Phosphorylation Of Tyrosine Hydroxylase Research Articles

Effects of propofol on presynaptic synapsin phosphorylation in the mouse brain in vivo

The commonly used general anesthetic propofol can enhance the γ-aminobutyric acid-mediated inhibitory synaptic transmission and depress the glutamatergic excitatory synaptic transmission to achieve general anesthesia and other outcomes. In addition to the actions at postsynaptic sites, the modulation of presynaptic activity by propofol is thought to contribute to neurophysiological effects of the anesthetic, although potential targets of propofol within presynaptic nerve terminals are incompletely studied at present. In this study, we explored the possible linkage of propofol to synapsins, a family of neuron-specific phosphoproteins which are the most abundant proteins on presynaptic vesicles, in the adult mouse brain in vivo. We found that an intraperitoneal injection of propofol at a dose that caused loss of righting reflex increased basal levels of synapsin phosphorylation at the major representative phosphorylation sites (serine 9, serine 62/67, and serine 603) in the prefrontal cortex (PFC) of male and female mice. Propofol also elevated synapsin phosphorylation at these sites in the striatum and S9 and S62/67 phosphorylation in the hippocampus, while propofol had no effect on tyrosine hydroxylase phosphorylation in striatal nerve terminals. Total synapsin protein expression in the PFC, hippocampus, and striatum was not altered by propofol. These results reveal that synapsin could be a novel substrate of propofol in the presynaptic neurotransmitter release machinery. Propofol possesses the ability to upregulate synapsin phosphorylation in broad mouse brain regions.

The effects of phenylalanine and tyrosine levels on dopamine production in rat PC12 cells. Implications for treatment of phenylketonuria, tyrosinemia type 1 and comorbid neurodevelopmental disorders

Phenylketonuria (PKU) is an autosomal recessive metabolic disorder caused by mutations in the phenylalanine hydroxylase (PAH) gene, resulting in phenylalanine accumulation and impaired tyrosine production. In Tyrosinemia type 1 (TYRSN1) mutations affect fumarylacetoacetate hydrolase, leading to accumulation of toxic intermediates of tyrosine catabolism. Treatment of TYRSN1 with nitisinone results in extreme tissue levels of tyrosine. Although PKU and TYRSN1 have opposite effects on tyrosine levels, both conditions have been associated with neuro-psychiatric symptoms typically present in ADHD, possibly indicating an impaired dopamine (DA) synthesis. However, concrete in vivo data on the possible molecular basis for disrupted DA production under disease mimicking conditions have been lacking. In pursuit to uncover associated molecular mechanisms, we exposed an established, DA producing cell line (PC12) to different concentrations of phenylalanine and tyrosine in culture media. We measured the effects on viability, proteomic composition, tyrosine, DA and tyrosine hydroxylase (TH) levels and TH phosphorylation. TH catalyzes the rate-limiting step in DA synthesis. High extracellular levels of phenylalanine depleted cells of intracellular tyrosine and DA. Compared to physiological levels (75 μM), either low (35 μM) or high concentrations of tyrosine (275 or 835 μM) decreased cellular DA, TH protein, and its phosphorylation levels. Using deep proteomic analysis, we identified multiple proteins, biological processes and pathways that were altered, including enzymes and transporters involved in amino acid metabolism. Using this information and published data, we developed a mathematical model to predict how extracellular levels of aromatic amino acids can affect the cellular synthesis of DA via different mechanisms. Together, these data provide new information about the normal regulation of neurotransmitter synthesis and how this may be altered in neurometabolic disorders, such as PKU and TYRSN1, with implications for the treatment of cognitive symptoms resulting from comorbid neurodevelopmental disorders.

Tyrosine hydroxylase phosphorylation is under the control of serine 40.

