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Background: The insoluble tangles of alpha-synuclein (α-syn) protein in the nigrostriatal circuit, characteristic of synucleinopathy, originate from low molecular weight oligomers, whose appearance and dissemination are related to neuroinflammation. These oligomeric forms of α-syn are considered highly cytotoxic but transient, so knowing the timing in which they appear remains challenging. Therefore, this study aimed to analyze the abundance of oligomeric forms of α-syn and tyrosine hydroxylase (TH) between 3 and 7 days after inducing neuroinflammation with lipopolysaccharide (LPS)., Methods and Results: LPS (2.5 µg/2.5 µL) was stereotaxically injected in the substantia nigra (SN) of adult male Wistar rats, which were sacrificed 3, 5 and 7 days after this intervention. The brains were processed for semi quantitative Western blot, along with brains from control and sham animals. Our results show an increased expression of α-syn monomer (15 kDa) only 3 days after LPS infusion, and the formation of 50 KDa and 60 kDa α-syn oligomers in the SN and striatum (STR) between 3 and 7 days after LPS infusion. Furthermore, the presence of these oligomers was accompanied by a decrease in the expression of nigral TH., Conclusion: These findings highlight the rapidity with which potentially toxic forms of α-syn appear in the nigrostriatal circuit after a neuroinflammatory challenge, in addition to allowing us to identify specific oligomers and a temporal relation with neurodegeneration of TH-positive cells. Knowledge of the timing and location in which these small oligomers appear is essential to developing therapeutic strategies to prevent its formation., (© 2024. The Author(s), under exclusive licence to Springer Nature B.V.)

Innovative therapeutic strategies are urgently needed for Parkinson's disease due to limited efficacy of current treatments and a weak therapeutic pipeline. In this forum article, we propose targeting tyrosine hydroxylase phosphorylation as a novel mechanism of action to address this critical need., Competing Interests: Declaration of interests M.P.S. and L.P.v.d.H. are cofounders and shareholders of Parkinnova Therapeutics and Macrobian Biotech. The University of Amsterdam licenses patents related to this work (16/490,192 and 17/271,978) to Parkinnova Therapeutics., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

Parkinson's disease (PD) is one of the fastest-growing neurodegenerative diseases and has been linked to the exposure to numerous environmental neurotoxins. Although lead (Pb) exposure has been related to the development of PD, the molecular target of Pb to cause the onset of PD is insufficiently investigated. Herein, we explored the effects of Pb exposure on behavior, pathophysiology, and gene expression of wild-type (WT) fly (Drosophila melanogaster) by comparison with its PD model. After exposure to Pb, the WT flies showed PD-like locomotor impairments and selective loss of dopaminergic (DAergic) neurons, displaying similar phenotypes to fly PD model (PINK1). Transcriptomic analysis showed the similarity in gene expression profiles between Pb treatment WT flies and PINK1 mutant flies. Moreover, Pb exposure resulted in endogenous dopamine deficits in WT flies. Analyses of gene expression and enzyme activity confirmed that Pb exposure reduced tyrosine hydroxylase (TH) activity and led to failure of dopamine synthesis. Furthermore, molecular dynamics simulation confirmed that Pb was adsorbed by TH and subsequently inhibited the enzymatic activity. Exogenous injection of L-dopa and melatonin could partially rescue the pathological phenotypes of Pb-exposed flies and PD fly model. Antagonist injection of microRNA-133, which negatively regulated the expression of TH gene, ultimately rescued in the manifestation of PD phenotypes in flies. Involvement of TH overexpression mutants of fly strongly promoted the resistance to Pb exposure and rescued both behavior and the number of DAergic neurons. Therefore, our study elucidates the Pb molecular target in dopamine pathway and mechanism underlying the risks of Pb exposure on the occurrence of PD at environmentally-relevant concentrations., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

Physiology and behavior are structured temporally to anticipate daily cycles of light and dark, ensuring fitness and survival. Neuromodulatory systems in the brain-including those involving serotonin and dopamine-exhibit daily oscillations in neural activity and help shape circadian rhythms. Disrupted neuromodulation can cause circadian abnormalities that are thought to underlie several neuropsychiatric disorders, including bipolar mania and schizophrenia, for which a mechanistic understanding is still lacking. Here, we show that genetically depleting serotonin in Tph2 knockout mice promotes manic-like behaviors and disrupts daily oscillations of the dopamine biosynthetic enzyme tyrosine hydroxylase (TH) in midbrain dopaminergic nuclei. Specifically, while TH mRNA and protein levels in the Substantia Nigra (SN) and Ventral Tegmental Area (VTA) of wild-type mice doubled between the light and dark phase, TH levels were high throughout the day in Tph2 knockout mice, suggesting a hyperdopaminergic state. Analysis of TH expression in striatal terminal fields also showed blunted rhythms. Additionally, we found low abundance and blunted rhythmicity of the neuropeptide cholecystokinin (Cck) in the VTA of knockout mice, a neuropeptide whose downregulation has been implicated in manic-like states in both rodents and humans. Altogether, our results point to a previously unappreciated serotonergic control of circadian dopamine signaling and propose serotonergic dysfunction as an upstream mechanism underlying dopaminergic deregulation and ultimately maladaptive behaviors.

Tyrosine hydroxylase (TH) catalyzes hydroxylation of L-tyrosine to L-3,4-dihydroxyphenylalanine, the initial and rate-limiting step in the synthesis of dopamine, noradrenaline, and adrenaline. Mutations in the human TH gene are associated with hereditary motor disorders. The common C886T mutation identified in the mouse Th  gene results in the R278H substitution in the enzyme molecule. We investigated the impact of this mutation on the TH activity in the mouse midbrain. The TH activity in the midbrain of Mus musculus castaneus (CAST) mice homozygous for the 886C allele was higher compared to C57BL/6 and DBA/2 mice homozygous for the 886T allele. Notably, this difference in the enzyme activity was not associated with changes in the Th gene mRNA levels and TH protein content. Analysis of the TH activity in the midbrain in mice from the F2 population obtained by crossbreeding of C57BL/6 and CAST mice revealed that the 886C allele is associated with a high TH activity. Moreover, this allele showed complete dominance over the 886T allele. However, the C886T mutation did not affect the levels of TH protein in the midbrain. These findings demonstrate that the C886T mutation is a major genetic factor determining the activity of TH in the midbrain of common laboratory mouse strains. Moreover, it represents the first common spontaneous mutation in the mouse Th gene whose influence on the enzyme activity has been demonstrated. These results will help to understand the role of TH in the development of adaptive and pathological behavior, elucidate molecular mechanisms regulating the activity of TH, and explore pharmacological agents for modulating its function.

Tyrosine hydroxylase (TH) is the rate-limiting enzyme in dopamine biosynthesis catalyzing the tetrahydrobiopterin (BH 4 )-dependent hydroxylation of tyrosine to L-DOPA. Here, we analyzed 25 TH variants associated with various degrees of dopa-responsive dystonia and evaluate the effect of each variant on protein stability, activity and cellular localization. Furthermore, we investigated the physical interaction between TH and human wildtype (wt) GTP cyclohydrolase 1 (GTPCH) and the effect of variants on this interaction. Our in vitro results classify variants according to their resistance to proteinase K digestion into three groups (stable, intermediate, unstable). Based on their cellular localization, two groups of variants can be identified, variant group one with cytoplasmic distribution and variant group two forming aggregates. These aggregates do not correlate with loss of enzymatic activity but nevertheless might be a good target for molecular chaperones. Unfortunately, no obvious correlation between the half-life of a variant and its enzymatic activity or between solubility, stability and enzymatic activity of a given variant could be found. Excitingly, some variants disrupt the physical interaction between TH and human wildtype GTPCH, thereby interfering with enzymatic activity and offering new druggable targets for therapy. Taken together, our results highlight the importance of an in-depth molecular analysis of each variant in order to be able to classify groups of disease variants and to find specific therapies for each subgroup. Stand-alone in silico analyses predict less precise the effect of specific variants and should be combined with other in vitro analyses in cellular model systems., (© 2023 The Authors. Journal of Inherited Metabolic Disease published by John Wiley & Sons Ltd on behalf of SSIEM.)

