bluuebunny/crossref_metadata_2025 huggingface dataset:
Regulation of lysophosphatidic acid receptor 1 stability modulates cytokine release in lung injury
Authors: Zhao Yutong , Wei Jianxin , Dong Su , Bowser Rachel K , Zhang Lina , Jacko Anastasia M , Zhao Jing
Published: November 2023

Lysophosphatidic acid receptor 1 (LPA1), a member of G‐protein‐coupled receptors (GPCRs), is a druggable target for treating pulmonary inflammatory diseases, such as acute lung injury and pulmonary fibrosis. However, the molecular regulation of LPA1 stability, a factor that critically impacts its biological activity, remains largely unknown.

To investigate the molecular regulation of LPA1 stability, we focus on investigating ubiquitination of LPA1. We identified two enzymes that catalyze ubiquitination and deubiquitination of LPA1. The ubiquitin E3 ligase Nedd4L targets LPA1 for its site specific ubiquitination and degradation in the lysosome. Nedd4L negatively regulates LPA/LPA1‐mediated cytokine release, suggesting targeting LPA1 stability is potential strategy for regulating LPA signaling. The stability of LPA1 is up‐regulated by ubiquitin‐specific protease 11 (USP11), which deubiquitinates and stabilizes LPA1, thus enhancing LPA1‐mediated pro‐inflammatory effects. LPA1 is associated with USP11 in quiescent cells, while LPA treatment triggers LPA1 dis‐association with USP11 and in turn binding to Nedd4L. We have shown that LPA1 contributes to LPS‐induced cytokine release through interaction with CD14. Here, we show that knockdown of USP11 reduces LPA1 stability, levels of LPA1, and LPA1/CD14 interaction complex; thereby diminishing both LPA‐ and LPS‐induced inflammatory responses. C57/BL mice were infected with USP11 shRNA lentivirus for 7 days, and then mice were challenged with intratracheal LPS. USP11 shRNA reduced USP11 levels in lungs and attenuated LPS‐increased BAL protein, IL‐6, KC, and neutrophil infiltration.

Our findings identify an ubiquitin E3 ligase and a deubiquitinating enzyme responsible for regulation of LPA1 stability and biological activities. This study provides new targets for the development of anti‐inflammatory molecules to lessen LPA1‐mediated lung injury.

Support or Funding Information

This work was supported by the US National Institutes of Health (R01HL091916 and R01HL112791 to Y.Z, R01GM115389 to J.Z.), American Heart Association 12SDG9050005 (J.Z.), and American Lung Association Biomedical Research Grant RG350146 (J.Z.).
Kinetics and Implications of Germinal Center Formation in Induced Bronchus‐Associated Lymphoid Tissue During Influenza Infection
Authors: Gibson‐Corley Katherine N. , Boyden Alexander W. , Tygrett Lorraine , Waldschmidt Thomas J.
Published: November 2023

Induced bronchus‐associated lymphoid tissue (iBALT) develops within the lung of both humans and mice following an adaptive immune response upon exposure to certain classes of antigens. iBALT has been documented to develop with Influenza A virus (IAV) infection but the kinetics of this localized immune response have yet to be documented. Using both flow cytometry and immunohistochemistry we have identified the structure, composition and development of B cells and iBALT over time in mice intranasally infected with an H1N1 IAV. Early in infection (days 6–12) B cells accumulate within the lung, primarily around blood vessels, and are very loosely organized. By flow cytometry, these are mature B cells and there is no evidence of germinal center cells. As time progresses to days 18 and 24, B cells more tightly organize around medium to large airways and have a well‐defined lymphoid structures complete with Bcl‐6 positive, PNAhi, CD95hi, IgMhi germinal center B cells which persist up to and likely beyond day 50 of infection. These findings are of interest as Influenza viruses are constantly undergoing antigenic changes, which make the development of vaccines very challenging. Understanding the kinetics of iBALT development can help us identify key triggers for a protective adaptive immune response and novel targets for vaccine development.

Support or Funding Information

Departmental Funding; Department of Pathology, University of Iowa
Pulmonary Dipeptidyl Peptidase‐4 Expression in Lung: Relevance in Experimental Models and for Middle East Respiratory Syndrome Clinical Disease
Authors: Meyerholz David K. , McCray Paul B.
Published: November 2023

Dipeptidyl peptidase‐4 (DPP4, CD26) is a type II transmembrane exopeptidase and functions as a receptor for Middle East Respiratory Syndrome coronavirus (MERS‐CoV). Respiratory disease is a common presentation in people clinically affected by MERS‐CoV infection and likely contributes in a significant manner to MERS‐CoV morbidity and mortality. Unfortunately, the study of MERS‐CoV related pulmonary pathology is limited because of the lack of autopsy examination and leaves imaging technologies as the main evaluation of lung disease. Because of this constraint, we compared pulmonary distribution of DPP4 in humans to clinical MERS‐CoV disease in experimental animal models and to that reported in humans to evaluate for common features of disease pathogenesis. DPP4 is present in small amounts in airways – but is localized more extensively in cells of the pulmonary acinus in epithelial, immune cell and mesenchymal compartments. This anatomic distribution ostensibly correlates with ground‐glass and edematous changes seen by imaging in MERS‐CoV patients and in lung disease seen some, but not all animal models of MERS‐CoV infection. The putative lesions in humans and animal models with lung disease predominantly consist of diffuse alveolar disease‐like changes with a lack of airway centric disease suggesting that the receptor cellular distribution could be a relevant feature in MERS‐CoV pathogenesis. Furthermore, recent studies that show DPP4 expression levels might be influenced by comorbidities (concurrent lung disease, diabetes, etc.) that are known to predispose to severe disease make its relevance to pathogenesis and possible development of therapeutics even more compelling.
Investigating the Mechanism Of Autoimmune Disease Associated LQTS; anti‐Ro52 IgG Decreases hERG Channel Density
Authors: Szendrey John Alexander , Zhang Shetuan
Published: November 2023

