bluuebunny/crossref_metadata_2025_split huggingface dataset:
The Role of Monoamines on MPP+ and H2O2 Induced Oxidative Stress Response
Authors: Cobb Shelley , Shepherd Kennie 
Published: June 2021

Parkinson's Disease is a neurodegenerative disorder characterized by the depletion of monoamines, degeneration of dopaminergic neurons, and subsequent symptoms of bradykinesia, muscle rigidity, and other movement deficiencies. The production of reactive oxygen species has also been implicated in PD. It was previously demonstrated in vivo, that monoamines are depleted in the brain prior to the degeneration of dopamine neurons induced by the Parkinson's inducing agent MPTP. The current study seeks to examine the effects of monoamines such as dopamine, norepinephrine, and serotonin in buffering the effects oxidative stress and cell death mediated by hydrogen peroxide and the Parkinson's inducing agent MPTP/MPP+. The study also aims to elucidate how the monoamines provide this neuroprotective effect. The data has shown differing effects between the different monoamines which may be due to their differing structures. It is hypothesized that they interact with the Nrf2/KEAP1 pathway which regulates the antioxidant enzyme system. The data from these studies will provide potential insight into the role that monoamines play in the neurodegenerative process.Support or Funding InformationThis work was supported by NIH Grant U54NS083932 and NIMHD Grant #5S21MD000101. The investigation was conducted in a facility constructed with support from Research Facilities Improvement Grant #C06 RR‐07571 from the National Center for Research Resources, NIH.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Methylene Blue Protects The Dopaminergic Neurons through Upregulation of BDNF
Authors: Bhurtel Sunil , Choi Dong‐Young , Katila Nikita 
Published: June 2021

Parkinson's disease is a relatively common neurodegenerative disease without known causes. There are no effective therapies to halt or heal the neurodegeneration. Methylene blue (MB) possesses neuroprotective property, and has been tested in a clinical trial for Alzheimer's disease treatment. However, few studies have elucidated the effects of MB in Parkinson's disease. In this investigation, we evaluated effects of MB in the 1‐methyl‐4‐phenyl‐1,2,3,6‐tetrahydropyridine (MPTP) mouse model of Parkinson's disease. Mice received MB (20 mg/kg/day, i.p.) for four consecutive days, and then MPTP (30 mg/kg/day, i.p.) for the next four days. Immunohistochemical stainings displayed that MB significantly attenuated MPTP‐induced loss of the dopaminergic neurons in the nigrostriatal region. Activation of glial cells was observed in the substantia nigra and striatum challenged with MPTP, and pretreatment of MB markedly decreased the glial reaction. Moreover, MB restored MPTP‐induced depletion of dopamine in the striatum. To illuminate the underlying mechanism for the neuroprotection of MB, we analyzed the level of neurotrophic factors such as brain derived neurotrophic factor (BDNF). The western blotting showed MB as a strong inducer of BDNF and its downstream signaling pathways, suggesting BDNF might be a critical contributor to the beneficial effects. In study with SH‐SY5Y cells, inhibition of the receptor tyrosine kinase B, a receptor for BDNF reversed MB‐mediated protection against MPP+ toxicity implying that BDNF was responsible for the preventive effect. These results indicate that MB protects neurons against MPTP neurotoxicity through induction of BDNF, representing MB as a potential modulating agent for Parkinson's disease.Support or Funding InformationKorea Foundation for the Advancement of Science & Creativity (KOFAC)This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Impact of early life sleep deprivation on later life behaviors in rats
Authors: Atrooz Fatin , Liu Hesong , Kochi Camila , Salim Samina 
Published: June 2021

