Tyrosine Hydroxylase Expression Research Articles

Bile duct ligation increased dopamine levels in the cerebral cortex of rats partly due to induction of tyrosine hydroxylase.

Liver failure is associated with psychiatric alterations, partly resulting from the increased brain dopamine levels. We investigated the relationship between increased dopamine levels and mental abnormalities using bile duct ligation (BDL) rats and the mechanism by which liver failure increased dopamine levels in SH-SY5Y cells. Behavioural tests were carried out on day 13 and 27 following BDL, along with measurements of dopamine and metabolites, expressions of enzymes and transporters related to dopamine metabolism, and its transport into the cortex and the hippocampus. SH-SY5Y cells were used to investigate whether NH4 Cl, bile acids and bilirubin affected expression of tyrosine hydroxylase or not. Tyrosine hydroxylase (TH) expression in SH-SY5Y cells co-incubated with bilirubin and signal pathway inhibitors was measured. Open-field test results demonstrated BDL rats showed anxiety-like behaviour, accompanied by increased dopamine levels and expression of TH protein in the cortex. Membrane bound long form (MB)-COMT, slightly but significantly decreased. SH-SY5Y cells indicated that increased bilirubin levels was a factor in inducing TH expression. Both inhibitor of NF-κB pathway BAY 11-7082 and silencing NF-κB p65 reversed bilirubin-induced upregulation of TH protein. NF-κB activator TNF-α increased expression of TH protein. Roles of bilirubin in increases of TH protein expressions and dopamine levels were measured using hyperbilirubinemia rats. Anxiety-like behaviour, was associated with increased dopamine levels and TH protein expressions in hyperbilirubinemia rats. BDL significantly increased dopamine levels in rat cortex partly due to bilirubin-mediated TH induction. Increased bilirubin induced TH expression via activating NF-κB signalling pathway.

Neuronal Blockade of Thyroid Hormone Signaling Increases Sensitivity to Diet-Induced Obesity in Adult Male Mice.

Thyroid hormone increases energy expenditure. Its action is mediated by TR, nuclear receptors present in peripheral tissues and in the central nervous system, particularly in hypothalamic neurons. Here, we address the importance of thyroid hormone signaling in neurons, in general for the regulation of energy expenditure. We generated mice devoid of functional TR in neurons using the Cre/LoxP system. In hypothalamus, which is the center for metabolic regulation, mutations were present in 20% to 42% of the neurons. Phenotyping was performed under physiological conditions that trigger adaptive thermogenesis: cold and high-fat diet (HFD) feeding. Mutant mice displayed impaired thermogenic potential in brown and inguinal white adipose tissues and were more prone to diet-induced obesity. They showed a decreased energy expenditure on chow diet and gained more weight on HFD. This higher sensitivity to obesity disappeared at thermoneutrality. Concomitantly, the AMPK pathway was activated in the ventromedial hypothalamus of the mutants as compared with the controls. In agreement, sympathetic nervous system (SNS) output, visualized by tyrosine hydroxylase expression, was lower in the brown adipose tissue of the mutants. In contrast, absence of TR signaling in the mutants did not affect their ability to respond to cold exposure. This study provides the first genetic evidence that thyroid hormone signaling exerts a significant influence in neurons to stimulate energy expenditure in some physiological context of adaptive thermogenesis. TR function in neurons to limit weight gain in response to HFD and this effect is associated with a potentiation of SNS output.

Testing the extreme male hypothesis in the valproate mouse model; sex-specific effects on plasma testosterone levels and tyrosine hydroxylase expression in the anteroventral periventricular nucleus, but not on parental behavior.

