Charles F. Zorumski

Neuroactive steroids modulate GABA _A and NMDA receptors allosterically, typically requiring specific structural features for their activity. In this study, we characterize YX84, a novel neuroactive steroid bearing a 3β sulfate and p- trifluoroacetylbenzyl alcohol attached in an ether linkage to a hydroxyl group at steroid carbon 17. This compound and similar analogues exhibit an atypical pharmacological profile, with three distinct actions at GABA _A receptors. First, YX84 is a full agonist, with EC ₅₀ near 1 µM and comparable efficacy to GABA at GABA _A receptors in native hippocampal neurons. It presents as a full agonist relative to GABA at α4/δ subunit-containing receptors. Second, YX84 acts as a slow-onset, potent positive allosteric modulator (PAM) of GABA _A receptors at concentrations below those that gate a response. Finally, YX84 exhibits rapid desensitizing and/or blocking kinetics; voltage dependence is consistent with a contribution of channel block. Structure– activity relationship analyses reveal that both functional groups are essential for gating activity, while classical requirements such as carbon 3 hydroxyl stereoselectivity and carbon 5 reduction are dispensable. YX84 also modestly inhibits NMDA receptor currents, suggesting weak negative allosteric modulation. Behavioral assays show that intraperitoneal administration of YX84 (30 mg/kg) does not impair sensorimotor function, unlike allopregnanolone. These findings identify YX84 as a structurally distinct neuroactive steroid with dual receptor activity and favorable behavioral tolerability, offering a promising scaffold for therapeutic development targeting excitatory/inhibitory imbalance in neuropsychiatric disorders if pharmacokinetic considerations can be overcome.

Neurosteroids are powerful endogenous modulators of inhibition and emerging therapeutics for anxiety, epilepsy, and mood disorders, yet their actions at defined receptor subtypes and within specific neuronal populations remain poorly resolved. Here, we engineer a neurosteroid DART (Drug Acutely Restricted by Tethering) platform to deliver neuroactive steroid (NAS) activity with cellular precision and receptor-subunit selectivity. From a screen of seventeen NAS analogs, we identified seven scaffolds suitable for further engineering, and we discovered that linker attachment at the steroid C11 position uniquely preserves NAS positive allosteric modulation of GABA _A receptors, whereas C2 and C17 attachment abolished activity. C11-linked NAS-DARTs slowed IPSC decay kinetics and showed variable off-target modulation of NMDA and AMPA EPSCs. The lead DART compound, YX85.1 ^DART.2 , enhanced GABA-evoked currents in neurons expressing engineered α4/δ-containing GABA _A receptors but spared γ2-containing receptors. A complementary benzodiazepine DART BZP.1 ^DART.2 showed the opposite selectivity. Together, these tools enable cell-restricted, subunit-resolved interrogation of neurosteroid action on inhibitory microcircuits and provide a strategy to dissect how distinct GABA _A receptor subclasses contribute to circuit function and therapeutic outcomes.

The success of ketamine, a dissociative anesthetic and non-competitive N-methyl-D-aspartate receptor (NMDAR) antagonist, as a rapidly acting antidepressant has ignited efforts to identify other novel depression treatments. In recent years, several clinical trials indicated that nitrous oxide (N₂O), an inhalational dissociative anesthetic in clinical use for over 150 years, also has rapid and durable antidepressant effects in patients with major depressive disorder (MDD) and treatment resistant major depression (TRMD). N₂O is a non-competitive NMDAR inhibitor but acts on NMDARs by mechanisms distinct from ketamine. Cellular and neuronal circuit studies of N₂O-induced psychotropic and antidepressant effects are in their infancy and suggest that N₂O shares at least some downstream mechanisms with ketamine, while also having unique effects on neurophysiology and signaling. Human neuroimaging and brain network connectivity studies of N₂O have begun to identify acute and persisting effects of the drug on brain circuits likely relevant for antidepressant responses. In this review, we highlight the current state of clinical and preclinical research into the effects of N₂O and emphasize major unanswered questions, some of which are currently being explored. We emphasize future directions and potential barriers to clinical use of N₂O for treatment of patients with psychiatric illnesses.

