The Etv1/Er81 transcription factor orchestrates activity-dependent gene regulation in the terminal maturation program of cerebellar granule cells

Abstract

In the postnatal period, cerebellar granule cells express a set of the maturation gene battery in an activity-dependent manner and establish synaptic function in the cerebellar circuitry. Using primary cultures combined with specific inhibition of signaling cascades, the present investigation revealed that the expression of the maturation genes, including the NMDA glutamate receptor NR2C and GABA A receptor GABA A Rα6 genes, is controlled by strikingly unified signaling mechanisms that operate sequentially through stimulation of AMPA and NMDA receptors, Na + channels [voltage-gated Na channel type II (Nav1.2)], and voltage-dependent Ca 2+ channels. This signaling then induces the Ets variant gene 1 (Etv1/Er81) transcription factor of the ETS family in an activity-dependent manner. Consistent with the culture study, the ChIP assay indicated that Etv1 up-regulates the maturation genes in a developmentally regulated manner. This activation, as revealed by the luciferase assay, occurrs by interacting with the Etv1-interacting motifs present in the promoter region. Importantly, in vivo knockdown of Etv1 by DNA electroporation in the developing cerebellum prevents the upregulation of the maturation genes but has no effects on preceding developmental processes occurring in the granule cells. Etv1 thus orchestrates the activity-dependent gene regulation in the terminal maturation program and specifies the identity of cerebellar granule cells.cell culture | membrane potential | neuronal cell excitation | synaptic maturation | transcriptional regulation

Gene regulation via excitation and BDNF is mediated by induction and phosphorylation of the Etv1 transcription factor in cerebellar granule cells

“…To address whether, and if so how, BDNF and the activity-dependent signaling cascades synergistically regulate the expression of the maturation genes, we examined the effects of an inhibitor or blocker of each activity-dependent signaling component on the BDNF-mediated up-regulation of the representative maturation genes, namely the NR2C and Tiam1 genes. The following inhibitors/blockers were used: for glutamate receptors, an AMPA receptor-selective antagonist, 2, 3-dioxo-6-nitro-1,2,3,4-tetrahydrobenzo [f] quinoxaline-7-sulfonamide (NBQX) and an NMDA receptor-selective antagonist, 3-(2-carboxypiperazin-4-yl)-propyl-1-phosphonic acid (CPP) in combination; for Na + channels, either tetrodotoxin (TTX) or siRNA against Na + channel type II mRNA (Nav1.2 siRNA); for Ca 2+ channels, 4 Ca 2+ channel blockers in combination: that is, nifedipine (L-type), ω-agatoxin TK (P/Q-type), ω-conotoxin GVIA (N-type), and NNC 55-0396 (T-type) (7,13) (Fig. 1A).…”

“…The BDNF-TrkBErk cascade thus contributes to the up-regulation of the NR2C and other maturation genes in maturing granule cells (10, 11). However, how the BDNF-Erk signaling cascade and the activitydependent sequential events converge to control up-regulation of these maturation genes remains to be clarified.Granule cells in primary culture are capable of recapitulating many properties characteristic of maturing granule cells in vivo (6,7,12,13). The present study addressed how the expression of maturation genes is controlled by the BDNF-Erk signaling and activity-dependent mechanisms in cultured granule cells.…”

“…Granule cells in primary culture are capable of recapitulating many properties characteristic of maturing granule cells in vivo (6,7,12,13). The present study addressed how the expression of maturation genes is controlled by the BDNF-Erk signaling and activity-dependent mechanisms in cultured granule cells.…”

“…This shift of the membrane potential plays a key role in activity-dependent regulation of the expression of a set of maturation genes in maturing granule cells (5,6). The hyperpolarized state of maturing granule cells enhances responsiveness to glutamatergic excitation and leads to the Nav1.2 Na + channel-mediated action potential, thereby stimulating Ca 2+ entry via voltage-dependent Ca 2+ channels (7). Ets variant gene 1 (Etv1/Er81), which is a transcription factor of the ETS family, is then induced by this sequential activity-dependent signaling mechanism and commonly up-regulates the battery of maturation genes, including those of the NMDA receptor subunit NR2C and Tiam1 proteins in both primary cultures and the developing cerebellum (7).…”

“…The hyperpolarized state of maturing granule cells enhances responsiveness to glutamatergic excitation and leads to the Nav1.2 Na + channel-mediated action potential, thereby stimulating Ca 2+ entry via voltage-dependent Ca 2+ channels (7). Ets variant gene 1 (Etv1/Er81), which is a transcription factor of the ETS family, is then induced by this sequential activity-dependent signaling mechanism and commonly up-regulates the battery of maturation genes, including those of the NMDA receptor subunit NR2C and Tiam1 proteins in both primary cultures and the developing cerebellum (7). During cerebellar development, BDNF and its TrkB receptor are highly expressed in granule cells at the internal granular layer after migration (8,9), and the activation of the TrkB receptor and its downstream MEK-Erk signaling cascade up-regulates the NR2C and other maturation genes (10,11).…”