miR-137 attenuates Aβ-induced neurotoxicity through inactivation of NF-κB pathway by targeting TNFAIP1 in Neuro2a cells

https://doi.org/10.1016/j.bbrc.2017.06.144Get rights and content

Highlights

  • 25-35 downregulated miR-137 and upregulated TNFAIP1 in cortical neurons and N2a cells.

  • miR-137 suppressed TNFAIP1 expression through direct binding at the 3′UTR.

  • miR-137 overexpression contributed to Aβ25-35-induced cell toxicity and apoptosis.

  • miR-137 overexpression on Aβ25-35-induced neurotoxicity activated NF-κB pathway.

Abstract

Background

Accumulation of β-amyloid (Aβ) and neuroinflammation are implicated in the pathogenesis and development of Alzheimer's disease (AD). Neuron-enriched miR-137 was aberrantly downregulated and may be associated with the pathogenesis of AD. However, the detailed function of miR-137 in AD pathogenesis and the molecular mechanism have not been elucidated.

Methods

The expressions of miR-137 and tumor necrosis factor alpha (TNFα)-induced protein 1 (TNFAIP1) at mRNA and protein levels in primary mouse cortical neurons and Neuro2a (N2a) cells exposed to different concentrations of Aβ25-35 were examined by qRT-PCR and western blot. Luciferase reporter assay was used to confirm the potential target of miR-137. MTT assay, flow cytometry analysis, caspase-3 activity assay, Enzyme-linked immunosorbent assay (ELISA), and western blot were used to detect cell viability, apoptosis, caspase-3 activity, Nuclear factor-kappa B (NF-κB) activity and level, respectively.

Results

25-35 downregulated miR-137 and upregulated TNFAIP1 in primary mouse cortical neurons and N2a cells. In addition, miR-137 was found to directly target TNFAIP1 and suppress its mRNA and protein levels. Moreover, miR-137 restoration and TNFAIP1 knockdown facilitate Aβ25-35-induced cell toxicity, apoptosis, caspase-3 activity, and activated NF-κB in N2a cells, which was partially abolished by TNFAIP1 overexpression.

Conclusion: miR-137 attenuated Aβ-induced neurotoxicity through inactivation of NF-κB pathway by targeting TNFAIP1 in N2a cells, shedding light on the molecular mechanism of miR-137 underlying Aβ-induced neurotoxicity.

Introduction

Alzheimer's disease (AD), a major cause of dementia, is a devastating neurodegenerative disorder characterized by progressively debilitating memory impairment, cognitive dysfunction and behavioral damage [1]. The existence of amyloid plaques, intracellular neurofibrillary tangles (NFTs), massive neuronal cell death and uncontrolled microglial activation is the main pathological hallmarks in AD pathogenesis [2]. It is widely accepted that amyloid plaques are extracellular accumulation of the β-amyloid (Aβ) peptides, which are 39–43 amino acid peptides originally derived from proteolytic cleavages of the amyloid precursor protein (APP) in the brains of AD patients [3]. Even though the exact etiologies of AD remain unknown, convincing evidence from experimental models and clinical research studies suggests neuroinflammation and neuronal apoptosis are causatively implicated in the pathogenesis and development of AD, all of which involve Aβ peptides [4].

Emerging evidence has suggested that Aβ plays a crucial role in stimulating the production of pro-inflammatory cytokines through Nuclear factor-kappa B (NF-κB) pathway, which is widely expressed in brain cells including neurons and microglia cells [5]. NF-κB is a family of transcription factors which play an important role in multiple cellular pathways associated with the inflammatory response, immune responses, cell division, cell growth and apoptosis [6]. It has been proposed that activation of NF-κB pathway and its subsequent activation of expressions of a cellular defense program, contribute to the degeneration of neurons in AD patients [7]. Therefore, inhibition of NF-κB pathway may be of great benefit for the treatment of AD.

MicroRNAs (miRNAs) are a class of endogenous and evolutionarily non-coding RNA molecules (18–22 nucleotides in length), which cause translation inhibition or mRNA degradation by binding to the 3’-untranslated region (UTR) of target gene mRNA [8]. Multiple miRNAs are observed to be aberrantly expressed in AD patients and transgenic animal models of AD [9]. Accumulating evidence suggests that dysregulation of several miRNAs participates in the pathogenesis and neuropathology of AD by regulating the production of Aβ and inflammatory responses [10]. miR-137, localizing on human chromosome 1p22, has received tremendous attention over the past couple of decades due to its important regulatory role in brain function and association with the etiology of neuropsychiatric disorders [11]. Functionally, neuron-enriched miR-137 was aberrantly downregulated and may be associated with the pathogenesis and development of AD [12]. However, the detailed function of miR-137 in the pathogenesis of AD and the underlying molecular mechanism remain elusive.

In the present study, we demonstrated that miR-137 was downregulated and tumor necrosis factor alpha (TNFα)-induced protein 1 (TNFAIP1) was upregulated in primary cortical neurons and neuroblastoma cell line Neuro2a (N2a). Furthermore, miR-137 overexpression attenuated Aβ25–35-induced neurotoxicity through inactivation of NF-κB pathway by targeting TNFAIP1 in N2a cells.

Section snippets

Cell culture and treatment

This study was approved by the Ethic Committee of Huaihe Hospital of Henan University and performed in accordance with the NIH Guide for the Care and Use of Laboratory Animals. Primary mouse cortical neurons were isolated from 17-day-old embryonic cortex obtained from newborn mice (C57BL/6) as described previously [13]. Neurons were grown in Neurobasal medium (Gibco, Carlsbad, CA, USA) supplemented with 2% B27 (Gibco), 0.8 mM l-Glutamine (Gibco) and 100 μg/ml penicillin/streptomycin (Gibco) at

25–35 downregulated miR-137 and upregulated TNFAIP1

To explore the functional role of miR-137 and TNFAIP1 in AD pathogenesis, we determined the effect of Aβ25–35 on the expressions of miR-137 and TNFAIP1 at mRNA and protein levels by treated mouse cortical neurons and N2a cells with indicated concentration of Aβ25–35 (0, 5, 10 and 20 μM) for 24 h, which was considered to be a causative factor in the pathogenesis of AD [16]. qRT-PCR analysis showed that miR-137 expression was significantly decreased by Aβ25–35 in a dose-dependent manner in mouse

Discussion

Several lines of evidence highlighted that Aβ-induced neurotoxicity including inflammation and neuronal apoptosis in vitro and in vivo play a fatal role in the pathogenic process of AD [18]. Many experiments have demonstrated that Aβ25-35 can induce neurotoxicity and AD-like pathology and has been extensively used to induce in vitro and in vivo AD models [19]. In the present study, we provided the evidence that Aβ25-35 treatment decreased miR-137 expression and increased TNFAIP1 expression in

Conflicts of interest

The authors have no conflict of interest to declare.

Acknowledgment

Not applicable.

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