Different oxysterols have opposing actions at N-methyl-d-aspartate receptors

Highlights

• Oxysterols represent new modulators of NMDARs that increase agonist efficacy.

• Oxysterols do not need direct access to the NMDAR; are ineffective from the cytosol.

• 24(S)-HC and 25-HC may compete in a physiological “tug-of-war” for NMDAR modulation.

Abstract

Oxysterols have emerged as important biomarkers in disease and as signaling molecules. We recently showed that the oxysterol 24(S)-hydroxycholesterol, the major brain cholesterol metabolite, potently and selectively enhances NMDA receptor function at a site distinct from other modulators. Here we further characterize the pharmacological mechanisms of 24(S)-hydroxycholesterol and its synthetic analog SGE201. We describe an oxysterol antagonist of this positive allosteric modulation, 25-hydroxycholesterol. We found that 24(S)-hydroxycholesterol and SGE201 primarily increased the efficacy of NMDAR agonists but did not directly gate the channel or increase functional receptor number. Rather than binding to a direct aqueous-accessible site, oxysterols may partition into the plasma membrane to access the NMDAR, likely explaining slow onset and offset kinetics of modulation. Interestingly, oxysterols were ineffective when applied to the cytosolic face of inside-out membrane patches or through a whole-cell pipette solution, suggesting a non-intracellular site. We also found that another natural oxysterol, 25-hydroxycholesterol, although exhibiting slight potentiation on its own, non-competitively and enantioselectively antagonized the effects of 24(S)-hydroxycholesterol analogs. In summary, we suggest two novel allosteric sites on NMDARs that separately modulate channel gating, but together oppose each other.

Introduction

The N-methyl-d-aspartate receptor (NMDAR) is a heterotetrameric ligand-gated ion channel. Its role in synaptic plasticity, specifically in long-term potentiation (LTP) and long-term depression (LTD), makes it a target of intense focus in the study of learning and memory. However, activation of NMDARs can also be detrimental, as NMDARs are implicated in multiple neuropsychiatric disorders including schizophrenia, epilepsy, depression, Alzheimer's disease, and ischemic brain injury. As such, NMDARs remain potentially important targets for drug development (Collingridge et al., 2013, Coyle, 2006, Olivares et al., 2012).

Neurosteroids represent one of several known classes of endogenous positive and negative allosteric modulators of NMDAR function. These natural or synthetic compounds directly and rapidly modulate several types of ion channels, including NMDARs and GABA_A receptors (GABA_ARs). Pregnenolone sulfate (PREGS) is one such neuroactive steroid, acting to positively modulate NMDARs and negatively modulate GABA_ARs (Majewska and Schwartz, 1987, Wu et al., 1991). Though PREGS is an endogenous neuroactive steroid, it requires supraphysiological concentrations to affect NMDARs, and thus its relevance to brain function is controversial (Liere et al., 2009, Reddy, 2010, Wu et al., 1991).

The brain harbors other cholesterol derivatives, including oxysterols. Oxysterols are direct metabolic bi-products of cholesterol (Mast et al., 2010). The enzyme responsible for production of the major brain oxysterol is cholesterol-24-hydroxylase, localized primarily to postsynaptic compartments of neurons (Lund et al., 1999, Ramirez et al., 2008). Cholesterol-24-hydroxylase produces 24(S)-hydroxycholesterol (24(S)-HC), the most abundant cholesterol metabolite found in the brain (Russell et al., 2009). Other oxysterols, including 25-hydroxycholesterol (25-HC), are also present in brain, though at lower concentrations (Griffiths et al., 2006). The role of oxysterols in nervous system functioning has only begun to be investigated (Freemantle et al., 2013, Wong et al., 2007).

We have recently shown that 24(S)-HC, as well as its synthetic derivatives, SGE201 and SGE301, are both potent positive allosteric modulators (PAMs) of NMDARs (Paul et al., 2013). 24(S)-HC appears to be a highly selective NMDAR modulator and interacts with a site that is distinct from that of other neuroactive steroids and known NMDAR modulators. Its relatively high brain concentration suggests that 24(S)-HC may endogenously modulate NMDARs, although its physiological relevance remains unknown. Synthetic oxysterol analogs also represent important lead compounds for treating NMDAR-related neuropsychiatric disorders. Here, we describe details of the pharmacological mechanisms of oxysterol modulation of NMDARs and provide evidence that 25-HC, another natural oxysterol, non-competitively antagonizes the actions of both natural and synthetic oxysterol PAMs. Taken together, we provide evidence for a rather intricate bidirectional modulation of NMDARS by oxysterols.