Comprehensive proteomics of CSF, plasma, and urine identify DDC and other biomarkers of early Parkinson's disease.

Parkinson's disease (PD) starts at the molecular and cellular level long before motor symptoms appear, yet there are no early-stage molecular biomarkers for diagnosis, prognosis prediction, or monitoring therapeutic response. This lack of biomarkers greatly impedes patient care and translational research-L-DOPA remains the standard of care more than 50 years after its introduction. Here, we performed a large-scale, multi-tissue, and multi-platform proteomics study to identify new biomarkers for early diagnosis and disease monitoring in PD. We analyzed 4877 cerebrospinal fluid, blood plasma, and urine samples from participants across seven cohorts using three orthogonal proteomics methods: Olink proximity extension assay, SomaScan aptamer precipitation assay, and liquid chromatography-mass spectrometry proteomics. We discovered that hundreds of proteins were upregulated in the CSF, blood, or urine of PD patients, prodromal PD patients with DAT deficit and REM sleep behavior disorder or anosmia, and non-manifesting genetic carriers of LRRK2 and GBA mutations. We nominate multiple novel hits across our analyses as promising markers of early PD, including DOPA decarboxylase (DDC), also known as L-aromatic acid decarboxylase (AADC), sulfatase-modifying factor 1 (SUMF1), dipeptidyl peptidase 2/7 (DPP7), glutamyl aminopeptidase (ENPEP), WAP four-disulfide core domain 2 (WFDC2), and others. DDC, which catalyzes the final step in dopamine synthesis, particularly stands out as a novel hit with a compelling mechanistic link to PD pathogenesis. DDC is consistently upregulated in the CSF and urine of treatment-naïve PD, prodromal PD, and GBA or LRRK2 carrier participants by all three proteomics methods. We show that CSF DDC levels correlate with clinical symptom severity in treatment-naïve PD patients and can be used to accurately diagnose PD and prodromal PD. This suggests that urine and CSF DDC could be a promising diagnostic and prognostic marker with utility in both clinical care and translational research.

Comparing the effect of intravenous versus intracranial grafting of mesenchymal stem cells against parkinsonism in a rat model: Behavioral, biochemical, pathological and immunohistochemical studies.

Parkinson's disease (PD) is one of the most common neurodegenerative diseases worldwide. Currently applied therapeutic protocols are limited to improve the motor functions of patients. Therefore, seeking alternative regimes with better therapeutic impact is crucial. This study aims to validate the therapeutic impact of mesenchymal stem cell injection using two delivery methods, intracranial administration and intravenous administration, on rotenone (ROT)-induced PD model in rats. Our work included behavioral, biochemical, histological, and molecular investigations. Open field test (OFT) and rotarod tests were applied. Important oxidative stress, antioxidant and proinflammatory markers were monitored. Substantia Nigra and Striatum tissues were examined histologically and the molecular expression of DOPA decarboxylase, Tyrosine hydroxylase, and α-synuclein in neurons in these tissues were investigated. Our results showed that MSC grafting improved motor and memory impairments and oxidative stress status that were observed after ROT administration. Additionally, BM-MSCs application restored SOD and CAT activities and the levels of DA, L-Dopa, IL6, IL1ß, and TNFα. Moreover, MSC grafting overwhelmed the pathological changes induced by ROT and normalized the expression of Tyrosine hydroxylase, DOPA decarboxylase, and α-synuclein towards the control values in the Nigral and Striatal tissues of male rats. Conclusively, both administration routes improved motor function, protection of the nigrostriatal system, and improved striatal dopamine release. The observed beneficial effect of applying MSCs suggests potential benefits in clinical applications. No significant differences in the outcomes of the treatment would favor a certain way of MSC application over the other. However, the intravenous delivery method seems to be safer and more feasible compared to the intrastriatal method.

Detection of hidden intronic DDC variant in aromatic L-amino acid decarboxylase deficiency by adaptive sampling.

