The signaling molecule RGS9-2 is a potent modulator of G-protein-coupled receptor function in striatum. Our earlier work revealed a critical role for RGS9-2 in the actions of the μ-opioid receptor (MOR) agonist morphine. In this study, we demonstrate that RGS9-2 may act as a positive or negative modulator of MOR-mediated behavioral responses in mice depending on the agonist administered. Paralleling these findings we use coimmunoprecipitation assays to show that the signaling complexes formed between RGS9-2 and Gα subunits in striatum are determined by the MOR agonist, and we identify RGS9-2 containing complexes associated with analgesic tolerance. In striatum, MOR activation promotes the formation of complexes between RGS9-2 and several Gα subunits, but morphine uniquely promotes an association between RGS9-2 and Gαi3. In contrast, RGS9-2/Gαq complexes assemble after acute application of several MOR agonists but not after morphine application. Repeated morphine administration leads to the formation of distinct complexes, which contain RGS9-2, Gβ5, and Gαq. Finally, we use simple pharmacological manipulations to disrupt RGS9-2 complexes formed during repeated MOR activation to delay the development of analgesic tolerance to morphine. Our data provide a better understanding of the brain-region-specific signaling events associated with opiate analgesia and tolerance and point to pharmacological approaches that can be readily tested for improving chronic analgesic responsiveness.

Regulators of G-protein signaling (RGS proteins) comprise a large family of signal transduction molecules that modulate G-protein-coupled-receptor (GPCR) function. Among the RGS proteins expressed in the brain, RGS9-2 is very abundant in the striatum, a brain region involved in movement, motivation, mood and addiction. This protein negatively modulates signal transduction thus playing a key part in striatal function and resultant behavioral responses. In particular, there is evidence of important interactions with μ-opioid- and dopamine D2-receptor signaling pathways. Several studies indicate that manipulations of RGS9-2 levels in the striatum might greatly affect pharmacological responses. These findings indicate that treatment strategies targeting RGS9-2 levels or activity might be used to enhance responses to drugs acting at GPCRs and/or prevent undesired drug actions.

No abstract

The striatal protein Regulator of G-protein signaling 9-2 (RGS9-2) plays a key modulatory role in opioid, monoamine, and other G-protein-coupled receptor responses. Here, we use the murine spared-nerve injury model of neuropathic pain to investigate the mechanism by which RGS9-2 in the nucleus accumbens (NAc), a brain region involved in mood, reward, and motivation, modulates the actions of tricyclic antidepressants (TCAs). Prevention of RGS9-2 action in the NAc increases the efficacy of the TCA desipramine and dramatically accelerates its onset of action. By controlling the activation of effector molecules by G protein α and βγ subunits, RGS9-2 affects several protein interactions, phosphoprotein levels, and the function of the epigenetic modifier histone deacetylase 5, which are important for TCA responsiveness. Furthermore, information from RNA-sequencing analysis reveals that RGS9-2 in the NAc affects the expression of many genes known to be involved in nociception, analgesia, and antidepressant drug actions. Our findings provide novel information on NAc-specific cellular mechanisms that mediate the actions of TCAs in neuropathic pain states.desipramine | duloxetine | HDAC5 | spared nerve injury | gene expression R egulator of G-protein signaling 9-2 (RGS9-2) is an intracellular modulator of G-protein-coupled receptor (GPCR) function, which is expressed in medium spiny neurons and cholinergic interneurons of the striatum (1, 2). RGS9-2 influences the magnitude and time course of GPCR signaling by promoting GTPase activity of the Gα subunit and by preventing activation of Gα effectors (3). This modulation can also influence the duration of interactions between the Gβγ subunits and their effector molecules. In addition to Gα subunits, RGS9-2 interacts with several scaffolds and signal transduction proteins that affect its function, expression, and cellular localization. Interactions with the Gβ5 subunit and the adaptor protein R7BP determine the stability and cellular localization of RGS9-2, respectively (3, 4). Several recent studies have provided information on the regulation and function of RGS9-2 complexes in the striatum and how these complexes affect pharmacologic responses (2, 5, 6). In particular, RGS9-2 has been shown to modulate the actions of various psychotropic, antiparkinsonian, neuroleptic, and opiate analgesic drugs (1, 7). The nucleus accumbens (NAc) is a striatal brain region that is a major site of antidepressant drug action (8). Recent studies provided information on signal transduction events triggered by tricyclic antidepressants (TCAs) in NAc neurons and identified several second messengers, transcription factors, and epigenetic molecules involved in their therapeutic actions (9-11). TCAs, such as desipramine (DMI) and nortriptyline (NTL), and selective serotonin/norepinephrine reuptake inhibitors (SNRIs) have also been used to treat neuropathic pain, a complex chronic disorder that is highly comorbid with anxiety and depression (12, 13) and is characterized by thermal hyperalgesia, mech...