Human tyrosine hydroxylase (hTH) has key role in the production of catecholamine neurotransmitters. The structure, function and regulation of hTH has been extensively researched area and the possibility of enzyme replacement therapy (ERT) involving hTH through nanocarriers has been raised as well. However, our understanding on how hTH may interact with nanocarriers is still lacking. In this work, we attempted to investigate the immobilization of hTH on magnetic nanoparticles (MNPs) with various surface linkers in quantitative and mechanistic detail. Our results showed that the activity of hTH was retained after immobilization via secondary and covalent interactions as well. The colloidal stability of hTH could be also enhanced proved by Dynamic light scattering and Zeta potential analysis and a homogenous enzyme layer could be achieved, which was investigated by Raman mapping. The covalent attachment of hTH on MNPs via aldehyde or epoxy linkers provide irreversible immobilization and 38.1 % and 16.5 % recovery (ER). The hTH-MNPs catalyst had 25 % ER in average in simulated nasal electrolyte solution (SNES). This outcome highlights the relevance of immobilization applying MNPs as a potential formulation tool of sensitive therapeutic enzymes offering new opportunities for ERT related to neurodegenerative disorders., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Author(s). Published by Elsevier B.V. All rights reserved.)

Amphetamines (Amph) are psychostimulants broadly used as physical and cognitive enhancers. However, the long-term effects of prenatal exposure to Amph have been poorly investigated. Here, we show that continuous exposure to Amph during early development induces long-lasting changes in histone methylation at the C. elegans tyrosine hydroxylase (TH) homolog cat-2 and the vesicular monoamine transporter (VMAT) homologue cat-1 genes. These Amph-induced histone modifications are correlated with enhanced expression and function of CAT-2/TH and higher levels of dopamine, but decreased expression of CAT-1/VMAT in adult animals. Moreover, while adult animals pre-exposed to Amph do not show obvious behavioral defects, when challenged with Amph they exhibit Amph hypersensitivity, which is associated with a rapid increase in cat-2 /TH mRNA. Because C. elegans has helped reveal neuronal and epigenetic mechanisms that are shared among animals as diverse as roundworms and humans, and because of the evolutionary conservation of the dopaminergic response to psychostimulants, data collected in this study could help us to identify the mechanisms through which Amph induces long-lasting physiological and behavioral changes in mammals.

The gene encoding 5'-nucleotidase domain-containing protein 2 (NT5DC2) has been associated with neuropsychiatric disorders related to the abnormality of dopamine activity in the brain. However, its physiological functions remain unclear. In this study, we analyzed the features of NT5DC2 that influence its binding with tyrosine hydroxylase (TH) and its effects on dihydroxyphenylalanine (DOPA) synthesis, using NT5DC2 overexpressed in PC12D cells by the pCMV vector. Western blot analysis revealed that the purified NT5DC2-DYKDDDDK-tag (NT5DC2-tag) protein can bind with the phosphorylated form of recombinant human TH type 1 (rhTH1), apart from the endogenous TH in PC12D cells. Proteomic analysis by mass spectrometry revealed that the purified NT5DC2-tag protein has the potential to bind to 41 proteins with multiple phosphorylation sites in PC12D cells (NT5DC2 binding proteins: positive, 391 sites/41 proteins; and negative, 85 sites/27 proteins). Overexpression of NT5DC2 in PC12D cells decreased DOPA levels in the medium. When the lysate of PC12D cells overexpressing NT5DC2 was incubated at 37 °C, the phosphorylated form of endogenous TH in PC12D cells decreased. This decrease was also detected when phosphorylated rhTH1 was incubated with purified NT5DC2-tag. Overall, our results suggest that NT5DC2 regulates DOPA synthesis by promoting the dephosphorylation of TH, similar to a phosphatase. Therefore, our study provides useful information for understanding various disorders associated with abnormalities in dopamine levels in the brain., Competing Interests: Declaration of competing interest The named authors have no conflict of interest, financial or otherwise., (Copyright © 2024 Elsevier Inc. All rights reserved.)

Background: Parkinson's disease (PD) is the second most common neurodegenerative disorder, with genetic factors accounting for about 15% of cases. There is a significant challenge in tracking disease progression and treatment response, crucial for developing new therapies. Traditional methods like imaging, clinical monitoring, and biomarker analysis have not conclusively tracked disease progression or treatment response in PD. Our previous research indicated that PD patients with increased dopamine transporter (DAT) and tyrosine hydroxylase (TH) in peripheral blood mononuclear cells (PBMCs) might show disease progression and respond to levodopa treatment., Objective: This study evaluates whether DAT- and TH-expressing PBMCs can monitor motor progression in a PD patient with a heterozygous TH mutation., Methods: We conducted a longitudinal follow-up of a 46-year-old female PD patient with a TH mutation, assessing her clinical features over 18 months through DaT scans and PBMC immunophenotyping. This was compared with idiopathic PD patients (130 subjects) and healthy controls (80 age/sex-matched individuals)., Results: We found an increase in DAT+ immune cells concurrent with worsening motor scores (UPDRS-III). Following levodopa therapy, unlike idiopathic PD patients, TH+ immune cell levels in this patient remained high even as her motor scores improved., Conclusions: Longitudinal immunophenotyping in this PD patient suggests DAT+ and TH+ PBMCs as potential biomarkers for tracking PD progression and treatment efficacy, supporting further exploration of this approach in PD research.

CHIR99021, also known as laduviglusib or CT99021, is a Glycogen-synthase kinase 3β (GSK3β) inhibitor, which has been reported as a promising drug for cardiomyocyte regeneration or treatment of sensorial hearing loss. Since the activation of dopamine (DA) receptors regulates dopamine synthesis and they can signal through the β-arrestin pathway and GSK3β, we decided to check the effect of GSK3β inhibitors (CHIR99021, SB216763 and lithium ion) on the control of DA synthesis. Using ex vivo experiments with minces from rat brain striatum, we observed that CHIR99021, but not SB216763 or lithium, causes complete abrogation of both DA synthesis and accumulation, pointing to off-target effects of CHIR99021. This decrease can be attributed to tyrosine hydroxylase (TH) inhibition since the accumulation of l-DOPA in the presence of a DOPA decarboxylase inhibitor was similarly decreased. On the other hand, CHIR99021 caused a dramatic increase in the DOPAC/DA ratio, an indicator of DA metabolization, and hindered DA incorporation into striatum tissue. Tetrabenazine, an inhibitor of DA vesicular transport, also caused DA depletion and DOPAC/DA ratio increase to the same extent as CHIR99021. In addition, both CHIR99021 or SB216763, but not lithium, decreased TH phosphorylation in Ser19, but not in Ser31 or Ser40. These results demonstrate that CHIR99021 can lead to TH inactivation and DA depletion in brain striatum, opening the possibility of its use in DA-related disorders, and shows effects to be considered in future clinical trials. More work is needed to find the mechanism exerted by CHIR99021 on DA accumulation., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Compensatory mechanisms that augment dopamine (DA) signaling are thought to mitigate onset of hypokinesia prior to major loss of tyrosine hydroxylase (TH) in striatum that occurs in Parkinson's disease. However, the identity of such mechanisms remains elusive. In the present study, the rat nigrostriatal pathway was unilaterally-lesioned with 6-hydroxydopamine (6-OHDA) to determine whether differences in DA content, TH protein, TH phosphorylation, or D 1 receptor expression in striatum or substantia nigra (SN) aligned with hypokinesia onset and severity at two time points. In striatum, DA and TH loss reached its maximum (>90%) 7 days after lesion induction. However, in SN, no DA loss occurred, despite ∼60% TH loss. Hypokinesia was established at 21 days post-lesion and maintained at 28 days. At this time, DA loss was ∼60% in the SN, but still of lesser magnitude than TH loss. At day 7 and 28, ser31 TH phosphorylation increased only in SN, corresponding to less DA versus TH protein loss. In contrast, ser40 TH phosphorylation was unaffected in either region. Despite DA loss in both regions at day 28, D 1 receptor expression increased only in lesioned SN. These results support the concept that augmented components of DA signaling in the SN, through increased ser31 TH phosphorylation and D 1 receptor expression, contribute as compensatory mechanisms against progressive nigrostriatal neuron and TH protein loss, and may mitigate hypokinesia severity., Competing Interests: Declaration of Competing Interest The authors declare that they have no competing interests relevant to the content of this article, no financial or proprietary interests in any material discussed in this article, or affiliations with or involvement in any organization or entity with any financial interest or non-financial interest in the subject matter or materials discussed in this manuscript., (Copyright © 2023 Elsevier Inc. All rights reserved.)

