DA Content Research Articles

Polyvinylpyrrolidone‐Stabilized Blue‐Emitting Copper Nanoclusters as Fluorescence Probe for Selective Detection of Dopamine

AbstractDopamine (DA), a critical catecholamine in mammals, functions as a significant biological indicator for specific diseases. The precise and timely identification of DA is essential for disease monitoring and prevention. In this study, copper nanoclusters (Cu NCs) were synthesized using polyvinyl pyrrolidone (PVP) as the protective ligand and L‐ascorbic acid (L‐AA) as the reducing agent via a simple hydrothermal process. The produced PVP−Cu NCs displayed strong blue emission at 427 nm when excited at 365 nm. In alkaline conditions, DA was able to selectively inhibit the fluorescence of PVP−Cu NCs. The investigation of the mechanism revealed that dopamine self‐polymerization resulted in the formation of polydopamine, which inhibited the emission of PVP−Cu NCs at 427 nm due to the inner filter effect (IFE). Based on the above phenomenon, a simple and selective analytical method for DA determination was developed. The fluorescence intensity of PVP−Cu NCs demonstrated a reliable response to DA concentrations within the range of 5 to 200 μM, with a detection limit (LOD) of 1.32 μM. The proposed method was successfully utilized for the precise detection of DA in human urine samples, indicating its potential application in biomedical dopamine detection.

Dose-Ranging Effects of the Intracerebral Administration of Atsttrin in Experimental Model of Parkinson’s Disease Induced by 1-Methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) in Mice

Parkinson’s disease is one of the most common neurodegenerative disorders characterized by a multitude of motor and non-motor clinical symptoms resulting from the progressive and long-lasting abnormal loss of nigrostriatal dopaminergic neurons. Currently, the available treatments for patients with Parkinson’s disease are limited and exert only symptomatic effects, without adequate signs of delaying or stopping the progression of the disease. Atsttrin constitutes the bioengineered protein which ultrastructure is based on the polypeptide chain frame of the progranulin (PGRN), which exerts anti-inflammatory effects through the inhibition of TNFα. The conducted preclinical studies suggest that the therapeutic implementation of Atsttrin may be potentially effective in the treatment of neurodegenerative diseases that are associated with the occurrence of neuroinflammatory processes. The aim of the proposed study was to investigate the effect of direct bilateral intracerebral administration of Atsttrin using stereotactic methods in the preclinical C57BL/6 mouse model of Parkinson’s disease inducted by 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) intoxication. The analysis of the dose dependency effects of the increasing doses of Atsttrin has covered a number of parameters and markers regarding neurodegenerative processes and inflammatory responses including IL-1α, TNFα, IL-6, TH, and TG2 mRNA expressions. Accordingly, the evaluation of the changes in the neurochemical profile included DA, DOPAC, 3-MT, HVA, NA, MHPG, 5-HT, and 5-HIAA concentration levels. The intracerebral administration of Atsttrin into the striatum effectively attenuated the neuroinflammatory reaction in evaluated neuroanatomical structures. Furthermore, the partial restoration of monoamine content and its metabolic turnover were observed. In this case, taking into account the previously described pharmacokinetic profile and extrapolated bioavailability as well as the stability characteristics of Atsttrin, an attempt was made to describe as precisely as possible the quantitative and qualitative effects of increasing doses of the compound within the brain tissue microenvironment in the presented preclinical model of the disease. Collectively, this findings demonstrated that the intracerebral administration of Atsttrin may represent a potential novel therapeutic method for the treatment of Parkinson’s disease.

Preparation of carbon quantum dot fluorescent probe from waste fruit peel and its use for the detection of dopamine.

Carbon quantum dots (CQDs), as a new type of fluorescent nanomaterial, are widely used in the detection of small molecules. Abnormal dopamine secretion can lead to diseases such as Parkinson's disease and schizophrenia. Therefore, it is highly significant to detect dopamine levels in the human body. Using discarded fruit peels to prepare carbon quantum dots can achieve the reuse of kitchen waste, reduce pollution, and create value. Nitrogen-doped carbon quantum dots (N-CQDs) were prepared using the hydrothermal method, with orange peel as the raw material. The fluorescence quantum yield of N-CQDs reached a high value of 35.37% after optimizing the temperature, reaction time, and ethylenediamine dosage. N-CQDs were characterized using various techniques, including ultraviolet visible (UV-vis) spectroscopy, fluorescence spectrophotometer (PL), transmission electron microscopy (TEM), and Fourier transform infrared spectroscopy (FT-IR). These analyses confirmed the successful doping of nitrogen in the CQDs. The DA concentration ranged from 0 to 300 μmol L-1, and the linear equation for fluorescence quenching of N-CQDs was F/F0 = -0.0056c + 0.98647, with an R2 value of 0.99071. The detection limit was 0.168 μmol L-1. The recovery and precision of dopamine in rabbit serum were 98% to 103% and 2% to 6%, respectively. The prepared N-CQDs could be used as a fluorescent probe to effectively detect DA.

