Stephen J. Gold

Neurobiology of depression.

Current treatments for depression are inadequate for many individuals, and progress in understanding the neurobiology of depression is slow. Several promising hypotheses of depression and antidepressant action have been formulated recently. These hypotheses are based largely on dysregulation of the hypothalamic-pituitary-adrenal axis and hippocampus and implicate corticotropin-releasing factor, glucocorticoids, brain-derived neurotrophic factor, and CREB. Recent work has looked beyond hippocampus to other brain areas that are also likely involved. For example, nucleus accumbens, amygdala, and certain hypothalamic nuclei are critical in regulating motivation, eating, sleeping, energy level, circadian rhythm, and responses to rewarding and aversive stimuli, which are all abnormal in depressed patients. A neurobiologic understanding of depression also requires identification of the genes that make individuals vulnerable or resistant to the syndrome. These advances will fundamentally improve the treatment and prevention of depression.

Regulators of G-protein signaling (RGS) proteins: Region-specific expression of nine subtypes in rat brain

The recently discovered regulators of G-protein signaling (RGS) proteins potently modulate the functioning of heterotrimeric G-proteins by stimulating the GTPase activity of G-protein α subunits. The mRNAs for numerous subtypes of putative RGS proteins have been identified in mammalian tissues, but little is known about their expression in brain. We performed a systematic survey of the localization of mRNAs encoding nine of these RGSs, RGS3–RGS11, in brain by in situhybridization. Striking region-specific patterns of expression were observed. Five subtypes, RGS4, RGS7, RGS8, RGS9, and RGS10 mRNAs, are densely expressed in brain, whereas the other subtypes (RGS3, RGS5, RGS6, and RGS11) are expressed at lower density and in more restricted regions. RGS4 mRNA is notable for its dense expression in neocortex, piriform cortex, caudoputamen, and ventrobasal thalamus. RGS8 mRNA is highly expressed in the cerebellar Purkinje cell layer as well as in several midbrain nuclei. RGS9 mRNA is remarkable for its nearly exclusive enrichment in striatal regions. RGS10 mRNA is densely expressed in dentate gyrus granule cells, superficial layers of neocortex, and dorsal raphe. To assess whether the expression of RGS mRNAs can be regulated, we examined the effect of an acute seizure on levels of RGS7, RGS8, and RGS10 mRNAs in hippocampus. Of the three subtypes, changes in RGS10 levels were most pronounced, decreasing by ∼40% in a time-dependent manner in response to a single seizure. These results, which document highly specific patterns of RGS mRNA expression in brain and their ability to be regulated in a dynamic manner, support the view that RGS proteins may play an important role in determining the intensity and specificity of signaling pathways in brain as well as their adaptations to synaptic activity.

RGS9 modulates dopamine signaling in the basal ganglia.

Regulators of G protein signaling (RGS) modulate heterotrimeric G proteins in part by serving as GTPase-activating proteins for Galpha subunits. We examined a role for RGS9-2, an RGS subtype highly enriched in striatum, in modulating dopamine D2 receptor function. Viral-mediated overexpression of RGS9-2 in rat nucleus accumbens (ventral striatum) reduced locomotor responses to cocaine (an indirect dopamine agonist) and to D2 but not to D1 receptor agonists. Conversely, RGS9 knockout mice showed heightened locomotor and rewarding responses to cocaine and related psychostimulants. In vitro expression of RGS9-2 in Xenopus oocytes accelerated the off-kinetics of D2 receptor-induced GIRK currents, consistent with the in vivo data. Finally, chronic cocaine exposure increased RGS9-2 levels in nucleus accumbens. Together, these data demonstrate a functional interaction between RGS9-2 and D2 receptor signaling and the behavioral actions of psychostimulants and suggest that psychostimulant induction of RGS9-2 represents a compensatory adaptation that diminishes drug responsiveness.

Essential role for RGS9 in opiate action

Regulators of G protein signaling (RGS) are a family of proteins known to accelerate termination of effector stimulation after G protein receptor activation. RGS9-2, a brain-specific splice variant of the RGS9 gene, is highly enriched in striatum and also expressed at much lower levels in periaqueductal gray and spinal cord, structures known to mediate various actions of morphine and other opiates. Morphine exerts its acute rewarding and analgesic effects by activation of inhibitory guanine nucleotide-binding regulatory protein-coupled opioid receptors, whereas chronic morphine causes addiction, tolerance to its acute analgesic effects, and profound physical dependence by sustained activation of these receptors. We show here that acute morphine administration increases expression of RGS9-2 in NAc and the other CNS regions, whereas chronic exposure decreases RGS9-2 levels. Mice lacking RGS9 show enhanced behavioral responses to acute and chronic morphine, including a dramatic increase in morphine reward, increased morphine analgesia with delayed tolerance, and exacerbated morphine physical dependence and withdrawal. These findings establish RGS9 as a potent negative modulator of opiate action in vivo, and suggest that opiate-induced changes in RGS9 levels contribute to the behavioral and neural plasticity associated with chronic opiate administration.

