bluuebunny/crossref_metadata_2025 huggingface dataset:
Loss of cortactin causes endothelial barrier dysfunction via disturbed adrenomedullin secretion and actomyosin contractility
Authors: Ponce Alexander Garcia , Madrid Alí Francisco Citalán , Robles Hilda Vargas , Betanzos Abigail , Nava Porfirio , Zarbock Alexander , Rottner Klemens , Vestweber Dietmar , Schnoor Michael
Published: November 2023

Inflammatory processes lead to changes in vascular permeability via the opening and closure of cell‐cell contacts. The actin cytoskeleton plays a crucial role in the regulation of endothelial cell contacts and thus permeability. Several molecules regulate actin cytoskeleton dynamics, one of these being cortactin, an actin nucleator protein capable of directly bind to Arp2/3 complex, filamentous‐(F)‐actin and additionally, to molecules involved in the regulation of actin dynamics such as myosin light chain kinase (MLKC) and ezrin, but also, proteins from cell contacts such as ZO‐1. These capabilities render cortactin as a potential regulator of endothelial cell actin dynamics and thus cell‐cell contacts. We already showed that cortactin regulates vascular permeability in vivo via Rap1, part of this regulation depends on Rap1 ability to regulate the actin cytoskeleton by blocking RhoA activity, a very well‐studied enhancer of vascular permeability. However, the exact mechanism that contributes to increased permeability without cortactin is not known. In a mass spectrometric analysis, we found that ROCK1 is induced ~3‐fold in cortactin‐KO endothelium. This was confirmed by Western blot and immunofluorescence of tissues from cortactin‐KO mice and human HMEC‐1 cells stably depleted for cortactin. Concomitantly, we observed increased phosphorylation of myosin light chain (MLC) and more stress fibres without cortactin. Other molecules involved in stress fibres formation and stabilization such as mDia1, MLCK and ezrin, were not affected by cortactin absence. Secretion of the endogenous hormone adrenomedullin (ADM), that is known to activate Rap1 and counteract formation of stress fibres, is reduced in cortactin‐KO mouse serum and supernatant of cortactin‐depleted HMEC‐1 cells. Importantly, adrenomedullin administration or ROCK1 inhibition reduced actomyosin contractility and rescued the effect on permeability provoked by cortactin deficiency in vitro and in vivo. Our data suggest a new role for cortactin in controlling ctomyosin contractility with consequences for endothelial barrier integrity.

Support or Funding Information

CONACyT grants 179895 and 233395 to MS and an international, bilateral grant of CONACyT and the German Ministry for Education and Research (BMBF); 207268/01DN14039 to MS and AZ)
Inhibiting Cardiac Fibrosis in Myocardial Infarction by CXCL10 Agonist Peptide
Authors: Espinoza Liliana , Jaynes Jesse , Bodnar Richard , Willis Monte S. , Yates Cecelia C.
Published: November 2023

Objective

Heart failure is the leading cause of death and disability in the world, with 7.4 million deaths occurring in 2012 alone. Despite marked improvements in nearly every cardiovascular disease including heart attacks, heart failure has uniquely increased in prevalence and lacks therapeutic options targeting the progressive fibrotic response that commonly characterizes its pathogenesis. Much like the compensatory process seen in scarring of wounded skin, the heart responds to ischemia, hypertension, and common genetic mutations by undergoing extensive remodeling. However, overcompensation of the remodeling process often leads to fibrosis—an excessive accumulation of extracellular matrix (ECM) components. This accumulation is the results of activated fibroblasts that release collagen, fibronectin, and other ECM components. While a minimum of fibrosis is sometimes critical for structure (e.g. myocardial infarction), beyond this it has numerous negative consequences in the heart, including decreasing pump function and contributing to the development of cardiac arrhythmias. Outside of the heart, the beneficial role of the chemokine CXCL10 and its receptor CXCR3 in reducing fibrosis is well established. In the present study, we test the hypothesis that a newly developed CXCL10 agonist (IP10 peptide) similarly reduces fibrosis in the heart using a co‐culture model in vitro, and determine its relevance to MI in vivo.

Method

Cardiac tissue from mice challenged with permanent LAD ligation model (or sham controls) at 2 weeks post‐myocardial infarction were stained for CXCL10, its receptor CXCR3, and ECM components by immunohistochemistry to determine the temporal regulation of these components in vivo. In parallel, cardiomyocytes (CM), cardiac fibroblasts (CF), or CM/CF co‐cultures were challenged with transforming growth factor beta (TGF‐β) and/or treatment with CXCL10 or IP‐10 peptide. The in vitro cellular fibrotic response was then assessed for ECM expression, migration pattern, and apoptosis by immunocytochemistry (IHC) and immunoblot for specific components.

Results

IHC analysis of the heart two weeks post‐MI demonstrate a decrease in CXCL10, yet an increase in its receptor CXCR3 vs. sham controls. Treatment of CM/CF co‐culture with IP‐10 peptide in combination with TGF‐β resulted in reduced expression of α‐smooth muscle actin, collagen I, fibronectin, and tenascin C, compared to TGF‐β alone. These findings were not observed in single cells (CM or CF) cultures.

Conclusion

Our data suggests that chemokine CXCL10 plays an anti‐fibrotic role in remodeling, myocardial infarction‐induced cardiac tissue.
Genetic Inactivation of DNMT3b Protects Against Activation of Osteogenic Signaling and Slows Progression of Aortic Valve Stenosis in Mice
Authors: Sritharen Yoginee , Roos Carolyn M. , Nwachukwu Nneka , Kafa Rami , Hagler Michael A. , Verzosa Grace C. , Zhang Bin , Miller Jordan D.
Published: November 2023

Background

While recent work from our laboratory showed that DNA (cytosine‐5)‐methyltransferase 3 beta (DNMT3b) is significantly increased in tissue from humans with calcific aortic valve stenosis and is associated with increases in global genomic methylation, it is unknown whether the net effects of these changes are protective or deleterious. The objective of this study was to test the hypothesis that genetic reduction of DNMT3b allows for increased anti‐osteogenic gene expression, and consequently results in suppression of osteogenic signaling and slower progression of valvular stenosis.