Background

The human ether‐a‐go‐go‐related gene (hERG) is the pore forming subunit of Kv11.1, which is responsible for the rapidly activating delayed rectifier potassium current (IKr). Reductions to IhERG through mutations or drug interactions can cause LQTS, which predisposes individuals to ventricular arrhythmia's and sudden cardiac death. hERG has been speculated to be involved in autoimmune disease‐associated LQTS, however the mechanism is poorly understood. In 2006, a Japanese group reported an anti‐Ro52 IgG positive patient with LQTS and accompanying torasdes de pointes. The patient's serum was found to reduce the human ether‐a‐go‐related gene (hERG) channel density in a stable cell line. The authors speculated that anti‐Ro52 underwent a cross reaction with hERG, leading the production of anti‐hERG IgG. Here we investigate in detail this proposed mechanism, and provide strong evidence that anti‐Ro52 IgG reduces hERG channel density and current.

Methods

Human embryonic kidney (HEK 293) cell lines stably expressing the channel of interest were treated with anti‐Ro52 IgG (Sigma, 4 μg/ml) for 24 h, while saline treated cells served as control. Western blot analysis was used to quantify changes in protein expression, and whole cell patch clamp was performed to quantify changes in current.

Results

We report that anti‐Ro52 IgG reduced hERG channel density and current. This was unique to hERG in that Kv1.5 and EAG channel density and current were not affected by IgG. Replacing the extracellular S5‐pore linker with that of EAGs instilled immunity to IgG treatment. Furthermore, anti‐Ro52 IgG treatment reduced IKr in rat neonatal cardiomyocytes, significantly increased the APD90, but did not affect other native currents. Our data suggest that IgG decreased hERG membrane expression through inducing channel internalization. Digestion of anti‐Ro52 and isolation of the Fab and Fc fragments revealed that the Fc region of IgG is responsible for reducing hERG. Finally, we are currently testing the effects of IgG positive and negative autoimmune patient serum on the QTc interval of rabbits.

Conclusion

Our findings provide strong evidence that anti‐Ro52 IgG is responsible for autoimmune disease associated‐LQTS, which mechanistically occurs through a reduction of the hERG current due to IgG‐mediated channel internalization.

Support or Funding Information

Funded by CIHR and Heart and Stroke
Generating Hypertrophic Adipocytes from 3T3 L1 Cell Line and Characterizing the Phenotypic Transition
Authors: Yuan Xinxu , Xu Xiaoyang , Li Ningjun , Dewey William L. , Li Pin‐Lan , Zhang Fan
Published: November 2023

3T3 L1 is a classical preadipocyte cell line that has been extensively used for the study of adipogenesis, a physiological process giving rise to the adipocytes. Adipocytic hypertrophy, a pathological enlargement of adipocytes, underscores the occurrences of obesity‐associated metabolic syndrome. Currently, it is lack of a reliable cell model to simulate the hypertrophic transition of adipocytes. This study aims to develop adipocytic hypertrophy from 3T3 L1 and characterize the related phenotypic transition by examining the genes key to the pathophysiology of adipocytes as well as their functional outcomes. To generate hypertrophic adipocytes, we grew the post‐differentiated 3T3 L1 cells in DMEM/F12 medium (the starting date designated as day 0), supplemented with 10% FBS and 0.1% insulin, to their maturation approximately at day 10; and then continued loading lipids to the matured adipocytes by culturing these cells in the same media. The hypertrophic phenotype appeared around day 24. Morphologically, the further enlargement of lipid droplets during the hypertrophy was confirmed by Bodipy‐stained fluorescence images. Quantitative RT‐PCR analysis showed the expression of PPARγ, an adipogenesis‐promoting nuclear factor, and adiponectin, an adipogenetic indicator, were first progressively increased from day 0 to reach their maximum at the day 10, and then regressively declined to day 32, an end point in experimental observations. Consistent with the decline in adipogenesis, the mRNA levels of pro‐inflammatory factor MCP‐1 were markedly elevated in the hypertrophic adipocytes after day 24, as well, their MCP‐1‐containing culture media displayed significant macrophage‐attracting activity in cell migration assays. But the expression and activity of cytochrome C oxidase, a critical enzyme for energy generation, were reduced during the hypertrophy. Functionally, the viability of adipocytes, as determined by colorimetrically reading the formation of formazan from the metabolized tetrazolium, was gradually decreased after day 16. In situ TUNEL (Terminal Deoxynucleotidyl Transferase dUTP Nick End Labeling) analysis showed the adipocyte apoptosis was significantly increased during the hypertrophy. These results for the first time demonstrate that hypertrophic adipocytes can be generated from 3T3 L1 cell line. And this hypertrophic transition may be able to serve as a model to develop adipocytic hypertrophy‐targeting remedy in the treatment of obesity‐associated metabolic syndrome.

