bluuebunny/crossref_metadata_2025 huggingface dataset:
Activation of Mitochondrial Calpains Contributes to the Selective Degradation of Specific Mitochondrial Proteins
Authors: Chen Qun , Thompson Jeremy , Hu Ying , Hollander John M. , Lesnefsky Edward J.
Published: June 2021

INTRODUCTION

Calpain 1 (CPN1) and 2 (CPN2) are ubiquitous calpains that exist in both cytosol and mitochondria. Calpains are calcium‐activated proteases. Pharmacologic inhibition of CPN1 and CPN2 decreases cardiac injury by improving mitochondrial function during ischemia‐reperfusion. Since both CPN1 and CPN2 include one large subunit and a small regulatory subunit (calpain 4, CPN4), genetic removal of CPN4 eliminates the activities of CPN1 and CPN2. CPN4 knockout mice were used in this study to test if activation of mitochondrial calpains leads to mitochondrial damage by proteolysis of mitochondrial proteins.

HYPOTHESIS

We hypothesize that exogenous calcium‐mediated activation of mitochondrial CPN1 and CPN2 impairs mitochondrial function through degradation of mitochondrial proteins.

METHODS

Purified cardiac mitochondria were isolated from wild type and CPN4 knockout mice. Exogenous calcium (25 uM) was used to activate mitochondrial CPN1 and CPN2. Mitochondria were subjected to proteomic analysis using tryptic digestion, liquid chromatography, and mass spectrometry with iTRAQ quantification.

RESULTS

We focused on the proteins that were decreased by exogenous calcium exposure in mitochondria from wild type but not in CPN4 knockout mice as specific targets of mitochondrial CPN1 and CPN2. In wild type mice, calcium treatment significantly decreased the contents of multiple metabolic enzymes involving fatty acid oxidation and oxidative phosphorylation (electron transport chain components and ATP synthase) compared to non‐calcium treated mitochondria. Knockout of CPN4 preserved these enzyme contents in calcium‐treated mitochondria (Table). Decreases in ATPase factor 6 and very long chain acyl‐coA dehydrogenase were confirmed by Western blot.

CONCLUSION

Activation of mitochondrial calpains leads to decreased mitochondrial function by impairing metabolic enzyme activity. Prevention of CPN1 and CPN2 activation provides an attractive approach to protect mitochondria during pathologic conditions including ischemia‐reperfusion and heart failure.

Support or Funding Information

This work was supported by the Office of Research and Development, Medical Research Service Merit Review Award (2IO1BX001355‐01A2) and Department of Veterans Affairs (EJL); National Institutes of Health NIH R21AG054975‐01 (QC).

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Morphometric Evaluation of Gross Pulmonary Indices in the Red Sokoto Goats for Pulmonary Consolidation Assessment
Authors: Alaka Olugbenga Olayinka , Unuoah Precious Adetokunbo , Jarikre Theophillus Aghogho , Emipke Benjamin Obukowho
Published: June 2021

Goats are widely adapted to different climates and are found in all production systems. However, very little is known on the pulmonary morphometry of indigenous goats in Africa. The study aims to determine the standard morphometry and dimensions of the Red Sokoto RS goat lungs, which may serve as basis for clinical and radiographic assessment of pulmonary consolidation. The goats were sourced from a Municipal abattoir considering age and sex. A total of 35 non‐pneumonic RS lungs were collected including 22 males and 8 females, 6 between <1 years, 15 between 1–2 years and 9 > 2 years. Weight (gram), length (cm) and diameter (cm) of each lung lobe, distance from trachea bronchus to point of trachea bifurcation, distance between left and right trachea bifurcation were taken using standard measurements. The right lung contributes the most (61.67%) while the left lung contributes the least weight (38.32%). Of the 61.67% contributed by the right lung, the caudal lobe contributes 38.9% while the cranial (14.71%), middle (5.83%) and accessory (1.53%) lobes makes up the remaining. It was also discovered that the caudal lobe of the right lung contributes more than the left lung. The percentage contribution of right lung lobes increased with increase in age except for the accessory lobe which decreased with increasing age. The percentage contribution of the left lung lobes decreased with increasing age except for the middle lobe which was increasing with age. The right lung is longer (6.70±0.57) than the left lung (13.40±0.82). The right cranial lobe is longer and wider than the left cranial lobe. The right middle lobe is insignificantly longer (1.20±0.57) than the left (1.10±0.22) but significantly less wide (6.7±0.57) than the left (7.72±1.14). The right caudal lobe is longer (8.76±0.25) than the left (8.50±0.50) but less wider (4.86±0.20) than the left (5.5±0.61). The males have higher weight and dimensions than females except that the values of the right lung, the caudal lobe of the right lung and the middle lobe of the left lung are higher in females than in males. In conclusion, this study established the standard as regarding the normal size of each lung lobe and lobule in relation to the age and sex of the RS goat. These parameters will aid diagnosis of lung disease and assessment of pulmonary consolidation at postmortem in a poor resource setting.

