[Improvement effect of cinnamaldehyde on reserpine-induced Parkinson's disease rat model].

In order to study the neuroprotective mechanism of cinnamaldehyde on reserpine-induced Parkinson's disease(PD) rat models, 72 male Wistar rats were randomly divided into blank group, model group, Madopar group, and cinnamaldehyde high-, medium-, and low-dose groups. Except for the blank group, the other groups were intraperitoneally injected with reserpine of 0.1 mg·kg~(-1) once every other morning, and cinnamaldehyde and Madopar solutions were gavaged every afternoon. Open field test, rotarod test, and oral chewing movement evaluation were carried out in the experiment. The brain was taken and fixed. The positive expression of dopamine receptor D1(DRD1) was detected by TSA, and the changes in neurotransmitters such as dopamine(DA) and 3,4-dihydroxyphenylacetic acid(DOPAC) in the brain were detected by enzyme-linked immunosorbent assay(ELISA). The protein and mRNA expression levels of tyrosine hydroxylase(TH) and α-synuclein(α-Syn) in substantia nigra(SN) were detected by RT-PCR and Western blot. The results showed that after the injection of reserpine, the hair color of the model group became yellow and dirty; the arrest behavior was weakened, and the body weight was reduced. The spontaneous movement and exploration behavior were reduced, and the coordination exercise ability was decreased. The number of oral chewing was increased, but the cognitive ability was decreased, and the proportion of DRD1 positive expression area in SN was decreased. The expression of TH protein and mRNA was down-regulated, and that of α-Syn protein and mRNA was up-regulated. After cinnamaldehyde intervention, it had an obvious curative effect on PD model animals. The spontaneous movement behavior, the time of staying in the rod, the time of movement, the distance of movement, and the number of standing times increased, and the number of oral chewing decreased. The proportion of DRD1 positive expression area in SN increased, and the protein and mRNA expression levels of α-Syn were down-regulated. The protein and mRNA expression levels of TH were up-regulated. In addition, the levels of DA, DOPAC, and homovanillic acid(HVA) neurotransmitters in the brain were up-regulated. This study can provide a new experimental basis for clinical treatment and prevention of PD.

Tyrosine hydroxylase plays crucial roles in larval cuticle formation and larval-pupal tanning in the rice stem borer, Chilo suppressalis.

The striped stem borer, Chilo suppressalis (Walker), a notorious pest infesting rice, has evolved a high level of resistance to many commonly used insecticides. In this study, we investigate whether tyrosine hydroxylase (TH), which is required for larval development and cuticle tanning in many insects, could be a potential target for the control of C. suppressalis. We identified and characterized the full-length cDNA (CsTH) of C. suppressalis. The complete open reading frame of CsTH (MW690914) was 1683 bp in length, encoding a protein of 560 amino acids. Within the first to the sixth larval instars, CsTH was high in the first day just after molting, and lower in the ensuing days. From the wandering stage to the adult stage, levels of CSTH began to rise and reached a peak at the pupal stage. These patterns suggested a role for the gene in larval development and larval-pupal cuticle tanning. When we injected dsCsTH or 3-iodotyrosine (3-IT) as a TH inhibitor or fed a larva diet supplemented with 3-IT, there were significant impairments in larval development and larval-pupal cuticle tanning. Adult emergence was severely impaired, and most adults died. These results suggest that CsTH might play a critical role in larval development as well as larval-pupal tanning and immunity in C. suppressalis, and this gene could form a potential novel target for pest control.

Tyrosine hydroxylase inhibits HCC progression by downregulating TGFß/Smad signaling.

The alteration of metabolic processes has been found to have significant impacts on the development of hepatocellular carcinoma (HCC). Nevertheless, the effects of dysfunction of tyrosine metabolism on the development of HCC remains to be discovered. This research demonstrated that tyrosine hydroxylase (TH), which responsible for the initial and limiting step in the bio-generation of the neuro-transmitters dopamine and adrenaline, et al. was shown to be reduced in HCC. Increased expression of TH was found facilitates the survival of HCC patients. In addition, decreased TH indicated larger tumor size, much more numbers of tumor, higher level of AFP, and the presence of cirrhosis. TH effectively impairs the growth and metastasis of HCC cells, a process dependent on the phosphorylation of serine residues (S19/S40). TH directly binds to Smad2 and hinders the cascade activation of TGFß/Smad signaling with the treatment of TGFß1. In summary, our study uncovered the non-metabolic functions of TH in the development of HCC and proposes that TH might be a promising biomarker for diagnosis as well as an innovative target for metastatic HCC.

