ABSTRACT
We have demonstrated previously that exogenously expressed Brn-3a is capable of protecting neurons of the peripheral nervous system against apoptosis. In these previous studies Brn-3a showed a degree of neuronal sub-type specificity, in that while it could promote survival in NGF-dependent sensory neurons, no effect was observed in NGF-dependent neurons of the sympathetic nervous system. In this report, we show that Brn-3a delivered using a herpes simplex virus is capable of protecting cultures of rat cerebrocortical neurons of the central nervous system against two types of cell death stimuli, including glutamate neurotoxicity. Hence the protective effect of Brn-3a is not confined to neurons of the peripheral nervous system but can also occur in neurons of the CNS.
Subject(s)
Apoptosis/physiology , Cerebral Cortex/metabolism , DNA-Binding Proteins/genetics , Nerve Degeneration/genetics , Neurons/metabolism , Transcription Factors/genetics , Animals , Animals, Newborn , Apoptosis/drug effects , Camptothecin/pharmacology , Cell Survival/genetics , Cerebral Cortex/cytology , Cerebral Cortex/drug effects , Enzyme Inhibitors/pharmacology , Excitatory Amino Acid Antagonists/pharmacology , Genetic Therapy , Genetic Vectors/genetics , Glutamic Acid/metabolism , Glutamic Acid/pharmacology , Green Fluorescent Proteins , Immunohistochemistry , Indicators and Reagents/metabolism , Luminescent Proteins/genetics , Nerve Degeneration/physiopathology , Nerve Degeneration/therapy , Neurons/cytology , Neurons/drug effects , Neurotoxins/pharmacology , Protein Structure, Tertiary/genetics , Rats , Rats, Sprague-Dawley , Simplexvirus/genetics , Staurosporine/pharmacology , Transcription Factor Brn-3ABSTRACT
The determination of cell fate plays a critical role during the later stages of embryogenesis and the early postnatal period-a time during which approximately half of neurons born during neurogenesis undergo programmed cell death. It has previously been reported that the type IV POU domain transcription factor Brn-3a plays a role in the maturation and survival of sensory neuronal populations. Indeed we have shown that the long form of Brn-3a is capable of activating expression of the antiapoptotic Bcl-2 gene and enhancing neuronal survival in cultures of sensory neurons. In this study, we report the identification of another antiapoptotic family member, Bcl-x(L), as a target gene of Brn-3a in sensory neurons, providing a further mechanism by which Brn-3a determines sensory neuronal fate during development. Bcl-x(L) gene expression is activated by Brn-3a in sensory but not in sympathetic neurons and its expression is reduced by antisense inhibition of Brn-3a expression in sensory neurons. Most importantly, both Bcl-x(L) expression and neuronal survival are enhanced by the overexpression of Brn-3a in dorsal root ganglion in vivo in a model of sciatic nerve injury in the intact animal.
Subject(s)
DNA-Binding Proteins/genetics , Neurons/cytology , Neurons/physiology , Proto-Oncogene Proteins c-bcl-2/genetics , Transcription Factors/genetics , Animals , Cell Survival/physiology , Cells, Cultured , DNA, Antisense , Disease Models, Animal , Ganglia, Spinal/cytology , Gene Expression/physiology , In Vitro Techniques , Promoter Regions, Genetic/physiology , RNA, Messenger/metabolism , Rats , Rats, Sprague-Dawley , Sciatic Nerve/injuries , Sciatic Nerve/physiology , Superior Cervical Ganglion/cytology , Transcription Factor Brn-3 , Trigeminal Ganglion/cytology , bcl-X ProteinABSTRACT
NGFI-A is an immediate early gene (IEG) that is transcriptionally induced by nerve growth factor (NGF) in PC12 cells and has been implicated in a number of cellular responses. Studies have shown that elements within the first 106 base pairs of the NGFI-A promoter contribute to its induction by NGF in PC12 cells. One element, within the serum response element (SRE) bridge region, bears strong homology to a motif previously identified in promoters regulated by the Brn-3a POU domain transcription factor. We report here that Brn-3a activates the NGFI-A promoter in neurons (both primary and cell lines). Analysis revealed that this response requires sequences between positions -49 and -106. Whilst DNA-protein interaction studies failed to identify a site bound directly by Brn-3a, the data presented here suggest that Brn-3a may cooperate in the regulation of NGFI-A gene expression in neurons, possibly during the developmental switch between neurotrophin dependency that occurs during neurogenesis.
Subject(s)
DNA-Binding Proteins/genetics , DNA-Binding Proteins/physiology , Immediate-Early Proteins , Transcription Factors/genetics , Transcription Factors/physiology , 3T3 Cells , Animals , Base Sequence , Cell Line , Cells, Cultured , Chlorocebus aethiops , DNA-Binding Proteins/metabolism , Dexamethasone/pharmacology , Early Growth Response Protein 1 , Gene Expression Regulation/drug effects , Hybrid Cells , Mice , Mice, Inbred BALB C , Molecular Sequence Data , Neurons/cytology , Neurons/drug effects , Neurons/metabolism , Neurons, Afferent/cytology , Neurons, Afferent/metabolism , PC12 Cells , Promoter Regions, Genetic/genetics , Protein Binding , Rats , Rats, Sprague-Dawley , Recombinant Fusion Proteins/genetics , Recombinant Fusion Proteins/metabolism , Recombinant Fusion Proteins/physiology , Response Elements/genetics , Transcription Factor Brn-3 , Transcription Factor Brn-3A , Transcription Factors/metabolism , Vero CellsABSTRACT
The BCL-2 protein is able to protect neuronal and other cell types from apoptotic programmed cell death and plays a key role in regulating the rate of apoptosis during development of the nervous system. We have previously demonstrated that the Brn-3a POU domain transcription factor protects sensory neurons from apoptotic programmed cell death induced by nerve growth factor withdrawal. We report here that Bcl-2 transcription is predominantly initiated from the Bcl-2 P2 promoter in both the ND7 neuronal cell line and primary dorsal root ganglion neurons, in contrast to the predominant use of the Bcl-2 P1 promoter in other cell types. Moreover, Bcl-2 transcription initiated from the P2 region increases in ND7 cells stably overexpressing Brn-3a, resulting in enhanced BCL-2 protein levels. Similarly, the Bcl-2 P2 promoter is directly activated by Brn-3a in co-transfection assays in both ND7 cells and dorsal root ganglion neurons. Analysis of the Bcl-2 regulatory sequence revealed a binding site for Brn-3a that is required for maximal activation by Brn-3a both in transfected cells and during differentiation of ND7 cells. Together these data identify Brn-3a as the first transcription factor regulating Bcl-2 activity specifically in neuronal cells and indicate that the anti-apoptotic effect of Brn-3a is likely to be mediated, at least in part, via the up-regulation of Bcl-2 expression.