Autism is a neurodevelopmental disorder with a strong male bias in prevalence and severity. The extreme male hypothesis proposed that autism is a manifestation of extreme male traits as evidenced by increased masculine behaviors, hypermasculinization of some brain regions, and alterations in androgen metabolism. In the present study, the extreme male hypothesis was tested in the valproate (VPA) mouse model. Females of the C57BL/6JOlaHsd mouse strain were treated with 500 mg/kg VPA on gestational day 12. Offspring of both sexes were tested at 3 to 4 months of age in the elevated plus maze (EPM), open field, sociability tests, and for parental behavior. After sacrifice at 5 to 6 months of age, plasma testosterone was measured in males, while the brains of both sexes were examined for tyrosine hydroxylase (TH) expression in the anteroventral periventricular nucleus (AVPV). VPA treatment significantly increased plasma testosterone levels and decreased AVPV TH expression in males, whereas the expression of TH in females remained at the same level. In parental behavior test none of the pup-oriented behavior was affected by VPA treatment in both sexes, the exception was nest quality which was lower after VPA exposure in males, but not in females. Our results suggest a hypermasculinizing effect of VPA that occurred specifically in males but not in females, and this effect could be related to changes in androgen physiology. Nevertheless, a generalized interpretation of the extreme male hypothesis on brain and behavior should be avoided due to the complex effects of VPA.

TGFβ3, dibutyryl cAMP and a notch inhibitor modulate phenotype late in stem cell-derived dopaminergic neuron maturation.

The generation of midbrain dopaminergic neurons (mDAs) from pluripotent stem cells (hPSC) holds much promise for both disease modelling studies and as a cell therapy for Parkinson's disease (PD). Generally, dopaminergic neuron differentiation paradigms rely on inhibition of smad signalling for neural induction followed by hedgehog signalling and an elevation of β-catenin to drive dopaminergic differentiation. Post-patterning, differentiating dopaminergic neuron cultures are permitted time for maturation after which the success of these differentiation paradigms is usually defined by expression of tyrosine hydroxylase (TH), the rate limiting enzyme in the synthesis of dopamine. However, during maturation, culture media is often supplemented with additives to promote neuron survival and or promote cell differentiation. These additives include dibutyryl cyclic adenosine monophosphate (dbcAMP), transforming growth factor β3 (TGFβ3) and or the γ-secretase inhibitor (DAPT). While these factors are routinely added to cultures, their impact upon pluripotent stem cell-derived mDA phenotype is largely unclear. In this study, we differentiate pluripotent stem cells toward a dopaminergic phenotype and investigate how the omission of dbcAMP, TGFβ3 or DAPT, late in maturation, affects the regulation of multiple dopaminergic neuron phenotype markers. We now show that the removal of dbcAMP or TGFβ3 significantly and distinctly impacts multiple markers of the mDA phenotype (FOXA2, EN1, EN2, FOXA2, SOX6), while commonly increasing both MSX2 and NEUROD1 and reducing expression of both tyrosine hydroxylase and WNT5A. Removing DAPT significantly impacted MSX2, OTX2, EN1, and KCNJ6. In the absence of any stressful stimuli, we suggest that these culture additives should be viewed as mDA phenotype-modifying, rather than neuroprotective. We also suggest that their addition to cultures is likely to confound the interpretation of both transplantation and disease modelling studies.

Derivatives of 3, 4, 5-Trimethoxycinnamic Acid Ameliorate Stress-Induced Anxiety in Mice and Rats.

Stress is an overwhelming problem associated with neuronal damage leading to anxiety and depression. The compound 3, 4, 5-trimethoxycinnamic acid (TMCA) has shown anti-stress effects; however, its derivatives remained unknown for their anxiolytic properties. Here, therefore, we investigated derivatives of TMCA (dTMCA) for their anxiolytic effects using immobilization and electric shock-induced stress in rats. Derivatives of TMCA ameliorated anxiety in mice and rats revealed by extended period of time spent in the open arms of elevated plus maze. Stress-mediated repression of tyrosine hydroxylase (TH) protein expression in the amygdala regions of rat brain and dopamine levels in the PC12 cells was restored by two selected derivatives (TMCA#5 and TMCA#9). Unlike TH expression, stress-induced protein expression of phospho-extracellular signal-regulated kinase (pERK) was unaffected by both derivatives in rats. Given the preferential inhibitory activity of dTMCA on dopamine and serotonin receptors, serotonergic road map of cellular signaling could be their target for anxiolytic effects. Thus, dTMCA would be promising agents to prevent neuronal damage associated with rampant stressful conditions.