The signal transduction modulator Rgs9-2 (Regulator of G protein signaling 9-2) plays a key role in dopaminergic and opioidergic transmission in the striatum. Rgs9-2 is a potent modulator of opiate reward and analgesia, but its role in chronic pain remains unknown. Here, we use the spared nerve injury model (SNI), to evaluate the influence of Rgs9-2 in sensory symptoms, as well as in anxiety and depression-like behaviors observed under neuropathic pain conditions. Our data demonstrate that knockout of the Rgs9 gene reduces the intensity of thermal hyperalgesia and mechanical allodynia the first few days after nerve injury. This small, but significant effect is only observed at early time points after nerve injury, whereas after the first week of SNI, Rgs9 knockout (Rgs9KO) and Rgs9 wildtype (Rgs9WT) mice show similar levels of mechanical allodynia and thermal hyperalgesia. Furthermore, Rgs9-2 deletion exacerbates anxiety and depression like behaviors several weeks after the emergence of the neuropathic pain symptoms. Our findings also reveal a temporal and regional regulation of Rgs9-2 protein expression by neuropathic pain, as Rgs9-2 levels are reduced in the spinal cord a few days after nerve injury, whereas decreased Rgs9-2 levels in the Nucleus Accumbens (NAc) are only observed several weeks after nerve injury. Thus, adaptations in Rgs9-2 activity in the spinal cord and in the NAc may contribute to sensory and affective components of neuropathic pain.

The adaptor protein R7 family binding protein (R7BP) modulates G protein coupled receptor (GPCR) signaling and desensitization by controlling the function of regulator of G protein signaling (RGS) proteins. R7BP is expressed throughout the brain and appears to modulate the membrane localization and stability of three proteins that belong to R7 RGS family: RGS6, RGS7, and RGS9-2. RGS9-2 is a potent negative modulator of opiate and psychostimulant addiction and promotes the development of analgesic tolerance to morphine, whereas the role of RGS6 and RGS7 in addiction remains unknown. Recent studies revealed that functional deletion of R7BP reduces R7 protein activity by preventing their anchoring to the cell membrane and enhances GPCR responsiveness in the basal ganglia. Here, we take advantage of R7BP knockout mice in order to examine the way interventions in R7 proteins function throughout the brain affect opiate actions. Our results suggest that R7BP is a negative modulator of the analgesic and locomotor activating actions of morphine. We also report that R7BP contributes to the development of morphine tolerance. Finally, our data suggest that although prevention of R7BP actions enhances the analgesic responses to morphine, it does not affect the severity of somatic withdrawal signs. Our data suggest that interventions in R7BP actions enhance the analgesic effect of morphine and prevent tolerance, without affecting withdrawal, pointing to R7BP complexes as potential new targets for analgesic drugs.

Regulator of G protein signaling 9–2 (RGS9–2) is a protein that is highly enriched in the striatum, a brain region that mediates motivation, movement and reward responses. We identified a naturally occurring 5 nucleotide deletion polymorphism in the human RGS9 gene and found that the mean body mass index (BMI) of individuals with the deletion was significantly higher than those without. A splicing reporter minigene assay demonstrated that the deletion had the potential to significantly decrease the levels of correctly spliced RGS9 gene product. We measured the weights of rats after virally transduced overexpression of RGS9–2 or the structurally related RGS proteins, RGS7, or RGS11, in the nucleus accumbens (NAc) and observed a reduction in body weight after overexpression of RGS9–2 but not RGS7 or 11. Conversely, we found that the RGS9 knockout mice were heavier than their wild-type littermates and had significantly higher percentages of abdominal fat. The constituent adipocytes were found to have a mean cross-sectional area that was more than double that of corresponding cells from wild-type mice. However, food intake and locomotion were not significantly different between the two strains. These studies with humans, rats and mice implicate RGS9–2 as a factor in regulating body weight.

Over the last few years, a large number of preclinical and clinical studies have demonstrated the potential of gene therapy applications using adeno-associated viral (AAV) vectors. Gene transfer via AAV vectors has been particularly successful for the treatment or adjunct therapy of several CNS disorders. The present review summarizes the progress on AAV gene delivery models for three different CNS disorders. In particular, we discuss advances in AAV-mediated gene transfer strategies in animal models of Parkinson's disease, Alzheimer's disease and spinal cord trauma and summarize the results from the first clinical studies using AAV systems.