RGS9–2 Negatively Modulates l-3,4-Dihydroxyphenylalanine-Induced Dyskinesia in Experimental Parkinson's Disease

Chronic l-dopa treatment of Parkinsons disease (PD) often leads to debilitating involuntary movements, termed l-dopa-induced dyskinesia (LID), mediated by dopamine (DA) receptors. RGS9–2 is a GTPase accelerating protein that inhibits DA D2 receptor-activated G proteins. Herein, we assess the functional role of RGS9–2 on LID. In monkeys, Western blot analysis of striatal extracts shows that RGS9–2 levels are not altered by MPTP-induced DA denervation and/or chronic l-dopa administration. In MPTP monkeys with LID, striatal RGS9–2 overexpression – achieved by viral vector injection into the striatum – diminishes the involuntary movement intensity without lessening the anti-parkinsonian effects of the D1/D2 receptor agonist l-dopa. In contrasts, in these animals, striatal RGS9–2 overexpression diminishes both the involuntary movement intensity and the anti-parkinsonian effects of the D2/D3 receptor agonist ropinirole. In unilaterally 6-OHDA-lesioned rats with LID, we show that the time course of viral vector-mediated striatal RGS9–2 overexpression parallels the time course of improvement of l-dopa-induced involuntary movements. We also find that unilateral 6-OHDA-lesioned RGS9−/− mice are more susceptible to l-dopa-induced involuntary movements than unilateral 6-OHDA-lesioned RGS9+/+ mice, albeit the rotational behavior – taken as an index of the anti-parkinsonian response – is similar between the two groups of mice. Together, these findings suggest that RGS9–2 plays a pivotal role in LID pathophysiology. However, the findings also suggest that increasing RGS9–2 expression and/or function in PD patients may only be a suitable therapeutic strategy to control involuntary movements induced by nonselective DA agonist such as l-dopa.

Regulation of RGS proteins by chronic morphine in rat locus coeruleus

The present study explored a possible role for RGS (regulators of G protein signalling) proteins in the long term actions of morphine in the locus coeruleus (LC), a brainstem region implicated in opiate physical dependence and withdrawal. Morphine influences LC neurons through activation of µ‐opioid receptors, which, being Gi/o‐linked, would be expected to be modulated by RGS proteins. We focused on several RGS subtypes that are known to be expressed in this brain region. Levels of mRNAs encoding RGS2, ‐3, ‐4, ‐5, ‐7, ‐8 and ‐11 are unchanged following chronic morphine, but RGS2 and −4 mRNA levels are increased 2–3‐fold 6 h following precipitation of opiate withdrawal. The increases in RGS2 and −4 mRNA peak after 6 h of withdrawal and return to control levels by 24 h. Immunoblot analysis of RGS4 revealed a striking divergence between mRNA and protein responses in LC: protein levels are elevated twofold following chronic morphine and decrease to control values by 6 h of withdrawal. In contrast, levels of RGS7 and −11 proteins, the only other subtypes for which antibodies are available, were not altered by these treatments. Intracellular application of wild‐type RGS4, but not a GTPase accelerating‐deficient mutant of RGS4, into LC neurons diminished electrophysiological responses to morphine. The observed subtype‐ and time‐specific regulation of RGS4 protein and mRNA, and the diminished morphine‐induced currents in the presence of elevated RGS4 protein levels, indicate that morphine induction of RGS4 could contribute to aspects of opiate tolerance and dependence displayed by LC neurons.

Differentially Regulated Expression of Endogenous RGS4 and RGS7

Regulators of G protein signaling (RGS proteins) constitute a family of newly appreciated components of G protein-mediated signal transduction. With few exceptions, most information available on mammalian RGS proteins was gained by transfection/overexpression or in vitro experiments, with relatively little known about the endogenous counterparts. Transfection studies, typically of tagged RGS proteins, have been conducted to overcome the low natural abundance of endogenous RGS proteins. Because transfection studies can lead to imprecise or erroneous conclusions, we have developed antibodies of high specificity and sensitivity to focus study on endogenous proteins. Expression of both RGS4 and RGS7 was detected in rat brain tissue and cultured PC12 and AtT-20 cells. Endogenous RGS4 presented as a single 27–28-kDa protein. By contrast, cultured cells transfected with a plasmid encoding RGS4 expressed two observable forms of the protein, apparently due to utilization of distinct sites of initiation of protein synthesis. Subcellular localization of endogenous RGS4 revealed predominant association with membrane fractions, rather than in cytosolic fractions, where most heterologously expressed RGS4 has been found. Endogenous levels of RGS7 exceeded RGS4 by 30–40-fold, and studies of cultured cells revealed regulatory differences between the two proteins. We observed that RGS4 mRNA and protein were concomitantly augmented with increased cell density and decreased by exposure of PC12M cells to nerve growth factor, whereas RGS7 was unaffected. Endogenous RGS7 was relatively stable, whereas proteolysis of endogenous RGS4 was a strong determinant of its lower level expression and short half-life. Although we searched without finding evidence for regulation of RGS4 proteolysis, the possibility remains that alterations in the degradation of this protein could provide a means to promptly alter patterns of signal transduction.

