Dysfunctions of dopaminergic homeostasis leading to either low or high dopamine (DA) levels are causally linked to Parkinson's disease, schizophrenia, and addiction. Major sites of DA synthesis are the mesencephalic neurons originating in the substantia nigra and ventral tegmental area; these structures send major projections to the dorsal striatum (DSt) and nucleus accumbens (NAcc), respectively. DA finely tunes its own synthesis and release by activating DA D2 receptors (D2R). To date, this critical D2R-dependent function was thought to be solely due to activation of D2Rs on dopaminergic neurons (D2 autoreceptors); instead, using site-specific D2R knock-out mice, we uncover that D2 heteroreceptors located on non-DAergic medium spiny neurons participate in the control of DA levels. This D2 heteroreceptor-mediated mechanism is more efficient in the DSt than in NAcc, indicating that D2R signaling differentially regulates mesolimbic-versus nigrostriatal-mediated functions. This study reveals previously unappreciated control of DA signaling, shedding new light on region-specific regulation of DA-mediated effects.

SummaryDopamine (DA) signaling controls many physiological functions ranging from locomotion to hormone secretion, and plays a critical role in addiction. DA elevation, for instance in the response to drugs of abuse, simultaneously activates neurons expressing different DA receptors; how responses from diverse neurons/receptors are orchestrated in the generation of behavioral and cellular outcomes, is still not completely defined. Signaling from DA D2 receptors (D2Rs) is a good example to illustrate this complexity. D2Rs have presynaptic and postsynaptic localization and functions, which are shared by two isoforms in vivo. Recent results from knockout mice are clarifying the role of site and D2 isoform-specific effects thereby increasing our understanding of how DA modulates neuronal physiology.

REV-ERBα and REV-ERBβ nuclear receptors regulate several physiological processes, including circadian rhythm and metabolism. A previous study reported the REV-ERBα gene to be co-overexpressed with ERBB2 in breast cancer cell lines. Surprisingly, we found that several tumor types, including a number of breast cancer cell lines, predominantly express the REV-ERBβ variant. This pattern was independent of ERBB2 and ER status, and opposite to that of non-cancer mammary epithelial HMEC cells, in which REV-ERBα was the major variant. Consistent with this molecular profile, REV-ERB target genes in both circadian and metabolic pathways were derepressed upon silencing of REV-ERBβ, but not REV-ERBα. Strikingly, we found that REV-ERBβ is a determinant of sensitivity to chloroquine, a clinically relevant lysosomotropic agent that suppresses autophagy. The cytoprotective function of REV-ERBβ appears to operate downstream of autophagy blockade. Through compound screening, we identified ARN5187, a novel lysosomotropic REV-ERBβ ligand with a dual inhibitory activity toward REV-ERB-mediated transcriptional regulation and autophagy. Remarkably, although ARN5187 and chloroquine share similar lysosomotropic potency and have a similar effect on autophagy inhibition, ARN5187 is significantly more cytotoxic. Collectively, our results reveal that dual inhibition of REV-ERBβ and autophagy is an effective strategy for eliciting cytotoxicity in cancer cells. Furthermore, our discovery of a novel inhibitor compound of both REV-ERB and autophagy may provide a scaffold for the discovery of new multifunctional anticancer agents.

N-acylethanolamine acid amidase (NAAA), a cysteine hydrolase highly expressed in macrophages and B lymphocytes, catalyzes the degradation of palmitoylethanolamide. Palmitoylethanolamide is an agonist of PPAR-α and an important regulator of pain and innate immunity. In this study, we investigated the properties of the NAAA inhibitor, ARN077, in a mouse model of allergic contact dermatitis. Acute topical applications of ARN077 attenuated key signs of DNFB-induced dermatitis in a dose-dependent manner. Moreover, ARN077 increased tissue palmitoylethanolamide content and normalized circulating levels of cytokines and immunoglobulin E. No such effect was seen in PPAR-α-deficient mice. Moreover, mice lacking NAAA failed to develop edema or scratching behavior after challenge with DNFB, confirming that this enzyme plays an important role in dermatitis. Consistent with this conclusion, subchronic applications of ARN077 suppressed DNFB-induced inflammation when administered either before or after the DNFB challenge. The effects of subchronic ARN077 were dose dependent and comparable in size to those produced by the steroids clobetasol and dexamethasone. Unlike the latter, however, ARN077 did not cause skin atrophy. The results identify NAAA as a promising target for the development of effective and safe treatments for atopic dermatitis and other inflammatory disorders of the skin.