Mice Expressing A53T/A30P Mutant Alpha-Synuclein in Dopamine Neurons Do Not Display Behavioral Deficits.

Alpha-synuclein has been implicated in neurodegenerative diseases such as Parkinson's disease and dementia with Lewy bodies, with A53T and A30P mutations shown to be disease causing. It has been reported that hemizygous transgenic mice with tyrosine hydroxylase promotor-driven expression of A53T/A30P mutant alpha-synuclein in dopamine neurons provide a useful preclinical model of these conditions by virtue of developing behavioral deficits. Here, we report a lack of replication of this finding. Despite detecting robust overexpression of A53T/A30P mutant alpha-synuclein in dopamine neurons, we did not observe decreased tyrosine hydroxylase immunofluorescence or behavioral deficits in these mice. Our results demonstrate that preclinical models of synucleinopathy need careful validation in the field.

Role of NT5DC2 in tyrosine hydroxylase phosphorylation based on the analysis of NT5DC2-binding proteins.

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.

Effects of Bradykinin B2 Receptor Ablation from Tyrosine Hydroxylase Cells on Behavioral and Motor Aspects in Male and Female Mice.

The kallikrein-kinin system is a versatile regulatory network implicated in various biological processes encompassing inflammation, nociception, blood pressure control, and central nervous system functions. Its physiological impact is mediated through G-protein-coupled transmembrane receptors, specifically the B1 and B2 receptors. Dopamine, a key catecholamine neurotransmitter widely distributed in the CNS, plays a crucial role in diverse physiological functions including motricity, reward, anxiety, fear, feeding, sleep, and arousal. Notably, the potential physical interaction between bradykinin and dopaminergic receptors has been previously documented. In this study, we aimed to explore whether B2R modulation in catecholaminergic neurons influences the dopaminergic pathway, impacting behavioral, metabolic, and motor aspects in both male and female mice. B2R ablation in tyrosine hydroxylase cells reduced the body weight and lean mass without affecting body adiposity, substrate oxidation, locomotor activity, glucose tolerance, or insulin sensitivity in mice. Moreover, a B2R deficiency in TH cells did not alter anxiety levels, exercise performance, or motor coordination in female and male mice. The concentrations of monoamines and their metabolites in the substantia nigra and cortex region were not affected in knockout mice. In essence, B2R deletion in TH cells selectively influenced the body weight and composition, leaving the behavioral and motor aspects largely unaffected.

Mutual Effects of Orexin and Bone Morphogenetic Proteins on Catecholamine Regulation Using Adrenomedullary Cells.

Orexins are neuronal peptides that play a prominent role in sleep behavior and feeding behavior in the central nervous system, though their receptors also exist in peripheral organs, including the adrenal gland. In this study, the effects of orexins on catecholamine synthesis in the rat adrenomedullary cell line PC12 were investigated by focusing on their interaction with the adrenomedullary bone morphogenetic protein (BMP)-4. Orexin A treatment reduced the mRNA levels of key enzymes for catecholamine synthesis, including tyrosine hydroxylase (Th), 3,4-dihydroxyphenylalanie decarboxylase (Ddc) and dopamine ß-hydroxylase (Dbh), in a concentration-dependent manner. On the other hand, treatment with BMP-4 suppressed the expression of Th and Ddc but enhanced that of Dbh with or without co-treatment with orexin A. Of note, orexin A augmented BMP-receptor signaling detected by the phosphorylation of Smad1/5/9 through the suppression of inhibitory Smad6/7 and the upregulation of BMP type-II receptor (BMPRII). Furthermore, treatment with BMP-4 upregulated the mRNA levels of OX1R in PC12 cells. Collectively, the results indicate that orexin and BMP-4 suppress adrenomedullary catecholamine synthesis by mutually upregulating the pathway of each other in adrenomedullary cells.

