The formation and plasticity of neuronal circuits relies on dynamic activity-dependent gene expression. Although recent work has revealed the identity of important transcriptional regulators and of genes that are transcribed and translated in response to activity, relatively little is known about the cell biological mechanisms by which activity alters the nuclear proteome of neurons to link neuronal stimulation to transcription. Using nucleus-specific proteomic mapping in silenced and stimulated neurons, we uncovered an understudied mechanism of nuclear proteome regulation: activity-dependent proteasome-mediated degradation. We found that the tumor suppressor protein PDCD4 undergoes rapid stimulus-induced degradation in the nucleus of neurons. We demonstrate that degradation of PDCD4 is required for normal activity-dependent transcription and that PDCD4 target genes include those encoding proteins critical for synapse formation, remodeling, and transmission. Our findings highlight the importance of the nuclear proteasome in regulating the activity-dependent nuclear proteome and point to a specific role for PDCD4 as a regulator of activity-dependent transcription in neurons.
Calcium activity has been implicated in many neurodevelopmental events, including the specification of neurotransmitter phenotypes. Higher levels of calcium activity lead to an increased number of inhibitory neural phenotypes, while lower levels of calcium activity lead to excitatory neural phenotypes. Voltage-gated calcium channels (VGCCs) allow for rapid calcium entry and are expressed during early neural stages, making them likely regulators of activity-dependent neurotransmitter phenotype specification. To test this hypothesis, multiplex fluorescent in situ hybridization was used to characterize the coexpression of eight VGCC α1 subunits with the excitatory and inhibitory neural markers, xVGlut1 and xVIAAT, in Xenopus laevis embryos. VGCC coexpression was higher with xVGlut1 than xVIAAT, especially in the hindbrain, spinal cord, and cranial nerves. Calcium activity was also analyzed on a single-cell level and spike frequency was correlated with the expression of VGCC α1 subunits in cell culture. Cells expressing Cav2.1 and Cav2.2 displayed increased calcium spiking compared to cells not expressing this marker. VGCC antagonist diltiazem and agonist (−)BayK 8644 were used to manipulate calcium activity. Diltiazem exposure increased the number of glutamatergic cells and decreased the number of GABAergic cells, while (−)BayK 8644 exposure decreased the number of glutamatergic cells without having an effect on the number of GABAergic cells. Given that the expression and functional manipulation of VGCCs is correlated with neurotransmitter phenotype in some, but not all, experiments, VGCCs likely act in combination with a variety of other signaling factors to determine neuronal phenotype specification.
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