Sehyoun Yoon

Enhanced Hypothalamic Leptin Signaling in Mice Lacking Dopamine D2 Receptors

The dopamine D2 receptor (D2R) plays a critical role in diverse neurophysiological functions. D2R knock-out mice (D2R−/−) show reduced food intake and body weight while displaying an increased basal energy expenditure level, compared with their wild type littermates. Thus, these mice show a lean phenotype. D2R−/− mice displayed increased leptin sensitivity, and leptin injection induced increased phosphorylation of the hypothalamic signal transducer and activator of transcription 3 (STAT3) in D2R−/− mice relative to wild type littermates. Using double immunofluorescence histochemistry, we have demonstrated that D2Rs are present in leptin-sensitive STAT3-positive cells in the arcuate nucleus of the hypothalamus and that leptin injection induces STAT3 phosphorylation in hypothalamic neurons expressing D2Rs. Stimulation of D2R by the D2R agonist quinpirole suppressed the leptin-induced STAT3 phosphorylation and nuclear trans-localization of phospho-STAT3 in the hypothalamus of wild type mice. However, this regulation was not detected in the D2R−/− mice. Treatment of D2R agonist and antagonist could modulate the leptin-induced food intake and body weight changes in wild type mice but not in D2R−/− mice. Together, our findings suggest that the interaction between the dopaminergic system and leptin signaling in hypothalamus is important in control of energy homeostasis.

Role of dopamine D2 receptors in plasticity of stress-induced addictive behaviours

Dopaminergic systems are implicated in stress-related behaviour. Here we investigate behavioural responses to chronic stress in dopamine D2 receptor knockout mice and find that anxiety-like behaviours are increased compared with wild-type mice. Repeated stress exposure suppresses cocaine-induced behavioural sensitization, cocaine-seeking and relapse behaviours in dopamine D2 receptor knockout mice. Cocaine challenge after drug withdrawal in cocaine-experienced wild-type or dopamine D2 receptor knockout mice is associated with inhibition of long-term depression in the nucleus accumbens, and chronic stress during withdrawal prevents inhibition after cocaine challenge in cocaine-experienced dopamine D2 receptor knockout mice, but not in wild-type mice. Lentiviral-induced knockdown of dopamine D2 receptors in the nucleus accumbens of wild-type mice does not affect basal locomotor activity, but confers stress-induced inhibition of the expression of cocaine-induced behavioural sensitization. Stressed mice depleted of dopamine D2 receptors do not manifest long-term depression inhibition. Our results suggest that dopamine D2 receptors have roles in regulating synaptic modification triggered by stress and drug addiction.

Wnt5a-dopamine D2 receptor interactions regulate dopamine neuron development via extracellular signal-regulated kinase (ERK) activation.

The dopamine D2 receptor (D2R) plays an important role in mesencephalic dopaminergic neuronal development, particularly coupled with extracellular signal-regulated kinase (ERK) activation. Wnt5a protein is known to regulate the development of dopaminergic neurons. We analyzed the effect of Wnt5a on dopaminergic neuron development in mesencephalic primary cultures from wild-type (WT) and D2R knock-out (D2R−/−) mice. Treatment with Wnt5a increased the number and neuritic length of dopamine neurons in primary mesencephalic neuronal cultures from WT mice, but not from D2R−/− mice. The effect of Wnt5a was completely blocked by treatment with D2R antagonist or inhibitors of MAPK or EGFR. Wnt5a-mediated ERK activation in mesencephalic neuronal cultures was inhibited by treatment of D2R antagonist and EGFR inhibitors in WT mice. However, these regulations were not observed for D2R−/− mice. Co-immunoprecipitation and displacement of [3H]spiperone from D2R by Wnt5a demonstrated that Wnt5a could bind with D2R. This interaction was confirmed by GST pulldown assays demonstrating that the domain including transmembrane domain 4, second extracellular loop, and transmembrane domain 5 of D2R binds to Wnt5a. These results suggest that the interaction between D2R and Wnt5a has an important role in dopamine neuron development in association with EGFR and the ERK pathway.

