Objective: To establish a stable human neuronal model defective in PanK2.

Background: Pantothenate Kinase-Associated Neurodegeneration (PKAN), is caused by mutations in PANK2, which encodes one isoform of the pantothenate kinases (PanK). PanK catalyzes the phosphorylation of pantothenic acid (PA, vitamin B5), the rate-limiting step in the synthesis of CoA. Since PKAN is caused by mutations in the PANK2 gene, our focus is on PanK2, which is expressed in neuronal tissue and postulated to be localized in mitochondria. A deficiency in CoA biosynthesis has been linked with the PKAN disorder. Human cellular models of PKAN are not available.

Methods: The human neuroblastoma model of PKAN was developed by selectively silencing PanK2 with a short-hairpin RNA. Four different gene-specific shRNA expression vectors were used for transduction of IMR32 cells. Target cells were transduced and stable clones selected. Effects of PanK2 knockdown over time were evaluated. PANK2 mRNA was quantified by real-time RT-PCR (TaqMan). Tubulin and histone acetylation were determined by western blot. Intracellular CoA was quantified by LC-MS/MS. Intracellular PanK2 localization was determined by immunofluorescence.

Results: We established a stable PanK2 defective cell model using shRNAs specific for PANK2. shRNAs were delivered into a human neuronal cell line IMR32 via infection with a suitable lentiviral vector. A reliable, sensitive and reproducible LC-MS/MS method, without derivatization, for the quantification of free CoA was established. PanK2-silenced neuroblastoma IMR32 cells exhibited reduced proliferation; CoA levels were ∼ 4-fold lower than in non-silenced cells. The reduced CoA levels correlated with decreased levels of tubulin and histone H3 acetylation. Incubation with PPA in silenced IMR32 cells did not restore CoA levels, unless cells were permeabilized with alamethicin. Intracellular CoA levels were rescued after treatment with RE-024, a permeable PPA precursor converted to CoA intracellularly.

Conclusions: This model is the first reported human cellular model for PKAN. It allows the evaluation of drug candidates designed to restore CoA in the context of a PanK2 deficient pathway. Effects on both the level of CoA and on metabolic pathways requiring CoA can be monitored.

ACMG, March 2016.

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MDS Abstracts - https://www.mdsabstracts.org/abstract/development-of-a-human-neuroblastoma-model-of-pantothenate-kinase-associated-neurodegeneration/