods
Cell culture
Progenitor-derived astrocyte (PDAs) were generated from neural progenitor cells (kindly provided by Dr. Eugene Major, NINDS, NIH, MD) as previously described [
14]. Briefly, PDAs were maintained in progenitor medium consisting of Neurobasal media (Invitrogen) supplemented with 0.5 % bovine albumin (Sigma-Aldrich), neurosurvival factor (NSF; Lonza), N2 components (Invitrogen), 25 ng/ml basic fibroblast growth factor (bFGF), 20 ng/ml EGF (R&D Systems), 50 μg/ml gentamicin (Lonza), and 2 mM L-glutamine (Invitrogen). To induce differentiation, progenitor medium was replaced with PDA medium containing DMEM supplemented with 10 % heat-inactivated fetal bovine serum, 2 mM L-glutamine, and 50 μg/ml gentamicin. Cultures were >90 % positive for glial fibrillary acidic protein (GFAP) after 30 days of differentiation. Fetal-derived Normal Human Astrocytes (NHAs, Lonza) were maintained in Astrocyte Growth Media (AGM) BulletKit (Lonza) supplemented with 0.3 % heat-inactivated fetal bovine serum, 1 ml/ml ascorbic acid, 1 ml/ml rhEGF, 1 ml/ml AG-1000 (30 mg/ml gentamicin and 15 μg/ml amphotericin), 2.5 ml/ml insulin, and 10 ml/ml L-glutamine. Adherent primary cells were removed by treatment with 1 mM EDTA for 5 min with gentle scraping or pipetting multiple times.
Plasmids, transfection, and small interfering RNAs (siRNAs)
The TOPflash plasmid (Millipore) detecting β-catenin signaling contains multiple TCF/LEF binding sites linked to a firefly luciferase reporter. An approximate 2.5-kb human EAAT2 promoter linked to firefly luciferase plasmid (EAAT2-prom) was a kind gift from Dr. Paul B. Fisher (Virginia Common University, Virginia, USA). A pcDNA plasmid harboring the constitutively active β-catenin gene termed as pABC (S33Y mutation; 19286) or control pcDNA (10792) was obtained from Addgene. Plasmids were transfected using Lipofectamine 2000 as per manufacturer’s instruction (Invitrogen). ON-TARGET plus SMART pool siRNAs specific for TCF-1 (L-019735-00), TCF-3 (L-014703-00), TCF-4 (L-003816-00), LEF1 (L-015396-00), β-catenin (L-003482-00), and scrambled (D-001810-10) all from Thermo Fisher Dharmacon were transfected using Lipofectamine siRNAmax (Invitrogen Life Technologies) according to the reagent protocol. Cells were approximately 70–80 % confluent at the time of transfection.
Firefly luciferase reporter assay
Twenty-four hours post transfection, the culture medium was removed, cells were gently washed with PBS once, and 100 μl of passive lysis buffer (PLB) was added and incubated at 37 °C for 5 min. Cells were lysed by pipetting up and down several times and spun at 5000 rpm for 4 min to remove debris, and 10–20 μl was used to assay for luciferase activity using luciferase assay system (Promega) in a single injector luminometer. Total protein concentration was measured using bicinchoninic acid assay (BCA) protein assay kit (Thermo Fisher), and relative light units were normalized to micrograms of protein. Graphs were plotted from data obtained as a mean of three independent experiments with standard deviation as error bars.
Western blotting
In vitro cell samples were lysed by incubating with 50 μl of PLB for 5 min at 37 °C and pipetting up and down several times. In vivo brain tissue samples were lysed by incubating with 100 μl Radioimmunoprecipitation Assay (RIPA) buffer and homogenized. Both lysates were cleared for cell debris by spinning at 5000 rpm for 5 min. Total protein content of the cleared lysate was estimated by BCA, and 5–20 μg of total protein was separated on a 10 % SDS PAGE, transferred to a nitrocellulose membrane, blocked with superblock (Thermo Fisher) containing 0.1 % Tween 20 (T20) for 1 h, and incubated with appropriate primary antibodies overnight at 4 °C in superblock-0.1 % T20. Membranes were washed extensively with Tris buffer saline containing 0.1 % T20 (TBST) and incubated with appropriate secondary antibody in Superblock-0.1 % T20 for 45 min at room temperature (RT). Membranes were again washed extensively in TBST and developed with SuperSignal West Femto Maximum Sensitivity Substrate (Thermo Fisher) according to instructions. Relative quantification of proteins were carried out using ImageJ software and normalized to GAPDH (Glyceraldehyde 3-phosphate dehydrogenase). Primary antibodies used were as follows: anti-rabbit β-catenin antibody (Sigma-Aldrich; C 2206), anti-mouse EAAT2 antibody (Cell Signaling; 3838), anti-mouse GS antibody (Thermo Fisher; PA5-29737), and anti-rabbit GAPDH antibody (Sigma-Aldrich; G9545). Secondary antibodies were enhanced chemiluminescence horseradish peroxidase (ECL HRP)-linked rabbit IgG whole antibody (7074) or ECL HRP-linked mouse IgG whole antibody (7076) from Cell Signaling.