CRISPR/sgRNA-directed synergistic activation mediator (SAM) as a therapeutic tool for Parkinson´s disease.

Parkinson`s disease (PD) is the second most prevalent neurodegenerative disease, and different gene therapy strategies have been used as experimental treatments. As a proof-of-concept for the treatment of PD, we used SAM, a CRISPR gene activation system, to activate the endogenous tyrosine hydroxylase gene (th) of astrocytes to produce dopamine (DA) in the striatum of 6-OHDA-lesioned rats. Potential sgRNAs within the rat th promoter region were tested, and the expression of the Th protein was determined in the C6 glial cell line. Employing pseudo-lentivirus, the SAM complex and the selected sgRNA were transferred into cultures of rat astrocytes, and gene expression and Th protein synthesis were ascertained; furthermore, DA release into the culture medium was determined by HPLC. The DA-producing astrocytes were implanted into the striatum of 6-OHDA hemiparkinsonian rats. We observed motor behavior improvement in the lesioned rats that received DA-astrocytes compared to lesioned rats receiving astrocytes that did not produce DA. Our data indicate that the SAM-induced expression of the astrocyte´s endogenous th gene can generate DA-producing astrocytes that effectively reduce the motor asymmetry induced by the lesion.

Effect of electroacupuncture on oxidative stress and cell apoptosis induced by ferroptosis in mice with Parkinson's disease. / ç”µé’ˆå¯¹å¸•é‡‘æ£®ç— å°é¼ é“æ­»äº¡è¯±å¯¼çš„æ°§åŒ–åº”æ¿€åŠç»†èƒžå‡‹äº¡çš„å½±å“.

OBJECTIVES: To observe the effect of electroacupuncture (EA) on ferroptosis and apoptosis-related proteins in the substantia nigra of midbrain in mice with Parkinson's disease (PD), so as to explore its possible mechanisms in the treatment of PD. METHODS: Twenty-four C57BL/6 mice were randomly divided into blank, model and EA groups, with 8 mice in each group. The PD model was established by continuous gavage of rotenone for 4 weeks. EA was applied at "Baihui" (GV20), "Quchi" (LI11) and "Zusanli" (ST36) for 20 min, once a day for 14 days, with 2-day rest after every 5-day treatment. The open field test was used to evaluate the residence time in the central area, ave-rage movement speed, and total distance of the open field. Western blot was used to detect the protein expression le-vels of divalent metal ion transporter 1 (DMT1), membrane ferroportin 1 (FPN1), glutathione peroxidase 4 (GPX4), proapoptotic protein Bax, and anti apoptotic protein Bcl-2 in the substantia nigra. Immunohistochemical method was used to detect the morphological changes of neurons and the positive expression of tyrosine hydroxylase (TH) in the substantia nigra of mice. RESULTS: After 4 weeks of modeling, compared with the blank group, the residence time in the central area, average speed and total distance of open field were significantly lower (P<0.000 1, P<0.01, P<0.001)；the protein expression levels of DMT1 and Bax in the substantia nigra were increased (P<0.001, P<0.000 1), while the protein expression levels of FPN1, GPX4 and Bcl-2, and the optical density of TH+ cells in the substantia nigra were decreased (P<0.000 1, P<0.001) in the model group. In comparison with the model group, the residence time in the central area, average speed, and total distance of the EA group were increased (P<0.01, P<0.05)；the protein expression levels of DMT1 and Bax in the substantia nigra were decreased (P<0.01, P<0.001), while the protein expression levels of FPN1, GPX4, and Bcl-2, and the optical density of TH+ cells in the substantia nigra were increased (P<0.000 1, P<0.01, P<0.001, P<0.05). CONCLUSIONS: EA has a protective effect on dopaminergic neurons in the substantia nigra of midbrain in PD model mice, which may be related with its effect in regulating oxidative stress and cell apoptosis induced by ferroptosis.

Compensatory Processes in Striatal Neurons Expressing the Tyrosine Hydroxylase Gene in Transgenic Mice in a Model of Parkinson's Disease.