Tyrosine hydroxylase catalyzes the initial and rate-limiting step in the biosynthesis of the neurotransmitter dopamine. The phosphorylation state of Ser40 and Ser31 is believed to exert a direct effect on the enzymatic activity of tyrosine hydroxylase. Interestingly, some studies report that Ser31 phosphorylation affects Ser40 phosphorylation, while Ser40 phosphorylation has no effect on Ser31 phosphorylation, a process named hierarchical phosphorylation. Here, we provide a detailed investigation into the signal transduction mechanisms regulating Ser40 and Ser31 phosphorylation in dopaminergic mouse MN9D and Neuro2A cells. We find that cyclic nucleotide signaling drives Ser40 phosphorylation, and that Ser31 phosphorylation is strongly regulated by ERK signaling. Inhibition of ERK1/2 with UO126 or PD98059 reduced Ser31 phosphorylation, but surprisingly had no effect on Ser40 phosphorylation, contradicting a role for Ser31 in the regulation of Ser40. Moreover, to elucidate a possible hierarchical mechanism controlling tyrosine hydroxylase phosphorylation, we introduced tyrosine hydroxylase variants in Neuro2A mouse neuroblastoma cells that mimic either phosphorylated or unphosphorylated serine residues. When we introduced a Ser40Ala tyrosine hydroxylase variant, Ser31 phosphorylation was completely absent. Additionally, neither the tyrosine hydroxylase variant Ser31Asp, nor the variant Ser31Ala had any significant effect on basal Ser40 phosphorylation levels. These results suggest that tyrosine hydroxylase is not controlled by hierarchical phosphorylation in the sense that first Ser31 has to be phosphorylated and subsequently Ser40, but, conversely, that Ser40 phosphorylation is essential for Ser31 phosphorylation. Overall our study suggests that Ser40 is the crucial residue to target so as to modulate tyrosine hydroxylase activity.

Nigral-specific increase in ser31 phosphorylation compensates for tyrosine hydroxylase protein and nigrostriatal neuron loss: Implications for delaying parkinsonian signs

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 D1 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, D1 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 D1 receptor expression, contribute as compensatory mechanisms against progressive nigrostriatal neuron and TH protein loss, and may mitigate hypokinesia severity.

Short-term hindlimb unloading negatively affects dopaminergic transmission in the nigrostriatal system of mice.

The nigrostriatal system composed of the dorsal striatum and the substantia nigra (SN) is highly involved in the control of motor behavior. Various extremal and pathological conditions as well as social isolation (SI) may cause an impairment of locomotor function; however, corresponding alterations in the nigrostriatal dopaminergic pathway are far from full understanding. Here, we analyzed the effect of 3-day hindlimb unloading (HU) and SI on the key players of dopamine transmission in the nigrostriatal system of CD1 mice. Three groups of mice were analyzed: group-housed (GH), SI, and HU animals. Our data showed a significant decrease in the expression and phosphorylation of tyrosine hydroxylase (TH) in the SN and dorsal striatum of HU mice that suggested attenuation of dopamine synthesis in response to HU. In the dorsal striatum of HU mice, the downregulation of TH expression was also observed indicating the effect of unloading; however, TH phosphorylation at Ser40 was mainly affected by SI pointing on an impact of isolation too. Expression of dopamine receptors D1 in the dorsal striatum of HU mice was increased suggesting a compensatory response, but the activity of downstream signaling pathways involving protein kinase A and cAMP response element-binding protein was inhibited. At the same time, SI alone did not affect expression of DA receptors and activity of downstream signaling in the dorsal striatum. Obtained data let us to conclude that HU was the main factor which impaired dopamine transmission in the nigrostriatal system but SI made some contribution to its negative effects.

Dehydroepiandrosterone (DHEA) increases undirected singing behavior and alters dopaminergic regulation of undirected song in non-breeding male European starlings (Sturnus vulgaris).

It has been proposed that in species that defend territories across multiple life history stages, brain metabolism of adrenal dehydroepiandrosterone (DHEA) regulates aggressive behavior at times when gonadal androgen synthesis is low (i.e. the non-breeding season). To date, a role for DHEA in the regulation of other forms of social behavior that are expressed outside of the context of breeding remains unknown. In this experiment, we used the European starling (Sturnus vulgaris) model system to investigate a role for DHEA in the neuroendocrine regulation of singing behavior by males in non-breeding condition. Starling song in a non-breeding context is spontaneous, not directed towards conspecifics, and functions to maintain cohesion of overwintering flocks. Using within-subjects design, we found that DHEA implants significantly increase undirected singing behavior by non-breeding condition male starlings. Given that DHEA is known to modulate multiple neurotransmitter systems including dopamine (DA) and DA regulates undirected song, we subsequently used immunohistochemistry for phosphorylated tyrosine hydroxylase (pTH, the active form of the rate-limiting enzyme in DA synthesis) to investigate the effect of DHEA on dopaminergic regulation of singing behavior in a non-breeding context. Pearson correlation analysis revealed a positive linear association between undirected singing behavior and pTH immunoreactivity in the ventral tegmental area and midbrain central gray of DHEA-implanted, but not control-implanted, males. Taken together, these data suggest that undirected singing behavior by non-breeding starlings is modulated by effects of DHEA on dopaminergic neurotransmission. More broadly, these data expand the social behavior functions of DHEA beyond territorial aggression to include undirected, affiliative social communication.

