Dopamine D4 receptor (D4R) signaling affects decision-making, memory formation, cognition, and attention. Previously developed D4R-selective ligands were metabolically unstable in vivo due to amide bond linker hydrolysis. In this study, analog compounds were synthesized using click chemistry, bioisosterically replacing amides with a 1,2,3-triazole linker. Herein, we report 1,2,3-triazole analogs maintained high D4R affinity and subtype selectivity but had slightly reduced functional efficacy in cAMP and β-arrestin recruitment assays. Using rat and human liver microsomes to evaluate phase I metabolism, we determined that amide ligands were more metabolically unstable in rat microsomes, and the triazole substitutions enhanced compound stability. Four compounds were evaluated in rat pharmacokinetics studies. In particular, 17 (antagonist) and 18 (low-efficacy partial agonist) had desirable results in plasma half-life and brain exposure measures. These new analogs are suitable for behavioral studies in rats and represent improved molecular tools to further explore D4R signaling in rodent models.

The D1 dopamine receptor (D1R) ligand BMS Compound A is a positive allosteric modulator (PAM) of D1R signaling. BMS Compound A binds specifically to one of three known allosteric sites on the D1R and is a useful chemical tool for the investigation of D1R pharmacology. Despite the utility of BMS Compound A, even small quantities of the molecule are not commercially available. Here we report two variations on a convergent synthetic approach to construct this tool molecule. The first route is a four-step sequence to afford the four stereoisomers of BMS Compound A. The second route leverages a diastereoselective cyclopropanation on a chiral ketal substrate. Carrying this single enantiomer intermediate through our previous convergent reaction sequence affords two separable diastereomers of BMS Compound A-each a single enantiomer. We characterized the four stereoisomers of BMS Compound A, assigned their absolute configuration, specific rotation, 13C nuclear magnetic resonance (NMR) spectra. We further evaluated their ability to activate D2R signaling or potentiate dopamine-mediated D1R signaling. We found the greatest difference in D1R PAM activity for syn vs. anti configurations and less drastic differences between enantiomers. We also determined that the four stereoisomers exhibited agonist activity at the D2R.

Allosteric modulation of G protein-coupled receptors (GPCRs) is an emerging therapeutic paradigm that has proven effective, yet the cooperative action of multiple modulators remains unexplored. Here, we reveal how positive allosteric modulators (PAMs) synergistically enable extraordinary signal amplification through the D 1 dopamine receptor (D1R). We developed UNC9815 and UNC10062 as enhanced D1R PAMs from the parent compound MLS6585, and then employed cryo-electron microscopy to reveal concurrent occupancy of three distinct allosteric sites by PAMs of different scaffolds, including LY3154207, BMS-A1, and our UNC compounds. Remarkably, we discovered two adjacent allosteric pockets at the transmembrane helix (TM) 1-7 interface: BMS-A1 occupies an intracellular site promoting activation through TM7 conformational shifts, while the UNC compounds complementarily stabilize the extracellular side of the interface. When combined with LY3154207, this cooperative architecture enhances dopamine potency by more than 1,000-fold. These findings elucidate the first structural blueprint for multi-site GPCR cooperativity, unlocking transformative therapeutic strategies inaccessible to orthosteric and single- site allosteric drugs.

To identify novel D3 dopamine receptor (D3R)-selective antagonist scaffolds, we conducted a high-throughput screen of a small-molecule library using a β-arrestin recruitment assay. The lead hit compound, MLS6357, displayed unprecedented D3R selectivity as well as unusual positive allosteric modulator (PAM)-antagonist activity, which may confer unique therapeutic advantages to this scaffold. Iterative medicinal chemistry was used to synthesize and characterize 137 analogues, with several demonstrating both high D3R selectivity and improved D3R potency in β-arrestin recruitment and G protein activation assays. Two of the more promising analogues with 10-fold or greater improvements in potency, 6a and 10aa, were further characterized and found to recapitulate both the allosteric PAM-antagonism and global D3R selectivity of MLS6357. 6a and 10aa also demonstrated favorable pharmacokinetics in mice suggesting that these compounds may serve as both research tools and therapeutic leads for the treatment of neuropsychiatric disorders, including substance use disorder.