Environmental levels of azoxystrobin disturb male zebrafish behavior: Possible roles of oxidative stress, cholinergic system, and dopaminergic system

A widely applied pesticide of azoxystrobin, is increasingly detected in the water environment. Concern has been raised against its potential detriment to aquatic ecosystems. It has been shown that exposure to azoxystrobin interfere with the locomotor behavior of zebrafish larvae. This study aims to investigate whether exposure to environmental levels of azoxystrobin (2 μg/L, 20 μg/L, and 200 μg/L) changes the behavior of male adult zebrafish. Herein, we evaluated behavioral response (locomotor, anxiety-like, and exploratory behaviors), histopathology, biochemical indicators, and gene expression in male adult zebrafish upon azoxystrobin exposure. The study showed that exposure to azoxystrobin for 42 days remarkably increased the locomotor ability of male zebrafish, resulted in anxiety-like behavior, and inhibited exploratory behavior. After treatment with 200 μg/L azoxystrobin, vasodilatation, and congestion were observed in male zebrafish brains. Exposure to 200 μg/L azoxystrobin notably elevated ROS level, MDA concentration, CAT activity, and AChE activity, while inhibiting SOD activity, GPx activity, ACh concentration, and DA concentration in male zebrafish brains. Moreover, the expression levels of genes related to the antioxidant, cholinergic, and dopaminergic systems were significantly changed. This suggests that azoxystrobin may interfere with the homeostasis of neurotransmitters by causing oxidative stress in male zebrafish brains, thus affecting the behavioral response of male zebrafish.

Electroanalytical sensor based on CuInSe2/carbon sphere towards the non‐invasive determination of dopamine

AbstractDopamine (DA) is known as a crucial neurotransmitter synthesized and secreted by dopaminergic neurons and plays a role in regulating human emotions and behaviors. The determination of DA content is of great significance in clinical application and physiological function research. An electroanalytical sensor based on the ternary semiconductor material copper indium selenide (CuInSe2) coated carbon spheres functionalized glassy carbon electrode (CuInSe2@CS/GCE) towards the non‐invasive determination of DA was constructed in this study. The performances of the sensor were characterized using cyclic voltammetry, impedance method, and differential pulse voltammetry. The results show that wrinkled CuInSe2 completely coated on CS. The sensor exhibits good electrochemical kinetics and response signal towards DA, which results from the good electrochemical activity and excellent electroconductibility of transition metal centers of the composite. The determination limit of the proposed sensor towards DA is 0.59 μM (S/N=3), and the linear measuring range is between 1–800 μM. In addition, the sensor exhibits desirable long‐term robustness, reproducibility, repetitiveness, and interference immunity. In the non‐invasive determination towards real samples of human urine and sweat, the sensor provides good recovery rate and small determination deviation.

Explication of polydopamine modified g-C3N4 as effective additive in PVDF nanocomposite membrane fabrication for enhanced ultrafiltration and self-cleaning performance

Nanocomposite membranes (NCMs) incorporated with nanomaterials have recently received increasing attention, but the preparation of high-performance membranes is still challenging due to the hydrophilicity and self-aggregation of the nanoparticles. In this study, graphite carbon nitride containing nitrogen defects was modified using dopamine (PDA@DCN) for effective alleviation of the agglomeration of nanoparticles resulting in a uniform casting solution. A non-solvent-induced phase separation method was adapted to prepare PVDF NCMs by using PDA@DCN as an effective additive. Superior permeability and selectivity were exhibited by the PDA0.25 @DCN/PVDF2 membrane (where a DA concentration of 0.25 g/100 mL was used in PDA@DCN preparation and 2% PVP was added during the membrane preparation) with the pure water flux of 390 L/(m2·h) and Bovine serum albumin (BSA) rejection of 95.9%. The extended Derjaguin-Landau-Verwey-Overbeek (XDLVO) theory revealed that the improvement of the flux recovery rate of PDA0.25 @DCN/PVDF2 was due to the decreased attractive interaction for fouling. Ternary phase diagram and viscosity experiments confirmed that the addition of PDA0.25 @DCN accelerated phase inversion rate (as evident from thermodynamics and kinetics) and increased the porosity with smaller average surface pore size, which is expected to have good permeability and selectivity as an ultrafiltration membrane. Moreover, an excellent self-cleaning performance was demonstrated by PDA0.25 @DCN/PVDF2, with a flux recovery rate of 84.5%, simply under visible light irradiation, which indicates its potential for environment-friendly practical application.