The mammalian striatum is known to contain non-dopaminergic neurons that express dopamine (DA)-synthesizing enzymes and produce DA, responsible for the regulation of motor function. This study assessed the expression of DA-synthesizing enzymes in striatal neurons and their role in DA synthesis in transgenic mice expressing the green fluorescent protein (GFP) gene under the tyrosine hydroxylase (TH) gene promoter in a 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) model of Parkinson's disease (PD). We showed that, in Parkinsonian animals, the number of neurons expressing the TH gene increased by 1.9 times compared with the control (0.9% NaCl), which indicates a compensatory response to the DAergic denervation of the striatum. This assumption is supported by a 2.5-fold increase in the expression of genes for TH and transcription factor Nurr1 and a 1.45-fold increase in the expression of the large amino acid transporter 1 gene. It is noteworthy that, in Parkinsonian mice, in contrast to the controls, DA-synthesizing enzymes were found not only in nerve fibers but also in neuronal cell bodies. Indeed, TH or TH and aromatic L-amino acid decarboxylase (AADC) were detected in GFP-positive neurons, and AADC was detected in GFP-negative neurons. These neurons were shown to synthesize DA, and this synthesis is compensatorily increased in Parkinsonian mice. The above data open the prospect of improving the treatment of PD by maintaining DA homeostasis in the striatum.

[Clinical and genetic analysis of tyrosine hydroxylase deficiency of six cases].

The clinical and molecular genetic data of 6 patients with genetically confirmed tyrosine hydroxylase deficiency(THD) diagnosed in Department of Neurology, Qilu Hospital of Shandong University from March 2017 to February 2022 were retrospectively collected and analyzed. The 6 patients were from 5 families. Among them, 5 patients had persistent or paroxysmal abnormal walking posture, 4 patients had dystonia of head and face, including spasm of perioral and oculopharyngeal muscles, hyperactivity, and binocular upvision, 4 patients showed obvious morning light and evening heavy phenomenon, 2 patients had postural tremor of limbs, 2 patients had psychomotor retardation from childhood, 1 patient only had limb and cervical muscle weakness, 1 patient had epileptic seizures. Of the 6 patients, only 1 was adult-onset, and the rest were child-onset. Four patients had good response to low-dose dopa preparation, 2 patients from the same family had poor response to dopamine treatment, requiring extremely low dose initiation and multi-frequency titration treatment. However, the long-term treatment effect was poor with obvious abnormalities. Gene testing of 5 families revealed 8 mutations in the TH gene, with c.698G>A (p.R233H) being the hot spot mutation site. The clinical manifestations of THD are complex. Besides paroxysmal or persistent dystonia, it can also be accompanied by eye movement crisis, muscle weakness, epilepsy, and delayed mental and motor development. Most patients respond well to low-dose dopamine preparations, but a small number of patients require titration treatment with extremely low-dose dopamine preparations, and the long-term effect is not satisfactory.

Generation of a tyrosine hydroxylase-2A-Cre knockin non-human primate model by homology-directed-repair-biased CRISPR genome editing.

Non-human primates (NHPs) are the closest animal model to humans; thus, gene engineering technology in these species holds great promise for the elucidation of higher brain functions and human disease models. Knockin (KI) gene targeting is a versatile approach to modify gene(s) of interest; however, it generally suffers from the low efficiency of homology-directed repair (HDR) in mammalian cells, especially in non-expressed gene loci. In the current study, we generated a tyrosine hydroxylase (TH)-2A-Cre KI model of the common marmoset monkey (marmoset; Callithrix jacchus) using an HDR-biased CRISPR-Cas9 genome editing approach using Cas9-DN1S and RAD51. This model should enable labeling and modification of a specific neuronal lineage using the Cre-loxP system. Collectively, the current study paves the way for versatile gene engineering in NHPs, which may be a significant step toward further biomedical and preclinical applications.

Post-Mortem Studies of Neurturin Gene Therapy for Parkinson's Disease: Two Subjects with 10 Years CERE120 Delivery.

