Estrogen deficiency results in apoptosis in the frontal cortex of adult female aromatase knockout mice

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Abstract

The aromatase knockout (ArKO) mouse is completely estrogen deficient. We previously detected apoptosis in the hypothalamus of 1 year-old male ArKO mice. This study shows that 12 week-old female ArKO mice display spontaneous apoptosis of pyramidal neurons in the frontal cortex while wild-type (WT) littermates show no signs of apoptosis. Concomitantly, bcl-2 related anti-apoptotic genes are down-regulated whereas the pro-apoptotic gene TRADD is up-regulated in the female ArKO frontal cortex. This phenotype can be rescued by 3-week replacement of 17β-estradiol. Furthermore, the apoptosis phenotype is exacerbated in 12–15 month-old female ArKO mice, which have 30% less neurons in the frontal cortex and lower brain weights than WT counterparts. These data show that estrogens are essential for the survival of female cortical neurons even in the absence of pathological conditions or external assaults. Our observations also demonstrate the sexually dimorphic susceptibility of neurons to estrogen deficiency.

Introduction

The presence of aromatase (the enzyme that converts androgens to estrogens) and estrogen receptor (ER) mRNA and protein expression in several regions of the mammalian brain, including the cortex, suggests a strong functional role for estradiol in the brain (Balthazart et al., 1991, Foidart et al., 1995, Jakab et al., 1994). Indeed several studies have reported on the neuroprotective effects of estradiol including anti-apoptotic effects, protection from free radicals, anti-inflammatory effects, regulation of calcium channels and protection via increasing cerebral blood flow (see review Amantea et al., 2005). Such neuroprotective properties of estradiol have been implicated in the treatment of several neurological dysfunctions including cognitive and memory deficits, brain injury and stroke (Garcia-Segura et al., 2001, Wise et al., 2001) and in addition neurodegenerative diseases such as Alzheimer's disease (Tang et al., 1996, Xu et al., 2006) and Parkinson's disease (Cyr et al., 2002).

While in most cases, the neuroprotective actions of estradiol are ER mediated, non-genomic actions of estradiol are also beneficial — the unique phenolic A ringed structure of estradiol renders it an effective scavenger of free radicals (Moosmann and Behl, 1999). One mechanism of ER-mediated estrogenic neuroprotection is via regulation of anti- and pro-apoptotic genes. Several in vitro studies have demonstrated the anti-apoptotic effects of 17β-estradiol treatment via down-regulation of pro-apoptotic genes such as BAD (Gollapudi and Oblinger, 1999) and up-regulation of anti-apoptotic genes such as Bcl-2 (Dubal et al., 1999, Zhao et al., 2004). In addition, neuroprotective effects of estradiol upon neurodegenerative disease processes such as Alzheimer's and Parkinson's may be mediated through the regulation of anti- and pro-apoptotic gene expression (Pike, 1999, Morissette et al., 2008). Clinically, the topic of estrogen replacement therapy in postmenopausal women has received much attention, and although the literature has been, for the most part, contradictory, it now seems evident that early in menopause, when neurons are still in a healthy state, estrogen therapy can be effective, however if this brief window of opportunity is missed, estradiol treatment is relatively ineffective (Amantea et al., 2005, Maki, 2005, Maki et al., 2007, Stephens et al., 2006). The use of animal models to effectively replicate problems such as hormone deficiency, neurological diseases, brain injury and stroke, is an effective tool for investigating the beneficial effects of estradiol on the brain. Studies using ovariectomy procedures followed by 17β-estradiol replacement have effectively demonstrated the positive effects that estradiol provides in cognitive performances and memory tasks (Gibbs, 2000, Vaucher et al., 2002).

