Estrogen receptor subtypes alpha and beta contribute to neuroprotection and increased Bcl-2 expression in primary hippocampal neurons

Abstract

Estrogen receptor (ER) mediated neuroprotection has been demonstrated in both in vitro and in vivo model systems. However, the relative contribution by either ER subtype, ERα or ERβ, to estrogen-induced neuroprotection remains unresolved. To address this question, we investigated the impact of selective ER agonists for either ERα, PPT, or ERβ, DPN, to prevent neurodegeneration in cultured hippocampal neurons exposed to excitotoxic glutamate. Using three indicators of neuronal viability and survival, we demonstrated that both the ERα selective agonist PPT and the ERβ selective agonist DPN protected hippocampal neurons against glutamate-induced cell death in a dose-dependent manner, with the maximal response occurring at 100 pM. Further analyses showed that both PPT and DPN enhanced Bcl-2 expression in hippocampal neurons, with an efficacy comparable to their neuroprotective capacity. Collectively, the present data indicate that activation of either ERα or ERβ can promote neuroprotection in hippocampal neurons, suggesting that both receptor subtypes could be involved in estrogen neuroprotection. As ERβ is highly expressed in the brain and has little or no expression in the breast or uterus, discovery and design of ERβ selective molecules could provide a strategy for activating the beneficial effects of estrogen in the brain without activating untoward effects of estrogen in reproductive organs.

Introduction

Two subtypes of the estrogen receptor, ERα and ERβ, exist and exhibit distinct cellular and tissue distribution patterns. Weihua et al. [50] demonstrated that ERα is highly expressed in the classical estrogen target tissues such as uterus, mammary gland, bone and cardiovascular systems, whereas ERβ is mainly expressed in non-classical tissues such as prostate, ovary and urinary tract. In the central nervous system (CNS), although both receptors similarly coexpress in some brain regions, including the preoptic area, the bed nucleus of the stria terminalis and the medial and cortical amygdaloid nuclei, some regions show selective expression such as the detection of only ERα in the ventromedial hypothalamic nucleus and subfornical organ [39]. In contrast, ERβ is predominant in the cerebral cortex and hippocampus in rat brain [32], [39], though others show that ERα is predominant in the hippocampus in mouse [26] and human brain [46], indicating that expression of ERs may be species specific.

Estrogen and its cognate receptors can regulate gene transcription via both direct genomic and indirect genomic mechanisms. The direct genomic mechanism requires estrogen binding to the estrogen receptor, followed by the induction of conformational change in the receptor leading to dimerization and translocation of ER into the nucleus where ER interacts with estrogen response elements (EREs) located within the regulatory region of target genes and coadaptor proteins leading to either activation or suppression of transcription of a target gene in a promoter- and cell-specific manner [23], [47]. The indirect genomic mechanism, proposed to account for the neuroprotective effects of estrogen in the brain, requires rapid activation of mitogen-activated protein kinases (MAPK) [27], [30], [42] and akt signaling pathways [43], activation of a transcriptional factor cAMP-responsive element binding protein (CREB) [7], [24], [55], and modulation of antiapoptotic Bcl-2 family proteins [12], [15], [28], [29].

Recent evidence reveals the requirement of ERs in mediating estrogen neuroprotection. The differential roles of estrogen receptor subtypes ERα and ERβ for neuroprotection remains unresolved. For instance, Gollapudi and Oblinger [16] showed that in PC12 cells transfected with only ERα, 17β-estradiol (E₂) enhanced the viability of cells against serum deprivation. Moreover, in studies using ERα and β knockout mice, Dubal et al. [13] found that ERα was required for the protective effects of E₂ against brain injury. However, the findings of Wang et al. [48] indicated the importance of ERβ in neuronal survival as manifested by developmental abnormalities in the brains of ERβ knockout mice. Rissman et al. [35] also proposed that ERβ plays a pivotal role for mediating E₂-induced learning and memory.

One strategy to address the relative contribution of ERα and ERβ to estrogen-inducible neuroprotection in neurons expressing both receptors is through the use of receptor selective agonists. 1,3,5-tris(4-hydroxyphenyl)-4-propyl-1H-pyrazole (PPT) and 2,3-bis(4-hydroxyphenyl) propionitrile (DPN) are two nonsteroidal ER subtype-selective agonists synthesized by Katzenellenbogen et al. [25], [44]. PPT is a highly selective and specific ERα agonist displaying a 410-fold higher binding selectivity for ERα over ERβ and activates gene transcription only through ERα (Fig. 1)[44]. Furthermore, PPT increased progesterone receptor mRNA and suppressed experimentally induced hot flushes, both of which are known to be estrogen regulated, suggesting its estrogenic effects in the brain evoked by the activation of ERα [18]. In contrast, the estrogen receptor agonist DPN displays a 70-fold higher binding selectivity for ERβ over ERα and is a full ERβ agonist (Fig. 1)[25]. In transcriptional activation studies, DPN exhibited a 170-fold higher relative potency for ERβ over ERα [25]. Based on their ER subtype-selective characteristics, PPT and DPN offer a pharmacological strategy to elucidate the differential contributions of the two ER subtypes in estrogen-induced neuroprotection.

In the present study, using PPT and DPN, we sought to determine the potential role of ER subtypes ERα and ERβ in mediating estrogen neuroprotection and their underlying cellular mechanisms. Since glutamate excitotoxicity is thought to be one of the contributing factors to the neurodegeneration associated with Alzheimer's disease and other neurodegenerative disorders, the neuroprotective efficacy of PPT and DPN against supraphysiological glutamate-induced excitotoxicity was investigated in primary hippocampal neurons. Three independent measures of neuronal response to neurotoxic glutamate were evaluated. Furthermore, in the pursuit of the molecular mechanisms mediating PPT and DPN neuroprotection, their impact on the expression of antiapoptotic protein Bcl-2 in primary hippocampal neurons was also investigated. Results of these analyses indicate that agonists for either ERα or ERβ protect neurons against excitotoxic-induced damage and death.