Ophthalmate is a new regulator of motor functions via CaSR: implications for movement disorders

Abstract

Dopamine’s role as the principal neurotransmitter in motor functions has long been accepted. We broaden this conventional perspective by demonstrating the involvement of non-dopaminergic mechanisms. In mouse models of Parkinson’s disease, we observed that L-DOPA elicited a substantial motor response even when its conversion to dopamine was blocked by inhibiting the enzyme aromatic amino acid decarboxylase (AADC). Remarkably, the motor activity response to L-DOPA in the presence of an AADC inhibitor (NSD1015) showed a delayed onset, yet greater intensity and longer duration, peaking at 7 h, compared to when L-DOPA was administered alone. This suggests an alternative pathway or mechanism, independent of dopamine signalling, mediating the motor functions. We sought to determine the metabolites associated with the pronounced hyperactivity observed, using comprehensive metabolomics analysis.

Our results revealed that the peak in motor activity induced by NSD1015/L-DOPA in Parkinson’s disease mice is associated with a surge (20-fold) in brain levels of the tripeptide ophthalmic acid (also known as ophthalmate in its anionic form). Interestingly, we found that administering ophthalmate directly to the brain rescued motor deficits in Parkinson’s disease mice in a dose-dependent manner. We investigated the molecular mechanisms underlying ophthalmate’s action and discovered, through radioligand binding and cAMP-luminescence assays, that ophthalmate binds to and activates the calcium-sensing receptor (CaSR).

Additionally, our findings demonstrated that a CaSR antagonist inhibits the motor-enhancing effects of ophthalmate, further solidifying the evidence that ophthalmate modulates motor functions through the activation of the CaSR. The discovery of ophthalmate as a novel regulator of motor function presents significant potential to transform our understanding of brain mechanisms of movement control and the therapeutic management of related disorders.

Alhassen et al. show that L-DOPA elicits a delayed motor response in a mouse model of Parkinson’s disease, even when its conversion to dopamine is blocked. The motor response is accompanied by a surge in brain ophthalmate, suggesting that ophthalmate may be a novel neuromodulator of motor function and could have therapeutic potential.

Introduction

The dopamine theory of movement control has been the prevailing model for explaining the symptoms and treatment responses seen in individuals with Parkinson’s disease (PD) for over five decades. This widely accepted theory suggests that dopamine neurons in the basal ganglia regions are involved in initiating and modulating voluntary movement, with dopamine acting as a ‘go’ signal that activates the neural pathways that control movement.

Individuals affected by PD have low levels of dopamine in the basal ganglia as a result of degenerative damage to dopamine neurons in the substantia nigra pars compacta. This brings about tremors and difficulty initiating and controlling movement, as well as rigidity and slowed mobility (bradykinesia). Treatment with agents that raise dopamine levels can significantly improve movement-related symptoms in PD patients. Since dopamine is unable to cross the blood–brain barrier (BBB), L-DOPA (L-3, 4, hydroxypheylalanine, levodopa)—dopamine precursor—is regarded as the gold standard treatment for PD.^1,2

L-DOPA is converted to dopamine by the enzyme aromatic amino acid decarboxylase (AADC). AADC is found in the brain and a number of peripheral organs. The conversion of L-DOPA to dopamine peripherally can lead to two main issues. First, the amount of L-DOPA that reaches the brain, where it is needed to replace dopamine in PD patients, is reduced.^1-3 This can diminish the therapeutic effects of L-DOPA in relieving PD movement symptoms. Second, the increased levels of dopamine can cause a range of side effects, such as nausea, vomiting, and orthostatic hypotension. To reduce the peripheral metabolism of L-DOPA to dopamine, peripheral AADC inhibitors such as carbidopa can be used in combination with L-DOPA.⁴

Despite being the most effective treatment for PD for over 50 years, the precise mechanisms of L-DOPA action remain uncertain. Initial treatments with L-DOPA can provide great relief from motor symptoms, however, over time its therapeutic effects tend to diminish and dyskinesia, abnormal involuntary movements, can increase in PD patients.⁵ This phenomenon, known as L-DOPA-induced dyskinesia (LID), can be difficult to treat and may require a change in treatment approach.^6,7 The mechanisms underlying LID are also not fully understood.

A plethora of data suggest that L-DOPA has more functions than merely being a precursor for dopamine creation in the brain. Our earlier research showed that L-DOPA produces hyperkinesia in the presence of a central AADC inhibitor (NSD1015) in the reserpine-treated rat model of PD.⁸ We speculated therein that this hyperkinesia is mediated through the direct action of a metabolite(s)—other than dopamine- on non-dopamine receptors. We based our speculation on the following observations: (i) the onset of motor activity induced by the administration of L-DOPA and a potent central AADC inhibitor (3-hydroxybenzyl hydrazine, NSD1015) was delayed (110 min) compared to that with L-DOPA alone (45 min), suggesting that NSD1015, at least initially, inhibited central AADC activity and prevented the enzymatic conversion of L-DOPA to dopamine or any other effective metabolites^9-11; and (ii) 110 min following L-DOPA administration, motor activity was greatly increased and hyperkinesia occurred for a prolonged duration of time. We argued that L-DOPA alone could not be responsible for this hyporeactivity solely through its conversion to dopamine, as one would expect that the motor behaviour would be abolished subsequent to the inhibition by AADC. These observations provoked our investigation into the potential molecule(s) behind such a remarkable boost and extension of motor activity. We here conducted a series of behavioural, metabolomic, binding and functional studies, and identified the tripeptide ophthalmic acid (OA, also known as ophthalmate in its anionic form) that modulates motor functions, through activating calcium sensing receptors (CaSRs).

Materials and methods

Mouse models of Parkinson’s disease

Swiss Webster mice (8–10 weeks old) were obtained from Charles River Laboratories. Animals were group housed with a maximum of four to five animals per cage and acclimated to the vivarium for a week prior to treatments. Animals were kept in a normal 12:12 h light/dark cycle with free access to food and water. For the reserpine treatment, animals were lightly anaesthetized and then injected subcutaneously (s.c.) with either reserpine (1 mg/kg, dissolved in 1% (v/v) glacial acetic acid) or vehicle (1% (v/v) glacial acetic acid), as we previously described.⁸ For the MPTP treatment, mice were injected intraperitoneally (i.p.) for 3 days with MPTP (20 mg/kg) or saline per day, based on previous studies, with slight modifications.^12,13 All experimental procedures were approved by the Institutional Animal Care and Use Committee (IACUC) of the University of California, Irvine, and were conducted in accordance with national and institutional guidelines for the care and use of laboratory animals.

Global metabolomics profiling

Metabolomic analysis was performed by Metabolon (Durham, NC, USA).

