Theobromine improves hyperactivity, inattention, and working memory via modulation of dopaminergic neural function in the frontal cortex of spontaneously hypertensive rats.

Attention-deficit/hyperactivity disorder (ADHD) is a developmental disorder and dopaminergic dysfunction in the prefrontal cortex (PFC) may play a role. Our previous research indicated that theobromine (TB), a methylxanthine, enhances cognitive function in rodents via the PFC. This study investigates TB's effects on hyperactivity and cognitive function in stroke-prone spontaneously hypertensive rats (SHR), an ADHD animal model. Male SHRs (6-week old) received a diet containing 0.05% TB for 40 days, while control rats received normal diets. Age-matched male Wistar-Kyoto rats (WKY) served as genetic controls. During the TB administration period, we conducted open-field tests and Y-maze tasks to evaluate hyperactivity and cognitive function, then assessed dopamine concentrations and tyrosine hydroxylase (TH), dopamine receptor D1-5 (DRD1-5), dopamine transporter (DAT), vesicular monoamine transporter-2 (VMAT-2), synaptosome-associated protein-25 (SNAP-25), and brain-derived neurotrophic factor (BDNF) expressions in the PFC. Additionally, the binding affinity of TB for the adenosine receptors (ARs) was evaluated. Compared to WKY, SHR exhibited hyperactivity, inattention and working memory deficits. However, chronic TB administration significantly improved these ADHD-like behaviors in SHR. TB administration also normalized dopamine concentrations and expression levels of TH, DRD2, DRD4, SNAP-25, and BDNF in the PFC of SHR. No changes were observed in DRD1, DRD3, DRD5, DAT, and VMAT-2 expression between SHR and WKY rats, and TB intake had minimal effects. TB was found to have affinity binding to ARs. These results indicate that long-term TB supplementation mitigates hyperactivity, inattention and cognitive deficits in SHR by modulating dopaminergic nervous function and BDNF levels in the PFC, representing a potential adjunctive treatment for ADHD.

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.

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.

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.

[Improved immunity and mean lifespan in mature mice genetically deficient in catecholamine synthesis after living with wild type for two months]. / Mejoría de la inmunidad y la esperanza de vida en ratones maduros, genéticamente deficientes en la síntesis de catecolaminas, tras convivir con controles durante dos meses.

INTRODUCTION: Mice hemizygous in tyrosine hydroxylase (TH-HZ), the limiting enzyme in catecholamine synthesis, show premature immunosenescence, which in females is associated with a shorter lifespan than the corresponding controls (WT). The coexistence of TH-Hz with WT improves the immune function in both males and females in adulthood. OBJECTIVE: To test whether cohabitation for two months of mature male TH-HZ with WT improves the immune function of the former and whether this impacts the lifespan. MATERIAL AND METHODS: Mature male ICR-CD1 mice (13 ± 1 months) TH-HZ coexisted with WT (2:4 ratio in each cage) for two months. Peritoneal leukocytes were extracted from all animals at baseline, one month, and two months after cohabitation, and macrophage phagocytic capacity, macrophage and lymphocyte chemotaxis, natural killer (NK) antitumor activity, and lymphoproliferative capacity in response to the mitogens concanavalin A and lipopolysaccharide (LPS) were assessed. The animals were maintained under these conditions until their natural death. RESULTS: The TH-HZ, which start, in general, with lower values than the WT in the immune functions studied, improved them after two months of cohabitation, becoming similar to those of the controls. This improvement was already observed in NK activity after one month of cohabitation. The TH-HZ presented lower mean longevity than WT, but when they cohabited with WT, it was similar to the latter. CONCLUSION: The coexistence of TH-HZ male mice with WT mice for two months at mature age improves these genetically modified animals' immune response and longevity.

[Effects of early electroacupuncture on the expression of Iba-1 and TNF-α in Parkinson's disease mice].

