Retinoids and glucocorticoids have opposite effects on actin cytoskeleton rearrangement in hippocampal HT22 cells

Highlights

• Retinoic acid and glucocorticoids affect synaptic plasticity.

• Retinoic acid increases and dexamethasone decreases CaMKII mRNA expression in hippocampal cells.

• Actin cytoskeleton change after retinoic acid and dexamethasone treatments in neuronal cells in vitro.

• Retinoic acid increases calpain activity whereas dexamethasone decreases calpain activity.

Abstract

A chronic excess of glucocorticoids elicits deleterious effects in the hippocampus. Conversely, retinoic acid plays a major role in aging brain plasticity. As synaptic plasticity depends on mechanisms related to cell morphology, we investigated the involvement of retinoic acid and glucocorticoids in the remodelling of the HT22 neurons actin cytoskeleton.

Cells exhibited a significantly more elongated shape with retinoic acid and a rounder shape with dexamethasone; retinoic acid reversed the effects of dexamethasone.

Actin expression and abundance were unchanged by retinoic acid or dexamethasone but F-actin organization was dramatically modified. Indeed, retinoic acid and dexamethasone increased (70 ± 7% and 176 ± 5%) cortical actin while retinoic acid suppressed the effect of dexamethasone (90 ± 6%). Retinoic acid decreased (−22 ± 9%) and dexamethasone increased (134 ± 16%) actin stress fibres. Retinoic acid also suppressed the effect of dexamethasone (−21 ± 7%).

Spectrin is a key protein in the actin network remodelling. Its abundance was decreased by retinoic acid and increased by dexamethasone (−21 ± 11% and 52 ± 10%). However, retinoic acid did not modify the effect of dexamethasone (48 ± 7%). Calpain activity on spectrin was increased by retinoic acid and decreased by dexamethasone (26 ± 14% and −57 ± 5%); retinoic acid mildly but significantly modified the effect of dexamethasone (−44 ± 7%). The calpain inhibitor calpeptin suppressed the effects of retinoic acid and dexamethasone on cell shape and actin stress fibres remodelling but did not modify the effects on cortical actin.

Retinoic acid and dexamethasone have a dramatic but mainly opposite effect on actin cytoskeleton remodelling. These effects originate, at least partly, from calpain activity.

Introduction

Retinoic acid (RA, the main vitamin A metabolite) apart from playing a major role in brain development (Maden et al., 1998), is critically implicated in adult brain because of its involvement in cellular and synaptic plasticity (Chen et al., 2014, Lane and Bailey, 2005, McCaffery et al., 2006, Mey and McCaffery, 2004). Notably, it has been shown that the decreased retinoid signalling with age correlates with cognitive alterations (Bonhomme et al., 2014a, Etchamendy et al., 2001, Mingaud et al., 2008). By contrast, aging is marked by an increased signalling of the glucocorticoid (GC) pathway (McEwen, 2007, Mohler et al., 2011, Yau et al., 2007). In humans and animals, this is correlated with hippocampus-dependent memory impairment (Lupien et al., 2009, Yau et al., 2007). In mice, GCs affect synaptic potentiation (Alfarez et al., 2002) and impair synaptic plasticity (Krugers et al., 2006) while RA seems to counteract some of the deleterious cognitive effects of GCs (Bonhomme et al., 2014a, Bonhomme et al., 2014b, Touyarot et al., 2013). RA and GCs both act, mainly but not exclusively, through dimers of nuclear receptors (RAR and RXR, GR, MR) and RA and GC-response elements (RARE and GRE) (Bamberger et al., 1996, Piskunov et al., 2014). Interestingly, RA and GR signalling pathways may interact. For instance, RA potentiates GC-induced thymocytes apoptosis (Toth et al., 2011) and reduces GC sensitivity of skeletal muscle (Aubry and Odermatt, 2009) while dexamethasone (Dex) a GR-specific agonist enhances the RA-dependent increase of RARβ expression in hepatocytes (Yamaguchi et al., 1999). More recently, we have shown an interaction between these two pathways in the hippocampal HT22 cell line (Brossaud et al., 2013). Of note, this interaction targets the transcription and secretion of the neurotrophin brain-derived neurotrophic factor (BDNF).

Brain aging is marked by a decline of cognitive functions apparently related to a decreased neuronal plasticity, the basis of learning and memory process. Previous studies have shown that stress is associated with reduced dendritic branching and decreased spine density (Liston and Gan, 2011, McEwen et al., 1999). By contrast, other studies have shown that hippocampal neurons spine formation is induced by RA (Chen and Napoli, 2008) and by BDNF, a major regulator of synaptic plasticity of adult synapses (Bramham and Messaoudi, 2005, Magarinos et al., 2011). RA increases, whereas Dex reduces, the expression and abundance of the BDNF (Brossaud et al., 2013) whereas other factors contribute to normal brain function and plasticity. For instance, physiological levels of reactive oxygen and nitrogen species (ROS and RNS, respectively) contribute to synaptic plasticity and memory consolidation (Munnamalai and Suter, 2009, Wilson and Gonzalez-Billault, 2015). For various reasons the brain is particularly sensible to oxidation and any modification of the redox balance may have consequences on physiological responses. It appears that both retinoic acid and GCs interact with the redox balance particularly in neurons (da Frota Junior et al., 2011, Guleria et al., 2006, Spiers et al., 2014).

It can be argued that the molecular and cellular mechanisms underlying learning and memory are an adaptation of the mechanisms used by all cells to regulate cell motility (Baudry and Bi, 2013) and that cell motility involves cellular cytoskeleton remodelling. The cytoskeleton is composed of three major elements: microtubules, intermediate filaments and actin microfilaments. The latter are formed by polymerization of globular actin (G-actin) into filamentous actin (F-actin). The redox balance is one component of this cytoskeleton organisation. ROS target actin directly through the semaphorin signalling and ROS/RNS may act by opening calcium channels activating calcium-dependent processes (Munnamalai and Suter, 2009, Tiago et al., 2011, Wilson and Gonzalez-Billault, 2015). BDNF is another player where its effects on neurons involve at least two pathways: (i) regulation of the expression of genes involved in cellular plasticity such as activity-regulated cytoskeleton-associated protein (Arc) and Ca²⁺/calmodulin-dependent protein kinase II (CaMKII) (Bramham and Messaoudi, 2005, Larsen et al., 2007, Rao et al., 2006, Soule et al., 2006), and (ii) modifications of cytoskeleton organization (Gehler et al., 2004, Rex et al., 2007). Part of the F-actin cytoskeleton regulation depends on the activation of calcium-dependent endoproteases and the calpains (Baudry and Bi, 2013, Sato and Kawashima, 2001). The activity of calpains is mainly regulated by calpastatin which is their specific endogenous inhibitor.

On the basis of our recent results related to the different effects of RA and Dex treatments on hippocampal cells (Brossaud et al., 2013) we explored some pathways of neuroplasticity upon administration of retinoids and glucocorticoids pathway agonists. We then investigated the influence of RA and GC in the hippocampal HT22 cell line on (i) the expression of CaMKII and Arc genes and (ii) F-actin cytoskeleton organization including the implication of calpains.