Brain tissue harvesting

Mice were injected (s.c.) with reserpine 1 mg/kg. After 18 h, the mice were treated with NSD1015 or saline, followed by L-DOPA administration 30 min later. Brain tissues were collected from the two treatment groups at two time points (2 and 7 h after L-DOPA administration). The brains were divided into two hemispheres, with one hemisphere homogenized entirely, and the other hemisphere used to extract the striatum. Global metabolic profiles were determined from the experimental groups outlined in Supplementary Table 1.

Sample preparation

Samples were prepared using the automated MicroLab STAR^® system from Hamilton Company. Several recovery standards were added prior to the first step in the extraction process for quality control (QC) purposes. To remove protein, dissociate small molecules bound to protein or trapped in the precipitated protein matrix, and to recover chemically diverse metabolites, proteins were precipitated with methanol under vigorous shaking for 2 min (Glen Mills GenoGrinder 2000) followed by centrifugation. The resulting extract was divided into five fractions: two for analysis by two separate reverse phase (RP) ultra-high performance tandem mass spectrometry (UPLC-MS/MS) methods with positive ion mode electrospray ionization (ESI): one for analysis by RP/UPLC-MS/MS with negative ion mode ESI; one for analysis by HILIC/UPLC-MS/MS with negative ion mode ESI; and one sample was reserved for backup. Samples were placed briefly on a TurboVap^® (Zymark) to remove the organic solvent. The sample extracts were stored overnight under nitrogen before preparation for analysis.

Quality analysis/quality control

Several types of controls were analysed in concert with the experimental samples: a pooled matrix sample generated by taking a small volume of each experimental sample served as a technical replicate throughout the dataset; extracted water samples served as process blanks; and a cocktail of QC standards, carefully chosen not to interfere with the measurement of endogenous compounds, were spiked into every analysed sample, allowing instrument performance monitoring and aiding chromatographic alignment. Instrument variability was determined by calculating the median relative standard deviation (RSD) for the standards that were added to each sample prior to injection into the mass spectrometers. Overall process variability was determined by calculating the median RSD for all endogenous metabolites (i.e. non-instrument standards) present in 100% of the pooled matrix samples. Experimental samples were randomized across the platform run with QC samples spaced evenly among the injections.

Ultra-high performance liquid chromatography-tandem mass spectroscopy

All methods used a Waters ACQUITY UPLC and a Thermo Scientific Q-Exactive high resolution/accurate mass spectrometer interfaced with a heated electrospray ionization (HESI-II) source and Orbitrap mass analyser operated at 35 000 mass resolution. The sample extract was dried then reconstituted in solvents compatible to each of the four methods. Each reconstitution solvent contained a series of standards at fixed concentrations to ensure injection and chromatographic consistency. One aliquot was analysed using acidic positive ion conditions, chromatographically optimized for more hydrophilic compounds. In this method, the extract was gradient eluted from a C18 column (Waters UPLC BEH C18-2.1 × 100 mm, 1.7 µm) using water and methanol, containing 0.05% perfluoropentanoic acid (PFPA) and 0.1% formic acid (FA). Another aliquot was also analysed using acidic positive ion conditions; however, it was chromatographically optimized for more hydrophobic compounds. In this method, the extract was gradient eluted from the same afore mentioned C18 column using methanol, acetonitrile, water, 0.05% PFPA and 0.01% FA and was operated at an overall higher organic content. Another aliquot was analysed using basic negative ion optimized conditions using a separate dedicated C18 column. The basic extracts were gradient eluted from the column using methanol and water, however with 6.5 mM ammonium bicarbonate at pH 8. The fourth aliquot was analysed via negative ionization following elution from a HILIC column (Waters UPLC BEH Amide 2.1 × 150 mm, 1.7 µm) using a gradient consisting of water and acetonitrile with 10 mM ammonium formate, pH 10.8. The MS analysis alternated between MS and data-dependent MSⁿ scans using dynamic exclusion. The scan range varied slighted between methods but covered 70–1000 m/z. Raw data files were archived and extracted as described below.

Bioinformatics

The informatics system consisted of four major components, the Laboratory Information Management System (LIMS), the data extraction and peak-identification software, data processing tools for QC and compound identification, and a collection of information interpretation and visualization tools for use by data analysts. The hardware and software foundations for these informatics components were the LAN backbone, and a database server running Oracle 10.2.0.1 Enterprise Edition.

Data extraction and compound identification

Raw data were extracted, peak-identified and QC processed using Metabolon’s hardware and software. These systems are built on a web-service platform using Microsoft’s .NET technologies, which run on high-performance application servers and fiber-channel storage arrays in clusters to provide active failover and load-balancing. Compounds were identified by comparison to library entries of purified standards or recurrent unknown entities. Metabolon maintains a library based on authenticated standards that contains the retention time/index (RI), mass to charge ratio (m/z) and chromatographic data (including MS/MS spectral data) on all molecules present in the library. Furthermore, biochemical identifications are based on three criteria: retention index within a narrow RI window of the proposed identification; accurate mass match to the library ±10 ppm; and the MS/MS forward and reverse scores between the experimental data and authentic standards. The MS/MS scores are based on a comparison of the ions present in the experimental spectrum to the ions present in the library spectrum. While there may be similarities between these molecules based on one of these factors, the use of all three data points can be used to distinguish and differentiate biochemicals. More than 3300 commercially available purified standard compounds have been acquired and registered into LIMS for analysis on all platforms for determination of their analytical characteristics. Additional mass spectral entries have been created for structurally unnamed biochemicals, which have been identified by virtue of their recurrent nature (both chromatographic and mass spectral).

Metabolite quantification and data normalization

Peaks were quantified using area under the curve. For studies spanning multiple days, a data normalization step was performed to correct variation resulting from instrument inter-day tuning differences. Essentially, each compound was corrected in run-day blocks by registering the medians to equal one (1.00) and normalizing each data point proportionately (termed the ‘block correction’).

Synthesis of deuterated ophthalmate

The detailed methods of deuterated ophthalmate (D5-OA) synthesis are described in a paper in preparation. Briefly, the synthesis of D5-OA as a mixture of isomers was accomplished with standard peptide coupling reagents. Glycine benzyl ester was first coupled with racemic tert-butyloxycarbonyl protected D5-2-aminobutyrate. This dipeptide was deprotected and N-carbobenzyloxy-L-glutamate 1-methyl ester (Z-L-Glu-OMe) was added. Two deprotection steps, hydrolysis of the methyl ester, and hydrogenolysis of the benzyl ester and the N-carbobenzyloxy groups gave the acetate salt of D5-OA.