Objectives: Previously, we identified four open-angle glaucoma (OAG)-associated autoantibodies (anti-ETNK1, anti-VMAC, anti-NEXN, and anti-SUN1) using proteome-wide autoantibody screening by wet protein arrays. The objective of this exploratory study was to evaluate the diagnostic performance of these four glaucoma-associated autoantibodies using automated machine learning. Methods: Plasma samples from 119 patients with OAG and 35 patients with cataracts as controls were enrolled for the study. All machine-learning analyses were performed in Python 3.9.16 (GCC 11.2.0) using scikit-learn 1.2.2 and PyCaret 3.0.1. Variables included plasma levels of the autoantibodies, age, sex, and intra-ocular pressure (IOP). Probability calibration (Platt/sigmoid and isotonic) was assessed with reliability curves and Brier scores. Model explainability was examined with permutation importance, SHAP values, and an ablation analysis removing one autoantibody at a time. Results: The tuned random forest achieved an out-of-fold (OOF) area under the receiver-operating characteristic curve (ROC–AUC) of 0.852 (±0.040), an average precision (AP) of 0.950, and an F1 score of 0.865. Isotonic mapping improved agreement between predicted and empirical probabilities. Among these four autoantibodies, VMAC was the most important factor for the model’s prediction. Conclusions: A machine learning model using four autoantibodies from blood samples showed potential for diagnosing OAG.

Voltage-dependent anion channels (VDACs) are the most abundant proteins in the outer mitochondrial membrane (OMM) and key regulators of mitochondrial function under physiological and pathological conditions. These channels are modulated by multiple agents and are known binding sites for neuroactive steroids (NAS) including allopregnanolone (AlloP). Using erastin, an agent that promotes VDAC activation by preventing inhibition by tubulin, we assessed the impact of VDAC activation on hippocampal function. Brief erastin administration had no effect on basal transmission but completely inhibited induction of long-term potentiation (LTP) in the Schaffer collateral pathway of rat hippocampal slices. This LTP inhibition was prevented by VBIT-4, an agent that inhibits VDAC oligomerization. VDAC-mediated LTP inhibition was also prevented by inhibitors of the NLRP3 inflammasome and caspase-1, downstream effectors of VDACs, but not by inhibition of cGAS-STING, narrowing the neuroinflammatory pathways involved. Similarly, effects of erastin were prevented by AlloP and at lower concentrations by its unnatural enantiomer ( ent -AlloP), an agent that unlike AlloP has little effect on GABA _A receptors. Erastin also inhibited memory formation in a hippocampal-dependent form of one-trial learning and these effects were prevented by VBIT-4 and ent -AlloP, but not by a non-sedating dose of AlloP. These results have relevance for understanding the role of VDACs as mediators of neuronal stress and for the further development of NAS as neurotherapeutics and modulators of cellular stress. SIGNIFICANCE STATEMENT: Voltage-dependent anion channels (VDACs) are important regulators of mitochondrial function, playing roles in cellular metabolism, stress responses and neuroinflammation, and contribute to the pathogenesis of neuropsychiatric illnesses. Here we show that erastin, an agent that activates VDACs initiates specific neuroinflammatory responses to acutely disrupt synaptic plasticity in the rodent hippocampus and abrogate learning. These adverse effects are prevented by the neuroactive steroids allopregnanolone, which binds VDACs and is used clinically for postpartum depression, and its unnatural enantiomer, suggesting that these agents could have therapeutic effects in a broad range of brain illnesses.