Hypothalamic GnRH expression and pulsatility depends on a balance of prolactin receptors in the plains vizcacha, Lagostomus maximus.

In mammals, gestation is considered a physiological hyperprolactinemia status. Prolactin (PRL) is one of the modulators of gonadotropin-releasing hormone (GnRH) neurons function. The South American plains vizcacha (Lagostomus maximus) is a unique model to study the regulation of hypothalamic GnRH neurons by direct and indirect steroid-dependent pathways. The aim was to characterize the hypothalamic expression of endocrine markers in vizcacha during gestation as well as their response to experimental induced hyperprolactinemia. The possible involvement of PRL regulatory pathways on GnRH in the context of hypothalamic and pituitary reactivation in mid-gestating vizcachas was discussed. Using two in vivo approaches, we determined changes in the hypothalamic expression and distribution of prolactin receptor (PRLR), tyrosine hydroxylase (TH), and dopamine type 2 receptor. A significant increment in the number of tuberoinfundibular dopaminergic (TIDA) neurons was determined in the arcuate nucleus from early to term pregnancy. On the other hand, at preoptic area, the number of both TH+PRLR+ and GnRH+PRLR+ double-labeled neurons significantly decreased at mid-pregnancy probably allowing the recovery of GnRH expression indicating that both types of neurons may represent the key points of PRL indirect and direct pathways modulating GnRH. Moreover, in a model of induced hyperprolactinemic vizcachas, the inhibitory effect of PRL on GnRH at both expression and delivery levels were confirmed. These results suggest the concomitant participation of both PRL regulatory pathways on GnRH modulation and pinpoint the key role of PRL on GnRH expression enabling the recovery of the hypothalamic activity during the gestation in this species.

The Stressed Gut: Region-specific Immune and Neuroplasticity Changes in Response to Chronic Psychosocial Stress

Chronic psychological stress affects gastrointestinal physiology which may underpin alterations in the immune response and epithelial transport, both functions are partly regulated by enteric nervous system. However, its effects on enteric neuroplasticity are still unclear. This study aims to investigate the effects of chronic unpredictable psychological stress on intestinal motility and prominent markers of enteric function. Adult male C57BL/6J mice were exposed to 19 day of unpredictable stress protocol schedule of social defeat and overcrowding. We investigated the effects on plasma corticosterone, food intake, and body weight. In vivo gastrointestinal motility was assessed by fecal pellet output and by whole-gastrointestinal transit (using the carmine red method). Tissue monoamine level, neural and glial markers, neurotrophic factors, monoamine signaling, and Toll-like receptor expression in the proximal and distal colon, and terminal ileum were also assessed. Following chronic unpredictable psychological stress, stressed mice showed increased food intake and body weight gain (P < 0.001), and reduced corticosterone levels (P < 0.05) compared to control mice. Stressed mice had reduced stool output without differences in water content, and showed a delayed gastrointestinal transit compared to control mice (P < 0.05). Stressed mice exhibited decreased mRNA expression of tyrosine hydroxylase (Th), brain-derived neurotrophic factor (Bdnf) and glial cell-derived neurotrophic factor (Gdnf), as well as Toll-like receptor 2 (Tlr2) compared to control (P < 0.05), only proximal colon. These molecular changes in proximal colon were associated with higher levels of monoamines in tissue. Unpredictable psychological chronic stress induces region-specific impairment in monoamine levels and neuroplasticity markers that may relate to delayed intestinal transit.

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.

Lipid profiles in the cerebrospinal fluid of rats with 6-hydroxydopamine-induced lesions as a model of Parkinson's disease.