Aromatic l-amino acid decarboxylase (AADC) deficiency is an autosomal recessive neurotransmitter disorder caused by pathogenic DOPA decarboxylase (DDC) variants. We previously reported Japanese siblings with AADC deficiency, which was confirmed by the lack of enzyme activity; however, only a heterozygous missense variant was detected. We therefore performed targeted long-read sequencing by adaptive sampling to identify any missing variants. Haplotype phasing and variant calling identified a novel deep intronic variant (c.714+255 C > A), which was predicted to potentially activate the noncanonical splicing acceptor site. Minigene assay revealed that wild-type and c.714+255 C > A alleles had different impacts on splicing. Three transcripts, including the canonical transcript, were detected from the wild-type allele, but only the noncanonical cryptic exon was produced from the variant allele, indicating that c.714+255 C > A was pathogenic. Target long-read sequencing may be used to detect hidden pathogenic variants in unresolved autosomal recessive cases with only one disclosed hit variant.

L-Dopa decarboxylase modulates autophagy in hepatocytes and is implicated in dengue virus-caused inhibition of autophagy completion.

The enzyme L-Dopa Decarboxylase (DDC) synthesizes the catecholamine dopamine and the indolamine serotonin. Apart from its role in the brain as a neurotransmitter biosynthetic enzyme, DDC has been detected also in the liver and other peripheral organs, where it is implicated in cell proliferation, apoptosis, and host-virus interactions. Dengue virus (DENV) suppresses DDC expression at the later stages of infection, during which DENV also inhibits autophagosome-lysosome fusion. As dopamine affects autophagy in neuronal cells, we investigated the possible association of DDC with autophagy in human hepatocytes and examined whether DDC mediates the relationship between DENV infection and autophagy. We performed DDC silencing/overexpression and evaluated autophagic markers upon induction of autophagy, or suppression of autophagosome-lysosome fusion. Our results showed that DDC favored the autophagic process, at least in part, through its biosynthetic function, while knockdown of DDC or inhibition of DDC enzymatic activity prevented autophagy completion. In turn, autophagy induction upregulated DDC, while autophagy reduction by chemical or genetic (ATG14L knockout) ways caused the opposite effect. This study also implicated DDC with the cellular energetic status, as DDC silencing reduced the oxidative phosphorylation activity of the cell. We also report that upon DDC silencing, the repressive effect of DENV on the completion of autophagy was enhanced, and the inhibition of autolysosome formation did not exert an additive effect on viral proliferation. These data unravel a novel role of DDC in the autophagic process and suggest that DENV downregulates DDC expression to inhibit the completion of autophagy, reinforcing the importance of this protein in viral infections.

Large-scale proximity extension assay reveals CSF midkine and DOPA decarboxylase as supportive diagnostic biomarkers for Parkinson's disease.

BACKGROUND: There is a need for biomarkers to support an accurate diagnosis of Parkinson's disease (PD). Cerebrospinal fluid (CSF) has been a successful biofluid for finding neurodegenerative biomarkers, and modern highly sensitive multiplexing methods offer the possibility to perform discovery studies. Using a large-scale multiplex proximity extension assay (PEA) approach, we aimed to discover novel diagnostic protein biomarkers allowing accurate discrimination of PD from both controls and atypical Parkinsonian disorders (APD). METHODS: CSF from patients with PD, corticobasal syndrome (CBS), progressive supranuclear palsy (PSP), multiple system atrophy and controls, were analysed with Olink PEA panels. Three cohorts were used in this study, comprising 192, 88 and 36 cases, respectively. All samples were run on the Cardiovascular II, Oncology II and Metabolism PEA panels. RESULTS: Our analysis revealed that 26 and 39 proteins were differentially expressed in the CSF of test and validation PD cohorts, respectively, compared to controls. Among them, 6 proteins were changed in both cohorts. Midkine (MK) was increased in PD with the strongest effect size and results were validated with ELISA. Another most increased protein in PD, DOPA decarboxylase (DDC), which catalyses the decarboxylation of DOPA (L-3,4-dihydroxyphenylalanine) to dopamine, was strongly correlated with dopaminergic treatment. Moreover, Kallikrein 10 was specifically changed in APD compared with both PD and controls, but unchanged between PD and controls. Wnt inhibitory factor 1 was consistently downregulated in CBS and PSP patients in two independent cohorts. CONCLUSIONS: Using the large-scale PEA approach, we have identified potential novel PD diagnostic biomarkers, most notably MK and DDC, in the CSF of PD patients.

