| Abstract |
Opioids are among the most effective medications used for the alleviation of severe pain conditions, but their long-term use is hindered by the development of analgesic tolerance, dependence and addiction. Understanding the molecular mechanisms underlying their analgesic effects and the adaptations leading to the development of the undesirable side-effects would be a significant contribution towards improving chronic pain treatment. Falling within this rational, the present thesis focused on examining the role of regulator of G-protein signaling (RGS) proteins in opiate actions. RGS proteins possess a wide and diverse family of proteins known to mediate G-protein coupled receptor (GPCR) signaling duration and direction, through their interaction with heterotrimeric Gα subunits. Here, we study the role of two RGS family members that modulate Mu opioid receptor (MOPR) mediated signaling, RGS9-2 and RGSz1. Particularly, the thesis is consisting of three specific aims:
Aim I examined if and how RGS9-2 modulates responses to oxycodone both in pain-free conditions and under chronic pain states. RGS9-2 is predominantly expressed in the striatum and it has been shown to exert a critical role on the MOPR signaling. It is a negative regulator of morphine analgesia and reward, while at the same time it promotes the development of morphine tolerance. In contrast, RGS9-2 appears as a positive modulator of other MOPR agonist, such as methadone and fentanyl. Here we investigate its role as a regulator of another MOPR agonist widely used and abused over the last years, oxycodone. Using transgenic mice lacking the Rgs9 gene (RGS9KO), we observed that RGS9-2 positively regulates the rewarding effects of oxycodone both in pain-free states and in a model of neuropathic pain. Furthermore, although RGS9-2 does not affect the acute analgesic efficacy of the drug, it opposes the development of oxycodone tolerance. Overall, these results provide new information on the signal transduction mechanisms underlying the analgesic and rewarding actions of oxycodone.
Aim II is focused on the role of RGSz1 in opiate actions. RGSz1 is a small RGS protein primarily expressed in brain tissue, that exhibits high specificity for Gαz subunits and has been shown to act downstream of MOPR and serotonin receptor 1A (Htr1A). Using genetic mouse models for constitutive or conditional/brain region-targeted manipulations of RGSz1 expression, we demonstrated that the analgesic efficacy of several MOPR agonists is increased by preventing RGSz1 actions both in male and female mice. In addition, prevention of RGSz1 action delays the development of morphine tolerance while decreasing the sensitivity to rewarding and locomotor activating effects. Using next-generation RNA sequencing combined with further biochemical assays examining protein expression and localization, we identified a key role of RGSz1 in the periaqueductal gray (PAG) in morphine tolerance. We show that chronic morphine administration promotes RGSz1 activity in the PAG, which in turn modulates transcription mediated by the Wnt/β-catenin signaling pathway and promotes analgesic tolerance to morphine. Conversely, prevention of RGSz1 action stabilizes Axin2-Gαz complexes near the membrane, promoting β-catenin activation, thereby delaying the development of analgesic tolerance. These data highlight that regulation of RGS complexes, particularly those involving RGSz1-Gαz, represent a promising target for optimizing the analgesic actions of opioids without increasing the risk of dependence or addiction.
Finally, aim III examined the role of RGSz1 in behavioral and biochemical adaptations to chronic pain. Using well established murine pain models, we show that deletion of the Rgsz1 gene, as well as conditional knockdown of RGSz1 in the mouse PAG, lead to prolonged sensitized behaviors only in female mice undergoing inflammatory or neuropathic pain. In addition, by combining standard molecular biology techniques (RT-PCR, Western Blot) with next-generation RNA sequencing we showed that chronic inflammatory pain-like states promote a number of unique adaptations in the female PAG, including changes in the expression of molecules involved in serotonin synthesis and release. Further upstream regulator analysis, together with behavioral and biochemical data, revealed the involvement of estrogen receptor mediated signaling in nociceptive hypersensitivity. These findings provide insight to the sex-specific intracellular mechanisms underlying nociceptive responses under chronic pain conditions, and point to RGSz1-regulated pathways as promising targets for the treatment of chronic pain.
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