Extracellular H+ Ions are a Novel Signal for Tyrosine Hydroxylase Activation in Catecholaminergic Cells.

Tyrosine hydroxylase catalyzes the rate-limiting step in the catecholamine biosynthetic pathway. Short-term TH activity is proposed to be regulated by the phosphorylation/dephosphorylation of regulatory domains Ser 40, 31, and/or 19 in response to membrane depolarization coupled increase in intracellular Ca2+. Here, we present in situ evidence to support that extracellular H+ ions ([H+]o) are an intracellular or extracellular Ca2+-independent novel signal for TH activation in catecholaminergic MN9D and PC12 cells. [H+]o-mediated TH activation is a short-term process coupled with a Na+-independent Cl-/HCO3- exchanger-mediated increase of intracellular hydrogen ions ([H+]i). While extracellular Ca2+ is not required for [H+]o-mediated TH activation, [H+]o does not increase the cytosolic Ca2+ levels in neuronal or non-neuronal cells in the presence or absence of extracellular Ca2+. Although [H+]o-mediated TH activation is associated with a significant increase in Ser 40 phosphorylation, major protein kinases proposed to be responsible for this process appear to be not involved. However, we have not been able to identify the protein kinase(s) involved in [H+]o-mediated phosphorylation of TH at present. Studies with a pan-phosphatase inhibitor, okadaic acid (OA), appear to indicate that the inhibition of phosphatase activities may not play a significant role in H+-mediated activation of TH. The relevance of these findings to the physiological TH activation mechanism and hypoxia, ischemia, and trauma-induced selective dopaminergic neural death is being discussed in this paper.

Electroacupuncture intervention on stress-induced cardiac autonomic imbalance in rats involves corticotropin-releasing hormone system activity.

Autonomic imbalance is a core aspect of stress response that strongly correlates to cardiovascular diseases. Enhanced activity of the central corticotropin-releasing hormone (CRH) system may result in autonomic imbalance to cause cardiovascular responses in a stress state. Electroacupuncture at PC6 acupoints has been demonstrated to prevent and treat cardiovascular diseases. In this study, we aim to demonstrate the protective role of electroacupuncture at PC6 in ameliorating cardiac autonomic imbalance and investigate the underlying mechanisms in immobilization stress rats. Four groups were subjected. Immobilization stress was applied to three groups. And the rats in two electroacupuncture-intervened groups exerted electroacupuncture at PC6 or tail respectively. Then, we performed ECG recording for heart rate variability (HRV) analysis, and rats were sacrificed after experiments for biological analysis. HRV analysis indicated that electroacupuncture at PC6 improved the enhanced low-frequency band of the power spectrum (LF), the reduced high-frequency band of the power spectrum (HF), and the enhanced LF/HF ratio caused by immobilization stress. Besides, electroacupuncture at PC6 significantly decreased phosphorylated tyrosine hydroxylase expression and increased acetylcholine esterase expression in heart of immobilization stress rats. Furthermore, electroacupuncture at PC6 significantly decreased CRH level and CRH 1 type receptor and CRH 2 type receptor (CRHR2) expressions in the rostral ventrolateral medulla (RVLM), and CRH level and CRHR2 expression in the nucleus of the solitary tract (NTS) of immobilization stress rats. Our findings suggest that electroacupuncture at PC6 can ameliorate stress-induced cardiac autonomic imbalance by modulating the CRHergic input in the RVLM and NTS.

Partial loss of arcuate kisspeptin neurons in female rats stimulates luteinizing hormone and decreases prolactin secretion induced by estradiol.