Aberrant dopamine (DA) signaling is implicated in schizophrenia, bipolar disorder (BPD), autism spectrum disorder (ASD), substance use disorder, and attention-deficit/hyperactivity disorder (ADHD). Treatment of these disorders remains inadequate, as exemplified by the therapeutic use of d-amphetamine and methylphenidate for the treatment of ADHD, agents with high abuse liability. In search for an improved and non-addictive therapeutic approach for the treatment of DA-linked disorders, we utilized a preclinical mouse model expressing the human DA transporter (DAT) coding variant DAT Val559, previously identified in individuals with ADHD, ASD, or BPD. DAT Val559, like several other disease-associated variants of DAT, exhibits anomalous DA efflux (ADE) that can be blocked by d-amphetamine and methylphenidate. Kappa opioid receptors (KORs) are expressed by DA neurons and modulate DA release and clearance, suggesting that targeting KORs might also provide an alternative approach to normalizing DA-signaling disrupted by perturbed DAT function. Here we demonstrate that KOR stimulation leads to enhanced surface trafficking and phosphorylation of Thr53 in wildtype DAT, effects achieved constitutively by the Val559 mutant. Moreover, these effects can be rescued by KOR antagonism of DAT Val559 in ex vivo preparations. Importantly, KOR antagonism also corrected in vivo DA release as well as sex-dependent behavioral abnormalities observed in DAT Val559 mice. Given their low abuse liability, our studies with a construct valid model of human DA associated disorders reinforce considerations of KOR antagonism as a pharmacological strategy to treat DA associated brain disorders.

Abstract ID 98801Poster Board 159Dopamine receptors (DARs) are G-protein coupled receptors (GPCRs) that regulate diverse physiological functions including cognition, mood, movement, and reward-related behaviors. They are classified as either D1-like (D1R and D5R) or D2-like (D2R, D3R, and D4R) based on structural homology and pharmacological properties. The D1-like DARs couple to Gs and Golf to increase cAMP levels, while the D2-like DARs couple to Gi/o to decrease cAMP levels. All DARs also recruit β-arrestin which activates distinct signaling cascades and can also initiate receptor desensitization and internalization. There are seven GRK isoforms (GRKs1-7), with GRK2, GRK3, GRK5, and GRK6 being widely expressed. Using systematic mutational analyses, our lab has previously mapped the GRK-mediated phosphorylation sites on both the D1R and D2R, with phosphorylation occurring exclusively on ICL3 for the D2R, and both ICL3 and the C-terminus for the D1R. GRK-mediated phosphorylation of the D1R was found to be required for subsequent β-arrestin recruitment. In contrast, while GRKs play a role in β-arrestin recruitment to the D2R, GRK-mediated D2R phosphorylation is dispensable for this process. We have now sought to determine which GRK isoform(s) are involved in regulating β-arrestin recruitment to these receptors using cell lines in which the expression of specific GRK isoforms were selectively eliminated via CRISPR, as well as by utilizing isoform-selective GRK inhibitors. β-arrestin recruitment to both the D1R and D2R was severely impaired in cells lacking all GRKs (total KO), whereas selective expression of GRK isoforms 2, 3, 5 and 6 in the total KO cells was able to rescue β-arrestin recruitment. The kinase activity of each GRK was required for this recue for the D1R. Interestingly, the GRK2/3 and GRK5/6 isoforms differed in their requirement of kinase activity for the rescue of β-arrestin recruitment to the D2R. Individual and double GRK KO cells revealed that the GRK5/6 subfamily is more important for β-arrestin recruitment to the D1R, while the GRK2/3 subfamily is more important for the D2R. Treatment with GRK2/3-selective inhibitors recapitulated these findings with the GRK KO cells in that they impaired β-arrestin recruitment to the D2R, but had limited effect on this process for the D1R. Intriguingly, in cells with endogenous levels of GRK expression, β-arrestin recruitment to both the D1R and D2R were unaffected by inhibitors of GRK5/6. However, these inhibitors could block the rescue of β-arrestin recruitment to the D2R observed with GRK5 or GRK6 overexpression but had little effect on the rescue of β-arrestin recruitment to the D1R or the Gs-coupled β2 adrenergic receptor. As GRK distribution varies by tissue and brain region, it is intriguing to postulate that DAR regulation, and GPCR regulation in general, by specific GRK isoforms can add layers of regulatory fine-tuning through differentially directing signaling or trafficking outcomes.National Institute of Neurological Disorders and Stroke Intramural Research Program, National Institutes of Health (ZIA NS002263)