Support or Funding Information

Supported by NIH R01HL115068
Effects of Melatonin and Beta‐Amyloid on Mitochondrial Function in a Model of Aging Mouse Astrocytes
Authors: Debner Emily K , King Jonathan M
Published: November 2023

Background

Astrocytes provide support for neurons, however, with age, astrocytes’ ability to provide neuroprotection decreases. This decrease in neuroprotection increases the likelihood of neurodegenerative diseases, that compromise function. One hallmark of neurodegeneration is accumulation of β‐amyloid (βA) plaques, which disrupts cellular function and often leads to cellular death. As astrocytes age, they have been shown to naturally have lower mitochondrial membrane potential (MMP) and more frequent with larger amplitude calcium waves. Treatment with melatonin has been shown to decrease βA production and prevent plaque formation in the brain, suggesting melatonin plays a role during neurodegeneration. The interplay between melatonin and βA effects on astrocyte function was examined by measuring MMP and calcium waves.

Methods

Primary mouse astrocyte cultures obtained from young (4 month) and old (28 month) animals were used in the current study. Astrocytes were cultured for 24hrs with 100μM melatonin before loading with either MitoID or Fluo3. Measures were obtained using galvano or resonant scanning modes on a confocal microscope system, respectively. Astrocytes were then treated with 0.5μM βA (AggreSure) and imaged for an additional 30 minutes. MMP and calcium waves were analyzed to indicate cellular stress and change in cellular function.

Results

Melatonin significantly elevated baseline MMP in young astrocytes while protecting MMP loss due to βA exposure. Baseline MMP levels were unaffected by melatonin in old astrocytes although melatonin preserved MMP levels during βA exposure. This suggests young astrocytes capacity to alter cellular activities due to melatonin exposure is more robust. Calcium waves in young astrocytes persisted for a longer duration during βA exposure.

Conclusions

These results indicate that melatonin has protective effects on preserving MMP in the presence of βA, but produced greater effects in younger astrocytes. These data indicate effects are related to cellular vitality and not due to accumulation of βA.

Support or Funding Information

Support:

Trinity University Murchison Undergraduate Research Fellowship (EKD), Trinity University Biology Department and Neuroscience Program.
Novel Synthetic Cannabinoid AB‐PINACA Produces THC‐Like Behavioral and Cellular Effects Through activation of the Cannabinoid CB1 Receptor
Authors: Tai Sherrica , Crane Jasmine , Ford Benjamin M , Prather Paul L , Fantegrossi William E
Published: November 2023

Novel synthetic cannabinoid AB‐PINACA has been identified in herbal products marketed as K2/Spice. Limited information exists on the affinity, intrinsic efficacy, or acute and repeated in vivo effects elicited by AB‐PINACA. In in vitro receptor binding studies, AB‐PINACA displayed high affinity at the CB1R where it functioned as a full agonist. In the tetrad assay, mice injected with 3 mg/kg AB‐PINACA exhibited hypothermia, antinociception, catalepsy, and hypolocomotion, all of which are classical cannabinoid agonist effects. Importantly, these effects were attenuated by rimonabant, a CB1R antagonist/inverse agonist, suggesting that AB‐PINACA mediates these behavioral effects through activation of the CB1R. Mice previously prepared with radiotelemetry probes capable of simultaneously monitoring core body temperature and locomotor activity were repeatedly administered 3 mg/kg AB‐PINACA once every 24 hrs for 5‐days. Mice repeatedly administered AB‐PINACA developed rapid and progressive tolerance to hypothermic effects, and this tolerance was still evident when re‐challenged with the same dose after a 14‐day drug abstinence period. However, no tolerance developed to hypolocomotion. Lastly, mice were administered 30 mg/kg THC repeatedly for 4 days. We have previously shown that this dose regimen produces downregulation and desensitization of CB1Rs and elicits tolerance to cannabinoid induced hypothermia. On Day 5 these mice were challenged with 3 mg/kg AB‐PINACA to determine cross‐tolerance. These data demonstrate that – similar to other high‐efficacy synthetic cannabinoid drugs of abuse – the intrinsic efficacy, acute and repeated in vivo effects of AB‐PINACA are similar to those of THC, and are mediated through agonist interactions with CB1Rs.

Support or Funding Information

This work was supported by RR029884, RR020146 and UAMS Department of Pharmacology and Toxicology.
Behavioral Characterization of Spiradoline and CP55,940 in Rats: Potential of Drug Mixtures for Treating Pain
Authors: Minervini Vanessa , France Charles Patrick
Published: November 2023

Pain is a significant clinical problem and there is a need for drugs that are more effective with less adverse effects. Previous studies have shown that cannabinoid receptor agonists enhance the antinociceptive effects of mu opioid receptor agonists. It is unknown whether the well‐documented antinociceptive effects of kappa opioid receptor agonists are enhanced by cannabinoids and, therefore, whether kappa opioid receptor agonists, which are devoid of abuse liability, might be clinically useful in a drug mixture for treating pain. Cumulative dose‐effect functions (30‐min inter‐injection interval) were determined for the kappa opioid receptor agonist spiradoline (0.032 – 32.0 mg/kg) and the cannabinoid receptor agonist CP55,940 (0.0032 – 1.0 mg/kg) in 8 male Sprague Dawley rats for antinociception, schedule‐controlled behavior, body temperature, and diuresis. Spiradoline and CP55,940 dose dependently increased the latency for rats to remove their tails from warm (50°C) water to 100% of the maximum possible effect (20 s), while eliminating responding for food and decreasing body temperature by 5°C. CP55,940 was approximately 20‐ and 6‐fold more potent than spiradoline in the antinociception and the schedule‐controlled responding procedures, respectively. Spiradoline was 18‐fold and CP55,940 was 5‐fold more potent in the schedule‐controlled responding procedure than in the antinociception procedure. For both drugs, the rank order potencies across assays were schedule‐controlled responding > body temperature > antinociception. Spiradoline significantly increased (7‐fold) diuresis at doses that eliminated schedule‐controlled responding, whereas CP55,940 did not have a significant effect on urine output. These results provide the basis for examining mixtures of spiradoline and CP55,940 to test whether the preferred and potentially clinically useful effects of kappa opioid receptor agonists (antinociception) are selectively enhanced by cannabinoid receptor agonists.