Kisspeptin, neurokinin, and dynorphin (KNDy) neurons in the arcuate nucleus (ARC) control luteinizing hormone (LH) and prolactin (PRL) release, although their role in conveying the effects of estradiol (E2 ) to these hormones is not well understood. We performed a longitudinal evaluation of female rats in which KNDy neurons were ablated using a neurokinin-3 receptor agonist conjugated with saporin (NK3-SAP) to investigate the impact of the reduction of KNDy neurons on the E2 regulation of gonadal and PRL axes. NK3-SAP rats, bearing a moderate loss of ARC kisspeptin-immunoreactive (-IR) neurons (50%-90%), displayed irregular estrous cycles but essentially unaltered follicular development and a normal number of corpora lutea. Rats were then ovariectomized (OVX) and treated with a positive-feedback dose of E2 (OVX + E2 ). LH and PRL were measured in the tail blood by an enzyme-linked immunosorbent assay. The E2 -induced LH surge was amplified, whereas the PRL rise was decreased in NK3-SAP rats compared to Blank-SAP control. After 10 days of no hormonal treatment, basal LH levels were equally elevated in NK3-SAP and controls. Tyrosine hydroxylase (TH) phosphorylation in the median eminence, in turn, was increased in NK3-SAP rats, with no change in the number of ARC TH-IR neurons. Thus, KNDy neurons exert concurrent and opposite roles in the E2 -induced surges of LH and PRL. The partial loss of KNDy neurons disrupts ovarian cyclicity but does not preclude ovulation, consistent with the disinhibition of the LH preovulatory surge. Conversely, KNDy neurons tonically inhibit the enzymatic activity of tuberoinfundibular dopaminergic neurons, which appears to facilitate PRL release in response to E2 .

Striatal miR-183-5p inhibits methamphetamine-induced locomotion by regulating glucocorticoid receptor signaling.

MicroRNA (miRNA)-mediated striatal gene regulation may play an important role in methamphetamine (METH) addiction. This study aimed to identify changes in novel miRNAs and their target genes during METH self-administration and investigate their roles in METH-induced locomotion. RNA sequencing analysis revealed that mir-183-5p was upregulated in the striatum of METH self-administered rats, and target gene prediction revealed that the glucocorticoid receptor (GR) gene, Nr3c1, was a potential target gene for mir-183-5p. We confirmed that single and repeated METH administrations increased METH-induced locomotion and plasma corticosterone levels in rats. Additionally, increased miR-185-5p expression and decreased GR gene expression were observed only in the repeated-METH-injection group but not in the single-injection group. We then investigated the effects of miR-183-5p on METH-induced locomotion using a miR-183-5p mimic and inhibitor. Injection of a mir-183-5p mimic in the striatum of rats attenuated METH-induced locomotion, whereas injection of a miR-183-5p inhibitor enhanced the locomotor activity in METH-administered rats. Furthermore, the miR-183-5p mimic reduced the phosphorylation of tyrosine hydroxylase (TH) whereas the inhibitor increased it. Taken together, these results indicate that repeated METH injections increase striatal miR-183-5p expression and regulate METH-induced locomotion by regulating GR expression in rats, thereby suggesting a potential role of miR-183-5p as a novel regulator of METH-induced locomotion.

Spontaneous changes in brain striatal dopamine synthesis and storage dynamics ex vivo reveal end-product feedback-inhibition of tyrosine hydroxylase

Synaptic events are important to define treatment strategies for brain disorders. In the present paper, freshly obtained rat brain striatal minces were incubated under different times and conditions to determine dopamine biosynthesis, storage, and tyrosine hydroxylase phosphorylation. Remarkably, we found that endogenous dopamine spontaneously accumulated during tissue incubation at 37 °C ex vivo while dopamine synthesis simultaneously decreased. We analyzed whether these changes in brain dopamine biosynthesis and storage were linked to dopamine feedback inhibition of its synthesis-limiting enzyme tyrosine hydroxylase. The aromatic-l-amino-acid decarboxylase inhibitor NSD-1015 prevented both effects. As expected, dopamine accumulation was increased with l-DOPA addition or VMAT2-overexpression, and dopamine synthesis decreased further with added dopamine, the VMAT2 inhibitor tetrabenazine or D2 auto-receptor activation with quinpirole, accordingly to the known synaptic effects of these treatments. Phosphorylation activation and inhibition of tyrosine hydroxylase on Ser31 and Ser40 with okadaic acid, Sp-cAMP and PD98059 also exerted the expected effects. However, no clear-cut association was found between dopamine feedback inhibition of its own biosynthesis and changes of tyrosine hydroxylase phosphorylation, assessed by Western blot and mass spectrometry. The later technique also revealed a new Thr30 phosphorylation in rat tyrosine hydroxylase. Our methodological assessment of brain dopamine synthesis and storage dynamics ex vivo could be applied to predict the in vivo effects of pharmacological interventions in animal models of dopamine-related disorders.

