Sequential induction of embryonic and adult forms of glutamic acid decarboxylase during in vitro-induced neurogenesis in cloned neuroectodermal cell-line, NE-7C2 Patricia Varju,* Zoya Katarova, Emı ´lia Madara ´sz* and Ga ´bor Szabo ´   *Laboratory of Neural Cell Biology and  Laboratory of Molecular Biology and Genetics, Institute of Experimental Medicine, Hungarian Academy of Sciences, Budapest, Hungary Abstract The expression of different forms of glutamate decarboxy- lases and GABA was investigated in the course of retinoic acid-induced neuronal differentiation of NE-7C2 cell-line established from brain vesicles of 9-day-old mouse embryos lacking functional p53 gene. Non-induced NE-7C2 cells expressed embryonic GAD mRNAs with a low level of embryonic GAD25 protein and did not contain detectable amounts of GABA. Addition of 10 )6 M retinoic acid induced the expression of N-tubulin and a significant increase in the level of embryonic GAD messages and GAD25 protein in early stage differentiating neurones. The enzymatically active embryonic GAD44 was detected at later stages of induction in neurone-like cells and showed a maximum of expression at the time of neurite elongation and network formation. With the advance of neuronal maturation, the expression of embryonic forms declined while the adult GAD65 and GAD67 transcripts became dominant. GABA-containing neurones were first demonstrated on the sixth day of induction coinciding with the peak of GAD44 expression and the beginning of GAD65 expression. The sequential induction of different GAD forms and the stage-dependent GABA synthesis in NE-7C2 cells is highly reminiscent of the temporal pattern found in vivo and suggests that these processes might be involved in the differentiation of neuronal progenitors. Keywords: GABA, GAD isoforms, neuronal differentiation, retinoic acid. J. Neurochem. (2002) 80, 605–615. GABA, a classical inhibitory neurotransmitter in the adult nervous system, plays an important role in neural differen- tiation well before synapses become functionally active (Meier et al. 1991; Nguyen et al. 2001; Varju et al. 2001a). The trophic effects of GABA are thought to be mediated via the elevation of intracellular Ca 2+ concentration through GABA A receptor channels, exerting depolarizing effects on developing neurones (Cherubini et al. 1991; Owens et al. 1999). GABA-mediated signalling may be regulated, in part, by the modulation of GABA-production by glutamic acid decarboxylases (GAD; 4.1.1.15), the key enzymes in GABA synthesis (Martin and Rimvall 1993; Soghomonian and Martin 1998; Varju et al. 2001a). In the adult nervous system, the two known GAD isoforms – GAD65 and GAD67 – are encoded by indepen- dent genes (Erlander et al. 1991; Bu et al. 1992), and differ in enzyme-kinetic properties and cofactor binding (Kaufman et al. 1991; Martin and Rimvall 1993; Martin et al. 2000). The GAD67 form is evenly distributed in the cytoplasm (Erlander et al. 1991; Kaufman et al. 1991) and binds also to the plasma membrane (Kanaani et al. 1999), while the 65-kDa GAD form is mainly anchored to various membranes and synaptic vesicles (Hsu et al. 2000). During embryonic development, two additional alterna- tively spliced transcripts are produced from the rat and mouse GAD67 gene (Bond et al. 1990; Szabo ´ et al. 1994). The embryo-specific GAD mRNAs (I-80 and I-86) include two, almost identical alternatively spliced exons (7A and 7B) inserted into the coding sequence of GAD67 (Szabo ´ et al. Received July 3, 2001; revised manuscript received November 15, 2001; accepted November 15, 2001. Address correspondence and reprint requests to G. Szabo ´ , Laboratory of Molecular Biology and Genetics, Institute of Experimental Medicine, Hungarian Academy of Sciences, Budapest, PO Box 67, Hungary 1450. E-mail: szabog@koki.hu Abbreviations used: ATRA, all-trans retinoic acid; dpi, days post induction; GAD, glutamic acid decarboxylase; GFAP, glial fibrillary acidic protein; MAP2, microtubule associated protein 2; NFM, neuro- filament mezo; N-tubulin, neurone-specific IIIb-tubulin; ORF, open reading frame. Journal of Neurochemistry , 2002, 80, 605–615 Ó 2002 International Society for Neurochemistry, Journal of Neurochemistry , 80, 605–615 605