Increasing body of epidemiological studies suggest that sleep deprivation at early life stages (childhood and adolescence) is associated with behavioral and cognitive impairment. While these studies are important, yet, the relationship between early life sleep deprivation and development of later life psychiatric symptoms is not clearly understood. While examining the link between early life sleep deprivation and later life behavioral and cognitive well‐being is important, conducting such clinical studies in children are difficult. Therefore, animal models are valuable in studying the behavioral and biochemical consequences of early life sleep deprivation across the developmental course. Extended wakefulness increases cellular metabolism and induces reactive oxygen species (ROS) formation leading to oxidative stress. Hence, we examined the role of oxidative stress on behavioral impairment caused by early life SD. We employed two groups of male Sprague Dawley rats; control (CON) and sleep deprivation (SD) groups. Rats, at postnatal day (PND) 19 were subjected to SD for 14 days, (6–8 hours/day) using Pinnacle automated sleep deprivation system. Behavioral and cognitive tests were performed at three stages; early adolescent (PND32), late adolescent (PND60) and adult stage (PND90). Rats in the SD group exhibited anxiety‐like behavior at PND32 and 60 but not at PND90. Interestingly, SD rats did not exhibit depression‐like behavior at PND32 or at PND60 but developed depression‐like behavior at PND90, as indicated by increased immobility time in forced swim test compared to control group. Plasma levels of corticosterone (indicator of stress) were significantly increased in SD rats compared to control rats. We conclude that early life sleep deprivation during postnatal brain development, by engaging oxidative stress cascades, might alter the normal synapse formation and pruning causing anxiety‐like behavior early in life (at PND32 and 60), which in later life transforms into depression‐like behavior (PND 90).Support or Funding Information2R15MH093918‐02This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
VOLTAGE‐GATED SODIUM CHANNEL ACTIVATION TRIGGERS CALCIUM INFLUX AND HIPPOCAMPAL NEURON COMPLEXITY
Authors: Gomez Dina Akasheh , Murray Thomas F. 
Published: June 2021

Background and significanceAfter stroke, the molecular mechanisms underlying recovery are similar to the development of the nervous system. During development, neuronal activity regulates both the morphology and connectivity of neurons. Neuronal activity primarily involves voltage‐gated sodium channel (VGNaC) activation. Calcium signaling through the N‐methyl‐D‐aspartate receptor (NMDAR) drives morphological changes such as, dendritic arborization, synaptogenesis, and spinogenesis. VGNaC activation influences NMDAR function making this pathway an attractive therapeutic target for stroke recovery.HypothesisActivation of VGNaCs by PbTx‐2 augments NMDAR signaling resulting in and increased Ca2+ influx leading to dendritic arborization and spinogenesis in hippocampal neurons.Experimental design and MethodsWe used the VGNaC gating modifier, brevetoxin (PbTx‐2), to assess the effects of VGNaC activation on neuronal morphology. We defined the calcium‐signaling pathway triggered by PbTx‐2 in a population of hippocampal neurons (HNs), and evaluated PbTx‐2‐induced effects on dendritic arborization and spinogenesis in organotypic hippocampal slice culture (OHSC).HNscells were prepared from E18 Swiss Webster mouse embryos and plated in 96‐well plates. All experiments were run on (10 Days in vitro). A FlexStation2 was used to monitor PbTx‐2‐induced Ca2+ influx.OHSCSlices were prepared from P5, Thy‐1 YFP transgenic, mouse pups. Slices were treated with Pbtx‐2 18H after plating. A Leica SP8 confocal microscope was used to generate Z‐stack images of neurons or dendrites. Imaris‐XT software was used to reconstruct 3D‐images of the dendritic arbor, run Sholl analysis, and measure spine density.Results and ConclusionIn HNs, the EC50 for PbTx‐2‐induced Ca2+ influx was 376 nM (95% CI, 281–504 nM). The PbTx‐2 triggered Ca2+ influx in HNs was through the reverse mode of operation of Na+/Ca2+ exchanger, the NMDAR and the L‐type Ca2+ channel pathways. In OHSC, four‐day treatment with PbTx‐2 (100 nM) produced significant increases in dendritic arbor complexity as determined by Sholl analysis (p<0.005, ANOVA). PbTx‐2 treatment (300 nM) produced significant increases in spine density (p<0.001, ANOVA). Modifying the gating properties of VGNaC using PbTx‐2 enhanced the morphology of hippocampal neurons. Overall, these studies are consistent with the hypothesis that VGNaC activation may represent a novel pharmacological strategy to regulate NMDAR function and aid in recovery post‐stroke.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Comparison of agonist occupancy of M2 muscarinic receptor‐G protein complexes in myocardial homogenates with functional estimates of active receptor‐state affinity in isolated atria
Authors: Ehlert Frederick J. 
Published: June 2021