Measurement of deuterated ophthalmate using tandem mass spectrometry

Adult male Swiss Webster mice, weighing approximately 20–30 g, were sacrificed by asphyxiation. After decapitation, the blood was collected and the brains were rapidly removed on a cold surface, frozen in isopentane at −40°C and then stored at −80°C. The protocol for metabolite extraction from the brain tissue and serum was adapted from a previously reported protocol.¹⁴ Frozen tissue was immediately plunged into methanol (1 ml) containing internal standards and homogenized for 1 min to inactivate enzymes. Five hundred microlitres of deionized water was added and 300 μl of the solution was transferred to another tube and 200 μl of chloroform was added and mixed thoroughly. The solution was centrifuged at 12 000g for 15 min at 4°C. The upper aqueous layer was filtered and the filtrate was lyophilized and dissolved in 50 μl of methanol. For the serum, 200 μl of serum was plunged into 1.8 ml of methanol. An 800 μl volume of deionized water was added with an additional 2 ml of chloroform. The solution was centrifuged at 2500g for 5 min at 4°C. The upper aqueous layer was filtered, lyophilized and dissolved in 50 μl of methanol. Ophthalmate measurements were performed using a TSQ Quantum Ultra Mass Spectrometer (Thermo Finnigan).

Perfusion and immunohistochemistry

Adult male Swiss Webster mice were anaesthetized using isoflurane, and using sterile PBS, the blood from the tissues and brain was flushed out. A 4% paraformaldehyde (PFA) solution was then flushed through the animal until the limbs became rigid. The brain was removed and kept in a 4% PFA solution for 24–48 h at 4°C. The brain was then transferred to a solution of 30% sucrose where it was kept until it was ready to be sectioned. Coronal sections were cut at 20 μm thickness. Sections were blocked with 4% goat serum in PBS with 0.3% Triton X-100 for 1 h and were then incubated with the tyrosine hydroxylase (TH) antibody overnight. The sections were then washed three times with PBS, and were then incubated with the secondary antibody for 1 h, washed three times again, and then mounted onto gelatin-coated glass slides. Sections were then imaged using a BZ-9000 fluorescence microscope (Keyence), and TH-positive cells were counted.

Cannulation surgery

Swiss Webster mice were anaesthetized with isoflurane (5% for induction; 1%–3% to sustain). Once anaesthetized, each mouse was placed on the stereotaxic device and implanted with a stainless-steel cannula (20G, 2.5 mm length) into the brain ventricle (0.2 mm posterior and 1 mm lateral to bregma and 2.3 mm below the surface of the skull). Mice were allowed to recover for 7 days.

Assessment of locomotor activity

Mice were placed in a locomotor test chamber (40 × 40 × 38 cm³) and the horizontal locomotor activity was monitored with a 16 × 16 photobeam array (San Diego Instruments) located 1.25 cm above the floor of the enclosure. Mice were transported to the activity chamber room at least 30 min prior to placement into the activity chambers. All mice were given time to acclimatize to the chamber prior to injections, and locomotor activity was recorded for 20–24 h following drug administration. Mice were randomly distributed to each treatment group with all proper controls being run in parallel. OA peripheral doses were selected based on previous studies that established the pharmacokinetics of OA in the plasma and peripheral tissues.^15,16 For central administration, OA was initially delivered to the brain through intracerebroventricular (i.c.v.) injection at a high dose of 20 µM, chosen empirically based on doses typically used for glutathione administration.^17-19 Subsequent dose-response curve analysis was performed with OA doses below 20 µM, specifically targeting those near the EC50 and the lowest effective dose. The doses of NPS2143 were selected based on previous studies, substantiating that these specific doses effectively block the CaSR in mice.^20-22

Cyclic AMP assay

Human embryonic kidney 203 (HEK293T) cells were grown in Dulbecco’s modified Eagle Medium (DMEM) adjusted to contain 10% fetal bovine serum (FBS), 1% penicillin and streptomycin at 5%–10% CO₂ and 37°C. HEK293T cells were transiently transfected to express both the pGloSensor-22F cAMP plasmid and a CaSR plasmid. Cells were transfected using the jetPRIME transfection reagent using the standard conditions of 5 μg of each plasmid and 20 μl of the reagent. After an overnight incubation in 37°C with 5%–10% CO₂, the cells were then ready to be used. On the day of the experiment, cells were washed with PBS and detached using 0.05% trypsin. The cells were then pelleted and resuspended in CO₂-independent media, 10% FBS and the GloSensor cAMP reagent. The cells were incubated at room temperature for 2 h and gently mixed every 15 min to prevent the cells from settling. The cells were then transferred to a 96-well plate and luminescence was measured using the MicroBeta2 2450 Microplate counter (Perkin Elmer). Basal luminescence level was recorded prior to assay.

Radioligand binding assay

Sections were pre-incubated in buffer (50 mM Tris), pH 7.4 for 30 min at 4°C. Total binding was determined by incubating sections in buffer containing varying concentrations of ³H-OA (Moravek, Inc) for 60 min at room temperature. Non-specific binding of ³H-OA was defined by that seen in the presence of 10μM NPS-2143. The incubation was terminated by washing the sections in an ice-cold (4°C) incubation buffer for 10 min. The sections were then dipped in ice-cold distilled water and dried in a stream of cold air. Sections were scraped off of the slides and placed into vials containing 4.5 ml of scintillation fluid and were subsequently measured at a scintillation counter.

Molecular docking

To predict the experimental binding modes and affinities of L-DOPA and ophthalmate to the CaSR, the following steps were conducted. First, the crystal structure of the calcium sensing receptor was retrieved from a protein database (PBD ID #7M3F),²³ and UCSF Chimera^24,25 was used to prepare the structure by removing water molecules and other unwanted entities. The chemical structures of L-DOPA (PubChem SID 3648) and ophthalmate (PubChem SID 254741470) were obtained, protonated and converted into PDBQT format using Open Babel. Autodock Vina^26,27 was used for molecular docking, and simulations conducted under experimental conditions of pH 4.5 (for X-ray) and in an aqueous buffer. Docking was performed with L-DOPA and ophthalmate targeting chain A of 7M3F in the active state. The docking configurations are based on the Trp ligand binding site reported in the previous report,²³ with a grid box size of 40 × 40 × 40, coordinates of 190.442, 212.175, 134.085 for L-DOPA and 210.238, 186.017, 134.231 for ophthalmate and spacing of 0.375 Å. The genetic algorithm was run 100 times for each ligand. After completing molecular docking, the predicted binding poses, interaction and ligand binding affinities were determined.

Statistical analysis

Statistical analyses of data were carried out using GraphPad Prism (GraphPad Software, Inc.). Data were presented as means ± standard error means (SEM). Results were analysed by unpaired Student’s t-test, one-way and two-way ANOVA, followed by the appropriate post hoc comparisons, and P < 0.05 was considered statistically significant.

