The Etv1 transcription factor activity-dependently downregulates a set of genes controlling cell growth and differentiation in maturing cerebellar granule cells

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Highlights

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    Immaturation genes of granule cells are downregulated in an activity-dependent manner during cerebellar maturation.

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    The downregulation of immaturation genes is controlled by a unified sequential CaMKII-mediated signaling cascade.

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    Etv1 is the primary transcription factor that directs the downregulation of immaturation genes.

Abstract

In the early postnatal period, cerebellar granule cells exhibit an activity-dependent downregulation of a set of immaturation genes involved in cell growth and migration and are shifted to establishment of a mature network formation. Through the use of a granule cell culture and both pharmacological and RNA interference (siRNA) analyses, the present investigation revealed that the downregulation of these immaturation genes is controlled by strikingly unified signaling mechanisms that operate sequentially through the stimulation of AMPA and NMDA receptors, tetrodotoxin-sensitive Na+ channels and Ca2+/calmodulin-dependent protein kinase II (CaMKII). This signaling cascade induces the Etv1 transcription factor, and knockdown of Etv1 by a siRNA technique prevented this activity-dependent downregulation of immaturation genes. Thus, taken into consideration the mechanism that controls the upregulation of maturation genes involved in synaptic formation, these results indicate that Etv1 orchestrates the activity-dependent regulation of both maturation and immaturation genes in developing granule cells and plays a key role in specifying the identity of mature granule cells in the cerebellum.

Introduction

In the early postnatal stage of cerebellar development, granule cells proliferate in the outer part of the external granular layer, postmitotically differentiate in the inner part of this layer and migrate into the internal granular layer, where they extend dendrites and form refined synaptic connections with mossy fibers [1]. During these developmental processes, the resting membrane potential of maturing granule cells significantly shifts from a relatively depolarized state to a more hyperpolarized state [2], [3], [4]. The depolarized state of granule cells activates Ca2+/calmodulin-dependent protein phosphatase calcineurin (CaN) and this activation not only upregulates the expression of a large number of immaturation genes associated with cell growth, differentiation and migration, but also suppresses the expression of a set of maturation genes that are associated with synapse formation [4], [5]. When the membrane potential decreases below the threshold of Nav1.2 Na+ channels during the development of granule cells, maturing cells become permissive to excitation by glutamatergic stimulation, which leads to Ca2+ entry via voltage-dependent Ca2+ channels [6]. This signaling then induces the expression of the ets variant gene 1 (Etv1/Ev81) transcription factor of the ETS family and Etv1 transcriptionally upregulates a set of the maturation gene battery in both primary culture and the developing cerebellum in vivo [6]. However, little is known about the mechanism that underlies the downregulation of immaturation genes, such as those encoding NR2B, Dcx, VGLUT2 and Homer2 [7], [8], [9], [10]. It is unclear how the expression of immaturation genes is downregulated in an activity-dependent manner and whether or not such downregulation is controlled by a mechanism that is the same as or different from that which underlies the upregulation of maturation genes.

In primary cultures of the postnatal cerebellum, granule cells represent more than 90% of a cerebellar cell population and cultured granule cells are capable of recapitulating the characteristic features of developing processes of granule cells in vivo [11]. This study using cultured granule cells addressed how immaturation genes are regulated by sequential signaling mechanisms in an activity-dependent manner. Here we report that immaturation genes are downregulated through activity-dependent Etv1 expression via the signaling cascade of Ca2+/calmodulin-dependent protein kinase II (CaMKII).

Section snippets

Granule cell culture

All procedures for animal handling were performed according to the guidelines of the Osaka Bioscience Institute and National Cerebral and Cardiovascular Center. Primary cultures containing ∼90% cerebellar granule cells were prepared from 8-day-old (PD 8) ICR mice as previously reported [5], [12], [13]. Granule cells were cultured for 24 h in medium containing serum and then for 96 h in serum-free medium containing a physiological concentration of KCl (5 mM KCl). Microarray analysis was

Activity-dependent downregulation of immaturation genes

Our previous study demonstrated that granule cell culture is capable of recapitulating an activity-dependent developmental signaling cascade that operates sequentially through the stimulation of α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) and N-methyl-d-aspartate (NMDA) glutamate receptors, voltage-gated Na+ channels and Ca2+ channels [6]. Therefore, to examine whether the downregulation of immaturation genes is controlled in an activity-dependent manner, we cultured granule

Discussion

In the terminal maturation of the cerebellum, granule cells exhibit not only the upregulation of maturation genes responsible for functional synaptic formation but also the downregulation of immaturation genes involved in cell proliferation and differentiation [5]. In these developmental processes, granule cells become permissive to responding to excitatory glutamatergic transmission, and in turn evoking TTX-sensitive, Na+ channel-mediated action potential, which leads to the entry of Ca2+ via

Conflict of interest

The authors declare no conflict of interest.

Acknowledgments

This work was supported by Grants KAKENHI (24790300 and 25118731 to M.O., 22220005 to S.N.) from the Ministry of Education, Culture, Sports, Science, and Technology of Japan.

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Cited by (6)

1

Present address: Department of Emerging Infectious Diseases, Institute of Tropical Medicine, Nagasaki University, Nagasaki 852-8523, Japan.

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