The serotonergic system and in particular serotonin 1A receptor (5-HT1AR) are implicated in major depressive disorder (MDD). Here we demonstrated that 5-HT1AR is palmitoylated in human and rodent brains, and identified ZDHHC21 as a major palmitoyl acyltransferase, whose depletion reduced palmitoylation and consequently signaling functions of 5-HT1AR. Two rodent models for depression-like behavior show reduced brain ZDHHC21 expression and attenuated 5-HT1AR palmitoylation. Moreover, selective knock-down of ZDHHC21 in the murine forebrain induced depression-like behavior. We also identified the microRNA miR-30e as a negative regulator of Zdhhc21 expression. Through analysis of the post-mortem brain samples in individuals with MDD that died by suicide we find that miR-30e expression is increased, while ZDHHC21 expression, as well as palmitoylation of 5-HT1AR, are reduced within the prefrontal cortex. Our study suggests that downregulation of 5-HT1AR palmitoylation is a mechanism involved in depression, making the restoration of 5-HT1AR palmitoylation a promising clinical strategy for the treatment of MDD.

The precise regulation of synaptic integrity is critical for neuronal network connectivity and proper brain function. Essential aspects of the activity and localization of synaptic proteins are regulated by posttranslational modifications. S-palmitoylation is a reversible covalent modification of the cysteine with palmitate. It modulates affinity of the protein for cell membranes and membranous compartments. Intracellular palmitoylation dynamics are regulated by crosstalk with other posttranslational modifications, such as S-nitrosylation. S-nitrosylation is a covalent modification of cysteine thiol by nitric oxide and can modulate protein functions. Therefore, simultaneous identification of endogenous site-specific proteomes of both cysteine modifications under certain biological conditions offers new insights into the regulation of functional pathways. Still unclear, however, are the ways in which this crosstalk is affected in brain pathology, such as stress-related disorders. Using a newly developed mass spectrometry-based approach Palmitoylation And Nitrosylation Interplay Monitoring (PANIMoni), we analyzed the endogenous S-palmitoylation and S-nitrosylation of postsynaptic density proteins at the level of specific single cysteine in a mouse model of chronic stress. Among a total of 813 S-PALM and 620 S-NO cysteine sites that were characterized on 465 and 360 proteins, respectively, we sought to identify those that were differentially affected by stress. Our data show involvement of S-palmitoylation and S-nitrosylation crosstalk in the regulation of 122 proteins including receptors, scaffolding proteins, regulatory proteins and cytoskeletal components. Our results suggest that atypical crosstalk between the S-palmitoylation and S-nitrosylation interplay of proteins involved in synaptic transmission, protein localization and regulation of synaptic plasticity might be one of the main events associated with chronic stress disorder, leading to destabilization in synaptic networks. Graphical Abstract Highlights Using mass spectrometry-based proteomic approach, PANIMoni, we analyzed endogenous S-nitrosylation and S-palmitoylation of postsynaptic density proteins in mouse models of chronic stress. We provided differential analysis of S-palmitoylation and S-nitrosylation at the level of exact sites of modifications. We show that affected mechanism of S-palmitoylation and S-nitrosylation interplay of proteins involved in synaptic transmission, protein localization and regulation of synaptic plasticity is associated with chronic stress disorder.

Ex vivo expanded CD4⁺CD25^highCD127^- T regulatory cells (Tregs) are recognized as a promising candidate for immunosuppressive therapy in humans. However, due to the plasticity of Tregs lineage and artificial environment present during ex vivo expansion, Tregs easily lose suppressive activity. Here, we followed expanding CD4⁺CD25^highCD127^- Tregs and their naive (CD45RA⁺) and memory-like (CD45RA^-) subsets in order to establish the best conditions of the expansion. We found that, regardless of the phenotype sorted, expanding Tregs were undergoing changes resembling homeostatic proliferation and transformed into effector memory-like cells which produced not only suppressive interleukin-10 (IL-10) but also IL-6, IL-17, and interferon-γ (IFN-γ). With the time ex vivo, Tregs were losing the expression of FoxP3 and suppressive activity both when stimulated and when at rest. The only variable that helped preserve suppressive abilities of Tregs was the limitation of the time of ex vivo cultures to 2 weeks only. According to our study, the highest number of highly suppressive Tregs could be yielded with CD4⁺CD25^highCD127^- Tregs cultured no longer than 2 weeks. Thorough quality check, preferentially with the assessment of FoxP3 expression and IFN-γ suppression assay, should be applied to assess suppressive activity of the cells.

Ketamine is an N-methyl-d-aspartate receptor antagonist that has gained wide attention as a potent antidepressant. It has also been recently reported to have prophylactic effects in animal models of depression and anxiety. Alterations of neuroplasticity in different brain regions; such as the hippocampus; prefrontal cortex; and amygdala; are a hallmark of stress-related disorders; and such changes may endure beyond the treatment of symptoms. The present study investigated whether a prophylactic injection of ketamine has effects on structural plasticity in the brain in mice that are subjected to chronic unpredictable stress followed by an 8-day recovery period. Ketamine administration (3 mg/kg body weight) 1 h before stress exposure increased the number of resilient animals immediately after the cessation of stress exposure and positively influenced the recovery of susceptible animals to hedonic deficits. At the end of the recovery period; ketamine-treated animals exhibited significant differences in dendritic spine density and dendritic spine morphology in brain regions associated with depression compared with saline-treated animals. These results confirm previous findings of the prophylactic effects of ketamine and provide further evidence of an association between the antidepressant-like effect of ketamine and alterations of structural plasticity in the brain