OBJECTIVE: To observe the effect of early electroacupuncture(EA) intervention on ionized calcium binding adapter molecule 1 (Iba-1), tyrosine hydroxylase (TH) and tumor necrosis factor-α (TNF-α) in Parkinson's disease (PD) mice, so as to explore its neuroinflammation mechanism in treating PD. METHODS: A total of 24 male C57BL/6J mice (9 weeks old) were randomly divided into control, model and EA groups, with 8 mice in each group. The PD model was established by long-term low dose subcutaneous injection of rotenone. Started at the same time with modeling, EA (2 Hz/100 Hz, 1 mA) was applied to "Shenting"(GV24), bilateral "Tianshu"(LI11), "Quchi"(ST25), and "Shangjuxu"(ST37) for 15 min, once a day for 8 weeks. The motor function was assessed by rotorod test and step length test. The expression levels of Iba-1 and TH proteins in substantia nigra pars compacta (SNpc) was detected by Western blot and immunohistochemistry. The expression level of TNF-α protein in colon tissue was examined by Western blot and immunofluorescence staining. RESULTS: Compared with the control group, the fall latency shortened at 4, 6, and 8 weeks after modeling (P<0.01) and the step length of hind limbs shortened at 5 and 7 weeks after modeling (P<0.01), the expression levels of Iba-1 in SNpc and TNF-α in colon tissue were increased (P<0.01), and the expression level of TH in SNpc was decreased (P<0.01) in the model group. Compared with the model group, the fall latency prolonged at 6 and 8 weeks after modeling (P<0.01) and the step length of hind limbs prolonged at 5 and 7 weeks after modeling (P<0.01), the expression levels of Iba-1 in SNpc and TNF-α in colon tissue were decreased (P<0.01, P<0.05), and the expression level of TH in SNpc was increased (P<0.05, P<0.01) in the EA group. CONCLUSION: Early EA intervention can delay the occurring time of motor disfunction, alleviated the loss of substantia nigra dopaminergic neurons, and exerted a good neuroprotective effect on the degenerative changes in rotenone-induced PD mice, which may be related to its effects in alleviating the intestinal inflammation, inhibiting the activation of microglia, thereby reducing the neuroinflammation.

Loss of Pleckstrin homology like domain, family A, member 1 promotes type â ¡ alveolar epithelial cell apoptosis in chronic obstructive pulmonary disease emphysematous phenotype via interaction with tyrosine 3-monooxygenase/tryptophan 5-monooxygenase activation protein epsilon.

Emphysematous phenotype is the most important phenotypic component of chronic obstructive pulmonary disease and is associated with substantial morbidity and mortality. The current pharmaceutical treatments and therapeutic procedures do not reduce pulmonary damage in patients with emphysematous phenotype. Therefore, it is important to identify effector molecules that can be used as interfering targets in such patients. Apoptosis of type II alveolar epithelial cells plays a key role in the phenotypic formation. This study aimed to further explore the molecular mechanisms involved in this process. The number of type II alveolar epithelial cells was significantly reduced due to increased apoptosis in patients with emphysematous phenotype compared to those with non-emphysematous phenotype. Pleckstrin homology like domain, family A, member 1 (PHLDA1) was mainly distributed in type II alveolar epithelial cells in both groups but was markedly reduced in patients with emphysematous phenotype. Overexpression of PHLDA1 prevented cigarette smoke extract-stimulated apoptosis of type II alveolar epithelial cells, whereas its knockdown worsened the apoptosis. PHLDA1 binding ability to tyrosine 3-monooxygenase/tryptophan 5-monooxygenase activation protein epsilon (YWHAE) was weakened after exposure to cigarette smoke extract, with decreased PHLDA1 level lowering the abundance of YWHAE and attenuating the binding ability of YWHAE to p-Bad. These results demonstrate that considerable apoptosis of type II alveolar epithelial cells occurs in patients with emphysematous phenotype, and PHLDA1 may act as an effective antiapoptotic factor via YWHAE. Moreover, PHLDA1 may serve as a potential interfering target, providing insights into therapeutic strategies for emphysematous phenotype.