Results

Brain AADC inhibition alters L-DOPA-induced motor activity in reserpine-treated PD mice

Building upon our prior observations in rats, we investigated the effects of central AADC inhibition, mediated by NSD1015, on L-DOPA-induced motor behaviours in reserpine-treated mouse models of PD. As expected, reserpine treatment led to hypoactivity in mice (Supplementary Fig. 1). We first administered L-DOPA (100 mg/kg) with and without NSD1015 (100 mg/kg), which prevents its conversion to dopamine inhibiting AADC^28,29 (Fig. 1A). All treatments were administered in combination with the peripheral AADC inhibitor benserazide (25 mg/kg). No alteration of motor activity was found in control animals (Fig. 1B and C). In the control group, motor activity remained largely unchanged (Fig. 1B and C). However, in the reserpine-treated mice, L-DOPA treatment noticeably increased activity, persisting up to 4 h (Fig. 1C). Remarkably, reserpinized-mice pre-administered NSD1015 showed a more pronounced motor response to L-DOPA than their counterparts treated with L-DOPA alone (Fig. 1D and E). This amplified response had an onset delay of approximately 120 min and was sustained for a longer duration than that induced by L-DOPA alone (Fig. 1D). While the last-duration motor effects of L-DOPA remained dose-dependent, the modulating effects of varying NSD1015 doses were less distinct (Fig. 1F–K).

Figure 1.

Motor response to L-DOPA is enhanced by inhibition of DOPA decarboxylase in mouse model of Parkinson’s disease. (A) The biosynthesis of dopamine from L-DOPA and its proposed inhibition by benserazide and NSD1015 (NSD) in the periphery and the CNS, respectively. (B and C) Effects of L-DOPA and NSD on normal mice. The mice were injected subcutaneously (s.c.) with a vehicle, and 18 h later, injected intraperitoneally (i.p.) with either NSD1015 or saline, followed by an L-DOPA/benserazide (100/25 mg/kg) or saline injection 30 min after. Motor activity was monitored for 20 h. Data represent (B) the time course and (C) the area under the curve (AUC) of the effects of L-DOPA, NSD, and combination of L-DOPA and NSD. One-way ANOVA, followed by Tukey’s post hoc test, ns = not significant. Values are expressed as mean ± standard error of the mean (SEM), n = 8 for each group. (D and E) Effects of L-DOPA and NSD on reserpinized mice: Mice were injected (s.c.) with reserpine 1 mg/kg, and 18 h later, mice were injected with NSD or saline, followed by an L-DOPA injection 30 min after. Locomotion was monitored for 20 h. Data represent the (D) time course of the effect of L-DOPA alone and L-DOPA in conjunction with NSD, and (E) the AUC. One-way ANOVA, followed by Tukey’s post hoc test, ****P < 0.0001. Values are expressed as mean ± SEM, n = 6–8 for each group. (F–K) Effects of varying doses of L-DOPA and NSD on motor activity in reserpine-treated mice. Mice were injected (s.c.) with reserpine 1 mg/kg and 18 h later, mice were injected (i.p.) with varying doses of NSD, followed by varying doses of L-DOPA 30 min after. Locomotion was monitored for 20 h. The same data are presented in two clusters of figures. (F–H) Data represent the time course of the effect of varying doses of NSD with fixed doses of L-DOPA (F: 50 mg/kg, G: 100 mg/kg, H: 200 mg/kg). (I–K ) Data represent the timecourse of the effect of varying doses of L-DOPA with fixed doses of NSD (I: 50 mg/kg, J: 100 mg/kg, K: 200 mg/kg). Values are expressed as mean ± SEM, n = 6–8 for each group.

To determine whether the enhanced activity with the L-DOPA and NSD1015 combination was exclusive to L-DOPA or applicable to other dopamine agonists, we administered a dopamine-releasing agent (amphetamine) and a dopamine receptor D1/D2 agonist (apomorphine), along with NSD1015, to reserpine-treated mice. We found that both agents displayed motor activity patterns consistent with their expected individual effects, indicating NSD1015-associated hyperactivity is linked to L-DOPA (Supplementary Fig. 2A and B). Additionally, to explore the potential role of dopamine receptors in this effect, mice were pre-treated with a D2 receptor antagonist (haloperidol). Notably, haloperidol failed to block the hyperactivity induced by the L-DOPA/NSD1015 combination (Supplementary Fig. 2C and D), implying the possibility of alternative mechanisms or pathways being triggered by the L-DOPA and NSD1015 combination.

NSD1015/L-DOPA-induced hyperactivity in PD mice is associated with a surge in ophthalmate levels

To elucidate the neurochemical consequences of combined NSD1015 and L-DOPA administration in PD mice, we used both targeted and untargeted metabolomic analyses on whole brain and striatal tissues (Fig. 2A–G, Supplementary Tables 2 and 3 and Supplementary Fig. 3). Our observations revealed that L-DOPA administration, when combined with AADC disruption, caused remarkable changes in the levels of L-DOPA and its associated metabolites within the brain and striatum (Figs 2C–G, 3A and B and Supplementary Table 3). Interestingly, dopamine levels in the L-DOPA-treated group correlated closely with motor activity patterns, notably dropping at the 7-h mark compared to the levels observed at 2 h (Fig. 3B). In contrast, dopamine concentrations in the NSD1015/L-DOPA group remained consistent between these time points. This lends credence to the idea that dopamine may not be the primary agent inducing hyperactivity in these conditions, hinting at the possible involvement of alternative, non-dopamine pathways. Our data also showed that AADC inhibition via NSD1015 shifts the metabolic processing of aromatic amino acids, prioritizing methylation, deamination and hydroxylation over decarboxylation (Supplementary Fig. 4A–C and Supplementary Table 3). This metabolic profile mirrors that observed in cases of AADC deficiency.^30,31 Although we noticed a 13-fold rise in 3-O-methyldopamine (3-MT) levels at the hyperactivity peak (7 h) in the NSD1015/L-DOPA-treated mice, its reduced concentrations at the 7-h mark (compared to the 2-h mark) suggest that 3-MT is not the sole contributor to the heightened activity observed at 7 h (Fig. 2C–G and Supplementary Fig. 4B). An increase in the stress hormone corticosterone was observed at 7 h in the NSD1015/L-DOPA-treated mice, accompanied by alterations in energy pathways, notably in glycolysis and the mitochondrial tricarboxylic acid (TCA) cycle metabolism pathways (Supplementary Fig. 5A and B and Supplementary Table 3).

Figure 2.