Whereas the in vivo effects of estrogen on the brain were primarily investigated using gonadectomised animals (Fukuda et al., 2000, Miller et al., 1999), these animal models are not ideal as aromatase cytochrome P450 (the enzyme that synthesises estrogens from androgens) is known to be expressed in numerous regions of the brain (Lephart et al., 2001, Wagner and Morrell, 1997) such as hypothalamus, amygdala and hippocampus. In studies using aromatase inhibitors, complete blockage in all sites is uncertain. Therefore the total estrogen deficiency can be achieved by knocking out the aromatase gene. Our laboratory has developed an aromatase knockout (ArKO) mouse model of complete estrogen deficiency by deleting exon IX (Fisher et al., 1998) of the aromatase gene (Cyp19A1). Single point mutations in this region have resulted in total estrogen ablation in humans. Neurologically, female ArKO mice show significantly increased ischemic damage following insult in all brain areas, when compared to WT controls (McCullough et al., 2003). In addition, our laboratory has previously determined that 1 year-old male, but not female ArKO mice display apoptosis in the dopaminergic neurons of the arcuate nucleus and medial preoptic area (Hill et al., 2004), indicating that estradiol is not only neuroprotective upon brain injury, but is actually required for the maintenance and integrity of dopaminergic neurons in the hypothalamic region of male mice. Furthermore, we found that ERα agonist treatment prevented the appearance of apoptosis in the arcuate nucleus, while ERβ agonist treatment was neuroprotective in the MPO region (Hill et al., 2007). These data suggest that the role of estradiol in the regulation of dopaminergic neurons is ER mediated.

Another ArKO model was generated by deleting exons 1 and 2 of the Cyp19A1, this female ArKO mouse has been shown to exhibit abnormal sexual behaviours (Bakker et al., 2002), and in addition displays depressive-like behaviours that may not be reversed by estradiol treatment (Dalla et al., 2004), suggesting a developmental role for estradiol in the female brain. Furthermore, when crossed with the APP23 Alzheimer's disease model, the combined ArKO/APP23 mice exhibit earlier onset and increased β-amyloid peptide deposition (Yue et al., 2005), suggesting an essential role for estrogens as neuroprotectants from such insults as ischemia and amyloid deposition. This neuroprotective role for estradiol against neurotoxic insults also holds true in the male ArKO which shows enhanced hippocampal damage following domoic acid induced excitotoxicity when compared to WT (Azcoitia et al., 2001).

Herein, we present a comprehensive study on consequences of estrogen deficiency on the morphology of the brain of female mice.

Section snippets

Histological studies

The brain tissues from ArKO mice were noticeably more friable than those of WT mice. Preliminary histological analysis by NeuN revealed there were less neurons in the frontal cortex in the 1 year-old female ArKO mouse (Fig. 1A) as compared to the 1 year-old female WT (Fig. 1B). No such differences were noticed in the younger animals (14 week-old). Subsequently, semi-quantitation of NeuN immunostained cells of the frontal cortex (Bregma 3.0 mm to 2.10 mm; Paxinos and Franklin 2004) showed that

Discussion

The results of histological studies (TUNEL and immunostaining), RNase protection assay and 17β-estradiol replacement showed that a consequence of estrogen deficiency in female ArKO mice is apoptosis and cell loss in the frontal cortex of the brain. Our results demonstrated that estrogen likely exerts its survival effect on the neurons through up-regulation of anti-apoptotic genes (bfl-1, bcl-W, bcl-xL and bcl-2) and down-regulation of at least one pro-apoptotic gene TRADD. It is interesting to

Animals

ArKO mice (129SV/J X C57BL/6J) were generated by disruption of the Cyp19 gene by homologous recombination (Fisher et al., 1998). Homozygous null or wild type offspring were bred by crossing mice heterozygous for the disrupted gene. The pups were genotyped by PCR as described (Robertson et al., 1999). Animals were housed under SPF conditions and had ad libitum access to water and soy-free mouse chow (Glen Forrest Stockfeeders, Western Australia). Mice were killed by CO2 asphyxiation, brain

Acknowledgments

This work was supported by the Australian National Health and Medical Research Council, project grant numbers 338510 and 494813.

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