Abstract ID 90930Poster Board 516Abstract Text Parkinson’s disease (PD) is a prevalent neurodegenerative condition characterized by degeneration of dopaminergic neurons in the substantia nigra of the midbrain. This results in bradykinesia, tremors, rigidity, and other motor and non-motor deficits. The prevalence of PD is increasing, in part, due to the aging population. Despite this growing burden, the development of effective therapies to halt disease progression remains an unmet need. This challenge is due to gaps in our understanding of the molecular mechanisms underlying the disease and deficiencies of preclinical models in recapitulating all aspects of the pathobiology of PD. Consequently, deepening our understanding of pathobiology and discovering druggable targets remain a research priority. Herein, we describe assay development and optimization of a high throughput phenotypic screen in the roundworm Caenorhabditis elegans for discovery of novel druggable targets and genetic influencers of neurodegeneration and neuroprotection. We utilized two transgenic C. elegans strains expressing human PD-linked genes—one with mutant (A53T) alpha-synuclein (SNCA) and the other expressing mutant (G2019S) leucine-rich repeat kinase 2 (LRRK2). These strains exclusively express the PD-related transgenes and green fluorescent protein (GFP) in their dopaminergic neurons, facilitating the tracking of neurodegeneration through measurable changes in the GFP fluorescence. Using laser cytometry and high content imaging with worms growing in liquid culture within 384-well plates, we assessed the health of the dopamine neurons via GFP intensity, number of neurons, and area of green objects. We observed a 30-50% and 75-85% decrease in GFP intensity in the SNCA and LRRK2 worms, respectively, by day seven compared to wild-type controls lacking the PD genes but expressing GFP exclusively in the dopaminergic neurons. We crossed the worms to RNAi hypersensitive backgrounds carrying eri-1, rrf-3, and eri-1;lin-15B mutations to overcome the typical resistance to neuronal RNAi knockdown in C. elegans. We observed a robust RNAi knockdown in the control and mutant worms with a 50-75% knockdown on day seven with RNAi against GFP, ama-1, ceh-43, and unc-62 compared to empty vector control. RNAi specifically engineered against the LRRK2 transgene slows neurodegeneration in the LRRK2 mutant worms. Additionally, we have identified selective LRRK2 kinase inhibitors that confer neuroprotection in the LRRK2-carrying worms with promising properties to serve as controls for high throughput molecular library screening. Further optimization studies are ongoing with the goal of conducting a screen for genes influencing neuroprotection in these models. Additionally, we plan to conduct a small molecule library screen for novel neuroprotective compounds using these PD models. These lines of inquiry may identify new molecules or potential druggable targets for novel therapeutics for influencing neurodegeneration and PD progression.Supported by the NINDS Intramural Research Program.

Dopamine release in striatal circuits, including the nucleus accumbens (NAc), tracks separable features of reward such as motivation and reinforcement. However, the cellular and circuit mechanisms by which dopamine receptors transform dopamine release into distinct constructs of reward remain unclear. Here, we show that dopamine D3 receptor (D3R) signaling in the NAc drives motivated behavior by regulating local NAc microcircuits. Furthermore, D3Rs co-express with dopamine D1 receptors (D1Rs), which regulate reinforcement, but not motivation. Paralleling dissociable roles in reward function, we report non-overlapping physiological actions of D3R and D1R signaling in NAc neurons. Our results establish a novel cellular framework wherein dopamine signaling within the same NAc cell type is physiologically compartmentalized via actions on distinct dopamine receptors. This structural and functional organization provides neurons in a limbic circuit with the unique ability to orchestrate dissociable aspects of reward-related behaviors that are relevant to the etiology of neuropsychiatric disorders.