Parkinson's disease (PD) is a progressive neurodegenerative disease with characteristic pathological abnormalities, including the loss of dopaminergic (DA) neurons, a dopamine-depleted striatum, and microglial activation. Lipid accumulation exhibits a close relationship with these pathologies in PD. Here, 6-hydroxydopamine (6-OHDA) was used to construct a rat model of PD, and the lipid profile in cerebrospinal fluid (CSF) obtained from model rats was analyzed using lipidomic approaches. Establishment of this PD model was confirmed by apomorphine-induced rotation behaviors, loss of DA neurons, depletion of dopamine in the striatum, and microglial activation after 6-OHDA-induced lesion generation. Unsupervised and supervised methods were employed for lipid analysis. A total of 172 lipid species were identified in CSF and subsequently classified into 18 lipid families. Lipid families, including eicosanoids, triglyceride (TG), cholesterol ester (CE), and free fatty acid (FFA), and 11 lipid species exhibited significantly altered profiles 2 weeks after 6-OHDA administration, and significant changes in eicosanoids, TG, CE, CAR, and three lipid species were noted 5 weeks after 6-OHDA administration. During the period of 6-OHDA-induced lesion formation, the lipid families and species showed concentration fluctuations related to the recovery of behavior and nigrostriatal abnormalities. Correlation analysis showed that the levels of eicosanoids, CE, TG families, and TG (16:0_20:0_18:1) exhibited positive relationships with apomorphine-induced rotation behaviors and negative relationships with tyrosine hydroxylase (TH) expression in the midbrain. These results revealed that non-progressive nigrostriatal degeneration induced by 6-OHDA promotes the expression of an impairment-related lipidomic signature in CSF, and the level of eicosanoids, CE, TG families, and TG (16:0_20:0_18:1) in CSF may reveal pathological changes in the midbrain after 6-OHDA insult.

Brief Communication: Histological Assessment of Nonhuman Primate Brown Adipose Tissue Highlights the Importance of Sympathetic Innervation.

The objective of this study was to functionally analyze the correlation of key histological features in brown adipose tissue (BAT) with clinical metabolic traits in nonhuman primates. Axillary adipose tissue biopsies were collected from a metabolically diverse nonhuman primate cohort with clinical metabolism-related data. Expression of tyrosine hydroxylase (TH), uncoupling protein 1 (UCP1), cluster of differentiation 31 (CD31), cytochrome c oxidase subunit 4 (COX IV), beta-3 adrenergic receptor (β3-AR), and adipose cell size were quantified by immunohistochemical analysis. Computed tomography scans were performed to assess body composition. Tyrosine hydroxylase was negatively correlated with whole body fat mass as a percentage of body weight (p = 0.004) and was positively correlated with the density of UCP1 (p = 0.02), COX IV (p = 0.006), CD31 (p = 0.007), and cell density (p = 0.02) of the BAT samples. Beta-3 adrenergic receptor abundance had a weak positive correlation with COX IV (p = 0.04) in BAT but did not significantly correlate to UCP1 or TH expression in BAT. Our findings highlight that there is a disparity in innervation provided to BAT based on body composition, as seen with the negative association between TH, a marker for innervation, and adiposity. These findings also support the importance of innervation in the functionality of BAT, as TH abundance not only supports leaner body composition but is also positively correlated with known structural elements in BAT (UCP1, COX IV, CD31, and cell density). Based on our observations, β3-AR abundance does not strongly drive these structural elements or TH, all of which are known to be important in the function of brown adipose tissue. In effect, while the role of other receptors, such as β2-AR, should be reviewed in BAT function, these results support the development of safe sympathetic nervous system stimulants to activate brown adipose tissue for obesity treatment.

Luteolin Attenuates Hypertension via Inhibiting NF-κB-Mediated Inflammation and PI3K/Akt Signaling Pathway in the Hypothalamic Paraventricular Nucleus.

