Astrocytic transcription factor REST upregulates glutamate transporter EAAT2, protecting dopaminergic neurons from manganese-induced excitotoxicity

Edward Pajarillo,Alexis Digman,Ivan Nyarko-Danquah,Deok-Soo Son,Karam F.A Soliman,Michael Aschner,Eunsook Lee

doi:10.1016/j.jbc.2021.101372

Abstract

Chronic exposure to high levels of manganese (Mn) leads to manganism, a neurological disorder with similar symptoms to those inherent to Parkinson's disease. However, the underlying mechanisms of this pathological condition have yet to be established. Since the human excitatory amino acid transporter 2 (EAAT2) (glutamate transporter 1 in rodents) is predominantly expressed in astrocytes and its dysregulation is involved in Mn-induced excitotoxic neuronal injury, characterization of the mechanisms that mediate the Mn-induced impairment in EAAT2 function is crucial for the development of novel therapeutics against Mn neurotoxicity. Repressor element 1-silencing transcription factor (REST) exerts protective effects in many neurodegenerative diseases. But the effects of REST on EAAT2 expression and ensuing neuroprotection are unknown. Given that the EAAT2 promoter contains REST binding sites, the present study investigated the role of REST in EAAT2 expression at the transcriptional level in astrocytes and Mn-induced neurotoxicity in an astrocyte–neuron coculture system. The results reveal that astrocytic REST positively regulates EAAT2 expression with the recruitment of an epigenetic modifier, cAMP response element-binding protein–binding protein/p300, to its consensus binding sites in the EAAT2 promoter. Moreover, astrocytic overexpression of REST attenuates Mn-induced reduction in EAAT2 expression, leading to attenuation of glutamate-induced neurotoxicity in the astrocyte–neuron coculture system. Our findings demonstrate that astrocytic REST plays a critical role in protection against Mn-induced neurotoxicity by attenuating Mn-induced EAAT2 repression and the ensuing excitotoxic dopaminergic neuronal injury. This indicates that astrocytic REST could be a potential molecular target for the treatment of Mn toxicity and other neurological disorders associated with EAAT2 dysregulation.

Highlights

(EAAT2) (glutamate transporter 1 (GLT-1) in attenuates Mn-induced reduction in EAAT2 rodents) is predominantly expressed in expression, leading to attenuation of glutamateastrocytes and its dysregulation is involved in induced neurotoxicity in the astrocyte-neuron
Our findings demonstrate characterization of the mechanisms that mediate that astrocytic REST plays a critical role in the Mn-induced impairment in EAAT2 function protection against Mn-induced neurotoxicity by is crucial for the development of novel attenuating Mn-induced EAAT2 repression and therapeutics against Mn neurotoxicity. the ensuing excitotoxic dopaminergic neuronal
We found that REST Since Mn decreases EAAT2 mRNA and interacted with CREB, as well as histone acetyltransferases (HAT) such as CBP and p300, which are known to enhance EAAT2 transcription in the nuclear region by coprotein levels in vitro as well as in vivo models [27,63], and REST increases EAAT2 expression, we tested whether REST modulates at the transcriptional level the Mninduced reduction in EAAT2 expression

Summary

Astrocyte cultures were transfected with the

Primary cultures of human and mouse plasmids using lipofectamine 3000 or by astrocytes and a human H4 astrocyte cell line electroporation as described previously [50]. The following primers were PBS pH 7.4 for 10 min at room temperature; used: human REST 5'-GTG AGC GAG TAT cells were washed three times with ice-. ATG GGA TAC AAT G-3' (reverse); human overnight with primary antibodies for EAAT2, EAAT1 5'-GGA GCA AAA CAA AGC CAG REST, CBP/p300 or CREB at 1:250 dilution at CA-3' (forward) and 5'-ATT CCC CAG CAG 4 °C. Western blot analysis For protein analysis, astrocytes (106-107 with primary antibodies for REST, CBP/p300, and/or CREB (1:250 dilution) at 4 °C overnight. Real-time qPCR was carried out with ° C for 1 h, followed by incubation with ligation the RE1 primer pairs for -663 site: 5′-GAG CTG solution containing two oligonucleotides and AAG CGG GTG CTC-3′ (forward) and 5′-AAT ligase at 37 °C for 30 min to hybridize two TAG CCA AAT AAG AAA AGA GG-3′. (Leica biotinylated oligonucleotides were incubated with 50 μg of nuclear extract as described previously [50] in a binding buffer for 20 min

ChIP assay

LUHMES cells were treated with toxic

Generation of ROS as an indicator of oxidative

Findings

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