Aim: In our pilot study, we found an increase in tyrosine hydroxylase (Th) mRNA expression in the prefrontal cortex of 72-h REM sleep-deprived (SD) rats, a mania model. Additionally, the expression levels of miR-325-3p, miR-326-3p, and miR-330-5p, the predicted target miRNAs on TH, were significantly decreased. Based on these results, in this study, we investigated whether miRNA-325-3p, miR-326-3p, and miR-330-5p modulate TH and manic-like behaviors in SD rats., Methods: Manic-like behaviors were assessed using the open field test (OFT) and elevated plus-maze (EPM) test. The direct binding activity of miRNAs to the 3'-untranslated region (3'-UTR) of the Th gene was measured in HEK-293 cells using a luciferase reporter system. We also examined mRNA and protein expression of TH after intracerebroventricular (ICV) injection of miR-330-5p agomir to SD rats, along with manic-like behaviors., Results: We observed an upregulation in mRNA and protein expression of TH and downregulation in miRNA-325-3p, miR-326-3p, and miR-330-5p expressions in the prefrontal cortex of SD rats, together with increased manic-like behaviors. The luciferase reporter assay showed that miR-330-5p could repress TH expression through direct binding to its target site in the 3'-UTR of Th, whereas miR-326-3p and miR-330-5p could not. In addition, ICV injection of miR-330-5p agomir alleviated the increase in TH expression in the prefrontal cortex of SD rats and manic-like behaviors., Conclusions: TH expression regulation through miR-330-5p may be implicated in the pathophysiology of mania in SD rats., (© 2023 The Authors. CNS Neuroscience & Therapeutics published by John Wiley & Sons Ltd.)

Background: Cell groups containing catecholamines provide a useful model to study the molecular and cellular mechanisms underlying the morphogenesis, physiology, and pathology of the central nervous system. For this purpose, it is necessary to establish a system to induce catecholaminergic group-specific expression of Cre recombinase. Recently, we introduced a gene cassette encoding 2A peptide fused to Cre recombinase into the site between the C-terminus and translational termination codons of the rat tyrosine hydroxylase (TH) open reading frame by the Combi-CRISPR technology, which is a genomic editing method to enable an efficient knock-in (KI) of long DNA sequence into a target site. However, the expression patterns of the transgene and its function as well as the effect of the mutation on the biochemical and behavioral phenotypes in the KI strains have not been characterized yet., New Method: We aimed to evaluate the usefulness of TH-Cre KI rats as an experimental model for investigating the structure and function of catecholaminergic neurons in the brain., Results: We detected cell type-specific expression of Cre recombinase and site-specific recombination activity in the representative catecholaminergic groups in the TH-Cre KI rat strains. In addition, we measured TH protein levels and catecholamine accumulation in the brain regions, as well as motor, reward-related, and anxiety-like behaviors, indicating that catecholamine metabolism and general behavior are apparently normal in these KI rats., Conclusions: TH-Cre KI rat strains produced by the Combi-CRISPR system offer a beneficial model to study the molecular and cellular mechanics for the morphogenesis, physiology, and pathology of catecholamine-containing neurons in the brain., Competing Interests: Declaration of Competing Interest The authors are aware of no conflicts of interest., (Copyright © 2022 Elsevier B.V. All rights reserved.)

Tyrosine hydroxylase is the rate-limiting enzyme of catecholamine biosynthesis that catalyzes the conversion of L-tyrosine to L-3,4-dihydroxyphenylalanine. The tyrosine hydroxylase gene is regulated by extracellular signaling molecules such as epidermal growth factor, nerve growth factor and steroids. Here, we investigated whether the activity of the tyrosine hydroxylase gene promoter is upregulated by activation of G protein-coupled receptors, the largest group of plasma membrane receptors. We used catecholaminergic neuroblastoma cells as a cellular model and chromatin-integrated tyrosine hydroxylase promoter-luciferase reporter genes. The results show that stimulation of Rαq, a Gαq-coupled designer receptor, triggered transcription of a reporter gene driven by the tyrosine hydroxylase promoter. Transcription was attenuated by overexpression of regulator of G-protein signaling-2, which activates the GTPase activity of the G protein α-subunit, and by a truncated, dominant-negative mutant of phospholipase Cβ3. Extracellular signal-regulated protein kinase was identified as the signal transducer. At the transcriptional level, tyrosine hydroxylase promoter activity was found to be controlled by the transcription factor CREB. Expression experiments with the adenoviral regulator protein E1A, an inhibitor of CBP/p300 histone acetyltransferases, showed that transcription of the reporter gene controlled by the tyrosine hydroxylase is under epigenetic control. We identified the protein phosphatases MAP kinase phosphatase-1 and calcineurin as part of a shutdown device of the signaling cascade linking Rαq designer receptor activation to tyrosine hydroxylase gene transcription. We conclude that tyrosine hydroxylase promoter activity is controlled by Gαq-coupled receptors., Competing Interests: Declaration of competing interest The authors declare that they have no conflict of interest., (Copyright © 2022 Elsevier Ltd. All rights reserved.)

Senescence is a natural and progressive physiological event that leads to a series of morphophysiological alterations in the organism. The brain is the most vulnerable organ to both structural and functional changes during this process. Dopamine is a key neurotransmitter for the proper functioning of the brain, directly involved in circuitries related with emotions, learning, motivation and reward. One of the main dopamine- producing nuclei is the substantia nigra pars compacta (SNpc), which establish connections with the striatum forming the so-called nigrostriatal pathway. S100B is a calcium binding protein mainly expressed by astrocytes, involved in both intracellular and extracellular processes, and whose expression is increased following injury in the nervous tissue, being a useful marker in altered status of central nervous system. The present study aimed to analyze the impact of senescence on the cells immunoreactive for tyrosine hydroxylase (TH) and S100B along the nigrostriatal pathway of the rat. Our results show an decreased expression of S100B + cells in SNpc. In addition, there was a significant decrease in TH immunoreactivity in both projection fibers and TH + cell bodies. In the striatum, a decrease in TH immunoreactivity was also observed, as well as an enlargement of the white matter bundles. Our findings point out that senescence is related to the anatomical and neurochemical changes observed throughout the nigrostriatal pathway., (Copyright © 2022 Elsevier B.V. All rights reserved.)

Since the discovery of striatal neurons expressing dopamine-synthesizing enzymes, researchers have attempted to identify their phenotype and functional significance. In this study, it was shown that in transgenic mice expressing green fluorescent protein (GFP) under the tyrosine hydroxylase (TH) gene promoter, (i) there are striatal neurons expressing only TH, only aromatic L-amino acid decarboxylase (AADC), or both enzymes of dopamine synthesis; (ii) striatal neurons expressing dopamine-synthesizing enzymes are not dopaminergic since they lack a dopamine transporter; (iii) monoenzymatic neurons expressing individual complementary dopamine-synthesizing enzymes produce this neurotransmitter in cooperation; (iv) striatal nerve fibers containing only TH, only AADC, or both enzymes project into the lateral ventricles, providing delivery pathways for L-3,4-dihydroxyphenylalanine and dopamine to the cerebrospinal fluid; and (v) striatal GFP neurons express receptor genes for various signaling molecules, i.e., classical neurotransmitters, neuropeptides, and steroids, indicating fine regulation of these neurons. Based on our data, it is assumed that the synthesis of dopamine by striatal neurons is a compensatory response to the death of nigral dopaminergic neurons in Parkinson's disease, which opens broad prospects for the development of a fundamentally novel antiparkinsonian therapy.

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., (© 2022 Society of Chemical Industry.)

A time span of 60 years covers the detection of catecholamines in the brain, their function in movement and correlation to Parkinson's disease (PD). The clinical findings that orally given L-DOPA can alleviate or even prevent akinesia gave great hope for the treatment of PD. Attention focused on the role of tyrosine hydroxylase (TH) as the rate-limiting enzyme in the formation of catecholamines. It became evident that the enzyme driven formation is lowered in PD. Such results could only be obtained from studying human brain samples demonstrating the necessity for human brain banks. Originally, a TH enzyme deficiency was suspected in PD. Studies were conducted on the enzyme properties: its induction and turnover, the complex regulation starting with cofactor requirements as tetrahydrobiopterin and ferrous iron, and the necessity for phosphorylation for activity as well as inhibition by toxins or regulatory feedback inhibition by catecholamines. In the course of time, it became evident that neurodegeneration and cell death of dopaminergic neurons is the actual pathological process and the decrease of TH a cophenomenon. Nevertheless, TH immunochemistry has ever since been a valuable tool to study neuronal pathways, neurodegeneration in various animal models of neurotoxicity and cell cultures, which have been used as well to test potential neuroprotective strategies., (© 2022. The Author(s).)

