AUTHOR=Ishikawa Makoto , Nakazawa Toru , Kunikata Hiroshi , Sato Kota , Yoshitomi Takeshi , Krishnan Kathiresan , Covey Douglas F. , Zorumski Charles F. , Izumi Yukitoshi 

TITLE=The Enantiomer of Allopregnanolone Prevents Pressure-Mediated Retinal Degeneration Via Autophagy

JOURNAL=Frontiers in Pharmacology

VOLUME=13

YEAR=2022

URL=https://www.frontiersin.org/journals/pharmacology/articles/10.3389/fphar.2022.855779

DOI=10.3389/fphar.2022.855779

ISSN=1663-9812

ABSTRACT=<p>In an <italic>ex vivo</italic> rat ocular hypertension (OHT) model, the neurosteroid allopregnanolone (AlloP) exerts neuroprotective effects via enhancement of both GABA<sub>A</sub> receptors and autophagy. We now examine whether its enantiomer (<italic>ent-</italic>AlloP), which is largely inactive at GABA receptors, offers similar neuroprotection in <italic>ex vivo</italic> and <italic>in vivo</italic> rat OHT models. <italic>Ex vivo</italic> rat retinal preparations were incubated in a hyperbaric condition (10 and 75 mmHg) for 24 h. An <italic>in vivo</italic> ocular hypertension (OHT) model was induced by intracameral injection of polystyrene microbeads. We examined pharmacological effects of AlloP, <italic>ent-</italic>AlloP, picrotoxin (a GABA<sub>A</sub> receptor antagonist), and 3-MA (an autophagy inhibitor) histologically and biochemically. We found that both AlloP and <italic>ent</italic>-AlloP have marked neuroprotective effects in the retina, but effects of the unnatural enantiomer are independent of GABA<sub>A</sub> receptors. Electron microscopic analyses show that pressure elevation significantly increased autophagosomes (APs) in the nerve fiber layer and addition of AlloP also increased APs and degenerative autophagic vacuoles (AVds). <italic>ent</italic>-AlloP markedly increased APs and AVds compared to AlloP. Examination of LC3B-II and SQSTM1 protein levels using immunoblotting revealed that AlloP increased LC3B-II, and <italic>ent</italic>-AlloP further enhanced LC3B-II and suppressed SQSTM1, indicating that autophagy is a major mechanism underlying neuroprotection by <italic>ent</italic>-AlloP. In an rat <italic>in vivo</italic> OHT model, single intravitreal <italic>ent</italic>-AlloP injection prevented apoptotic cell death of retinal ganglion cells similar to AlloP. However, even in this model, <italic>ent</italic>-AlloP was more effective in activating autophagy than AlloP. We conclude that <italic>ent</italic>-AlloP may be a prototype of potential therapeutic for treatment of glaucoma as an autophagy enhancer without affecting GABA receptors.</p>