Luteolin is widely distributed among a number of vegetal species worldwide. The pharmacological effects of luteolin are diverse and amongst antioxidant, free radical scavenging, and anti-inflammatory activities. Preliminary study showed that luteolin can ameliorate hypertension. However, the precise mechanism needs further investigation. There is no evidence that luteolin affects the paraventricular nucleus of the hypothalamus (PVN), a brain nucleus associated with a critical neural regulator of blood pressure. Our main aim was to explore the effect of luteolin on the PI3K/Akt/NF-κB signaling pathway within the PVN of hypertensive rats. spontaneously hypertensive rats (SHRs) and corresponding normotensive control rats, the Wistar Kyoto (WKY) rats were divided into four groups and subsequently treated for 4 weeks with bilateral PVN injections of either luteolin (20 µg/0.11 µL, volume: 0.11 µL/h) or vehicle (artificial cerebrospinal fluid). luteolin infusion to the PVN significantly decreased some hemodynamic parameters including the mean arterial pressure (MAP), heart rate (HR), circulating plasma norepinephrine (NE) and epinephrine (EPI). Additionally, there was a decrease in the expressions of the phosphatidylinositol 3-kinase (p-PI3K) and phosphorylated protein kinase-B (p-AKT), levels of reactive oxygen species (ROS), NAD(P)H oxidase subunit (NOX2, NOX4) in the PVN of SHRs. Meanwhile, the expression of inflammatory cytokines and the activity of nuclear factor κB (NF-κB) p65 in the PVN of SHRs were lowered. Furthermore, immunofluorescence results showed that injection of luteolin in the PVN reduced the expression of tyrosine hydroxylase (TH), and increased that of superoxide dismutase (SOD1) and the 67-kDa isoform of glutamate decarboxylase (GAD67) in the PVN of SHRs. Our novel findings revealed that luteolin lowered hypertension via inhibiting NF-κB-mediated inflammation and PI3K/Akt signaling pathway in the PVN.

Dopaminergic and cholinergic modulation of the amygdala is altered in female mice with oestrogen receptor β deprivation

The amygdala is modulated by dopaminergic and cholinergic neurotransmission, and this modulation is altered in mood disorders. Therefore, this study was designed to evaluate the presence/absence of quantitative alterations in the expression of main dopaminergic and cholinergic markers in the amygdala of mice with oestrogen receptor β (ERβ) knock-out which exhibit increased anxiety, using immunohistochemistry and quantitative methods. Such alterations could either contribute to increased anxiety or be a compensatory mechanism for reducing anxiety. The results show that among dopaminergic markers, the expression of tyrosine hydroxylase (TH), dopamine transporter (DAT) and dopamine D2-like receptor (DA2) is significantly elevated in the amygdala of mice with ERβ deprivation when compared to matched controls, whereas the content of dopamine D1-like receptor (DA1) is not altered by ERβ knock-out. In the case of cholinergic markers, muscarinic acetylcholine type 1 receptor (AChRM1) and alpha-7 nicotinic acetylcholine receptor (AChRα7) display overexpression while the content of acetylcholinesterase (AChE) and vesicular acetylcholine transporter (VAChT) remains unchanged. In conclusion, in the amygdala of ERβ knock-out female the dopaminergic and cholinergic signalling is altered, however, to determine the exact role of ERβ in the anxiety-related behaviour further studies are required.

The effect of vitamin D on morphine preference in rats: Possible biochemical and DRD2-GDNF signaling.

