Oxytocin accelerates tight junction formation and impairs cellular migration in 3D spheroids: evidence from Gapmer-induced exon skipping

Abstract

Oxytocin (OXT) is a neuropeptide that has been associated with neurological diseases like autism, a strong regulating activity on anxiety and stress-related behavior, physiological effects during pregnancy and parenting, and various cellular effects in neoplastic tissue. In this study, we aimed to unravel the underlying mechanism that OXT employs to regulate cell-cell contacts, spheroid formation, and cellular migration in a 3D culture model of human MLS-402 cells. We have generated a labeled OXT receptor (OXTR) overexpressing cell line cultivated in spheroids that were treated with the OXTR agonists OXT, Atosiban, and Thr4-Gly7-oxytocin (TGOT); with or without a pre-treatment of antisense oligos (Gapmers) that induce exon skipping in the human OXTR gene. This exon skipping leads to the exclusion of exon 4 and therefore a receptor that lost its intracellular G-protein-binding domain. Sensitive digital PCR (dPCR) provided us with the means to differentiate between wild type and truncated OXTR in our cellular model. OXTR truncation differentially activated intracellular signaling cascades related to cell-cell attachment and proliferation like Akt, ERK1/2-RSK1/2, HSP27, STAT1/5, and CREB, as assessed by a Kinase Profiler Assay. Digital and transmission electron microscopy revealed increased tight junction formation and well-organized cellular protrusions into an enlarged extracellular space after OXT treatment, resulting in increased cellular survival. In summary, OXT decreases cellular migration but increases cell-cell contacts and therefore improves nutrient supply. These data reveal a novel cellular effect of OXT that might have implications for degenerating CNS diseases and tumor formation in various tissues.

An AAV-CRISPR/Cas9 strategy for gene editing across divergent rodent species: Targeting neural oxytocin receptors as a proof of concept

“…However, the relative ease with which gRNAs can be multiplexed in AAV vectors should make it possible to target a wide variety of species with one single strategy. It is also important to consider that while two-thirds of the mutated gene products are likely to be nonfunctional because of frameshift mutations, one-third of mutated gene products might retain some functionality (e.g., receptor dimerization) ( 55 ). While our tool induces a near-complete loss of a specific functionality of the OXT receptor (i.e., ligand binding), we cannot exclude the possibility that a small part of the mutated gene products retains some form of residual activity.…”

Advances in current in vitro models on neurodegenerative diseases

“…However, these junctions differ from synaptic junctions which are essential for neural communication and signaling. It is then important to assess the formation of tight junctions and synaptic activity for a complete characterization of the brain model (Wilson et al, 2020;Jurek et al, 2022).…”