Pratap Karki

Mechanism of raloxifene-induced upregulation of glutamate transporters in rat primary astrocytes

Raloxifene (RX), a selective estrogen receptor modulator (SERM), exerts neuroprotection in multiple clinical and experimental settings. Astrocytic glutamate transporters GLT‐1 (EAAT2) and GLAST (EAAT1) are the main glutamate transporters in the central nervous system, taking up most of excess glutamate from the synaptic cleft to prevent excitotoxic neuronal death. Since drugs targeting astrocytic glutamate transporters to enhance their expression and function represent potential therapeutics for neurodegenerative disorders associated with excitotoxicity, we tested if RX modulates the expression and function of GLT‐1 and GLAST in rat primary astrocytes. The results showed that RX significantly increased glutamate uptake and expression of GLT‐1 mRNA and protein levels. RX enhanced GLT‐1 expression by the activation of multiple signaling pathways including ERK, EGFR, and CREB mediated by estrogen receptors (ERs) ER‐α, ER‐β, and GPR30. At the transcriptional level, NF‐κB played a critical role in RX‐induced GLT‐1 expression as RX increased NF‐κB reporter activity and induced binding of NF‐κB p65 and p50 to the GLT‐1 promoter. RX attenuated the reduction of GLT‐1 expression and glutamate uptake induced by manganese (Mn) whose chronic high levels of exposure cause manganism. RX also upregulated GLAST by increasing its promoter activity and protein levels via the NF‐κB pathway and ERs. Our findings provide new insight into the mechanism of RX‐induced enhancement of GLT‐1 and GLAST expression, as well as the attenuation of Mn‐reduced expression of these transporters. These findings will be highly valuable for developing therapeutics of neurodegenerative diseases associated with impaired astrocytic glutamate transporters. GLIA 2014;62:1270–1283

cAMP Response Element-binding Protein (CREB) and Nuclear Factor κB Mediate the Tamoxifen-induced Up-regulation of Glutamate Transporter 1 (GLT-1) in Rat Astrocytes

Background: Tamoxifen (TX), a selective estrogen receptor modulator, enhances glutamate transporter (GLT-1) expression in astrocytes. Results: TX up-regulated GLT-1 expression via the CREB and NF-κB pathways. Conclusion: TX enhanced GLT-1 expression at the transcriptional level. Significance: Understanding the mechanisms of TX action on GLT-1 will contribute to the development of neuroprotectants against excitotoxicity. Tamoxifen (TX), a selective estrogen receptor modulator, exerts antagonistic effects on breast tissue and is used to treat breast cancer. Recent evidence also suggests that it may act as an agonist in brain tissue. We reported previously that TX enhanced the expression and function of glutamate transporter 1 (GLT-1) in rat astrocytes, an effect that was mediated by TGF-α. To gain further insight into the mechanisms that mediate TX-induced up-regulation of GLT-1 (EAAT2 in humans), we investigated its effect on GLT-1 at the transcriptional level. TX phosphorylated the cAMP response element-binding protein (CREB) and recruited CREB to the GLT-1 promoter consensus site. The effect of TX on astrocytic GLT-1 was attenuated by the inhibition of PKA, the upstream activator of the CREB pathway. In addition, the effect of TX on GLT-1 promoter activity was abolished by the inhibition of the NF-κB pathway. Furthermore, TX recruited the NF-κB subunits p65 and p50 to the NF-κB binding domain of the GLT-1 promoter. Mutation of NF-κB (triple, −583/-282/-251) or CRE (-308) sites on the GLT-1 promoter led to significant repression of the promoter activity, but neither mutant completely abolished the TX-induced GLT-1 promoter activity. Mutation of both the NF-κB (-583/-282/-251) and CRE (-308) sites led to a complete abrogation of the effect of TX on GLT-1 promoter activity. Taken together, our findings establish that TX regulates GLT-1 via the CREB and NF-κB pathways.