Dopamine receptors (DARs) are G protein-coupled receptors (GPCRs) that regulate diverse physiological functions and are involved in the treatment and/or etiology of many neuropsychiatric disorders including schizophrenia and substance use disorder (SUD). DARs are classified as either D1-like (D1R and D5R) or D2-like (D2R, D3R, and D4R) based on structural homology and pharmacological profiles. Antagonists of D2-like DARs are currently used in the therapies for many neuropsychiatric disorders. D3R-selective antagonists have the potential to be better therapeutics for schizophrenia or SUD as they could attenuate psychotic or drug craving symptoms without the motor side effects frequently produced by D2R-preferring antagonists. Unfortunately, discovery of subtype-selective compounds for the D3R and D2R has been challenging due to high sequence homology within the orthosteric binding sites of DARs. However, compounds that modulate receptor activities through interactions with less conserved allosteric sites have the potential to be highly selective. To find highly selective allosteric antagonists of the D3R, we screened the NIH Molecular Libraries Program 400,000+ small molecule library with a D3R-mediated β-arrestin recruitment assay. We found that one compound, MLS6357, was selective for the D3R over the D2R and D4R in several functional outputs including β-arrestin recruitment and G-protein activation. Radioligand binding and functional assays using closely related GPCRs revealed that MLS6357 has very limited cross-reactivity with other GPCRs. Additionally, Schild-type functional assays showed that MLS6357 acts as a purely non-competitive negative allosteric modulator (NAM) of the D3R. We synthesized and characterized >60 analogs of MLS6357 using iterative medicinal chemistry approaches, which revealed structure-activity relationships and enabled further optimization of the scaffold. These efforts produced analogs that are 10-fold and 30-fold more potent than the parent compound in D3R-mediated β-arrestin recruitment and G-protein activation assays, respectively. Moreover, some analogs appear to display functional selectivity for inhibition of G-protein activation versus inhibition of β-arrestin recruitment, and vice versa, and some also display inverse agonist activity. To identify the allosteric binding site for the MLS6357 scaffold on the D3R, we utilized various D3R/D2R chimeras, receptor mutants, and molecular modeling techniques to reveal and characterize receptor regions necessary for compound efficacy. Further refinement of the binding pocket for MLS6357 will inform future medicinal chemistry efforts. Ultimately, this novel scaffold may be of benefit as a pharmacological probe or therapeutic lead for D3R-related pathophysiology.

Rational drug design for G protein-coupled receptors (GPCRs) remains a challenging area. A new study from the Xu, Roth, and Zhang groups provides a complete set of active structures for the entire dopamine receptor family bound with rotigotine that will aid in designing selective agonists for these important therapeutic targets.

The D1 dopamine receptor (D1R) is a G protein-coupled receptor that signals through activating adenylyl cyclase and raising intracellular cAMP levels. When activated, the D1R also recruits the scaffolding protein β-arrestin, which promotes receptor desensitization and internalization, as well as additional downstream signaling pathways. These processes are triggered through receptor phosphorylation by G protein-coupled receptor kinases (GRKs), although the precise phosphorylation sites and their role in recruiting β-arrestin to the D1R remains incompletely described. In this study, we have used detailed mutational and in situ phosphorylation analyses to completely identify the GRK-mediated phosphorylation sites on the D1R. Our results indicate that GRKs can phosphorylate 14 serine and threonine residues within the C-terminus and the third intracellular loop (ICL3) of the receptor, and that this occurs in a hierarchical fashion, where phosphorylation of the C-terminus precedes that of the ICL3. Using β-arrestin recruitment assays, we identified a cluster of phosphorylation sites in the proximal region of the C-terminus that drive β-arrestin binding to the D1R. We further provide evidence that phosphorylation sites in the ICL3 are responsible for β-arrestin activation, leading to receptor internalization. Our results suggest that distinct D1R GRK phosphorylation sites are involved in β-arrestin binding and activation.

