Abstract
Oxysterol analogs that modulate NMDA receptor function are candidates for therapeutic development to treat neuropsychiatric disorders. However, the cellular actions of these compounds are still unclear. For instance, how these compounds are compartmentalized or trafficked in neurons is unknown. In this study, we utilized a chemical biology approach combining photolabeling and click chemistry. We introduce a biologically active oxysterol analog that contains: (1) a diazirine group, allowing for the permanent labeling of cellular targets, and (2) an alkyne group, allowing for subsequent in situ visualization using Cu2+ catalyzed cycloaddition of an azide-conjugated fluorophore. The physiological properties of this analog at NMDA receptors resemble those of other oxysterols, including occlusion with other oxysterol-like compounds. Fluorescent imaging reveals that the analog accumulates diffusely in the cytoplasm of neurons through an energy-independent mechanism. Overall, this work introduces a novel chemical biology approach to investigate oxysterol actions and introduces a tool useful for further cell biological and biochemical studies of oxysterols.
Highlights
N-methyl-D-aspartate receptors (NMDARs) are glutamate-gated ion channels that play important roles in neurobiological function
We demonstrate that MQ-182 is a potent potentiator of NMDARs, increases the open channel probability of the NMDAR, and occludes the effect of another oxysterol-like modulator but not a sulfated steroid modulator
The intracellular accumulation of this probe may be relevant to cellular actions of oxysterols aside from an effect on surface NMDARs, our work revealed only limited evidence for off target effects at high concentrations
Summary
N-methyl-D-aspartate receptors (NMDARs) are glutamate-gated ion channels that play important roles in neurobiological function. Normal NMDAR function is associated with synaptic plasticity important for encoding new memories, but abnormal NMDAR function is implicated in a multitude of neuropsychiatric defects, including depression, autism, schizophrenia, Alzheimer’s disease, and epilepsy. These receptors represent important therapeutic targets for many CNS disorders, and both negative and positive regulators have potential roles. Analogs of the natural cholesterol metabolite 24S-hydroxycholesterol (24S-HC), are pharmacologically efficacious positive allosteric modulators of NMDARs (Paul et al, 2013; Linsenbardt et al, 2014; Warikoo et al, 2018). The mechanisms by which oxysterols are trafficked or compartmentalized in neurons are unknown
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