LIM kinase mediates estrogen action on the actin depolymerization factor Cofilin

Abstract

The ovarian hormone estrogen increases the axospinous synapse density in the hippocampal CA1 region of young female rats but fails to do so in aged rats. This estrogen-mediated alteration of spine synapse structures suggests the coincident requirement for the structural reorganization of the underlying actin cytoskeleton network. Actin reorganization is known to require the deactivation of Cofilin, an actin depolymerization factor. Cofilin is deactivated by LIM kinase (LIMK), and LIMK activity is modulated by the phosphorylation of specific residues. We have previously demonstrated that estrogen is able to increase phosphorylated LIMK (pLIMK) immunoreactivity (IR) in the hippocampus in vivo and that this estrogen-stimulated pLIMK-IR is decreased in the aged brain. Because Cofilin phosphorylation allows for actin filament elongation and spine synapse growth, we sought to determine if estrogen acts through Cofilin and if such estrogen action requires the observed LIMK activity. Using both hippocampal neurons and the NG108-15 neuroblastoma cell line, we demonstrate here that estrogen stimulates the phosphorylation of Cofilin in vitro. Furthermore, this estrogen action on Cofilin requires LIMK. Lastly, while initiating the phosphorylation of LIMK and Cofilin, estrogen can also stimulate the formation of filopodial extensions, an early step in the formation of nascent spines, demonstrating that estrogen can alter the actin-dependent neuronal morphology. This linkage of estrogen communication to Cofilin via LIMK provides the functionality to the age-sensitive pLIMK-IR that we have observed in vivo.

Introduction

The hippocampal formation remains receptive and responsive to experience and to stimuli throughout adulthood. This responsiveness includes a sensitivity to circulating steroid hormones that results in the morphological remodeling of hippocampal neurons in vivo (McEwen, 2002). Across the estrous cycle of adult female rats, hippocampal CA1 pyramidal neurons respond to cyclical increases in the circulating estrogen levels with corresponding increases in dendritic spine density and axospinous synapse number (McEwen and Milner, 2007, Woolley and McEwen, 1992). The observation of such estrogen-induced spinogenesis in vivo has been reiterated in vitro with early video microscopy studies on cultured primary hippocampal neurons where stimulation with supra-physiological levels of estrogen induces the formation of immature spines, or neurite filopodia (Brinton, 1993). Together, these in vivo and in vitro observations of estrogen-modulated morphologic plasticity suggest that estrogen directly communicates with and affects the structural reorganization of the cytoskeletal network in hippocampal neurons (Dillon and Goda, 2005, Matus, 2000, Schubert and Dotti, 2007). However, this ability of estrogen to modulate dendritic spine plasticity is lost in the aged female brain (Adams and Morrison, 2003, Morrison et al., 2006).

In the aged female brain, estrogen fails to increase CA1 spine synapse density, presumably due to a loss of the required molecular communication from estrogen to the underlying actin cytoskeletal network (Yildirim et al., 2008). Actin filaments, the major cytoskeletal protein structures in the dendritic spine, affect synaptic plasticity by regulating the formation of synaptic structures as well as by affecting dendritic spine size and shape (Cingolani and Goda, 2008, Sekino et al., 2007). Estrogen may be involved in regulating spine synapse formation by regulating the actin cytoskeletal network. In support of this possibility, we have recently observed that estrogen can increase phosphorylated (p) LIM Kinase (LIMK) immunoreactivity (IR) in the hippocampus in vivo (Spencer et al., 2008a), and that this estrogen-stimulated pLIMK-IR in neurons is diminished in the aged brain (Yildirim et al., 2008).

LIMK is a signal transduction intermediate that is essential for spine formation and synapse function (Sarmiere and Bamburg, 2002) and plays a key role in regulating actin polymerization and depolymerization (Bernard, 2007). LIMK functions as a serine/threonine kinase, and one LIMK target substrate of interest is Cofilin (Yang et al., 1998). Cofilin, a member of the actin depolymerization factor (ADF) family, is a key actin binding protein that promotes the turnover and severing of actin filaments (Bernstein and Bamburg, 2010, Maciver and Hussey, 2002). Cofilin plays an important role in neuronal shape (Sarmiere and Bamburg, 2004), and Cofilin is localized to neuronal structures with high actin filament turnover, including the growth cone of axons and dendrites (Fass et al., 2004, Gungabissoon and Bamburg, 2003). Importantly, Cofilin is present in CA1 spines and is enriched near the membrane and in the postsynaptic density (PSD) where it can regulate actin dynamics in the context of synapse activity and plasticity (Racz and Weinberg, 2006). LIMK phosphorylates Cofilin, rendering Cofilin severing inactive and thereby promoting actin filament extension and dendritic spine formation (Arber et al., 1998, Endo et al., 2007, Meng et al., 2003).

To identify a functional role for the estrogen-stimulated pLIMK-IR that is observed in vivo, we asked whether estrogen affects the phosphorylation of the LIMK substrate Cofilin in cultured hippocampal neurons and in NG108-15 neuronal cells in vitro. We determined that estrogen can stimulate the phosphorylation of both Cofilin and LIMK in vitro, and we demonstrate here that the phospho-modulation of Cofilin by estrogen is LIMK-dependent.