Attenuation of the Counter-Regulatory Glucose Response in CVLM C1 Neurons: A Possible Explanation for Anorexia of Aging

This study aimed to determine the effect of age on CVLM C1 neuron glucoregulatory proteins in the feeding pathway. Male Sprague Dawley rats aged 3 months and 24 months old were divided into two subgroups: the treatment group with 2-deoxy-d-glucose (2DG) and the control group. Rat brains were dissected to obtain the CVLM region of the brainstem. Western blot was used to determine protein expression of tyrosine hydroxylase (TH), phosphorylated TH at Serine40 (pSer40TH), AMP-activated protein kinase (AMPK), phosphorylated AMPK (phospho AMPK), and neuropeptide Y Y5 receptors (NPY5R) in CVLM samples. Immunofluorescence was used to determine TH-, AMPK-, and NPY5R-like immunoreactivities among other brain coronal sections. Results obtained denote a decrease in basal TH phosphorylation levels and AMPK proteins and an increase in TH proteins among aged CVLM neurons. Increases in the basal immunoreactivity of TH+, AMPK+, NPY5R+, TH+/AMPK+, and TH+/NPY5R+ were also observed among old rats. Young treatment-group rats saw a decrease in TH phosphorylation and AMPK proteins following 2DG administration, while an increase in AMPK phosphorylation and a decrease in TH proteins were found among the old-treatment-group rats. These findings suggest the participation of CVLM C1 neurons in counter-regulatory responses among young and old rats. Altering protein changes in aged CVLM C1 neurons may attenuate responses to glucoprivation, thus explaining the decline in food intake among the elderly.

Calpain activation and progression of inflammatory cycles in Parkinson's disease.

Parkinson's disease (PD) is a progressive, neurodegenerative condition of the central nervous system (CNS) affecting 6.3 million people worldwide with no curative treatments. Current therapies aim to mitigate PD's effects and offer symptomatic relief for patients. Multiple pathways are involved in the pathogenesis of PD, leading to neuroinflammation and the destruction of dopaminergic neurons in the CNS. This review focuses on PD pathology and the role of calpain, a neutral protease, as a regulator of various immune cells such as T-cells, microglia and astrocytes which lead to persistent neuroinflammatory responses and neuronal loss in both the brain and spinal cord (SC). Calpain plays a significant role in the cleavage and aggregation of toxic α-synuclein (α-syn), a presynaptic neural protein, and other organelles, contributing to mitochondrial dysfunction and oxidative stress. α-Syn aggregation results in the formation of Lewy bodies (LB) that further contribute to neuronal damage through lipid bilayer penetration, calcium ion (Ca2+) influx, oxidative stress and damage to the blood brain barrier (BBB). Dysfunctional mitochondria destabilize cytosolic Ca2+ concentrations, raising intracellular Ca2+; this leads to excessive calpain activation and persistent inflammatory responses. α-Syn aggregation also results in the disruption of dopamine synthesis through phosphorylation of tyrosine hydroxylase (TH), a key enzyme involved in the conversion of tyrosine to levodopa (L-DOPA), the amino acid precursor to dopamine. Decreased dopamine levels result in altered dopamine receptor (DR) signaling, ultimately activating pro-inflammatory T-cells to further contribute to the inflammatory response. All of these processes, together, result in neuroinflammation, degeneration and ultimately neuronal death seen in PD. 1-Methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP-a prodrug to the neurotoxin 1-methyl-4-phenylpyridinium (MPP+)), rotenone (an environmental neurotoxin), and 6-hydroxydopamine (6-OHDA - a neurotoxic synthetic organic compound) induce PD-like conditions when injected into rodents. All three agents work through similar mechanisms and lead to degeneration of dopaminergic neurons in the substantia nigra (SN) and more recently discovered in motor neurons of the spinal cord (SC). These neurotoxins also increase calpain activity, furthering the neuroinflammatory response. Hence, calpain inhibitors have been posited as potential therapeutics for PD to prevent calpain-related inflammation and neurodegenerative responses in not only the SN but the SC as well.