DOPA decarboxylase is an emerging biomarker for Parkinsonian disorders including preclinical Lewy body disease.

The diagnosis of Parkinsonian disorders is currently based on clinical criteria, which have limited sensitivity until most dopaminergic neurons are lost. Here we show that cerebrospinal fluid levels of DOPA decarboxylase (DDC) (also known as aromatic L-amino acid decarboxylase) can accurately identify patients with Lewy body disease (LBD) (area under the curve (AUC) = 0.89; PFDR = 2.6 × 10-13) and are associated with worse cognitive performance (P < 0.05). We also found that DDC can detect preclinical LBD stages in clinically unimpaired individuals with a positive seed amplification α-synuclein assay (AUC = 0.81, P = 1.0 × 10-5) and that this biomarker could predict progression to clinical LBD over a 3-year period in preclinical cases (hazard ratio = 3.7 per s.d. change, confidence interval = 1.1-12.7). Moreover, DDC levels were also increased in atypical Parkinsonian disorders but not in non-Parkinsonian neurodegenerative disorders. These cerebrospinal fluid results were replicated in an independent cohort, where we also found that DDC levels in plasma could identify both LBD and atypical Parkinsonian disorders (AUC = 0.92, P = 1.3 × 10-14). Our results show that DDC might have a future role in clinical practice as a biomarker of dopaminergic dysfunction to detect Parkinsonian disorders even during the preclinical disease stages and predict their progression to clinical LBD.

DDC-Promoter-Driven Chemogenetic Activation of SNpc Dopaminergic Neurons Alleviates Parkinsonian Motor Symptoms.

Parkinson's disease (PD) is a neurodegenerative disorder with typical motor symptoms. Recent studies have suggested that excessive GABA from reactive astrocytes tonically inhibits dopaminergic neurons and reduces the expression of tyrosine hydroxylase (TH), the key dopamine-synthesizing enzyme, in the substantia nigra pars compacta (SNpc). However, the expression of DOPA decarboxylase (DDC), another dopamine-synthesizing enzyme, is relatively spared, raising a possibility that the live but non-functional TH-negative/DDC-positive neurons could be the therapeutic target for rescuing PD motor symptoms. However, due to the absence of a validated DDC-specific promoter, manipulating DDC-positive neuronal activity has not been tested as a therapeutic strategy for PD. Here, we developed an AAV vector expressing mCherry under rat DDC promoter (AAV-rDDC-mCherry) and validated the specificity in the rat SNpc. Modifying this vector, we expressed hM3Dq (Gq-DREADD) under DDC promoter in the SNpc and ex vivo electrophysiologically validated the functionality. In the A53T-mutated alpha-synuclein overexpression model of PD, the chemogenetic activation of DDC-positive neurons in the SNpc significantly alleviated the parkinsonian motor symptoms and rescued the nigrostriatal TH expression. Altogether, our DDC-promoter will allow dopaminergic neuron-specific gene delivery in rodents. Furthermore, we propose that the activation of dormant dopaminergic neurons could be a potential therapeutic strategy for PD.

Paraganglioma with High Levels of Dopamine, Dopa Decarboxylase Suppression, Dopamine ß-hydroxylase Upregulation and Intra-tumoral Melanin Accumulation: A Case Report with a Literature Review.