Support or Funding Information

This work was supported, in part, by NIDA grants K05DA017918 and T32DA031115.
Daily Synthetic Cannabinoid Agonist (JWH‐018) Treatment: Tolerance to Δ9‐Tetrahydrocannabinol‐Like Discriminative Stimulus Effects in Rhesus Monkeys
Authors: Cocke Morgan L , McMahon Lance R
Published: November 2023

Abused synthetic cannabinoids are used repeatedly, yet the consequences of repeated treatment on sensitivity to abuse‐related effects have not been established. This study assessed sensitivity to the discriminative stimulus effects of JWH‐018 and Δ9‐tetrahydrocannabinol (Δ9‐THC) before and immediately after daily JWH‐018 treatment in rhesus monkeys. Rhesus monkeys discriminated Δ9‐THC (0.1 mg/kg i.v.) under an FR5 schedule of stimulus‐shock termination. Cumulative dose‐response functions for JWH‐018 and Δ9‐THC were determined before and the day after 7 days of twice daily treatment with 0.32 mg/kg of JWH‐018 s.c. 6 h apart. Drug discrimination sessions were suspended during daily JWH‐018 treatment. Δ9‐THC and JWH‐018 dose‐dependently increased Δ9‐THC lever responding; the respective ED50 values were 0.017 mg/kg and 0.0077 mg/kg. After daily JWH‐018 treatment, the ED50 values of JWH‐018 and Δ9‐THC were significantly increased, as evidenced by potency ratios (95% confidence limits) of 4.1 (2.1–8.1) and 6.6 (5.0–8.8), respectively. The magnitudes of increases in ED50 value were not significantly different between JWH‐018 and Δ9‐THC. Twice daily vehicle treatment for 7 days did not significantly alter sensitivity to JWH‐018, i.e., the potency ratio (95% confidence limits) comparing the ED50 value after vs. before vehicle treatment was 0.4 (0.2–1.2). Full substitution of JWH‐018 for Δ9‐THC, coupled with the similar tolerance to JWH‐018 and cross‐tolerance to Δ9‐THC as a function of daily JWH‐018 treatment, are consistent with a common pharmacological mechanism. Daily synthetic cannabinoid agonist use can result in tolerance and a likely need to increase the frequency and quantity of use to maintain subjective effects.

Support or Funding Information

USPHS grant R01 DA19222 and T32 NS082145
Comparison of Heroin‐ and Δ9‐Tetrahydrocannabinol‐Induced Antinociception and Physical Dependence in Monkeys
Authors: Ding Huiping , Kiguchi Norikazu , Kishioka Shiroh , Ko Mei‐Chuan
Published: November 2023

Both the opioid heroin and the cannabinoid Δ9‐Tetrahydrocannabinol (Δ9‐THC) exhibit analgesic effects, but both drugs produce undesirable abuse liability. Abuse of opioids and cannabis is a serious global problem that affects all societies on multiple levels. However, little is known about the development of physical dependence on these two classes of drugs in primates. Thus, the aim of this study was to compare the potency of heroin‐ and Δ9‐THC‐induced analgesic effects and the development of physical dependence on these two drugs in rhesus monkeys. Systemic administration of either heroin (0.03–0.18 mg/kg) or Δ9‐THC (0.3–1.8 mg/kg) dose‐dependently produced antinociceptive effects against acute thermal nociceptive stimulus. The opioid receptor antagonist naltrexone (0.01 mg/kg) and the cannabinoid receptor antagonist SR141716A (0.3 mg/kg) produced the same degree of a rightward shift of the dose‐response curve for heroin‐ and Δ9‐THC‐induced antinociception, respectively. To study acute physical dependence on heroin and Δ9‐THC, monkeys were subjected to a short‐term repeated administration (two injections per day for 2 to 3 days) of either heroin (0.18 mg/kg) or Δ9‐THC (1.8 mg/kg). Then, monkeys were treated with either naltrexone (0.01 mg/kg) or SR141716A (0.3 mg/kg), i.e., antagonist‐precipitated withdrawal. We found that surgically implanted telemetric probes (DSI, model D70‐PCTR) are sensitive in detecting physiological withdrawal signs. Administration of naltrexone increased heart rate and blood pressure in heroin‐treated monkeys, indicating a fast development of physical dependence on heroin. Depending upon the duration of repeated administration of Δ9‐THC and the dose of SR141716A, development of physical dependence on Δ9‐THC can be detected too. Collectively, these results demonstrate that in nonhuman primates, systemic heroin is more potent than Δ9‐THC in producing antinociception, and physical dependence on heroin can occur following a short‐term exposure.