Tyrosine hydroxylase (TH) catalyzes the rate-limiting step in the catecholamine (CA) biosynthesis pathway, making TH a molecular target for controlling CA production, specifically dopamine. Dysregulation of dopamine is correlated with neurological diseases such as Parkinson's disease (PD) and post-traumatic stress disorder (PTSD), among others. Previously, we showed that a 49-nucleotide guanine (G)-rich sequence within the human TH promoter adopts two different sets of G-quadruplex (GQ) structures (5'GQ and 3'GQ), where the 5'GQ uses G-stretches I, II, IV, and VI in TH49, which enhances TH transcription, while the 3'GQ utilizes G-stretches II, IV, VI, and VII, which represses transcription. Herein, we demonstrated targeted switching of these GQs to their active state using rationally designed DNA GQ Clips (5'GQ and 3'GQ Clips) to modulate endogenous TH gene expression and dopamine production. As a translational approach, we synthesized a targeted nanoparticle delivery system to effectively deliver the 5'GQ Clip in vivo. We believe this strategy could potentially be an improved approach for controlling dopamine production in a multitude of neurological disorders, including PD., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2021. Published by Elsevier Inc.)

Background: The sympathetic nervous system plays an important role in the regulation of pancreatic exocrine and endocrine secretion. The results of experimental studies also demonstrate the involvement of the sympathetic nervous system in the regulation of endocrine cell differentiation and islet formation during the development of the pancreas. However, the prenatal development of sympathetic innervation of the human pancreas has not yet been studied., Material and Methods: Pancreatic autopsy samples from 24 human fetuses were examined using immunohistochemistry with antibodies to tyrosine hydroxylase (TH). The density, concentration, and size (width, length, perimeter and area) of the TH-positive sympathetic nerves were compared in four developmental periods: pre-fetal (8-11 weeks post conception (w.p.c.), n = 6), early fetal (13-20 gestational weeks (g.w.), n = 7), middle fetal (21-28 g.w., n = 6) and late fetal (29-40 g.w., n = 5) using morphometric methods and statistical analysis (Multiple Comparisons p values). Double immunofluorescence with antibodies to TH and either insulin or glucagon and confocal microscopy were applied to analyze the interaction between the sympathetic nerves and endocrine cells, and the co-localization of TH with hormones., Results: TH-positive sympathetic nerves were detected in the fetal pancreas starting from the early stages (8 w.p.c.). The developmental dynamics of sympathetic nerves was follows: from the pre-fetal period, the amount of TH-positive nerves gradually increased and their branching occurred reaching the highest density and concentration in the middle fetal period, followed by a decrease in these parameters in the late fetal period. From the 14th g.w. onwards, thin TH-positive nerve fibers were mainly distributed in the vicinity of blood vessels and around the neurons of intrapancreatic ganglia, which is similar in adults. There were only rare TH-positive nerve fibers adjacent to acini or located at the periphery of some islets. The close interactions between the TH-positive nerve fibers and endocrine cells were observed in the neuro-insular complexes. Additionally, non-neuronal TH-containing cells were found in the pancreas of fetuses from the pre-fetal and early fetal periods. Some of these cells simultaneously contained glucagon., Conclusions: The results demonstrate that sympathetic innervation of the human pancreas, including the formation of perivascular and intraganglionic nerve plexuses, extensively develops during prenatal period, while some processes, such as the formation of sympathetic innervation of islet capillaries, may occur postnatally. Non-neuronal TH-containing cells, as well as the interactions between the sympathetic terminals and endocrine cells observed in the fetal pancreas may be necessary for endocrine pancreas development in humans., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2021 Elsevier GmbH. All rights reserved.)

The Parkinson's disease (PD) risk gene GTP cyclohydrolase 1 (GCH1) catalyzes the rate-limiting step in tetrahydrobiopterin (BH4) synthesis, an essential cofactor in the synthesis of monoaminergic neurotransmitters. To investigate the mechanisms by which GCH1 deficiency may contribute to PD, we generated a loss of function zebrafish gch1 mutant ( gch1 -/- zebrafish develop marked monoaminergic neurotransmitter deficiencies by 5 d postfertilization (dpf), movement deficits by 8 dpf and lethality by 12 dpf. Tyrosine hydroxylase (Th) protein levels were markedly reduced without loss of ascending dopaminergic (DAergic) neurons. L-DOPA treatment of gch1 -/- zebrafish develop marked monoaminergic neurotransmitter deficiencies by 5 d postfertilization (dpf), movement deficits by 8 dpf and lethality by 12 dpf. Tyrosine hydroxylase (Th) protein levels were markedly reduced without loss of ascending dopaminergic (DAergic) neurons. L-DOPA treatment of gch1 -/- Genome-wide association studies have now identified at least 90 genetic risk factors for sporadic Parkinson's disease (PD). Zebrafish are an ideal tool to determine the mechanistic role of genome-wide association studies (GWAS) risk genes in a vertebrate animal model. The discovery of GTP cyclohydrolase 1 (GCH1) as a genetic risk factor for PD was counterintuitive, GCH1 is the rate-limiting enzyme in the synthesis of dopamine (DA), mutations had previously been described in the non-neurodegenerative movement disorder dopa-responsive dystonia (DRD). Rather than causing DAergic cell death (as previously hypothesized by others), we now demonstrate that GCH1 impairs tyrosine hydroxylase (Th) homeostasis and activates innate immune mechanisms in the brain and provide evidence of microglial activation and phagocytic activity.gch1 -/- larval brain tissue identified highly upregulated transcripts involved in innate immune response. Subsequent experiments provided morphologic and functional evidence of microglial activation in gch1 -/- The results of our study suggest that GCH1 deficiency may unmask early, subclinical parkinsonism and only indirectly contribute to neuronal cell death via immune-mediated mechanisms. Our work highlights the importance of functional validation for genome-wide association studies (GWAS) risk factors and further emphasizes the important role of inflammation in the pathogenesis of PD. SIGNIFICANCE STATEMENT Genome-wide association studies have now identified at least 90 genetic risk factors for sporadic Parkinson's disease (PD). Zebrafish are an ideal tool to determine the mechanistic role of genome-wide association studies (GWAS) risk genes in a vertebrate animal model. The discovery of GTP cyclohydrolase 1 (GCH1) as a genetic risk factor for PD was counterintuitive, GCH1 is the rate-limiting enzyme in the synthesis of dopamine (DA), mutations had previously been described in the non-neurodegenerative movement disorder dopa-responsive dystonia (DRD). Rather than causing DAergic cell death (as previously hypothesized by others), we now demonstrate that GCH1 impairs tyrosine hydroxylase (Th) homeostasis and activates innate immune mechanisms in the brain and provide evidence of microglial activation and phagocytic activity., (Copyright © 2022 the authors.)

Tyrosine hydroxylase (TH) is the rate-limiting enzyme in the biosynthesis of dopamine (DA), and the regulation of its activity is important for DA homeostasis. In this study, we focused on the modification of TH through a cysteine residue. We found that incubation with N-ethylmaleimide (NEM), a cysteine modification reagent, inactivated TH. The responsible cysteine was identified as Cys176 of human TH with recombinant mutant proteins. We further examined how NEM modification was affected by the states of TH. DA binding, a feedback inhibition mechanism of TH, delayed the modification and inactivation of TH by NEM. In contrast, the S40E mutant, which mimics the phosphorylation of Ser40 that suppresses DA binding and is thus considered as an active state of TH, did not affect modification and inactivation. These results suggest that the modification of Cys176 can inhibit even phosphorylated active TH. In addition, we found that DA oxides, which are generated by oxidative stress in dopaminergic neurons, reacted with TH through Cys176 and inhibited its activity, similar to NEM. These results suggest that the modification of Cys176 of TH could be involved in the mechanisms of neurotoxicity caused by DA oxides., (Copyright © 2021 Elsevier Inc. All rights reserved.)

Parkinson's disease (PD) occurs when dopaminergic cells in the substantia nigra (SN) region are destroyed; however, the cause of the destruction of dopamine cells has not yet been determined. This study was performed to investigate whether changes in the hormones that cause benign prostatic hyperplasia (BPH) are related to pathological changes in PD. The pathological findings were examined by observing the lesion sites related to PD in a BPH rat model. BPH was induced in rats by subcutaneous injection of testosterone propionate for 4 weeks after castration. To investigate the changes in the SN regions, tyrosine hydroxylase (TH) and α-synuclein (α-syn) expression were analyzed by western blotting. TH expression, expressed in dopaminergic cells and used as a dopaminergic cell detection marker, decreased, whereas α-syn expression increased at the SN site. These results are quite similar to the pathological changes observed in patients with PD and Parkinsonism animal models. Our results showed an increased expression of inducible nitric oxide synthase and cyclooxygenase-2 in the SN regions in the BPH group. Additionally, a decreased expression of B-cell lymphoma protein 2 and an increased expression of B-cell lymphoma protein 2-associated X, suggesting increased apoptosis, were observed in the BPH group. These results suggest that the pathological changes associated with PD may be caused by BPH or factors related to BPH. Thus, this study has presented a new avenue for an approach related to hormonal changes as a method to determine the cause of PD, for which the exact cause is not yet known., (Copyright © 2021 Elsevier B.V. All rights reserved.)