Object Exclusively dopamine-producing pheochromocytoma/paraganglioma (PPGL) is an extremely rare subtype. In this condition, intratumoral dopamine ß-hydroxylase (DBH), which controls the conversion of norepinephrine from dopamine, is impaired, resulting in suppressed norepinephrine and epinephrine production. However, the rarity of this type of PPGL hampers the understanding of its pathophysiology. We therefore conducted genetic and immunohistological analyses of a patient with an exclusively dopamine-producing paraganglioma. Methods Paraganglioma samples from a 52-year-old woman who presented with a 29.6- and 41.5-fold increase in plasma and 24-h urinary dopamine, respectively, but only a minor elevation in the plasma norepinephrine level was subjected to immunohistological and gene expression analyses of catecholamine synthases. Three tumors carrying known somatic PPGL-related gene variants (HRAS, EPAS1) were used as controls. Whole-exome sequencing (WES) was also performed using the patient's blood and tumor tissue. Results Surprisingly, the protein expression of DBH was not suppressed, and its mRNA expression was clearly higher in the patient than in the controls. Furthermore, dopa decarboxylase (DDC), which governs the conversion of 3,4-dihydroxyphenyl-L-alanine (L-DOPA) to dopamine, was downregulated at the protein and gene levels. In addition, melanin, which is synthesized by L-DOPA, accumulated in the tumor. WES revealed no PPGL-associated pathogenic germline variants, but a missense somatic variant (c.1798G>T) in CSDE1 was identified. Conclusion Although pre-operative plasma L-DOPA was not measured, our histological and gene expression analyses suggest that L-DOPA, rather than dopamine, might have been overproduced in the tumor. This raises the possibility of pathophysiological heterogeneity in exclusively dopamine-producing PPGL.

Phytoconstituents of Datura metel extract improved motor coordination in haloperidol-induced cataleptic mice: Dual-target molecular docking and behavioural studies.

ETHNOPHARMACOLOGICAL RELEVANCE: Parkinson's disease (PD) is a prominent health challenge characterized by complex aetiology and limited therapeutic breakthroughs. Datura metel (DM) is a medicinal plant containing active phytoconstituents with neuropharmacological potentials. In traditional medicine, it exerts anticholinergic, anti-inflammatory and antioxidant effects, and protection from organophosphate poisoning inclusively involved in the pharmacotherapy of PD. Its other PD-related medicinal potency includes treatment of motor sickness and bradycardia. However, the exact mechanisms of anti-PD effects of its phytoconstituents remain underexplored. MATERIALS AND METHODS: In this study, methanolic extract of DM was evaluated for anti-PD behavioural effects in vivo haloperidol-induced cataleptic mice. The GC-MS-identified phytochemicals were studied for one-drug-multi-target inhibitory mechanisms against some key targets for PD treatment, alpha-synuclein (ASN) and dopa decarboxylase (DDC) using molecular docking. RESULTS: and discussion: Chronic administration of 50, 100 and 200 mg/kg of DM extract improved the 14-s latency time induced by haloperidol to 54, 54 and 57 s respectively, whereas levodopa (30 mg/kg) produced 47 s in rotarod tests. Similarly, the descending times for haloperidol-induced cataleptic mice were significantly reduced from 110 s to 17.7, 17.7 and 12.5 s by the respective chronic doses of DM extract, whereas levodopa-administered mice spent 17.5 s descending the same 30 cm pole. The interesting motor coordination enhancements are suggestively due to synergistic inhibition of ASN and DCC by the phytoconstituents of DM, especially, atropine and scopolamine. From the docking analysis, the two phytochemicals interacted more potently with the active therapeutic sites of the dual targets than levodopa and carbidopa. CONCLUSION: Methanolic extract of DM contains active phytochemicals for multi-target-directed antiparkinsonian mechanisms amenable for further studies.

Spectrum of DDC variants causing aromatic l-amino acid decarboxylase (AADC) deficiency and pathogenicity interpretation using ACMG-AMP/ACGS recommendations.