Yin Yang 1 is a Repressor of Glutamate Transporter EAAT2 and it Mediates Manganese-induced Decrease of EAAT2 Expression in Astrocytes

ABSTRACT Impairment of astrocytic glutamate transporter (GLT-1; EAAT2) function is associated with multiple neurodegenerative diseases, including Parkinsons disease (PD) and manganism, the latter being induced by chronic exposure to high levels of manganese (Mn). Mn decreases EAAT2 promoter activity and mRNA and protein levels, but the molecular mechanism of Mn-induced EAAT2 repression at the transcriptional level has yet to be elucidated. We reveal that transcription factor Yin Yang 1 (YY1) is critical in repressing EAAT2 and mediates the effects of negative regulators, such as Mn and tumor necrosis factor alpha (TNF-α), on EAAT2. YY1 overexpression in astrocytes reduced EAAT2 promoter activity, while YY1 knockdown or mutation of the YY1 consensus site of the EAAT2 promoter increased its promoter activity and attenuated the Mn-induced repression of EAAT2. Mn increased YY1 promoter activity and mRNA and protein levels via NF-κB activation. This led to increased YY1 binding to the EAAT2 promoter region. Epigenetically, histone deacetylase (HDAC) classes I and II served as corepressors of YY1, and, accordingly, HDAC inhibitors increased EAAT2 promoter activity and reversed the Mn-induced repression of EAAT2 promoter activity. Taken together, our findings suggest that YY1, with HDACs as corepressors, is a critical negative transcriptional regulator of EAAT2 and mediates Mn-induced EAAT2 repression.

Manganese Neurotoxicity: a Focus on Glutamate Transporters

Manganese (Mn) is an essential element that is required in trace amount for normal growth, development as well maintenance of proper function and regulation of numerous cellular and biochemical reactions. Yet, excessive Mn brain accumulation upon chronic exposure to occupational or environmental sources of this metal may lead to a neurodegenerative disorder known as manganism, which shares similar symptoms with idiopathic Parkinson’s disease (PD). In recent years, Mn exposure has gained public health interest for two primary reasons: continuous increased usage of Mn in various industries, and experimental findings on its toxicity, linking it to a number of neurological disorders. Since the first report on manganism nearly two centuries ago, there have been substantial advances in the understanding of mechanisms associated with Mn-induced neurotoxicity. This review will briefly highlight various aspects of Mn neurotoxicity with a focus on the role of astrocytic glutamate transporters in triggering its pathophysiology.

Astrocyte-derived growth factors and estrogen neuroprotection: role of transforming growth factor-α in estrogen-induced upregulation of glutamate transporters in astrocytes.

Extensive studies from the past decade have completely revolutionized our understanding about the role of astrocytes in the brain from merely supportive cells to an active role in various physiological functions including synaptic transmission via cross-talk with neurons and neuroprotection via releasing neurotrophic factors. Particularly, numerous studies have reported that astrocytes mediate the neuroprotective effects of 17β-estradiol (E2) and selective estrogen receptor modulators (SERMs) in various clinical and experimental models of neuronal injury. Astrocytes contain two main glutamate transporters, glutamate aspartate transporter (GLAST) and glutamate transporter-1 (GLT-1), that play a key role in preventing excitotoxic neuronal death, a process associated with most neurodegenerative diseases. E2 has been shown to increase expression of both GLAST and GLT-1 mRNA and protein and glutamate uptake in astrocytes. Growth factors such as transforming growth factor-α (TGF-α) appear to mediate E2-induced enhancement of these transporters. These findings suggest that E2 exerts neuroprotection against excitotoxic neuronal injuries, at least in part, by enhancing astrocytic glutamate transporter levels and function. Therefore, the present review will discuss proposed mechanisms involved in astrocyte-mediated E2 neuroprotection, with a focus on glutamate transporters.

