The Neuroactive Steroid Pregnenolone Sulfate Stimulates Trafficking of FunctionalN-Methyl D-Aspartate Receptors to the Cell Surface via a Noncanonical, G Protein, and Ca²⁺-Dependent Mechanism

Abstract

N-methyl D-aspartate (NMDA) receptors (NMDARs) mediate fast excitatory synaptic transmission and play a critical role in synaptic plasticity associated with learning and memory. NMDAR hypoactivity has been implicated in the pathophysiology of schizophrenia, and clinical studies have revealed reduced negative symptoms of schizophrenia with a dose of pregnenolone that elevates serum levels of the neuroactive steroid pregnenolone sulfate (PregS). This report describes a novel process of delayed-onset potentiation whereby PregS approximately doubles the cell's response to NMDA via a mechanism that is pharmacologically and kinetically distinct from rapid positive allosteric modulation by PregS. The number of functional cellsurface NMDARs in cortical neurons increases 60-100% within 10 minutes of exposure to PregS, as shown by surface biotinylation and affinity purification. Delayed-onset potentiation is reversible and selective for expressed receptors containing the NMDAR subunit subtype 2A (NR2A) or NR2B, but not the NR2C or NR2D, subunits. Moreover, substitution of NR2B J/K helices and M4 domain with the corresponding region of NR2D ablates rapid allosteric potentiation of the NMDA response by PregS but not delayed-onset potentiation. This demonstrates that the initial phase of rapid positive allosteric modulation is not a first step in NMDAR upregulation. Delayed-onset potentiation by PregS occurs via a noncanonical, pertussis toxin-sensitive, G protein-coupled, and Ca 21 -dependent mechanism that is independent of NMDAR ion channel activation. Further investigation into the sequelae for PregS-stimulated trafficking of NMDARs to the neuronal cell surface may uncover a new target for the pharmacological treatment of disorders in which NMDAR hypofunction has been implicated.

Proof-of-concept randomized controlled trial of pregnenolone in schizophrenia

“…Potentially important to the pathophysiology of schizophrenia, pregnenolone sulfate positively modulates NMDA receptors (Bowlby 1993;Irwin et al 1994;Wu et al 1991) and prevents cognitive deficits induced by NMDA antagonists (Cheney et al 1995;Mathis et al 1994Mathis et al , 1996Romeo et al 1994). Pregnenolone sulfate also stimulates NMDA receptor trafficking (Kostakis et al 2013) and enhances learning and memory in rodent models (Akwa et al 2001;Darnaudery et al 2002;Flood et al 1992;Ladurelle et al 2000;Mayo et al 1993;Meziane et al 1996;Pallares et al 1998;Vallee et al 1997Vallee et al , 2001 at physiologically relevant concentrations that have been reported in human brain (Weill-Engerer et al 2002).…”

A Role for Picomolar Concentrations of Pregnenolone Sulfate in Synaptic Activity-Dependent Ca2+ Signaling and CREB Activation

“…We previously reported the phenomenon of delayed onset potentiation of the NMDAR response by PregS and that this was a Ca 21 -dependent process that results in the movement of functional receptors to the cell surface of cortical neurons and Xenopus oocytes stimulated via a noncanonical G-protein coupled mechanism (Kostakis et al, 2013). Here we sought to determine whether the effect of PregS could exert functionally significant effects on excitatory synaptic transmission at physiologic concentrations and couple to a downstream signal transduction mechanism associated with learning and memory.…”

“…We recently reported that PregS, at high nanomolar to low micromolar concentrations, releases Ca 21 from intracellular stores in Xenopus oocytes expressing NR1/NR2A or NR1/NR2B receptors via a noncanonical mechanism that requires NMDARs but does not involve activation of the NMDAR ion channel (Kostakis et al, 2013 ] i increase by PregS (Weaver et al, 2000), but the EC 50 for PregS potentiation of [Ca 21 ] i is over six orders of magnitude lower (higher potency), which argues for a distinct binding site. On the other hand, PregS is lipophilic and charged so the observed potency could be substantially enhanced if PregS accesses its recognition site from within or at the lipid bilayer.…”

“…PregS enhances excitatory synaptic transmission by both presynaptic and postsynaptic mechanisms with potencies in the micromolar range (Wu et al, 1991;Park-Chung et al, 1997;Wagner et al, 2008). PregS, but not PREG, potentiates NMDAR responses (Wu et al, 1991, Malayev et al, 2002, enhances NMDAR trafficking to the cell surface (Kostakis et al, 2013) via a Ca 21 dependent G-protein coupled mechanism, and enhances LTP (Sliwinski et al, 2004;Chen et al, 2007) in an NMDARdependent fashion. It is also known that PregS can be released in a retrograde messenger-like fashion to increase presynaptic glutamate release (Mameli et al, 2005).…”

Neuroactive Steroids and Related Steroids in Autism Spectrum Disorders

“…Pregnenolone sulfate has been reported to stimulate the trafficking of NMDA glutamate receptors to the neuronal surface [90,91]. Allopregnanolone, a metabolite of pregnenolone (via progesterone), is produced de novo in both neurons and glia [92] and is also synthesized in ovaries and adrenal glands [12].…”