Motor response to L-DOPA and aromatic amino acid decarboxylase (AADC) inhibitor correlates with alterations in brain and striatum metabolites, with highest alteration in ophthalmate levels. (A) The study’s experimental design and timeline. Mice were injected subcutaneously (s.c.) with reserpine 1 mg/kg. After 18 h, the mice were treated with NSD1015 (NSD) or saline, followed by L-DOPA/benserazide administration 30 min later. Brain tissues were collected from the two treatment groups at two time points (2 and 7 h after L-DOPA administration). The brains were divided into two hemispheres, with one hemisphere homogenized entirely, and the other hemisphere was used to extract the striatum, n = 6 for each group. Diagram created using images from BioRender.com. (B) Unsupervised principal component analysis (PCA) of the metabolomics data of mouse whole brain and striatum from the two treatment groups at the two time points. (C) Volcano plot illustrating the differential expression of metabolites in the brains of L-DOPA + NSD treated mice compared to L-DOPA treated mice, with brain samples taken 2 h post-DOPA injection. (D) Volcano plot showing the differential expression of metabolites in the brains of l-DOPA + NSD treated mice versus L-DOPA treated mice, with brain samples obtained 7 h after L-DOPA injection. One-way ANOVA contrasts: significantly increased metabolites in red, and significantly decreased metabolites in blue. (E) Volcano plot displaying the differential expression of metabolites in the brains of L-DOPA + NSD treated mice at 7 h compared to L-DOPA + NSD treated mice at 2 h post-DOPA injection. One-way ANOVA contrasts: significantly increased metabolites in red, and significantly decreased metabolites in blue. (F) Volcano plot demonstrating the differential expression of metabolites in the striatum tissues of L-DOPA + NSD treated mice in comparison to L-DOPA treated mice, with brain samples collected 7 h following L-DOPA injection. One-way ANOVA contrasts: significantly increased metabolites in red, and significantly decreased metabolites in blue. (G) List of top altered metabolites in the brains of different treatment groups (≥2-fold change, q < 0.05, one-way ANOVA contrasts), x-axis represents fold changes.

Figure 3.

Motor response to L-DOPA and aromatic amino acid decarboxylase (AADC) inhibitor is associated with alterations in dopamine and ophthalmate synthesis pathways. (A) Box plot legend showing the range, median and quartiles. (B) The fold-changes in the major components of dopamine synthesis pathway after L-DOPA and L-DOPA/NSD1015 (NSD) administration. (C) The fold-changes in ophthalmate and the key metabolites of the proposed pathways leading to its synthesis. The asterisk is used to compare metabolite levels within the same group at the two different time points, whereas the hashtag is used to compare metabolite levels between the two treatment groups at the same time point. ^*,#q < 0.05, ^**,##q < 0.01, ^***,###q < 0.001, ^****,####q < 0.0001. 2-AB = 2-aminobutyrate, α-KB = α-ketobutyrate; α-KG = α-ketoglutarate; GCS = glutamate-cysteine synthase; Glu = glutamate; Gly = glycine; GS = glutathione synthase; Hcy = homocysteine; SAH = S-adenosylhomocysteine; SAM = S-adenosylmethionine.

The most striking finding was the pronounced surge in ophthalmic acid (OA; L-γ-glutamyl-L-2-aminobutyryl-glycine, γ-Glu-2AB-Gly) following NSD1015 pretreatment. At the hyperactivity peak (7 h), ophthalmate levels in the whole brain of L-DOPA/NSD1015-treated animals rose 20-fold compared to the 2-h measurement and exhibited an 8-fold increase relative to L-DOPA-treated mice (Fig. 2D–G and Fig. 3C).

Ophthalmate restores motor activity in MPTP PD mouse model

We investigated whether the observed hyperactivity following L-DOPA/NSD1015 treatment in PD mouse models was directly caused by elevated OA levels. In our first set of experiments on the MPTP model of PD (Fig. 4A–C), peripheral administration (i.p.) of OA did not elicit any motor response in MPTP-treated mice (Fig. 4D). Given the limited data on OA’s pharmacokinetics, we were uncertain if the absence of a motor response was due to OA being intrinsically inactive or its inability to cross the BBB. We, therefore, administered OA directly to the brain through intracerebroventricular injection in MPTP-treated mice. Remarkably, OA significantly enhanced motor activity in these mice in a dose-dependent manner (Fig. 4E–H). Notably, this enhanced motor activity persisted for around 24 h, peaking during the initial 10 h (Fig. 4G and H). To further explore ophthalmate’s capability to cross the BBB, we injected deuterated ophthalmate D5-OA into mice and utilized MS to monitor its distribution across various tissues, including blood, liver, kidney and brain. Our findings showed that, following peripheral administration, D5-OA was detectable in the plasma and other organs but was absent from the brain (Fig. 4I). This highlights that ophthalmate does not cross the BBB, suggesting that the surge in ophthalmate levels following NSD1015/L-DOPA treatment was likely a result of its synthesis in the brain.

Figure 4.

Central but not peripheral ophthalmate rescues motor activity in MPTP mouse model of Parkinson’s disease. Mice were given MPTP injections intraperitoneally (i.p.) at 20 mg/kg over 3 days. Ophthalmate was administered 24 h after the final MPTP injection, and motor activity was monitored for the following 20 h. For the central ophthalmate experiments, mice underwent a surgical procedure to implant a cannula for intracerebroventricular (i.c.v.) injections a week prior to receiving the MPTP injection. (A) Representative immunostaining of tyrosine hydroxylase (TH, green) and DAPI (blue), showing the effect of MPTP on dopamine neuronal loss in the substantia nigra; (B) the time course of the effect of MPTP; (C) the area under the curve (AUC) of the effect of MPTP during the 20-h experiment time. Unpaired t-test, ****P < 0.0001. (D) Peripheral ophthalmate injection did not induce motor activity in MPTP-treated mice. Data represent the time course of the effect of ophthalmate injected i.p. at three different doses and are expressed as mean ± standard error of the mean (SEM), n = 8 for each group. (E–H) The effect of central administration of ophthalmate on the motor activity of MPTP-treated mice. The data presented include (E) the time course of the effect of ophthalmate injected (i.c.v.) at four different doses; (F) the AUC of the effect of different doses of ophthalmate during the 20-h experiment time; (G) the AUC of the effect of different doses of ophthalmate during the first 10 h of the experiment time; and (H) the AUC of the effect of different doses of ophthalmate during the 11–20 h of the experiment time. In F–H, one-way ANOVA, followed by Tukey’s post hoc test, *P < 0.05, ****P < 0.0001, ns = not significant. Data are expressed as mean ± SEM, n = 6–8 for each group. (I) Deuterated ophthalmate (D5-OA) levels following peripheral administration. Mice were injected (i.p.) with D5-OA and the brain, blood, kidney and liver samples were collected 10 min after injection. Data represent the interpolated concentrations of D5-OA using mass spectrometry (MS) and are expressed as mean ± SEM, n = 6 for each group.