Methods

We used Ldlr−/−/ApoB100/100 (LA) mice that were wild‐type for DNMT3b (LA‐DNMT3b+/+) or deficient in DNMT3b (LA‐DNMT3b+/−) and fed a western diet for six months.

Results

Using quantitative real‐time RT‐PCR, we confirmed that expression of DNMT3b was reduced by ~50% in valves from haploinsufficient mice. In line with our hypothesis, reductions in DNMT3b were associated with increases in expression of FABP4 (which opposes osteogenesis) and Smad6 (which opposes canonical BMP signaling). Similarly, expression of the osteogenic genes Msx2 and Spp1 were substantially reduced in DNMT3b‐deficient mice. Critically, aortic valve cusp separation distance was significantly greater in DNMT3b‐deficient mice, demonstrating slower progression of valvular dysfunction. No other gross abnormalities were noted in the mice.

Conclusions

Collectively, these data suggest that selectively reducing DNMT3b activity may be a viable therapeutic approach to slowing progression of calcific aortic valve stenosis in humans.

Support or Funding Information

Grant Funding Source: Supported by NHLBI
Effects of physical exercise and thyroid hormone on myosin heavy chain expression in doxorubicin‐induced cardiotoxicity in rats
Authors: Campos Erica C , Souza Fernanda R , Freitas Ana C S , Ramos Simone G , Resende Elmiro S
Published: November 2023

The cardiomyocytes membrane integrity and the presence of structural proteins play an essential role for cellular function. The structural proteins that maintain the sarcolemma integrity has been associated with cardiotoxic effects of doxorubicin, as example, the loss of actin‐myosin interaction. Two therapeutic options could be used to minimize the deleterious effects of cardiotoxic effects in contractile apparatus: the use of thyroid hormone and exercise. For analysis of myosin expression the animals were divided into seven groups identified as: control (C), exercise control (EC), thyroid hormone control (CHT), doxorubicin control (CDX), hormone and doxorubicin (HTDX), exercise and doxorubicin (EDX) and the hormone plus exercise and doxorubicin (HTEDX). The animals received intraperitoneal injections of doxorubicin hydrochloride three times a week, for two weeks, reaching a cumulative dose of 7.5 mg / kg. To the CHT groups, and HTDX HTEDX hormone was administered daily for four weeks, by oro gastric probe. The dose was 10 mg / 100g body weight. CE groups, EDX and HTEDX performed exercise. The swimming exercise was used, and with additional cumulative loading up to 5% of body weight. The training was conducted for 4 weeks with three weekly sessions of swimming. The expression of myosin heavy chain evaluated by Western blotting showed increase in myosin expression in HTDX and HTEDX groups. The results suggest that exposure to higher doses of thyroid hormone significantly increases the expression of myosin both in the presence and absence of exercise. Additional effects of exercise can contribute to other favorable results in cardiovascular system.

Support or Funding Information

CAPES
Neonatal and Juvenile Ocular Development in Sprague‐Dawley Rats: a Histomorphological Study
Authors: Vrolyk Vanessa , Apreutese Ancuta , Gordon Cedric , Forster Roy , Graham Andrew , Palate Bernard , Haruna Julius , Benoit‐Biancamano Marie‐Odile
Published: November 2023

This study aimed at evaluating the histomorphological developmental features of the eye in Sprague‐Dawley rats, with focus on the postnatal and juvenile periods. Tissues were collected from 51 rats, from both sexes, aged 1 to 30 days old. As in other altricial species, the eyes of newborn rats are underdeveloped and the eyelids remain closed for approximately two weeks after birth. During the postnatal development, dramatic changes occurred within the retina, when programmed cell death and cell differentiation coexisted. A complete separation of the inner nuclear layer from outer nuclear layer was first observed close to the end of the first week of life, while the nerve fiber, ganglion cell and inner plexiform layers almost reached maturity concurrently with the opening of the eyes, around the end of the second week. At birth, apoptotic bodies were frequently seen within the undifferentiated nuclear layer and peaked at post‐natal day (PND) 8 within the inner nuclear layer. In newborn animals, mitoses formed a contiguous band of proliferating cells within the undifferentiated nuclear layer, adjacent to the photoreceptors layer. Perinatally, the corneal stroma and sclera exhibited high cellularity and became thinner and less cellular with maturity. The corneal epithelium consisted of a single layer of non‐stratified columnar cells during the first ten PNDs, and transformed into 4 to 5 cell layers of stratified squamous epithelium at about PND 14. The lens continuously developed by addition of new lens fibers from the lens epithelium. Between PND8 and PND17, a band‐like region of apoptotic bodies was seen centrally within the zone of nucleated lens fibers. We conclude that, during the first month of life, the rat's eyes undergo a rapid phase of growth and almost reach adult maturity at PND30. These observations will serve as an historical database, useful in pediatric ocular drug development.