Despite half a century of research on vitamin D (Vit. D), its link to substance abuse and dependence has only been discussed in recent decades. Evidence also shows the involvement of Vit. D in the evolution of dopaminergic neurons in the nucleus accumbens, an increase in the expression of tyrosine hydroxylase, and the regulation of dopaminergic processes. The novel idea for this work is taken from a hypothesis given about the effectiveness of Vit. D on dopamine signaling pathway. It is therefore presumed that Vit. D can be considered an effective therapeutic approach for narcotic addiction and substance abuse. The animals were assigned into six groups (control, vehicle, Morphine [Mor.], and Vit. D [250, 500, and 1000IU/kg, i.p.]). Following each conditioning session in a conditioned place preference (CPP) model, the animals received Vit. D. Afterward, the locomotor activity of the animals was assessed using open-field apparatus. Malondialdehyde (MDA), nitric oxide (NO), catalase (CAT), superoxide dismutase (SOD), thiol, and total antioxidant capacity (TAC) were measured in the brain. The relative DRD2 and GDNF expressions (%) were also measured in the hippocampus. Vit. D administration after Mor. caused a significant increase in the place preference index in the acquisition phase (p<.05). Vit. D altered the oxidation/antioxidation profiles (CAT, SOD, MDA, NO, TAC, and Thiol). Vit. D was more effective than Mor. in the expression of GDNF (p<.0001); however, in the expression of DRD2, this was only the case for 1000IU Vit. D (p<.0001). Considering the increased place preference index induced by Mor., it can be concluded that Vit. D interacts via the oxidative pathway and DRD2-GDNF signaling to potentiate the Mor. effect.

Identification and evaluation of midbrain specific longevity-related genes in exceptionally long-lived but healthy mice.

Brain aging is a complex biological process that is affected by both genetic background and environment. The transcriptomic analysis of aged human and rodent brains has been applied to identify age-associated molecular and cellular processes for which intervention could possibly restore declining brain functions induced by aging. However, whether these age-associated genetic alterations are indeed involved in the healthy aging of the brain remains unclear. We herein characterized a naturally occurring, extremely long-lived (34 months of age) but healthy mouse group retaining well-preserved motor functions. Strikingly, these long-lived mice maintained tyrosine hydroxylase expression and dopaminergic fiber densities, even in the presence of persistent neuroinflammation and expression of aging markers. Combined with Endeavor gene prioritization, we identified the following midbrain-specific longevity-associated genes in the midbrain of these mice: aimp2, hexb, cacybp, akt2, nrf1, axin1, wwp2, sp2, dnajb9, notch, traf7, and lrp1. A detailed biochemical analysis of the midbrain of these long-lived mice confirmed the increased expression of Nrf1 and the activation of Akt1 and 2. Interestingly, dopaminergic neuroprotective and age-associated E3 ubiquitin ligase parkin expression was retained at high levels in the aforementioned midbrains, possibly supporting the suppression of its toxic substrates AIMP2 and PARIS. In contrast, the 24-month-old mice with dopaminergic neurite deficits failed to maintain parkin expression in the midbrain. AIMP2-induced cytotoxicity, mitochondrial stress, and neurite toxicity can be prevented by overexpression of parkin, Akt1, and Nrf1 in SH-SY5Y and PC12 cells, and basal expression of parkin, Akt1, and Nrf1 is required for maintenance of mitochondrial function and neurite integrity in PC12 cells. Taken together, this longevity-associated pathway could be a potential target of intervention to maintain nigrostriatal dopaminergic fibers and motor ability to ensure healthy longevity.

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

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

Identification of molecular network of gut-brain axis associated with neuroprotective effects of PPARδ-ligand erucic acid in rotenone-induced Parkinson's disease model in zebrafish.

