Pregnenolone sulfate as a synaptic modulator
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Pregnenolone (PREG), the precursor of all neurosteroids, is synthesized in the nervous system from cholesterol and recent clinical studies indicate that reduced cognitive symptoms of schizophrenia correlate with elevated serum levels of pregnenolone sulfate (PregS), its immediate sulfated metabolite. PregS fulfills most of the classical criteria for an endogenous modulator of excitatory synaptic transmission, including: presence in nervous tissue at physiologically relevant concentrations, potentiation of N-methyl-D-aspartate receptor (NMDAR) mediated synaptic activity, and a mechanism for its inactivation. As NMDAR hypoactivity has been implicated in the pathophysiology of schizophrenia, defects in neurosteroid metabolism might play a role in its associated cognitive dysfunction. PregS improves memory performance in rodents and augments long-term potentiation (LTP), an electrophysiological correlate of synaptic plasticity that is stabilized by phosphorylation of the cAMP response element binding protein (CREB). We have previously demonstrated that PregS at low picomolar (pM) concentrations increases intracellular Ca2+ and CREB via synaptic NMDARs. Therefore, we hypothesized that low pM concentrations of PregS might potentiate spontaneous excitatory postsynaptic currents (sEPSCs) and promote molecular events underlying synaptic plasticity. Here, using whole-cell patch clamp recordings, we report that PregS enhances the frequency of sEPSCs of cultured hippocampal neurons by about 2-fold while not altering their amplitude or passive membrane properties. This suggests that PregS acts presynaptically by increasing the frequency of neurotransmitter release or postsynaptically by activating silent synapses. We then investigated the hypothesis that PregS increases α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor (AMPAR) and NMDAR subtypes at synapses as a molecular switch for this enhancement. We measured receptor redistribution and phosphorylation using fluorescence imaging and Western blot technology. The results demonstrate that PregS (50pM, 10min): (1) Increases AMPAR (GluA1)/PSD95 colocalization (dependent on L-type voltage-gated Ca+2 channel and synaptic NMDAR activity), and increases phosphorylation of GluA1 at serine-831/845; (2) Increases casein kinase 2 (CK2) dependent surface NMDAR2A (GluN2A) but not GluN1 or GluN2B; and (3) Increases GluN2B serine-1480 phosphorylation. The results show that PregS increases the frequency of excitatory synaptic transmission and increases surface/synaptic AMPARs and surface GluN2A (but not GluN1 or GluN2B) NMDARs, shifting the molecular composition of young glutamatergic synapses toward the adult GluN2A enriched synaptic phenotype.