BACKGROUND: Neurturin is a member of the glial cell line-derived neurotrophic factor family of neurotrophic factors and has the potential to protectdegenerating dopaminergic neurons. OBJECTIVE: Here, we performed post-mortem studies on two patients with advanced Parkinson's disease that survived 10 years following AAV-neurturin gene (Cere120) delivery to verify long-term effects of trophic factor neurturin. METHODS: Cere120 was delivered to the putamen bilaterally in one case and to the putamen plus substantia nigra bilaterally in the second. Immunohistochemistry was used to examine neurturin, Rearranged during transfection(RET), phosphor-S6, and tyrosine hydroxylase expressions, inflammatory reactions, and α-synuclein accumulation. RESULTS: In both patients there was persistent, albeit limited, neurturin expression in the putamen covering 1.31% to 5.92% of the putamen. Dense staining of tyrosine hydroxylase-positive fibers was observed in areas that contained detectable neurturin expression. In substantia nigra, neurturin expression was detected in 11% of remaining melanin-containing neurons in the patient with combined putamenal and nigral gene delivery, but not in the patient with putamenal gene delivery alone. Tyrosine hydroxylase positive neurons were 66% to 84% of remaining neuromelanin neurons in substantia nigra with Cere120 delivery and 23% to 24% in substantia nigra without gene delivery. More RET and phosphor-S6 positive neurons were observed in substantia nigra following nigral Cere120. Inflammatory and Lewy pathologies were similar in substantia nigra with or without Cere120 delivery. CONCLUSIONS: This study provides evidence of long-term persistent transgene expression and bioactivity following gene delivery to the nigrostriatal system. Therefore, future efforts using gene therapy for neurodegenerative diseases should consider means to enhance remaining dopamine neuron function and stop pathological propagation. © 2023 The Authors. Movement Disorders published by Wiley Periodicals LLC on behalf of International Parkinson and Movement Disorder Society.

Schistosoma japonicum Tyrosine Hydroxylase is promising targets for immunodiagnosis and immunoprotection of Schistosomiasis japonica.

Identification of promising schistosome antigen targets is crucial for the development of anti-schistosomal strategies. Schistosomes rely on their neuromuscular systems to coordinate important locomotory behaviors. Tyrosine hydroxylase (TH) is critical in the initial rate-limiting step in biosynthesis of catecholamine, the important neuroactive agents, which promote the lengthening of the worm through muscular relaxation and are therefore of great importance to the movement of the organism both within and between its hosts. THs from both Schistosoma mansoni and Schistosoma japonicum and their enzyme activities have been discovered; however, the role of these proteins during infection have not been explored. Herein, a recombinant protein of the nonconserved fragment of S. japonicum TH (SjTH) was produced and the corresponding polyclonal antibody was generated. The expression and antigenicity of SjTH were detected by qRT-PCR, western blotting, immunofluorescence assays, and ELISA. Mice immunized with the recombinant SjTH were challenged with cercariae to evaluate the immunoprotective value of this protein. Our results showed SjTH not only distributed in the head associated with the central nervous system, but also expressed along the tegument and the intestinal intima, which are involved in the movement, coupling and digestion of the parasites and associated with the peripheral nervous system. This protein can effectively stimulate humoral immune responses in mammalian hosts and has high potential as a biomarker for schistosomiasis immunodiagnosis. Furthermore, immunization with recombinant SjTH showed to reduce the worm and egg burden of challenged mice, and to contribute to the systemic balance of the Th1/Th2 responses. Taken together, these results suggest that SjTH is an important pathogenic molecule in S. japonicum and may be a possible target for anti-schistosomal approaches.

CRISPR/Cas9-Mediated genomic knock out of tyrosine hydroxylase and yellow genes in cricket Gryllus bimaculatus.

