Identification of the C-terminal activation domain of the NeuroD-related factor (NDRF)

NeuroD-related factor (NDRF) is a basic helix–loop–helix (bHLH) protein whose expression is restricted to the central nervous system, and is considered to be responsible for maintenance of differentiated neurons as well as neurogenesis. NDRF structurally resembles NeuroD in the bHLH region and can induce neurogenesis ectopically in ectodermal cells of the Xenopus embryo. In this study, we delineated the functional domains of NDRF. Using GAL4/NDRF fusion proteins, we identified the C-terminal activation domain (C-AD) in NDRF between amino acid positions 294 and 383. This region was highly homologous to one part of the activation domain of NeuroD. We further investigated the transactivational function of C-AD in the mouse type 1 inositol 1,4,5-trisphosphate receptor promoter, which has an NDRF site. Truncation of C-AD resulted in reduction of the activation function, whereas the DNA-binding specificity was not affected. These results suggest that C-AD has a stimulatory function in the mammalian nervous system.     

Translational Regulation of NeuroD1 Expression by FMRP: Involvement in Glutamatergic Neuronal Differentiation of Cultured Rat Primary Neural Progenitor Cells

Fragile X mental retardation protein (FMRP) is encoded by Fmr1 gene in which mutation is known to cause fragile X syndrome characterized by mental impairment and other psychiatric symptoms similar to autism spectrum disorders. FMRP plays important roles in cellular mRNA biology such as transport, stability, and translation as an RNA-binding protein. In the present study, we identified potential role of FMRP in the neural differentiation, using cortical neural progenitor cells from Sprague–Dawley rat. We newly found NeuroD1, an essential regulator of glutamatergic neuronal differentiation, as a new mRNA target interacting with FMRP in co-immunoprecipitation experiments. We also identified FMRP as a regulator of neuronal differentiation by modulating NeuroD1 expression. Down-regulation of FMRP by siRNA also increased NeuroD1 expression along with increased pre- and post-synaptic development of glutamatergic neuron, as evidenced by Western blot and immunocytochemistry. On the contrary, cells harboring FMRP over-expression construct showed decreased NeuroD1 expression. Treatment of cultured neural precursor cells with a histone deacetylase inhibitor, valproic acid known as an inducer of hyper-glutamatergic neuronal differentiation, down-regulated the expression of FMRP, and induced NeuroD1 expression. Our study suggests that modulation of FMRP expression regulates neuronal differentiation by interaction with its binding target mRNA, and provides an example of the gene and environmental interaction regulating glutamatergic neuronal differentiation.     

Promotion of Both Proliferation and Neuronal Differentiation in Pluripotent P19 Cells with Stable Overexpression of the Glutamine Transporter slc38a1

Background

We previously demonstrated the functional expression in newborn rat neocortical astrocytes of glutamine transporter (GlnT = slc38a1) believed to predominate in neurons over astroglia in the brain. In order to evaluate the possible role of this transporter in neurogenesis, we attempted to establish stable transfectants of GlnT in mouse embryonal carcinoma P19 cells endowed to proliferate for self-renewal and differentiate into progeny cells such as neurons and astroglia, in addition to in vitro pharmacological profiling of the green tea ingredient theanine, which is shown to be a potent inhibitor of glutamine transport mediated by GlnT in cultured neurons and astroglia.

Methodology/Principal Findings

The full-length coding region of rat GlnT was inserted into a vector for gene transfection along with selection by G418, followed by culture with all-trans retinoic acid under floating conditions and subsequent dispersion for spontaneous differentiation under adherent conditions. Stable overexpression of GlnT led to marked increases in the size of round spheres formed during the culture for 4 days and 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide reduction, with concomitant promotion of subsequent differentiation into cells immunoreactive for a neuronal marker protein. In these stable GlnT transfectants before differentiation, drastic upregulation was seen for mRNA expression of several proneural genes with a basic helix-loop-helix domain such as NeuroD1. Although a drastic increase was seen in NeuroD1 promoter activity in stable GlnT transfectants, theanine doubled NeuroD1 promoter activity in stable transfectants of empty vector (EV), without affecting the promoter activity already elevated in GlnT transfectants. Similarly, theanine promoted cellular proliferation and neuronal differentiation in stable EV transfectants, but failed to further stimulate the acceleration of both proliferation and neuronal differentiation found in stable GlnT transfectants.