Support or Funding Information

Mitacs
Characterization of Toll‐like Receptor 4 Neuroinflammatory Signaling and the Effects of Fentanyl Citrate on CHME‐5 Human Microglial Cells
Authors: Figueroa‐Hall Leandra K , Das Subhas , Stevens Craig W , Davis Randall L
Published: November 2023

Neuroinflammation is a key mediator of neuronal damage observed in various neuropathologies, thus understanding pro‐inflammatory pathways of human glia is important in identifying potential pharmacological strategies to modulate inflammatory signaling in these cells. Microglia‐derived cytokines and chemokines are instrumental in neuroinflammation and several pro‐inflammatory mediators activate these cells to include bacterial lipopolysaccharide (LPS) that signals through toll‐like receptor 4 (TLR4). Even though peripheral TLR4 signaling is well defined, human microglial TLR4 signaling is not. Furthermore, recent studies suggest the ability of opioid agents to modulate immune function in peripheral and central nervous system cells. Therefore, our goals are to characterize the molecular mechanisms of LPS‐induced TLR4 signaling and elucidate the effects of the mu opioid receptor (MOR) agonist, fentanyl citrate, on this signaling in human microglial cells, CHME‐5. In keeping with these goals, LPS‐induced TLR4 gene expression was significantly increased at 3, 6 and 18 hours, whilst TNFα was significantly increased at 6 hours. Additionally, activation of p65 NF‐κB and IκBα expression revealed optimal LPS dosage of 1000 ng/ml and significant up‐regulation at 10 and 30 minutes, respectively. Lastly, dose‐dependent co‐exposure to LPS and fentanyl citrate (3–100 μM) down‐regulated LPS‐induced NF‐κB binding activity. To date, our data suggest that CHME‐5 human microglial cells serve as an experimental model for analyzing the inflammatory actions of human microglial TLR4 signaling, which may be modulated by opioid compounds, specifically, fentanyl citrate. Thus, we have also initiated studies to elucidate the effects of LPS, morphine and other pharmacological agents on key TLR4 signaling proteins. Together, these ongoing studies are expected to further elucidate the mechanisms of LPS‐induced TLR4 signaling and the effects of opioid agents in human microglia.

Support or Funding Information

Support: NIH‐NS 062664 (RLD); OCAST HR10‐31 (CWS)
Mitochondrial dysfunction in iPSC‐derived neurons in subjects with Chronic Mountain Sickness
Authors: Zhao Huiwen , Perkins Guy , Callacondo David , Appenzeller Otto , Haddad Gabriel
Published: November 2023

Chronic mountain sickness (CMS) is a disease that potentially threatens a large segment of the high altitude population (more than 140 million highlanders) during extended living over 2500m. Patients with CMS suffer from severe hypoxemia, erythrocytosis and neurologic manifestations including migraine, headache, mental fatigue, confusion, and memory loss. The only therapy, which is inadequate, is periodic phlebotomy. By understanding the mechanisms of CMS neuropathology, we wish to be able to develop better therapeutic strategies, not only for CMS but also for those diseases that are relevant at the sea level as well.

In the current study, we used induced pluripotent stem cells (iPSCs)‐derived neurons from subjects with CMS and others without any evidence of CMS (non‐CMS) living at the same altitude, as a model to study CMS in vitro. We noticed that CMS neurons are more sensitive to the culture environment than non‐CMS, with more blebbing or beadings of dendrites after 3 weeks in culture. Although intracellular ATP level is significantly decreased in the CMS group, there was no difference in LDH release between the two groups. Transmission electron microscopic images illustrated that CMS neurons have a decreased mitochondrial volume density, cristae and mitochondrial length. Real‐time PCR analysis confirmed that CMS neurons have 1) decreased mitochondrial biogenesis gene such as PGC1 and increased mitophagy genes including PARKIN and PINK; 2) decreased fission and fusion genes including DRP1, FIS, OPA, and MFN2, as well as 3) many altered nuclear and mitochondrial gene including UCP2, ANT1, VDAC, BCL2‐XL, NMNAT1, and VEGF, suggesting the present of mitochondrial dysfunction in CMS neurons. Within these genes, NMNAT1, a central enzyme of NAD biogenesis, attracts attention because overexpression of NMNAT1 is believed to be neuroprotective in neuronal morphogenesis. Decreased NAD level, due to a down‐regulation of NMNAT1, has been shown to disrupt the nuclear‐mitochondrial communication and lead to mitochondrial dysfunction and render cells in a pseudohypoxic state under normoxia. Thus, we speculate that a similar phenomenon takes place in CMS neurons; indeed we have confirmed that both NMNAT1 gene expression and NAD level are lower in CMS neurons as compared to non‐CMS neurons. We conclude that a decreased NAD level might be one of reasons for mitochondrial dysfunction and results in a vulnerable survival phenotype in CMS neurons.

Support or Funding Information

supported by NIH 5P01HL098053
Globally monitoring protein synthesis in time and space through ribosome profiling
Authors: Weissman Jonathan
Published: November 2023

The ability to sequence genomes has far outstripped approaches for deciphering the information they encode. We have developed a suite of techniques based on ribosome profiling (deep sequencing of ribosome protected fragments) that dramatically expand our ability to follow translation in vivo. I will present recent applications of our ribosome profiling approach including the following: (1) Development of ribosome profiling protocols for a wide variety of eukaryotic and prokaryotic organisms. (2) Uses of ribosome profiling to globally monitor when chaperones, targeting factors or processing enzymes engage nascent chains. (3) Using ribosome profiling to define the protein coding potential of complex genomes. (4) Monitoring localized translation including translation on the surface of the endoplasmic reticulum and mitochondria.
Characterizing the role of D2–like dopamine receptors in cocaine self‐administration in rats
Authors: Verduguez Randy Edgar , Wetzel Hanna , Norman Andrew
Published: November 2023