Quantitative real-time PCR
RNA was isolated using RNeasy mini kit (Qiagen). Digested with DNaseI (Sigma-Aldrich) for 15 min at RT to remove DNA contamination, and subsequently, DNaseI was inactivated by heating at 70 °C for 10 min. cDNA was synthesized using Qscript supermix (Quanta Biosciences). Real-time PCR was performed using SSO fast SYBR green supermix (Bio-Rad) in a 7500 real-time PCR system (Applied Biosystems) using 7500 software v2.0.1. Melting curve analysis was performed to ensure the amplification of a single product. Primers used were the following: TCF-1-F-5′-AGGCCAAGAAGCCAACCATCAAGA and TCF-1-R-5′-ACTCTGCAATGACCTTGGCTCTCA; TCF-3-F-5′-TGCAGTGAGCGTGAAATCACCAGT and TCF-3-R-5′-AATGGCTGCACTTTCCTTCAGGGT; TCF-4-F-5′-TCGGCAGAGAGGGATTTAGCTGATGT and TCF-4-R-5′-CTTTCCCGGGATTTGTCTCGGAAACT; LEF1-F-5′-AAGCATCCAGATGGAGGCCTCTACAA and LEF1-R-5′-TGATGTTCTCGGGATGGGTGGAGAAA; β-catenin-F-5′-TCTTGCCCTTTGTCCCGCAAATCA and β-catenin-R-5′-TCCACAAATTGCTGCGTCCCA; EAAT2-F-5′-CCAAGCTTGGATCACTGCCCTGG and EAAT2-R-5′-CCAGCCCCAAAAGAGTCACCCACAA; GS-F-5′-TTGAGAAACTAAGCAAGCGGCACC and GS-R-5′-ATCCAGTTAGACGTCGGGCATTGT; and GAPDH-F-5′-CTTCAACGACCACTTTGT and GAPDH-R-5′-TGGTCCAGGGGTCTTACT. Fold change in messenger RNA (mRNA) expression was calculated by relative quantification using the comparative C
T
method with GAPDH as the endogenous control.
Chromatin immunoprecipitation (ChIP)
PDAs were grown for 24–48 h and ChIPed for the presence of TCF-1 and β-catenin on the endogenous promoter region of EAAT2 or GS using a Millipore kit with antibodies for TCF-1, TCF-3 (anti-mouse, Cell Signaling; 9383), and β-catenin (anti-rabbit, Sigma-Aldrich; C 2206). A suitable mouse IgG1 isotype control (5415) or rabbit IgG control (3900) from Cell Signaling was included in the experiments. Similarly, ChIP was performed on PDA cells transfected with EAAT2-prom for 24 h using anti-TCF-1 antibody. Per immunoprecipitation, approximately 2 × 106 cells and 5 μg of antibody were used. Samples were analyzed by real-time PCR as explained earlier. Primers used to amplify EAAT2 promoter regions were divided into three regions: proximal (F-AAGACACACACTTACCCTTGACGG and R-CGTCTTAGGGCATTTGACTTTGGG), middle (F-TGTGGCCCTCCAAGTGAGTTCTTT and R-TGACGAGACCTGTGCAGCTTTGAT), and distal (F-GGGTGATGTCAGCTCTCGACGAA and R-AGGGAGGGATTGCAAGGTTTAGC). Primers used to amplify the putative GS promoter region were GS-F-TCTCTTGATGGTGCTGCTGTCACT and GS-R-AGAACGGAATGGTAAGGCGTGAGT. Data was normalized to IgG and represented as fold change with respect to IgG.
