Sequential induction of embryonic and adult forms of glutamic acid decarboxylase during in vitro‐induced neurogenesis in cloned neuroectodermal cell‐line, NE‐7C2

Abstract

The expression of different forms of glutamate decarboxylases 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.

GAD isoforms exhibit distinct spatiotemporal expression patterns in the developing mouse lens: Correlation with Dlx2 and Dlx5

“…For amplification of different GAD transcripts, we used PCR primerpairs specific for embryonic GADs and GAD65, as described previously (Varju et al, 2002). Adult GAD67 amplification was performed using a forward primer spanning the exon 6 -8 border region (Table 1), thus precluding amplification of exon 7A/B-containing embryonic cDNAs.…”

“…EGAD (GAD25 and GAD44) showed a transient temporal expression pattern similar to that characteristic for the embryonic nervous system (Szabo et al, 1994): it was low at the earliest stages (E15.5); peaked at E17.5, the stage of most intensive primary fiber cell terminal differentiation (Kaufman, 1992); gradually decreased until a The positions are relative to the ATG codons of the corresponding cDNAs. Primers for actin, EGAD, and GAD65 were as published in Varju et al (2002). RT-PCR, reverse transcriptase-polymerase chain reaction; F, forward; R, reverse primers; GAD, glutamic acid decarboxylase.…”

“…To detect the GAD67 gene-encoded GAD isoforms, we used an anti-GAD antibody (serum #6799), which was raised against the recombinant mouse GAD67, but also recognizes the truncated GAD25 and GAD44 and to a lesser extent, the GAD65 on Western blots (Katarova et al, 1990;Szabo et al, 1994;Varju et al, 2002). This antibody does not bind to GAD65 on tissue sections under the conditions used in this study (Z.D.…”

“…During mouse and rat embryonic development, two additional, alternatively spliced forms are synthesized from the GAD67 gene (Bond et al, 1990;Szabo et al, 1994) encoding the truncated 25-kDa leader (GAD25) and enzymatically active GAD44. GAD25 and GAD44 are transiently expressed during neuronal differentiation (Szabo et al, 1994;Varju et al, 2001Varju et al, , 2002 being more abun-dant in proliferating progenitors and down-regulated in postmitotic neurons concomitant with up-regulation of GAD67 expression (Ma et al, 1993;Behar et al, 1994;Katarova et al, 2000). This has led to the idea that the embryonic GAD proteins are predominantly involved in the synthesis of nonsynaptic GABA acting as a paracrine (autocrine) signaling molecule, while the adult GAD forms provide GABA used for synaptic transmission (Varju et al, 2001).…”

GABAergic lineage differentiation of AF5 neural progenitor cells in vitro

“…Whereas cell lines can also be manipulated to over-express GAD65 Thompson et al 2000), little has been reported regarding inherent GAD65 expression or putative GABAergic function for immortalized cell lines during neuronal differentiation. Promoter studies have been performed in the P19 embryonic carcinoma line (Pinal et al 1997), and GAD65 expression has been reported in human fibroblasts and in neuroectodermal and insulinoma cell lines (Kono et al 2001;Salazar et al 2001;Varju et al 2002). The neuronal cell line NTera-2N is negative for GAD65 mRNA (Yoshioka et al 1997), and although PC12 cells have been found to modulate GAD65 and GAD67 expression in response to hypoxia, they are not otherwise GABAergic (Kobayashi and Millhorn 2001).…”