Support or Funding Information

Supported by US‐PHS grants DA‐032568 and DA‐035359.
Impact of Chronic Ethanol Self‐Administration on Kappa Opioid Receptor Regulation of Dopamine Signaling in Nonhuman Primates
Authors: Siciliano Cody A , Calipari Erin S , Fordahl Steven C , Melchior James R , Yorgason Jordan T , Mateo Yolanda , Helms Christa M , Jimenez Vanessa A , Lovinger David M , Grant Kathleen A , Jones Sara R
Published: November 2023

Although alcoholism is one of the most prevalent disorders in the United States, with over 18 million individuals meeting the criteria for an alcohol use disorder, the exact neurobiological bases of this condition remain obscure. Recently, it has been demonstrated that kappa‐opioid receptor (KOR) signaling in the striatum plays a critical role in the increased reinforcing efficacy of ethanol following ethanol vapor exposure in rodent models. Here we examined the effects of chronic voluntary ethanol self‐administration in macaques on dopamine neurotransmission and the ability of KORs to regulate dopamine release in the nucleus accumbens core. Three cohorts of nonhuman primates were given free access to 4% ethanol (w/v) for 22 hr/day. These cohorts were composed of male cynomolgus, female rhesus or male rhesus macaques, and were given access to ethanol for 6, 12, or 18 months, respectively. Ex vivo fast‐scan cyclic voltammetry was then conducted the nucleus accumbens core to determine dopamine signaling kinetics as well as the ability of U50,488 (KOR agonist) to inhibit dopamine release. We found that chronic ethanol drinking increased dopamine uptake rates, which could have implications for reductions in basal dopamine tone in vivo during ethanol withdrawal. Further, across sex, strain and exposure length, ethanol use augmented the ability of KORs to inhibit dopamine release, demonstrating that ethanol‐induced increases in KOR sensitivity are widespread and independent of other factors. Finally, KOR sensitivity was positively correlated with lifetime ethanol intake, suggesting that changes in KOR regulation of dopamine release may be a determinant of aberrant drinking behaviors. Nonhuman primate models of ethanol abuse represent a highly translational avenue for identifying molecular targets for pharmacotherapeutic compounds, and here we show, for the first time, that voluntary ethanol self‐administration has a unique effect on KOR sensitivity and regulation of dopamine release directly at the dopamine terminal that was positively correlated with drinking behavior. Together, these data provide novel insight into ethanol‐induced dysregulation of opioid signaling and suggest that dopaminergic dysfunction may be mediating increases in voluntary drinking. Importantly, KOR antagonists may be efficacious in reducing drinking behaviors in alcoholics.

Support or Funding Information

This work was funded by NIH grants U01 AA014091, P01 AA021099 (SRJ), F31 DA031533 (ESC), F31 DA037710, T32 AA007565 (CAS), F31 AA020439 (JTY), P51 OD011092, R24 AA019431, P60 AA10760 (KAG), Division of Intramural Clinical and Biomedical Research NIAAA (DML), Integrative Neuroscience Initiative on Alcoholism AA 13510 (KAG).
The Serotonin 2A Receptor Agonist 2,5‐dimethoxy‐4‐iodoamphetamine (DOI) Reduces Ethanol Consumption and Place Conditioning
Authors: Murnane Kevin Sean , Nakhla Peter , Curry Kristen , Phillips John , Daphney Cedrick
Published: November 2023

17 million adults in the United States ages 18 and older had an alcohol use disorder (AUD) in 2012. Current FDA‐approved treatments for AUD are limited to disulfiram, naltrexone, and acamprosate, and many patients are refractory to these treatments. Hallucinogenic drugs were used in the 1960s to treat AUD, and a number of studies supporting their use were published. Moreover, recent studies have documented impressive effects of psilocybin for tobacco cessation and ayahuasca for the prevention of ethanol‐induced behavioral sensitization. Therefore, herein, we investigated the effects of the serotonin 2A receptor agonist and hallucinogen 2,5‐dimethoxy‐4‐iodoamphetamine (DOI) in ethanol‐induced place conditioning and 2‐bottle choice ethanol consumption in mice, 2 widely accepted animal models of ethanol abuse. We found that DOI pretreatment prevented the development of an ethanol‐induced place preference, and moreover, DOI treatment after ethanol prevented the consolidation of such conditioning. DOI treatment robustly decreased ethanol consumption without affecting total fluid intake, and interestingly, this effect was most profound in subjects that exhibited high ethanol consumption. As there are clear clinical limitations to the use of hallucinogenic drugs, we went on to explore whether computational chemistry approaches could be used to develop biased agonists of serotonin 2A receptors with reduced hallucinogenic activity, and whether such compounds suppressed ethanol consumption. These studies document a new model system in which we can now investigate the neurobiological and pharmacological mechanisms through which hallucinogens reduce ethanol consumption and appetitive conditioning.

Support or Funding Information

Support was provided by Mercer University and the Mercer University College of Pharmacy.
Tianeptine Co‐administration with Alcohol to Adolescent Rats Modifies the Alcohol Withdrawal Syndrome
Authors: Rhoads Dennis E. , Martin Bryan , Mauterer Madelyn , Orlando Krystal
Published: November 2023

