Additive neuroprotective effects of 24(S)-hydroxycholesterol and allopregnanolone in an ex vivo rat glaucoma model

Makoto Ishikawa,Douglas F Covey,Charles F Zorumski,Yukitoshi Izumi,Takeshi Yoshitomi

doi:10.1038/s41598-018-31239-2

Makoto Ishikawa, Douglas F Covey + Show 3 more

Open Access

https://doi.org/10.1038/s41598-018-31239-2

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Abstract

In a rat ex vivo acute glaucoma model, high pressure (75 mmHg) causes swelling of ganglion cell axons and elevates levels of the endogenous steroids 24(S)-hydroxycholesterol (24SH) and allopregnanolone (AlloP). Furthermore, 24SH (0.1 µM) alone elevates AlloP levels via NMDA receptors. With this model, we now investigate possible interactions between 24SH and AlloP. We found that inhibition of AlloP synthesis with dutasteride under high pressure results in severe excitotoxicity in addition to axonal swelling. The excitotoxicity is prevented by exogenous AlloP but not 24SH, indicating that endogenous AlloP is crucial for protection. However, inhibition of 24SH synthesis with voriconazole induces severe excitotoxicity under normal pressure. Paradoxically, the excitotoxicity by voriconazole is better prevented by AlloP than 24SH. These findings suggest that inhibition of 24SH synthesis becomes excitotoxic in the absence of AlloP. We also observed that co-administration of sub-micromolar 24SH (0.1 µM) and AlloP (0.1 µM), concentrations that are only partially effective when administered alone, prevents axonal swelling under high pressure. This apparent enhanced protection indicates strong interaction between the two neurosteroids to preserve neuronal integrity, with 24SH contributing to AlloP synthesis via NMDA receptors and with AlloP playing an essential role in neuroprotection via GABAA receptors.

Highlights

Glaucoma is a leading cause of irreversible blindness[1,2], and is characterized by apoptotic death of retinal ganglion cells (RGC)
Picrotoxin induced excitotoxic degeneration characterized by bull’s eye formation in the inner nuclear layer (INL) and edematous changes in the inner plexiform layer (IPL)[19] along with axonal degeneration in the nerve fiber layer (NFL) under hyperbaric conditions (Fig. 2D). This excitotoxic damage was blocked by 50 μM APV, an NMDA receptor antagonist, some nuclear degeneration in the ganglion cell layer (GCL) and axonal swelling in the NFL remained (Fig. 2E)
With regard to pressure-induced axonal swelling, the present study revealed that the simultaneous administration of AlloP and 24SH is able to inhibit pressure-induced axonal swelling at sub-micromolar concentrations (Fig. 8D), resulting in apparent additive neuroprotection

Summary

Introduction

Glaucoma is a leading cause of irreversible blindness[1,2], and is characterized by apoptotic death of retinal ganglion cells (RGC). Under physiological conditions the amounts of 27HC and pregnenolone are much lower than 24SH12, suggesting that CYP46A1 is the main enzyme responsible for cholesterol metabolism in the brain. AlloP, a potent and effective positive modulator of γ-aminobutyric acid (GABA) receptors, is an endogenous protectant of RGC against pressure-induced damage, and exogenously administered AlloP (1 μM) ameliorates pressure-induced retinal injury[14,15]. Because 24SH is a selective positive modulator of N-methyl-D-aspartate (NMDA) receptors16, 24SH was expected to worsen pressure-induced excitotoxicity in our ex vivo glaucoma model. Against this expectation, exogenously administered 24SH (1 μM) ameliorated pressure-induced retinal injury[15]. We examined functional interactions between AlloP and 24SH using inhibitors of key synthetic enzymes along with various concentrations of exogenous AlloP and 24SH, alone and together, examining whether these two neurosteroids (NSs) provide additive neuroprotection in the retina

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