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PO3-8-5

Effect of 5-aminolevulinic acid against low inorganic phosphate in neuroblastoma SH-SY5Y cells

[Speaker] Naoko Takase:1
[Co-author] Masatoshi Inden:1, Shin-Ichiro Sekine:1, Yuki Kaneko:1, Hisaka Kurita:1, Isao Hozumi:1
1:Laboratory of Medical Therapeutics and Molecular Therapeutics, Gifu Pharmaceutical University, Japan

[Purpose] Idiopathic basal ganglia calcification (IBGC) is a rare genetic condition characterized by symmetric calcification in the basal ganglia and other brain regions, a wide spectrum of neuropsychiatric symptoms. Recently, SLC20A2 mutations have been found in patients with IBGC. This mutation was predicted to be unable to transport inorganic phosphate (Pi) from the extracellular environment. PiT-2 encoded by SLC20A2, and PiT-1 encoded by SLC20A1 is classified as the type-III sodium-dependent phosphate cotransporter (NaPiTs). However, the roles of type-III NaPiTs under low Pi loading is unknown. 5-Aminolevulinic acid (5-ALA) is an amino acid which is localized in the mitochondria and has an important role for life support involving the production of ATP. In this study, we investigated the effect of low Pi loading on the human neuroblastoma cell line SH-SY5Y. We also investigated whether 5-ALA protects mitochondrial dysfunction by low phosphate loading.
[Method] SH-SY5Y cells were incubated overnight in FBS-free / Pi-free DMEM. An appropriated amounts of 5-ALA and sodium phosphate buffer (0.1 M Na2HPO4/NaH2PO4, pH 7.4) was administered in the cell culture. LIVE/DEAD Cell Imaging Kit and Cell Count Reagent SF were used for cytotoxicity assays. Mitochondrial membrane potential was measured by JC-1 regent. Cell Rox and Mito Sox regents were used to carry out the evaluation of oxidative stress. To investigate the signaling pathway of 5-ALA, phosphorylation ERK and p38 MAPK were measured by western blot, and the expressive level of HO-1 was measured by real time RT-PCR.
[Result/Discussion] Long term treatment with low phosphate (for 24, 48, and 72h) caused cell death, which increased with treatment time. The expression level of type-III NaPiTs by low Pi loading did not change. 5-ALA inhibited the low Pi loading-induced neurotoxicity. 5-ALA also prevented mitochondrial dysfunction and ROS production caused by low Pi loading. Moreover, our study demonstrated that 5-ALA inhibited the low Pi loading-induced neurotoxicity via the induction of HO-1 by p38 MAPK1). The findings provide us with novel viewpoints to understand the pathophysiology of IBGC and pave the way for clinical prevention and treatment of IBGC via type-III NaPiTs.
1) Takase N et al., Sci Rep. 2017; 7: 5768.
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