Toxic and Phenotypic Effects of AAV_Cre Used to Transduce Mesencephalic Dopaminergic Neurons.

A popular approach to spatiotemporally target genes using the loxP/Cre recombination system is stereotaxic microinjection of adeno-associated virus (AAV) expressing Cre recombinase (AAV_Cre) in specific neuronal structures. Here, we report that AAV_Cre microinjection in the ventral tegmental area (VTA) of ErbB4 Cyt-1-floxed (ErbB4 Cyt-1fl/fl) mice at titers commonly used in the literature (~1012-1013 GC/mL) can have neurotoxic effects on dopaminergic neurons and elicit behavioral abnormalities. However, these effects of AAV_Cre microinjection are independent of ErbB4 Cyt-1 recombination because they are also observed in microinjected wild-type (WT) controls. Mice microinjected with AAV_Cre (1012-1013 GC/mL) exhibit reductions of tyrosine hydroxylase (TH) and dopamine transporter (DAT) expression, loss of dopaminergic neurons, and they behaviorally become hyperactive, fail to habituate in the open field and exhibit sensorimotor gating deficits compared to controls microinjected with AAV_GFP. Importantly, these AAV_Cre non-specific effects are: (1) independent of serotype, (2) occur with vectors expressing either Cre or Cre-GFP fusion protein and (3) preventable by reducing viral titers by 1000-fold (1010 GC/mL), which retains sufficient recombination activity to target floxed genes. Our studies emphasize the importance of including AAV_Cre-injected WT controls in experiments because recombination-independent effects on gene expression, neurotoxicity and behaviors could be erroneously attributed to consequences of gene ablation.

Tyrosine hydroxylase gene promoter activity is upregulated in female catecholaminergic neuroblastoma cells following activation of a Gαq-coupled designer receptor.

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 Cß3. 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.

MicroRNAs miR-14 and miR-2766 regulate tyrosine hydroxylase to control larval-pupal metamorphosis in Helicoverpa armigera.

BACKGROUND: The cotton bollworm, Helicoverpa armigera, is a worldwide polyphagous pest, causing huge economic losses in vegetable, cotton and corn crops, among others. Owing to long-term exposure to Bacillus thuringiensis (Bt) toxins, evolution of resistance has been detected in this pest. As a conservative and effective neurotransmitter, dopamine (DA) has an important role in insect growth and development. In this study, we investigated the regulatory functions of DA and its associated non-coding RNA in metamorphosis in H. armigera. RESULTS: Expression profiles indicated that DA and DA pathway genes were highly expressed during larval-pupal metamorphosis in H. armigera. RNA interference and pharmacological experiments confirmed that tyrosine hydroxylase (TH), dopa decarboxylase, vesicular amine transporter and DA receptor 2 are critical genes related to the development of H. armigera from larvae to pupae. We also found that miR-14 and miR-2766 targeted the 3' untranslated region to post-transcriptionally regulate HaTH function. Application of miR-2766 and miR-14 antagomirs significantly increased levels of HaTH transcripts and proteins, while injection of miR-2766 and miR-14 agomirs not only suppressed messenger RNA and protein levels of HaTH, but also resulted in defective pupation in H. armigera. CONCLUSION: These results suggest that DA deficiency inhibits larval-pupal metamorphosis in H. armigera. Potentially, DA pathway genes and their microRNAs could be used as a novel target for H. armigera management. © 2022 Society of Chemical Industry.

Developmental exposure to the A6-pesticide causes changes in tyrosine hydroxylase gene expression, neurochemistry, and locomotors behavior in larval zebrafish.