We have recently developed methods for estimating the active (Kact) and inactive (Kinact) receptor‐state affinity constants of ligands in functional studies on G protein‐coupled receptors. Our approach is valid for in vitro responses measured downstream from receptor activation, such as second messenger signaling in cell lines and responses in isolated tissues. The data required for our analysis include agonist‐mediated responses measured in the absence and presence of reduced receptor expression and modulation with an allosteric agonist (J Pharmacol Toxicol Methods 69:253–279, 2014). We also described an analogous technique that employs a constitutively active receptor mutant (J Pharmacol Toxicol Methods 83:94–106, 2017) instead of an allosteric agonist. In this study, we sought to validate our functional determination of Kact by using an independent biophysical method for estimating Kact. It is well known that muscarinic agonists exhibit high affinity for a subset of M2 muscarinic receptors in myocardial homogenates as measured in competitive binding experiments with [3H]N‐methylscopolamine. This subset of muscarinic binding sites is thought to represent a receptor‐Gi/o protein complex. We measured agonist binding to this site directly using the agonist radioligand, [3H]oxotremorine‐M, and completed agonist/[3H]oxotremorine‐M competitive binding experiments to estimate ligand affinity for this site in the guinea pig myocardium. We estimated a log affinity constant of approximately 8.2 for oxotremorine‐M in competitive binding experiments with [3H]oxotremorine‐M in the presence of 140 mM Na+. The allosteric agonist, LY211,9620 caused about a twofold increase in the affinity of oxotremorine‐for this site. In the presence of low sodium, LY211,9620 had little effect on oxotremorine‐M affinity, suggesting that in presence of both LY211,9620 and low sodium, the observed affinity of oxotremorine‐M is nearly the same as the active state affinity. Under these conditions, the log affinity constant of oxotremorine‐M was approximately 8.8. This value was similar to that estimated in functional studies on the atria (log Kact, 9.1 ± 0.10). These functional studies also provided estimates of the log Kact and Kinact values of the allosteric agonist LY211,9620 (6.3 and 5.1, respectively), the log isomerization constant of the unoccupied M2 muscarinic receptor (Kq, −3.18 ± 0.11), and the log sensitivity constant of the signaling pathway (log KE, 1.87 ± 0.27). Receptor‐state affinity constants provide a more powerful approach to structure activity relationships and to the measurement of agonist bias.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
The Mechanistic Role of Metal Ions, Ca2+ and Mg2+, in RGS: G‐Protein Interactions
Authors: O'Brien Joseph , Sieng Monita , Hayes Michael , Fowler Andrew , Lyon Angeline , Roman David 
Published: June 2021

Regulator of G protein signaling (RGS) proteins are negative regulators of G protein‐coupled receptor (GPCR) signaling through their ability to act as GTPase activating proteins (GAPs) for activated Gα subunits. The RZ subfamily, of which RGS17 is a member, binds to activated Gαo, Gαz, and Gαi1–3 proteins to modulate downstream pathways, including those involved in formation of cyclic AMP and Ca2+‐dependent signaling. In contrast to other RGS proteins, less is known about the regulation of RZ family members. Both crystallization and 1H‐15N 2D HSQC NMR experiments revealed a putative interaction of the metal ion Ca2+ with RGS17 at a defined binding site. Subsequent protein‐protein interaction experiments, using AlphaScreen were used to assess the impact on the ions Ca2+ and Mg2+ on the RGS17 interaction with activated Gαo. The results indicate that both calcium and magnesium have an effect of promoting the RGS17 ‐ Gαo interaction. These studies will extend to examining the selectivity and affinity of RGS17 for other physiologically relevant divalent metal cations, such as Zn2+, Cu2+, and Mn2+. In addition, the residues of RGS17 that bind Ca2+ are conserved in multiple RGS proteins. The functional impact of metal ion binding is likely not limited to RGS17 and a more in‐depth evaluation of these proteins for metal binding deserves further attention.Support or Funding InformationThis work was supported by The University of Iowa Center for Biocatalysis and Bioprocessing and the NIH‐sponsored Predoctoral Training Program in Biotechnology (2 T32 GM008365).This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Dynamin internalizes tyrosine phosphorylated sphingosine 1 phosphate receptor 1 and impair downstream signaling
Authors: Anwar Mumtaz , Amin Md Ruhul , Mehta Dolly 
Published: June 2021