ZNF313 is a novel cell cycle activator with an E3 ligase activity inhibiting cellular senescence by destabilizing p21WAF1

ZNF313 encoding a zinc-binding protein is located at chromosome 20q13.13, which exhibits a frequent genomic amplification in multiple human cancers. However, the biological function of ZNF313 remains largely undefined. Here we report that ZNF313 is an ubiquitin E3 ligase that has a critical role in the regulation of cell cycle progression, differentiation and senescence. In this study, ZNF313 is initially identified as a XIAP-associated factor 1 (XAF1)-interacting protein, which upregulates the stability and proapoptotic effect of XAF1. Intriguingly, we found that ZNF313 activates cell cycle progression and suppresses cellular senescence through the RING domain-mediated degradation of p21WAF1. ZNF313 ubiquitinates p21WAF1 and also destabilizes p27KIP1 and p57KIP2, three members of the CDK-interacting protein (CIP)/kinase inhibitor protein (KIP) family of cyclin-dependent kinase inhibitors, whereas it does not affect the stability of the inhibitor of CDK (INK4) family members, such as p16INK4A and p15INK4B. ZNF313 expression is tightly controlled during the cell cycle and its elevation at the late G1 phase is crucial for the G1-to-S phase transition. ZNF313 is induced by mitogenic growth factors and its blockade profoundly delays cell cycle progression and accelerates p21WAF1-mediated senescence. Both replicative and stress-induced senescence are accompanied with ZNF313 reduction. ZNF313 is downregulated during cellular differentiation process in vitro and in vivo, while it is commonly upregulated in many types of cancer cells. ZNF313 shows both the nuclear and cytoplasmic localization in epithelial cells of normal tissues, but exhibits an intense cytoplasmic distribution in carcinoma cells of tumor tissues. Collectively, ZNF313 is a novel E3 ligase for p21WAF1, whose alteration might be implicated in the pathogenesis of several human diseases, including cancers.

Dopamine D2 Receptor-mediated Epidermal Growth Factor Receptor Transactivation through a Disintegrin and Metalloprotease Regulates Dopaminergic Neuron Development via Extracellular Signal-related Kinase Activation

Background: Dopamine D2R-mediated ERK activation regulates dopaminergic neuronal development. Results: D2R activation induces shedding of heparin-binding EGF by activating a disintegrin and metalloproteinase (ADAM) 10 or 17, causing EGFR transactivation in mesencephalic neurons. Conclusion: D2R-mediated ERK activation regulates mesencephalic dopaminergic neuron development via EGFR transactivation through ADAM10/17. Significance: Dopaminergic system alteration through D2R-ADAM-EGFR signaling maybe associated with dopamine-related neurological/psychiatric disorders. Dopamine D2 receptor (D2R)-mediated extracellular signal-regulated kinase (ERK) activation plays an important role in the development of dopaminergic mesencephalic neurons. Here, we demonstrate that D2R induces the shedding of heparin-binding epidermal growth factor (EGF) through the activation of a disintegrin and metalloprotease (ADAM) 10 or 17, leading to EGF receptor transactivation, downstream ERK activation, and ultimately an increase in the number of dopaminergic neurons and their neurite length in primary mesencephalic cultures from wild-type mice. These outcomes, however, were not observed in cultures from D2R knock-out mice. Our findings show that D2R-mediated ERK activation regulates mesencephalic dopaminergic neuron development via EGF receptor transactivation through ADAM10/17.

Striatal-enriched protein tyrosine phosphatase regulates dopaminergic neuronal development via extracellular signal-regulated kinase signaling

The striatal-enriched protein tyrosine phosphatase (STEP) is highly expressed within dopaminoceptive neurons, suggesting the possibility that STEP may interact with dopaminergic signaling. We have previously shown that signaling through dopamine D2 receptor (D2R)-mediated extracellular signal-regulated kinase (ERK) activation plays a critical role in mesencephalic dopaminergic neuronal development. Here, we investigate the role of STEP in D2R-mediated ERK signaling, especially in dopaminergic neuronal development. Analyses of developmental expression of STEP and tyrosine hydroxylase (TH) in mouse brain demonstrate that STEP- and TH-positive cells are co-localized in the substantia nigra compacta of brains of postnatal 8-day-old mice, displaying STEP expression in dopaminergic neurons at this stage. Stereological analysis demonstrates a dynamic change in the number of STEP-expressing cells from midbrain to striatum during development in WT mice and significantly decreased number of STEP-expressing cells in mice lacking D2R (D2R(-/-) mice). The knockdown of STEP expression by treatment with oligomeric STEP siRNA significantly decreased the number of mesencephalic TH cells and inhibited D2R-mediated development of dopaminergic neurons on primary mesencephalic culture from WT mice, but not in primary cultures from D2R(-/-) mice. Furthermore, knockdown of STEP expression perturbed D2R-mediated ERK signaling in dopaminergic neuronal cells from WT mice, but not from D2R(-/-) mice. These results suggest that STEP is an important mediator in the dopamine D2R-mediated activation of ERK signaling and in the regulation of dopaminergic neuronal development.