Activation of MAPK and FoxO by Manganese (Mn) in Rat Neonatal Primary Astrocyte Cultures

Environmental exposure to manganese (Mn) leads to a neurodegenerative disease that has shared clinical characteristics with Parkinsons disease (PD). Mn-induced neurotoxicity is time- and dose-dependent, due in part to oxidative stress. We ascertained the molecular targets involved in Mn-induced neurodegeneration using astrocyte culture as: (1) Astrocytes are vital for information processing within the brain, (2) their redox potential is essential in mitigating reactive oxygen species (ROS) levels, and (3) they are targeted early in the course of Mn toxicity. We first tested protein levels of Mn superoxide dismutase -2 (SOD-2) and glutathione peroxidase (GPx-1) as surrogates of astrocytic oxidative stress response. We assessed levels of the forkhead winged-helix transcription factor O (FoxO) in response to Mn exposure. FoxO is highly regulated by the insulin-signaling pathway. FoxO mediates cellular responses to toxic stress and modulates adaptive responses. We hypothesized that FoxO is fundamental in mediating oxidative stress response upon Mn treatment, and may be a biomarker of Mn-induced neurodegeneration. Our results indicate that 100 or 500 µM of MnCl2 led to increased levels of FoxO (dephosphorylated and phosphorylated) compared with control cells (P<0.01). p-FoxO disappeared from the cytosol upon Mn exposure. Pre-treatment of cultured cells with (R)-(−)-2-oxothiazolidine-4-carboxylic acid (OTC), a cysteine analog rescued the cytosolic FoxO. At these concentrations, MAPK phosphorylation, in particular p38 and ERK, and PPAR gamma coactivator-1 (PGC-1) levels were increased, while AKT phosphorylation remained unchanged. FoxO phosphorylation level was markedly reduced with the use of SB203580 (a p38 MAPK inhibitor) and PD98059 (an ERK inhibitor). We conclude that FoxO phosphorylation after Mn exposure occurs in parallel with, and independent of the insulin-signaling pathway. FoxO levels and its translocation into the nucleus are part of early events compensating for Mn-induced neurotoxicity and may serve as valuable targets for neuroprotection in the setting of Mn-induced neurodegeneration.

Role of transcription factor yin yang 1 in manganese-induced reduction of astrocytic glutamate transporters: Putative mechanism for manganese-induced neurotoxicity

Astrocytes are the most abundant non-neuronal glial cells in the brain. Once relegated to a mere supportive role for neurons, contemporary dogmas ascribe multiple active roles for these cells in central nervous system (CNS) function, including maintenance of optimal glutamate levels in synapses. Regulation of glutamate levels in the synaptic cleft is crucial for preventing excitotoxic neuronal injury. Glutamate levels are regulated predominantly by two astrocytic glutamate transporters, glutamate transporter 1 (GLT-1) and glutamate aspartate transporter (GLAST). Indeed, the dysregulation of these transporters has been linked to several neurodegenerative diseases such as amyotrophic lateral sclerosis (ALS), Alzheimers disease (AD) and Parkinsons disease (PD), as well as manganism, which is caused by overexposure to the trace metal, manganese (Mn). Although Mn is an essential trace element, its excessive accumulation in the brain as a result of chronic occupational or environmental exposures induces a neurological disorder referred to as manganism, which shares common pathological features with Parkinsonism. Mn decreases the expression and function of both GLAST and GLT-1. Astrocytes are commonly targeted by Mn, and thus reduction in astrocytic glutamate transporter function represents a critical mechanism of Mn-induced neurotoxicity. In this review, we will discuss the role of astrocytic glutamate transporters in neurodegenerative diseases and Mn-induced neurotoxicity.

