Cristina Missale

Dopamine receptors and brain function

In the central nervous system (CNS), dopamine is involved in the control of locomotion, cognition, affect and neuroendocrine secretion. These actions of dopamine are mediated by five different receptor subtypes, which are members of the large G-protein coupled receptor superfamily. The dopamine receptor subtypes are divided into two major subclasses: the D1-like and D2-like receptors, which typically couple to Gs and Gj mediated transduction systems. In the CNS, the various receptor subtypes display specific anatomical distributions, with D1-like receptors being mainly post-synaptic and D2-like receptors being both pre- and post-synaptic. D1 and D2 dopamine receptors, the most abundant subtypes in the CNS, appear to be expressed largely in distinct neurons. Substance P and dynorphin, which are expressed in D1 receptor-containing neurons, as well as pre-proenkephalin in D2 receptor-containing neurons, have been used as monitors of dopaminergic activity in the CNS. Expression of immediate early genes, in particular fos, has also been found to correlate with dopaminergic transmission. Dopamine released from the hypothalamus controls the synthesis and secretion of prolactin from the anterior pituitary via D2 dopamine receptors. As yet none of the dopamine receptor subtypes have been associated with the etiology of psychotic disorders, such as schizophrenia. However, the recent characterization of D3 and D4 receptors which are, interestingly, expressed in areas of the CNS mediating cognition and affect or showing increased affinity for certain neuroleptics, have renewed the interest and hope of finding effective neuroleptics devoid of side effects. Finally, the recent production of genetically-derived animals lacking several of these receptor genes should help elucidate which specific physiological paradigms the receptors mediate.

Anterior Pituitary Hypoplasia and Dwarfism in Mice Lacking the Dopamine Transporter

Deletion of the dopamine transporter (DAT) results in increased dopaminergic tone, anterior pituitary hypoplasia, dwarfism, and an inability to lactate. DAT elimination alters the spatial distribution and dramatically reduces the numbers of lactotrophs and somatotrophs in the pituitary. Despite having normal circulating levels of growth hormone and prolactin in blood, hypoplastic glands from DAT-/- mice fail to respond to secretagog stimulation. The effects of DAT deletion on pituitary function result from elevated DA levels that down-regulate the lactotroph D2 DA receptors and depress hypothalamic growth hormone-releasing hormone content. These results reveal an unexpected and important role or DA in the control of developmental events in the pituitary gland and assign a critical role for hypothalamic DA reuptake in regulating these events.

Retinoic acid prevents experimental Cushing syndrome

Cushing syndrome is caused by an excess of adrenocorticotropic hormone (ACTH) production by neuroendocrine tumors, which subsequently results in chronic glucocorticoid excess. We found that retinoic acid inhibits the transcriptional activity of AP-1 and the orphan receptors Nur77 and Nurr1 in ACTH-secreting tumor cells. Retinoic acid treatment resulted in reduced pro-opiomelanocortin transcription and ACTH production. ACTH inhibition was also observed in human pituitary ACTH-secreting tumor cells and a small-cell lung cancer cell line, but not in normal cells. This correlated with the expression of the orphan receptor COUP-TFI, which was found in normal corticotrophs but not in pituitary Cushing tumors. COUP-TFI expression in ACTH-secreting tumor cells blocked retinoic acid action. Retinoic acid also inhibited cell proliferation and, after prolonged treatment, increased caspase-3 activity and induced cell death in ACTH-secreting cells. In adrenal cortex cells, retinoic acid inhibited corticosterone production and cell proliferation. The antiproliferative action and the inhibition of ACTH and corticosterone produced by retinoic acid were confirmed in vivo in experimental ACTH-secreting tumors in nude mice. Thus, we conclude that the effects of retinoic acid combine in vivo to reverse the endocrine alterations and symptoms observed in experimental Cushing syndrome.

Reciprocal regulation of dopamine D1 and D3 receptor function and trafficking by heterodimerization.