Both D1–like and D2–like dopamine receptor families appear to be involved in cocaine self‐administration behavior, but the ability of agonists selective for these dopamine receptor subtypes to maintain self‐administration behavior is not well established. In rats that reliably self‐administered cocaine i.v., it was not initially possible to determine whether the selective D2‐like dopamine receptor agonist quinpirole (LY171555), across a range of unit doses, reliably maintained self‐administration behavior. Therefore, the effects of single injections of quinpirole on cocaine self‐administration sessions were studied. Quinpirole (0.8–1.6 umol/kg i.v.) extended the interval during maintained cocaine self‐administration behavior consistent with it having a cocaine‐mimetic effect. Then the effect of single i.v. doses of quinpirole on extinction of cocaine‐induced lever‐pressing behavior was investigated. Typically, once access to cocaine was terminated, lever pressing behavior accelerated and then extinguished after approximately 26 min, presumably until the cocaine concentration was no longer sufficient to induce this behavior. In contrast, after a single dose of quinpirole was administered and then access to cocaine was terminated lever pressing behavior stopped and then after a delay resumed and continued for approximately 1.5–2 hours. This indicates that quinpirole can maintain lever‐pressing behavior and should be self‐administered. However, quinpirole appears to have a long elimination half‐life and inter‐injection intervals are likely to be long requiring very long sessions to measure. These data imply that D2‐like receptors have a role in mediating self‐administration behavior but both the pharmacodynamics potency and the pharmacokinetics of agonists must be considered when measuring this behavior.

Support or Funding Information

Randy Verduguez was supported as a Dalton Zannoni Fellow of the American Society of Pharmacology and Experimental Therapeutics (ASPET) with funds generously provided by ASPET and the University of Cincinnati.

Grant Award: DP1DA031386s

UC 2015 ASPET SURF Fellowship
Methamphetamine Self‐Administration in Mice Results in Both Pre‐ and Postsynaptic Changes in Dendritic Dopamine Neurotransmission in Midbrain Dopaminergic Neurons
Authors: Hager Audrey M. , Branch Sarah Y. , Dominguez‐Lopez Sergio , Sharpe Amanda L. , Beckstead Michael J.
Published: November 2023

Dopaminergic neurons of the ventral midbrain are key mediators of substance abuse and addiction‐related behaviors. In the midbrain, dopamine release activates inhibitory postsynaptic currents through D2 autoreceptors (D2‐IPSCs) and G protein‐coupled potassium (GIRK) channels. Methamphetamine is a highly addictive psychomotor stimulant that acutely enhances D2‐IPSCs by decreasing uptake of the neurotransmitter dopamine. Chronic methamphetamine exposure can induce neurophysiological adaptations in midbrain dopaminergic neurons, but data regarding specific synaptic adaptations following self‐administration of methamphetamine is limited.

In this study we investigated the effects of methamphetamine self‐administration in mice on synaptic determinants of dopamine neuron excitability. Adult mice were trained to nose‐poke under a fixed ratio 3 (FR3) schedule for intravenous infusions of methamphetamine (0.05 mg/kg/infusion) in daily 2 h sessions for 8 to 12 days. Their average intake of methamphetamine was 1.3 mg/kg/day. Subsequently, patch clamp electrophysiology was performed in brain slices containing substantia nigra and ventral tegmental area dopamine neurons to examine both pre‐ and postsynaptic changes. Interestingly, self‐administration of methamphetamine produced an overall downward shift in the methamphetamine (100nM‐3μM) concentration‐response curve of D2‐IPSCs compared to drug‐naïve controls. In addition, self‐administration of methamphetamine produced an increase in the paired‐pulse ratio of D2‐IPSCs that persists with subsequent stimulus events, indicating presynaptic effects on dopamine release. These results indicate an overall decrease in the strength of inhibitory synaptic transmission between midbrain dopamine neurons following methamphetamine self‐administration. This would be expected to produce an increase in neuronal firing that could enhance the reinforcing properties of methamphetamine in experienced individuals.

Support or Funding Information

5R01DA032701‐03
Cocaine‐Induced Alterations in Blood Lipid Profiles for the Study of Addictive Behaviors
Authors: Pati Sumitra , Scholpa Natalie E , Wagner John J , Cummings Brian S
Published: November 2023

NBIB EB016100‐01 BSC

Cocaine‐dependence is a worldwide public health concern, affecting more than a million users in the United States alone. Much research has focused on the development of pharmacotherapies for the treatment of cocaine‐dependence; however, less emphasis has been placed on strategies for the clinical determination of a patient's susceptibility to drug‐seeking behavior and relapse. We utilized a lipidomics approach to assess cocaine‐induced alterations of blood lipid profiles in Sprague‐Dawley rats exposed to cocaine in a non‐contingent model, conditioned place preference (CPP). Following drug‐paired conditioning, extinction and reinstatement tests were also performed to further assess drug‐seeking behavior. Lipids were extracted using the Bligh‐Dyer method from whole blood samples obtained throughout the behavioral protocol from cocaine‐ and saline‐treated rats. Blood lipid extracts were analyzed by electrospray ionization‐mass spectrometry (ESI‐MS) to characterize lipid profiles. The resulting data were assessed using principal component analysis (PCA), which indicated differences in the lipid profiles between cocaine‐ and saline‐treated rats. Significant m/z features indicated alterations in glycerophospholipids, glycerolipids, and fatty acyls. Several of these features, including 18:1 phosphatidylcholine (PC), 22:1 PC, 20:3 phosphatidic acid (PA), tetratriacontapentaenoic acid, eicosenoic acid, and 36:0 diacylglycerol (DG) correlated to drug‐seeking behavior in cocaine‐treated animals, specifically the initial CPP. Interestingly, the relative expression of these species increased during extinction and reinstatement in cocaine‐treated rats. Moreover, PCA within each treatment group suggested a role for cocaine in remodeling the lipidome over time. These findings indicate a potential effect of cocaine on lipidome remodeling that could be exploited for the identification of lipid species related to the occurrence of cocaine‐induced responses and drug‐seeking behaviors in human cocaine addicts.