Pathogenic variants in dopa decarboxylase (DDC), the gene encoding the aromatic l-amino acid decarboxylase (AADC) enzyme, lead to a severe deficiency of neurotransmitters, resulting in neurological, neuromuscular, and behavioral manifestations clinically characterized by developmental delays, oculogyric crises, dystonia, and severe neurologic dysfunction in infancy. Historically, therapy has been aimed at compensating for neurotransmitter abnormalities, but response to pharmacologic therapy varies, and in most cases, the therapy shows little or no benefit. A novel human DDC gene therapy was recently approved in the European Union that targets the underlying genetic cause of the disorder, providing a new treatment option for patients with AADC deficiency. However, the applicability of human DDC gene therapy depends on the ability of laboratories and clinicians to interpret the results of genetic testing accurately enough to diagnose the patient. An accurate interpretation of genetic variants depends in turn on expert-guided curation of locus-specific databases. The purpose of this research was to identify previously uncharacterized DDC variants that are of pathologic significance in AADC deficiency as well as characterize and curate variants of unknown significance (VUSs) to further advance the diagnostic accuracy of genetic testing for this condition. DDC variants were identified using existing databases and the literature. The pathogenicity of the variants was classified using modified American College of Medical Genetics and Genomics/Association for Molecular Pathology/Association for Clinical Genomic Science (ACMG-AMP/ACGS) criteria. To improve the current variant interpretation recommendations, in silico variant interpretation tools were combined with structural 3D modeling of protein variants and applied comparative analysis to predict the impact of the variant on protein function. A total of 422 variants were identified (http://biopku.org/home/pnddb.asp). Variants were identified on nearly all introns and exons of the DDC gene, as well as the 3' and 5' untranslated regions. The largest percentage of the identified variants (48%) were classified as missense variants. The molecular effects of these missense variants were then predicted, and the pathogenicity of each was classified using a number of variant effect predictors. Using ACMG-AMP/ACGS criteria, 7% of variants were classified as pathogenic, 32% as likely pathogenic, 58% as VUSs of varying subclassifications, 1% as likely benign, and 1% as benign. For 101 out of 108 reported genotypes, at least one allele was classified as pathogenic or likely pathogenic. In silico variant pathogenicity interpretation tools, combined with structural 3D modeling of variant proteins and applied comparative analysis, have improved the current DDC variant interpretation recommendations, particularly of VUSs.

Clinical Features in Aromatic L-Amino Acid Decarboxylase (AADC) Deficiency: A Systematic Review.

Aromatic L-amino acid decarboxylase (AADC) deficiency is a rare congenital autosomal recessive metabolic disorder caused by pathogenic homozygous or compound heterozygous variants in the dopa decarboxylase (DDC) gene. Adeno-associated viral vector-mediated gene transfer of the human AADC gene into the putamina has become available. This systematic review on PubMed, Scopus databases, and other sources is aimed at describing the AADC whole phenotypic spectrum in order to facilitate its early diagnosis. Literature reviews, original articles, retrospective and comparative studies, large case series, case reports, and short communications were considered. A database was set up using Microsoft Excel to collect clinical, molecular, biochemical, and therapeutic data. By analysing 261 patients from 41 papers with molecular and/or biochemical diagnosis of AADC deficiency for which individuality could be determined with certainty, we found symptom onset to occur in the first 6 months of life in 93% of cases. Hypotonia and developmental delay are cardinal signs, reported as present in 73.9% and 72% of cases, respectively. Oculogyric crises were seen in 67% of patients while hypokinesia in 42% and ptosis in 26%. Dysautonomic features have been revealed in 53% and gastrointestinal symptoms in 19% of cases. With 37% and 30% of patients reported being affected by sleep and behavioural disorders, it seems to be commoner than previously acknowledged. Although reporting bias cannot be excluded, there is still a need for comprehensive clinical descriptions of symptoms at onset and during follow-up. In fact, our review suggests that most of the neurological and extraneurological symptoms and signs reported, although quite frequent in this condition, are not pathognomonic, and therefore, ADCC deficiency can remain an underdiscovered disorder.

Eladocagene Exuparvovec: First Approval.

Eladocagene exuparvovec (Upstaza™) is a gene therapy developed by PTC Therapeutics for the treatment of human aromatic L-amino acid decarboxylase (AADC) deficiency. Eladocagene exuparvovec comprises an adeno-associated virus vector that delivers the dopa decarboxylase (DDC) gene, the gene for human AADC. Eladocagene exuparvovec was approved in July 2022 in the EU for the treatment of patients aged 18 months and older with a clinical, molecular, and genetically confirmed diagnosis of AADC deficiency with a severe phenotype (i.e. patients who cannot sit, stand or walk). This article summarizes the milestones in the development of eladocagene exuparvovec leading to this first approval for the treatment of patients aged 18 months and older with AADC deficiency.

