DIFFERENCE IN TREATMENT-RESPONSE OF IPSC-DERIVED NEURONS TO REELIN AND (2R,6R)-HNK FROM PARTICIPANTS WITH TREATMENT- RESISTANT DEPRESSION
Authors: Johnston *Jenessa , Quintanilla Brandi , Yuan Peixiong , Peng Shiyong , Yavi Mani , Caruncho Hector , Kadriu Bashkim , Zarate Jr Carlos 
Published: February 2025

Abstract

Background
Treatment-resistant depression (TRD) presents a significant clinical challenge, with around 30% of patients failing to respond to first-line treatments. Ketamine, administered at subanesthetic doses, has demonstrated rapid and robust antidepressant effects in TRD patients. Some research has suggested that ketamine's fast-acting antidepressant effects are linked to the transient activation of the mammalian target of rapamycin (mTOR), which enhances synaptic signaling by upregulating proteins such GluA1. A major ketamine metabolite, (2R,6R)-hydroxynorketamine (HNK), appears to produce similar effects without the adverse side effects or N-methyl-d-asparate receptor inhibition. Additionally, reelin, a glycoprotein crucial for proper cortical development, seems to mirror some of the fast-acting antidepressant-like effects of ketamine in pre-clinical models, warranting further exploration.

Aims & Objectives
Characterize neurons derived from induced pluripotent stem cells (iPSCs) collected from participants with TRD. Baseline differences in protein expression were analyzed, as well as varying treatment-response to (2R,6R)-HNK and reelin.

Method
iPSCs were generated from peripheral mononuclear blood cells collected from individuals with TRD (n=5) and healthy controls (HCs) (n=6). The STEMdiffTM embryoid body protocol was followed for 19 days to develop single-cell neural progenitor cells. After 10 weeks of cultivation, cells were exposed to five different conditions (vehicle + DMSO, 5nM reelin, 10nM reelin, 50nM reelin, 1μ M (2R,6R)-HNK) at two time points (1 hour and 24 hours) to assess short- and long-term effects of reelin and (2R,6R)-HNK. Western blotting analyses were employed to measure mTOR, phosphorylated-mTOR (p-mTOR), post-synaptic density 95 (PSD95), GluA1, Synapsin 1 (Syn-I), Disabled-1 (Dab1), tyrosine kinase receptor B (TrkB), extracellular signal-regulated kinase (ERK), and p-ERK.

Results
In TRD cell lines at 1-hour, both (2R,6R)-HNK and reelin significantly increased levels of PSD-95 (50nM reelin, p&lt;0.05; HNK 1μ M, p&lt;0.01), Syn-I (10nM reelin, p&lt;0.05; 50nM reelin, p&lt;0.01; HNK 1μ M, p&lt;0.01), Dab1 (10nM reelin, p&lt;0.05; 50nM reelin, p&lt;0.01; HNK 1μ M, p&lt;0.01), and p-ERK (50nM reelin, p&lt;0.05; HNK 1μ M, p&lt;0.05). After 24 hours, these effects were reversed, showing significant down- regulation of PSD-95 (10nM reelin, p&lt;0.05; 50nM reelin, p&lt;0.01) and Syn-I (10nM reelin, p&lt;0.05; 50nM reelin, p&lt;0.01; HNK 1μ M, p&lt;0.01). However, no significant differences were found in any treatment or time group in healthy controls.

Discussion & Conclusion
The high rate of treatment failure in TRD necessitates the exploration of novel antidepressants with reduced long-term side effects, higher efficacy, and rapid therapeutic action. This preliminary study indicates that both reelin and (2R,6R)-HNK show promise in activating cellular pathways crucial for fast-acting antidepressant responses. The disparities observed between TRD and HC cell lines warrant further investigation. Additionally, the translation of novel antidepressants into clinical practice has been hindered by the lack of in vitro or in vivo models capable of representing the heterogeneity of depression. While still in early stages, iPSCs could offer a more precise proxy for evaluating treatment responsiveness, contributing to the development of innovative antidepressant therapeutics and improved translation in pharmacological research.