Abstract
N-methyl-D-aspartate receptors (NMDARs), a major subtype of glutamate receptor mediating excitatory transmission throughout the CNS, participate in ischemia-induced neuronal death. Unfortunately, undesired side effects have limited the strategy of inhibiting/blocking NMDARs as therapy. Targeting endogenous positive allosteric modulators of NMDAR function may offer a strategy with fewer downsides. Here, we explored whether 24S-hydroxycholesterol (24S-HC), an endogenous positive NMDAR modulator characterized recently by our group, participates in NMDAR-mediated excitotoxicity following oxygen-glucose deprivation (OGD) in primary neuron cultures. 24S-HC is the major brain cholesterol metabolite produced exclusively in neurons near sites of glutamate transmission. By selectively potentiating NMDAR current, 24S-HC may participate in NMDAR-mediated excitotoxicity following energy failure, thus impacting recovery after stroke. In support of this hypothesis, our findings indicate that exogenous application of 24S-HC exacerbates NMDAR-dependent excitotoxicity in primary neuron culture following OGD, an ischemic-like challenge. Similarly, enhancement of endogenous 24S-HC synthesis reduced survival rate. On the other hand, reducing endogenous 24S-HC synthesis alleviated OGD-induced cell death. We found that 25-HC, another oxysterol that antagonizes 24S-HC potentiation, partially rescued OGD-mediated cell death in the presence or absence of exogenous 24S-HC application, and 25-HC exhibited NMDAR-dependent/24S-HC-dependent neuroprotection, as well as NMDAR-independent neuroprotection in rat tissue but not mouse tissue. Our findings suggest that both endogenous and exogenous 24S-HC exacerbate OGD-induced damage via NMDAR activation, while 25-HC exhibits species dependent neuroprotection through both NMDAR-dependent and independent mechanisms.
Highlights
Ischemic stroke is a major cause of death and disability in the United States, and excitotoxicity is a major outcome of the bioenergetic failure associated with stroke [1]
Cell death induced by hypoxia and oxygen-glucose deprivation (OGD) in hippocampal cultures is N-methylD-aspartate receptors (NMDARs) dependent [14,15,16,17]
Effects of 24S-HC on NMDAR function saturate at ~10 μM [3]. Consistent with these observations, exogenous application of 24S-HC at 2 μM to WT rat hippocampal cultures 14 days in vitro enhanced OGD-induced cell death (Fig 1A and 1B). This cell death was rescued by co-treatment with an NMDAR antagonist, APV, prior to and during OGD, confirming that the exacerbation of cell death was NMDAR dependent [14,15,16,17]
Summary
Ischemic stroke is a major cause of death and disability in the United States, and excitotoxicity is a major outcome of the bioenergetic failure associated with stroke [1]. Energy disruption triggers depolarization, which initiates a positive-feedback cycle of release of glutamate, an important excitatory (depolarizing) transmitter, and subsequent overactivation of N-methylD-aspartate receptors (NMDARs), a major subtype of glutamate receptors mediating excitatory transmission throughout the CNS. NMDARs participate in ischemia-induced neuronal injury and death by admitting Ca2+ and promoting excitotoxic cell death [2]. Diminishing NMDAR-mediated excitotoxicity may be neuroprotective and benefit stroke outcome. Undesired side effects have limited the strategy of directly inhibiting/blocking NMDARs as therapy. Understanding and targeting endogenous positive allosteric modulators of NMDAR function may produce fewer downsides since basal (orthosteric) NMDAR function remains unaltered
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