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The role of the TLR4/NF-κB signaling pathway in Aβ accumulation in primary hippocampal neurons

WU Dan, ZHANG Xian, ZHAO Min, ZHOU Ai-Ling

Department of Pathophysiology, Medical School of Nantong University, Nantong 226001, China； Department of Neurology, Affiliated Hospital of Nantong University, Nantong 226001, China； Department of Infectious Disease, Affiliated Hospital of Nantong University, Nantong 226001, China； Department of Pathology, Nantong Tumour Hospital, Nantong 226001, China

Abstract

The present study aimed to investigate the role of the Toll-like receptor 4 (TLR4)/nuclear factor κB (NF-κB) signaling pathway in the accumulation of amyloid β protein (Aβ) in primary hippocampal neurons of rats. The purity of these cultured neurons was determined by using immunofluorescence techniques. Lipopolysaccharide (LPS, a TLR4 ligand) or CLI-095 (a TLR4 inhibitor) was used to activate or inhibit TLR4 signaling, respectively. Pyrrolidine dithiocarbamate (PDTC), on the other hand, was used to inhibit NF-κB, a downstream effector of the TLR4 signaling pathway. The contents of tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β) and Aβ1–42 in the supernatant were assessed by enzyme-linked immunosorbent assay (ELISA). The mRNA levels of TNF-α, IL-1β, a disintegrin and metalloproteinase domain-containing protein 10 (ADAM10), β-site APP cleaving enzyme 1 (BACE-1), Presenilin-1 (PS-1), and β-amyloid precursor protein (β-APP) were examined by real-time quantitative PCR (RT-qPCR). The protein levels of ADAM10, BACE-1, PS-1 and β-APP were examined by Western blotting. Meanwhile, the levels of TLR4 mRNA and protein in hippocampal neurons were tested by RT-qPCR and Western blotting, respectively, after stimulation with Aβ1–42 at different concentrations. We observed that the purity of cultured hippocampal neurons after being cultured for 7 days was above 95%. Compared with untreated neurons, LPS-treated neurons showed higher expression levels of TNF-α, IL-1β, BACE-1, PS-1, β-APP, and Aβ1–42, but a lower expression level of ADAM10. These effects were reversed upon pre-treatment with CLI-095 or PDTC. Furthermore, TLR4 expression was upregulated in the presence of Aβ1–42. Taken together, these results provide evidence that elevation in the level of inflammatory cytokines accompanies the activation of TLR4 signaling, and that the consequent downregulation of ADAM10 and upregulation of BACE-1/PS-1 are likely responsible for the accumulation of β-APP and Aβ, which in turn increases TLR4 level to create a positive feedback loop that may constitute the basis for the progression of Alzheimer’s disease.

Key words: Hippocampal neurons; Toll-like receptor 4; NF-κB; Aβ; Alzheimer’s disease; neuroinflammation

Received: 2014-10-31　　Accepted: 2015-04-07

Corresponding author: 周爱玲　　E-mail: ALZ@ntu.edu.cn

Citing This Article：

WU Dan, ZHANG Xian, ZHAO Min, ZHOU Ai-Ling. The role of the TLR4/NF-κB signaling pathway in Aβ accumulation in primary hippocampal neurons. Acta Physiol Sin 2015; 67 (3): 319-328 (in Chinese with English abstract).