Recent evidence identifies a potent role for aerobic exercise to modulate the activity of hypothalamic neurons related to appetite; however, these studies have been primarily performed in male rodents. Since females have markedly different neuronal mechanisms regulating food intake, the current study aimed to determine the effects of acute treadmill exercise on hypothalamic neuron populations involved in regulating appetite in female mice. Mature, untrained female mice were exposed to acute sedentary, low- (10 m/min), moderate- (14 m/min), and high (18 m/min)-intensity treadmill exercise in a randomized crossover design. Mice were fasted 10 h before exercise, and food intake was monitored for 48 h after bouts. Immunohistochemical detection of cFOS was performed 3 h post-exercise to determine the changes in hypothalamic neuropeptide Y (NPY)/agouti-related peptide (AgRP), pro-opiomelanocortin (POMC), tyrosine hydroxylase (TH), and SIM1-expressing neuron activity concurrent with the changes in food intake. Additionally, stains for pSTAT3tyr705 and pERKthr202/tyr204 were performed to detect exercise-mediated changes in intracellular signaling. Briefly, moderate- and high-intensity exercises increased 24-h food intake by 5.9 and 19%, respectively, while low-intensity exercise had no effects. Furthermore, increases in NPY/AgRPARC, SIM1PVN, and TH neuron activity were observed 3 h after high-intensity exercise, with no effects on POMCARC neurons. While no effects of exercise on pERKthr202/tyr204 were observed, pSTAT3tyr705 was elevated specifically in NPY/AgRP neurons 3 h post-exercise. Overall, aerobic exercise increased the activity of several appetite-stimulating neuron populations in the hypothalamus of female mice, which may provide insight into previously reported sexual dimorphisms in post-exercise feeding.

Paragangliomas (PGLs) are neural-crest-derived, non-epithelial neuroendocrine tumors distributed along the parasympathetic and sympathetic nerves. Head-and-neck PGLs (HNPGLs) have been recognized as nonchromaffin, nonfunctional, parasympathetic tumors. By contrast, thoracoabdominal paragangliomas and pheochromocytomas (PPGLs) are chromaffin, functional, sympathetic tumors. Although HNPGLs and PPGLs have the same histological structure, the zellballen pattern, composed of chief and sustentacular cells surrounded by abundant capillaries, the pathobiological differences between these types of PGLs remain unclarified. To determine the phenotypic features of these PGLs, we performed an immunohistochemical study using specific antibodies against choline acetyltransferase (ChAT), an enzyme involved in acetylcholine synthesis, and enzymes for the catecholamine-synthesis, tyrosine hydroxylase (TH), and dopamine beta-hydroxylase (DBH), in 34 HNPGLs from 31 patients, 12 thoracoabdominal PGLs from 12 patients, and 26 pheochromocytomas from 22 patients. The expression of ChAT, TH, and DBH was 100%, 23%, and 10% in the HNPGLs; 12%, 100%, and 100% in the pheochromocytomas; and 25%, 67%, and 100% in the thoracoabdominal PGLs, respectively. These results designate HNPGLs as acetylcholine-producing parasympathetic tumors, in contrast to PPGLs being catecholamine-producing tumors. The other most frequently used neuroendocrine markers are synaptophysin and chromogranin A expressed 100% and 80%, respectively, and synaptophysin was superior to chromogranin A in HNPGLs. This is the first report of HNPGLs being acetylcholine-producing tumors. Immunohistochemistry of ChAT could be greatly useful for pathologic diagnosis of HNPGL. Whether measurement of acetylcholine levels in the blood or urine could be a tumor marker of HNPGLs should be investigated soon., (© 2021. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

Dopamine (DA) is an important signal mediator in the brain as well as in the periphery. The term "dopamine homeostasis" occasionally found in the literature refers to the fact that abnormal DA levels can be associated with a variety of neuropsychiatric disorders. An analysis of the negative feedback inhibition of tyrosine hydroxylase (TH) by DA indicates, with support from the experimental data, that the TH-DA negative feedback loop has developed to exhibit 3,4-dihydroxyphenylalanine (DOPA) homeostasis by using DA as a derepression regulator. DA levels generally decline when DOPA is removed, for example, by increased oxidative stress. Robust DOPA regulation by DA further implies that maximum vesicular DA levels are established, which appear necessary for a reliable translation of neural activity into a corresponding chemical transmitter signal. An uncontrolled continuous rise (windup) in DA occurs when Levodopa treatment exceeds a critical dose. Increased oxidative stress leads to the successive breakdown of DOPA homeostasis and to a corresponding reduction in DA levels. To keep DOPA regulation robust, the vesicular DA loading requires close to zero-order kinetics combined with a sufficiently high compensatory flux provided by TH. The protection of DOPA and DA due to a channeling complex is discussed.

Aims: The neurotropic growth factor PDGF-BB was shown to have vital neurorestorative functions in various animal models of Parkinson's disease (PD). Previous studies indicated that the regenerative property of PDGF-BB contributes to the increased intensity of tyrosine hydroxylase (TH) fibers in vivo. However, whether PDGF-BB directly modulates the expression of TH, and the underlying mechanism is still unknown. We will carefully examine this in our current study., Method: MPTP-lesion mice received PDGF-BB treatment via intracerebroventricular (i.c.v) administration, and the expression of TH in different brain regions was assessed by RT-PCR, Western blot, and immunohistochemistry staining. The molecular mechanisms of PDGF-BB-mediated TH upregulation were examined by RT-PCR, Western blot, ChIP assay, luciferase reporter assay, and immunocytochemistry., Results: We validated a reversal expression of TH in MPTP-lesion mice upon i.c.v administration of PDGF-BB for seven days. Similar effects of PDGF-BB-mediated TH upregulation were also observed in MPP + -treated primary neuronal culture and dopaminergic neuronal cell line SH-SY5Y cells. We next demonstrated that PDGF-BB rapidly activated the pro-survival PI3K/Akt and MAPK/ERK signaling pathways, as well as the downstream CREB in SH-SY5Y cells. We further confirmed the significant induction of p-CREB in PDGF-BB-treated animals in vivo. Using a genetic approach, we demonstrated that the transcription factor CREB is critical for PDGF-BB-mediated TH expression. The activation and nucleus translocation of CREB were promoted in PDGF-BB-treated SH-SY5Y cells, and the enrichment of CREB on the promoter region of TH gene was also increased upon PDGF-BB treatment., Conclusion: Our data demonstrated that PDGF-BB directly regulated the expression of TH via activating the downstream Akt/ERK/CREB signaling pathways. Our finding will further support the therapeutic potential of PDGF-BB in PD, and provide the possibility that targeting PDGF signaling can be harnessed as an adjunctive therapy in PD in the future., (© 2021 The Authors. CNS Neuroscience & Therapeutics Published by John Wiley & Sons Ltd.)

Parkinson's disease (PD) is characterized by Lewy bodies (composed predominantly of alpha-synuclein [aSyn]) and loss of pigmented midbrain dopaminergic neurons comprising the nigrostriatal pathway. Most PD patients show significant deficiency of gangliosides, including GM1, in the brain, and GM1 ganglioside appears to keep dopaminergic neurons functioning properly. Thus, supplementation of GM1 could potentially provide some rescuing effects. In this study, we demonstrate that intranasal infusion of GD3 and GM1 gangliosides reduces intracellular aSyn levels. GM1 also significantly enhances expression of tyrosine hydroxylase (TH) in the substantia nigra pars compacta of the A53T aSyn overexpressing mouse, following restored nuclear expression of nuclear receptor related 1 (Nurr1, also known as NR4A2), an essential transcription factor for differentiation, maturation, and maintenance of midbrain dopaminergic neurons. GM1 induces epigenetic activation of the TH gene, including augmentation of acetylated histones and recruitment of Nurr1 to the TH promoter region. Our data indicate that intranasal administration of gangliosides could reduce neurotoxic proteins and restore functional neurons via modulating chromatin status by nuclear gangliosides., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2021. Published by Elsevier Inc.)

[Figure: see text].