Purpose: In recent years, the increase in the biopesticides synthesis for alternative agricultural uses has required their impacts study. Among these compounds, several of them are known to exert endocrinedisrupting (EDs) effects causing deregulation of physiological functions affecting cell signaling pathways involved in neural cell differentiation leading to developmental neurotoxicity. The objective of our study was to determine the impact of the biopesticide A6 structurally related to estrogenic EDs on zebrafish larvae, to define its toxicity, the mechanisms responsible, and to monitor the locomotors activity at nanomolar concentrations (0. 0.5, 5 and 50 nM).Materials and methods: Using imaging analysis tools, immunohistochemistry, quantitative PCR, and an automated behavior recording system (Zebrabox) we were able to assess these effects.Results: We have shown through its blue fluorescence properties that it accumulates in different parts of the body such as the intestine, adipose tissue, muscles, yolk sac and head. A6 also disrupted swimming behavior by affecting the expression of tyrosine hydroxylase (TH) in dopaminergic neurons.Conclusions: In conclusion, our study provided a mechanistic understanding of the A6 neurotoxic effect which could be the result of its binding to the estrogen receptor.

Naltrexone/bupropion modifies weight, food intake, and Drd2 gene expression in rats.

Obesogenic diets are known to induce obesity and changes in food intake in experimental animals. Obesity negatively affects the peripheral metabolism and neural aspects, such as changes in eating behavior. In obese animals, dopamine (DA) receptor levels are reduced. DA is one of the main peptides involved in the motivation and pleasure of eating. A combination of naltrexone/bupropion (NB) has shown promise in controlling metabolic alterations, but there are few studies on how they modulate dopaminergic expression. NB, in addition to reducing food intake and body weight, can modify tyrosine hydroxylase (Th) and DA receptor D2 (Drd2) levels in the mesolimbic areas of rats submitted to a high-fat diet (HF). The study evaluated the effect of NB on food intake, body weight, and expression levels of Th, Drd1a, and Drd2, in the nucleus accumbens and striatum of rats fed on HF diet. Wistar rats were grouped according to diet: standard (n = 20) and HF diet (n = 20). The food intake and body weight were analyzed. The gene expression of Th, Drd1a, and Drd2 was evaluated using real-time PCR. NB combination of 1 mg/kg and 20 mg/kg reduced food intake and body weight, increased Drd2 expression in rats on HF diet, and increased Th in rats on both experimental diets. The level of Drd1a was unchanged. We concluded that bodyweight reduction may be associated with decreased food intake in response to the increased Drd2 expression in the mesolimbic areas of rats that received an HF diet.

Expression Quantitative Trait Locus rs6356 Is Associated with Susceptibility to Heroin Addiction by Potentially Influencing TH Gene Expression in the Hippocampus and Nucleus Accumbens.

Opioid addiction is a complicated and highly heritable brain disease. Dysfunction in dopaminergic signaling is involved in the pathogenesis of addictive disorders. Encoding a dopamine synthetase, the tyrosine hydroxylase (TH) gene has long been an interesting candidate in genetic association studies for opioid addiction. However, the mechanisms underlying associations of risk gene variants and opioid addiction remain unknown. In the present study, we first analyzed the association between TH gene variants and susceptibility and traits of heroin addiction in 801 patients with heroin addiction and 930 healthy controls. Methylation levels in the promoter region of the TH gene were detected and compared between the heroin addiction and healthy control groups. To reveal the potential mechanism of the association of TH gene variants and heroin addiction, correlations between the risk TH single nucleotide polymorphism (SNPs) for heroin addiction and the methylation and expression levels of the TH gene were examined. Our results demonstrated that SNP rs6356 was associated with susceptibility to heroin addiction. CpG TH_15 was hypermethylated in the heroin addiction group compared with the healthy control group. Notably, SNP rs6356 was correlated in an allele-specific manner with expression of the TH gene in the hippocampus and nucleus accumbens but not with methylation levels of CpG TH_15. Our findings suggest that the eQTL rs6356 was associated with susceptibility to heroin addiction by potentially affecting the expression of the TH gene in brain regions in the mesocorticolimbic dopamine system, including the hippocampus and nucleus accumbens.