Surface‐enhanced Raman spectroscopy combined with multivariate analysis for the detection and differentiation of dopamine and DOPAC in cerebrospinal fluid

AbstractDopamine (DA) is a vital neurotransmitter for brain functions, including pleasure, memory, motivation, and movement control. Dopaminergic neuron degradation is correlated to neurological disorders such as Parkinson's disease, and changes in DA concentration are related to mental health disorders. Currently, the gold standard for monitoring DA concentrations in the brain is electrochemistry. However, although electrochemistry has high sensitivity, it has low specificity for molecules with similar molecular structures, such as DA and its metabolites. Here, we demonstrate that surface‐enhanced Raman spectroscopy (SERS), a vibrational spectroscopy that provides molecule‐specific information with excellent sensitivity, requires small sample sizes/volumes, and is non‐destructive, is optimal for the detection and differentiation of DA and its metabolites. We show that SERS combined with multivariate analysis allows for the detection and differentiation of DA and its metabolites, including 3‐O‐methyldopamine (3‐OMD), homovanillic acid (HVA), 3,4‐dihydroxyphenylacetic acid (DOPAC), and 3‐methoxytyramine (3‐MT) at physiologically relevant concentrations in artificial cerebrospinal fluid (aCSF).

Design of DMAP functionalized MoS2-GO hybrid for targeted visual detection of dopamine: a step towards chromogenic substrate free point of care testing

Abnormal release of DA, a crucial neurotransmitter, can cause serious neurological disorders such as Parkinson's and Alzheimer's disease. Thus, sensitive DA detection becomes critical for the investigation of physiological functions and disease diagnosis. Specific DA quantification in real samples is a tedious task due to the presence of analogous neurotransmitters. Therefore, aiming at on-site selective monitoring of DA, a high-performance colorimetric probe has been designed using a DMAP-modified MoS2-GO hybrid for highly selective visual detection of DA, especially for POC testing. The efficient DA recognition can be attributed to the large surface area with potentially active sites for greater electron transport and oxidation ability owing to the synergistic effect of MoS2 and GO. The hybrid has been modified with DMAP, which allows extremely selective and specific DA recognition against potential interferents, especially other neurotransmitters. The proposed mechanism relies on the attachment of DA to the hybrid surface, facilitating the oxidation and complexation processes that induce aggregation causing instant color change. The designed colorimetric method can recognize DA in neutral medium at room temperature without any reagents or supplementary operations. A good linear relationship was observed between the absorbance of the hybrid and DA concentrations in the range 1–100 μM, with a low detection limit of 0.17 μM. Furthermore, the applicability of the present approach was also verified by the analysis of DA in human serum samples and proved to be promising for sequential DA detection in biological samples with good precision and accuracy. From a POC perspective, easy-to-use paper-based test strips were prepared for visual recognition of DA in just 3 min. Results indicate that the designed DA assay provides a powerful, low-cost, convenient, and reagentless colorimetric platform for instant and highly selective DA detection in aqueous solution, strips, and human serum that is predicted to find extended applications in the fields of biosensing, clinical diagnosis, and in-vitro disease monitoring.

Self-demise of soft rot bacteria by activation of microbial predators by pectin-based carriers.

Soft rot pectobacteria (SRP) are phytopathogens of the genera Pectobacterium and Dickeya that cause soft rots on a wide range of crops and ornamental plants. SRP produce plant cell wall degrading enzymes (PCWDEs), including pectinases. Bdellovibrio and like organisms are bacterial predators that can prey on a variety of Gram-negative species, including SRP. In this research, a low methoxyl pectin (LMP)-based immobilization system for B. bacteriovorus is established. It takes advantage that pectin residues induce PCWDE secretion by the pathogens, bringing upon the release of the encapsulated predators. Three commercial LMPs differing in the degree of esterification (DE) and amidation (DA) were tested as potential carriers, by examining their effect on SRP growth, enzymes secretion and substrate breakdown. A clear advantage was observed for pectin 5 CS with the lowest DE and DA content. The degradation of 5 CS pectin-based carriers was further optimized by reducing cross-linker and pectin concentration, by adding gelatin and by dehydration. This resulted in SRP-induced disintegration of the carrier within 72 h. The released encapsulated predator caused a large decrease in SRP population while its own significantly increased, demonstrating the efficiency of this system in which the pathogen brings about its own demise.