Previous research advocates that exercise is a non-pharmacological therapy for Parkinson's disease (PD). However, few studies have investigated the effects of exercise on central nervous system structures other than the nigrostriatal pathway by using PD animal models. This study investigated the effects of exercise on tyrosine hydroxylase (TH)- and cerebral dopamine neurotrophic factor (CDNF)-containing spinal-cord neurons. Male Swiss mice were divided into 4 groups: sedentary control (SEDCONT), exercise control (EXERCONT), sedentary Parkinson (SEDPD), and exercise Parkinson (EXERPD). The PD groups were submitted to a surgical procedure for stereotaxic bilateral injection of 6-hydroxydopamine into the striatum. TH- and CDNF-containing spinal-cord neurons were evaluated in all groups, using immunohistochemistry and western-blotting. TH content in the ventral horn differed notably between the SEDPD and EXERPD groups. CDNF content was highest in the EXERPD group. SEDPD and EXERPD groups differed the most, as shown by immunohistochemistry and western-blotting. The EXERPD group showed the most intense labeling in immunohistochemistry compared to the SEDCONT and EXERCONT groups. Therefore, we showed here that exercise increased the content of both TH and CDNF in the spinal-cord neurons of a bilateral PD mouse model. We may assume that the spinal cord is affected in a PD model, and therefore this central nervous system region deserves more attention from researchers dealing with PD., (Copyright © 2021 Elsevier B.V. All rights reserved.)

In Parkinson's disease, dopamine-containing nigrostriatal neurons undergo profound degeneration. Tyrosine hydroxylase (TH) is the rate-limiting enzyme in dopamine biosynthesis. TH increases in vitro formation of reactive oxygen species, and previous animal studies have reported links between cytosolic dopamine build-up and oxidative stress. To examine effects of increased TH activity in catecholaminergic neurons in vivo, we generated TH-over-expressing mice (TH-HI) using a BAC-transgenic approach that results in over-expression of TH with endogenous patterns of expression. The transgenic mice were characterized by western blot, qPCR, and immunohistochemistry. Tissue contents of dopamine, its metabolites, and markers of oxidative stress were evaluated. TH-HI mice had a 3-fold increase in total and phosphorylated TH levels and an increased rate of dopamine synthesis. Coincident with elevated dopamine turnover, TH-HI mice showed increased striatal production of H 2 O 2 and reduced glutathione levels. In addition, TH-HI mice had elevated striatal levels of the neurotoxic dopamine metabolites 3,4-dihydroxyphenylacetaldehyde and 5-S-cysteinyl-dopamine and were more susceptible than wild-type mice to the effects of amphetamine and methamphetamine. These results demonstrate that increased TH alone is sufficient to produce oxidative stress in vivo, build up autotoxic dopamine metabolites, and augment toxicity., (© 2021 The Authors. Journal of Neurochemistry published by John Wiley & Sons Ltd on behalf of International Society for Neurochemistry.)

Parkinson's disease (PD) is characterized by a progressive loss of dopamine-producing neurons in the midbrain, which results in decreased dopamine levels accompanied by movement symptoms. Oral administration of l-3,4-dihydroxyphenylalanine (L-dopa), the precursor of dopamine, provides initial symptomatic relief, but abnormal involuntary movements develop later. A deeper understanding of the regulatory mechanisms underlying dopamine homeostasis is thus critically needed for the development of a successful treatment. Here, we show that p21-activated kinase 4 (PAK4) controls dopamine levels. Constitutively active PAK4 (caPAK4) stimulated transcription of tyrosine hydroxylase (TH) via the cAMP response element-binding protein (CREB) transcription factor. Moreover, caPAK4 increased the catalytic activity of TH through its phosphorylation of S 40 , which is essential for TH activation. Consistent with this result, in human midbrain tissues, we observed a strong correlation between phosphorylated PAK4 S474 , which represents PAK4 activity, and phosphorylated TH S40 , which reflects their enzymatic activity. Our findings suggest that targeting the PAK4 signaling pathways to restore dopamine levels may provide a new therapeutic approach in PD.

Reserpine (RES) is an irreversible inhibitor of VMAT2 used to study Parkinson's disease (PD) and screening for antiparkinsonian treatments in rodents. Recently, the repeated treatment with a low dose of reserpine was proposed as a model capable of emulating progressive neurochemical, motor and non-motor impairments in PD. Conversely, compared to Wistar rats, Spontaneously Hypertensive Rats (SHR) are resistant to motor changes induced by repeated treatment with a low dose of RES. However, such resistance has not yet been investigated for RES-induced non-motor impairments. We aimed to assess whether SHR would have differential susceptibility to the object recognition deficit induced by repeated low-dose reserpine treatment. We submitted male Wistar and SHR rats to repeated RES treatment (15 s.c. injections of 0.1 mg/kg, every other day) and assessed object memory acquisition and retrieval 48 h after the 6 th RES injection (immediately before the appearance of motor impairments). Only RES Wistar rats displayed memory impairment after reserpine treatment. On the other hand, untreated SHR rats displayed object recognition memory deficit, but RES treatment restored such deficits. We also performed immunohistochemistry for tyrosine hydroxylase (TH) and α-synuclein (α-syn) 48 h after the last RES injection. In a different set of animals submitted to the same treatment, we quantified DA, 5-HT and products of lipid peroxidation in the prefrontal cortex (PFC) and hippocampus (HPC). SHR presented increased constitutive levels of DA in the PFC and reduced immunoreactivity to TH in the medial PFC and dorsal HPC. Corroborating the behavioral findings, RES treatment restored those constitutive alterations in SHR. These findings indicate that the neurochemical, molecular and genetic differences in the SHR strain are potentially relevant targets to the study of susceptibility to diseases related to dopaminergic alterations., (Copyright © 2021 Elsevier B.V. All rights reserved.)

Inflammation plays a role in neuropathology. We hypothesised that inflammation, induced by a single intraperitoneal injection of lipopolysaccharide (LPS), would induce long-term changes in the regulation of tyrosine hydroxylase (TH) in the rat midbrain. The level of 12 cytokines was initially analysed from one day to six months after LPS injection to confirm that peripheral inflammation led to neuroinflammatory changes in the midbrain. In the substantia nigra (SN), the levels of 8 of the 12 measured cytokines was significantly increased at one day. Granulocyte-macrophage colony-stimulating factor showed a threefold increased level at 6 months. The ventral tegmental area (VTA) showed a completely different pattern, with no increases in the levels of the 12 cytokines at one day and no changes beyond one week. TH activity was determined using a tritiated water release assay, TH protein and phosphorylation levels (Ser19, Ser31 and Ser40) were determined using western blotting. TH-specific activity in the SN was unchanged at one day but was substantially increased at one week and one month with no concomitant increase in TH phosphorylation. Substantial changes in TH activation without changes in TH phosphorylation have not previously been observed in the brain in response to a range of stressors. TH-specific activity was increased in the SN, and in the caudate putamen, at 6 months and was associated with increased TH phosphorylation at Ser19 and Ser40 at both locations. TH-specific activity in the VTA showed only a transient increase at day one associated with increased phosphorylation at Ser19 and Ser31 but thereafter showed no changes. This study shows that inflammation induced by LPS generated two distinct long-term changes in TH activity in the SN that are caused by different mechanisms, but there were no long-term changes in the adjacent VTA., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

Methylmercury (MeHg) is a persistent environmental neurotoxicant that may cause adverse neurodevelopmental effects. Previous studies showed that developmental MeHg exposure caused damage to brain functions that were unmasked after a silent period of years or decades. However, the underlying mechanisms of the latent neurotoxicity associated with MeHg exposure from earlier developmental stages have yet to be fully understood. Herein, we established a Caenorhabditis elegans (C. elegans) model of developmental MeHg latent toxicity. Synchronized L1 stage worms were exposed to MeHg (0, 0.05, 0.5 and 5 μM) for 48 h. Swimming moving speeds at adulthood were analyzed in worms exposed to MeHg exposure at early larvae stages. Worms developmentally exposed to MeHg had a significant decline in swimming moving speed on day 10 adult stage, but not on day 1 or 5 adult stage, even though the mercury level in the worms exposed to 0.05 or 0.5 μM MeHg were below the quantification limit on day 10 adult. Day 10 adult worms treated with MeHg showed a significant decrease in bending angle and bending frequency during swimming. Furthermore, their reduced moving speeds tended to increase during the 300-s swimming experiment. Dopamine signaling is known to be involved in the regulation of worms' moving speed. Accordingly, the moving speed of worms with cat-2 (mammalian tyrosine hydroxylase homolog) mutation or dat-1 deletion were assayed on day 10 adult. The cat-2 mutant worms did not show a decline in moving speeds, body bends or bending angles during swimming on day 10 adult stage. Analyses of moving speeds of worms with dat-1 deletion showed that the moving speeds were further reduced after MeHg exposure. However, the effects of MeHg and dat-1 deletion were not synergistic, as the interaction between these parameters did not attain statistical significance. Altogether, our results suggest that developmental MeHg exposure reduced moving speed, and this latent toxicity was less pronounced in the context of deficient production of dopamine synthesis. Tyrosine hydroxylase plays an important role in regulating dopamine-mediated modulation of neurobehavioral functions. These findings uncovered a pivotal role of dopamine and its metabolism in the latent neurotoxic effects of MeHg., (Copyright © 2021 Elsevier Inc. All rights reserved.)