Support or Funding Information

Georgia Research Alliance VentureLab (BSC and JJW)
Amphetamine Enhances Impulsive Behavior in Rats Responding under a Stop Signal Procedure
Authors: Maguire David Richard , France Charles P
Published: November 2023

Impulsivity might contribute to the persistence of stimulant (e.g., amphetamine) abuse and stimulant use can enhance impulsivity. The dynamic relationship between stimulant use and impulsivity has been well‐documented; however, the exact nature of the relationship remains unclear, in part, because of the multidimensionality of impulsivity. This study examined effects of amphetamine in rats (n=9) responding under a stop signal procedure which measured two different aspects of motor impulsivity: 1) responding before presentation of a go signal; and 2) responding after presentation of a stop signal. Rats were trained to press one lever (e.g., left) and then another lever (e.g., right) in order to obtain food. On 80% of trials (go trials), completion of the second response within a limited hold delivered food; failure to complete the second response initiated a timeout. On the remaining 20% of trials (stop trials), a tone was presented after the first response signaling that food would be presented only if the second response was withheld; responses after presentation of the tone initiated a timeout. Trials were separated by an inter‐trial interval; responses during the inter‐trial interval initiated a timeout, and responses during a timeout extended the timeout. Responding on stop trials (false alarms) and during the inter‐trial interval (premature responses) were measures of motor impulsivity. Under baseline conditions, rats responded on at least 90% of go trials and on no more than 10% of stop trials, while making fewer than 10 premature responses per session. Amphetamine (0.1–1.78 mg/kg) impaired response accuracy by significantly decreasing responses during go trials and increasing responses during stop trials. Amphetamine also significantly increased premature responding but did not impact timeout responding. Increased premature responding occurred with doses of amphetamine (0.32 and 1.0 mg/kg) smaller than the dose (1.78 mg/kg) that significantly increased false alarms. These data indicate that amphetamine enhances two types of motor impulsivity as measured with a stop signal procedure. However, increased premature responding occurred with doses of amphetamine that were 5‐fold smaller than the smallest dose that increased stop trial responding. Taken together, these data suggest that although amphetamine increases different behaviors thought to reflect motor impulsivity, sensitivity to amphetamine differs across measures. Thus, the impact of stimulant use on impulsivity might depend upon which aspect is being measured.

Support or Funding Information

Supported, in part, by NIH grant K05DA017918 (CPF).
Insulin Enhancement of Dopamine Reuptake in the Nucleus Accumbens Is Blocked by Consumption of a High Fat diet in C57BL/6 Mice
Authors: Fordahl Steven , Jones Sara
Published: November 2023

Systemically released insulin crosses the blood brain barrier and binds to insulin receptors on dopamine and other neurons. Insulin is thought to contribute to satiety by reducing dopamine signaling in response to food, and has been shown to decrease dopamine neuron firing, increase dopamine reuptake, and reduce the intake of palatable food in sated animals. Here we show that high fat (HF) diet‐induced insulin resistance blocks insulin's novel facilitatory effect on dopamine reuptake in the nucleus accumbens core (NAcc). Mice fed a HF diet (60% kcals from fat) were considered insulin resistant compared to chow fed littermates by displaying significantly higher fasting blood glucose (175 and 99 mg/dL, respectively; p < 0.001) and reduced blood glucose clearance after an intraperitoneal glucose tolerance test (p < 0.001). Using fast scan cyclic voltammetry to measure dopamine in brain slices containing the NAcc, HF fed mice exhibited reduced maximal dopamine reuptake (Vmax) compared to chow fed mice (2.3 ± 0.1μM and 3.2 ± 0.2μM, respectively) (p < 0.001). Moreover, reductions in glucose clearance were negatively correlated with Vmax (r2=0.68; p=0.0019). Insulin (10nM‐1μM) dose dependently increased Vmax in chow fed mice by a maximal 77% compared with 4% in the HF group (p < 0.0001); however, the small molecule insulin receptor sensitizing agent (TCS 401, 300nM) restored Vmax in HF fed mice to chow levels, and a small molecule inhibitor of the insulin receptor (BMS 536924, 300nM) reproduced the low Vmax HF phenotype in chow fed mice. These data show that a HF diet impairs dopamine reuptake by attenuating insulin signaling at dopamine terminals. This suggests that prolonged consumption of a HF diet may block homeostatic mechanisms that contribute to satiety, thereby promoting excessive food intake.

Support or Funding Information

5T32AA007565‐22
Dietary Supplementation with Fish Oil Prevents High Fat Diet‐Induced Enhancement of Sensitivity to the Locomotor Stimulating Effects of Cocaine
Authors: Labay Caitlin , Serafine Katherine , France Charles
Published: November 2023