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Dopamine acting through D2 receptors (D2Rs) controls lactotroph proliferation and prolactin (PRL) levels. Ablation of this receptor in mice results in lactotroph hyperplasia and prolactinomas in aged females. Alternative splicing of the Drd2 gene generates 2 independent isoforms, a long (D2L) and a short (D2S) isoform, which are present in all D2R-expressing cells. Here, we addressed the role of D2L and D2S on lactotroph physiology through the generation and analysis of D2S-null mice and their comparison with D2L-null animals. These mice represent a valuable tool with which to investigate dopamine-dependent isoform-specific signaling in the pituitary gland. We sought to assess the existence of a more prominent role of D2L or D2S in controlling PRL expression and lactotroph hyperplasia. Importantly, we found that D2L and D2S are specifically linked to independent transduction pathways in the pituitary. D2L-mediated signaling inhibits the AKT/protein kinase B kinase activity whereas D2S, in contrast, is required for the activation of the ERK 1/2 pathway. Under normal conditions, presence of only 1 of the 2 D2R isoforms in vivo prevents hyperprolactinemia, formation of lactotroph's hyperplasia, and tumorigenesis that is observed when both isoforms are deleted as in D2R-/- mice. However, the protective function of the single D2R isoforms is overridden when single isoform-knockout mice are challenged by chronic estrogen treatments as they show increased PRL production and lactotroph hyperplasia. Our study indicates that signaling from each of the D2R isoforms is sufficient to maintain lactotroph homeostasis in physiologic conditions; however, signaling from both is necessary in conditions simulating pathologic states.

Autophagy inhibition is emerging as a promising anticancer strategy. We recently reported that the circadian nuclear receptor REV-ERBβ plays an unexpected role in sustaining cancer cell survival when the autophagy flux is compromised. We also identified 4-[[[1-(2-fluorophenyl)cyclopentyl]amino]methyl]-2-[(4-methylpiperazin-1-yl)methyl]phenol, 1 (ARN5187), as a novel dual inhibitor of REV-ERBβ and autophagy. 1 had improved cytotoxicity against BT-474 breast cancer cells compared to chloroquine, a clinically relevant autophagy inhibitor. Here, we present the results of structure-activity studies, based around 1, that disclose the first class of dual inhibitors of REV-ERBβ and autophagy. This study led to identification of 18 and 28, which were more effective REV-ERBβ antagonists than 1 and were more cytotoxic to BT-474. The combination of optimal chemical and structural moieties of these analogs generated 30, which elicited 15-fold greater REV-ERBβ inhibitory and cytotoxic activities compared to 1. Furthermore, 30 induced death in a panel of tumor cell lines at doses 5-50 times lower than an equitoxic amount of chloroquine but did not affect the viability of normal mammary epithelial cells.

Oligodendrocytes are the glial cells responsible for myelin formation. Myelination occurs during the first postnatal weeks and, in rodents, is completed during the third week after birth. Myelin ensures the fast conduction of the nerve impulse; in the adult, myelin proteins have an inhibitory role on axon growth and regeneration after injury. During brain development, oligodendrocytes precursors originating in multiple locations along the antero-posterior axis actively proliferate and migrate to colonize the whole brain. Whether the initial interactions between oligodendrocytes and neurons might play a functional role before the onset of myelination is still not completely elucidated. In this article, we addressed this question by transgenically targeted ablation of proliferating oligodendrocytes during cerebellum development. Interestingly, we show that depletion of oligodendrocytes at postnatal day 1 (P1) profoundly affects the establishment of cerebellar circuitries. We observed an impressive deregulation in the expression of molecules involved in axon growth, guidance and synaptic plasticity. These effects were accompanied by an outstanding increase of neurofilament staining observed 4 hours after the beginning of the ablation protocol, likely dependent from sprouting of cerebellar fibers. Oligodendrocyte ablation modifies localization and function of ionotropic glutamate receptors in Purkinje neurons. These results show a novel oligodendrocyte function expressed during early postnatal brain development, where these cells participate in the formation of cerebellar circuitries, and influence its development.