Ophthalmate binds to and activates the CaSR

To elucidate the mechanism underlying ophthalmate’s influence on motor activity, we probed ophthalmate’s interactions with potential receptor sites in brain sections, using radioligand binding saturation experiment with increasing concentrations of labelled ophthalmate (³H-OA). A saturating concentration of unlabelled ophthalmate was used to assess non-specific binding. Our data revealed reversible binding of ³H-OA to specific and saturable sites in the brain, with a single moderate-affinity population (Kd 2.61 ± 0.36 µM) (Fig. 5A). Given the structural similarities between ophthalmate and GSH, and past evidence suggesting modulation of CaSR by GSH and its gamma-glutamyl-tri-peptides analogues,^32,33 we sought to explore whether ophthalmate might interact with brain CaSR. CaSR is a class C G-protein-coupled receptor (GPCR) that is activated by extracellular calcium (Ca²⁺),^34-37 and can activate multiple signalling pathways through Gq/11, Gi/o, G12/13 and Gs proteins.^34,38-40 Aromatic L-amino acids such as L-phenylalanine, L-tyrosine and L-tryptophan increase the sensitivity of CaSR to Ca²⁺ and, thus, are considered positive allosteric modulators of the receptor.^41-46 Ca²⁺ ions and aromatic L-amino acids have also been proposed to act as co-agonists of the receptor.^42,45,47 We, therefore, sought to determine whether the saturable sites to which ophthalmate binds in brain sections are CaSR. We conducted a similar radioligand binding saturation experiment in the presence of a saturating concentration of an unlabelled CaSR antagonist (NPS-2143). At low concentrations of ³H-OA (0.1–0.5 µM), 80–85% of the total binding of the labelled peptide to brain sites was inhibited by NPS-2143 (Fig. 5B). The specific component of binding was saturable, while the non-specific binding was linearly dependent on ophthalmate concentration. The calculated K_d of ophthalmate binding to CaSR was 1.62 ± 0.22 µM. ³H-OA binding was also inhibited by a saturating concentration of Ca²⁺ and L-DOPA, rendering a K_d for ³H-OA binding of 4.06 ± 1.47 µM and 5.55 ± 0.85 µM, respectively (Fig. 5B). While this finding does not conclusively prove that L-DOPA binds to CaSR, it indicates that ophthalmate and L-DOPA can bind to the same target in the brain. ³H-OA binding was also inhibited by combinations of saturating concentrations of L-DOPA/NPS-2143, Ca²⁺/L-DOPA and Ca²⁺/NPS-2143, rendering K_d values for ³H-OA binding of 3.99 ± 0.05, 5.28 ± 0.57, and 5.99 ± 0.1, respectively (Fig. 5C). Lastly, using a radioligand competitive (displacement) experiment, with a fixed concentration of ³H-OA (2.5 µM), we found that Ca²⁺ inhibited the binding of ³H-OA in a dose-dependent manner, with a K_i of 0.98 ± 0.13 µM (Fig. 5D), thereby verifying the binding of ophthalmate to calcium binding sites in the brain. Neither SKF-82958, a D1 dopamine receptor ligand, nor haloperidol, a D2 dopamine receptor ligand, displaced ³H-OA binding (Supplementary Fig. 6).

Figure 5.

Ophthalmate binds to, and activates, CaSRs. (A and B) Saturation curve of ³H-OA binding to mouse brain sections in the presence of unlabelled ophthalmate, NPS2143, Ca²⁺ and L-DOPA. ³H-OA binding was carried out as described in the methods section. (A) Reprehensive plot of total, specific and non-specific binding of ³H-OA to mouse brain sections. Non-specific binding was determined as the levels of ³H-OA binding in the presence of 100 μM unlabelled ophthalmate. (B) Representative plot of specific binding of ³H-OA binding to mouse brain sections, where non-specific bindings were defined as the levels of ³H-OA binding in the presence of 10 μM NPS-2143, 100 μM calcium and 100 μM L-DOPA. (C) Representative plot of specific binding of ³H-OA binding to mouse brain sections, where non-specific binding was defined as the level of ³H-OA binding in the presence of combinations of NPS-2143+ Ca²⁺, L-DOPA + Ca²⁺, and NPS-2143 + L-DOPA. In all binding experiments, the degree of binding is expressed in disintegrations per minute (dpm). Data represent binding from three experiments for each point (total 36 sections repeat for each point). (D) Inhibition of ³H-OA binding to mouse brain sections by Ca²⁺. Competition experiments were carried out as described in the methods. Radioligand binding assay was performed in the presence of 2.5 µM ³H-OA and increasing concentrations of Ca²⁺. Each point represents the mean ± standard error of the mean (SEM) of at least three measurements from three experiments. (E–G) Forskolin- stimulated cyclic AMP (cAMP) Glo-sensor luminescence responses in Human embryonic kidney 203 (HEK293) cells transiently transfected with the Glo-sensor cAMP biosensor and the CaSR plasmid. [E(i–iii)] Representative dose-response curves of cAMP signal for (i) Ca²⁺, (ii) OA and (iii) L-DOPA, with and without CaSR antagonist NPS-2143. [F(i–iii)] Maximum signalling capacity (Emax) of (i) Ca²⁺, (ii) OA and (iii) L-DOPA at CaSR in the absence and presence of CaSR antagonist NPS-2143. [G(i–iii)] EC50 of (i) Ca²⁺, (ii) OA and (iii) L-DOPA at CaSR in the presence and absence of CaSR antagonist NPS-2143. Unpaired t-test, *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001, ns = not significant. (H–K) Docking models ligand-bound states in CaSR binding site in domain A (named A in amino acid hereinafter). (H) Cartoon presentation of ligand-bound CaSR structure (domain A) in the closed-closed conformation (5 Å). (I) Interface analysis of tryptophan (TRP)-bound state in CaSR; binding is shown with Ser170A, Ser147A, Ala298A, and Thr 145A and Ala168A. (J) Interface analysis of OA bound state in CaSR; binding is shown with Ser147A, Gly148A, Tyr218A, Ser170A, Asp216A, and Ala168A, Val149A. (K) Interface analysis of L-DOPA bound state in CaSR; binding is shown with Tyr218A, Ser170A, Asp216A, and Ala168A. Green curve shows the transition state of Ala298A and Thr 145A in the Trp-bound state, Gly146A in the OA-bound state and Tyr218A in DOPA-bound state.