Transcriptional Regulation of the Astrocytic Excitatory Amino Acid Transporter 1 (EAAT1) via NF-κB and Yin Yang 1 (YY1)

Background: The mechanism for transcriptional regulation of EAAT1 remains to be elucidated. Results: EGF-activated NF-κB is a positive regulator of EAAT1, whereas manganese-activated YY1, with HDACs acting as co-repressors, is a negative regulator. Conclusion: NF-κB and YY1 are two critical transcriptional regulators of EAAT1. Significance: Identifying the molecular targets of EAAT1 regulation is crucial to develop therapeutics against neurological disorders associated with impairment of EAAT1. Astrocytic glutamate transporter excitatory amino acid transporter (EAAT) 1, also known as glutamate aspartate transporter (GLAST) in rodents, is one of two glial glutamate transporters that are responsible for removing excess glutamate from synaptic clefts to prevent excitotoxic neuronal death. Despite its important role in neurophysiological functions, the molecular mechanisms of EAAT1 regulation at the transcriptional level remain to be established. Here, we report that NF-κB is a main positive transcription factor for EAAT1, supported by the following: 1) EAAT1 contains two consensus sites for NF-κB, 2) mutation of NF-κB binding sites decreased EAAT1 promoter activity, and 3) activation of NF-κB increased, whereas inhibition of NF-κB decreased EAAT1 promoter activity and mRNA/protein levels. EGF increased EAAT1 mRNA/protein levels and glutamate uptake via NF-κB. The transcription factor yin yang 1 (YY1) plays a role as a critical negative regulator of EAAT1, supported by the following: 1) the EAAT1 promoter contains multiple consensus sites for YY1, 2) overexpression of YY1 decreased EAAT1 promoter activity and mRNA/protein levels, and 3) knockdown of YY1 increased EAAT1 promoter activity and mRNA/protein levels. Manganese decreased EAAT1 expression via YY1. Epigenetic modifiers histone deacetylases (HDACs) served as co-repressors of YY1 to further decrease EAAT1 promoter activity, whereas inhibition of HDACs reversed manganese-induced decrease of EAAT1 expression. Taken together, our findings suggest that NF-κB is a critical positive regulator of EAAT1, mediating the stimulatory effects of EGF, whereas YY1 is a negative regulator of EAAT1 with HDACs as co-repressors, mediating the inhibitory effects of manganese on EAAT1 regulation.

Chapter 10:Mechanism of Manganese-Induced Impairment of Astrocytic Glutamate Transporters

Manganese (Mn) is an essential trace element, playing a vital role in numerous biochemical and cellular reactions; however, chronic exposure to high Mn levels from environmental and occupational sources causes a neurological disorder with shared features of Parkinsons disease (PD), referred to as manganism. Despite well-established pathological signs, the molecular mechanism(s) by which Mn induces these neurological disorders still remain to be established. In addition to oxidative stress and impairment of mitochondria, Mn dysregulates astrocytic glutamate transporters (GLAST [glutamate aspartate transporter] and GLT-1 [glutamate transporter 1]) by decreasing their promoter activity, mRNA, and protein levels as well as astrocytic glutamate uptake. The Mn-induced impairment in glutamate transporters is directly associated with excitotoxic neuronal death because the astrocytic glutamate transporters, GLAST and GLT-1, are mainly responsible for maintaining optimal glutamate levels in the synaptic clefts, thereby preventing glutamate-induced neuronal excitotoxicity. It is widely recognized that reduced expression and function of astrocytic glutamate transporters, in particular GLT-1, are associated with various neurodegenerative diseases, including PD and amyotrophic lateral sclerosis (ALS). Therefore, Mn-induced impairment of astrocytic glutamate transporters might be a critical mechanism for Mn neurotoxicity. Our latest studies have uncovered a novel mechanism of Mn-induced repression of GLT-1 at the transcriptional level. It appears that the transcription factor yin yang 1 (YY1) plays a critical role in Mn-induced repression of GLT-1 promoter activity and expression. Herein, we will discuss the cellular and molecular mechanisms by which Mn induces neurotoxicity, such as oxidative stress, mitochondrial impairment, inflammation, and dysregulation of glutamate transporters.