**1. Introduction**

Neuronal cell death is an important feature of human neurodegenerative diseases such as Alzheimer's disease (AD). This cell death is considered to occur as a consequence of aberrant activation of the cell cycle in neurodegeneration [1]. Under normal conduction, the cell cycle is tightly controlled by specific regulatory proteins. For instance, cyclins and cyclin-dependent kinases (CDKs) are two key classes of regulatory molecules that determine a cell's progress in the cell cycle [2]. As a key regulator of the G1-S transition, cyclin D1 interacts with CDK4 to form the cyclin D1-CDK4 complex and moves to the nuclei, thereby promoting cell cycle progression. Normal adult neuron cells never reenter the cell cycle (but stay in the G0 stage) and are thus recognized as permanently postmitotic cells [3]. Conversely, neurons reenter the cycle, undergo DNA replication, and die after they are exposed to DNA-damaging

agents, oxidative stress, or certain neurotoxins such as beta-amyloid (Aβ) aggregates [3]. The Aβ peptide is the major component of senile plaque derived from the Aβ precursor protein (APP); this peptide is a neuropathological hallmark of AD [4]. There are numerous different Aβ species including Aβ (1–40), Aβ (1–42), and Aβ (25–35). The Aβ (25–35) fragment is universally used in research as it has been found to elicit profound toxic manifestations in elderly people and to physiologically play a role in AD [5]. It has been previously shown that cell cycle activation accompanied by the upregulation of cyclin D1 in primary cultured rat cortical neurons was observed in response to exposure to Aβ (25–35) and that such activation was followed by apoptotic neuronal death [6]. To elucidate the possible intracellular signaling pathway involved in the activation of the cell cycle by Aβ, extracellular signal-regulated kinase (ERK) 1/2-related pathways are the major focus of the present study because there is evidence of the involvement of ERK1/2 activation in Aβ-induced neuronal cell death [7]. It has been documented that activation of ERK 1/2 appears to be critical for G1 to S phase progression in cell cycle regulation [8]. A previous study showed that the overexpression of ERK 1/2 in cells exposed to Aβ was followed by an elevation in cyclin D1 expression, which resulted in changes in the cell-cycle distribution, particularly in the G1-S phase [9].

ERK1/2 is also the target of the regulatory action of estrogen and its regulation requires interaction with the known estrogen receptors (ERs), ERα and ERβ [10]. In addition to the reproductive system, both ERα and ERβ are broadly expressed in nonreproductive systems including the central nervous system [11]. Particularly, brain regions such as the hypothalamus, amygdala, and hippocampus appear to have distinct expression patterns of both ER subtypes [12]. Although it is recognized that ERβ is the predominant receptor in the hippocampus, where its absence has an impact on memory and cognitive function [13], ERα co-exists, and its coregulation may be important for ERβ to fulfill its cellular roles [14]. In other words, ERβ collaborating with ERα in its molecular actions is crucial for estrogen-mediated beneficial effects on hippocampus-dependent memory and cognition. The ERα subtype is of particular interest in the present study as it exhibits stronger transcription activity than ERβ and thus appears to be functionally superior to ERβ in the modulation of age-related memory decline [13–15]. It is noteworthy that ERα diminishing in the hippocampus with age leads to a decrease in the relative expression of ERα and ERβ, and nuclear ERα-mediated effects, all of which are putative molecular mechanisms for age-related memory decline in the presence of low estrogen levels [13]. In this regard, the molecular actions of both ER subtypes have been reported to be involved in the neuroprotection of estrogen against the pathogenic processes of AD [16]. Evidence suggests that estrogen is capable of protecting against Aβ-induced toxicity through ERα-mediated signaling pathways [17]. Moreover, the other major neuropathological hallmark of AD is intracellular aggregates of hyperphosphorylated Tau protein, which has recently been found to interact with ERα potentiating the reduction of ERα's transcriptional activity [18]. SRC-1 is one of the nuclear receptor coactivators which enhance the transcriptional activity of ERs to manipulate the relevant molecular events [19]. Studies performed in a human astrocytoma cell line demonstrated that estradiol treatment increased the cell number through the mediation of ERα, whereas the coactivator silencing by RNA interference of SRC-1 was able to block this effect [19].

Equol is a metabolite of daidzein, one of the major isoflavones in soybean food products, and is known as an ERs agonist [20]. Equol is capable of inducing transcriptional responses, especially through the binding of ERα [21]. The oral bioavailability of equol in humans seems to be high, resulting in a plasma concentration of 0.4~2 μM after taking a single bolus of 2 mg of equol [22]. Consumption of phytoestrogens has been found to avoid many side effects from estrogens [23]. Intriguingly, equol has been shown to be a promising neuroprotectant in in vitro models, and its neuroprotective effects are exerted through anti-neuroinflammatory mechanisms with the regulation of relevant signaling pathways at molecular levels [24]. However, whether the cell cycle regulatory event and ER-dependent signaling pathways involve the neuroprotective properties of equol remains an enigma. Thus, in this study, we investigated the effects of equol on protecting SH-SY5Y cells against

Aβ-induced perturbations and the cellular mechanisms underlying equol's neuroprotective action in cell cycle events and ER pathways.
