Estrogen receptor ß activity modulates synaptic signaling and structure

Abstract

Brain-synthesized estrogen has been shown to influence synaptic structure, function, and cognitive processes. However, the molecular mechanisms underlying the rapid effects of estrogen on the dendritic spines of cortical neurons are not clear. Estrogen receptor β (ERβ) is expressed in cortical neurons, and ERβ knock-out mice display impaired performance in cortically mediated processes, suggesting that signaling via this receptor has profound effects on cortical neuron function. However, the effect of rapid signaling via ERβ on dendritic spines and the signaling pathways initiated by this receptor in cortical neurons are unknown. Here, we show that activation of ERβ with the specific agonist WAY-200070 results in increased spine density and PSD-95 (postsynaptic density-95) accumulation in membrane regions. Activation of ERβ by WAY-200070 also resulted in the phosphorylation of p21-activated kinase (PAK) and extracellular signal-regulated kinase 1/2 (ERK1/2) in cultured cortical neurons, suggesting a mechanism for the regulation of the actin cytoskeleton. Moreover, we found that aromatase, an enzyme critical for estrogen production, is present at presynaptic termini, supporting a role for brain-synthesized estrogen as a neuromodulator in the cortex. These results implicate ERβ signaling in controlling dendritic spine morphology, in part via a PAK/ERK1/2-dependent pathway, and provide mechanistic insight into the rapid cellular effects of estrogen on brain function.

Figure 1.

Activation of ERβ with 070 results in dendritic spine remodeling. a, Time course of dendritic spine morphogenesis in response to 10 nm 070. Cultured cortical neurons (DIV25) expressing EGFP were treated with 10 nm 070 in artificial CSF containing 2-amino-5-phosphonovaleric acid for 0–60 min. b, c, Quantification of dendritic spine morphogenesis in a. d, e, Histograms of spines area and breadth-to-length ratio show that 070-treated neurons have an increase in spines with smaller areas and lower breadth-to-length ratios. f, g, 070 treatment results in an increase in total spine density and number of spines forming contacts with the presynaptic termini. ^#,*p < 0.001. Scale bars, 5 μm.

Figure 2.

ERβ activation by 070 mobilizes cytosolic PSD-95, resulting in increased membrane association. a, Cortical neurons (DIV24) were treated for 0–60 min with 10 nm 070 in artificial CSF containing 2-amino-5-phosphonovaleric acid. Cell fractions were prepared and analyzed by SDS-PAGE. This revealed an increase in PSD-95 in the membrane fraction and a reduction in PSD-95 in the cytosolic fraction after treatment. b, c, Treatment with 070 increased total spine numbers and number of spines containing PSD-95. The dendritic shaft is outlined by yellow dashed lines. d, e, Analysis of PSD-95 cluster number and intensity in spines and dendritic shaft. ^#bassoon positive spines, *total spines, p < 0.001. Scale bars, 5 μm.

Figure 3.

ERβ activation by 070 induces PAK and ERK1/2 phosphorylation in a time-dependent manner. a, p-PAK levels in the dendrites and spines of cortical neurons after 070 treatment. White dotted lines indicate dendritic shaft, and white arrows indicate dendritic spines. b, Representative Western blot of p-PAK levels after treatment with 070. c, p-ERK1/2 levels in the dendrites and spines increases with time after treatment with 070. White dotted lines indicate dendritic shaft, and white arrows indicate dendritic spines. d, Representative Western blot of p-ERK1/2 levels after treatment with 070. *p < 0.001. Scale bars: 5 μm.

Figure 4.

Aromatase subcellular localization in cultured cortical neurons. a, b, Cultured cortical neurons (DIV25) were costained for aromatase and PSD-95 or bassoon. White arrows indicate colocalization; yellow open arrowheads indicate aromatase puncta lacking colocalization. c, d, Quantification of colocalization in a, b. e, Colocalization of aromatase with bassoon in areas lacking dendrites. f, Overlap of aromatase with axonal marker tau5 in areas lacking dendrites. White arrows indicate colocalization. Scale bars: 5 μm.