Abstract
Recent studies of the hippocampus have suggested that a network of genes is associated with the regulation of the GAD67 (GAD1) expression and may play a role in γ-amino butyric acid (GABA) dysfunction in schizophrenia (SZ) and bipolar disorder (BD). To obtain a more detailed understanding of how GAD67 regulation may result in GABAergic dysfunction, we have developed an in vitro model in which GABA cells are differentiated from the hippocampal precursor cell line, HiB5. Growth factors, such as PDGF, and BDNF, regulate the GABA phenotype by inducing the expression of GAD67 and stimulating the growth of cellular processes, many with growth cones that form appositions with the cell bodies and processes of other GAD67-positive cells. These changes are associated with increased expression of acetylated tubulin, microtubule-associated protein 2 (MAP2) and the post-synaptic density protein 95 (PSD95). The addition of BDNF, together with PDGF, increases the levels of mRNA and protein for GAD67, as well as the high affinity GABA uptake protein, GAT1. These changes are associated with increased concentrations of GABA in the cytoplasm of “differentiated” HiB5 neurons. In the presence of Ca2+ and K+, newly synthesized GABA is released extracellularly. When the HiB5 cells appear to be fully differentiated, they also express GAD65, parvalbumin and calbindin, and GluR subtypes as well as HDAC1, DAXX, PAX5, Runx2, associated with GAD67 regulation. Overall, these results suggest that the HiB5 cells can differentiate into functionally mature GABA neurons in the presence of gene products that are associated with GAD67 regulation in the adult hippocampus.
Highlights
Decreased expression of the 67 kDalton isoform of glutamic acid decarboxylase (GAD67) in the hippocampus has been reported in numerous studies on schizophrenia and bipolar disorder [1,2,3,4], two disorders that are thought to be neurodevelopmental in nature
We have established a novel in vitro model in which multipotent progenitor cells in HiB5 hippocampal cultures are differentiated into mature neurons that express GAD67 and other genes associated with the GABA phenotype in the adult hippocampus
To evaluate which growth factors drive HiB5 cell differentiation towards a GABA phenotype when applied acutely, cells were kept under differentiation conditions (39uC, N2 medium) for 2 days, incubated for 2 more days in the presence of platelet derived growth factor (PDGF) to drive differentiation towards a neuronal fate and stimulated with different growth factors (BDNF, NRG-1 or VEGF) two more days in the presence of PDGF
Summary
Decreased expression of the 67 kDalton isoform of glutamic acid decarboxylase (GAD67) in the hippocampus has been reported in numerous studies on schizophrenia and bipolar disorder [1,2,3,4], two disorders that are thought to be neurodevelopmental in nature. GABA cells in the hippocampus develop in response to a finely tuned temporal and spatial pattern of signals emanating from the surrounding cells/tissues at different stages of phenotypic differentiation [7]. In using a cell culture model to study GABA neurons, it is imperative that these cells are phenotypically similar to those endogenously present in the region under study Toward this end, we have established a novel in vitro model in which multipotent progenitor cells in HiB5 hippocampal cultures are differentiated into mature neurons that express GAD67 and other genes associated with the GABA phenotype in the adult hippocampus
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