Adolescence is a time when many individuals have their first experiences with alcohol and other drugs. It is a time when the brain undergoes important developmental changes that may be altered by drug use. We have shown previously that adolescent Long‐Evans rats consume high levels of an alcohol‐containing liquid diet, achieve correspondingly high blood alcohol concentrations, and develop a subsequent severe alcohol withdrawal syndrome. Using adult rats, tianeptine has been shown by other groups to decrease anxiety‐like behaviors and the onset and severity of convulsions during alcohol withdrawal. The present study was designed to test the effectiveness of tianeptine against the severe alcohol withdrawal syndrome seen with adolescent rats and to test the effects of tianeptine on upregulation of glutamate receptors during adolescent alcohol consumption. Liquid diets were used to administer alcohol and tianeptine, alone and in combination, using a modified chronic intermittent design. After 10 days of co‐administration of tianeptine and alcohol and 6 hours of withdrawal, motor activity showed little or none of the hypoactivity characteristic of alcohol withdrawal. Tianeptine alone had no effect on motor activity under these conditions. However, continued out to 21 days of co‐administration, adolescent rats showed the same high frequency of induced running episodes and convulsions during withdrawal as when alcohol was administered alone. Western blotting was used to determine expression levels for forebrain glutamate receptors, AMPA and NMDA. Both receptors were upregulated during chronic alcohol consumption. During co‐administration with alcohol, tianeptine delayed this upregulation so that at 10 days there was no significant upregulation of AMPA or NMDA whereas significant increases in expression were observed by 21 days. These results indicate that tianeptine may dampen symptoms of adolescent alcohol withdrawal in the short term by delaying the upregulation of excitatory glutamate receptors induced by chronic alcohol. However, tianeptine was ineffective in maintaining this delay and in preventing more severe withdrawal associated with longer term alcohol consumption. Thus, we conclude that tianeptine may be of limited effectiveness against alcohol withdrawal severity in adolescents compared to that suggested previously for adult rats.

Support or Funding Information

This work was supported by the Biology Department and School of Science of Monmouth University.
Role of extracellular matrix protein fibronectin‐integrin signaling in the hippocampal dentate gyrus in epilepsy
Authors: Wu Xin , Clossen Bryan , Muthuchamy Mariappan , Reddy Doodipala
Published: November 2023

Epilepsy affects ~3 million people in the United States alone. There is no specific drug for curing epilepsy or preventing epileptogenesis, a process whereby a normal brain becomes progressively epileptic due to precipitating factors. Integrins as membrane receptors on neurons play an important role in the development of neuroplasticity in the brain. Integrin recruitment and relocalization, and extracellular matrix (ECM) protein‐integrin attachments have been reported to dynamically change their position during cell spreading and migration. We propose that ECM protein fibronectin‐integrin interaction directly affects epileptogenesis, as well as fibronectin‐integrin bond formation and cell elasticity (remodeling) are altered in epilepsy. Here, we tested this hypothesis in adult mouse hippocampal dentate granule cells (DGCs) in the kindling model of epilepsy by direct intra‐hippocampal drug infusion and immunocytochemistry, and nanoscale atomic force microscopy (AFM). The fibronectin receptor α5β1‐integrin staining intensity in DGCs from epilepsy mice was about 22% stronger than in control. Mice were treated after kindling stage 1–2 with intra‐hippocampal injection of β1‐integrin monoclonal antibody (mAb, 100 μg/ml). The normalized electrographic after discharge threshold was 2.25‐fold higher than control group. The duration of seizure behavior and the duration of electrographic afterdischarge were inhibited by 23% and 20% after β1‐integrin application, respectively (p<0.05). In AFM study, the unbinding force (25.6 ± 0.2 pN) and binding probability between fibronectin‐coated AFM probe and membrane integrins in epilepsy DGCs were 49% and 18% lower than control, respectively (p<0.05). Moreover, the cell elasticity, partially representing changes in cytoskeletal reorganization, was 66% increased in epilepsy. Treatment with α5β1‐integrin mAb significantly reversed this membrane dysfunction. These results indicate that fibronectin‐adhesive activities directly modulate development of epilepsy, and are functional dynamic and primarily bound to α5β1 integrin. Integrin signaling pathway may offer new targets for the prevention or treatment of epilepsy.

Support or Funding Information

(1) 2014–2015, Special Seed grant from Dr. George C.Y. Chiou and the Institute of Ocular Pharmacology, Texas A&M Health Science Center (Xin Wu ‐ PI). (2) 2013–2018, CounterACT Program, NIH, NINDS U01 NS083460 (Doodipala Reddy ‐ PI)
Neurotensin Depresses GABA Type‐B Receptor‐Mediated Neurotransmission in the Substantia Nigra through Pre‐ and Postsynaptic Mechanisms
Authors: Tschumi Christopher W , Beckstead Michael J
Published: November 2023

Midbrain dopamine neurons play physiological roles in many processes including reward learning and motivated behavior. Conversely, dysfunction of these neurons is implicated in disorders such as schizophrenia and drug abuse. Midbrain dopamine neurons are tonically inhibited by γ‐Aminobutyric acid (GABA)ergic inputs from multiple brain regions. Neurotensin (NT) is a neuropeptide which modulates midbrain dopamine neuron excitability through multiple mechanisms, including a decrease of GABA mediated inhibition of dopamine neurons. However, it is not known if NT acts post‐synaptically on GABA type‐B receptor signaling, pre‐synaptically at GABA terminals to alter GABA release, or through a combination of synaptic mechanisms. Here we utilize whole cell patch‐clamp electrophysiology of dopamine neurons in brain slices to show that neurotensin acts both pre‐ and post synaptically to decreases GABA type‐B receptor‐mediated currents in the substantia nigra in mice. Bath perfusion of neurotensin produced a sustained depression of GABA type‐B receptor‐mediated currents that was more pronounced when GABA was released endogenously (due to electrical stimulation) compared to when GABA was directly applied to the cell (via iontophoresis). GABA type‐B receptor mediated currents exhibited paired pulse depression when currents were elicited with electrical stimulation, but not GABA iontophoresis. Bath application of neurotensin increased the paired pulse ratio with electrical stimulation but had no effect on paired pulse currents elicited by GABA iontophoresis, providing further evidence neurotensin modulates GABA release. As NT is an endogenous peptide present at high levels in the midbrain, determining the mechanism of action by which NT alters midbrain dopamine neuron excitability is a crucial step in understanding the importance of NT in dopamine mediated behavior and related disorders.