Dopamine (DA) exerts well-known functions in the brain as a neurotransmitter. In addition, it plays important physiological roles in peripheral organs, but it is largely unknown how and where peripheral DA is synthesized and regulated. Catecholamines in peripheral tissues are either produced within the tissue itself and/or derived from sympathetic neurons, which release neurotransmitters for uptake by peripheral tissues. To evaluate DA-producing ability of each peripheral tissue, we generated conditional KO mice (cKO mice) in which the tyrosine hydroxylase (TH) gene is ablated in the sympathoadrenal system, thus eliminating sympathetic neurons as a DA source. We then examined the alterations in the noradrenaline (NA), DA, and 3,4-dihydroxyphenylalanine (DOPA) contents in peripheral organs and performed immunohistochemical analyses of TH-expressing cells. In the heart and pancreas of cKO mice, both the TH protein and NA levels were significantly decreased, and the DA contents were decreased in parallel with NA contents, indicating that the DA supply originated from sympathetic neurons. We found TH-immunoreactive cells in the stomach and lung, where the TH protein showed a decreasing trend, but the DA levels were not decreased in cKO mice. Moreover, we found a significant correlation between the DA content in the kidney and the plasma DOPA concentration, suggesting that the kidney takes up DOPA from blood to make DA. The aforementioned data unravel differences in the DA biosynthetic pathway among tissues and support the role of sympathetic neurons as a DA supplier., Competing Interests: Conflict of interest The authors declare that they have no conflicts of interest with the contents of this article., (Copyright © 2021 The Authors. Published by Elsevier Inc. All rights reserved.)

Rationale: Transgenic mouse lines expressing Cre-recombinase under the regulation of either dopamine transporter (DAT) or tyrosine hydroxylase (TH) promoters are commonly used to study the dopamine (DA) system. While use of the TH promoter appears to have less liability to changes in native gene expression, transgene insertion in the DAT locus results in reduced DAT expression and function. This confound is sometimes overlooked in genetically targeted behavioral experiments., Objectives: We sought to evaluate the suitability of DAT-Ires-Cre and TH-Cre transgenic lines for behavioral pharmacology experiments with DA agonists. We hypothesized that DAT-Ires-Cre expression would impact DAT-mediated behaviors, but no impact of TH-Cre expression would be observed., Methods: DAT-Ires-Cre and TH-Cre mice bred on mixed 129S6/C57BL/6 and pure C57BL/6 backgrounds were evaluated for novelty-induced, baseline, and amphetamine (AMPH)-induced locomotion, and for AMPH and D1 agonist (SKF-38393)-induced preservative behaviors., Results: DAT-Ires-Cre mice on both mixed 129S6/C57BL/6 and pure C57BL/6 backgrounds displayed increased novelty-induced activity and decreased AMPH-induced locomotion, with mixed results for AMPH-induced stereotypy. TH-Cre mice on both backgrounds showed typical baseline activity and AMPH-induced stereotypy, with a difference in AMPH-induced locomotion observed only on the mixed background. Both transgenic lines displayed unaltered SKF-38393-induced grooming behavior., Conclusions: Our findings indicate that the DAT-Ires-Cre transgenic line may lead to confounds for experiments that are dependent on DAT expression. The TH-Cre transgenic line studied here may be a more useful option, depending on background strain, because of its lack of baseline and drug-induced phenotypes. These data highlight the importance of appropriate controls in studies employing transgenic mice.

5'-Nucleotidase domain-containing protein 2 (NT5DC2) has been revealed by genome-wide association studies (GWAS) as a gene implicated in neuropsychiatric disorders related to the abnormality of dopamine (DA) activity in the brain. Based on its amino acid sequence, NT5DC2 is assumed to be a member of the family of haloacid dehalogenase-type phosphatases; although there is no information about its function and structural conformation. We recently reported that NT5DC2 binds to tyrosine hydroxylase (TH) and that the down-regulation of NT5DC2 tended to increase DA synthesis. In this study, we investigated whether NT5DC2 could regulate the catalytic activity of TH, which converts tyrosine to DOPA, because the phosphorylation level of TH, controlled by protein kinases and phosphatases, is well known to regulate its catalytic activity. The down-regulation of NT5DC2 by siRNA increased mainly DOPA synthesis by TH in PC12D cells, although this down-regulation tended to increase the conversion of DOPA to DA by aromatic L-amino acid decarboxylase. The increased DOPA synthesis should be attributed to the catalytic activity of TH controlled by its phosphorylation, because Western blot analysis revealed that the down-regulation of NT5DC2 tended to increase the level of TH phosphorylated at its Ser residues, but not that of the TH protein. Moreover, the induction of kinase activity by forskolin markedly potentiated the phosphorylation of TH at its Ser40 in PC12D cells having down-regulated NT5DC2. Immunocytochemical analysis of PC12D cells demonstrated that NT5DC2, TH protein, and TH phosphorylated at its Ser40 were predominantly localized in the cytoplasm and that the localization of NT5DC2 and TH proteins partially overlapped. Collectively, our results indicate that NT5DC2 could work to inhibit the DOPA synthesis by decreasing the phosphorylation of TH at its Ser40. We propose that NT5DC2 might decrease this phosphorylation of TH by promoting dephosphorylation or by inhibiting kinase activity.

The hypothalamus contains catecholaminergic neurons marked by the expression of tyrosine hydroxylase (TH). As multiple chemical messengers coexist in each neuron, we determined if hypothalamic TH-immunoreactive (ir) neurons express vesicular glutamate or GABA transporters. We used Cre/loxP recombination to express enhanced GFP (EGFP) in neurons expressing the vesicular glutamate (vGLUT2) or GABA transporter (vGAT), then determined whether TH-ir neurons colocalized with native EGFP Vglut2 - or EGFP Vgat -fluorescence, respectively. EGFP Vglut2 neurons were not TH-ir. However, discrete TH-ir signals colocalized with EGFP Vgat neurons, which we validated by in situ hybridization for Vgat mRNA. To contextualize the observed pattern of colocalization between TH-ir and EGFP Vgat , we first performed Nissl-based parcellation and plane-of-section analysis, and then mapped the distribution of TH-ir EGFP Vgat neurons onto atlas templates from the Allen Reference Atlas (ARA) for the mouse brain. TH-ir EGFP Vgat neurons were distributed throughout the rostrocaudal extent of the hypothalamus. Within the ARA ontology of gray matter regions, TH-ir neurons localized primarily to the periventricular hypothalamic zone, periventricular hypothalamic region, and lateral hypothalamic zone. There was a strong presence of EGFP Vgat fluorescence in TH-ir neurons across all brain regions, but the most striking colocalization was found in a circumscribed portion of the zona incerta (ZI)-a region assigned to the hypothalamus in the ARA-where every TH-ir neuron expressed EGFP Vgat . Neurochemical characterization of these ZI neurons revealed that they display immunoreactivity for dopamine but not dopamine β-hydroxylase. Collectively, these findings indicate the existence of a novel mouse hypothalamic population that may signal through the release of GABA and/or dopamine., (© 2020 Wiley Periodicals, Inc.)

This work is aimed at studying the mechanisms of reciprocal humoral regulation of noradrenaline-producing organs in rats in the perinatal period of development. The activity of noradrenaline synthesis enzymes tyrosine hydroxylase and dopamine-beta-hydroxylase was measured in the brain and adrenal glands 48 and 72 h after the injection of immunotoxin (anti-dopamine-beta-hydroxylase-saporin) into the rat brain ventricles. It was shown that, 48 h after the immunotoxin injection into the brain, the activity of tyrosine hydroxylase in the brain decreased; however, 72 h after the injection it reached the control levels. This fact indicates that noradrenaline synthesis in the survived neurons increases. In the adrenal glands, 72 h after the immunotoxin injection into the brain, the activity of dopamine-beta-hydroxylase increased. This points to a compensatory increase in the rate of noradrenaline synthesis in the adrenal glands when the synthesis of noradrenaline in the brain is inhibited.