Melanin Synthesis Pathway Interruption: CRISPR/Cas9-mediated Knockout of dopa decarboxylase (DDC) in Harmonia axyridis (Coleoptera: Coccinellidae).

CRISPR/Cas9 technology is a very powerful genome editing tool and has been used in many insect species for functional genomics studies through targeted gene mutagenesis. Here, we successfully established CRISPR/Cas9 research platform in Asian multi-colored ladybird beetle, Harmonia axyridis, an important natural enemy in biological control. In this study, one pivotal gene dopa decarboxylase (DDC) in melanin synthesis was targeted by CRISPR/Cas9 to generate mutants in H. axyridis by CRISPR/Cas9 technology. Our results showed that injection of single guide RNA of the DDC and Cas9 protein into preblastoderm eggs induced one insertion and four deletion (indels) mutant H. axyridis. Mutations of HaDDC gene generated 25% mutant rate with melanin missing phenotype in larva, pupa,l and adult stage. The predation ability of the fourth instar larvae has no significant difference between wild (control) and mutant H. axyridis (G0), while these mutant fourth instar larvae had longer developmental period than that of the wild type. Consequently, the total predation of the fourth instar larvae was significantly increased in H. axyridis mutants comparing with the wild type. These results indicated that the success of CRISPR/Cas9 gene editing in H. axyridis. The gene editing platform in H. axyridis would facilitate the gene function research and promote special strain of predatory ladybird beetle generation.

Dopamine receptor (DAR) and dopa decarboxylase (DDC) mediate hepatopancreas antibacterial innate immune reactions in Procambarus clarkii.

Dopa is decarboxylated by dopa decarboxylase (DDC) to form dopamine, which is a significant signaling molecule in the neuroendocrine system. The dopamine receptor (DAR) is an important transmembrane receptor responsible for receiving extracellular signals in the DAR-mediated signaling pathway. In the present study, the expression patterns of Pc-dar were investigated after bacterial challenge. The obviously changed expression patterns showed Pc-dar was related to the antibacterial innate immunity. Endogenous Pc-DDC enzymatic activities were obviously downregulated after Pc-ddc dsRNA injection. The expression level of Pc-dar mRNA was obviously upregulated after bacterial injection when the expression level of Pc-ddc was knocked down. In addition, the upregulation trend of endogenous Pc-DDC enzymatic activities was obviously restrained after bacterial stimulation when Pc-ddc was knocked down. Finally, melanization was downregulated in crayfish hemolymph compared with the dsGFP injection group. In the RNAi assay, the results of qRT-PCR showed that Toll (TLRs) signaling pathway-related genes were activated in the early stages of bacterial stimulation when Pc-ddc was knocked down. Four tested ROS-related antioxidant enzyme genes were significantly upregulated after bacterial challenge compared with the dsGFP injection group. The above results indicated that Pc-DDC and Pc-DAR play important mediating roles in the neuroendocrine immune (NEI) system of crayfish.

Silencing tyrosine hydroxylase or dopa decarboxylase gene disrupts cuticle tanning during larva-pupa-adult transformation in Henosepilachna vigintioctopunctata.

BACKGROUND: The 28-spotted potato ladybird, Henosepilachna vigintioctopunctata, is a notorious defoliator of many solanaceous and cucurbitaceous plants. Tyrosine hydroxylase (TH) and dopa decarboxylase (DDC) are responsible for cuticle tanning pathway in insects. RESULTS: We identified HvTH and HvDDC in H. vigintioctopunctata, and found that high levels of them were accumulated just before or right after molting. Injection of dsHvTH or feeding 3-iodo-tyrosine (3-IT) at the third instar larval stage repressed tanning of the larval cuticle, reduced larval feeding, inhibited larval growth, and consequently caused 100% of larval mortality. Knockdown of HvDDC at the third instar larval stage hardly affected the coloration of larval head, and partially inhibited pigmentation of larval bodies and around 80% of the HvDDC RNAi larvae developed into albino pupae and adults. Moreover, depletion of HvTH or HvDDC at the fourth instar larval stage resulted in albino pupae and adults. The HvTH or HvDDC hypomorph adults fully or partially failed to remove the larval/pupal exuviae, possessed pale and abnormal wings, and poorly tanned heads and bodies, and eventually, struggled for several days without feeding on leaves before death. CONCLUSION: These results show that TH and DDC play key roles in larval and adult cuticle tanning and development in H. vigintioctopunctata. Also, these findings suggest that dopa- and dopamine-originated pigments are essential for larval and adult feeding behavior and the molting process during emergence. © 2022 Society of Chemical Industry.

