www.aging-us.com AGING 2021, Vol. 13, No. 11 Research Paper Repressor element-1 silencing transcription factor regulates glutamate receptors and immediate early genes to affect synaptic plasticity Chenhaoyi Xu1,*, Min Zhang1,*, Lu Zu1, Pei Zhang1, Letao Sun2, Xueyuan Liu1, Min Fang1 1Department of Neurology, Shanghai Tenth People's Hospital, Tongji University School of Medicine, Shanghai 200072, China 2Gordon F. Derner School of Psychology, Adelphi University, New York, NY 11530-0701, USA *Co-first authors Correspondence to: Min Fang, Xueyuan Liu; email: [email protected], https://orcid.org/0000-0001-6343-7726; [email protected] Keywords: repressor element-1 silencing transcription factor, immediate early genes, synaptic plasticity, synaptic genes Received: January 26, 2021 Accepted: May 17, 2021 Published: June 9, 2021 Copyright: © 2021 Xu et al. This is an open access article distributed under the terms of the Creative Commons Attribution License (CC BY 3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. ABSTRACT Objective: This study aimed to investigate the regulatory effects of repressor element-1 silencing transcription factor (REST) on the glutamate receptors and immediate early genes (IEGs) in the SH-SY5Y cells. Methods: The genes regulated by REST were screened by bioinformatics between AD patients and the control group. Then, SH-SY5Y cells were treated with 10 μM Aβ or REST siRNA/cDNA, and the expressions of synaptic genes and IEGs were detected. Moreover, the protein expression of synaptophysin and PSD-95 was detected by Western blotting in the primary mouse hippocampal neurons. Results: Firstly, 464 differentially expressed genes regulated by REST were identified between Alzheimer’s disease (AD) patients and controls, and REST was closely related to the glutamatergic synapses and long-term potentiation. GRIA1, GRIN2A, GRIN1, and ARC showed significant variations with the changes of REST. Moreover, the loss of REST reduced the expression of synaptophysin and PSD-95, which was related to synaptic plasticity. Conclusion: REST maintains synaptic plasticity by affecting both glutamate receptors and IEGs, and the imbalance between neural excitation and inhibition mediated by REST compromises neural function, contributing to cognitive impairment. INTRODUCTION synthesized subsequently through a series of signaling pathways for the maintenance of homeostatic synaptic Neurons engage in the processes of capturing, encoding, plasticity, and the synthesis and translation of the and saving information, to learn and form memories [1]. related proteins are important to maintain the synaptic It has been widely accepted that synapses can transmit plasticity [3]. For example, glutamate AMPA type information between neurons through electronic signals, subunit1 (GRIA1), glutamate NMDA type subunit 1 chemical signals, and electronic-chemical signals [2]. (GRIN1), and glutamate NMDA type subunit 2A Affected by the dynamic activity of neurons, the (GRIN2A) can encode glutamate AMPA receptor and structure of synapses can change specifically within glutamate NMDA receptor, respectively, which then seconds to dynamically regulate their ability to release bind to glutamate released from the pre-synapses, to neurotransmitters [1]. This close relationship between respond to long‐term potentiation (LTP) ‐ inducing or structure and function is regarded as synaptic plasticity. long-term depression (LTD) ‐ inducing stimuli [4]. The Once neurons are activated, numerous proteins are imbalance among the associated proteins involved in www.aging-us.com 15569 AGING the transcription, translation, and degradation may cause decrease REST expression, ultimately affecting the the abnormality of neural circuits, finally resulting in synaptic plasticity to cause the abnormal excitability of neurodegenerative diseases, such as Alzheimer’s the neural network. disease (AD), Parkinson’s disease, and others [5]. MATERIALS AND METHODS Synaptic plasticity participates in the informational storage in the brain, and it can be regulated by a number Cell culture and transfection of factors, including repressor element-1 silencing transcription factor (REST). REST can act on synaptic Human neuroblastoma SH-SY5Y cells were cultured in genes and immediate early genes (IEGs) to repress Dulbecco’s Modified Eagle’s Medium (DMEM) neural activity and avoid hyperexcitation in the aging containing 10% Fetal Bovine Serum (FBS), 1% brain [6, 7]. REST, a zinc-finger transcription factor, penicillin-streptomycin (PS) at 37°C with a mixed gas mainly binds to repressor element-1 (RE-1) motifs of of 21% O2, 5% CO2, and N2. the target genes to repress gene expression, and some studies also reveal that REST may also act as a Primary