A major unresolved issue in treating pain is the paradoxical hyperalgesia produced by the gold-standard analgesic morphine and other opiates. We show here that hyperalgesia-inducing treatment with morphine causes downregulation of the K+-Cl− cotransporter KCC2, impairing Cl− homeostasis in spinal lamina l neurons. Restoring Eanion reversed the morphine-induced hyperalgesia without affecting tolerance. The hyperalgesia was also reversed by ablating spinal microglia. Morphine hyperalgesia, but not tolerance, required μ opioid receptor-dependent expression of P2X4 receptors (P2X4Rs) in microglia and μ-independent gating of the release of brain-derived neurotrophic factor (BDNF) by P2X4Rs. Blocking BDNF-TrkB signalling preserved Cl− homeostasis and reversed the hyperalgesia. Gene-targeted mice in which BDNF was deleted from microglia did not develop hyperalgesia to morphine. Yet, neither morphine antinociception nor tolerance was affected in these animals. Our findings dissociate morphine-induced hyperalgesia from tolerance and unveil the microglia-to-neuron P2X4-BDNF-KCC2 pathway as a therapeutic target to prevent hyperalgesia without affecting morphine analgesia.

Inhibition of glycogen synthase kinase 3β (GSK3β) is a shared action believed to be involved in the regulation of behavior by psychoactive drugs such as antipsychotics and mood stabilizers. However, little is known about the identity of the substrates through which GSK3β affects behavior. We identified fragile X mental retardation-related protein 1 (FXR1P), a RNA binding protein associated to genetic risk for schizophrenia, as a substrate for GSK3β. Phosphorylation of FXR1P by GSK3β is facilitated by prior phosphorylation by ERK2 and leads to its down-regulation. In contrast, behaviorally effective chronic mood stabilizer treatments in mice inhibit GSK3β and increase FXR1P levels. In line with this, overexpression of FXR1P in the mouse prefrontal cortex also leads to comparable mood-related responses. Furthermore, functional genetic polymorphisms affecting either FXR1P or GSK3β gene expression interact to regulate emotional brain responsiveness and stability in humans. These observations uncovered a GSK3β/FXR1P signaling pathway that contributes to regulating mood and emotion processing. Regulation of FXR1P by GSK3β also provides a mechanistic framework that may explain how inhibition of GSK3β can contribute to the regulation of mood by psychoactive drugs in mental illnesses such as bipolar disorder. Moreover, this pathway could potentially be implicated in other biological functions, such as inflammation and cell proliferation, in which FXR1P and GSK3 are known to play a role.

Brain dopamine receptors have been preferred targets for numerous pharmacological compounds developed for the treatment of various neuropsychiatric disorders. Recent discovery that D2 dopamine receptors, in addition to cAMP pathways, can engage also in Akt/GSK3 signaling cascade provided a new framework to understand intracellular signaling mechanisms involved in dopamine-related behaviors and pathologies. Here we review a recent progress in understanding the role of Akt, GSK3, and related signaling molecules in dopamine receptor signaling and functions. Particularly, we focus on the molecular mechanisms involved, interacting partners, role of these signaling events in the action of antipsychotics, psychostimulants, and antidepressants as well as involvement in pathophysiology of schizophrenia, bipolar disorder, and Parkinson’s disease. Further understanding of the role of Akt/GSK3 signaling in dopamine receptor functions could provide novel targets for pharmacological interventions in dopamine-related disorders.

The delivery of biologic cargoes to airway epithelial cells is challenging due to the formidable barriers imposed by its specialized and differentiated cells. Among cargoes, recombinant proteins offer therapeutic promise but the lack of effective delivery methods limits their development. Here, we achieve protein and SpCas9 or AsCas12a ribonucleoprotein (RNP) delivery to cultured human well-differentiated airway epithelial cells and mouse lungs with engineered amphiphilic peptides. These shuttle peptides, non-covalently combined with GFP protein or CRISPR-associated nuclease (Cas) RNP, allow rapid entry into cultured human ciliated and non-ciliated epithelial cells and mouse airway epithelia. Instillation of shuttle peptides combined with SpCas9 or AsCas12a RNP achieves editing of loxP sites in airway epithelia of ROSAmT/mG mice. We observe no evidence of short-term toxicity with a widespread distribution restricted to the respiratory tract. This peptide-based technology advances potential therapeutic avenues for protein and Cas RNP delivery to refractory airway epithelial cells.