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Inhibition of Src Reduces Cortactin Phosphorylation and Transendothelial Migration of Acute Lymphoblastic Leukemia B cells
Authors: Camacho Karina Elizabeth Jimenez , Martinez Ramon Castellanos , Diaz Paola Briseño , Sevilla Jose Luis Flores , Schnoor Michael
Published: June 2021

B‐cell acute lymphoblastic leukemia (B‐ALL) is the most common leukemia in children and adults. B‐ALL cells have the ability to extravasate and infiltrate bone marrow and organs such as lungs, testis and central nervous system (CNS). Dynamic actin cytoskeletal remodeling is needed to accomplish transendothelial migration. Cortactin is an actin‐binding protein which accumulates in lamellipodia and invadopodia and regulates neutrophil transendothelial migration during inflammation. Recently, we showed that cortactin is overexpressed in B‐cells of B‐ALL patients, and significantly correlates with bone marrow colonization, organ infiltration, drug resistance and disease relapse. Cortactin is a target for several posttranslational modifications such as phosphorylation and acetylation, being first discovered as a substrate of Src kinase and recently as substrate of the histone deacetylases SIRT1 and HDAC6. Phosphorylation of cortactin is thought to lead to lamellipodia and invadopodia protusion, whereas acetylation diminishes the affinity of cortactin to F‐actin, decreases cell migration capability, and may lead to nuclear translocation. However, it remains to be explored whether these modifications are required for the aggressiveness of leukemic B‐ALL cells. Thus, in this study, we analyzed the importance of such modifications for transendothelial migration and bone marrow organoid colonization. A MTT (3‐(4, 5‐dimethylthiazolyl‐2)‐2, 5‐diphenyltetrazolium bromide) assay was performed to determine effects of certain pharmacological inhibitors of posttranslational modifications on cell viability. To this end, REH cells were incubated with the indicated pharmacological compounds for 24 hours at different concentrations (50 to 150 mM). The IC50 value for PP2 (Src inhibitor) was 190.82 mM, for EX‐527 (SIRT1 inhibitor) was 190.94 mM, for Tubacin (HDAC6 inhibitor) was 41.39 mM, and for Tubastatin‐A (HDAC6 inhibitor) was 94.09 mM. Importantly, after 20 min of treatment of only REH cells with PP2 at 20 μM, REH cells transmigrated significantly less across CXCL12‐stimulated HUVEC monolayers, which may be due to a reduction of phosphorylated cortactin that impairs cytoskeletal remodeling needed for migration of the cells. Taken together, these results suggest that cortactin phosphorylation may be needed by leukemic B cells to achieve transmigration and may reveal cortactin as a potential new therapeutic target for high risk B‐ALL patients to prevent disease relapse.

Support or Funding Information

CONACyT

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Association between Cadmium Air Exposure and Prostate Cancer Stage at Diagnosis
Authors: Vijayakumar Vish , Jagai Jyotsna S. , Abern Michael R. , Kajdacsy‐Balla Andre
Published: June 2021

Introduction

There is clear evidence that high levels of occupational exposure to cadmium (Cd) lead to a more aggressive prostate cancer (PC) tumor. At levels of exposure which are more relevant to the general population, there is disputing evidence as to whether Cd causes a PC tumor to become more aggressive. In this study, we assess the relationship between ambient exposure to air Cd and PC stage at diagnosis.

Methods

We collected outcome data from the Surveillance, Epidemiology, and End Results (SEER) for reported PC cases diagnosed from 2010 to 2014. PC stage at diagnosis was categorized as metastatic or localized. Exposure data was collected from the 2011 National Air Toxics Assessment (NATA) which was developed by the EPA to review air toxics in the United States. The total exposure concentration of Cd compounds in the air was a county‐level metric calculated by the EPA using weighted averages of air Cd concentration levels measured at various point emission sources. We calculated quintiles of Cd exposure concentration for ease of interpretation. Odds Ratios (OR) and 95% confidence intervals (CI) assessing the relationship between county‐level air Cd exposure and PC stage at diagnosis were calculated using multivariable logistic regression models. Analyses were adjusted for age at diagnosis, sociodemographic status, and overall air quality and were stratified by race.

Results

The study cohort consisted of 234,991 PC cases from 594 counties reported by SEER. Of these cases, 92.5% were localized and 7.5% metastatic at diagnosis. Higher levels of air Cd exposure were associated with an increased likelihood of a PC case being metastatic at diagnosis. The adjusted odds ratios for the 80th percentile vs 20th percentile of Cd exposure were: all races (OR 1.030, CI 1.019 – 1.040), White (1.049, CI 1.037 – 1.062), Black (1.032, CI 1.0072 – 1.057), and other races (0.965, CI 0.925 – 1.0062).