Eating a diet high in fat can lead to severe negative health consequences, including obesity, insulin resistance, and increased inflammation in both peripheral tissues as well as in the brain. For example, eating a diet high in fat causes inflammation as evidenced by increased concentrations of the pro‐inflammatory markers interleukin‐6 and tumor necrosis factor‐α in the hypothalamus. Dietary supplements that are rich in omega‐3 polyunsaturated fatty acids (e.g., fish oil) can reduce or prevent high fat diet‐induced obesity, insulin resistance, and hypothalamic inflammation in rats. Eating a high fat diet also enhances sensitivity of rats to the locomotor stimulating effects of cocaine, particularly in adolescent female rats. To test the hypothesis that dietary supplementation with fish oil prevents high fat diet‐induced enhanced sensitivity to a behavioral effect of cocaine caused by a possible reduction in inflammation, adolescent female rats ate standard laboratory chow (17% kcal from fat; n = 6), high fat chow (60% kcal from fat; n = 7), or high fat chow supplemented with 20% fish oil (w/w; 68% kcal from fat; n = 7). Similar to previous reports, the rats eating high fat chow without fish oil were more sensitive than rats eating standard chow to the locomotor stimulating effects of cocaine (1–17.8 mg/kg). Although sensitization to once weekly tests with cocaine for 6 weeks developed in all groups, there was no statistical difference in sensitization between rats eating the standard chow and those eating a high fat chow with fish oil, indicating that supplementation with fish oil prevented the greater sensitization that occurs in rats eating only high fat chow. However, there were no significant differences among groups in the pro‐inflammatory markers tumor necrosis factor‐α or interleukin‐6 in the hypothalamus, no difference in body weight or caloric intake, and no development of insulin resistance. Thus, fish oil supplementation prevented high fat diet‐induced enhancement in sensitivity to cocaine without altering inflammatory markers, body weight, or insulin status. These results suggest that while diets high in fat increase sensitivity of individuals to drugs acting on dopamine systems in ways that might be relevant to drug abuse, dietary supplementation with fish oil can prevent these effects, although the mechanism for this effect remains to be determined. These data add to a growing literature demonstrating the complex relationship between diet and drug abuse, and the health benefits of fish oil.

Support or Funding Information

Supported by USPHS Grants K05 DA017918 (CPF), T32 DA031115 (KMS), and the ASPET SURF program (CEL)
Cocaine‐induced chromatin modifications are associated with increased gene expression and DNA‐DNA interactions of AUTS2
Authors: Engmann Olivia , Labonte Benoit , Mitchell Amanda , Calipari Erin , Rabkin Jaclyn , Hurd Yasmin , Turecki Gustavo , Akbarian Sharam , Nestler Eric J
Published: November 2023

Exposure to drugs of abuse alters the epigenetic landscape of the brain's reward regions. We investigated how a combination of chromatin modifications (as previously measured by ChIP‐sequencing) affects genes that are relevant for cocaine response. Autism‐candidate 2 (AUTS2), a gene linked to human evolution and autism, is among the genes with most cocaine‐induced chromatin modifications. We observed by FACS sorting that Auts2 gene‐expression is increased specifically in nucleus accumbens (Nac) D2‐neurons of male cocaine‐IP injected mice. Auts2 mRNA is also up‐regulated potmortem in Nac of male human cocaine addicts. Additionally, by using chromatin conformation capture (3 and 4C) approaches, we found evidence that the Auts2 gene forms a cocaine‐inducible DNA‐loop which enables Auts2 to bind and potentially regulate the expression of Caln1. Cell‐type specific HSV‐mediated overexpression of Auts2 and Caln1 reveals a role for these genes in D2‐neurons during cocaine place preference. We are currently characterizing the character and function of these genes after cocaine exposure. Our results may help to identify how a given gene can mediate a drug‐induced phenotype on the mRNA as well as DNA‐level.
Atypical Dopamine Transporter Inhibitors R‐Modafinil and JHW007 Differentially Alter D2 Autoreceptor Currents and Firing Rate in Midbrain Dopamine Neurons
Authors: Avelar Alicia J , Newman Amy H , Beckstead Michael J
Published: November 2023

Psychostimulants increase attention and induce euphoria, but despite decades of work there still are no FDA approved clinical treatments for their abuse. Psychostimulants act on dopamine (DA) signaling of midbrain DA neurons to cause reinforcement. D2 autoreceptors on these neurons powerfully inhibit cell firing and have been inversely associated with psychostimulant use. D2 autoreceptor signaling is tightly controlled by DA transporters (DATs), which clear DA from the extracellular space. Cocaine, a typical DAT inhibitor, blocks DA reuptake and causes hyperlocomotion and reinforcement. Behavioral studies suggest that atypical DAT inhibitors are potential therapeutics to decrease cocaine abuse. Atypical DAT inhibitors bind to the transporter, but are less likely to cause hyperlocomotion and reinforcement than the cocaine. To date, there have been no investigations into the synaptic actions of these compounds. Here we determined the effects of atypical DAT inhibitors on D2 autoreceptor‐mediated currents and DA neuron excitability. To address this, we performed patch‐clamp electrophysiology of midbrain DA neurons in brain slices from DBA/2J mice. We utilized electrical stimulation, DA iontophoresis, and bath perfusion of atypical DAT inhibitors R‐Modafinil (10 and 100 μM) or JHW007 (100nM, 1 and 10 μM) to investigate their synaptic actions on D2 autoreceptor currents. Additionally, we recorded DA neuron firing before and after bath perfusion of R‐Modafinil (100 μM) and JHW007 (10 μM) and with or without the D2 receptor antagonist sulpiride (200 nM). These experiments show that, R‐Modafinil and JHW007 differentially affect D2 autoreceptor currents and DA neuron firing. R‐Modafinil increased D2 autoreceptor current amplitude and width and decreased DA neuron firing rate in a D2 autoreceptor dependent manner. Conversely, JHW007 primarily decreased D2 autoreceptor current amplitude, had little effect on width, and increased DA neuron firing rate in a D2 autoreceptor independent manner. These findings suggest that, despite both being atypical DAT inhibitors; these two compounds exhibit profoundly different effects on DA neuron excitability.