Support or Funding Information

Support provided by NIH R01 DA 32701
CANNABINOIDS ARE NEUROPROTECTIVE AGAINST HIV‐1 TAT AND OCCLUDE THE TAT‐INDUCED DECREASE IN GABAERGIC NEUROTRANSMISSION IN PREFRONTAL CORTEX SLICES
Authors: Xu Changqing , Hermes Douglas J , Mackie Ken , Lichtman Aron H , Ignatowska‐Jankowska Bogna M , Fitting Sylvia
Published: November 2023

In the era of combined antiretroviral therapy (cART), human immunodeficiency virus type 1 (HIV‐1) is now considered a chronic disease that specifically targets the brain and causes a high prevalence of mild forms of neurocognitive impairments, also referred to as HIV‐associated neurocognitive disorders (HAND). HAND is due in part to defects in synaptic neurotransmission that leads to synaptodendritic injury, including damage to synapses and dendrites. Endocannabinoids have been suggested to protect central nervous system (CNS) neurons in different disease models of toxicity, but their effect in HAND are poorly known. To address this issue primary prefrontal cortex (PFC) neuron culture studies examined the neuroprotective effects of the synthetic cannabinoid WIN55,212‐2 and the endocannabinoid N‐arachidonoyl ethanolamine (anandamide/AEA) on HIV‐1 Tat excitotoxicity and neuronal survival. Data demonstrated that WIN55,212‐2 and AEA are protective against HIV‐1 Tat‐induced increases in [Ca2+]i as well as neuron injury and cell death via a cannabinoid 1 receptor (CB1R)‐mediated mechanism. Further, whole‐cell patch‐clamp recordings were performed on PFC slices to assess cannabinoid effects on GABAergic neurotransmission in the presence of HIV‐1 Tat. Results indicated a HIV‐1 Tat concentration dependent (5 – 50 nM) decrease in the frequency and amplitude of miniature inhibitory postsynaptic currents (mIPSCs) but not spontaneous IPSCs (sIPSCs). Zero external calcium and cadium chloride (CdCl2, 200 μM), which blocks high and low threshold voltage‐dependent calcium currents, prevented the significant Tat‐induced decrease in mIPSCs. Interestingly, bath‐applied WIN55,212‐2 or AEA that significantly decreased the frequency but not amplitude of mIPSCs and/or spontaneous IPSCs (sIPSCs) blocked a further down‐regulation of IPSCs by Tat. Lastly, pretreatment with SR141716A, a CB1R antagonist, prevented the AEA‐induced decrease in IPSCs frequency without any further Tat effect. These results suggest that Tat significantly decreases GABAergic neurotransmission at the level of an action potential‐independent release of GABA due to the influx of extracellular calcium, partially via voltage‐dependent calcium currents. Additionally, bath application of cannabinoids following or prior Tat treatment occluded the Tat‐induced decrease in GABA release, with cannabinoids acting via a CB1R‐related mechanism. Understanding the effects of cannabinoids in the context of neuro AIDS may uncover novel therapeutic targets for HAND and other diseases in which cognitive deficits occur.

Support or Funding Information

NIDA R00 DA033878, T32 DA007244
Long term administration of methamphetamine in Tat mice causes altered behavior and neuroplasticity gene expression – Implications in neuroAIDS
Authors: Nookala Anantha Ram , Kumar Anil
Published: November 2023

Methamphetamine (MA) abuse is common among individuals infected with HIV‐1 and is shown to affect the disease progression towards AIDS. These HIV‐1 infected individual's exhibit greater neuronal injury and higher cognitive decline. Several HIV‐1 proteins, specifically gp120 and HIV‐1 Tat have been shown to affect neurocognition. HIV‐1 Tat, a viral protein released early during HIV‐1 replication has been implicated in HIV‐associated neurotoxicity mediated through production of pro‐inflammatory cytokines, reactive oxygen species and dysregulation of neuroplasticity among many other factors in the central nervous system. However, combined effect of methamphetamine and HIV‐1 Tat on the alteration of locomotor function, anxiety, memory and their correlation to neuroplasticity gene expression is not well documented. The present study was undertaken to determine the combined effect of methamphetamine and HIV‐1 Tat on behavior and neuroplasticity gene expression. Doxycycline (DOX)‐inducible HIV‐1 Tat (1–86) transgenic mice were administered 6mg/kg MA twice a day. Expression of Tat in various brain regions was confirmed by RT‐PCR. Behavior studies were performed in both males and female mice after 2 weeks of MA administration. As measured by open field assay, the ambulatory activity was decreased in Tat mice. However, administration of MA increased the ambulatory activity in both Tat −ve mice and Tat +ve mice. MA administered mice have showed decreased anxiety levels as assessed by the time spent in the light compartment during Light/dark box assay. In water maze task, there was a significant increase in escape latency in all the groups compared to control mice, particularly more in Tat transgenic mice that were administered MA. We looked at the alterations in behavior with correlation to neuroplasticity gene expression. We observed a significant decrease in the protein expression of CNTF, Lif in entorhinal cortex and prefrontal cortex. Furthermore, we observed decrease in mRNA expression of CNTF, IL‐11 and BDNF in parietal cortex. This study therefore provides novel insights into the interaction of HIV‐1 Tat and MA on the dysregulated expression of various neuroplasticity genes in different brain regions.

