Functional Expression of Choline Transporters in Human Neural Stem Cells and Its Link to Cell Proliferation, Cell Viability, and Neurite Outgrowth

Abstract

Choline and choline metabolites are essential for all cellular functions. They have also been reported to be crucial for neural development. In this work, we studied the functional characteristics of the choline uptake system in human neural stem cells (hNSCs). Additionally, we investigated the effect of extracellular choline uptake inhibition on the cellular activities in hNSCs. We found that the mRNAs and proteins of choline transporter-like protein 1 (CTL1) and CTL2 were expressed at high levels. Immunostaining showed that CTL1 and CTL2 were localized in the cell membrane and partly in the mitochondria, respectively. The uptake of extracellular choline was saturable and performed by a single uptake mechanism, which was Na+-independent and pH-dependent. We conclude that CTL1 is responsible for extracellular choline uptake, and CTL2 may uptake choline in the mitochondria and be involved in DNA methylation via choline oxidation. Extracellular choline uptake inhibition caused intracellular choline deficiency in hNSCs, which suppressed cell proliferation, cell viability, and neurite outgrowth. Our findings contribute to the understanding of the role of choline in neural development as well as the pathogenesis of various neurological diseases caused by choline deficiency or choline uptake impairment.

<em>Poria cocos</em> (Schw.) Wolf, a Traditional Chinese Edible Medicinal Herb, Promotes Neuronal Differentiation and Maturation of Neural Stem/Progenitor Cells

“…Maternal choline supplementation improves spatial learning and adult hippocampal neurogenesis in the Ts65Dn mouse model of Down syndrome [41]. Although the cellular mechanisms by which choline exerts these effects are unclear, numerous studies have implicated choline involves DNA methylation, altered gene expression, and associated changes in stem cell proliferation and differentiation [42,43]. Therefore, choline was considered to be the main active component of P. cocos that promotes the neuronal differentiation of NSPCs.…”

Poria cocos (Schw.) Wolf, a Traditional Chinese Edible Medicinal Herb, Promotes Neuronal Differentiation, and the Morphological Maturation of Newborn Neurons in Neural Stem/Progenitor Cells

“…In addition, it was also discovered that maternal choline supplementation significantly improves spatial learning and induces adult hippocampal neurogenesis in a Down syndrome mouse model [34]. Although the cellular mechanisms by which choline exerts these effects are unclear, numerous studies have implicated choline in the regulation of stem cell proliferation and differentiation through DNA methylation and altered gene expression [35,36]. We also found choline to have dramatic neuronal differentiation-promoting effects in Neuro-2a cells (Figure 5A-C) and obvious neuronal differentiation-promoting effects in NSPCs (Figure 5D-G).…”

Functional Expression of Choline Transporters in Microglia and Their Regulation of Microglial M1/M2 Polarization

“…Levels of mRNA expression were measured according to previously established methods [ 19 , 42 , 43 ]. Total RNA was extracted from SIM-A9 cells using QIA shredder and RNeasy Mini Kit (Qiagen Inc., Valencia, CA, USA) according to the manufacturer’s instructions.…”

“…The subcellular localization of CTL1 and CTL2 proteins in SIM-A9 cells was performed using immunocytochemistry with reference to previous publications [ 17 , 42 , 43 ]. SIM-A9 cells were washed with Dulbecco’s Phosphate Buffered Saline (D-PBS) (Wako), lysed in RIPA buffer (Santa Cruz Biotechnology, Inc., Dallas, TX, USA) including 1 mM ethylenediaminetetraacetic acid and a protease inhibitor cocktail on ice, and centrifuged at 14,000× g for 15 min at 4 °C.…”