Trace Amine-Associated Receptor 1 Contributes to Diverse Functional Actions of O-Phenyl-Iodotyramine in Mice but Not to the Effects of Monoamine-Based Antidepressants.

Trace Amine-Associated Receptor 1 (TAAR1) is a potential target for the treatment of depression and other CNS disorders. However, the precise functional roles of TAAR1 to the actions of clinically used antidepressants remains unclear. Herein, we addressed these issues employing the TAAR1 agonist, o-phenyl-iodotyramine (o-PIT), together with TAAR1-knockout (KO) mice. Irrespective of genotype, systemic administration of o-PIT led to a similar increase in mouse brain concentrations. Consistent with the observation of a high density of TAAR1 in the medial preoptic area, o-PIT-induced hypothermia was significantly reduced in TAAR1-KO mice. Furthermore, the inhibition of a prepulse inhibition response by o-PIT, as well as its induction of striatal tyrosine hydroxylase phosphorylation and elevation of extracellular DA in prefrontal cortex, were all reduced in TAAR1-KO compared to wildtype mice. O-PIT was active in both forced-swim and marble-burying tests, and its effects were significantly blunted in TAAR1-KO mice. Conversely, the actions on behaviour and prefrontal cortex dialysis of a broad suite of clinically used antidepressants were unaffected in TAAR1-KO mice. In conclusion, o-PIT is a useful tool for exploring the hypothermic and other functional antidepressant roles of TAAR1. By contrast, clinically used antidepressants do not require TAAR1 for expression of their antidepressant properties.

Enhanced tyrosine hydroxylase activity induces oxidative stress, causes accumulation of autotoxic catecholamine metabolites, and augments amphetamine effects in vivo.

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 H2O2 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.

Peripheral inflammation induces long-term changes in tyrosine hydroxylase activation in the substantia nigra

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.

Growth hormone receptor in dopaminergic neurones regulates stress-induced prolactin release in male mice.

Arcuate nucleus (ARH) dopaminergic neurones regulate several biological functions, including prolactin secretion and metabolism. These cells are responsive to growth hormone (GH), although it is still unknown whether GH action on ARH dopaminergic neurones is required to regulate different physiological aspects. Mice carrying specific deletion of GH receptor (GHR) in tyrosine hydroxylase (TH)- or dopamine transporter (DAT)-expressing cells were produced. We investigated possible changes in energy balance, glucose homeostasis, fertility, pup survival and restraint stress-induced prolactin release. GHR deletion in DAT- or TH-expressing cells did not cause changes in food intake, energy expenditure, ambulatory activity, nutrient oxidation, glucose tolerance, insulin sensitivity and counter-regulatory response to hypoglycaemia in male and female mice. In addition, GHR deletion in dopaminergic cells caused no gross effects on reproduction and pup survival. However, restraint stress-induced prolactin release was significantly impaired in DAT- and TH-specific GHR knockout male mice, as well as in pegvisomant-treated wild-type males, whereas an intact response was observed in females. Patch clamp recordings were performed in ARH DAT neurones and, in contrast to prolactin, GH did not cause acute changes in the electrical activity of DAT neurones. Furthermore, TH phosphorylation at Ser40 in ARH neurones and median eminence axonal terminals was not altered in DAT-specific GHR knockout male mice during restraint stress. In conclusion, GH action in dopaminergic neurones is required for stress-induced prolactin release in male mice, suggesting the existence of sex differences in the capacity of GHR signalling to affect prolactin secretion. The mechanism behind this regulation still needs to be identified.