Design, synthesis and evaluation of novel monoamine oxidase B (MAO-B) inhibitors with improved pharmacokinetic properties for Parkinson's disease

A series of novel ((benzofuran-5-yl)methyl)pyrrolidine-2-carboxamide derivatives were designed, synthesized and evaluated as MAO-B inhibitors. SAR studies indicated that cyclizing benzyl ether into benzofuran ring resulted in the most potent MAO-B inhibitor (IC50 = 0.037 μM), (2S,4S)-4-fluoro-1-((2-(4-fluorophenyl) benzofuran-5-yl)methyl)pyrrolidine-2-carboxamide (C14). PK properties of C14 in rats and mice were significantly improved compared to our previous candidate and safinamide, indicating that benzofuran moiety is essential for improving PK properties. Moreover, C14 displayed good metabolic stability and brain-blood barrier permeability, as well as favorable in vitro properties. Finally, C14 significantly inhibited MAO-B in the mouse brain. C14 exhibited a potential efficacy for DA deficits in the MPTP-induced mouse model and significantly increased DA concentration in the striatum. Thus, we identified that C14 may be a promising drug candidate for PD treatment.

Multi-applications of carbon dots and polydopamine-coated carbon dots for Fe3+ detection, bioimaging, dopamine assay and photothermal therapy

Carbon dots (CDs) or CDs/polymer composites have been applied in numerous fields. Here, novel CDs were synthesized by carbonization of egg yolk, and characterized by TEM, FTIR, XPS and photoluminescence spectra. The CDs were found to be approximate sphere in shape with an average size of 4.46 ± 1.17 nm, and emitted bright blue photoluminescence under UV irradiation. The photoluminescence of CDs was found selectively quenched by Fe3+ in a linear manner in the range of 0.05–0.45 mM, meaning they could be applied for Fe3+ detection in solution. Moreover, the CDs could be uptaken by HepG2 cells to exhibit bright blue photoluminescence. The intensity could reflect the level of intracellular Fe3+, indicating they could be further used for cell imaging and intracellular Fe3+ monitoring. Next, dopamine was polymerized on the surface of CDs to obtain the polydopamine (PDA)-coated CDs (CDs@PDA). We found PDA coating could quench the photoluminescence of CDs via inner filter effect, and the degree of quenching was linearly related to the logarithm of DA concentration (Log CDA). Also, the selectivity experiment indicated the method had a high selectivity for DA over a number of possible interfering species. This indicated the CDs in combination with Tris buffer could be potentially applied as the assay kit of dopamine. At last, the CDs@PDA exhibited excellent photothermal conversion capability, and they could efficiently kill HepG2 cells under NIR laser irradiation. Overall, the CDs and CDs@PDA in this work exhibited many excellent advantages, and could be potentially used for multi-applications, such as Fe3+ sensor in solution and cellular, cell imaging, dopamine assay kit, as well as photothermal agents for cancer therapy.

How does neurokinin 3 receptor agonism affect pathological and cognitive impairments in an Alzheimer's disease-like rat model?

Alzheimer's disease (AD) is accepted as a form of progressive dementia. Cholinergic systems are commonly affected in AD. Neurokinin 3 receptor (NK3R) is involved in learning memory-related processes. It is known that the activation of NK3R affects the release of many neurotransmitters. The aim of this project was to investigate the effects of NK3R agonist senktide administration on neurobehavioral mechanisms in the experimental AD-like rat model. 50 male Wistar albino rats were divided into Control (C), AD, Control + NK3R agonist (CS), AD + NK3R agonist (ADS), AD + NK3Ragonist + antagonist groups (ADSO). We designed AD-like model by intrahippocampal administration of Aβ1-42. After NK3R agonist + antagonist injections, open field (OF), Morris water maze (MWM) tests were applied. Cholinergic mechanism analysis from hippocampus-cortex tissues was performed by ELISA and catecholamine analysis from brain stem tissue were performed by HPLC method. The transitions from edge to center, rearing, grooming parameters were found to be reduced in final values of OF. While the group-time interaction was significant in the OF test findings, there was no significant difference between the groups. In MWM test, ADS group showed a learning level close to control group and animals in AD and ADSO groups could not learn target quadrant in MWM test. The brain stem NA and DA concentrations were not statistically significant. Hippocampal AChE-ChAT levels were supported by positive effects of senktide on learning via the cholinergic mechanisms. As a result, NK3R agonists were found to be effective in improving cognitive functions in rats with AD pathology. In the experimental AD model, positive effects of NK3R on learning memory may be mediated by cholinergic mechanisms.

