Two Pore Channel 2 Differentially Modulates Neural Differentiation of Mouse Embryonic Stem Cells

PLOS ONE, Dec 2019

Nicotinic acid adenine dinucleotide phosphate (NAADP) is an endogenous Ca2+ mobilizing nucleotide presented in various species. NAADP mobilizes Ca2+ from acidic organelles through two pore channel 2 (TPC2) in many cell types and it has been previously shown that NAADP can potently induce neuronal differentiation in PC12 cells. Here we examined the role of TPC2 signaling in the neural differentiation of mouse embryonic stem (ES) cells. We found that the expression of TPC2 was markedly decreased during the initial ES cell entry into neural progenitors, and the levels of TPC2 gradually rebounded during the late stages of neurogenesis. Correspondingly, TPC2 knockdown accelerated mouse ES cell differentiation into neural progenitors but inhibited these neural progenitors from committing to neurons. Overexpression of TPC2, on the other hand, inhibited mouse ES cell from entering the early neural lineage. Interestingly, TPC2 knockdown had no effect on the differentiation of astrocytes and oligodendrocytes of mouse ES cells. Taken together, our data indicate that TPC2 signaling plays a temporal and differential role in modulating the neural lineage entry of mouse ES cells, in that TPC2 signaling inhibits ES cell entry to early neural progenitors, but is required for late neuronal differentiation.

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Two Pore Channel 2 Differentially Modulates Neural Differentiation of Mouse Embryonic Stem Cells

Citation: Zhang Z-H, Lu Y-Y, Yue J ( Two Pore Channel 2 Differentially Modulates Neural Differentiation of Mouse Embryonic Stem Cells Zhe-Hao Zhang 0 Ying-Ying Lu 0 Jianbo Yue 0 Austin John Cooney, Baylor College of Medicine, United States of America 0 Department of Physiology, University of Hong Kong , Hong Kong , China Nicotinic acid adenine dinucleotide phosphate (NAADP) is an endogenous Ca2+ mobilizing nucleotide presented in various species. NAADP mobilizes Ca2+ from acidic organelles through two pore channel 2 (TPC2) in many cell types and it has been previously shown that NAADP can potently induce neuronal differentiation in PC12 cells. Here we examined the role of TPC2 signaling in the neural differentiation of mouse embryonic stem (ES) cells. We found that the expression of TPC2 was markedly decreased during the initial ES cell entry into neural progenitors, and the levels of TPC2 gradually rebounded during the late stages of neurogenesis. Correspondingly, TPC2 knockdown accelerated mouse ES cell differentiation into neural progenitors but inhibited these neural progenitors from committing to neurons. Overexpression of TPC2, on the other hand, inhibited mouse ES cell from entering the early neural lineage. Interestingly, TPC2 knockdown had no effect on the differentiation of astrocytes and oligodendrocytes of mouse ES cells. Taken together, our data indicate that TPC2 signaling plays a temporal and differential role in modulating the neural lineage entry of mouse ES cells, in that TPC2 signaling inhibits ES cell entry to early neural progenitors, but is required for late neuronal differentiation. - The in vitro generation of neural cells from ES cells promises to produce an almost unlimited supply of cells suitable for cell-based replacement therapies in the nervous system [15]. The most widely used method to trigger neural differentiation is to induce embryoid body (EB) formation followed by retinoic acid (RA) treatment [5,6], or, to culture ES cells with stroma conditioned medium [7,8]. Several studies have been able to direct ES cell differentiation and to generate specific neuronal populations, including spinal cord motor neurons, dorsal interneurons, cerebellar Purkinje and granule cells, and midbrain dopaminergic neurons [9,10]. Because ES cells are pluripotential and readily differentiate into almost any cell type in suspension culture, the efficiency of neural induction by these methods is low and the final cultures are always a heterogenous mixture of various cell types [1]. A simple and efficient way to induce ES cells into neural precursors and subsequently generate neuronal and glia cells is to culture ES cells in an adherent monolayer in defined medium [1,2]. In this method, ES cells are cultured in defined serum-free and feeder-free conditions, in the absence of bone morphogenetic protein (BMP) and Wnts signals. In these conditions, ES cells undergo neural commitment through an autocrine fibroblast growth factor (FGF) signaling mechanism. This method results in a more efficient neural differentiation. Yet, around 40% of cells still resist neural specification and adopt nonneural fates [1,2]. Therefore, to more efficiently induce neural commitment of ES cells, it is essential to define novel cellular and molecular events involved in neural differentiation. Mobilization of intracellular Ca2+ stores is involved in almost all the aspects of cellular processes, e.g. neural differentiation [1114]. Nicotinic adenine acid dinucleotide phosphate (NAADP) is one of the most potent endogenous Ca2+ mobilizing messengers. NAADP is formed by a base-exchange reaction that replaces the nicotinamidemoiety of NADP with nicotinic acid and is catalyzed by ADP-ribosyl cyclases (ARCs). Two enzymes have so far been shown to be capable of synthesizing NAADP from NADP in vitro, Aplysia ARC and CD38. Endogenous NAADP levels can be modulated by a variety of extracellular stimuli. NAADP mobilizes Ca2+ from an acidic lysosomes-related store, which can be subsequently amplified into global Ca2+ waves by CICR from ER/SR via IP3 receptors (IP3Rs) or ryanodine receptors (RyRs) [15,16]. Recently, two pore channel 2 (TPC2), a new member of the superfamily of voltage-gated ion channels containing 12 putative transmembrane segments, has been demonstrated to be the NAADP receptors in many cell types. TPC2 is located on lysosomal membranes when expressed in several cell types. TPC2 overexpression promotes NAADP-induced Ca2+ release from lysosome-related stores, whereas ablating or knocking-down TPC2 expression blocked NAADP-induced Ca2+ release [17 24]. Yet it has been recently shown that NAADP does not directly bind to TPC2 [2527]. In addition, TPC1, TRPML1, TRPM2, or RyRs has been reported to be NAADP receptor in different cell types [2833]. NAADP/Ca2+ signaling pathway regulates diverse cellular processes, including fertilization [34,35], receptor activation in lymphocytes [36], insulin secretion in pancreatic isl (...truncated)


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Zhe-Hao Zhang, Ying-Ying Lu, Jianbo Yue. Two Pore Channel 2 Differentially Modulates Neural Differentiation of Mouse Embryonic Stem Cells, PLOS ONE, 2013, Volume 8, Issue 6, DOI: 10.1371/journal.pone.0066077