Disruption of the gut-brain axis in Parkinson's disease (PD) may lead to motor symptoms and PD pathogenesis. Recently, the neuroprotective potential of different PPARδ-agonists has been shown. We aimed to reveal the effects of erucic acid, peroxisome proliferator-activated receptors (PPARs)-ligand in rotenone-induced PD model in zebrafish, focusing on the gut-brain axis. Adult zebrafish were exposed to rotenone and erucic acid for 30 days. Liquid chromatography-mass spectrometry and tandem mass spectrometry (LC-MS/MS) analysis was performed. Raw files were analysed by Proteome Discoverer 2.4 software; peptide lists were searched against Danio rerio proteins. STRING database was used for protein annotations or interactions. Lipid peroxidation (LPO), nitric oxide (No), alkaline phosphatase, superoxide dismutase, glutathione S-transferase (GST), acetylcholinesterase and the expressions of PD-related genes were determined. Immunohistochemical tyrosine hydroxylase (TH) staining was performed. LC-MS/MS analyses allowed identification of over 2000 proteins in each sample. The 2502 and 2707 proteins overlapped for intestine and brain. The 196 and 243 significantly dysregulated proteins in the brain and intestines were found in rotenone groups. Erucic acid treatment corrected the changes in the expression of proteins associated with cytoskeletal organisation, transport and localisation and improved locomotor activity, expressions of TH, PD-related genes (lrrk2, park2, park7, pink1) and oxidant-damage in brain and intestines in the rotenone group as evidenced by decreased LPO, No and increased GST. Our results showed beneficial effects of erucic acid as a PPARδ-ligand in neurotoxin-induced PD model in zebrafish. We believe that our study will shed light on the mechanism of the effects of PPARδ agonists and ω9-fatty acids in the gut-brain axis of PD.

The involvement of the noradrenergic system in the antinociceptive effect of cucurbitacin D on mice with paclitaxel-induced neuropathic pain.

Paclitaxel (sold under the brand name Taxol) is a chemotherapeutic drug that is widely used to treat cancer. However, it can also induce peripheral neuropathy, which limits its use. Although several drugs are used to attenuate neuropathy, no optimal treatment is available to date. In this study, the effect of cucurbitacins B and D on paclitaxel-induced neuropathic pain was assessed. Multiple paclitaxel injections (a cumulative dose of 8mg/kg, i. p.) induced cold and mechanical allodynia from days 10 to 21 in mice, and the i. p. administration of 0.025mg/kg of cucurbitacins B and D attenuated both allodynia types. However, as cucurbitacin B showed a more toxic effect on non-cancerous (RAW 264.7) cells, further experiments were conducted with cucurbitacin D. The cucurbitacin D dose-dependently (0.025, 0.1, and 0.5mg/kg) attenuated both allodynia types. In the spinal cord, paclitaxel injection increased the gene expression of noradrenergic (α 1-and α 2-adrenergic) receptors but not serotonergic (5-HT1A and 3) receptors. Cucurbitacin D treatment significantly decreased the spinal α 1- but not α 2-adrenergic receptors, and the amount of spinal noradrenaline was also downregulated. However, the tyrosine hydroxylase expression measured via liquid chromatography in the locus coeruleus did not decrease significantly. Finally, cucurbitacin D treatment did not lower the anticancer effect of chemotherapeutic drugs when co-administered with paclitaxel in CT-26 cell-implanted mice. Altogether, these results suggest that cucurbitacin D could be considered a treatment option against paclitaxel-induced neuropathic pain.

Alteration of monoaminergic systems in the caudal medulla and its possible link to diurnal increase of apnea in a mouse model of Rett syndrome.

Methyl-CpG binding protein 2 (MeCP2)-deficient (Mecp2-/y) mice exhibit apneas that resemble respiratory abnormalities observed in Rett syndrome (RTT) patients. The present study aimed to clarify whether Mecp2-/y mice show diurnal variations in apnea as seen in RTT and how the MeCP2 deficiency affects monoaminergic systems that control breathing. In 7-week-old Mecp2-/y mice, 24 h variation of apnea and effects of milnacipran, a serotonin/noradrenaline reuptake inhibitor, on the apnea were evaluated. The number of vesicular monoamine transporter 2 (VMAT2)-immunoreactive puncta in the caudal medulla was counted. Further, the effects of valproate (VPA) on the expression of tyrosine hydroxylase (TH) mRNA in the ventrolateral medulla of mice were assessed by RT-qPCR. Apnea occurred more frequently during the light phase under a 12:12 h light/dark environment in Mecp2-/y mice and milnacipran reduced apnea during the light phase but not during the dark phase. The number of VMAT2-immunoreactive puncta was reduced in Mecp2-/y mice. VPA treatment significantly increased TH mRNA expression in Mecp2-/y mice. Alteration of monoaminergic systems in the caudal medulla of Mecp2-/y mice is potentially relevant to the light-sensitive diurnal increase of apnea, and an improvement in monoaminergic neurotransmission can ameliorate the diurnal increase of apnea in Mecp2-/y mice.