Conclusion

Higher Cd air exposure concentration was associated with advanced PC stage at diagnosis with effects that vary somewhat by race. Since inhalation of Cd in the air only contributes to a small percentage of total Cd intake, further studies should be conducted to investigate the effects of other forms of Cd intake such as dietary and water exposures on PC aggressiveness.

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Effect of Beta‐amyloid on cell death‐related molecules: Is the co‐chaperone BAG2 a potential target for Alzheimer's disease?
Authors: Carrettiero Daniel Carneiro , Fardin Nubia M , do Carmo Ramon Torreglosa , Almeida Maria Camila
Published: June 2021

Alzheimer's disease is characterized by abnormal accumulation of extracellular beta‐amyloid plaques (Aβ). Aβ promotes cell death and NF‐κB signaling activation. Because BAG2 is repressed by NF‐κB signaling, the aim of the present study was to investigate the effect of Aβ/BAG2 on cell death‐related molecules such as NF‐κBp65, BAX, Bcl‐2, and Bcl‐3 in hippocampal primary cell culture and in differentiated and non‐differentiated SH‐SY5Y cells. In addition, we also investigate the effect of Aβ/BAG2 on NFκB1, NFκB2, RELA, and RELB gene expression. Western blotting and qPCR were used in the present project after Aβ stimulation with BAG2 overexpression or BAG2 knockdown.

Aβ toxicity was increased in BAG2 overexpressing cells and was decreased after mRNA BAG2 knockdown. Aβ treatment promotes an increase in pNF‐kBp65/NF‐kBp65 suggesting nuclear translocation of this molecule and possible gene activation independently of BAG2. Pro‐apoptotic BAX and anti‐apoptotic Bcl‐2 proteins and mRNA were analyzed in Aβ‐stimulated cells with BAG2 knockdown. Aβ treatment resulted in an increase in BAX which was reversed in mRNA BAG2 knockdown cells, but no significant effect was detected in Bcl‐2 or Bcl‐3 levels. The effect of Aβ in differentiated compared to non‐differentiated SH‐SY5Y cells were distinct. Aβ treatment, in differentiated SH‐SY5Y cells, resulted in an increase of NFKB1, NFKB2, and RELA which was reversed in mRNA BAG2 knockdown cells. No significant effect was detected in RELB levels. On the other hand, in non‐differentiated SH‐SY5Y cells, Aβ treatment resulted in a decrease in NFκB1, NFκB2 and increase in RELB which was reversed in mRNA BAG2 knockdown cells.

Our data suggests BAG2 an important molecule related to Aβ toxicity and NF‐κB signaling activation. BAG2 increased Aβ toxicity, probably interacting with the transcription factor NF‐κB in the NF‐κB nuclear translocation, and resulted in activation of pro‐apoptotic BAX leading to the expression of several cell death‐related molecules. Nevertheless, this effect seems to be differentially activated in differentiated and non‐differentiated SH‐SY5Y cells.

Support or Funding Information

FAPESP (grant 2015/23426‐9)

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Latent‐HIV‐1 Exosome Blockade Mitophagy Flux in Human Brain Microvascular Endothelial Cells
Authors: Chandra Partha K , Rutkai Ibolya , Kim Hogyoung , Braun Stephen E , Abdel‐Mageed Asim B , Mondal Debasis , Busija David W
Published: June 2021

Background

Insufficient removal of dysfunctional mitochondria, due to a defective mitophagy pathway, leads to increased ROS, oxidative stress, and neuroinflammation which triggers the pathogenesis of various neurodegenerative diseases. HIV‐1‐associated neurocognitive disorders (HAND) is a common disease with variable reported prevalence in different populations (20– 69%). We investigated how exosomes from latent‐HIV‐1 infected cells impaired mitophagy flux in the human brain microvascular endothelial cells (HBMVEC).

Methods

Exosomes were isolated by ultracentrifugation from latent‐HIV‐1 infected T‐cells [exo‐J Lat(9.2)] and monocytes (exo‐U1) and characterized by the qNano‐IZON and NanoSight 300 systems by measuring the concentrations (3.2×1012 particles/mL), particle diameter (Mean: 100.5±28.8 nm), and size‐distribution (Mode: 75.5±3.5 nm) of exosomes from J Lat(9.2). To determine whether exposure to exo‐J Lat(9.2) could induce mitophagy, HBMVECs were exposed to varying doses of HIV‐exosomes (10, 25, 50 and 100 μg/mL) for 24 and 48 h, after which the expression level of mitophagy markers PINK1, Parkin, and Drp1 were determined using western blotting. Carbonyl cyanide 4‐(trifluoromethoxy) phenylhydrazone (FCCP; 5 μM for 3 h), a known mitophagy inducer, was used as a positive control.

