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Luciferase complementation assays have emerged as a simple means of monitoring receptor-effector interactions in living cells in a time-resolved manner. Here, we describe a nanoluciferase complementation assay capable of reporting on β-arrestin2 recruitment to the human dopamine D₃ receptor (D₃R) upon its activation in intact HEK293T cells. Using this assay in time-resolved experiments, we detect differences in arrestin response termination rates between the endogenous agonist dopamine and the synthetic D3R agonist FAUC-73. We also investigate the influence of exogenous GRK2 on β-arrestin2 recruitment to the D₃R. We find that, in contrast to the D₂R and D₄R, the potency of dopamine to induce arrestin recruitment to D₃R is not significantly influenced by GRK2 overexpression. In further agreement with a lack of GRK2 regulation of D₃R signalling and again contrary to the D₂R and D₄R, we do not observe dopamine-induced recruitment of GRK2 to D₃R. Conversely, dopamine concentration-dependently decreases the interaction between GRK2 and D₃R. Additionally, we examine both the Ser-9 and Gly-9 variants of the human D₃R, which, according to some earlier reports, differ in terms of dopamine affinity and functional potency. However, we find no difference in the concentration-response relationships between these two variants, neither when arrestin recruitment nor GRK2 interactions are studied. In summary, the present report demonstrates the utility of nanoluciferase complementation for studying D₃R pharmacology in living cells.

The dopamine D1 receptor (D1R) is prominently expressed in the striatum and cerebral cortex and is an attractive target for treating Parkinson’s disease and cognitive impairment in schizophrenia. While newer, noncatechol D1R agonists such as tavapadon have shown promise in recent clinical trials, the therapeutic utility of earlier catechol agonists such as A77636 was hampered by tolerance development. The mechanism underlying tolerance induction was suggested to involve very slow A77636 dissociation from the D1R, promoting prominent arrestin recruitment and receptor internalization associated with delayed recycling to the cell surface. Here, we compared the signaling and binding kinetics of five D1R agonists─dopamine, dihydrexidine, apomorphine, A77636, and tavapadon─using two time-resolved assays of agonist-induced β-arrestin2 recruitment and G protein-coupled inward rectifier potassium (GIRK, also known as Kir3) channel activation, respectively. Additionally, D1R internalization was studied using cell-surface ELISA. Tavapadon and apomorphine did not induce significant D1R internalization, whereas pronounced internalization was observed with A77636, dopamine, and dihydrexidine. GIRK response deactivation time courses upon agonist washout were longer for A77636 and tavapadon compared to dopamine, dihydrexidine, and apomorphine. Similarly, in the β-arrestin2 assay, signal decay upon antagonist addition was slower for A77636 and tavapadon compared to the other three agonists. Tavapadon and apomorphine were partial agonists in both assays, whereas A77636 and dihydrexidine showed efficacies similar to dopamine. While our results do not provide evidence for a direct correlation between agonist dissociation and liability to tolerance induction, the possibility remains that certain combinations of agonist characteristics, such as high efficacy paired with slow dissociation, are associated with tolerance induction by D1R agonists.

ONC206 is a small molecule used in trials for treating diffuse midline glioma. The reported mechanisms of action include mitochondrial Clp protease activation and dopamine D2 receptor (D2R) antagonism. However, there is limited data in the literature on D2R engagement. Using a time-resolved functional assay, we assessed ONC206 interactions with D2R and observed low-potency antagonism, which was rapidly displaced in competition with dopamine. In vivo plasma concentrations of ONC206 support engagement of D2R and highlight this mechanism as a putative contributor to the cytotoxic effect.

Psychosis in Parkinson's disease is a common phenomenon associated with poor outcomes. To clarify the pathophysiology of this condition and the mechanisms of antipsychotic treatments, we have here characterized the neurophysiological brain states induced by clozapine, pimavanserin, and the novel prospective antipsychotic mesdopetam in a rodent model of Parkinson's disease psychosis, based on chronic dopaminergic denervation by 6-OHDA lesions, levodopa priming, and the acute administration of an NMDA antagonist. Parallel recordings of local field potentials from eleven cortical and sub-cortical regions revealed shared neurophysiological treatment effects for the three compounds, despite their different pharmacological profiles, involving reversal of features associated with the psychotomimetic state, such as a reduction of aberrant high-frequency oscillations in prefrontal structures together with a decrease of abnormal synchronization between different brain regions. Other drug-induced neurophysiological features were more specific to each treatment, affecting network oscillation frequencies and entropy, pointing to discrete differences in mechanisms of action. These findings indicate that neurophysiological characterization of brain states is particularly informative when evaluating therapeutic mechanisms in conditions involving symptoms that are difficult to assess in rodents such as psychosis, and that mesdopetam should be further explored as a potential novel antipsychotic treatment option for Parkinson psychosis.

