Carnitine Protects against MPP+-Induced Neurotoxicity and Inflammation by Promoting Primary Ciliogenesis in SH-SY5Y Cells

Abstract

Primary cilia help to maintain cellular homeostasis by sensing conditions in the extracellular environment, including growth factors, nutrients, and hormones that are involved in various signaling pathways. Recently, we have shown that enhanced primary ciliogenesis in dopamine neurons promotes neuronal survival in a Parkinson's disease model. Moreover, we performed fecal metabolite screening in order to identify several candidates for improving primary ciliogenesis, including L-carnitine and acetyl-L-carnitine. However, the role of carnitine in primary ciliogenesis has remained unclear. In addition, the relationship between primary cilia and neurodegenerative diseases has remained unclear. In this study, we have evaluated the effects of carnitine on primary ciliogenesis in 1-methyl-4-phenylpyridinium ion (MPP⁺)-treated cells. We found that both L-carnitine and acetyl-L-carnitine promoted primary ciliogenesis in SH-SY5Y cells. In addition, the enhancement of ciliogenesis by carnitine suppressed MPP⁺-induced mitochondrial reactive oxygen species overproduction and mitochondrial fragmentation in SH-SY5Y cells. Moreover, carnitine inhibited the production of pro-inflammatory cytokines in MPP⁺-treated SH-SY5Y cells. Taken together, our findings suggest that enhanced ciliogenesis regulates MPP⁺-induced neurotoxicity and inflammation.

Keywords: L-carnitine; MPP+; Mitochondria; SH-SY5Y cells; acetyl-L-carnitine; primary cilia.

Figure 1

Carnitines stimulate primary ciliogenesis in RPE and SH-SY5Y cells. (A–C), RPE cells stably expressing smo-GFP protein (RPE/Smo-GFP) were treated with Smoothened Agonist (SAG) (1 µM), L-carnitine (L-Car, 100 µM), or acetyl-L-carnitine (Ac-Car, 100 µM) for 24 h. Then, the Smo-GFP protein was imaged using fluorescence microscopy. White arrows indicate Smo-GFP (A). The ciliary cells and cilium lengths were measured using a fluorescence microscope (B). The RPE/Smo-GFP cells were harvested and analyzed by western blotting with indicated antibodies (C). (D–F), SH-SY5Y cells transiently transfected with either scrambled siRNA (Sc) or targeted siRNA for IFT88 (siIFT88) were treated with L-carnitine (100 µM) or Ac-L-carnitine (100 µM) for 24 h. Cells were stained with ARL13B antibody (red) and Hoechst dye (blue) (D). The ciliary cells and cilium lengths were measured (E). The cells were harvested and analyzed by western blotting with indicated antibodies (F). Data are presented as the mean ± SEM (n = 3, * p < 0.05, ** p < 0.01, *** p < 0.001, n.s. = not significant). Scale bar: 5 µm.

Figure 2

Carnitine inhibits excessive generation of mitochondrial ROS and mitochondrial fragmentation by MPP⁺ in SH-SY5Y cells. (A,B), SH-SY5Y cells stably expressing mito-HyPer protein (SY5Y/mito-HyPer cells) were treated with MPP⁺ (2.5 mM) in the presence or absence of L-carnitine (L-Car, 100 µM) or Ac-L-carnitine (Ac-Car, 100 µM) for 24 h. Next, the fluorescence intensity of mito-HyPer was imaged (A) and the fluorescence was measured (B). Scale bar: 20 µm. Data are presented as the mean ± SEM (n = 3, * p < 0.05, ** p < 0.01). (C–E), SH-SY5Y cells stably expressing mito-RFP protein (SY5Y/mito-RFP cells) were transiently transfected with either scrambled siRNA (Sc) or targeted siRNA for IFT88 (siIFT88). These cells were then treated with MPP⁺ (2.5 mM) in the presence or absence of L-Carnitine (100 µM) or Ac-L-Carnitine (100 µM) for 24 h. Then, Mito-RFP in the cells was imaged using fluorescence microscopy (C), and the mitochondrial length was assessed (D). The SH-SY5Y cells were harvested and analyzed by western blotting with indicated antibodies (E). Data are presented as the mean ± SEM (n = 3, *** p < 0.001, n.s. = not significant). Scale bar: 5 µm.

Figure 3

Carnitine inhibits MPP⁺-induced neurotoxicity by enhancing ciliogenesis in SH-SY5Y cells. (A,B), SH-SY5Y cells were treated with MPP⁺ (2.5 mM) in the presence or absence of L-carnitine (L-Car, 100 µM) or Ac-L-carnitine (Ac-Car, 100 µM) for 24 h. Then, cell viability was measured by a CCK-8 assay (A). The cells were further analyzed by western blotting with cleaved caspase-3 antibody (B). (C,D), SH-SY5Y cells were transiently transfected with either scrambled siRNA (Sc) or targeted siRNA for IFT88 (siIFT88). The cells were then treated with MPP⁺ (2.5 mM) in the presence or absence of L-carnitine (100 µM) or Ac-L-carnitine (100 µM) for an additional 24 h. Cell death was measured using a CCK-8 assay (C), and western blotting was performed with indicated antibodies (D). Data are presented as the mean ± SEM (n = 3, * p < 0.05, ** p < 0.01, *** p < 0.001, n.s. = not significant).

Figure 4

Carnitine inhibits the production of pro-inflammatory cytokines in MPP⁺-treated SH-SY5Y cells. SH-SY5Y cells transfected with either scrambled siRNA (Sc) or siRNA against IFT88 (siIFT88) were treated with MPP⁺ (2.5 mM) in the presence or absence of L-carnitine (L-Car, 100 µM) or Ac-L-carnitine (Ac-Car, 100 µM) for 24 h. The expression levels of TNF-α (A) and IL-6 (B) under the relevant culture conditions were measured using an ELISA kit. The treated cells were further analyzed by western blotting using indicated antibodies (C). Data are presented as the mean ± SEM (n = 3, * p < 0.05, ** p < 0.01, n.s. = not significant).