Results

Mitophagy is a two‐step process by which damaged mitochondria are first primed by PINK1/Parkin proteins and then eliminated via the autophagic machinery. We then examined the expression levels of autophagy (macroautophagy) proteins: the autophagosome initiation marker (Beclin 1), autophagosome formation marker (LC3B‐II), and autophagy degradation marker (SQSTM1 or p62) in HBMVECs exposed to latent‐HIV‐1‐exosomes. Interestingly, following exposure of HBMVECs to exo‐J Lat(9.2), the expression level of PINK1/Parkin proteins were upregulated in a dose dependent manner, with a concomitant increase in the formation of autophagosomes, shown by increased expression of Beclin 1 and LC3B‐II. FCCP treated cells showed decreased expression of p62 indicated by the active presence of mitophagy flux. Remarkably, cell exposure to HIV‐exosome resulted in increased expression of p62, indicating a possible blockage of the mitophagy flux, leading to the accumulation of mitophagosomes.

Conclusions

Our preliminary study indicated that latent‐HIV‐1 exosome increased mitochondrial damage via defective mitophagy.

Support or Funding Information

Grant Numbers: U54 GM104940, AHA 17SDG33410366, HL‐093554.

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Serum Estradiol/Testosterone Ratio Affects the Development of Benign Prostate Hyperplasia in Rats
Authors: Liu Xiangyun , Li Kai , Liu Zhiwei , Gui Shukang
Published: June 2021

Objective

Benign prostatic hyperplasia (BPH) develops more likely with increasing age and changing serum concentrations of circulating estradiol (E2) and/or testosterone (T).

Methods

In this study we explored the relationship between serum E2/T ratio and BPH risk in rats by fitting a mathematical model. A total of 176 male rats were randomized divided into normal control, castrated control, and 20 more groups of castrated animals were administered subcutaneously once daily with one of the four dose levels (0.06, 0.17, 0.5, and 1.5 mg per rat) of T combined with one of the five dose levels (2, 6, 17, 50, and 150μg/kg) of E2 in olive oil by different dose ratio. After 30 days, Serial blood samples were obtained to determine serum T and E2 levels by magnetic bead enzyme‐linked immunosorbent assay. The volume of prostate was measured by water displacement method. The prostate specimens were then fixed, embedded in paraffin, and subjected to hematoxylin and eosin staining for pathological examination. MATLAB software was used to simulate the relationship between prostate/body weight ratio (PBR) and E2/T ratio with a mathematical equation.

Results

Compared with the normal control group, the prostate volume decreased significantly (P < 0.05) in the castrated control group and the treatment groups receiving E2/T dose of 2/0.06 or 6/0.06, while the prostate volume increased significantly (P < 0.05) in the other treatment groups. Compared with the normal control group, the PBR increased statistically significant in the treatment groups receiving E2/T dose of 50/0.5, 150/0.5, 2/1.5, 6/1.5,17/1.5, 50/1.5, or 150/1.5 (P < 0.05). The values of PBR, E2 and T in the treatment groups were significantly higher than those in the control groups. Stepwise regression showed that PBR was a function of E2 and T. PBR=−0.1782 + 0.0081E2 + 0.063T − 0.6 × 10−5E2 2 − 0.28 × 10−3T2, (R2=0.6317)

Conclusion

E2 /T ratio change may be one of the risk factors for PBR, which is associated with the development of BPH.

Support or Funding Information

National Natural Science Foundation of China (No.: 81772429)

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Low‐intensity ultrasound differentially upregulates cell death regulators in cancer cells, and downregulates inflammatory pathways in non‐cancer cells via oxidative stresschromatin long‐range interaction signaling
Authors: Yang Qiao , Wang Jiwei , Lai Bin , Sun Yu , Yu Daohai , Yang William , Shao Ying , Zhang Chunquan , Liu Yanna , Wang Hong , Yang Xiaofeng
Published: June 2021

The mechanisms underlying low intensity ultrasound (LIUS) suppression of inflammations and tumorigenesis remain poorly determined. Here, we determined the mRNA expressions of 299 cell death regulators in 13 cell death types in the microarray data. We made the following findings: 1) LIUS upregulates more cell death regulators in cancer cells than in non‐cancer cells, and downregulates more inflammatory regulators in non‐cancer cells than cancer cells; 2) LIUS‐induced most top signaling pathways in cancer cells are not shared with that in non‐cancer cells. LIUS induces more cell death related pathways in cancer cells but inhibits the expressions of a list of inflammation‐related regulators via TP53‐, and SRF‐, mediated pathways, respectively, in non‐cancer cells; 3) LIUS shares 10–25% of the top pathways with mild hyperthermia in regulating cell death regulator expression regardless of cell types; and shares 15–45% of the top pathways with oscillatory shear stress (OSS) in regulating gene expressions in non‐cancer cells; and 4) LIUS may modulate chromatin long range interactions to regulate gene expressions in cells; upstream chromatin long‐range interaction sites (CLRISs) are more favorable than downstream CLRISs for LIUS modulation of gene expressions in cancer cells; and downstream CLRISs play more important roles than upstream CLRISs for LIUS modulation of gene expressions in non‐cancer cells. Our finding demonstrated that LIUS suppresses tumorigenesis and inhibits inflammation by modulating the expressions of cell death regulators, which leads to novel therapeutic targets for treating cancers and inflammation.