An Electrochemical Sensor Based on Carbon Quantum Dots and Ionic Liquids for Selective Detection of Dopamine

AbstractDopamine (DA) as a neurotransmitter has a pivotal role in the central nervous system. Because of altered levels of DA in various neuroscience diseases, development of a quick, sensitive, and simple analytical approach to determine DA in biological fluids could be very applicable. In this research, a novel electrochemical sensor based on a carbon paste electrode (CPE) modified with ionic liquid (IL) and carbon quantum dots (CQDs) for measuring DA with uric acid and ascorbic acid was developed. IL and CQDs were synthesized and characterized for their specific properties such as composition, emission, size distribution, and morphology structure. Then, the modified CPE and different DA concentration was determined via cyclic voltammetry. The modified electrode exhibited great electrocatalytic activity for DA oxidation. Under optimal conditions, the calibration diagram for DA was linear within the range of 0.1–50 μM in phosphate buffer (pH=7.4) and limit of detection was 0.046 μM. The electrode was successfully used in the determination of DA in real samples and generated acceptable outputs. The proposed electrochemical sensor could be an exceptional step forward in DA detection and pave the way for the molecular diagnosis of neurological illnesses.

Blocking the ErbB pathway during adolescence affects the induction of anxiety-like behavior in young adult maternal immune activation offspring

Epidemiological and experimental evidence demonstrates that maternal exposure to infection during gestation increases the offspring's risk of developing schizophrenia and other neurodevelopmental disorders. In addition, the NRG-ErbB4 signaling pathway is involved in brain development and neuropsychiatric disorders. Specifically, this pathway modulates the dopaminergic and GABAergic systems and is expressed in the early stages of prenatal development. We recently demonstrated that maternal immune activation (MIA) at late gestation altered the expression of NRG1, its receptor ErbB4, and the dopamine D2 receptor four hours post-injection of viral or LPS in the fetal brain. We also reported that blocking the ErbB pathway during adolescence resulted in increased striatal DA content and reduced preference for sweetness and alcohol that persists into adulthood. However, the combined effects of MIA, re-activation of the immune system, and disruption of the ErbB signaling during adolescence would affect young adult mice's behavioral phenotype is unknown. Here, we report that the expression levels of the NRG1, ErbB4, GAD67, and BDNF were changed as responses to MIA and blocked the ErbB signaling in the frontal cortex of adolescent mice. MIA-Offspring during late gestation and immune system re-activation during adolescence spent less time in the open arms of the elevated plus-maze in adulthood. At the same time, MIA-offspring administrated with the pan-ErbB inhibitor during adolescence spent the same amount of time in the opened arm as the control mice. Combining the ErbB signaling disruption during adolescence leads to a social interaction impairment in female offspring, but not male, without affecting the offspring's motor activity, long-term recognition, and working memory. These results imply that blocking the ErbB signaling during adolescence prevents the development of anxiety-like behavior of the MIA offspring later in life and suggest that this interaction does not reduce the risk of female MIA offspring developing impaired social behavior.

97 Maternal Nutrient Restriction and Melatonin Supplementation Alter Neurotransmitter Profiles in Bovine Fetal and Placental Tissues