Label-free LC-HRMS-based enzymatic activity assay for the detection of DDC, MAO and COMT inhibitors.

Pharmacological treatment of Parkinson's disease consists of a combined chemotherapy that mostly relies on levodopa (L-DOPA) administration together with inhibitors of dopa-decarboxylase (DDC), monoamine oxidase (MAO) and catechol-methyltransferase (COMT). Identification of inhibitors specifically targeting these enzymes is still a significative part of the development of new alternative antiparkinsonian drugs. Most of the available methods use measurement of enzymatic reactions through radioactive labeling, antibody-recognized products or coupled enzymatic assays. Mass spectrometry (MS) represents an interesting alternative approach as it allows direct and specific detection and quantification of enzymatic reactions. We describe the development of a simple, reliable, label-free assay based on high-resolution mass spectrometry (HRMS) for the detection and relative quantification of three different enzymatic reactions using non-isolated enzymes. The assay was applied both to reference drugs and plant crude extracts. This method can be used to detect hits in extracts libraries as well as determine relative IC50 of inhibitors.

The Role of Single Nucleotide Polymorphisms of Monoamine Oxidase B, Dopamine D2 Receptor, and DOPA Decarboxylase Receptors Among Patients Treated for Parkinson's Disease.

This study aimed to investigate the association between selected variants of genes related to dopamine metabolism pathways and the risk of and progression of Parkinson's disease (PD). This prospective cohort study was conducted in one academic teaching hospital. The study was conducted on 126 patients diagnosed with idiopathic Parkinson's disease. Blood samples were collected to conduct a genotyping of MAOB, DRD1, DRD2, and DDC genes. Genotype and allele frequencies of MAOB (rs1799836) variants were not associated with the course of PD. Genotype and allele frequencies of DRD2 (rs2283265) variants were associated with risk of dementia (p = 0.001) and resulted in parts II and III of the UPDRS scale (p = 0.001). Genotype and allele frequencies of DRD2 (rs1076560) variants were associated with risk of dementia (p = 0.001) and resulted in parts II and III of the UPDRS scale (p = 0.001). Genotype and allele frequencies of DDC (rs921451) variants were not associated with the course of PD.

Elucidating the Interaction between Pyridoxine 5'-Phosphate Oxidase and Dopa Decarboxylase: Activation of B6-Dependent Enzyme.

Pyridoxal 5'-phosphate (PLP), the active form of vitamin B6, serves as a cofactor for scores of B6-dependent (PLP-dependent) enzymes involved in many cellular processes. One such B6 enzyme is dopa decarboxylase (DDC), which is required for the biosynthesis of key neurotransmitters, e.g., dopamine and serotonin. PLP-dependent enzymes are biosynthesized as apo-B6 enzymes and then converted to the catalytically active holo-B6 enzymes by Schiff base formation between the aldehyde of PLP and an active site lysine of the protein. In eukaryotes, PLP is made available to the B6 enzymes through the activity of the B6-salvage enzymes, pyridoxine 5'-phosphate oxidase (PNPO) and pyridoxal kinase (PLK). To minimize toxicity, the cell keeps the content of free PLP (unbound) very low through dephosphorylation and PLP feedback inhibition of PNPO and PLK. This has led to a proposed mechanism of complex formation between the B6-salvage enzymes and apo-B6 enzymes prior to the transfer of PLP, although such complexes are yet to be characterized at the atomic level, presumably due to their transient nature. A computational study, for the first time, was used to predict a likely PNPO and DDC complex, which suggested contact between the allosteric PLP tight-binding site on PNPO and the active site of DDC. Using isothermal calorimetry and/or surface plasmon resonance, we also show that PNPO binds both apoDDC and holoDDC with dissociation constants of 0.93 ± 0.07 µM and 2.59 ± 0.11 µM, respectively. Finally, in the presence of apoDDC, the tightly bound PLP on PNPO is transferred to apoDDC, resulting in the formation of about 35% holoDDC.