Support or Funding Information

This work was supported by NIH grant to Drs. XF. Yang, H. Wang.

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
The Modulatory Effects of Cannabidiol on Microglial Function and Receptor Expression in Epilepsy
Authors: Victor Tanya R. , Nissen Jillian C. , Elmes Matthew W. , Deutsch Dale G. , Tsirka Stella E.
Published: June 2021

Epilepsy, a chronic disorder characterized by abnormal brain cell activity leading to recurrent seizures, has recently seen increased and FDA‐approved usage of cannabidiol (CBD) as a treatment for intractable epilepsies. CBD, a non‐psychotropic component of Cannabis sativa, is reported to lessen the severity of experimentally induced seizures in animals and to suppress neuroinflammation in culture. This compound has been able to provide therapeutic options for the nearly 30% of patients who do not respond to traditional epilepsy medications. Although CBD has been shown to increase intracellular levels of anandamide, an endogenous cannabinoid, its mechanism of action is still poorly understood. Our lab has shown that microglia, the immune cells of the central nervous system (CNS), act as important mediators of seizure severity. The ability of microglia to modulate seizures has been associated with the activation of Toll‐like receptors (TLRs), which play important roles in pathogen recognition and inflammation. Microglia also contribute to neurogenic processes and synapse maturation, aiding in the formation of functional neuronal circuits. Our preliminary data suggest that CBD functions partially through microglia. We postulate that CBD modulates the activation of microglial cells in the brain during experimental epilepsy. In this study, we evaluate the effects of CBD on cell death, inflammation and microglial receptor expression in vitro and in vivo post seizure.

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
A Peripherally Selective CB1 Receptor Inverse Agonist Improves Metabolic Syndrome in Mice
Authors: Khan Nayaab S , Amato George , Vasukuttan Vineetha , Wang Shaobin , Snyder Rodney , Rosa Taylor , Wiethe Robert , Hackney Aliah , Runyon Scott , Maitra Rangan
Published: June 2021

The type 1 cannabinoid receptor (CB1) can be targeted to treat major diseases like type 2 diabetes, fibrosis and steatohepatitis. This has led to the development and profiling of several CB1 antagonists; one such drug being rimonabant, a CNS penetrating inverse agonist, for the treatment of obesity. However, rimonabant was discontinued due to CNS‐related adverse psychiatric effects in a subset of patients. Accumulating evidence indicates that peripheral blockade of CB1 with antagonists that have limited brain penetration is an attractive strategy to treat various disorders. Our research has identified RTI‐1092663, a peripherally selective purine‐based inverse agonist with limited brain penetration (~1% of plasma concentration) and good ADME properties, as a therapeutic agent for the treatment of metabolic syndrome. C57/BL6J male mice were fed a standard chow or high fat diet (60% fat calories) from 6 – 24 weeks and were administered vehicle or RTI‐1092663 at doses of 0.1, 0.3 or 1.0mg/kg, PO/day from weeks 20–24. Oral glucose tolerance test was performed 3 days prior to euthanasia. High fat diet (HFD) significantly increased body weight, liver weights and blood glucose levels in both fasted and fed states. HFD also increased hepatic mRNA expression of CXCR1. Treatment with RTI‐1092663 resulted in a significant decline in body weight and liver weight. Further, RTI‐1092663 treatment lowered fasting glucose levels and demonstrated a dose dependent improvement in glucose tolerance. This compound also inhibited increased neutrophil infiltration as demonstrated by a significant decline in CXCR1 mRNA expression. These data suggest that compound RTI‐1092663 could be further developed for the treatment of metabolic syndrome and other diseases.

Support or Funding Information

1R01AA023256‐01

1R01DK100414‐01A1

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Mitochondrial Oxidative Metabolism and Dopamine Neurodegeneration in the Mesolimbic Pathway after Prolonged Methamphetamine Self‐Administration in Mice
Authors: Lopez Sergio Dominguez , Ahn Bumsoo , Premkumar Pavithra , Van Remmen Holly , Beckstead Michael J.
Published: June 2021