Support or Funding Information

This project was supported by NIH training grant T32 NS 082145, funded by the Jointly Sponsored NIH Predoctoral Training Program in the Neurosciences. Funding also provided by: NIH R01 DA 32701 and a diversity supplement.
Analogs of the designer drug, 3,4‐methylenedioxymethcathinone (methylone), differentially affect monoamine transporters in rat brain
Authors: Baumann Michael Howard , Bukhari Mohammad Omar , Lehner Kurt Richard , Partilla John S
Published: November 2023

The abuse of synthetic cathinone compounds, often sold as “bath salts” or “research chemicals”, is an emerging public health concern. 3,4‐Methylenedioxymethcathinone (methylone) is a popular synthetic cathinone that is now illegal, but new analogs are being marketed as replacements. The purpose of the present investigation was to examine the interaction of new methylone analogs with transporters for dopamine (DAT) and 5‐HT (SERT). β‐Keto‐N‐methylbenzodioxolylbutanamine (butylone) and β‐keto‐N‐methylbenzodioxolylpentanamine (pentylone) were the analogs studied. In vitro assays were carried out in rat brain synaptosomes to assess drug‐induced effects on transporter‐mediated uptake and release. In vivo microdialysis was carried out in the nucleus accumbens of conscious rats to assess drug‐induced changes in extracellular dopamine and 5‐HT. Methylone, butylone and pentylone were fully efficacious uptake blockers at DAT and SERT, but pentylone was more DAT‐selective than the other drugs. Methylone was a substrate‐type releaser at both transporters, while butylone evoked release at SERT but not at DAT. Pentylone failed to evoke release at either transporter. Administration of butylone (1–3 mg/kg, i.v.) increased extracellular concentrations of 5‐HT more than dopamine, while pentylone (1–3 mg/kg. i.v.) increased both neurotransmitters to the same extent. All drugs produced significant locomotor activation, but the effects of butylone were blunted compared to pentylone. Our data show that each of the compounds examined displays a unique profile of in vitro transporter activity. Butylone has “hybrid” transporter effects, acting as a substrate at SERT but a blocker at DAT. Pentylone is a DAT‐preferring uptake blocker which lacks substrate activity. Overall, the data show that increasing the α‐carbon chain length of methylone creates compounds with reduced releasing activity, converting them to DAT blockers. All of the compounds elevate extracellular dopamine and stimulate motor activity to some degree, suggesting the drugs possess a significant risk for abuse.

Support or Funding Information

This research was generously supported by the Intramural Research Program of NIDA, NIH, DHHS.
The Effects of Chronic Cigarette Smoke Extract Exposure on Somatic Withdrawal
Authors: Reynaga Daisy , Leslie Frances , Belluzzi James
Published: November 2023

Tobacco dependence is extremely difficult to treat and the vast majority of those who try to quit will relapse within the first year. Tobacco withdrawal is a major cause of relapse during the first few days of the quitting attempt. Adult rodent models of nicotine exposure show similar symptoms of withdrawal as humans. However, although human adolescents have an increased sensitivity to the effects of smoking cessation, rodent adolescents experience little to no somatic withdrawal symptoms after chronic nicotine exposure (DiFranza et al., 2007; O'Dell, 2009; Torres et al., 2013). This discrepancy may be due to the lack of other non‐nicotine smoke constituents in current animal models. We have shown that rats exposed to aqueous cigarette smoke extract (CSE), a solution containing nicotine and other cigarette smoke constituents, are more sensitive to stress‐induced reinstatement of self‐administration (Costello et al., 2014). The goal of this study is to determine whether adult and adolescents rats show enhanced spontaneous somatic withdrawal symptoms after chronic exposure to CSE, and to investigate the involvement of nicotinic acetylcholine receptors (nAChRs) in CSE cessation‐induced withdrawal using mecamylamine, a nonselective and noncompetitive nAChR antagonist, to precipitate withdrawal. To compare withdrawal effects, adult and adolescent rats were exposed to nicotine or CSE (1.5 mg nicotine/kg/day; i.v) for 10 days and analyzed for spontaneous or precipitated somatic withdrawal. We found that adult rats that received chronic CSE exposure showed enhanced spontaneous somatic withdrawal that emerged sooner than in animals that received chronic nicotine exposure. Adolescent rats showed significant spontaneous somatic withdrawal after chronic CSE exposure but not after nicotine. Mecamylamine (1 mg/kg; s.c.) administration precipitated withdrawal after chronic CSE exposure in both adult and adolescent rats. This provides evidence that non‐nicotine constituents in CSE mediate withdrawal processes, possibly via sensitization of nAChRs.

Support or Funding Information

Tobacco Related Disease Research Program (TRDRP): 21RT‐0136

National Institute on Drug Abuse (NIDA): 1RO1DA040440‐01
A Novel Structural Landscape for Ligand Binding to the α7 Nicotinic Acetylcholine Receptor
Authors: Camacho‐Bustamante Gisela Andrea , Kaczanowska Kasia , Harel Michal , Cornejo‐Bravo Jose , Taylor Palmer
Published: November 2023

In our previous study we identified a series of 4,6‐disubstituted 2‐aminopyrimidines interacting with Acetylcholine Binding Proteins (AChBP) in a cooperative fashion [Kaczanowska et al., Proc. Natl. Acad. Sci. USA 111, 10749 (2014)]. Based on X‐ray crystal structures of AChBP in complex with the cooperative ligands, we designed a new subset of these molecules that target the α7‐nicotinic acetylcholine receptor (nAChR). Seventeen compounds corresponding to this design were synthesized and characterized in cell based functional assays and radioligand binding assay using HEK cell lines overexpressing transfected cDNAs of human α7‐nAChRs, α4β2‐nAChRs and mouse 5HT3A receptors and Lymnaea (Ls) AChBP, respectively. We found a subset of structures that show Kd values in radioligand binding for AChBP between 51–68 nM and elicited responses for α7‐nAChRs in cell based assays (EC50's) as low as 60–70 nM, reflecting high affinity for α7‐nAChRs. Differential selectivity was characterized using α4β2‐nAChRs and 5HT3A receptors as references. Selected Ls‐AChBP and α7‐nAChRs ligands (AC‐171A, AC‐171A2, AC‐171C, AC‐171D) were then crystallized with the Ls‐AChBP revealing a unique binding pose at the five subunit interfaces. These 4,6‐disubsituted 2‐aminopyrimidines may hold potential in treatment of various CNS developmental disorders. For example, in schizophrenia nicotine addiction and resistance to abstinence prevails and may be modulated by these α7‐nAChRs ligands.