ID 17834 Poster Board 344 Parkinson’s disease (PD) is a neurodegenerative disorder characterized by the necrosis of midbrain dopaminergic neurons and subsequent deficiencies of dopamine (DA) signaling, resulting in tremors, rigidity, and bradykinesia among a range of other motor and non-motor complications. Despite its high prevalence and considerable economic burden, driven by a rapidly growing aging population, the underlying molecular mechanisms of PD remain poorly understood, and robust, translational models of the disease have yet to be fully established. These limitations in our collective understanding of PD warrant an urgent need for discovering and interrogating PD-associated druggable targets to address the lack of effective, neuroprotective therapeutics with minimal side effect profiles. Here, we report the development of two high throughput in vivo assays for the discovery of small molecule compounds with therapeutic potential and for probing genes potentially involved in dopaminergic neuroprotection. Transgenic mutant Caenorhabditis elegans (C.elegans) carrying human PD-linked genes were used, one expressing mutant (G2019S) leucine-rich repeat kinase 2 (LRRK2), and the other expressing mutant (A53T) α-synuclein (SNCA). Both strains express GFP exclusively within their dopaminergic neurons allowing for fluorescent signal intensity to serve as a proxy for monitoring dopaminergic neurodegeneration. A control strain (BY250) was used that expressed only dopaminergic neuronal GFP in the absence of PD-linked transgenes. Daily laser cytometry and high-content imaging readings of GFP intensity revealed a robust temporal dopaminergic neurodegeneration in both PD strains, mirroring that which is seen in human PD, within the first seven days of adulthood. By day seven, GFP fluorescent intensity had decreased by 30-50% and 75-85% in the SNCA and LRRK2 worms, respectively; such an effect was not observed in the wild-type control worms. In the LRRK2 mutant worms, we have identified a set of selective LRRK2 kinase inhibitors that may serve as positive controls for neuroprotection. Assay validation and optimization studies are ongoing with the goal of conducting a high throughput screen of small molecules that may confer neuroprotection in our LRRK2 model and serve as scaffolds for the development of drug leads. We have also established effective knockdown of GFP fluorescent signal intensity by the administration of RNA interference (RNAi) via engineered vector bacterial feeding. Due to the low penetrance of RNAi in neurons, we crossed our control and mutant worms into three RNAi hypersensitive backgrounds carrying eri-1, eri-1; lin-15B, and rrf-3 mutations. In a preliminary screen of RNAi vectors, three (ceh-43, unc-62, and ama-1) conferred robust knockdown of GFP signal in both the control and mutant α-synuclein-expressing strains, averaging ≥75% knockdown by day seven of treatment when compared to an empty vector control RNAi. In the future, we hope to use this assay to screen C.elegans RNAi libraries to elucidate genes that may be neuroprotective in worms expressing the PD transgenes and may serve as potential drug targets for PD therapeutics. A synergistic application of these two approaches may also prove fruitful in that we may deploy RNAi to interrogate potential targets of drugs identified in our high throughput screen or, conversely, use our neuroprotective controls to assess the efficacy of druggable targets related to genes identified by RNAi screening.