The dopaminergic mesolimbic pathway mediates the reinforcing properties of methamphetamine. Postmortem and brain imaging studies indicate that chronic methamphetamine users develop injury and neurodegeneration in the brain structures that form part of this pathway. Moreover, methamphetamine exposure produces an increase of oxidative molecules derived from increased release and metabolism of dopamine, which in turn is thought to initiate an imbalance of the redox metabolism in the mesolimbic pathway. Here we use a model of methamphetamine self‐administration in mice to determine how the oxidative environment produced by chronic methamphetamine exposure affects 1) mitochondrial oxygen consumption rate (OCR) and redox balance of the antioxidant molecule glutathione (GSH) in the ventral tegmental area (VTA) and striatum and, 2) electrophysiological function and survival of dopamine neurons in the VTA. Adult mice were implanted with a jugular catheter and were trained to nose poke to self‐administer methamphetamine in operant chambers in daily two hours sessions. Mice were trained with a schedule that gradually increased the number of nose pokes required to receive an infusion of methamphetamine from a fixed ratio (FR) 1 to a FR3. After training was complete, mice were placed in a FR3 schedule to complete 35–40 days of total methamphetamine self‐administration exposure. Our results showed that drug‐seeking behavior, evaluated with a progressive ratio protocol, was enhanced after 20 days of methamphetamine self‐administration exposure. Using high resolution respirometry and HPLC we detected an increase of mitochondrial OCR accompanied by a decrease of GSH levels in the VTA. Conversely, GSH levels were decreased in the striatum. A decrease in dopamine neuron firing rate and an increase of cell excitability was also detected in whole cell recordings in VTA slices. The number of immunoreactive tyrosine hydroxylase positive neurons was also decreased in the VTA. Our results suggest that prolonged methamphetamine self‐administration exposure produces an oxidative environment along the mesolimbic pathway that affects dopamine neurotransmission and dopamine cell survival in the VTA. The results are consistent with increased oxidative damage in VTA dopamine neurons contributing to neurodegeneration of the mesolimbic pathway and to the development of drug‐seeking behavior with prolonged methamphetamine exposure.

Support or Funding Information

This project was supported by National Institutes of Health Grants R01 DA32701 and AG52606 to MJB

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Green Apple Tobacco Flavorant Farnesol Enhances Nicotine Reward‐related Behavior
Authors: Avelar Alicia Jo , Akers Austin T , Cooper Skylar Y , Baumgard Zack J , Henderson Brandon J
Published: June 2021

Previous investigations have shown that menthol, the most popular tobacco flavor, can enhance nicotine reward and reinforcement. With the growing popularity of electronic nicotine delivery systems, more flavors are available to smokers and there is a critical need to understand how these flavors may alter nicotine's actions. In this study we examined the green apple flavorant and odorant farnesol for its ability to alter nicotine reward‐related behavior and nicotinic acetylcholine receptor (nAChR) upregulation.

Using a conditioned place preference assay we found that farnesol enhanced nicotine reward‐related behavior to a degree that is similar to menthol. When we examined farnesol alone, we observed significant reward‐related behavior. We then used confocal microscopy and α4‐ mCherryα6‐GFP mice to examine the upregulation of α4‐containing (α4*), α6*, and α4α6* nAChRs in ventral tegmental area (VTA) dopamine neurons. Here, we observed that farnesol, by itself, enhanced the upregulation of α6* nAChRs but had little effect on α4* nAChRs.

Together, these data show that farnesol alters nicotine reward‐related behavior. This is most likely accomplished by its ability to enhance α6* nAChR upregulation. These data highlight the need to study how tobacco flavors alter nicotine reward and reinforcement.

Support or Funding Information

Support: Marshall University Research Corporation and National Institutes of Health: DA040047 and DA043665 (BJH).

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Intermittent dietary fish oil supplementation prevents high fat diet‐induced enhanced sensitivity to the behavioral effects of quinpirole
Authors: Beltran Nina M , Galindo Kayla I , Echeverri Jose , Hernandez‐Casner Caroline , Serafine Katherine M
Published: June 2021

Eating a high fat diet can lead to obesity, type 2 diabetes, and dopamine system dysfunction. For example, rats eating high fat laboratory chow are more sensitive than rats eating standard chow to the behavioral effects of dopaminergic drugs. Specifically, drugs that act on dopamine systems (e.g., quinpirole and cocaine) produce unconditioned behavioral effects (e.g., yawning and locomotion) that are enhanced among rats eating high fat chow. Daily dietary supplementation with fish oil prevents this high fat diet‐induced effect; however, doctors recommend that patients take fish oil only 2–3 times a week for beneficial health effects. To test the hypothesis that intermittent (e.g., 2/7 days per week) dietary supplementation with fish oil prevents high fat diet‐induced effects (e.g., enhanced sensitivity to the behavioral effects of dopaminergic drugs) rats eating standard chow (17% kcal from fat), high fat chow (60% kcal from fat), or standard or high fat chow with 20% (w/w) intermittent (e.g., 2/7 days per week) dietary fish oil supplementation were tested once weekly with quinpirole (0.0032–0.32 mg/kg, i.p.) or cocaine (1–17.8 mg/kg, i.p.) using cumulative dosing procedures. Sensitivity to the behavioral effects of quinpirole was enhanced among rats eating high fat chow as compared to rats eating standard chow, and rats eating high fat chow with intermittent dietary fish oil supplementation were protected against this enhanced sensitivity. Intermittent access to fish oil significantly decreased cocaine‐induced locomotion in some groups, without impacting general locomotor activity (e.g., motor activity following saline injections). Future experiments will focus on understanding the mechanism(s) by which fish oil produces beneficial effects, by examining the specific omega‐3 polyunsaturated fatty acids found in fish oil.