Support or Funding Information

The work was supported by National Institute on Drug Abuse grants DA025528 & DA025011.
A multi‐herbal extract HT047 promotes long‐term functional recovery after focal cerebral ischemia in rats
Authors: Song Jungbin , Lee Donghun , Kim Young‐Sik , Kim Hyun Soo , Lee Hyun Jeong , Lee Seunggyeong , Lee Sung Hyun , Kim Hocheol
Published: November 2023

HT047 is a multi‐herbal extract consisting of Pueraria lobata root and Scutellaria baicalensis root which have been used to treat stroke in traditional Korean medicine. Since 2000, we screened more than 200 medicinal herb extracts for neuroprotective activities in animal stroke models. Pueraria lobata root and Scutellaria baicalensis root were identified as the most potent extracts in this screen and neuroprotective activities of individual herb were further enhanced by the cotreatment. Recently, bioactive constituents of HT047 have been reported to increase the expression of trophic factors in ischemic brain such as vascular endothelial growth factor (VEGF) and brain‐derived neurotrophic factor (BDNF) that are crucial for enhancing neurorestoration, leading to functional recovery after stroke. In the present study, we investigated the effects of delayed HT047 treatment on long‐term motor recovery after cerebral ischemia in rats. Sprague‐Dawley rats were subjected to 60 min of middle cerebral artery occlusion (MCAO). HT047 was orally administered to rats for consecutive 14 days at a dose of 300 mg/kg starting 24 h after MCAO and then rats were received chow diet containing 1.35 % HT047 up to 6 months. Long‐term functional outcomes were assessed by the rotarod test and the forelimb placing test during subacute (weekly intervals for one month) and recovery (monthly intervals up to 6 months) phases. HT047 accelerated recovery of rotarod performance during a subacute period and protected age‐related decline during a chronic recovery phase. The forelimb placing scores of HT047 group were significantly higher than those of control group during the whole testing period. These findings indicate that delayed HT047 treatment initiated at 24 h after stroke accelerates the recovery of motor function followed by sustained enhancement during a recovery phase. The enhancement of neurorestorative events including angiogenesis, dendritic branching, synaptogenesis, and axonal sprouting may contribute to the therapeutic benefits of HT047 in the treatment of stroke.

Support or Funding Information

This study was supported by a grant of the Korean Health Technology R&D Project, Ministry of Health & Welfare, Republic of Korea (HI15C0051).
The α2 Adrenergic Receptor as a Novel Target for Alzheimer's Disease
Authors: Gannon Mary , Peng Yin , Jiao Kai , Wang Qin
Published: November 2023

Alzheimer's disease (AD) is the most common form of dementia, and is the only cause of death within the top 10 in America that cannot be prevented, cured, or slowed. Alterations of the noradrenergic (NA) system occur early in AD. However, the relevance of NA dysfuntion to AD pathogenesis and the therapeutic potential of NA components for AD are unknown. We have previously shown that activation of the α2 adrenergic receptor (α2AR) increases amyloidogenic processing of amyloid precursor protein (APP), and therefore amyloid β (Aβ) load in the brain. In this study, we sought to investigate α2AR expression and activity with AD advancement, and test whether a blockade of the α2AR slows AD progression using an AD transgenic mouse model. We examined α2AR coupling to G‐protein by use of a [35S]GTPγS radioligand binding assay in both 5 week and 7.5 month old APP/PS1 mice and their nontransgenic littermates. At the earlier time point, prior to AD pathology, no difference was found between APP/PS1 and nontransgenic mice. However, at 7.5 months, after AD pathology is profound, increased α2AR‐G protein coupling efficiency was observed in APP/PS1 mice, suggesting that α2AR function is enhanced as AD progresses. Furthermore, we treated 8 month‐old APP/PS1 mice with an α2AR antagonist, idazoxan, for 8 weeks and evaluated their neuropathology and cognitive behaviors. Compared to saline treatment, treatment with idazoxan reduced Aβ load in both the hippocampus and cortex, and, more importantly, improved cognitive performance of APP/PS1 mice on the Morris water maze. In conclusion, our studies suggest that α2AR activity is upregulated in APP/PS1 mice with pathological progression and that a clinical α2AAR antagonist is effective in decreasing Aβ plaque load and improving cognition, even when started after the accumulation of Aβ plaques. Therefore, targeting the α2AR represents a promising therapeutic strategy for AD.

Support or Funding Information

This study is supported by NIH Grant AG042716 (to Q.W.).
Correcting memory deficits in Fragile X syndrome by targeting Rac1/PAK1 signaling
Authors: Martinez Luis , Tejada‐Simon Maria Victoria
Published: November 2023

Cognitive impairment in neurodevelopmental disorders like Fragile X syndrome (FXS) is thought to be due to abnormal neuroplasticity, altering synaptic connections. These synaptic modifications require reorganization of the actin cytoskeleton within dendritic spines, a dynamic process that is known to be regulated by the Rho family of small GTPases. In a mouse model of FXS we found increased levels of membrane‐bound small GTPase Rac1 in the hippocampus, a brain structure involved in learning. The FXS mouse hippocampus also exhibits impaired synaptic plasticity and morphologically distorted dendritic spines. In an effort to define whether small GTPases have a critical role in FXS‐associated learning deficiencies, Rac1 was pharmacologically targeted using novel agents that prevent its activation and membrane translocation. Mice treated with Rac1 inhibitors were subjected to behavioral tasks that examine contextual and cued memory. Performance on these tasks is dependent on the neuronal integrity of the hippocampus. Our results showed that FXS mice treated with the inhibitors improved their learning and memory skills. These findings suggest that Rac1 hyperactivity and membrane distribution may play an important, role in FXS and autism‐related molecular pathology contributing to cognitive complications.