The Effects of Acute Glucoprivation on Adrenomedullary Function in SHR and WKY Rats

We have shown previously, acute intraperitoneal administration of 2-deoxy-d-glucose (2DG) into Sprague-Dawley rats led to activation of the adrenal medulla chromaffin cells, indicated with increased protein kinase activity and increased tyrosine hydroxylase (TH) phosphorylation, as well as increased plasma adrenaline and glucose levels. Here we have used spontaneous hypertensive (SHR) and Wistar Kyoto (WKY) rats to investigate whether hypertension alters basal adrenal chromaffin cell function, or the response of these cells to acute 2DG treatment. At basal level, we found no differences in adrenal medulla TH protein, TH phosphorylation, TH activity or catecholamine levels between SHR and WKY despite a significant difference in the level of systolic blood pressure; nor were there differences in plasma catecholamine levels or blood glucose (BG). Furthermore, the vehicle animals evoked no significant changes in any parameter measured in SHR, but evoked significant increases in pSer19TH, plasma adrenaline and BG in WKY. Single episode of glucoprivation evoked increases in PKA and CDK/MAPK, pSer40TH, pSer31TH, TH activity, and plasma adrenaline and BG in SHR, and in addition evoked increases in PKC, CAMKII, and pSer19TH in WKY. These findings are significant which indicates hypertension does not impact catecholamine function in the adrenal gland. It also appears that hypertension does not alter the adrenal response to glucoprivation. The findings are also significant as WKY showed greater adrenal activation of protein kinases and TH phosphorylation in response to saline and 2DG when compared to SHR and possible reasons for these findings are further discussed.

Cell-penetrating, antioxidant SELENOT mimetic protects dopaminergic neurons and ameliorates motor dysfunction in Parkinson's disease animal models

Parkinson's disease (PD) is a neurodegenerative disorder characterized by motor dysfunction for which there is an unmet need for better treatment options. Although oxidative stress is a common feature of neurodegenerative diseases, notably PD, there is currently no efficient therapeutic strategy able to tackle this multi-target pathophysiological process. Based on our previous observations of the potent antioxidant and neuroprotective activity of SELENOT, a vital thioredoxin-like selenoprotein, we designed the small peptide PSELT from its redox active site to evaluate its antioxidant properties in vivo, and its potential polyfunctional activity in PD models. PSELT protects neurotoxin-treated dopaminergic neurons against oxidative stress and cell death, and their fibers against neurotoxic degeneration. PSELT is cell-permeable and acts in multiple subcellular compartments of dopaminergic neurons that are vulnerable to oxidative stress. In rodent models of PD, this protective activity prevented neurodegeneration, restored phosphorylated tyrosine hydroxylase levels, and led to improved motor skills. Transcriptomic analysis revealed that gene regulation by PSELT after MPP+ treatment negatively correlates with that occurring in PD, and positively correlates with that occurring after resveratrol treatment. Mechanistically, a major impact of PSELT is via nuclear stimulation of the transcription factor EZH2, leading to neuroprotection. Overall, these findings demonstrate the potential of PSELT as a therapeutic candidate for treatment of PD, targeting oxidative stress at multiple intracellular levels.

The Inhibition Effects of Shenmai Injection on Acetylcholine-Induced Catecholamine Synthesis and Secretion by Modulating Nicotinic Acetylcholine Receptor Ion Channels in Cultured Bovine Adrenal Medullary Cells.

Shenmai injection (SMI) has been widely used for the treatment of cardiovascular diseases in China. Cardiovascular disorders are often related to excessive catecholamine (CA) secretion. Here, we report the effects of SMI on CA secretion and synthesis in cultured bovine adrenal medullary cells. We found that SMI significantly reduced CA secretion induced by 300 μM acetylcholine (ACh). Cotreatment with SMI (10 μL/mL) and either of the ACh receptor α-subunit inhibitors, HEX (α3) or DhβE (α4β2), did not produce any further inhibition, indicating that SMI may play a role through α3 and α4β2 channels. Furthermore, SMI reduced tyrosine hydroxylase (TH) activity induced by ACh by inhibiting the phosphorylation of TH at Ser19 and Ser40. TH is phosphorylated at Ser19 by Ca2+/calmodulin-dependent protein kinase II (CaM kinase II) and at Ser40 by protein kinase A (PKA). KN-93 and H89, the antagonists of CaM kinase II and PKA, respectively, inhibited the ACh-induced phosphorylation at Ser19 and Ser40, and the addition of SMI did not augment the inhibitory effect. Taken together, our results show that SMI likely inhibits CA secretion by blocking TH activity at its Ser19 and Ser40 sites.