Support or Funding Information

Supported by NIH grant GM18360 and the Tobacco‐related Disease Research Program (TRDRP).
Pharmacology of a novel TRPM2 antagonist, JNJ‐28583113
Authors: Fourgeaud Lawrence , Faouzi Malika , Starkus John , Wang Qi , Coates Heather , He Yingbo , Lovenberg Timothy , Penner Reinhold , Dvorak Curt , Bhattacharya Anindya
Published: November 2023

TRPM2 is a calcium‐permeable non‐selective cation channel that is activated, in a synergistic manner, by both intracellular adenosine diphosphate ribose (ADPR) and calcium. It is also activated indirectly by reactive oxygen nitrogen species (ROS/NOS) due to increased ADPR production during oxidative/nitrosative stress. TRPM2 is primarily expressed in the immune cells and central nervous system (CNS) where it plays important roles in pathologies involving neuroimmune interactions, particularly in the context of oxidative stress. Within the CNS, TRPM2 is expressed mainly in neurons and microglia. Here, we present the pharmacological properties of JNJ‐28583113, a newly discovered compound representing the most potent TRPM2 antagonist described to date. Our data show that this compound has a potency range of 100–200 nM in blocking H2O2‐induced calcium flux in several recombinant systems over‐expressing human, chimpanzee and rat TRPM2. In whole‐cell patch clamp recordings, JNJ‐285831113 blocked intracellular ADPR‐induced human TRPM2 currents with a potency of 13 ± 3 nM. Similar pharmacology was observed for native TRPM2 in a human macrophage cell line (U937) and in a rat pancreatic cell line (INS‐1). In line with TRPM2 microglial expression, JNJ‐28583113 also modulated lipopolysaccharide (LPS)‐induced changes gliotransmitter secretion in mouse microglia in vitro, indicating a possible role of this channel in neuroinflammation. Using multi‐electrode arrays to record hippocampal field potentials in acute mouse brain slices, we observed that JNJ‐285831113 prevented hypoxia‐induced synaptic depression at CA3 to CA1 synapses, suggesting a role for TRPM2 in synaptic changes occurring during oxidative stress. Finally, JNJ‐28583113 inhibited H2O2‐induced dephosphorylation of glycogen synthase kinase‐3 (GSK3) alpha and beta in recombinant cells over‐expressing human TRPM2 channels, confirming the importance of TRPM2 in modulating GSK3 signaling. Together our results demonstrate that JNJ‐28583113 is a very potent TRPM2 antagonist that can be used to further elucidate TRPM2 physiological functions as well as to probe the role of TRPM2 in disease biology under conditions of oxidative stress.

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Janssen
The extent of vascular remodeling is dependent on the balance between ERα and GPER(GPR30)
Authors: Gros Robert , Ding Qingming , Hussain Yasin , Chorazyczewski Jozef , Pickering Geoffery , Feldman Ross D
Published: November 2023

Estrogens are important physiological and pathophysiological regulators of cardiovascular function. The traditional view of the mechanism for these cardiovascular effects has focused on the activation of “classical” steroid receptors (i.e., estrogen receptors‐ERs). However, recent studies have elucidated the mechanism of estrogen's cardiovascular effects mediated by an alternate GPCR‐based mechanism viz., via GPER (aka GPR30), formerly characterized as an “orphan receptor”. We are beginning to appreciate that to understand the effect of estradiol in cardiovascular regulation one must understand the balance between GPER‐ and ER‐mediated effects. In vascular smooth muscle cells, estrogen‐mediated regulation of apoptotic cell death and proliferation is divergently regulated by activation of ER vs. GPER. However, the significance of this divergence in the in vivo regulation of vascular growth processes was unknown. To determine the role of GPER in regulating vascular remodeling we studied the effects of altering GPER expression vs. altering the expression of ERα in a rat model of carotid ligation. Under baseline conditions, 1 week following carotid ligation and endothelial disruption with distilled water installation there was a 51±7.6% (n=3) increase in medial thickness, paralleling neointimal proliferation in female rats. This medial hypertrophy paralleled down‐regulation of GPER protein content (26±5% of control; n=3) and mRNA expression (29±13% of control; n=3). Re‐introduction of GPER, via abluminal adenoviral delivery, significantly attenuated the extent of medial hypertrophy (adeno‐GPER: 28±2.2% increase; n=10 vs. adeno‐GFP/control: 48±3.3% increase; n=16, p<0.05). Similarly, inhibition of ERα expression using an adeno‐shERαRNA construct resulted in an attenuation of injury‐mediated medial hypertrophy. In these studies we show that in vivo the balance between GPER and ERα is a significant regulator in the extent of vascular remodeling. Receptor‐specific modulation of estrogen's growth regulatory effects may be an important new approach in modifying the extent of vascular remodeling in both acute settings like vascular injury and perhaps in longer term models of regulation like in hypertension.

Support or Funding Information

These studies were supported by grants from the Heart and Stroke Foundation of Canada (RG and RDF).