Experimental evidence of a limited impact of new-generation perfluoroalkyl substance C6O4 on differentiating human dopaminergic neurons from induced pluripotent stem cells.

Perfluoroalkyl substances (PFASs) are persistent pollutants, raising concerns for human health. Legacy PFAS perfluoro-octanoic acid (PFOA) accumulate in brains of people at high environmental exposure, especially in areas enriched with dopaminergic neurons (DN). In vitro exposure to 10ng/mL PFOA for 24h was also associated with an altered molecular and functional phenotype of DN differentiated from human induced pluripotent stem cells (hiPSCs). Acetic acid, 2,2-difluoro-2-((2,2,4,5-tetrafluoro-5(trifluoromethoxy)-1,3-dioxolan-4-yl)oxy)-ammonium salt (1:1), known as C6O4, is a new generation PFAS proposed to have a safer profile. Here we investigated the effect of C6O4 exposure on the molecular phenotype of hiPSC-derived DN. Cells were exposed to C6O4 for 24h, at the concentration of 10ng/mL, at neuronal commitment (DP1), neuronal precursor (DP2) and the mature dopaminergic (DP3) phases of differentiation. Liquid-chromatography/mass-spectrometry showed negligible cell accumulation of C6O4 at each differentiation stage and by staining with Merocyanine-540 we observed unaltered cell membrane fluidity. Immunofluorescence showed that the expression of tyrosine hydroxylase (TH) and βIII-Tubulin was unaffected by the exposure to C6O4 at each differentiation phase (respectively: DP1, p=0.332; DP2, p=0.623; DP3, p=0.816, with respect to control unexposed conditions). Exposure to C6O4 is presumed to have minor effects on cell molecular/functional phenotype of developing human DN cells, requiring confirm on in vivo models.

Changes of Dopamine and Tyrosine Hydroxylase Levels in the Brain of Germ-free Mice.

Dopamine (DA) is one of the most important catecholamine neurotransmitters in the central nervous system. The degeneration and deletion of dopaminergic neurons are closely linked to Parkinson's disease (PD) and other psychiatric or neurological diseases. Several studies have been suggesting that intestinal microorganisms are associated with the occurrence of central nervous diseases, including diseases that are closely related to dopaminergic neurons. However, the intestinal microorganism's regulation of dopaminergic neurons in the brain is largely unknown. This study aimed to investigate the hypothetical differences of DA and its synthase tyrosine hydroxylase (TH) expression in different parts of the brain of germ free (GF) mice. Several studies in recent years have shown that commensal intestinal microbiota promotes changes in DA receptor expression, DA levels, and affects this monoamine turnover. Germ free (GF) and specific pathogen free (SPF) C57b/L male mice were used to analyze TH mRNA and expression levels, and DA levels in the frontal cortex, hippocampus, striatum, and cerebellum, using real time PCR, western blotting, and ELISA tools. Compared with SPF mice, the TH mRNA levels were decreased in the cerebellum of GF mice, while the TH protein expression was tended to increase in the hippocampus, and conversely showed significant decrease in the striatum. The average optical density (AOD) of TH immunoreactive nerve fibers and the number of axons in striatum of mice in GF group were significantly lower than that in SPF group. Compared with SPF mice, the DA concentration in the hippocampus, striatum and frontal cortex of GF mice was decreased in GF mice. The changes of DA and its synthase TH in the brain of GF mice showed that the absence of conventional intestinal microbiota had certain regulatory effects on central dopaminergic nervous system, which is considered helpful for studying the effect of commensal intestinal flora on diseases related to impaired dopaminergic nerve system.