Expression profiles of tyrosine metabolic pathway genes and functional analysis of DOPA decarboxylase in puparium tanning of Bactrocera dorsalis (Hendel).

BACKGROUND: Tanning is an important physiological process with critical roles in cuticle pigmentation and sclerotization. Previous studies have shown that insect cuticle tanning is closely associated with the tyrosine metabolism pathway, which consists of a series of enzymes. RESULTS: In this study, 24 tyrosine metabolism pathway genes were identified in the oriental fruit fly Bactrocera dorsalis (Hendel) genome. Gene expression profiles throughout 15 developmental stages of B. dorsalis were established based on our previous RNA sequencing data, and we found that 13 enzyme genes could be involved in the process of pupariation. Accordingly, a tyrosine-mediated tanning pathway during the pupariation of B. dorsalis was predicted and a critical enzyme, 3,4-dihydroxyphenylalanine (DOPA) decarboxylase (DDC), was used to explore its possible roles in formation of the puparium. First, a real-time quantitative polymerase chain reaction confirmed that BdDDC had an epidermis-specific expression pattern, and was highly expressed during larval metamorphosis in B. dorsalis. Subsequent disruption of BdDDC by feeding 5-day-old larvae with DDC inhibitor (l-α-methyl-DOPA) could lead to: (i) a significant decrease in BdDDC enzyme activity and dopamine concentration; (ii) defects in puparium pigmentation; (iii) impairment of the morphology and less thickness of the puparium; and (iv) lower pupal weight and obstacles to eclosion. CONCLUSION: This study provided a potential tyrosine metabolic pathway that was responsible for insect tanning during pupariation, and the BdDDC enzyme has been shown to have crucial roles in larval-pupal tanning of B. dorsalis. © 2021 Society of Chemical Industry.

Placental Changes in the serotonin transporter (Slc6a4) knockout mouse suggest a role for serotonin in controlling nutrient acquisition.

INTRODUCTION: The mouse placenta accumulates and possibly produces serotonin (5-hydroxytryptamine; 5-HT) in parietal trophoblast giant cells (pTGC) located at the interface between the placenta and maternal deciduum. However, the roles of 5-HT in placental function are unclear. This lack of information is unfortunate, given that selective serotonin-reuptake inhibitors are commonly used to combat depression in pregnant women. The high affinity 5-HT transporter SLC6A4 (also known as SERT) is the target of such drugs and likely controls much of 5-HT uptake into pTGC and other placental cells. We hypothesized that ablation of the Slc6a4 gene would result in morphological changes correlated with placental gene expression changes, especially for those involved in nutrient acquisition and metabolism, and thereby, provide insights into 5-HT placental function. METHODS: Placentas were collected at embryonic age (E) 12.5 from Slc6a4 knockout (KO) and wild-type (WT) conceptuses. Histological analyses, RNAseq, qPCR, and integrative correlation analyses were performed. RESULTS: Slc6a4 KO placentas had a considerable increased pTGC to spongiotrophoblast area ratio relative to WT placentas and significantly elevated expression of genes associated with intestinal functions, including nutrient sensing, uptake, and catabolism, and blood clotting. Integrative correlation analyses revealed upregulation of many of these genes was correlated with pTGC layer expansion. One other key gene was dopa decarboxylase (Ddc), which catalyzes conversion of L-5-hydroxytryptophan to 5-HT. DISCUSSION: Our studies possibly suggest a new paradigm relating to how 5-HT operates in the placenta, namely as a factor regulating metabolic functions and blood coagulation. We further suggest that pTGC might be functional analogs of enterochromaffin 5-HT-positive cells of the intestinal mucosa, which regulate similar activities within the gut. Further work, including proteomics and metabolomic studies, are needed to buttress our hypothesis.