Abstract The placenta modulates fetal brain development in mice and humans, with a paucity of information reported in cattle. The objective was to determine the effects of maternal nutrient restriction and melatonin supplementation on neurotransmitter concentrations in the cotyledon, fetal hypothalamus, and fetal blood. At day 160 of gestation, 29 spring-calving and 25 fall-calving Brangus heifers were assigned treatments: adequately fed (ADQ-CON; 100% NRC recommendation), nutrient restricted (RES-CON; 60% NRC recommendation), and ADQ or RES supplemented with 20 mg/d of melatonin (ADQ-MEL; RES-MEL). Fetuses were removed at day 240 of gestation for collection of placentomes, blood, and hypothalamic tissue. Neurotransmitters (serotonin, 5-HT; dopamine, DA; norepinephrine, NE; epinephrine, EPI) and metabolites (3,4-dihydroxyphenylacetic acid, DOPAC; 5-hydroxyindoleacetic acid, 5-HIAA; homovanillic acid HVA) were analyzed by LCMS or HPLC in blood or tissues, respectively. Data were analyzed by ANOVA using the mixed procedure in SAS with seasons analyzed separately. In spring-calving heifers, melatonin increased (P ≤ 0.05) concentrations of DOPAC, DA, 5-HIAA, and HVA in the cotyledon. Nutrient restriction and melatonin supplementation increased (P ≤ 0.05) blood 5-HT. In both calving seasons, there was a nutrition by treatment interaction in hypothalamic 5-HT (P ≤ 0.01), whereby RES-MEL was increased compared with ADQ-MEL. For fall-calving heifers, no differences were observed in neurotransmitter concentrations in the cotyledon. Hypothalamic NE was increased (P = 0.02) in RES vs. ADQ, while hypothalamic and blood DA were decreased (P ≤ 0.05) in RES vs. ADQ. Hypothalamic 5-HIAA was increased (P = 0.05) in the MEL vs. CON, while blood EPI was decreased (P = 0.04) in MEL vs. CON. In summary, nutrient restriction and melatonin supplementation alter concentrations of neurotransmitters and their metabolites in a seasonally dependent manner. Further investigation into the gene expression of these neurotransmitter pathways is necessary to better define the bovine placental-brain axis.

Protective effect of low-dose radiation on doxorubicin-induced brain injury in mice

BackgroundTo investigate the protective effect of low-dose radiation (LDR) on brain injury in mice induced by doxorubicin (DOX). MethodsSixty female BALB/C mice were randomly divided into the control (CTR) group, low-dose radiation (LDR) group, doxorubicin treatment (DOX) group and low-dose radiation before doxorubicin treatment (COM) group. After 72 h of exposure to 75 mGy, the mice were intraperitoneally injected with 7.5 mg/kg of doxorubicin and sacrificed 5 days later. Neuron-specific enolase (NSE), lactate dehydrogenase (LDH), adenosine triphosphate (ATP), neurotransmitters, inflammatory mediators, apoptosis- and oxidative stress-related mediators as well as mitochondrial dysfunction were examined. ResultsCompared to the DOX group, the concentrations of DA, 5-HT, EPI and GABA in the COM group were significantly decreased, and the number of TUNEL-positive cells was decreased. In addition, the expression of proapoptotic proteins was downregulated in the COM group compared to the DOX group. Low-dose radiation in advance reduced reactive oxygen species and activated the SOD antioxidant defense system as indicated by significantly reduced GSH expression, increased GSSG expression, increased GPx expression and activation of the Nrf2 redox pathway. After low-dose radiation, the expression levels of ATP5f1, NDUFV1 and CYC1 were close to normal, and the mitochondrial respiratory control rate (RCR) and activity of respiratory chain complex enzymes also tended to be normal. Low-dose radiation upregulated the expression levels of IL-2 and IL-4 but downregulated the expression levels of IL-10 and TGF-β. ConclusionLDR has a protective effect on brain injury in mice treated with DOX. The mechanism is related to LDR alleviating mitochondrial dysfunction and oxidative stress, which promotes the production of antioxidant damage proteins, thus exerting an adaptive protective effect on cells.

Effects of light color, photoperiod, and growth-related gene interference or overexpression on the survival, growth, or physiological and biochemical indices of red claw crayfish juveniles

To determine the optimal light color and photoperiod conditions for red claw crayfish juveniles, we studied the effects of these factors on their survival, growth, monoamine content, antioxidant capacity, nonspecific immune index, and the expression of EcR and α-AMY genes. Experiments involving five light color groups (red, green, blue, yellow, and white light), five photoperiod groups (0 L:24D, 6 L:18D, 12 L:12D, 18 L:6D, and 24 L:0D), besides, we also studied the effects of interference and overexpression of these genes on growth, survival, and their expression. (1) The red light group showed the highest survival rate, while the blue light group showed the fastest growth rate. Further, the red light group showed significantly higher superoxide dismutase [SOD], acid phosphatase [ACP], and alkaline phosphatase [AKP] activities, but significantly lower malondialdehyde [MDA], serotonin [5-HT], noradrenaline [NE], and dopamine [DA] concentrations. The yellow light group presented significantly higher SOD activity, and lower DA concentration. The blue light group showed the highest α-AMY expression level, while the EcR expression levels corresponding to the yellow and red light groups were significantly higher. (2) The 0 L:24D and 6 L:18D photoperiod groups showed significantly higher survival rates than the 24 L:0D group. Additionally, the SOD and ACP activities corresponding to the 6 L:18D and 0 L:24D group were significantly higher, while its MDA and DA concentrations were significantly lower. (3) The 6 L:18D, 12 L:12D, and 0 L:24D groups showed significantly higher α-AMY expression levels, while EcR expression was significantly higher in the 6 L:18D and 0 L:24D groups. (4) EcR or α-AMY gene interference or overexpression had no significant effect on juvenile crayfish survival. However, interfering with ECR expression reduced juvenile growth rate, while ECR or α-AMY gene overexpression enhanced their growth rate. Overall, red claw crayfish juveniles showed higher survival rates, greater antioxidant and immune activities, and lower monoamine contents under red light, 6 L:18D or 0 L:24D conditions. Thus, these conditions possibly improved digestion and absorption and also promoted molting in the juveniles. Additionally, ECR and α-AMY expression could reflect the growth rate of the juveniles, but not their survival.

