Inositol homeostasis and phosphoinositide signaling during the neuronal differentiation of NT2 cells.

Abstract

Although dysfunctional inositol homeostasis and phosphoinositide signaling in central nervous system (CNS) neurons have been implicated in neuropsychiatric disease, the study of inositol biochemistry in pure, human neuronal populations has been hindered by the lack of a suitable cell model. The goal of this thesis was to use the NT2-N neuronal cell system to test the hypotheses that (i) differentiated neurons attain a high concentration of inositol as a result of changes in inositol transport and that (ii) neuronal differentiation is associated with functional changes in the expression of phosphoinositide signaling proteins. Retinoic acid-mediated differentiation of NT2 precursors to the neuronal phenotype results in an increase in inositol concentration from &sim;2 to &sim;20 m<italic>M</italic>, the latter value which exceeds that of many other neural and non-neural cells and tissues. The high inositol concentration attained by NT2-N neurons is consistent with changes in inositol transport (2-fold increased uptake and 5-fold decreased efflux), whereas the enzymatic synthesis of inositol is not detected. Inositol transport is in turn regulated by signaling molecules in NT2-N neurons. Inositol uptake is refractory to modulation by most second messengers, although hypertonic stimulation of uptake may be mediated by p38 mitogen-activated protein kinase. In contrast, volume-sensitive inositol efflux is stimulated by protein kinase C activation and by elevation of the cytosolic Ca<super> 2+</super> concentration and is blocked by Cl<super>-</super> channel inhibitors. Neuronal differentiation of NT2 precursors also produces 5- to 15-fold increases in Galpha_q/11 and phospholipase C (PLC)-beta1/4 immunoreactivity. Increased expression of phosphoinositide signaling proteins in NT2-N neurons is induced by two chemically distinct agents and approximates the expression pattern of brain. In addition, neuronal differentiation is accompanied by increased rates of phosphoinositide hydrolysis in response to activation of either G proteins or PLC. In conclusion, neuronal differentiation of NT2 cells is associated with (i) an increased concentration of inositol, consistent with changes in polyol transport, and (ii) an increased rate of inositol lipid hydrolysis, consistent with changes in signaling protein expression. Further investigation of NT2-N cells and primary cultures of neuronal populations should facilitate more comprehensive characterization of inositol homeostasis and phosphoinositide signaling in human CNS neurons.