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

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.

Deletion of RE1‐silencing transcription factor in striatal astrocytes exacerbates manganese‐induced neurotoxicity in mice

“…Mn toxicity in the striatum is associated with glutamate excitotoxicity (Brouillet et al, 1993) and decreased in GLT‐1 mRNA and protein levels in the mouse brain (Johnson Jr. et al, 2018; Pajarillo et al, 2018). Since REST modulates astrocytic EAAT2 (GLT‐1) (Pajarillo, Digman, et al, 2021), we determined if modulation of REST affects GLT‐1 (EAAT2) expression in the mouse brain. The results showed that Mn decreased GLT‐1 mRNA and protein levels in the striatum of mice infused with the control vectors of AAV5‐GFAP‐GFP (Figure 8a,b), and striatal astrocytic REST deletion by infusion of the AAV5‐GFAP‐Cre‐GFP vectors further exacerbated the Mn‐induced decrease in GLT‐1 mRNA and protein levels in the striatum.…”

“…Empty vectors served as control. Briefly, PHA were transfected with EV (pCMV6‐entry and TetO‐FUW) and plasmid DNA vectors for REST, or DN‐REST by electroporation and incubated for 48 h as described in our previous studies (Pajarillo, Digman, et al, 2021). After incubation, REST or DN‐REST‐overexpressing PHA were exposed to Mn (250 μM, 6 h), followed by replacement of the Mn‐containing media with fresh experimental media for an additional of 6 h. Next, the PHA‐CM was applied to LUHMES cells to test the effects of astrocytic REST on dopaminergic neuronal cells.…”

“…Mn toxicity in the striatum is associated with glutamate excitotoxicity (Brouillet et al, 1993) and decreased in GLT-1 mRNA and protein levels in the mouse brain (Johnson Jr. et al, 2018;Pajarillo et al, 2018). Since REST modulates astrocytic EAAT2 (GLT-1) (Pajarillo, Digman, et al, 2021), we determined if modulation of REST affects F I G U R E 4 Deletion of striatal astrocytic REST exacerbates Mn-induced decrease of TH expression in the striatum and substantia nigra/ midbrain as well as GAD expression in the striatum of mice. (a,b) After Mn exposure (MnCl 2 , 30 mg/kg, nostril instillation, daily for 3 weeks), striatum (a,b) and midbrain (c,d) regions of mouse brains were analyzed for TH protein levels by western blotting and IHC.…”

“…REST binds to a 21‐bp cis‐regulatory element known as repressor element‐1/neuron‐restrictive silencer element (RE1/NRSE) to regulate more than 2000 target genes (Bruce et al, 2009). Although REST was initially known to repress target genes, growing evidence shows that REST also activates target genes in neurons and astrocytes (Abramovitz et al, 2008; Bessis et al, 1997; Pajarillo, Digman, et al, 2021; Pajarillo, Rizor, et al, 2020). We have recently reported that REST activated tyrosine hydroxylase (TH) in neuronal cells by direct binding to its cis ‐element of the TH promoter with parallel increases of TH promoter activities, mRNA and protein levels (Pajarillo, Rizor, et al, 2020).…”

“…We have recently reported that REST activated tyrosine hydroxylase (TH) in neuronal cells by direct binding to its cis ‐element of the TH promoter with parallel increases of TH promoter activities, mRNA and protein levels (Pajarillo, Rizor, et al, 2020). REST also directly activated excitatory amino acid transporter 2 (EAAT2) in astrocytes by increasing EAAT2 expression at the transcriptional level (Pajarillo, Digman, et al, 2021).…”