Post-translational modifications to hemidesmosomes in human airway epithelial cells following diacetyl exposure

Diacetyl (DA; 2,3-butanedione) is a highly reactive alpha (α)-diketone. Inhalation exposure to DA can cause significant airway epithelial cell injury, however, the mechanisms of toxicity remain poorly understood. The purpose of these experiments was to assess for changes in abundance and distribution of hemidesmosome-associated proteins following DA exposure that contribute to DA-induced epithelial toxicity. Human bronchial epithelial cells were grown in submerged cultures and exposed to three occupationally-relevant concentrations of DA (5.7, 8.6, or 11.4 mM) for 1 h. Following DA exposure, epithelial cells were cultured for 4 days to monitor for cell viability by MTT and WST-1 assays as well as for changes in cellular distribution and relative abundance of multiple hemidesmosome-associated proteins, including keratin 5 (KRT5), plectin (PLEC), integrin alpha 6 (ITGα6) and integrin beta 4 (ITGβ4). Significant toxicity developed in airway epithelial cells exposed to DA at concentrations ≥ 8.6 mM. DA exposure resulted in post-translational modifications to hemidesmosome-associated proteins with KRT5 crosslinking and ITGβ4 cleavage. Following DA exposure at 5.7 mM, these post-translational modifications to KRT5 resolved with time. Conversely, at DA concentrations ≥ 8.6 mM, modifications to KRT5 persisted in culture with decreased total abundance and perinuclear aggregation of hemidesmosome-associated proteins. Significant post-translational modifications to hemidesmosome-associated proteins develop in airway epithelial cells exposed to DA. At DA concentrations ≥ 8.6 mM, these hemidesmosome modifications persist in culture. Future work targeting hemidesmosome-associated protein modifications may prevent the development of lung disease following DA exposure.

Atorvastatin Modulates the Function and Trafficking of Dopamine Transporters

IntroductionDopaminergic signaling is involved in learning, memory, reward, movement, and emotion. The dopamine transporter (DAT) is the primary mechanism for terminating this signaling via DA uptake. A few lines of evidence indicate that DAT function is regulated by membrane cholesterol. Cholesterol is a critical component of plasma membranes and plays an essential role in regulating membrane fluidity, protein compartmentalization, and function. Statins are cholesterol‐lowering medications that have been linked to mild cognitive impairment. The goal of this project was to examine the effects of atorvastatin‐mediated reductions in cholesterol content on DAT function and trafficking.MethodsCultured cells & cholesterol measurement: HA‐tagged human DAT (hDAT) was stably expressed in neuroblastoma 2a (N2A) cells using G418 as selection. Cells were treated with atorvastatin (0.1, 0.3, 1.0, or 10 μM) and membrane cholesterol content was measured using Amplex Red.[3H]DA Uptake: After 24hr atorvastatin or vehicle treatment, cells were incubated with 15 nM [3H]DA in the presence of various concentrations of non‐labeled DA and [3H]DA uptake was measured to determine Vmax and Km.Cocaine inhibition of DA uptake & cholesterol replenishment: Cells were treated for 24hr with atorvastatin (1 μM) or vehicle and incubated with 15 nM [3H]DA and increasing concentrations of cocaine (100 nM‐10 μM). To determine if there was a causal effect of reduced cholesterol content and cocaine inhibition of DA uptake, atorvastatin‐treated cells were replenished with water soluble cholesterol (~40 mg per gram solid; MβCD) prior to cocaine inhibition.Amphetamine‐induced, DAT‐mediated DA efflux: Cells treated for 24hrs with either atorvastatin (1 μM) or vehicle were preloaded with 30 nM of [3H]DA. Then, cells were incubated with amphetamine (1 or 10 μM). AMPH‐induced DA efflux was determined by measuring DA content in the medium across time.Surface DAT Biotinylation: Basal surface levels of DAT in atorvastatin and vehicle treated cells, as well as DA‐induced DAT internalization, were measured by surface biotinylation using membrane non‐permeable sulfo‐NHS‐SS biotin.ImmunocytochemistryWe measured levels of F‐actin and G‐actin to determine the effects of atorvastatin treatment on actin dynamics using immunocytochemistry.ResultsWe found that atorvastatin dose dependently reduced membrane cholesterol content. This reduction was accompanied by decreases in the maximal rate of DA uptake and by decreases in the potency of cocaine to inhibit [3H]DA uptake. In contrast, atorvastatin treatment did not significantly alter AMPH‐induced DA efflux. Importantly, the reduced ability of cocaine to inhibit DA uptake was reversed by replenishing cholesterol in atorvastatin‐treated cells, suggesting a direct causal effect of reduced cholesterol content and cocaine inhibition. Lastly, we found that atorvastatin disrupted actin dynamics and abolished DA‐induced DAT internalization. The present study provides evidence for the first time that atorvastatin dysregulates DAT function and trafficking. Furthermore, this dysregulation may be associated with the cognitive deficits observed in patients receiving atorvastatin treatment.