Support or Funding Information

Nina Beltran is supported by the National Institute of General Medical Sciences of the National Institutes of Health (NIH) under the linked award number R25GM069621.

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Dendrobium nobile Lindl. alkaloids decreases the level of intracellular β‐amyloid by improving impaired autolysosomal proteolysis in APP/PS1 mice
Authors: Nie Jing , Jiang Lingshan , Lu Yanliu , Shi Jingshan
Published: June 2021

As the major degradation pathway for long‐lived proteins and organelles, macroautophagy is a decisive factor for the survival and longevity of cells. The existing evidence indicates that the disruption of substrate proteolysis in autolysosomes is the main mechanism underlying autophagy failure in Alzheimer's disease (AD). Thus, the restoration of normal lysosomal proteolysis and autophagy efficiency is a novel therapeutic strategy in the treatment of AD. In this study, 9‐month‐old APPswe/PS1ΔE9 transgenic (APP/PS1) mice were administered Dendrobium nobile Lindl. alkaloids (DNLA, 40 and 80 mg/kg) or Metformin (80 mg/kg), and age‐matched wild‐type mice were administered an isovolumic vehicle orally once a day for 4 months. The results demonstrated that DNLA significantly improved learning and memory in APP/PS1 transgenic mice in the Morris water maze. Furthermore, DNLA could increase the expression of the v‐ATPase A1 subunit to facilitate lysosomal acidification, prompt the dissociation of the cation independent‐mannose‐phosphate receptor from cathepsin (cat) D, promote the proteolytic maturation of cat D, increase the degradation of accumulated autophagic vacuoles (AVs) and β‐amyloid (Aβ) contained in the AVs, and alleviate neuronal and synaptic injury. These findings demonstrate that DNLA improves learning and memory function in APP/PS1 mice, and the mechanisms appear to be due to the promotion of intracellular Aβ degradation by increasing the protein level of v‐ATPase A1 and then improving autolysosomal acidification and proteolysis.

Support or Funding Information

This work has been supported by the National Natural Science Foundation of China (Grant No. 81473201); Fund of Key Discipline Construction of Zunyi Medical University; Science and Technology Foundation of State Administration of Traditional Chinese Medicine of Guizhou Province (QZYY2017‐016).

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
SMU‐A inhibits Aβ‐induced NLRP3 inflammasome via autophagy mediated by the AMPK/ULK1 and Raf/MEK/ERK pathways in microglia
Authors: Anguo Wu , Ya Zhao , Wenqiao Qiu , Jinfeng Teng , Rui Xiong , Jianming Wu , Dalian Qin
Published: June 2021

Accumulation of β‐amyloid (Aβ) and its induced inflammation are recognized as two major pathological features of Alzheimer disease (AD). Autophagy, an intracellular catabolic mechanism, plays a pivotal role in neurodegenerative disease for degrading the toxic aggregated proteins such as Aβ, α‐Synuclein and mHtt. Recently, autophagy activation in microglia was reported to not only degrade extracellular Aβ fribils but also inhibit Aβ‐induced NLRP3 inflmmasome. In the present experiment, the potential autophagy inducers from SMU, a traditional Chinese medicine, were identified in microglial cells (BV‐2) by using column chromatography, UHPLC‐TOF/MS and NMR technologies. Total four ellagitannin flavonoids including SMU‐A, SMU‐B, SMU‐C and SMU‐D were identified, and SMU‐A was proved to possess the best autophagic effect in BV‐2 cells. Furthermore, the autophagy was activated through the AMPK/ULK1, Raf/MEK/ERK and ATG7 dependent, but independent of mTOR signaling pathway. In Aβ1‐42 induced BV‐2 cells, SMU‐A inhibits inflammasome containing NLRP3, ASC, IL‐1β and caspase1 via autophagy, which was reversed by the autophagic inhibitors such as compound C, SCH772984, hydroxychloroquine and SBI‐0206965. Additionally, SMU‐A can improve cell viability of PC12 induced injury by cell culture medium of Aβ‐induced BV‐2. Taken together, enhanced microglial autophagy is a promising new therapeutic strategy for AD, and demonstrate the autophagy inducers in SMU exerts the potential neuroprotective mechanism in suppressing neuroinflammatory responses by enhancing autophagy in microglial. Therefore, uncovering the components in SMU contributing to autophagy activation and its molecular mechanism provide new insight of PCP into novel therapies for the amelioration of AD.