Regulator of G-Protein Signaling 10 Promotes Dopaminergic Neuron Survival via Regulation of the Microglial Inflammatory Response

Epidemiological studies suggest that chronic use of nonsteroidal anti-inflammatory drugs lowers the incidence of Parkinsons disease (PD) in humans and implicate neuroinflammatory processes in the death of dopamine (DA) neurons. Here, we demonstrate that regulator of G-protein signaling 10 (RGS10), a microglia-enriched GAP (GTPase accelerating protein) for Gα subunits, is an important regulator of microglia activation. Flow-cytometric and immunohistochemical analyses indicated that RGS10-deficient mice displayed increased microglial burden in the CNS, and exposure to chronic systemic inflammation induced nigral DA neuron loss measured by unbiased stereology. Primary microglia isolated from brains of RGS10-deficient mice displayed dysregulated inflammation-related gene expression profiles under basal and stimulated conditions in vitro compared with that of primary microglia isolated from wild-type littermates. Similarly, knockdown of RGS10 in the BV2 microglia cell line resulted in dysregulated inflammation-related gene expression, overproduction of tumor necrosis factor (TNF), and enhanced neurotoxic effects of BV2 microglia on the MN9D dopaminergic cell line that could be blocked by addition of the TNF decoy receptor etanercept. Importantly, ablation of RGS10 in MN9D dopaminergic cells further enhanced their vulnerability to microglial-derived death-inducing inflammatory mediators, suggesting a role for RGS10 in modulating the sensitivity of dopaminergic neurons against inflammation-mediated cell death. Together, our findings indicate that RGS10 limits microglial-derived TNF secretion and regulates the functional outcome of inflammatory stimuli in the ventral midbrain. RGS10 emerges as a novel drug target for prevention of nigrostriatal pathway degeneration, the neuropathological hallmark of PD.

RGS9-2 is a negative modulator of μ-opioid receptor function

Regulators of G‐protein signaling (RGS) 9‐2 is a striatal enriched protein that controls G protein coupled receptor signaling duration by accelerating Gα subunit guanosine triphosphate hydrolysis. We have previously demonstrated that mice lacking the RGS9 gene show enhanced morphine analgesia and delayed development of tolerance. Here we extend these studies to understand the mechanism via which RGS9‐2 modulates opiate actions. Our data suggest that RGS9‐2 prevents several events triggered by mu‐opioid receptor (MOR) activation. In transiently transfected PC12 cells, RGS9‐2 delays agonist induced internalization of epitope HA‐tagged μ‐opioid receptor. This action of RGS9‐2 requires localization of the protein near the cell membrane. Co‐immunoprecipitation studies reveal that RGS9‐2 interacts with HA‐tagged μ‐opioid receptor, and that this interaction is enhanced by morphine treatment. In addition, morphine promotes the association of RGS9‐2 with another essential component of MOR desensitization, β‐arrestin‐2. We also show that over‐expression of RGS9‐2 prevents opiate‐induced extracellular signal‐regulated kinase phosphorylation. Our data indicate that RGS9‐2 plays an essential role in opiate actions, by negatively modulating MOR downstream signaling as well as the rate of MOR endocytosis.

Regulation of regulators of G protein signaling mRNA expression in rat brain by acute and chronic electroconvulsive seizures

G protein‐coupled receptor (GPCR) signaling cascades may be key substrates for the antidepressant effects of chronic electroconvulsive seizures (ECS). To better understand changes in these signaling pathways, alterations in levels of mRNAs encoding regulators of G protein signaling (RGS) protein subtypes‐2, ‐4, ‐7, ‐8 and ‐10 were evaluated in rat brain using northern blotting and in situ hybridization. In prefrontal cortex, RGS2 mRNA levels were increased several‐fold 2 h following an acute ECS. Increases in RGS8 mRNA were of lesser magnitude (30%), and no changes were evident for the other RGS subtypes. At 24 h following a chronic ECS regimen, RGS4, ‐7, and ‐10 mRNA levels were reduced by 20–30%; only RGS10 was significantly reduced 24 h after acute ECS. Levels of RGS2 mRNA were unchanged 24 h following either acute or chronic ECS. In hippocampus, RGS2 mRNA levels were markedly increased 2 h following acute ECS. More modest increases were seen for RGS4 mRNA expression, whereas levels of the other RGS subtypes were unaltered. At 24 h following chronic ECS, RGS7, ‐8 and ‐10 mRNA levels were decreased in the granule cell layer, and RGS7 and ‐8 mRNA levels were decreased in the pyramidal cell layers. Only RGS8 and ‐10 mRNA levels were significantly reduced in hippocampus 24 h following an acute ECS. Paralleling neocortex, RGS2 mRNA content was unchanged in hippocampus 24 h following either acute or chronic ECS. In ventromedial hypothalamus, RGS4 mRNA content was increased 24 h following chronic ECS, whereas RGS7 mRNA levels were only increased 24 h following an acute ECS. The increased RGS4 mRNA levels in hypothalamus were significant by 2 h following an acute ECS. These studies demonstrate subtype‐, time‐, and region‐specific regulation of RGS proteins by ECS, adaptations that may contribute to the antidepressant effects of this treatment.