Induction of Parkinsonian-Like Changes via Targeted Downregulation of Astrocytic Glutamate Transporter GLT-1 in the Striatum.

BACKGROUND: Previous investigations have suggested that decreased expression of glutamate transporter-1 (GLT-1) is involved in glutamate excitotoxicity and contribute to the development of Parkinson's disease (PD), GLT-1 is decreased in animal models of PD. GLT-1 is mainly expressed in astrocytes, and the striatum is a GLT-1-rich brain area. OBJECTIVE: The aim was to explore the function and mechanism of astrocytic GLT-1 in PD-like changes. METHODS: In the study, PD-like changes and their molecular mechanism in rodents were tested by a behavioral assessment, micro-positron emission tomography/computed tomography (PET/CT), western blotting, immunohistochemical and immunofluorescence staining, and high performance liquid chromatography pre-column derivatization with O-pthaldialdehida after downregulating astrocytic GLT-1 in vivo and in vitro. RESULTS: In vivo, after 6 weeks of brain stereotactic injection of adeno-associated virus into the striatum, rats in the astrocytic GLT-1 knockdown group showed poorer motor performance, abnormal gait, and depression-like feature; but no olfactory disorders. The results of micro-PET/CT and western blotting indicated that the dopaminergic system was impaired in astrocytic GLT-1 knockdown rats. Similarly, tyrosine hydroxylase (TH) positive immune-staining in neurons of astrocytic GLT-1 knockdown rats showed deficit in cell count. In vitro, knockdown of astrocytic GLT-1 via RNA interference led to morphological injury of TH-positive neurons, which may be related to the abnormal calcium signal induced by glutamate accumulation after GLT-1 knockdown. Furthermore, the GLT-1 agonist ceftriaxone showed a protective effect on TH-positive neuron impairment. CONCLUSION: The present findings may shed new light in the future prevention and treatment of PD based on blocking glutamate excitotoxicity.

Reduction of tyrosine hydroxylase expression and increase of α-synuclein in the substantia nigra in a rat model of benign prostatic hyperplasia.

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.

Tyrosine hydroxylase activity is regulated through the modification of the 176th cysteine residue.

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.

Synaptotagmin-13 Is a Neuroendocrine Marker in Brain, Intestine and Pancreas.

Synaptotagmin-13 (Syt13) is an atypical member of the vesicle trafficking synaptotagmin protein family. The expression pattern and the biological function of this Ca2+-independent protein are not well resolved. Here, we have generated a novel Syt13-Venus fusion (Syt13-VF) fluorescence reporter allele to track and isolate tissues and cells expressing Syt13 protein. The reporter allele is regulated by endogenous cis-regulatory elements of Syt13 and the fusion protein follows an identical expression pattern of the endogenous Syt13 protein. The homozygous reporter mice are viable and fertile. We identify the expression of the Syt13-VF reporter in different regions of the brain with high expression in tyrosine hydroxylase (TH)-expressing and oxytocin-producing neuroendocrine cells. Moreover, Syt13-VF is highly restricted to all enteroendocrine cells in the adult intestine that can be traced in live imaging. Finally, Syt13-VF protein is expressed in the pancreatic endocrine lineage, allowing their specific isolation by flow sorting. These findings demonstrate high expression levels of Syt13 in the endocrine lineages in three major organs harboring these secretory cells. Collectively, the Syt13-VF reporter mouse line provides a unique and reliable tool to dissect the spatio-temporal expression pattern of Syt13 and enables isolation of Syt13-expressing cells that will aid in deciphering the molecular functions of this protein in the neuroendocrine system.

Zinc antagonizes iron-regulation of tyrosine hydroxylase activity and dopamine production in Drosophila melanogaster.