Conclusions/Significance

GlnT would promote both proliferation and neuronal differentiation through a mechanism relevant to the upregulation of particular proneural genes in undifferentiated P19 cells.     

Downregulation of CDC2 upon terminal differentiation of neurons.

The terminal differentiation of neurons occurs as precisely timed waves, with specific neuronal types differentiating in defined sequences. The precision of neuronal differentiation in the central nervous system offers an unusual opportunity to study terminal differentiation in vivo. The p34cdc2 kinase complex and the anti-oncogenes p53 and RB are central in the regulatory network that controls cell proliferation. We found high levels of expression of CDC2 mRNA and protein in proliferating neuronal precursor cells. The expression of both CDC2 and cyclin A was dramatically downregulated upon terminal differentiation of neurons in vivo and in a neuronal precursor cell line, ST15A. p53 mRNA expression was also downregulated but to a lesser extent; RB mRNA levels were unchanged during neuronal differentiation. Immunohistochemistry showed that p34cdc2 was expressed not only in the neuronal precursors of the cerebellar external granule layer but also in the early differentiating granule neurons. The expression of p34cdc2 in early neurons suggests a function for this enzyme in the events that occur soon after proliferation ceases. On the basis of the results reported here and other recent findings, we propose a model in which terminal differentiation is achieved by a switch in the neuronal precursors from p34cdc2-based proliferation to a differentiated state controlled by p34cdc2-related kinases.     

Activin receptor-like kinase 2 can mediate atrioventricular cushion transformation

The development of enteric and sympathetic neurons from neural crest precursor cells is regulated by signals produced by the embryonic environments to which the cells migrate. Bone morphogenetic proteins (BMPs) are present in the developing embryo and act to induce neuronal differentiation and noradrenergic properties of neural crest cells. We have investigated the role of BMP2 in regulating the appearance of distinct populations of autonomic neurons from postmigratory, HNK-1-positive neural crest precursor cells. BMP2 promotes neuronal differentiation of sympathetic and enteric precursor cells isolated from E14.5 rat. The effects of BMP2 change over time, resulting in a decrease in neuron number that can be attributed to apoptotic cell death. BMP2-dependent neuron death is rescued by gut-derived factors that provide trophic support to maturing neurons, indicating that BMP2 regulates the acquisition of trophic dependence of developing peripheral neurons. In addition to regulating neuron number, BMP2 promotes both panneuronal maturation and the acquisition of an enteric phenotype, as measured by lineage-specific changes in the expression of tyrosine hydroxylase and MASH-1. While BMP2 is sufficient to induce neuronal differentiation and panneuronal development, these results suggest that additional factors in the environment must collaborate with BMP2 to promote the final noradrenergic phenotype of sympathetic neurons.     

Immortalization and controlled in vitro differentiation of murine multipotent neural crest stem cells

To isolate mouse neural crest stem cells, we have generated a rat monoclonal antibody to murine neurotrophin receptor (p75). We have immortalized p75⁺ murine neural crest cells by expression of v-myc, and have isolated several clonal cell lines. These lines can be maintained in an undifferentiated state, or induced to differentiate by changing the culture conditions. One of these cell lines, MONC-1, is capable of generating peripheral neurons, glia, and melanocytic cells. Importantly, most individual MONC-1 cells are multipotent when analyzed at clonal density. The neurons that differentiate under standard conditions have an autonomic-like phenotype, but under different conditions can express markers of other peripheral neuronal lineages. These lines therefore exhibit a similar differentiation potential as their normal counterparts. Furthermore, they can be genetically modified or generated from mice of different genetic backgrounds, providing a useful tool for molecular studies of neural crest development. © 1997 John Wiley & Sons, Inc. J Neuroblol 32 : 722–746, 1997