Glutamate uptake assay
Cells either transfected with plasmids for 24 h or siRNAs for 72 h were spiked with 2 mM L-glutamic acid (glutamate) and incubated for 30 min, and the final concentration of glutamate in the supernatant was measured by glutamate assay kit (Biovision) as per instructions. A standard curve was generated by diluting the provided glutamate standard in complete DMEM media.
Vivo-morpholino injection in mice
Animal studies were conducted with approval from the Rush University Medical Center Institutional Animal Care and Use Committee (IACUC). Male 10C57bl/6J mice aged 4–6 weeks were purchased from Jackson Laboratories. Approximately 1 week after arrival, animals were anesthetized with ketamine/xylazine (90 mg/kg, 5 mg/kg, I.P.) and placed in a stereotaxic apparatus. Bilateral injections of vivo-morpholinos (Gene Tools, LLC) were targeted to the mPFC with coordinates relative to Bregma +2.0 A/P, +0.3 M/L, −1.0 D/V. The antisense (AS) sequences for mouse β-catenin was designed from the larger of the two mRNA variants, one near the 5′ region (AS) 5′-GCCGCACAAGGAGCGATTTATAAGC-3′ and one spanning the translational start site (AS2) 5′-CTTGAGTAGCCATTGTCCACGCAGC-3′. A control morpholino sequence 5′-CCTCTTACCTCAGTTACAATTTATA-3′ or PBS was injected as a control. All morpholinos were at a concentration of 500 nM per 1.0 μl. Mice received 1 μl/hemisphere microinjections on day 1 and day 3. Mice were sacrificed by CO2 inhalation followed by rapid decapitation 4 days following the second surgery, and brain punches were isolated from the region targeted with the injections. Punches were homogenized in RIPA buffer, and western blots were run as described above.
Discussion
The β-catenin signaling pathway is vital to various functions in the CNS ranging from memory consolidation in astrocytes, neurogenesis, and neurotransmitter release to induction of long-term potentiation and depolarization resulting in increased synaptic strengths [
21,
23]. Disruption of β-catenin signaling has profound biologic consequences on astrocyte/neuron communication. We previously determined that disruption of β-catenin in astrocytes led to the modification of 128 genes [
20]. We showed here, in vitro and in vivo, that disruption of β-catenin leads to dramatic inhibition of the glutamate transport network (EAAT2/GS) within astrocytes.
Through gain and loss of function studies as well as ChIP assays, we demonstrate the molecular mechanism by which β-catenin regulates EAAT2 and GS expression in astrocytes. We show that β-catenin partners with TCF-1 to induce EAAT2 expression and partners with TCF-3 to induce GS gene expression. Although we show that TCF-1 binds to EAAT2 promoter and regulates its expression, other members of TCF/LEF could also be binding to this promoter. In regulating GS gene expression, β-catenin may partner with additional transcriptional factors in addition to TCF-3. This is partly because, although we observed a statistically significant decrease in GS mRNA expression in TCF-3 KD astrocytes, this KD was only 35–40 % less than the control suggesting that other transcription factors may also be involved.
Our in vivo studies also confirmed that β-catenin positively regulates EAAT2/GLT-1 and GS expression. We used vivo-morpholinos to KD β-catenin in the prefrontal cortex of mice. Vivo-morpholinos are well established to effectively KD proteins of interest in many target organs in small rodents [
24‐
26]. However, there are few studies of vivo-morpholinos injected into the brain. We tested two morpholinos for their ability to efficiently KD β-catenin. Of the two morpholinos tested, only AS provided a high level of β-catenin KD resulting in decreased GLT-1 and GS expression. Interestingly, animals injected with the AS2 or animals that received AS but did not have KD of β-catenin had normal expression of GLT-1 and GS. Potential reasons why KD of β-catenin was not observed in the animal with AS could be that the tissue punch was not on the injection site or failure of the morpholino to KD β-catenin in that animal. Regardless, animals KD for β-catenin had subsequent KD of GLT-1 and GS to confirm our in vitro finding that β-catenin positively regulates EAAT2/GLT-1 and GS expression. Since vivo-morpholinos are not cell specific, treatment likely KD β-catenin expression in many CNS cell types. As such, an indirect result of β-catenin knockdown in neurons or microglia could potentially induce the changes in GLT-1 and GS expression through an unexplored mechanism. While this is possible given the vast number of gene targets for β-catenin that remain to be studied in all cell types, our in vivo data substantiates our in vitro findings which strongly supports astrocytic β-catenin transcriptional regulation of EAAT2 and GS protein expression. Further, GLT-1 and GS are predominately located in astrocytes suggesting astrocytic regulation of these proteins which are necessary for glutamate cycling and transmission.
