Regucalcin confers resistance to amyloid-β toxicity in neuronally differentiated PC12 cells

Abstract

Amyloid-β (Aβ), a primary component of amyloid plaques, has been widely associated with the pathogenesis of Alzheimer's disease. The Ca²⁺-binding protein regucalcin (RGN) plays multiple roles in maintaining cell functions by regulating intracellular calcium homeostasis, various signaling pathways, and gene expression systems. Here, we investigated the functional role of RGN against Aβ-induced cytotoxicity in neuronally differentiated PC12 cells. Overexpression of RGN reduced Aβ-induced apoptosis by reducing mitochondrial dysfunction and caspase activation. It also attenuated Aβ-induced reactive oxygen species production and oxidative damage and decreased Aβ-induced nitric oxide (NO) overproduction, upregulation of inducible NO synthase by nuclear factor-κB, and nitrosative damage. Interestingly, the genetic disruption of RGN increased the susceptibility of neuronally differentiated PC12 cells to Aβ toxicity. Thus, RGN possesses antioxidant activity against Aβ-induced oxidative and nitrosative stress and may play protective roles against Aβ-induced neurotoxicity in Alzheimer's disease.

Keywords: Alzheimer's disease; amyloid‐β; apoptosis; mitochondrial dysfunction; reactive nitrogen species; reactive oxygen species; regucalcin.

Figure 1

Regucalcin attenuates Aβ‐induced apoptosis by activating the mitochondrial caspase pathway in neuronally differentiated PC12 cells. NGF‐differentiated PC12 cells overexpressing β‐galactosidase (LacZ) or RGN were treated with 25 μm Aβ for the indicated periods. (A) Cells were incubated with the TUNEL reaction mixture to determine rates of apoptosis and then stained with the Hoechst 33342 nuclear stain. TUNEL‐positive apoptotic nuclei were identified using a fluorescent microscope. (B) Mitochondrial membrane potential was measured using the fluorescent probe TMRE. (C,D) Caspase‐9‐mediated cleavage of LEHD‐AFC and caspase‐3‐mediated cleavage of DEVD‐AFC were estimated in cell lysates using fluorometric assays with specific substrates. Data are expressed as mean a.u. of fluorescence/mg protein ± standard errors of the mean (SE) from three independent experiments performed in triplicate. (A) *P < 0.05, Aβ‐treated LacZ cells versus untreated LacZ cells at time 0; **P < 0.05, Aβ‐treated RGN cells versus Aβ‐treated LacZ cells at the same time point; (B) *P < 0.05, Aβ‐treated LacZ cells versus untreated LacZ cells; **P < 0.05, untreated RGN cells versus untreated LacZ cells; #P < 0.05, Aβ‐treated RGN cells versus Aβ‐treated LacZ cells; (C and D) *P < 0.05, Aβ‐treated LacZ cells versus untreated LacZ cells at time 0; **P < 0.05, Aβ‐treated RGN cells versus Aβ‐treated LacZ cells at the same time point.

Figure 2

RGN attenuates Aβ‐induced oxidative damage in neuronally differentiated PC12 cells. NGF‐differentiated PC12 cells overexpressing LacZ or RGN were treated with 25 μm Aβ for 24 h. (A) Mitochondrial ROS generation was detected using the fluorescent (${\mathrm{O}}_2^{-}$) probe MitoSOX Red. (B) Intracellular ROS generation was detected using fluorescent hydrogen peroxide indicator CM‐H₂ DCFDA. (C) Oxidative damage was estimated by measuring malondialdehyde contents as an index of lipid peroxidation. Data are presented as means ± SE of three independent experiments performed in triplicate. *P < 0.05, Aβ‐treated LacZ cells versus untreated LacZ cells; **P < 0.05, untreated RGN cells versus untreated LacZ cells; ^# P < 0.05, Aβ‐treated RGN cells versus Aβ‐treated LacZ cells.

Figure 3

RGN attenuates Aβ‐induced nitrosative damage in neuronally differentiated PC12 cells. NGF‐differentiated PC12 cells overexpressing LacZ or RGN were treated with 25 μm Aβ for the indicated time periods. (A) NO contents of medium were measured using the Griess method. (B) iNOS protein expression was determined using western blotting with a specific antibody. Actin expression was used as a protein loading control. (C) Cells were transiently cotransfected with a NF‐κB‐responsive reporter vector and the internal control vector pRL‐TK. After 12 h, transfected cells were treated with Aβ for the indicated periods and were then lysed for NF‐κB reporter assays with a dual‐luciferase reporter assay system. Data are presented as fold increases compared with luciferase activity at 0 h in LacZ cells, which was set at 1.0. (D) Nitrosative damage was estimated by measuring content of nitrotyrosine, which indicates the nitration of tyrosine by ONOO ⁻ generated from NO and ${\mathrm{O}}_2^{-}$. Data are presented as means ± SE of three independent experiments performed in triplicate. (A, C, and D) *P < 0.05, Aβ‐treated LacZ cells versus untreated LacZ cells at time 0; **P < 0.05, Aβ‐treated RGN cells versus Aβ‐treated LacZ cells at the same time point.

Figure 4

RGN‐deficient PC12 neuron‐like cells become vulnerable to Aβ toxicity. A lentivirus‐mediated CRISPR/Cas9 system was used to generate PC12 cells, in which the RGN gene is knocked out. PC12 cells were infected with lentiviruses harboring RGN sgRNA or control scramble sgRNA, selected with puromycin, and then neuronally differentiated using treatments with NGF. (A) Knockdown efficiency was examined by western blotting using β‐actin as a loading control. (B–F) Upon treatment with Aβ, apoptosis assay, caspase assay, mitochondrial ROS measurement, NO assay, and iNOS western blotting were performed as described in the legends to Figs 1A,C,D, 2A and 3A,B, respectively. Data are presented as means ± SE of three independent experiments performed in triplicate. (B) *P < 0.05, Aβ‐treated RGN sgRNA cells versus Aβ‐treated scramble sgRNA cells; (C,D) *P < 0.05, Aβ‐treated scramble sgRNA cells versus untreated scramble sgRNA cells; **P < 0.05, Aβ‐treated RGN sgRNA cells versus Aβ‐treated scramble sgRNA cells; (E) *P < 0.05, Aβ‐treated scramble sgRNA cells versus untreated scramble sgRNA cells; **P < 0.05, untreated RGN sgRNA cells versus untreated scramble sgRNA cells; ^# P < 0.05, Aβ‐treated RGN sgRNA cells versus Aβ‐treated scramble sgRNA cells; (F) *P < 0.05, Aβ‐treated scramble sgRNA cells versus untreated scramble sgRNA cells; **P < 0.05, Aβ‐treated RGN sgRNA cells versus Aβ‐treated scramble sgRNA cells.