BACKGROUND: Dopamine (DA) is a neurotransmitter that plays roles in movement, cognition, attention, and reward responses, and deficient DA signaling is associated with the progression of a number of neurological diseases, such as Parkinson's disease. Due to its critical functions, DA expression levels in the brain are tightly controlled, with one important and rate-limiting step in its biosynthetic pathway being catalyzed by tyrosine hydroxylase (TH), an enzyme that uses iron ion (Fe2+) as a cofactor. A role for metal ions has additionally been associated with the etiology of Parkinson's disease. However, the way dopamine synthesis is regulated in vivo or whether regulation of metal ion levels is a component of DA synthesis is not fully understood. Here, we analyze the role of Catsup, the Drosophila ortholog of the mammalian zinc transporter SLC39A7 (ZIP7), in regulating dopamine levels. RESULTS: We found that Catsup is a functional zinc transporter that regulates intracellular zinc distribution between the ER/Golgi and the cytosol. Loss-of-function of Catsup leads to increased DA levels, and we showed that the increased dopamine production is due to a reduction in zinc levels in the cytosol. Zinc ion (Zn2+) negatively regulates dopamine synthesis through direct inhibition of TH activity, by antagonizing Fe2+ binding to TH, thus rendering the enzyme ineffective or non-functional. CONCLUSIONS: Our findings uncovered a previously unknown mechanism underlying the control of cellular dopamine expression, with normal levels of dopamine synthesis being maintained through a balance between Fe2+ and Zn2+ ions. The findings also provide support for metal modulation as a possible therapeutic strategy in the treatment of Parkinson's disease and other dopamine-related diseases.

Protective effects of berberine against MPTP-induced dopaminergic neuron injury through promoting autophagy in mice.

Berberine, an isoquinoline alkaloid isolated from Coptis chinensis, has been widely studied for its efficacy in the treatment of neurodegenerative diseases. However, the detailed mechanisms are unknown. In this study, the effects of berberine on 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-induced mice model of Parkinson's disease were investigated. We showed that treatment with berberine significantly ameliorates the degeneration of dopaminergic neurons in substantia nigra compacta (SNc) and improves motor impairment in MPTP-treated mice. Berberine also significantly decreased the level of α-synuclein and enhanced the microtubule-associated protein light chain 3 (LC3-II)-associated autophagy in the SN of MPTP-treated mice. Furthermore, adenosine 5'-monophosphate (AMP)-activated protein kinase (AMPK) was activated by berberine. Berberine's actions were abolished by pre-treatment with 3-methyladenine (an autophagy inhibitor) or compound c (an AMPK inhibitor) in the MPP+-treated SH-SY5Y cells. These results suggested that the protective effects of berberine on the toxicity of MPTP could be attributed to berberine-enhanced autophagy via the AMPK dependent pathway.

Encounters between Cas9/dCas9 and G-Quadruplexes: Implications for Transcription Regulation and Cas9-Mediated DNA Cleavage.

Using the nuclease-dead Cas9 (dCas9), we targeted in cellulo a G-rich sequence, which contains multiple potentially G-quadruplex (GQ) forming sites, within the human tyrosine hydroxylase (TH) promoter. We demonstrate that transcription can be up or down regulated by targeting different parts of this G-rich sequence. Our results suggest that TH transcription levels correlate with stability of different GQs formed by this sequence and targeting them with dCas9 can modulate their stability. Unlike alternative approaches, regulating TH expression by targeting the promoter GQs with dCas9 enables a specific and potentially transient control and does not require mutations in the sequence. We also investigated whether the presence of GQs in target sequences impacts DNA cleavage activity of Cas9. We discovered significant reduction in cleavage activity when the vicinity of a high-stability GQ was targeted. Furthermore, this reduction is significantly more prominent for the G-rich strand compared to the complementary C-rich strand.