GABAergic lineage differentiation of AF5 neural progenitor cells in vitro

Abstract

We have previously described an immortal rat central-nervous-system progenitor cell line, AF5, which is able to exit the cell cycle and assume a differentiated state with neuronal properties. The phenotypic specification of differentiated AF5 cells, however, is not known. In the present study, when induced to differentiate by serum starvation in Neurobasal medium, AF5 cells down-regulate glial fibrillary acidic protein and up-regulate expression of beta-III-tubulin, medium-molecular-weight neurofilament protein, and neuronal growth-associated protein 43. Expression of the gamma-aminobutyric acid (GABA) lineage marker, glutamic acid decarboxylase 67 (GAD67), increases during differentiation, suggesting that AF5 cells adopt a GABAergic lineage. Time-course analysis of the GABAergic neuron specification transcription factor, Pitx2, by reverse transcription/polymerase chain reaction, has shown an increase in the Pitx2 transcript 48 h after initiation of differentiation. In differentiated AF5 cells, expression of the Pitx2 target gene products GAD65 and GABA transporter-1 increases. Cellular GABA levels in differentiated AF5 cells increase by about 26-fold, and GABA release into the medium is 150-fold higher compared with that of undifferentiated cells. Therefore, AF5 cells can be induced to differentiate to a neuronal phenotype with a GABAergic lineage.

Fig. 1

a) Confluent AF5 cells, b) Confluent E15 derived rat astrocytes, c) coculture of rat astrocytes and AF5 cells resulted in morphological changes. d) AF5 cells treated with 50μM dbcAMP for several days resulted in cell death and minor neurite extension. e) AF5 cells labeled with GFP (green) and cultured in the presence of rat cortical neurons did not undergo morphological changes. f) β-III-tubulin (red) expression increased in a minority of cells. *β-III-tubulin expression detected in a single AF5 cell.

Fig. 2

Phenotypic Changes in AF5 cells. (a, b) Cells transiently transfected with a plasmid vector encoding an enhanced green fluorescent protein/tau fusion protein (EGFP-Tau; pTG) and cultured under normal proliferative conditions or incubated in Neurobasal/B27 for 4 days. This resulted in morphological changes including neurite extension and cytosolic compaction. (c) Cells under proliferative conditions expressed high levels of nestin protein (red). (d) The number of cells that are nestin positive decreased with Neurobasal/B27 incubation supplemented with dbcAMP for 48 hr; nuclei were counterstained with DAPI (blue). (e, f) 48 hr in Neurobasal/B27/dbcAMP also modulated both cellular morphology and GAD67 expression. (g) Cells under logarithmic growth conditions were negative for GAD65, but (h) when allowed to grow undisturbed in complete media for 1 week, began to express GAD65 (red); nuclei were counterstained with DAPI (blue).

Fig. 3

(a) Neural marker expression during differentiation. AF5 cells were incubated to sub-confluence in DMEM/F12 media (log growth) or in Neurobasal/B27 media post-confluence for 4 days (differentiated). Immunoblot analysis indicates downregulation of glial fibrillary acidic protein (GFAP) and increased expression of neuron-specific β-III-tubulin, neuronal GAP43 and neurofilament-M proteins (NF-M). Immunoblot analysis of α-tubulin is shown to indicate protein loading. (b) AF5 cells upregulated the GABAergic marker, GAD67 after 4 days in Neurobasal/B27 media indicating a potential for expression of a GABAergic phenotype.

Fig. 4

AF5 cells modulated the GABAergic-specific Pitx2 transcription factor and target protein expression. (a) Real-time PCR time-course analysis was carried out on AF5 cells under normal proliferating conditions, growth to confluence or differentiated for 2 and 4 days in Neurobasal/B27 medium. A significant increase in Pitx2 transcription factor mRNA was detected 2 days after induction of differentiation. (b) AF5 cells were incubated to sub-confluence in DMEM/F12 media (log growth) or in Neurobasal/B27 media post-confluence for 4 days (differentiated). Immunoblot analysis indicates that GAD65 protein was undetectable in AF5 cells during logarithmic growth in DMEM/F12 media, and accumulated during differentiation. AF5 cells contain basal levels of GAT-1, which was up-regulated in response to differentiating conditions. Also shown is analysis of α-tubulin to indicate protein loading.

Fig. 5

AF5 cells contain and release GABA. (a) AF5 cells cultured after confluence in Neurobasal media contained ∼26-fold more GABA than undifferentiated cells, as measured by HPLC. Values of 13.6 +/- 0.6 and 8.07 +/- 0.06 nmol/mg protein were measured in differentiated cells following 24-hour and 48-hour exposure to fresh NB/B27 medium, respectively. (b) Differentiated AF5 cells also released >150-fold more GABA into the media than undifferentiated cells, generating media GABA levels of 115.0 +/- 6.0 and 194.0 +/- 11.0 nmol/mg protein after 24 and 48 hr in Neurobasal/B27 media, respectively.