ID 15411 Poster Board 351 Dopamine receptors (DARs) are G protein-coupled receptors (GPCRs) that regulate diverse physiological functions including movement, mood, cognition, and motivation, and are involved in the pathology and treatment of numerous neuropsychiatric disorders. The D1-like DARs (D1R and D5R) activate adenylyl cyclase and increase cAMP levels, while D2-like DARs (D2R, D3R, D4R) inhibit adenylyl cyclase and decrease cAMP levels. Additionally, all DARs recruit β-arrestin, leading to separate signaling cascades and the initiation of receptor desensitization and internalization. β-arrestin recruitment to the D1R is closely linked to receptor phosphorylation, and the D1R has been shown to be phosphorylated by various kinases including protein kinase A (PKA), protein kinase C (PKC), and G protein-coupled receptor kinases (GRKs). The D1R contains 32 intracellular serine and threonine residues within the C-terminus and third intracellular loop (ICL3) that serve as potential phosphorylation sites. Using mutational analyses, we previously identified the PKA- and PKC-mediated phosphorylation sites and showed that GRK4 constitutively phosphorylates the receptor. We have now identified the D1R residues that are phosphorylated by GRKs in response to dopamine (DA) stimulation and found that mutation of these residues severely impairs β-arrestin recruitment, but has little effect on G protein-mediated signaling. Our results also suggest that most of the DA-induced GRK phosphorylation of the D1R occurs at residues T360 and S362 in the proximal C-terminus, and that these residues are primarily responsible for β-arrestin recruitment to the receptor. We next sought to determine which GRK subtypes are involved in DA-induced β-arrestin recruitment to the D1R. Using CRISPR-edited HEK293 cells in which all the endogenous GRKs were knocked out, we found that DA-induced β-arrestin recruitment to the D1R is severely impaired in the absence of GRKs, but can be rescued by exogenous GRK overexpression. The kinase activity of the GRKs appears to be necessary for this effect, as a catalytically inactive K220R GRK2 mutant is unable to rescue DA-induced β-arrestin recruitment to the D1R. Using individual or combinatorial GRK KO cells, we found that GRK2 and GRK3 play little to no role in mediating DA-stimulated β-arrestin recruitment to the D1R, which contrasts with diminished β-arrestin recruitment to the D2R in GRK2 KO cells. However, we found that DA-induced β-arrestin recruitment to the D1R was significantly impaired in GRK5/6 and GRK6 KO cells. We found similar effects using multiple GRK2/3-selective inhibitors, in that they had no effect on β-arrestin recruitment to the D1R, while they severely diminished DA-induced β-arrestin recruitment to the D2R. We are currently using GRK5/6-selective inhibitors to determine if pharmacological inhibition of GRK5/6 imitates GRK5/6 knockout. Taken together, these results suggest that GRK6 is prominently involved in DA-induced β-arrestin recruitment to the D1R, and that different GRK subtypes regulate β-arrestin recruitment to the D1R vs. the D2R. Because GRK subtype distribution varies by tissue and brain region, it is intriguing to postulate that D1R phosphorylation by different GRKs may add layers of regulatory fine-tuning through differentially directing D1R signaling or trafficking outcomes.

Since its first use in treating Parkinson’s disease (PD), L‐DOPA has remained the gold standard of therapy for this disorder, defined by the progressive degeneration of dopaminergic neurons in the CNS leading to profound bradykinesia and tremor. The efficacy of L‐DOPA wanes over time and is associated with increasing side effects, including motor fluctuations and dyskinesias. Several dopaminergic agonists have also been introduced to treat PD, including pramipexole and ropinirole, which exhibit fewer motor side effects but are associated with impulse control disorders such as excessive gambling and hypersexuality. Notably, the dopamine receptor subtype(s) mediating the therapeutic actions and/or side effects in PD therapy remain unknown. However, the preference of pramipexole and ropinirole for the D3 dopamine receptor (D3R) suggests the involvement of this subtype, although these drugs also activate the D2R at therapeutic doses. Importantly, no drug currently employed to treat PD alters the course of the disease and the discovery of neuroprotective agents remain an unmet need in PD therapeutics. Recently, we discovered a novel, potent and highly selective agonist for the D3R, ML417, that is brain penetrant and was found to protect against 6‐OHDA‐induced neurodegeneration of dopaminergic neurons (Moritz et al., J. Med. Chem. 63: 5526, 2020). In the current study, we used ML417 to probe the role of the D3R in a rat model of PD. We initially sought to investigate the role of the D3R in ameliorating bradykinesia in a hemi‐parkinsonian rat model induced by 6‐OHDA infusion into the medial forebrain bundle. Using a validated cylindrical treadmill test of locomotion, doses of ML417 up to 20 mg/kg had no effect on improving impairments in walking as assessed by step counts in the hemi‐parkinsonian rats. In contrast, administration of L‐DOPA (6 mg/kg) significantly improves locomotion in the same model. Further, pretreatment with a D3R‐selective antagonist, SB277011A (30 mg/kg), did not attenuate the effects of L‐DOPA in reducing bradykinesia. These results suggest that the D3R does not mediate the anti‐bradykinetic effects of current PD therapeutics. However, we hypothesize that D3R stimulation may be beneficial for the treatment of L‐DOPA‐induced dyskinesias (LIDs) in PD. To test this, we used a chronic L‐DOPA administration paradigm (12 mg/kg/day for 7 days) to induce dyskinesias in the hemi‐parkinsonian rats. Subsequently, the effects of ML417 and SB277011A were assessed in these animals using an abnormal involuntary movement (AIMs) scoring method. Pretreatment with a single dose of ML417 (20 mg/kg) significantly reduced the intensity and duration of dyskinesias promoted with a single dose of L‐DOPA (6 mg/kg). Further, co‐administration of SB277011A (30 mg/kg) with ML417 attenuated the anti‐dyskinetic effects of ML417, suggesting that the benefit is D3R‐mediated. Overall, this study implies that D3R stimulation has no therapeutic effect on bradykinesia in PD, however, it may be beneficial in treating dyskinesias arising from L‐DOPA therapy.