To determine whether ophthalmate functions as an agonist or antagonist at CaSR, we employed a cAMP-luminescence assay using HEK cells that express CaSR and forskolin to amplify cAMP production. Our results demonstrated ophthalmate’s dose-dependent activation of the Gi-coupled CaSR, reducing forskolin-stimulated cAMP. The EC₅₀ for ophthalmate was determined 1.55 ± 0.1 µM, while for Ca²⁺, an orthosteric agonist of CaSR, the EC₅₀ was 0.7 ± 0.01 µM [Fig. 5E(i and ii)]. Under the same conditions, L-DOPA also activated the CaSR at an EC₅₀ of 2.44 ± 0.45 µM (Fig. 5Eiii). L-DOPA agonistic action on CaSR is noteworthy given that other aromatic amino acids, such as L-tryptophan and phenylalanine, are known to act as allosteric agonists at CaSR.^41-46 In the presence of NPS-2143, a negative allosteric modulator (NAM), the CaSR displayed a decrease in maximum signalling capacity (Emax) of ophthalmate and Ca²⁺ [Fig. 5E(i–iii) and F(i–iii)], and rightward shift of the dose-response curves (EC₅₀) of ophthalmate, L-DOPA and Ca²⁺ [Fig. 5G(i–iii)]. Our results provide evidence that both ophthalmate and L-DOPA bind to and activate CaSR at EC₅₀ values comparable to that of the orthosteric agonist (Ca²⁺).

While these experiments did not identify the specific binding sites of ophthalmate and L-DOPA to CaSR, we used computational modelling and structure-based docking to propose a model for their interaction. Our model predicted that ophthalmate binds to the CaSR in a manner similar to L-tryptophan (TRP), specifically targeting the L-aromatic acid binding pocket (Fig. 5H–K).

CaSR mediates the motor-enhancing effects of L-DOPA/NSD1015 and ophthalmate in PD mice

We lastly examined the involvement of ophthalmate binding to the CaSR in motor activity. Our findings showed that when we used the CaSR antagonist NPS-2143, the motor-enhancing effects of L-DOPA/NSD1015 in MPTP-treated mice were suppressed (Fig. 6A and B). This suggests that CaSR activation contributes to the increased motor activity observed. Moreover, using NPS-2143 as a pretreatment revealed a dose-dependent decrease in ophthalmate-induced motor activity in both the MPTP (Fig. 6C–F) and reserpine (Fig. 6G and H) PD models. This substantiates that ophthalmate’s effect on motor function is largely due to its interaction with the CaSR.

Figure 6.

CaSR mediates the motor-enhancing effects of L-DOPA/NSD1015 and ophthalmate in Parkinson’s disease mice. (A and B) CaSR antagonist NPS-2143 inhibition of motor response induced by L-DOPA/NSD1015 in MPTP-treated mice. Mice were injected intraperitoneally (i.p.) with MPTP 20 mg/kg for 3 days. The following day, mice were injected (i.p.) with NSD1015 or saline (Sal), followed by a Sal or L-DOPA injection 30 min after, with or without NPS2143. Locomotion was monitored for 20 h. Data represent (A) the time course and (B) the area under the curve (AUC) of the effect of NSD1015 and L-DOPA with or without NPS2143. One-way ANOVA, followed by Tukey’s post hoc test. Values are expressed as mean ± standard error of the mean (SEM), n = 8 for each group. ****P < 0.0001. (C–F) CaSR antagonist inhibits ophthalmate-induced motor response in MPTP-treated mice. Mice were anaesthetized and underwent surgery where a cannula was implanted for future intracerebroventricular (i.c.v.) injections. Following recovery, the mice were injected (i.p.) with MPTP 20 mg/kg for 3 days. On the day following the final MPTP treatment, mice were injected i.c.v. with ophthalmate at (C and D) 5 µM and (E and F) 10 µM with or without NPS2143 (10 µM and 20 µM). Locomotion was monitored for 20 h. Data represent (C and E) the time course and (D and F) the AUC of the effect of ophthalmate with and without NPS2143 on motor activity. In D and F, one-way ANOVA, followed by Tukey’s post hoc test. **P < 0.01, ****P < 0.0001. Values are expressed as mean ± SEM, n = 6–8 for each group. (G and H) CaSR antagonist NPS2143 inhibits ophthalmate-induced motor response in reserpine-treated mice. Mice were injected subcutaneously (s.c.) with reserpine 1 mg/kg, and 18 h later, mice were injected (i.c.v) with ophthalmate (10 µM) with or without NPS2143 (20 µM). The (G) time course and (H) the AUC of the effect of ophthalmate with and without NPS2143 on motor activity. In H, one-way ANOVA, followed by Tukey’s post hoc test. ****P < 0.0001.

Discussion

The central role of dopamine in governing motor functions has been deeply accepted in the scientific understanding for years. This view posits dopamine as the primary neurotransmitter mediating such functions. Our study introduces a paradigm shift in this understanding by highlighting the significant role of the tripeptide ophthalmic acid in mediating motor activity, particularly in conditions where the dopamine system is disrupted.

Dopamine-independent role of L-DOPA in movement

Earlier research proposed that L-DOPA may mediate its effects in part by acting directly, or via its metabolites, on alternative non-dopaminergic neurotransmitter systems involved in motor behaviour.^48-50 For example, the presence has long been demonstrated of neurons containing L-DOPA that release it in a calcium-dependent fashion upon stimulation.^51,52 Additionally, L-DOPA enhances the release of various neurotransmitters, including dopamine, noradrenaline, glutamate, and GABA,^53-55 suggesting it may act as a neurotransmitter or neuromodulator in the CNS.^56,57 Additionally, L-DOPA undergoes nonenzymatic conversion into biologically active compounds like 24,5-trihydroxyphenylalanine (TOPA), which can elicit neuronal responses in dopaminergic pathways independent of dopamine receptor stimulation, including neuronal firing and membrane depolarization.^58-60

Our earlier research in a rat model of PD demonstrated that L-DOPA, when its conversion to dopamine in the brain is inhibited, leads to delayed but significantly enhanced and prolonged motor activity.⁶ Our current study’s observations in a mouse reserpine model of PD mirror those from earlier rat studies and are substantiated using another mouse PD model (MPTP), suggesting the generalizability of our results. While L-DOPA does not seem to be the molecule that directly induces the long-lasting enhanced activity—evidenced by its negligible levels during hyperactive periods—its presence is indispensable for activating the pathways responsible for this motor activity. This is demonstrated by our findings that both the intensity and duration of the motor activity strongly correlate with L-DOPA doses.

Interestingly, the long-duration response of L-DOPA has been recognized in early studies.^61-63 Researchers distinguished between the short-duration response, which lasts a few hours and correlates with plasma L-DOPA concentrations and the long-duration response, which persists after the elimination of L-DOPA, gradually declining back to baseline over a period of days to weeks.^64,65 The long-duration response to L-DOPA has been largely overlooked for the past five decades, with only a handful of studies confirming early observations.^64-70 However, the mechanisms underlying the long-duration response remained poorly understood.