Nigrostriatal dopaminergic systems govern physiological functions related to locomotion, and their dysfunction leads to movement disorders, such as Parkinson's disease and dopa-responsive dystonia (Segawa disease). Previous studies revealed that expression of the gene encoding nigrostriatal tyrosine hydroxylase (TH), a rate-limiting enzyme of dopamine biosynthesis, is reduced in Parkinson's disease and dopa-responsive dystonia; however, the mechanism of TH depletion in these disorders remains unclear. In this article, we review the molecular mechanism underlying the neurodegeneration process in dopamine-containing neurons and focus on the novel degradation pathway of TH through the ubiquitin-proteasome system to advance our understanding of the etiology of Parkinson's disease and dopa-responsive dystonia. We also introduce the relation of α-synuclein propagation with the loss of TH protein in Parkinson's disease as well as anticipate therapeutic targets and early diagnosis of these diseases.

Neurotransmitters, such as oxytocin (OT), vasopressin (AVP), and dopamine (DA), within the mesolimbic system have deeply conserved roles in regulating mating-related behaviors. However, comparative studies among monogamous and polygamous animals focus mainly on Microtus; very little research has been done in gerbils. Here, we measured body weight, body length, tail length, serum hormone concentrations, and the immunoreactive (ir)-cells of OT, AVP, and tyrosine hydroxylase (TH) in the brain of the polygamous great gerbil (Rhombomys opimus), midday gerbil (Meriones meridianus), and monogamous Mongolian gerbil (Meriones unguiculatus). Body weight, body length, tail length, and serum AVP concentrations were greater in the great gerbil than in the midday gerbil and Mongolian gerbil. The number of OT and AVP cells in the para ventricular nucleus (PVN) and supra optic nucleus (SON) of the hypothalamus were greater in the Mongolian gerbil than in the great gerbil and midday gerbil. Similarly, the number of TH cells in the PVN, medial preoptic area (MPOA), and ventral tegmental area (VTA) was greater in the Mongolian gerbil than in the great gerbil and midday gerbil. To summarize, the number of OT and AVP cells in the PVN and SON and TH cells in the PVN, MPOA, and VTA in the monogamous Mongolian gerbil are greater than those in the great gerbil and midday gerbil., (Copyright © 2020. Published by Elsevier B.V.)

Background: Tyrosine hydroxylase (TH) catalyzes the rate-limiting step for the biosynthesis of the catecholamines dopamine, noradrenaline and adrenaline. Although its distribution in different organs, species and stages of development has been the subject of numerous studies, the recent emergence of 3D imaging techniques has created the potential to shed new light on the dynamics of TH expression during the development of the mammalian central and peripheral nervous systems., New Method: Here, we describe a flowchart summarizing different protocols adapted to developmental stage-specific tissues to generate a 3D atlas of the catecholaminergic system in the brain and peripheral nervous system in mice from embryonic to pre-weaning stages. The procedures described allowed a quantitative assessment of developing TH-positive neuronal populations and pathways, previously understudied due to dimensional limitations., Results: Our approach allowed us to reveal in 3D the dynamics of the onset and the establishment of the catecholaminergic system in embryonic and developing central and peripheral nervous system. Quantitative analyses applied to 3D images yielded accurate measurements of neuron population volumes and numbers, and tract pathway dimensions for selected TH-positive brain structures., Comparison With Existing Methods: We applied a set of different protocols to yield a comprehensive flowchart for 3D imaging and a precise quantitative assessment of specific neuronal populations during the course of their development up to adulthood in mice., Conclusion: The procedures described and the extensive 3D mapping of TH immunoreactivity at early embryonic and postnatal stages provide a comprehensive view of the onset and development of the catecholaminergic system in the mouse brain and sympathoadrenal nervous system., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Dopaminergic functions are important for various biological activities, and their impairment leads to neurodegeneration, a hallmark of Parkinson's disease (PD). Chronic manganese (Mn) exposure causes the neurological disorder manganism, presenting symptoms similar to those of PD. Emerging evidence has linked the transcription factor RE1-silencing transcription factor (REST) to PD and also Alzheimer's disease. But REST's role in dopaminergic neurons is unclear. Here, we investigated whether REST protects dopaminergic neurons against Mn-induced toxicity and enhances expression of the dopamine-synthesizing enzyme tyrosine hydroxylase (TH). We report that REST binds to RE1 consensus sites in the TH gene promoter, stimulates TH transcription, and increases TH mRNA and protein levels in dopaminergic cells. REST binding to the TH promoter recruited the epigenetic modifier cAMP-response element-binding protein-binding protein/p300 and thereby up-regulated TH expression. REST relieved Mn-induced repression of TH promoter activity, mRNA, and protein levels and also reduced Mn-induced oxidative stress, inflammation, and apoptosis in dopaminergic neurons. REST reduced Mn-induced proinflammatory cytokines, including tumor necrosis factor α, interleukin 1β (IL-1β), IL-6, and interferon γ. Moreover, REST inhibited the Mn-induced proapoptotic proteins Bcl-2-associated X protein (Bax) and death-associated protein 6 (Daxx) and attenuated an Mn-induced decrease in the antiapoptotic proteins Bcl-2 and Bcl-xL. REST also enhanced the expression of antioxidant proteins, including catalase, NF-E2-related factor 2 (Nrf2), and heme oxygenase 1 (HO-1). Our findings indicate that REST activates TH expression and thereby protects neurons against Mn-induced toxicity and neurological disorders associated with dopaminergic neurodegeneration., (© 2020 Pajarillo et al.)

Two billion people are chronically infected with Toxoplasma gondii worldwide with unknown consequences. Important neurological diseases have been associated to the brain infection, making essential to understand the neurophysiological changes associated with the neuronal encystment. T. gondii may subvert neuronal functions modifying neurotransmitter concentration in chronically infected mice but the molecular mechanisms involved are still unclear. Parasites were observed inside neuronal cells in cultures from 24-192 hs. The rate of infection increased with time. Neurite density decreased affecting network functionality. Neuronal survival was affected and we detected the presence of cysts inside neuronal bodies and dilated portions of neurites in association with a relative increase of TH-positive neuritic area without noticeable changes in DA immunofluorescence pattern. These results advance our knowledge of the interaction between T. gondii and the neuronal network of the host., (Copyright © 2019 Elsevier B.V. All rights reserved.)

Current pharmacological treatments for Parkinson's disease (PD) are focused on symptomatic relief, but not on disease modification, based on the strong belief that PD is caused by irreversible dopaminergic neuronal death. Thus, the concept of the presence of dormant dopaminergic neurons and its possibility as the disease-modifying therapeutic target against PD have not been explored. Here we show that optogenetic activation of substantia nigra pars compacta (SNpc) neurons alleviates parkinsonism in acute PD animal models by recovering tyrosine hydroxylase (TH) from the TH-negative dormant dopaminergic neurons, some of which still express DOPA decarboxylase (DDC). The TH loss depends on reduced dopaminergic neuronal firing under aberrant tonic inhibition, which is attributed to excessive astrocytic GABA. Blocking the astrocytic GABA synthesis recapitulates the therapeutic effect of optogenetic activation. Consistently, SNpc of postmortem PD patients shows a significant population of TH-negative/DDC-positive dormant neurons surrounded by numerous GABA-positive astrocytes. We propose that disinhibiting dormant dopaminergic neurons by blocking excessive astrocytic GABA could be an effective therapeutic strategy against PD., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

Adult female rats show greater running output compared with age-matched male rats, and the midbrain dopaminergic system may account for behavioral differences in running output. However, it is unknown if the lower running output in adult males can be regulated by wheel running experience during adolescence, and whether wheel running experience during adolescence will diminish the sex differences in running output during adulthood. We therefore determined and compared the exercise output in adult male and female rats that either had initiated voluntary wheel running only during adulthood or during adolescence. Our results demonstrate that running output in adult males were significantly higher when running was initiated during adolescence, and this higher running output was not significantly different from females. Running output did not differ during adulthood in females when wheel running was initiated during adolescence or during adulthood. Higher running output in females was associated with reduced expression of tyrosine hydroxylase and hyperactivation of calcium/calmodulin-dependent protein kinase II (CaMKII) in the dorsal striatum. Notably, running during adolescence-induced higher exercise output in adult males was associated with hyperactivation of CaMKII in the dorsal striatum, indicating a mechanistic role for CaMKII in running output. Together, the present results indicate sexually dimorphic adaptive biochemical changes in the dorsal striatum in rats that had escalated running activity, and highlight the importance of including sex as a biological variable in exploring neuroplasticity changes that predict enhanced exercise output in a voluntary physical activity paradigm., (Copyright © 2019 Elsevier B.V. All rights reserved.)