Parkinson’s disease (PD) is a common neurodegenerative disorder characterized by the selective death of dopaminergic neurons in the substantia nigra of the midbrain, resulting in bradykinesia, tremors, muscle rigidity, and other motor and non‐motor deficits. The prevalence and economic burden of Parkinson’s disease are increasing due to the aging population. Despite this increasing burden, the discovery of clinically useful therapies to halt the progression of the disease remains an unmet need. This is partly because of gaps in our knowledge of the underlying molecular mechanisms of the disease and the lack of accurate disease models. Therefore, understanding the pathobiology and discovery of potential druggable targets remain a priority in PD research. In the current study, we report the development of a high‐throughput assay in the nematode, Caenorhabditis elegans, to monitor neurodegeneration and to probe for genes potentially involved in mediating neuroprotection of dopaminergic neurons using RNA interference (RNAi) technology. Two transgenic strains of C. elegans carrying human PD‐linked genes were used, one expressing mutant (A53T) alpha‐synuclein, and the other expressing mutant (G2019S) leucine‐rich repeat kinase 2 (LRRK2). These strains express the PD‐transgenes and GFP exclusively in their dopaminergic neurons allowing for the monitoring of neurodegeneration via changes in GFP signal intensity. A third control strain was used that lacked the PD‐transgenes genes but expressed GFP in their dopaminergic neurons. We crossed these three strains of worms into neuronal RNAi‐sensitive backgrounds that express mCherry under a pharyngeal promoter thus allowing for accurate quantitation of the worms in liquid cultures within 384‐well microtiter plates. Using a combination of laser cytometry and high content imaging, we have established that these models recapitulate the age‐dependent degeneration of dopaminergic neurons observed in human PD. Neurodegeneration was seen in the SNCA (A53T) and LRRK2 (G2019S) transgenic models, as evidenced by a 30‐50% and 75‐85% loss of GFP intensity, respectively, after 7 days in culture. In preliminary studies, we have found that these worms show susceptibility to RNAi via bacterial feeding as evidenced by a loss of GFP signal from RNAi against GFP or from RNAi against growth factors supporting the differentiation of dopaminergic neurons. Further optimization studies of this RNAi screening assay are currently ongoing. The demonstrated susceptibility of C. elegans to neuronal RNAi in our screening assay, and the availability of chromosomal and genome‐wide RNAi libraries will allow for the interrogation of the entire C. elegans genome in these models. This line of inquiry, if successful, will improve our knowledge of the underlying mechanisms of neurodegeneration and may also identify genes encoding druggable targets for therapeutics directed at slowing the progression of PD.

