Abstract
The proximal enhancer of the cytochrome c gene (Cycs) contains binding sites for both cAMP response element binding proteins (CREB) and Nuclear Respiratory Factor 1 (NRF1). To investigate how neuronal activity regulates this enhancer region, a lentivirus was constructed in which a short-lived green fluorescent protein (GFP) was placed under the transcriptional control of the Cycs proximal enhancer linked to a synthetic core promoter. Primary hippocampal neurons were infected, and the synaptic strengths of individual neurons were measured by whole-cell patch clamping. On average the amplitude of miniature postsynaptic currents (mEPSCs) was higher in brighter GFP+ neurons, while the frequency of mEPSCs was not significantly different. Increasing neural activity by applying a GABAA receptor antagonist increased GFP expression in most neurons, which persisted after homeostatic synaptic scaling as evidenced by a decrease in the amplitude and frequency of mEPSCs. Removing the CREB binding sites revealed that calcium influx through L-type channels and NMDA receptors, and ERK1/2 activation played a role in NRF1-mediated transcription. CREB and NRF1, therefore, combine to regulate transcription of Cycs in response to changing neural activity.
Highlights
Transcriptional responses to strong activation of glutamatergic synapses are triggered by calcium influx through NMDA receptors and calcium channels (Bading et al, 1993)
DIFFERENTIAL EXPRESSION OF Cycs REPORTER IN INDIVIDUAL NEURONS The 235 bp Cycs proximal enhancer fragment was linked to a synthetic basal promoter and a green fluorescent protein (GFPpd2) gene, and inserted into a self-inactivating lentiviral vector containing a copy of the core sequence of the hypersensitive site 4 (HS4) insulator element in place of U3 (Chung et al, 1997)
We found that brighter neurons had statistically significant larger size miniature excitatory postsynaptic currents than dimly fluorescent neurons while there was no significant difference in the frequency of miniature postsynaptic currents (mEPSCs) (Figures 1B–F)
Summary
Transcriptional responses to strong activation of glutamatergic synapses are triggered by calcium influx through NMDA receptors and calcium channels (Bading et al, 1993). Many immediate early genes that are thought to drive synaptic remodeling are regulated by CREB, highlighting the importance of this transcription factor in the nervous system (Flavell and Greenberg, 2008). The promoter regions of a number of nuclear-encoded mitochondrial genes have CREB binding sites (Zhang et al, 2005; van Waveren and Moraes, 2008) indicating that energy metabolism and neural activity may be coordinated via this transcription factor. A second important link between neuronal activity and mitochondrial function is forged by Nuclear Respiratory Factor 1 (NRF1), which has recently been shown to regulate the transcription of NMDA receptor subunit genes (Dhar and Wong-Riley, 2009). Subsequent work has shown that NRF1 regulates all of the nuclear-encoded components of Complex IV (Dhar et al, 2008), and other genes critical for mitochondrial function (Scarpulla, 2008)
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