AN INVESTIGATION OF THE PUTATIVE ANTIDEPRESSANT MECHANISMS OF KETAMINE, REELIN, AND MU OPIOID RECEPTOR AGONISTS IN RATS AND IPSC-DERIVED CEREBRAL ORGANOIDS EXPOSED TO CHRONIC STRESS

Background

Major depressive disorder (MDD) is a leading cause of global disability, yet current treatment options do not provide symptom relief for many individuals, and delayed treatment onset increases suicide risk. Esketamine, an FDA-approved antidepressant, rapidly increases long-term potentiation (LTP) and functional synapse density, providing fast symptom relief for treatment-resistant depression (TRD). The opioid system is implicated in the pathogenesis of MDD, and mu opioid receptor (MOR) agonism demonstrates rapid antidepressant potential while inhibition of the opioid receptors attenuates ketamine’s antidepressant effects. Translatability, however, is hindered by the significant risk for addiction and dependence. Reelin, an endogenous extracellular matrix glycoprotein, produces rapid antidepressant-like effects that parallel ketamine’s timeline and mechanism in a preclinical rodent model of chronic stress. As a collaborative project between the University of Victoria (Canada) and the National Institute of Mental Health (USA), we designed a comprehensive preclinical evaluation of the potential rapid antidepressant effects of low-dose ketamine or reelin in combination with concurrent administration of a low dose of a MOR agonist with a favourable safety profile. We assessed this in both rodents and iPSC-derived cerebral organoids from TRD patients. We aim to develop a safe, tolerable, and effective rapid antidepressant treatment effective for individuals with TRD. The opioids used (oliceridine and a novel opioid compound) are G-protein-biased MOR agonists that provide effective analgesia with improved tolerability. in vivo: 32 male rats (n = 4/group) underwent daily injections of corticosterone (CORT; 21 d), followed by reelin (3 µg), oliceridine (2.5, 5, or 10 mg/kg), or a combination of reelin or ketamine (5 mg/kg) with low-dose oliceridine (1 mg/kg). Chronic CORT significantly increased immobility in the forced swim test, which was normalized by treatment with oliceridine (5 mg/kg) and the combination of oliceridine with ketamine, and partially by reelin alone and in combination with oliceridine. The prefrontal cortex (PFC) was microdissected and flash-frozen in liquid nitrogen. Synaptoneurosomes were created from PFC homogenate, and synaptic plasticity–related proteins were quantified using western blot. Protein expression did not differ significantly between treatments; however, trends towards increased levels of p-mTOR, m-TOR, p-AKT, and p-eEF2 (relative abundances to CV group ~1.4 - 1.75) following treatment with 2.5 mg/kg of oliceridine indicate MOR-dependent subtle synaptic alterations that warrant further investigation with a larger sample size. in vitro: iPSC-derived cerebral organoids were created using PBMCs collected from patients with TRD or healthy controls. Organoids were treated with vehicle or cortisol for 5 days, followed by treatment with vehicle, reelin (50, 100, or 200 nM), a novel MOR agonist, or a combination of low-dose novel MOR agonist with low-dose reelin (10 nM) or ketamine (0.5 nM). Media IL-6 levels were quantified with ELISA at baseline, 1h post-CORT, 1h post treatment, and 24h post treatment. IL-6 levels significantly increased 24h following treatment with 50 nM of reelin, but not at higher doses. IL-6 was unchanged for all treatments that included the novel MOR agonist compound, suggesting that this compound avoids proinflammatory effects at the administered doses. This preliminary investigation establishes a preclinical rationale for advancing ketamine or reelin in combination with a low-dose MOR agonist as a compelling antidepressant candidate and supports its progression towards clinical evaluation.