Given the dominant hypothesis on dopamine replacement therapy in PD has focused on centrally formed dopamine, research into the effects of central AADC inhibitors has been limited. A study from the 1970s showed that an AADC inhibitor with both peripheral and central effects, could potentiate the therapeutic effect of L-DOPA in PD patients.⁷¹ The study recommended extending the search for effective decarboxylase inhibitors to compounds that can cross the BBB, yet subsequent investigations have only focused on AADC inhibitors lacking this ability.⁷²

The neurobiological contexts of ophthalmate surge

Originally identified in the calf lens,⁷³ ophthalmate’s presence has since been detected in various tissues in advanced animals and even microorganisms.^74-77 Structurally, ophthalmate is analogous to glutathione (GSH; L-γ-glutamyl-L-cysteinyl-glycine, γ-Glu-Cys-Gly), which functions as an essential antioxidant and detoxifying agent in biological systems.⁷³ The synthesis pathways of both GSH and ophthalmate share common enzymes: glutamate-cysteine synthase (GCS) and glutathione synthase (GS), which sequentially produce ophthalmate (Fig. 3C).^78,79 The notable increase in ophthalmate levels in the brain and striatum of L-DOPA/NSD1015-treated mice suggests that these two drugs have an effect on glutathione metabolism and ophthalmate’s synthesis. GSH and ophthalmate synthesis is closely linked to methionine metabolism via the transsulfuration pathway, which is facilitated by cystathionine β-synthase (CBS) and cystathionine γ-lyase (CSE).⁸⁰ CSE catalyses the cleavage of cystathionine to generate equal amounts of cysteine (Cys) and 2-ketobutyric acid (2KB), which is subsequently converted to 2-aminobutryate (2-AB) through a transamination reaction with glutamate and other keto-carboxyl compounds as the donor substrate and aspartate aminotransferase (AST) as the catalytic enzyme.^81-83

Our study revealed a pronounced elevation in ophthalmate levels under conditions where the conversion of L-DOPA to dopamine was inhibited. This unexpected surge in ophthalmate is indicative of alternative metabolic or signalling pathways being activated when traditional dopaminergic pathways are disrupted. This discovery raises pertinent questions: What triggers the synthesis and release of ophthalmate under such conditions? Is there an intrinsic neural mechanism that compensates for reduced dopamine by increasing ophthalmate? Based on our findings, the elevations of ophthalmate levels observed in the NSD/L-DOPA group may be related to the alteration of methylation pathways, with a notable increase in methylated metabolites and hydroxyl-carboxyl metabolites observed within this group. The scarcity of research on ophthalmate implies that its physiological importance is yet to be established. Historically, two primary scenarios have been elucidated for ophthalmate synthesis: (i) in the context of Escherichia coli lacking PLP-dependent proteins^77,84; and (ii) under conditions necessitating GSH synthesis, positioning ophthalmate as a potential indicator for oxidative stress and the subsequent depletion of cysteine or GSH.^14,15 It is noteworthy that conditions leading to GSH depletion, such as the hepatotoxic effects of acetaminophen overdose, or systemic responses to starvation and acute stress, have been associated with elevated ophthalmate levels.^14,85-88 This is due to the fact that, in environments favoring reductive reactions, GSH inhibits GCS, suppressing ophthalmate synthesis. In contrast, during oxidative stress, the depletion of GSH activates GCS, leading to the production of ophthalmate.^14,87,88

In light of these two contexts, it can be inferred that the rise in ophthalmate levels observed in our study may be linked to the effects of the combination of L-DOPA and NSD1015 on AADC, oxidative stress, and GSH metabolism. First, NSD1015 inhibits AADC, a PLP-dependent enzyme,^28,29 mimicking the production of ophthalmate observed in E. coli lacking PLP-dependent proteins. Second, L-DOPA, due to its highly reactive nature, can induce oxidative stress. When its conversion to dopamine is inhibited, the excess L-DOPA interacts and conjugates with GSH, resulting in GSH depletion.^89,90 The observations of altered PLP and GSH levels in the NSD1015/L-DOPA group support these notions (Fig. 3B and C). These findings suggest that the rise in ophthalmate levels observed in our study may be a result of a combination of factors. These include shifts in PLP and GSH metabolism influenced by NSD1015’s impact on AADC and L-DOPA’s propensity for oxidative stress, along with enhanced methylation pathways that facilitate the synthesis of both ophthalmate and GSH. Further supporting this conclusion is the notable rise in the stress hormone corticosterone, along with shifts in energy pathways, specifically, the glycolysis and mitochondrial TCA cycle metabolism pathways.

Ophthalmate emergence in the regulation of motor function

Historically, dopaminergic pathways have been central to our comprehension of movement disorders, especially with dopamine’s connection to conditions like PD. The effectiveness of dopaminergic drugs in treating motor symptoms of PD further reinforced this understanding. Yet, our study highlights a notable observation: pronounced motor activity persists even in dopamine-deprived states. This suggests alternative mechanisms and pathways influencing motor activity. Our findings point toward ophthalmate’s significant role in influencing motor activity, specifically through its interaction with the CaSR signalling system. While CaSR has historically been recognized for its integral role in systemic calcium regulation,^91-94 its implications within the CNS and its potential influence on motor activities have largely remained underexplored. The direct connection between ophthalmate and CaSR, revealed by our study, presents a new perspective in understanding neural signalling beyond the conventional neurotransmitters. By elucidating the connection between ophthalmate and CaSR signalling, we have established a foundational basis for the development of potential new therapeutic strategies targeting this pathway. Leveraging this pathway holds the promise of refining therapeutic strategies for movement disorders, offering potential benefits with fewer complications compared to traditional dopaminergic approaches. If ophthalmate can indeed offer similar or superior therapeutic benefits with a reduced side-effect profile, it could revolutionize treatment approaches for movement disorders.

While our study has highlighted the significant role of ophthalmate in modulating motor functions, many questions remain. For example, are there other neurological conditions, beyond PD, where ophthalmate’s role might be crucial? Additionally, are there specific neurons that synthesize and release ophthalmate in the brain? Lastly, are there other receptors or pathways influenced by ophthalmate that haven not been identified yet? Examining into these questions will extend our understanding of ophthalmate’s role in the brain functions and open up avenues for therapeutic interventions. Future studies should also focus on confirming these results in primate models of PD to validate and broaden the applicability of our findings.

In summary, our study establishes the critical role of ophthalmate in regulating motor functions via its interaction with the CaSR. This discovery challenges the traditional understanding that primarily associates dopamine with motor functions. By identifying ophthalmate’s connection with the CaSR pathway, we open up new therapeutic opportunities for movement disorders.

Data availability

All data are available in the main text and Supplementary material. Data that support the findings of this study are available from the corresponding author upon request.

Funding

The work of G.W.A. was supported by the National Institutes of Health, National Institute of Neurological Disorders and Stroke (NS107671 to G.W.A.). The work of O.C. was supported by Eric L. and Lila D. Nelson Chair in Neuropharmacology.

Competing interests

A.A. and O.C. are inventors on a pending patent application that covers the role of ophthalmate and CaSR in motor functions. The remaining authors declare no competing interests.

Supplementary material

Supplementary material is available at Brain online.