NELL2, a Neuron-Specific EGF-Like Protein, Is Selectively Expressed in Glutamatergic Neurons and Contributes to the Glutamatergic Control of GnRH Neurons at Puberty

Abstract

NELL2, a protein containing EGF-like repeats, is almost exclusively expressed in the nervous system. In the mammalian brain, NELL2 expression is mostly neuronal. NELL2 was previously found to be a secreted protein that functions during embryonic development as a neuronal differentiation and survival factor. We now show that the Nell2 gene is selectively expressed in the two major subtypes of glutamatergic neurons described in the postnatal brain: those containing the vesicular glutamate transporter 1 and those expressing vesicular glutamate transporter 2. No Nell2 mRNA is detected in GABAergic neurons. Likewise, GnRH neurons are devoid of NELL2. During prepubertal development of the female rat, Nell2 mRNA abundance increases selectively in the medial basal hypothalamus, reaching maximal values at the end of the juvenile period, to decline at the time of puberty to intermediate levels. Similar, but less pronounced changes are observed in the preoptic area, but they are absent in the cerebral cortex. A well-established glutamatergic function in the neuroendocrine brain is to enhance release of GnRH, the neurohormone controlling sexual development and the time of puberty. In vivo disruption of NELL2 synthesis via intraventricular administration of antisense oligodeoxynucleotides reduced GnRH release from the medial basal hypothalamus and delayed the initiation of female puberty. These results identify NELL2 as a new component of glutamatergic neurons and provide evidence for its physiological involvement in a major, glutamate-dependent, process of neuroendocrine regulation.

Regulation of the Female Rat Estrous Cycle by a Neural Cell-Specific Epidermal Growth Factor-like Repeat Domain Containing Protein, NELL2

“…We also identified that NELL2 is specifically expressed in hypothalamic cells expressing ERα and is involved in E2-dependent hypothalamic sexual differentiation (Jeong et al, 2008a) and reproductive maturation (Ha et al, 2008), while GnRH neurons do not express ERα, a major feedback regulator for the GnRH neural apparatus (Herbison and Theodosis, 1992). Therefore, NELL2 might be essential to the E2-dependent control of neuroendocrine events related to reproduction, such as puberty initiation (Ha et al, 2008) and female reproductive cyclicity.…”

“…Proteins on the gel were transferred onto a nitrocellulose membrane (Amersham Biosciences, UK) by an electrophoretic transfer cell (Bio-Rad Laboratories, USA). The membrane was incubated with primary antibody (rabbit anti-NELL2, 1:1000) (Ha et al, 2008) for 1 h in hybridization buffer (1x Tris-buffered saline with 0.1% Tween 20, 1% milk) and followed by incubation with secondary antibody (anti-rabbit IgG, 1:2000; Amersham Biosciences). The immunoreactions were detected with an enhanced chemiluminescence detection kit (Amersham Biosciences) according to the supplied protocol.…”

“…However, we recently found that NELL2 is specifically expressed in glutamatergic neurons, but not in the gonadotropin-releasing hormone (GnRH) neurons of the rat hypothalamus (Ha et al, 2008). In vivo blockade of NELL2 synthesis results in decreased GnRH release (Ha et al, 2008), suggesting that NELL2 is involved in the GnRH release apparatus via regulating glutamate or other neuronal networks that control GnRH release.…”

“…However, we recently found that NELL2 is specifically expressed in glutamatergic neurons, but not in the gonadotropin-releasing hormone (GnRH) neurons of the rat hypothalamus (Ha et al, 2008). In vivo blockade of NELL2 synthesis results in decreased GnRH release (Ha et al, 2008), suggesting that NELL2 is involved in the GnRH release apparatus via regulating glutamate or other neuronal networks that control GnRH release. The transsynaptic networks controlling GnRH secretion are many (review, Levine et al, 1991) and subject to the modulatory influence of gonadal steroids such as estradiol (E2) (review, Horvath et al, 1997).…”

The Molecular Mechanism of NELL2 Movement and Secretion in Hippocampal Progenitor HiB5 Cells

“…Hybridization was performed using anti-green fluorescent protein (GFP) antibody [Santa Cruz Biotechnology Inc. (Germany), 1:1000] and anti-NELL2 antibody (1:500, see Ha et al, 2008 for the generation and verification of the antibody). The epitope of the anti-NELL2 antibody is amino acids 273-588 of the rat NELL2 protein.…”