We recently showed that S1P induces S1PR1 phosphorylation at tyrosine 143 residue impairing S1PR1 cell‐surface expression. Here we determined if phosphorylation alters receptor trafficking to the cell‐surface and if so what is the mechanism. The GTPase dynamin is essential for both clathrin‐dependent and clathrin‐independent‐endocytosis of receptors and thereby regulates the abundance of the receptor on the plasma membrane and downstream signaling. Thus, we inhibited dynamin using dynasore, which blocks dynamin GTPase activity in HEK cells transducing native S1PR1, phosphodefective (Y143F)‐S1PR1 and phosphomimicking (Y143D)‐S1PR1 and assessed S1PR1 spatial localization using confocal imaging. Interestingly, we found that inhibition of dynamin function blocked the internalization of Y143D‐S1PR1 leading to cell surface retention of the receptor. Cell‐surface retained S1PR1 induces signaling events by binding Gi heterotrimeric protein which includes calcium mobilization, cAMP generation, activation of MAP kinases. Thus, we assessed if phosphorylation altered S1PR1 downstream signaling. We observed that S1P‐induced enhanced Ca2+ entry in HEK cells expressing Y143D‐S1PR1 in Gi‐dependent manner indicating phosphorylated receptor binds Gi and dynamin and the internalized complex is functional. Further studies are under way to characterize the role of dynamin interaction with Y143 phosphorylated receptor in regulating S1PR1 signaling and endothelial barrier repair function.Support or Funding InformationNIHThis abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Crystal Structure of the C‐terminal Guanine Exchange Factor Module of Trio Reveals its Oncogenic Potential
Authors: Bandekar Sumit 
Published: June 2021

The C‐terminal guanine exchange factor module of Trio (TrioC) serves as a link between the heterotrimeric G proteins Gαq/11 and the small G‐protein RhoA, joining Gαq/11‐coupled GPCRs to downstream events of cell motility and gene transcription. This ancient signal transduction axis has been identified as crucial to the rise and spread of the fatal malignancy uveal melanoma. Previous studies have shown that TrioC is likely regulated via an autoinhibitory constraint that is released upon binding of Gαq/11. However, the structural determinants of this autoinhibition remain unclear. We have determined the crystal structure of TrioC in its basal state, revealing high‐resolution detail of autoinhibition mediated by the Pleckstrin Homology (PH) domain. We show that truncation of the PH domain activates the module in vitro, showcasing the importance of the αN helix found at the DH/PH junction. We then show using two orthogonal biochemical assays that the disruption of the autoinhibited conformation by point mutations destabilizes and activates the module. Finally, we show that mutations in αN found in cancer patients hyperactivate TrioC. These point mutations likely increase mitogenic signaling through the RhoA axis and may thus represent the first known cancer drivers identified in Trio, operating in an analogous, yet Gαq/11 independent manner. We are in the process of confirming this hypothesis using cell‐based assays.Support or Funding InformationRackham Graduate Student Research Grant, University of Michigan Horace H. Rackham Graduate SchoolPharmacological Sciences Training Program Fellow, 5T32GM007767–38This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Regulator of G‐protein Signaling (RGS10) regulates the expression of COX‐2 and TNF‐α in microglia via G‐protein independent mechanism
Authors: Wendimu Menbere Y. , Alqinyah Mohammed , Hooks Shelley 
Published: June 2021