Background: An acute angle-closure attack (AAC) is an ocular emergency that results from a rapid increase in intraocular pressure (IOP). Sustained IOP elevation induces severe degeneration of retinal ganglion cells (RGCs) without treatment. Overactivated microglia, key participants in innate immune responses, have critical roles in the pathogenesis of IOP-induced RGC death, although precise mechanisms remain unclear. In the present study, we used a rat ex vivo acute glaucoma model to investigate the role of microglial signaling in RGC death and examined whether pharmacological depletion of microglia using a CSF-1R inhibitor, PLX5622, exerts neuroprotection against pressure-induced retinal injury. Methods: Ex vivo rat retinas were exposed to hydrostatic pressure (10 mmHg or 75 mmHg) for 24 h. Pressure-dependent changes in retinal microglia and RGCs were detected by immunofluorescence. Morphological changes in the retina and RGC apoptosis were examined using light microscopy and TUNEL staining, respectively. The expression of NLRP3, active caspase-1, pro IL-1β, and IL-1β were examined using Western blotting. Effects of PLX5622, an agent that depletes microglia, were examined in morphology, apoptosis, and protein expression assays, while TAK-242, a TLR4 inhibitor, was examined against protein expression. Results: Pressure loading at 75 mmHg markedly increased activated microglia and apoptotic RGCs in the isolated retinas. Western blotting revealed increases in expression of NLRP3, active caspase-1, pro IL-1β, and IL-1β at 75 mmHg compared to 10 mmHg. Inhibition of pressure-induced increases in NLRP3 by TAK-242 indicates that pressure elevation induces RGC death via activation of the TLR4–NLRP3 inflammasome cascade. PLX5622 depleted microglia at 75 mmHg and significantly decreased expression of NLRP3, active caspase-1, pro IL-1β, and IL-1β at 75 mmHg, resulting in preservation of RGCs. Conclusions: These results indicate that pressure elevation induces proliferation of inflammatory microglia and promotes IL-1β production via activation of the TLR4–NLRP3 inflammasome cascade, resulting in RGC death. Pharmacological depletion of microglia with PLX5622 could be a potential neuroprotective approach to preserve RGCs from inflammatory cytokines in AAC eyes.

Clinical trials are increasingly using remote monitoring techniques at the patient's home. We conducted a secondary analysis of remote safety and tolerability monitoring from two fully‐remote clinical trials that tested fluvoxamine for the acute treatment of COVID‐19. Both trials assessed pulse and blood pressure daily, and one study assessed symptoms daily via Ecological Momentary Assessment. On average, patients provided data on vital signs on 93% of the study days and provided data on side effects on 81% of the study days. With respect to safety, patients taking fluvoxamine had reduced pulse rate compared to placebo, with the greatest difference—5 points—at treatment Day 4. In contrast, fluvoxamine showed little to no effect on blood pressure. With respect to tolerability, nausea was most frequent in the first 4–5 days, declining significantly thereafter, while anxiety and difficulty concentrating were uncommon with fluvoxamine compared to placebo. These findings show that remote assessment of safety and tolerability is feasible in clinical trials, and that frequent assessments can provide in‐depth data on the timecourse of safety or tolerability signals.

GABA_Areceptors containing δ subunits have been shown to mediate tonic/slow inhibition in the CNS. These receptors are typically found extrasynaptically and are activated by relatively low levels of ambient GABA in the extracellular space. In the mouse neocortex, δ subunits are expressed by some pyramidal cells as well as on parvalbumin-positive (PV+) interneurons. An important function of PV+ interneurons is the organization of coordinated network activity that can be measured by EEG. However, it remains unclear what role tonic/slow inhibitory control of PV+ neurons may play in shaping oscillatory activity. After validating expected functional loss of δ-associated current in cortex of PV δcKO mice of both sexes, we performed EEG recordings to survey network activity across wake and sleep states. PV δcKO mice showed altered spectral content of EEG during NREM and REM sleep that was a result of increased oscillatory activity in NREM and the emergence of transient high-amplitude bursts of theta-frequency activity during REM. Viral reintroduction ofGabrdto PV+ interneurons in PV δcKO mice rescued REM EEG phenotypes, supporting an important role for δ subunit-mediated inhibition of PV+ interneurons for maintaining normal REM cortical oscillations.

We reveal the critical role of δ-containing GABA_A receptors in parvalbumin interneurons in the medial prefrontal cortex, important for human neuropsychiatric disorders. We demonstrate these receptors’ importance in regulating neuronal excitability and network dynamics. δ-containing receptors act as a brake on interneuron activity, maintaining the excitation-inhibition balance in cortical circuits. Our findings provide insights into how disruptions in inhibitory signaling alter network function through a receptor subtype that is a target of neurotherapeutics.