mouse hippocampal neurons were collected transcriptional promoting factor [8]. In the post-natal from neonatal C57BL/6 mice as described previously and aging brains, the REST expression remains at a [16]. Then, 6 × 105 cells were plated in 6-well plates high level to ensure neuronal specificity and cell type- coated by 0.05 mg/ml poly-L-lysine (PLL, BBI Life specific gene expression. The regulatory effects of Science Corporation, China) and incubated with B-27 REST are complicated due to its various binding sites Serum-Free Supplement (Gibco, USA), GlutaMAX on the target genes and its environment-dependence Supplement (Gibco, USA) and Neurobasal Medium which is influenced by the locations and types of cells (Gibco, USA) at 37°C with a mixed gas of 21% O2, 5% [9]. Studies have shown that the reduced REST CO2 and N2. For the Aβ treatment, cells were incubated expression in aging adults is involved in the with 10 μM human Aβ1-42 (MCE, China) for 48 h. pathogenesis of AD. However, how REST regulates the epigenetic modification of targeted mRNAs remains For transfection, SH-SY5Y cells or primary mouse largely unknown. In our previous studies, our results hippocampal neurons were plated in 6-well plates at showed high Aβ load reduced REST expression [10]. the density of 3 × 105 cells/well. REST siRNA (Zorin This study aimed to explore the regulatory effects of Bio, Shanghai, China) was transfected into cells REST on the synaptic genes and IEGs, which may with Lipofectamine 3000 kit according to the provide evidence on the role of REST in synaptic manufacturer’s instructions (Thermo Fisher, USA). plasticity and on the potential mechanism underlying The target sequence of REST siRNA was the initiation of AD. GCGTACTCATTCAGGTGAGAA. IEGs, such as activity-regulated cytoskeleton-associated RNA isolation and real-time quantitative polymerase protein (ARC), neuronal Per/Arnt/Sim domain protein 4 chain reaction (RT-qPCR) (NPAS4), early growth response protein 1 (EGR1), and brain-derived neurotrophic factor (BDNF), are subsets Total RNA was extracted by Trizol reagent and of genes induced by stimuli in seconds [11]. In neurons, transcribed reversely into cDNA using the PrimeScript IEGs can be induced immediately and play critical roles RT reagent kit (Takara Bio, Japan). Real-time PCR was in the establishment and maintenance of permanent performed using the TB Green Premix Ex Taq II kit changes of neurons, forming learning and memory [12]. (Takara Bio, Japan). The primers were designed in The abnormal expression of IEGs may lead to the Primer Premier 5.0 software (Premier, Canada) and occurrence of cognitive disorders, schizophrenia, and synthesized by Sangon Bio (Shanghai, China). The other diseases [13]. Particularly, the induction of ARC sequences of primers are listed in the Supplementary is associated with the consolidation process of labile Materials. The reaction conditions were as follows: memories through its ability of prior history of neuronal 95°C for 30 s, and 50 cycles of 95°C for 3 s and 60°C activation to facilitate LTP or LTD, which has been for 30 s. The mRNA expression of target genes was termed as metaplasticity to influence synaptic plasticity normalized to that of β-actin as the internal reference. [14]. It has been revealed that the ARC expression reduces in the dentate gyrus and CA1 region of AD Western blotting mice, and the neurons with amyloid plaque-associated dystrophic neurites fail to express ARC mRNA [15]. SH-SY5Y cells were treated by RIPA Lysis Buffer (Epizyme, China) with 1 mM PMSF (Beyotime, China) We hypothesize that REST may regulate synaptic genes on ice for 5 s. Then, the lysate was centrifuged at 14,000 and IEGs to affect the synaptic plasticity, and Aβ can g for 5 min. The supernatant was collected and the www.aging-us.com 15570 AGING concentration of total protein was quantified by Shapiro-Wilk’s test. Subsequently, Levene’s test was bicinchoninic acid assay (Beyotime, China). 15 μg of used to test the equality of variances. The equal- proteins were loaded and separated on 10% SDS-PAGE. variances data between the two groups were compared Then, proteins were transferred onto PVDF membranes with the two-tailed student’s t-test and the unequal- which were blocked with 5% non-fat milk in TBST variances data with the corrected two-tailed student’s t- buffer at room temperature for 1 h. The membranes were test. Comparisons of data among 3 or more groups were incubated with appropriate primary antibodies at 4°C done with the analysis of variance (ANOVA). A value overnight and then with corresponding secondary of p < 0.05 was considered statistically significant. antibodies. The optical density of protein bands was quantified using Image J. Antibodies