Gryllus bimaculatus is an emerging model organism in various fields of biology such as behavior, neurology, physiology and genetics. Recently, application of reverse genetics provides an opportunity of understanding the functional genomics and manipulating gene regulation networks with specific physiological response in G. bimaculatus. By using CRISPR/Cas9 system in G. bimaculatus, we present an efficient knockdown of Tyrosine hydroxylase (TH) and yellow-y, which are involved in insect melanin and catecholamine-biosynthesis pathway. As an enzyme catalyzing the conversion of tyrosine to 3,4-dihydroxyphenylalanine, TH confines the first step reaction in the pathway. Yellow protein (dopachrome conversion enzyme, DCE) is also involved in the melanin biosynthetic pathway. The regulation system and molecular mechanism of melanin biogenesis in the pigmentation and their physiological functions in G. bimaculatus hasn't been well defined by far for lacking of in vivo models. Deletion and insertion of nucleotides in target sites of both TH and Yellow are detected in both F0 individuals and the inheritable F1 progenies. We confirm that TH and yellow-y are down-regulated in mutants by quantitative real-time PCR analysis. Compared with the control group, mutations of TH and yellow-y genes result in defects in pigmentation. Most F0 nymphs with mutations of TH gene die by the first instar, and the only adult had significant defects in the wings and legs. However, we could not get any homozygotes of TH mutants for all the F2 die by the first instar. Therefore, TH gene is very important for the growth and development of G. bimaculatus. When the yellow-y gene is knocked out, 71.43% of G. bimaculatus are light brown, with a slight mosaic on the abdomen. The yellow-y gene can be inherited stably through hybridization experiment with no obvious phenotype except lighter cuticular color. The present loss of function study indicates the essential roles of TH and yellow in pigmentation, and TH possesses profound and extensive effects of dopamine synthesis in embryonic development in G. bimaculatus.

In Response to a Punctual Stress Male and Female Tyrosine Hydroxylase Haploinsufficient Mice Show a Deteriorated Behavior, Immunity, and Redox State.

An inadequate stress response is associated with impaired neuroimmunoendocrine communication, increasing morbidity and mortality. Since catecholamines (CA) constitute one of the acute stress response pathways, female mice with an haploinsufficiency of the tyrosine hydroxylase gene (TH-HZ), the main limiting enzyme in CA synthesis, show low CA amounts, exhibiting an impairment of homeostatic systems. The aim of this study was to investigate the effect of a punctual stress in TH-HZ mice, determining the differences with wild-type (WT) mice and those due to sex by restraint with a clamp for 10 min. After restraint, a behavioral battery was performed, and several immune functions, redox state parameters, and CA amounts were evaluated in peritoneal leukocytes. Results show that this punctual stress impaired WT behavior and improved female WT immunity and oxidative stress, whereas in TH-HZ mice, all parameters were impaired. In addition, different responses to stress due to sex were observed, with males having a worse response. In conclusion, this study confirms that a correct CA synthesis is necessary to deal with stress, and that when a positive stress (eustress) occurs, individuals may improve their immune function and oxidative state. Furthermore, it shows that the response to the same stressor is different according to sex.

[Clinical and genetic characteristics of children with dopa-responsive dystonia caused by tyrosine hydroxylase gene variations].

Objective: To explore the clinical and genetic characteristics of children with dopa-responsive dystonia (DRD) caused by tyrosine hydroxylase (TH) gene variations. Methods: Clinical data of 9 children with DRD caused by TH gene variations diagnosed in the Department of Children Rehabilitation, the Third Affiliated Hospital of Zhengzhou University from January 2017 to August 2022 were retrospectively collected and analyzed, including the general conditions, clinical manifestations, laboratory tests, gene variations and follow-up data. Results: Of the 9 children with DRD caused by TH gene variations, 3 were males and 6 were females. The age at diagnosis was 12.0 (8.0, 15.0) months. The initial symptoms of the 8 severe patients were motor delay or degression. Clinical symptoms of the severe patients included motor delay (8 cases), truncal hypotonia (8 cases), limb muscle hypotonia (7 cases), hypokinesia (6 cases), decreased facial expression (4 cases), tremor (3 cases), limb dystonia (3 cases), diurnal fluctuation (2 cases), ptosis (2 cases), limb muscle hypertonia (1 case) and drooling (1 case). The initial symptom of the very severe patient was motor delay. Clinical symptoms of the very severe patient included motor delay, truncal hypotonia, oculogyric crises, status dystonicus, hypokinesia, decreased facial expression, and decreased sleep. Eleven TH gene variants were found, including 5 missense variants, 3 splice site variants, 2 nonsense variants, and 1 insertion variant, as well as 2 novel variants (c.941C>A (p.T314K), c.316_317insCGT (p.F106delinsSF)). Nine patients were followed up for 40 (29, 43) months, and no one was lost to follow-up. Seven of the 8 severe patients were treated by levodopa and benserazide hydrochloride tablets and 1 severe patient was treated by levodopa tablets. All the severe patients responded well to levodopa and benserazide hydrochloride tablets or levodopa tablets. Although the weight of the patients increased and the drug dosage was not increased, the curative effect remained stable and there was no obvious adverse reaction. One severe patient developed dyskinesia in the early stage of treatment with levodopa and benserazide hydrochloride tablets and it disappeared after oral administration of benzhexol hydrochloride tablets. Until the last follow-up, motor development of 7 severe patients returned to normal and 1 severe patient still had motor delay due to receiving levodopa and benserazide hydrochloride tablets for only 2 months. The very severe patient was extremely sensitive to levodopa and benserazide hydrochloride tablets and no improvement was observed in this patient. Conclusions: Most of the DRD caused by TH gene variations are severe form. The clinical manifestations are varied and easily misdiagnosed. Patients of the severe patients responded well to levodopa and benserazide hydrochloride tablets or levodopa tablets, and it takes a long time before full effects of treatment become established. Long-term effect is stable without increasing the drug dosage, and no obvious side effect is observed.