The finding that β-catenin positively regulates EAAT2 and GS has a broader applicability in understanding mechanisms of neurodegenerative diseases which can be related to chronic neuroinflammation. The role of astrocytic β-catenin in neuroinflammation has not been well characterized. Total β-catenin has been shown to be dysregulated in a number of neurodegenerative diseases including Alzheimer’s disease, Parkinson’s disease, amyotrophic lateral sclerosis, and neuroAIDS, in neuropsychiatric disorders such as schizophrenia as well as a decline in Wnt/β-catenin signaling due to aging processes [
21,
27‐
30]. Emerging studies have found a decrease in Wnt/β-catenin signaling in aged rats with an increase in neuroinflammation [
27]. Also, agents used to increase Wnt/β-catenin signaling have been shown to be neuroprotective in a Parkinsonian rat model by reducing glial activation and oxidative stress [
31]. Interestingly, Wnts, which can be released by astrocytes, have also been linked to inflammatory pathways and neurorepair in Parkinson’s models [
32]. Further, dysfunction in the glutamate/glutamine cycle, mostly through reduction or impairment of EAAT2 function, is linked to neuroAIDS and other neurodegenerative diseases such as ALS, AD, epilepsy, and ischemia/stroke [
4,
33‐
43]. Therefore, signals that diminish β-catenin will also impact EAAT2/GS expression and ultimately glutamate excitotoxicity, a common neuropathologic feature of neuroinflammation and neurodegeneration. Indeed, specific inflammatory signals can lead to the inhibition of β-catenin signaling in astrocytes and a subsequent reduction in EAAT2 and GS expression. We previously showed that interferon gamma (IFN-γ) down-regulates β-catenin signaling, which also leads to alteration in EAAT2 expression [
44]. Therefore, regardless of what the trigger(s) for neurodegeneration may be, inflammation is a common feature and this state of inflammation may reduce β-catenin and consequently EAAT2/GS expression, enhancing glutamate-mediated neurotoxicity.
Past strategies to eliminate excess glutamate signaling via antagonizing NMDAR receptor (memantine) have had many side effects [
4] with little clinical improvement, if any. Memantine is a low-affinity NMDAR antagonist, and complete blocking of NMDAR (N-methyl-D-aspartate receptor) will likely have detrimental effects in the brain. Over-expression of β-catenin can be used as a strategy to induce EAAT2 and GS expression in astrocytes to overcome excess glutamate and neuroinflammation in neurodegenerative diseases. There are a number of small molecules, mostly from the cancer research field, that can induce β-catenin activity [
45]. Such molecules can be explored for inducing EAAT2 and GS in the CNS. However, due to the many functions of β-catenin in cell homeostasis, targeting β-catenin for any type of therapeutic intervention will have to be carefully examined to avoid off-target effects. Nonetheless, enhancing glutamate clearance and metabolism remains a viable treatment strategy against excitotoxicity especially due to limited efficacy of glutamate receptor antagonists. A better understanding of glutamate cycling regulation including β-catenin signaling may lead to therapies to ameliorate or reduce neuropathology of numerous diseases linked to glutamate-induced excitotoxicity.
Conclusions
In summary, we demonstrate that β-catenin signaling in primary human astrocytes regulates key proteins of glutamate/glutamine cycling. β-Catenin partners with transcription factor TCF-1 to positively regulate EAAT2 protein expression. Further, β-catenin KD decreases GS in part through partnering with TCF-3 although there are likely other transcriptional binding partners as TCF-3 KD resulted in a partial decrease in GS expression. Using vivo-morpholinos to KD β-catenin expression in the prefrontal cortex of mice, we show that KD of β-catenin results in KD of GLT-1/GS. By showing here that β-catenin regulates glutamate/glutamine cycle, our findings can now be exploited as a novel and viable target to induce EAAT2/GS on astrocytes and reduce excess glutamate potentially diminishing neuroinflammation/neurodegeneration.
Acknowledgements
Not applicable.