Dopamine (DA) receptors (DARs) are G protein-coupled receptors (GPCRs) that regulate diverse physiological functions including movement, cognition, mood, and reward-related behaviors, as well as cardiovascular and renal physiology. Multiple diseases are linked to dysregulated dopaminergic functioning including Parkinson's disease, schizophrenia, substance use disorder, and hypertension. DARs are classified as either D1-like (D1R and D5R) or D2-like (D2R, D3R, and D4R) based on structural homology and pharmacological properties. The D1-like DARs (D1R and D5R) increase cAMP, while the D2-like DARs (D2R, D3R, D4R) decrease cAMP. All DARs also recruit β-arrestin which activates separate signaling cascades and also initiates receptor desensitization and internalization. Generally, agonist activation of GPCRs, including DARs, rapidly leads to receptor desensitization and a return to basal levels of signaling. This occurs even in the continued presence of agonist, ensuring homeostasis. This desensitization process is intimately linked with receptor phosphorylation. The D1R is highly phosphorylated, with 32 intracellular serine and threonine residues, and is known to be phosphorylated by several kinases including protein kinase A (PKA), protein kinase C (PKC), and G protein-coupled receptor kinases (GRKs). Previous studies by our lab indicate that the D1R is phosphorylated on its third intracellular loop (ICL3) and C-terminus in a hierarchical fashion, in that phosphorylation must first occur on the C-terminus before the ICL3 can be phosphorylated. Using systematic mutational analyses, we previously identified the PKC-mediated D1R phosphorylation sites. We have now extended these studies to completely identify the DA-induced, GRK-mediated phosphorylation sites on the D1R. We found that GRK-mediated phosphorylation involves several serine and threonine residues on the C-terminus and ICL3. Mutation of these residues to alanine or valine, respectively, abolishes DA-induced D1R phosphorylation and severely impairs β-arrestin recruitment, but causes little effect on G protein-mediated signaling. Our results indicate that a large fraction of DA-induced D1R phosphorylation occurs on residues T360 and S362 in the proximal C-terminus, and that these residues are also responsible for the majority of DA-induced β-arrestin recruitment to the D1R. Using HEK cells that have had their endogenous GRKs knocked out via CRISPR, we found that DA-induced β-arrestin recruitment to the D1R is severely impaired. However, β-arrestin recruitment can be restored by expressing exogenous GRKs in these cells, further suggesting that DA-induced β-arrestin recruitment to the D1R is highly dependent on GRK phosphorylation. As GRK distribution varies by tissue and brain region, it is intriguing to postulate that D1R phosphorylation by different GRKs can add layers of regulatory fine-tuning through differential effects on D1R signaling or trafficking outcomes.

Parkinson’s disease (PD) is a neurodegenerative disorder that is characterized by loss of dopaminergic neurons resulting in bradykinesia, tremor, gait abnormalities, and numerous non‐motor complications. The prevalence and economic burden of PD is increasing due to an aging population. However, there are currently no drugs to halt the progression of this disease. Although loss of dopamine in the basal ganglia is recognized as the hallmark of PD, the molecular mechanisms underlying this loss and subsequent brain dysfunction remain poorly characterized. The majority of PD animal models involve the administration of neurotoxins that target dopaminergic neurons leading to their degeneration, however these models correlate poorly with the disease progression in humans. More accurate models may utilize genetic modulation of PD‐related genes and exhibit progressive neurodegeneration. Here, we report the development of a high‐throughput assay for monitoring dopaminergic neurodegeneration in Caenorhabditis elegans (C. elegans) . Two strains of C. elegans containing human PD‐linked genes were used, one expressing mutant (A53T) alpha‐synuclein, and the other expressing mutant (G2019S) leucine‐rich repeat kinase 2 (LRRK2). Both strains express GFP in their dopaminergic neurons and the lines were further crossed into a neuronal RNAi‐sensitive background strain expressing mCherry under a pharyngeal promoter. This allows for the accurate measurement of dopaminergic neurons (via GFP) and a normalization and sorting control of the total number of worms plated (via mCherry). Daily measurements of GFP/mCherry fluorescence intensity were performed using laser cytometry of worms sorted into 384‐well microplates. Robust temporal degeneration of dopaminergic neurons was found to occur within the first eight days of adulthood in the C. elegans models of PD, but not in a wild‐type control strain. The LRRK2 model was particularly severe as total GFP intensity dropped by 75–85% during the time of assay. We determined a signal (cell loss) to baseline ratio of approximately 4‐fold, sufficient for screening applications. Our results indicate that this assay provides a reproducible high‐throughput measurement of dopaminergic neurodegeneration using an in vivo model. Future studies may exploit this model to conduct quantitative high‐throughput screens to identify small molecules capable of inhibiting this neurodegeneration or to use RNAi libraries to identify genes mediating the neurodegenerative response, and hence new drug targets for the treatment of PD. Support or Funding Information This study is supported by the intramural program of the NINDS/NIH