3,4‐methylenedioxymethamphetamine (MDMA) stimulates activation of TAAR1 and subsequent neurotransmitter transporter internalization in serotonin neurons

The use of 3,4‐methylenedioxymethamphetamine (MDMA) has recently gained interest as a potential therapeutic tool for the treatment of post‐traumatic stress disorder (PTSD). However, the mechanism of action of this drug has not yet been fully elucidated. Here we demonstrate that MDMA acts by targeting the trace amine associated receptor 1 (TAAR1), an intracellular G‐protein coupled receptor (GPCR) expressed throughout the brain and in peripheral tissues. In previous studies, we have shown that a closely‐related compound, amphetamine (AMPH), enters neurons through the dopamine and norepinephrine transporters (DAT and NET) and activates TAAR1. This activates the small GTPase RhoA that in turn increases internalization of the DAT and NET. We have also found that a neuronal glutamate transporter, EAAT3, is also internalized. These actions result in an increase in extracellular DA, NE and glutamate concentrations. In this study, we show that MDMA enters serotonin neurons through the 5‐HT transporter (SERT) and activates a similar signaling cascade.We isolated brain tissue encompassing the Raphe nucleus from E15 mice and prepared primary cultures of serotonin (5‐HT) neurons. 5‐HT neurons were identified by immunocytochemistry for 5‐HT. Functional SERT activity was confirmed by citalopram sensitive 3H‐5‐HT uptake. Pre‐treatment of these cultures for thirty minutes with 1 mM MDMA resulted in a decrease of SERT transport activity. This loss of function was blocked by an inhibitor of RhoA, indicating that MDMA regulates SERT through a Rho‐dependent signaling pathway.Using total internal reflection fluorescence (TIRF) microscopy in HEK293 cells we confirmed that the loss in SERT activity produced by MDMA was due to internalization of the SERT. The application of 1 mM MDMA led to a decrease in GFP‐tagged SERT at the cell membrane. Similarly, GFP‐tagged EAAT3 was also internalized in response to MDMA. Parallel studies in HEK293 cells in which the TAAR1 gene was deleted by CRISPR‐Cas9 showed no effects of MDMA treatment on either SERT or EAAT3 trafficking and confirmed that TAAR1 is an obligate target for the actions of MDMA,To examine the effects of MDMA on endogenous SERT and EAAT3 in situ, we prepared acute brain slices from wildtype and TAAR1 knockout mice. Slices containing the Raphe nucleus were treated with either vehicle control or MDMA (1 mM) and subsequently treated with a cell‐impermeable biotin reagent that would label all surface proteins. By western blot analysis, we observed internalization of SERT and EAAT3 in response to the MDMA treatment. However, neither transporter exhibited any change in cell‐surface expression in response to MDMA in the TAAR1 knockout tissue.Our data indicate that in serotonergic neurons MDMA potentiates the action of both serotonin and glutamate by activating TAAR1 signaling and triggering SERT and EAAT3 internalization. These observations point to new mechanisms to target for the development of drugs to treat PTSD.