Optogenetics reveals a role for accumbal medium spiny neurons expressing dopamine D2 receptors in cocaine-induced behavioral sensitization

Long-lasting, drug-induced adaptations within the nucleus accumbens (NAc) have been proposed to contribute to drug-mediated addictive behaviors. Here we have used an optogenetic approach to examine the role of NAc medium spiny neurons (MSNs) expressing dopamine D2 receptors (D2Rs) in cocaine-induced behavioral sensitization. Adeno-associated viral vectors encoding channelrhodopsin-2 (ChR2) were delivered into the NAc of D2R-Cre transgenic mice. This allowed us to selectively photostimulate D2R-MSNs in NAc. D2R-MSNs form local inhibitory circuits, because photostimulation of D2R-MSN evoked inhibitory postsynaptic currents (IPSCs) in neighboring MSNs. Photostimulation of NAc D2R-MSN in vivo affected neither the initiation nor the expression of cocaine-induced behavioral sensitization. However, photostimulation during the drug withdrawal period attenuated expression of cocaine-induced behavioral sensitization. These results show that D2R-MSNs of NAc play a key role in withdrawal-induced plasticity and may contribute to relapse after cessation of drug abuse.

Regulation of dopamine D2 receptor-mediated extracellular signal-regulated kinase signaling and spine formation by GABAA receptors in hippocampal neurons.

Dopamine (DA) signaling via DA receptors is known to control hippocampal activity that contributes to learning, memory, and synaptic plasticity. In primary hippocampal neuronal culture, we observed that dopamine D2 receptors (D2R) co-localized with certain subtypes of GABAA receptors, namely α1, β3, and γ2 subunits, as revealed by double immunofluorocytochemical analysis. Treatment with the D2R agonist, quinpirole, was shown to elicit an increase in phosphorylation of extracellular signal-regulated kinase (ERK) in hippocampal neurons. This phosphorylation was inhibited by pretreatment with the GABAA receptor agonist, muscimol. Furthermore, treatment of hippocampal neurons with quinpirole increased the dendritic spine density and this regulation was totally blocked by pretreatment with a MAP kinase kinase (MEK) inhibitor (PD98059), D2R antagonist (haloperidol), or by the GABAA receptor agonist, muscimol. These results suggest that D2R-mediated ERK phosphorylation can control spine formation and that the GABAA receptor negatively regulates the D2R-induced spine formation through ERK signaling in hippocampal neurons, thus indicating a potential role of D2R in the control of hippocampal neuronal excitability.

Effects of atypical antipsychotic drugs on body weight and food intake in dopamine D2 receptor knockout mice

Many atypical antipsychotic drugs cause weight gain, but the mechanism of this weight gain is unclear. To dissect the role of the dopamine D2 receptor (D2R), an important receptor in the pharmacology of antipsychotic drugs, we analyzed the effect of olanzapine, risperidone, and ziprasidone on changes in body weight and food intake in male wild-type (WT) and D2R knockout (D2R(-/-)) mice. The oral delivery of atypical antipsychotics, olanzapine (5 and 10mg/kg), risperidone (0.1 and 1.0mg/kg) and ziprasidone (10 and 20mg/kg) in both strains mice for 2 weeks suppressed body weight gain, except for olanzapine treatment in D2R(-/-) mice. Olanzapine treatment suppressed body weight gain and decreased food intake in WT mice, but also reduced fat body mass and locomotor activity, whereas D2R(-/-) mice did not show these changes. Ziprasidone and risperidone treatment produced similar responses in WT and D2R(-/-) mice. These data suggest the involvement of D2R in the effect of olanzapine on metabolic regulation. Further studies are required to explore the implications of D2R activity in antipsychotic-mediated metabolic complications.

Role of Dopamine D2 Receptor in Stress-Induced Myelin Loss

Dopaminergic systems play a major role in reward-related behavior and dysregulation of dopamine (DA) systems can cause several mental disorders, including depression. We previously reported that dopamine D2 receptor knockout (D2R−/−) mice display increased anxiety and depression-like behaviors upon chronic stress. Here, we observed that chronic stress caused myelin loss in wild-type (WT) mice, while the myelin level in D2R−/− mice, which was already lower than that in WT mice, was not affected upon stress. Fewer mature oligodendrocytes (OLs) were observed in the corpus callosum of stressed WT mice, while in D2R−/− mice, both the control and stressed group displayed a decrease in the number of mature OLs. We observed a decrease in the number of active β-catenin (ABC)-expressing and TCF4-expressing cells among OL lineage cells in the corpus callosum of stressed WT mice, while such regulation was not found in D2R−/− mice. Administration of lithium normalized the behavioral impairments and myelin damage induced by chronic stress in WT mice, and restored the number of ABC-positive and TCF4-positive OLs, while such effect was not found in D2R−/− mice. Together, our findings indicate that chronic stress induces myelin loss through the Wnt/β-catenin signaling pathway in association with DA signaling through D2R.