A five-year-old girl presented with headache attacks, clumsiness, and a history of transient gait disturbances. She and her father, mother, twin sister, and brother underwent neurological evaluation, neuroimaging, and exome sequencing covering 357 genes associated with movement disorders. Sequencing revealed the new variant KCND3 c.838G>A, p.E280K in the father and sisters, but not in the mother and brother. KCND3 encodes voltage-gated potassium channel D3 (Kv4.3) and mutations have been associated with spinocerebellar ataxia type 19/22 (SCA19/22) and cardiac arrhythmias. SCA19/22 is characterized by ataxia, Parkinsonism, peripheral neuropathy, and sometimes, intellectual disability. Neuroimaging, EEG, and ECG were unremarkable. Mild developmental delay with impaired fluid reasoning was observed in both sisters, but not in the brother. None of the family members demonstrated ataxia or parkinsonism. In Xenopus oocyte electrophysiology experiments, E280K was associated with a rightward shift in the Kv4.3 voltage-activation relationship of 11 mV for WT/E280K and +17 mV for E280K/E280K relative to WT/WT. Steady-state inactivation was similarly right-shifted. Maximal peak current amplitudes were similar for WT/WT, WT/E280K, and E280K/E280K. Our data indicate that Kv4.3 E280K affects channel activation and inactivation and is associated with developmental delay. However, E280K appears to be relatively benign considering it does not result in overt ataxia.

The dopamine D4 receptor (D4R) is expressed in the retina, prefrontal cortex, and autonomic nervous system and has been implicated in attention deficit hyperactivity disorder (ADHD), substance use disorders, and erectile dysfunction. D4R has also been investigated as a target for antipsychotics due to its high affinity for clozapine. As opposed to the closely related dopamine D2 receptor (D2R), dopamine-induced arrestin recruitment and desensitization at the D4R have not been studied in detail. Indeed, some earlier investigations could not detect arrestin recruitment and desensitization of this receptor upon its activation by agonist. Here, we used a novel nanoluciferase complementation assay to study dopamine-induced recruitment of β-arrestin2 (βarr2; also known as arrestin3) and G protein-coupled receptor kinase-2 (GRK2) to the D4R in HEK293T cells. We also studied desensitization of D4R-evoked G protein-coupled inward rectifier potassium (GIRK; also known as Kir3) current responses in Xenopus oocytes. Furthermore, the effect of coexpression of GRK2 on βarr2 recruitment and GIRK response desensitization was examined. The results suggest that coexpression of GRK2 enhanced the potency of dopamine to induce βarr2 recruitment to the D4R and accelerated the rate of desensitization of D4R-evoked GIRK responses. The present study reveals new details about the regulation of arrestin recruitment to the D4R and thus increases our understanding of the signaling and desensitization of this receptor.

BACKGROUND: Trace amine-associated receptor-1 (TAAR1) agonists have been proposed as potential antipsychotics, with ulotaront and ralmitaront having reached clinical trials. While ulotaront demonstrated efficacy in a recent Phase II trial, a corresponding study studies of ralmitaront failed to show efficacy as a monotherapy or as an adjunct to atypical antipsychotics. In addition to TAAR1 agonism, ulotaront is a partial agonist at the serotonin 1A receptor (5-HT1AR). However, little is known about ralmitaront.

METHODS: We compared ulotaront and ralmitaront at TAAR1, 5-HT1AR, and dopamine D2 using luciferase complementation-based G protein recruitment, cAMP accumulation, and G protein-coupled inward rectifier potassium channel activation assays.

RESULTS: Ralmitaront showed lower efficacy at TAAR1 in G protein recruitment, cAMP accumulation, and GIRK activation assays. Moreover, ralmitaront lacked detectable activity at 5-HT1AR and dopamine D2.

CONCLUSIONS: Compared with ulotaront, ralmitaront shows lower efficacy and slower kinetics at TAAR1 and lacks efficacy at 5-HT1AR. These data may be relevant to understanding differences in clinical profiles of these 2 compounds.

Dupuytren's disease is characterized by fingers becoming permanently bent in a flexed position. Whereas people of African ancestry are rarely afflicted by Dupuytren's disease, up to ∼30% of men over 60 years suffer from this condition in northern Europe. Here, we meta-analyze 3 biobanks comprising 7,871 cases and 645,880 controls and find 61 genome-wide significant variants associated with Dupuytren's disease. We show that 3 of the 61 loci harbor alleles of Neandertal origin, including the second and third most strongly associated ones (P = 6.4 × 10^-132 and P = 9.2 × 10^-69, respectively). For the most strongly associated Neandertal variant, we identify EPDR1 as the causal gene. Dupuytren's disease is an example of how admixture with Neandertals has shaped regional differences in disease prevalence.

Background and Purpose: Opioid-based drugs are the gold standard medicines for pain relief. However, tolerance and several side effects (i.e. constipation and dependence) may occur upon chronic opioid administration. Photopharmacology is a promising approach to improve the benefit/risk profiles of these drugs. Thus, opioids can be locally activated with high spatiotemporal resolution, potentially minimizing systemic-mediated adverse effects. Here, we aimed at developing a morphine photo-derivative (photocaged morphine), which can be activated upon light irradiation both in vitro and in vivo.

Experimental Approach: Light-dependent activity of pc-morphine was assessed in cell-based assays (intracellular calcium accumulation and electrophysiology) and in mice (formalin animal model of pain). In addition, tolerance, constipation and dependence were investigated in vivo using experimental paradigms.

Key results: In mice, pc-morphine was able to elicit antinociceptive effects, both using external light-irradiation (hind paw) and spinal cord implanted fibre-optics. In addition, remote morphine photoactivation was devoid of common systemic opioid-related undesired effects, namely, constipation, tolerance to the analgesic effects, rewarding effects and naloxone-induced withdrawal.

Conclusion and Implications: Light-dependent opioid-based drugs may allow effective analgesia without the occurrence of tolerance or the associated and severe opioid-related undesired effects.