Regulators of G‐protein signaling (RGS) are a family of proteins that classically act to inhibit signaling downstream of heterotrimeric G‐proteins. RGS10, a member of the R12 subfamily, has been shown to selectively target G alpha‐i and is enriched in immune cells, such as peripheral macrophages and microglia. Previous reports have characterized RGS10 as an anti‐inflammatory protein, playing a protective role in the nervous and immune system. Specifically, loss of RGS10 in microglia amplifies inflammatory cytokine production such as tumor necrosis factor alpha (TNF‐α) and leads to microglia‐induced neurotoxicity. However, the mechanism by which RGS10 mediates these effects is unknown and has not been linked to a GPCR pathway. In addition to inflammatory cytokines, inflammation and neurotoxicity are enhanced and maintained by the induction of the inflammatory enzyme cyclooxygenase‐2 (COX‐2) which is essential for the production of inflammatory molecules such as prostaglandin E2 (PGE2). However, it is not known if RGS10 influences COX‐2 and PGE2 production. The goal of this study was to directly investigate the role of RGS10 on the expression and function of COX‐2 in microglia, and further determine if RGS10 acts in a canonical G‐protein dependent or G‐protein independent mechanism to regulate inflammatory signaling. Our data revealed that RGS10 deficiency significantly elevates COX‐2 mRNA and protein levels, and enhances the release of PGE2 from microglia. Furthermore, the elevated TNF‐α and COX‐2 signal resulting from RGS10 loss was not affected by G‐protein inhibition, and a GAP‐deficient RGS10 mutant was as effective in inhibiting TNF‐alpha and COX‐2 expression as wild type RGS10, suggesting that the anti‐inflammatory functions of RGS10 are not mediated by its classic action on G‐proteins.Support or Funding InformationNational Institute of Health (NINDS)National Multiple Sclerosis SocietyThis abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Role of Protein Dynamics in Selectivity of Thiadiazolidinone Inhibition of RGS Proteins
Authors: Shaw Vincent , Mohammadiarani Hossein , Vashisth Harish , Neubig Richard 
Published: June 2021

BackgroundRegulator of G‐protein Signaling (RGS) proteins play a critical role in GPCR (G‐protein Coupled Receptor) signaling by binding to active, GTP‐bound Gα subunits and accelerating GTP hydrolysis, thus terminating signaling. There are multiple RGS isoforms with signature tissue distributions, so by targeting RGS proteins in an isoform‐specific manner, better tissue specificity of pharmacological effects may be achieved than by global application of a GPCR agonist. Thiadiazolidinones (TDZDs) are a series of compounds that inhibit RGS proteins by covalent modification of cysteine residues. They are very potent against RGS4, but also act less potently on several other cysteine‐containing RGS proteins. RGS4, RGS8, and RGS19, three proteins that are inhibited by TDZDs, share a cysteine on the α4 helix of the RGS homology domain. RGS19 has only this cysteine, while RGS4 and RGS8 have additional cysteines, including a shared one on the α6‐α7 interhelical loop. Interestingly, these shared cysteines are not exposed to solvent in crystal structures.HypothesisDifferences in potency due to covalent modification of the shared cysteine at the α4 helix of RGS proteins is driven by differences in flexibility.ApproachThe rate of covalent modification at individual cysteines can be determined by treating proteins with iodoacetamide, digestion, and detection of adduct by mass spectrometry. In addition, the effect of modification at different cysteines on protein function can be evaluated by measuring TDZD inhibition of binding between Gα and cysteine mutants of RGS proteins.ResultsDeuterium exchange and molecular dynamics studies have shown differential flexibility in key regions of these RGS proteins, which may be necessary for TDZD access to buried cysteines. RGS4 is more potently inhibited by CCG‐50014 (IC50: 81 nM) than RSG19 (IC50: 1.1 μM) or RGS8 (IC50: 1.8 μM). However, when RGS4 or RGS8 are mutated to contain only the α4 helical cysteine, the potency of TDZD inhibition is drastically reduced to below that of RGS19 (RGS4 IC50: 8.5 μM, RGS8 IC50 >100 μM). In accordance with previous molecular simulation data showing this α4 cysteine to be least solvent accessible in RGS4, it had a lower degree of covalent adduct formation by iodoacetamide than other cysteines.ConclusionThese results indicate that while additional cysteines in RGS4 may be responsible for its high potency of inhibition by TDZDs, differences in the rate of covalent modification and subsequent potency of inhibition at the conserved α4 cysteine is driven by differences in RGS protein flexibility.Support or Funding InformationNIH T32 GM092715NSF 1507588This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.