Colocalization of dopamine D1 (D1R) and D3 receptors (D3R) in specific neuronal populations suggests that their functional cross-talk might involve direct interactions. Here we report that the D1R coimmunoprecipitates with the D3R from striatal protein preparations, suggesting that they are clustered together in this region. Using bioluminescence resonance energy transfer (BRET2), we further suggest the existence of a physical interaction between D1R and D3R. Tagged D1R and D3R cotransfected in human embryonic kidney (HEK) 293 cells generated a significant BRET2 signal that was insensitive to agonist stimulation, suggesting that they form a constitutive heterodimer. D1R and D3R regulate adenylyl cyclase (AC) in opposite ways. In HEK 293 cells coexpressing D1R and D3R, dopamine stimulated AC with higher potency and displaced [3H]R-(+)-7-chloro-8-hydroxy-3-methyl-1-phenyl-2,3,4,5-tetrahydro-1H-3-benzazepine (SCH23390) binding with higher affinity than in cells expressing the D1R. In HEK 293 cells individually expressing D1R or D3R, agonist stimulation induces internalization of D1R but not of D3R. Heterodimerization with D3R abolishes agonist-induced D1R cytoplasmic sequestration induced by selective D1R agonists and enables internalization of the D1R/D3R complex in response to the paired stimulation of both D1R and D3R. This mechanism involves β-arrestin binding because it was blocked by mutant β-arrestinV53D. These data suggest that as a result of dimerization, the D3R is switched to the desensitization mechanisms typical of the D1R. These data give a novel insight into how D1R and D3R may function in an integrated way, providing a molecular mechanism by which to converge D1R- and D3R-related dysfunctions.

Dopamine Uptake is Differentially Regulated in Rat Striatum and Nucleus Accumbens

Abstract: Active uptake of 3,4‐dihydroxyphenylethylamine (dopamine) is sodium‐ and temperature‐dependent, strongly inhibited by benztropine and nomifensine, and present in corpus striatum and nucleus accumbens. In rat striatum dopamine uptake is related to a receptor that is specifically labelled by [3H]cocaine in the presence of Na+ and is located on dopaminergic terminals. The dopamine uptake is differentially affected in the two areas by single or repeated injections of cocaine. Cocaine inhibits dopamine uptake in slices of corpus striatum. Moreover Na+‐dependent [3H]cocaine binding is not detectable in nucleus accumbens. Nomifensine inhibits [3H]dopamine uptake by interacting with low‐ and high‐affinity sites in corpus striatum, but shows only low affinity for dopamine uptake in nucleus accumbens. The present data indicate that different mechanisms are involved in the regulation of dopamine uptake in corpus striatum and nucleus accumbens.

Afferent fibers mediate the increase of met-enkephalin elicited in rat spinal cord by localized pain

Abstract Met‐enkephalin levels were measured in various spinal cord regions of rats chronically suffering from the inflammation of a single paw following a treatment with Freunds adjuvant. The results indicate that chronic localized pain induces a selective increase of met‐enkephalin immunoreactive material (ME‐IR) in the dorsal horn of the spinal cord segment which receives a direct projection from the inflamed paw. In order to gain information on the functional meaning of these data, either the plexus brachialis or the sciatic nerve were sectioned peripherally before inducing inflammation. Denervation prevented the increase of ME‐IR concentration induced by the injection of Freunds adjuvant. Our observations suggest that chronic localized pain in a limb induces a change in ME‐IR content which is selective for the spinal cord segment receiving a direct projection from the inflamed paw. This increase depends on an intact innervation.