Downregulation of Plasma Membrane Ca2+ ATPase driven by tyrosine hydroxylase-Gal4 reduces Drosophila lifespan independently of effects in neurons.

In Drosophila melanogaster, several Gal4 drivers are used to direct gene/RNAi expression to different dopaminergic neuronal clusters. We previously developed a fly model of Parkinson's disease, in which dopaminergic neurons had elevated cytosolic Ca2+ due to the expression of a Plasma Membrane Ca2+ ATPase (PMCA) RNAi under the thyroxine hydroxylase (TH)-Gal4 driver. Surprisingly, TH-Gal4>PMCARNAi flies died earlier compared to controls and showed swelling in the abdominal area. Flies expressing the PMCARNAi under other TH drivers also showed such swelling and shorter lifespan. Considering that TH-Gal4 is also expressed in the gut, we proposed to suppress the expression specifically in the nervous system, while maintaining the activation in the gut. Therefore, we expressed Gal80 under the direction of the panneuronal synaptobrevin (nSyb) promoter in the context of TH-Gal4. nSyb-Gal80; TH-Gal4>PMCARNAi flies showed the same reduction of survival as TH-Gal4>PMCARNAi flies, meaning that the phenotype of abdomen swelling and reduced survival could be due to the expression of the PMCARNAi in the gut. In perimortem stages TH-Gal4>PMCARNAi guts had alteration in the proventriculi and crops. The proventriculi appeared to lose cells and collapse on itself, and the crop increased its size several times with the appearance of cellular accumulations at its entrance. No altered expression or phenotype was observed in flies expressing PMCARNAi in the dopaminergic PAM cluster (PAM-Gal4>PMCARNAi). In this work we show the importance of checking the global expression of each promoter and the relevance of the inhibition of PMCA expression in the gut.

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.

DNA Methylation-Dependent Restriction of Tyrosine Hydroxylase Contributes to Pancreatic ß-Cell Heterogeneity.

The molecular and functional heterogeneity of pancreatic ß-cells is well recognized, but the underlying mechanisms remain unclear. Pancreatic islets harbor a subset of ß-cells that co-express tyrosine hydroxylase (TH), an enzyme involved in synthesis of catecholamines that repress insulin secretion. Restriction of the TH+ ß-cells within islets is essential for appropriate function in mice, such that a higher proportion of these cells corresponds to reduced insulin secretion. Here, we use these cells as a model to dissect the developmental control of ß-cell heterogeneity. We define the specific molecular and metabolic characteristics of TH+ ß-cells and show differences in their developmental restriction in mice and humans. We show that TH expression in ß-cells is restricted by DNA methylation during ß-cell differentiation. Ablation of de novo DNA methyltransferase Dnmt3a in the embryonic progenitors results in a dramatic increase in the proportion of TH+ ß-cells, whereas ß-cell-specific ablation of Dnmt3a does not. We demonstrate that maintenance of Th promoter methylation is essential for its continued restriction in postnatal ß-cells. Loss of Th promoter methylation in response to chronic overnutrition increases the number of TH+ ß-cells, corresponding to impaired ß-cell function. These results reveal a regulatory role of DNA methylation in determining ß-cell heterogeneity.