Estrogen stimulates postsynaptic density-95 rapid protein synthesis via the Akt/protein kinase B pathway

Abstract

Estrogens induce synaptogenesis in the CA1 region of the dorsal hippocampus during the estrous cycle of the female rat. Functional consequences of such estrogen-mediated synaptogenesis include cyclic changes in neurotransmission and memory. At the molecular level, estrogen stimulates the rapid activation of specific signal transduction pathways, and of particular interest is the activation of Akt (protein kinase B), a key signal transduction intermediate that initiates protein translation by alleviating the downstream translational repression of eukaryotic initiation factor 4E-binding protein 1 (4E-BP1). Using a well established in vitro model system of differentiated NG108-15 neurons to investigate such rapid signaling effects of estrogen, we show that estrogen stimulates the phosphorylation of Akt, an indication of kinase activation, as well as the phosphorylation of 4E-BP1. In turn, the activation of these signaling intermediates suggests a non-genomic mechanism by which estrogen might likewise lead to protein translation of dendrite-localized mRNA transcripts in the hippocampus in vivo. We therefore considered the translation of the dendritic spine scaffolding protein postsynaptic density-95 (PSD-95). Although estrogen does not stimulate a rapid increase in PSD-95 mRNA levels in NG108-15 neurons, we show here that estrogen does however stimulate a rapid increase in PSD-95 new protein synthesis in vitro and that this new protein synthesis is Akt dependent. These results demonstrate an essential role for Akt in estrogen-stimulated dendritic spine protein expression, describe for the first time a signal transduction pathway in PSD-95 expression, and delineate a novel, molecular mechanism by which ovarian hormones might translationally regulate synaptogenesis via activating protein synthesis for dendritic function.

Fig. 1.

Estrogen rapidly stimulates the phosphorylation of Akt. dNG108-15 neurons were treated with either control (con) (β-cyclodextrin) or 10 nm17β-estradiol (Estrogen) for the times indicated.+ indicates positive control treatment of dNG108-15 neurons, 60 min exposure to 100 nm insulin.Ø indicates untreated dNG108-15 neurons. Phosphorylated Akt (pAkt) at Ser-473 (A) or total Akt (non-phosphospecific) (B) was measured by Western blot analysis of total cell lysates of cells solubilized in SDS loading buffer and run on 10% SDS-PAGE. C, Semiquantitative densitometry of scanned blots. pAkt levels were first normalized to total Akt levels, and then each normalized pAkt level was expressed as fold increase over pAkt level from untreated (Ø) cells. As early as 30 min, estrogen treatment rapidly stimulates an approximate fourfold increase in pAkt levels over control treatment. Graphed data represent averages and SD from three independent experiments. *Statistically significant over control from same time point (p < 0.05; two-tailed Student'st test).

Fig. 2.

Estrogen stimulates the phosphorylation of 4E-BP1. The same lysates from Figure 1 were analyzed for phosphorylation of 4E-BP1 (p4E-BP1) at Thr70 by Western blot analysis. A, Samples were run on 15% SDS-PAGE, and the blot shown is representative of three independent experiments. Control treatment did not lead to 4E-BP1 phosphorylation, but 10 nm17β-estradiol treatment lead to an increase in p4E-BP1 that increased steadily through 4 hr. B, Semiquantitative densitometric analysis of p4E-BP1 levels.

Fig. 3.

Estrogen does not affect PSD-95 mRNA levels. Equal amounts of total RNA isolated from control-treated (con) and 10 nm 17β-estradiol-treated (E) dNG108-15 neurons were analyzed by real-time RT-PCR to quantitate the amount of PSD-95 mRNA. The mRNA level from the single-copy ∂-opioid receptor gene was also measured to normalize for total RNA per sample. Data represent averages and SD from three independent experiments, each run in triplicate, and are expressed as fold increase over control-treated sample. After 6 hr estrogen treatment, there is no significant change in PSD-95 mRNA levels (Student's two-tailed t test;p > 0.05).

Fig. 4.

PSD-95 new protein synthesis. Metabolically [³⁵S-Met/Cys] pulse-labeled dNG108-15 neurons were preincubated for 1 hr with either inhibitor (lane 3, 100 nm ICI 182,780; lane 4, 50 μmLY294002; lane 5, 10 nm rapamycin;lane 6, 4 μm actinomycin D) or (—) inhibitor diluent control (equivalent volume DMSO, lanes 1 and 2). Then, the neurons were either treated for an additional 6 hr with β-cyclodextrin control (con, lane 1) or 10 nm17β-estradiol (Estrogen, lanes 2–6). The cells were harvested in ice-cold RIPA buffer, and the cleared extract was immunoprecipitated for PSD-95 protein. After stringent washing, the immunoprecipitate was run by 7.5% SDS-PAGE, and the dried acrylamide gel was then exposed to a phosphorimage screen for densitometry. Only newly synthesized PSD-95 protein with [³⁵S] incorporation is captured by the phosphorimaging screen. A, The PSD-95 immunoprecipitation (PSD-95 ip); B, the corresponding densitometry analysis. In the absence of inhibitors, estrogen stimulates an approximate threefold increase in new PSD-95 protein synthesis (significantly greater than control; Student's two-tailed t test; *p < 0.05). This protein synthesis is reduced to near control levels (lane 1) by either the ERα antagonist ICI 182,780 (lane 3) or the PI3K inhibitor LY294002 (lane 3). Rapamycin is a potent mTOR kinase inhibitor and can inhibit protein synthesis, and it reduces estrogen-stimulated PSD-95 protein synthesis to below control levels (lane 5). Actinomycin D inhibits mRNA transcription and decreases slightly estrogen-stimulated PSD-95 new protein synthesis (lane 6). However, this decrease is not statistically significant (Student's two-tailedt test; **p > 0.05) and suggests that PSD-95 protein synthesis is transcription independent.

Fig. 5.

Model of estrogen action on PSD-95 new protein synthesis. Estrogen stimulates rapid PSD-95 new protein synthesis via Akt and 4E-BP1 phosphorylation. This signal transduction pathway leading to PSD-95 protein translation can be inhibited by the ERα antagonist ICI 182,780, by the PI3K selective inhibitor LY294002, and by the mTOR kinase inhibitor rapamycin. FRAP, FKBP (FK506-binding protein) and rapamycin-associated protein.