CaMKII-dependent Phosphorylation Regulates SAP97/NR2A Interaction

Synapse-associated protein 97 (SAP97), a member of membrane-associated guanylate kinase protein family, has been implicated in the processes of targeting ionotropic glutamate receptors at postsynaptic sites. Here we show that SAP97 is enriched at the postsynaptic density where it co-localizes with both ionotropic glutamate receptors and downstream signaling proteins such as Ca2+/calmodulin-dependent protein kinase II (CaMKII). SAP97 and αCaMKII display a high co-localization pattern in hippocampal neurons as well as in transfected COS-7 cells. Metabolic labeling of hippocampal cultures reveals that N-methyl-d-aspartic acid (NMDA) receptor activation induces CaMKII-dependent phosphorylation of SAP97; co-incubation with the CaMKII-specific inhibitor KN-93 reduces SAP97 phosphorylation to basal levels. Our results show that SAP97 directly interacts with the NR2A subunit of NMDA receptor both in an in vitro “pull-out” assay and in co-immunoprecipitation experiments from homogenates and synaptosomes purified from hippocampal rat tissue. Interestingly, in the postsynaptic density fraction, SAP97 fails to co-precipitate with NR2A. We show here that SAP97 is directly associated with NR2A through its PDZ1 domain, and CaMKII-dependent phosphorylation of SAP97-Ser-232 disrupts NR2A interaction both in an in vitro pull-out assay and in transfected COS-7 cells. Moreover, expression of SAP97(S232D) mutant has effects similar to those observed upon constitutively activating CaMKII. Our findings suggest that SAP97/NR2A interaction is regulated by CaMKII-dependent phosphorylation and provide a novel mechanism for the regulation of synaptic targeting of NMDA receptor subunits.

Induction of the unfolded protein response by α-synuclein in experimental models of Parkinson's disease.

J. Neurochem. (2011) 116, 588–605.

Opposite effects of dopamine D2 and D3 receptors on learning and memory in the rat.

Mesolimbocortical dopamine plays a role in learning and memory. The specific receptor subtypes mediating the effects of dopamine, however, are still unknown. Dopamine D2, D3 and D4 receptors are expressed in the hippocampus and dopamine D3 receptors are present in the septal area, suggesting that these receptor subtypes can contribute to the behavioral effects of dopamine D2-like receptor agonists. We now investigated the role of dopamine D2 and D3 receptors in learning and memory by using the transient amnesia induced by scopolamine in the passive avoidance test as experimental model. The data strongly suggest that both dopamine D2 and D3 receptors mediate the effects of dopamine on the integrative function of learning and memory. In particular, we show that the non-selective dopamine agonist apomorphine prevents the scopolamine-induced disruption of consolidation of the previously acquired passive avoidance behavior. This effect is mediated by receptors belonging to the dopamine D2 family since it was antagonized by (-)-sulpiride and mimicked by quinpirole. Nafadotride, a relatively selective antagonist for dopamine D3 receptors, antagonized scopolamine-induced memory disruption and potentiated the facilitatory effect of quinpirole. Taken together, these results suggest that the effects of dopamine on memory consolidation are the result of a balance between dopamine D2 receptor-mediated facilitation and dopamine D3 receptor-mediated inhibition, and that dopamine D2 and D3 receptors play opposite roles in the control of the mechanisms leading to memory consolidation.

D2 dopamine receptors associated with inhibition of dopamine release from rat neostriatum are independent of cyclic AMP

The ability of the selective D2 dopamine (DA) receptor agonist bromocriptine to inhibit potassium-induced DA release from striatal slices was measured in rats, which had been unilaterally injected with kainic acid into the left striatum, with the aim of verifying whether the central nervous system contains DA receptors whose stimulation evokes intracellular events which do not involve cyclic AMP. It was found that increasing concentrations of bromocriptine inhibited the potassium-stimulated DA release from rat striatal slices of the kainic acid-treated side with the same potency as in control slices. On the contrary, bromocriptine and the selective D1 agonist SKF 82526 completely lost the ability to inhibit or stimulate, respectively, striatal adenylate cyclase activity from the lesioned side. Our conclusion asserts that inhibition of DA release from rat striatal slices is mediated by stimulation of D2 DA receptors which are fully operative in absence of both DA-stimulated and DA-inhibited adenylate cyclase activity. These data suggest that the intracellular events that follow D2 receptor stimulation in the nigrostriatal nerve terminals may be regulated by second messengers other than cyclic AMP.