Support or Funding Information

The Science and Technology Program of Luzhou” (grant numbers 2016LZXNYD‐T03 and 2017LZXNYD‐J28), “Educational Commission of Sichuan Province” (grant numbers 18ZA0528 and 18TD0051), “Science and Technology Planning Project of Sichuan Province” (grant numbers 2018JY0474 and 2018JY0237)

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Effects and Side Effects of Cannabinoid/Opioid Combination in a Chronic Pain Model in Rats
Authors: Alsalem Mohammad , Altarifi Ahmad , Aldaoud Nour
Published: June 2021

Chronic pain is considered a growing healthcare problem becoming a major cause of global burden. Prolonged use of the currently used analgesics such opioids is associated with adverse effects; urging the need for safer alternatives. Cannabinoids are a group of compounds structurally related to Δ9‐tetrahydrocannabinol (Δ9‐THC). Both cannabinoids and opioids produce antinociception in different chronic pain animal models. However, therapeutic doses of these drugs may be accompanied by undesirable side effects, such as abuse liability and decrease locomotion in animals. The aim of this study was to assess the outcomes of combining range of doses of opioids and cannabinoids that are sufficient to produce antinociception without producing unwanted side effects. Inflammatory pain was induced by intra‐plantar injection of Complete Freund's Adjuvant (CFA) into the left hindpaw of adult male Sprague Dawley rats. Von Frey filaments were applied to the mid‐plantar aspect of the left hind paw using the “up‐down” method to determine the CFA‐induced mechanical allodynia. Antinociceptive effect of morphine (0.32, 1 and 3.2 mg/kg; i.p), the synthetic cannabinoid CP55940 (0.032, 0.1 and 0.32 mg/kg; s.c.), their combinations, or their vehicles was evaluated 3–9 day post‐CFA administration. Intracranial self‐stimulation (ICSS) test was utilized to evaluate the abuse liability of these drugs or their combinations. Morphine produced significant antinociceptive effects at 1 and 3.2 mg/kg, but not at the lowest dose of 0.32 mg/kg with minimal changes in ICSS. CP55940 showed significant antinociceptive effects only at the highest administered dose (0.32 mg/kg), while producing depression in ICSS at 0.1 and 0.32 mg/kg. The sub‐theraputic dose of morphine (0.32 mg/kg) was co‐administered with the different doses of CP55940 (0.032, 0.1 and 0.32 mg/kg). 0.32 mg/kg morphine enhanced the antinocicpetive effects of CP55940, such that CP55940 produced antinociception at lower dose (0.1 mg/kg) compared to vehicle. However, in ICSS test, the aforementioned combinations did not change CP55940‐induced depression of ICSS. In conclusion morphine and CP55940 produce analgesic effects at lower doses if combined together with no additional risk of abuse liability side effects, which may be useful in clinical practice.

Support or Funding Information

Scientific Research Support Fund (MPH/1/29/2016)

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Arachidonoyl‐2‐chloroethylamide alleviates inflammatory pain in a sex‐dependent manner using the formalin test
Authors: Garcia Dianise Rodriguez , Lugo‐Rodriguez Ismael , Blanton Henry , Guindon Josee
Published: June 2021

Previous studies have shown that certain cannabinoids have analgesic effects. Specifically, activation of cannabinoid receptor (CB) 1 has been demonstrated to produce analgesia in multiple animal models. Arachidonyl‐2‐chloroethylamide (ACEA) is a synthetic agonist for CB1 receptors. The activation of CB1 with ACEA could enable chronic inflammatory pain patients to manage this discomfort. ACEA has not been extensively studied in animal models; therefore, not much is known about the dose effectiveness, development of tolerance, or sex‐specific effects.

To study the analgesic effects of ACEA, we treated male and female C57BL6J mice prior to the formalin pain test. Administration of formalin (2.5% intraplantar) produces a biphasic pain response, modeling both acute and inflammatory pain. We believe that administration of ACEA at a dose of 0.5 mg per kg intraperitoneal will reduce inflammatory pain in this model. Also, we suggest that daily pretreatment with ACEA prior to the formalin test will cause desensitization to this compound; therefore, the initial analgesic effect will not be seen. To examine sex‐specific effects, we also tested and compared male and female mice. Moreover, vaginal lavage was performed daily on female mice to track the estrous cycle.

Preliminary data suggests that females experience higher pain sensitivity compared with males when ACEA 0.5 mg per kg was used. Furthermore, our preliminary data shows that chronic administration in females results in estrous cycle changes. We concluded that there is a sex‐dependent difference in analgesic response to ACEA, and that it may be hormonally‐mediated.

Support or Funding Information

Funded by NIDA DA044999‐01A1 (JG) and Texas Tech University Health Sciences Center School of Medicine #121035 (JG).

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Comparison of the anti‐allodynic effects of gabapentin, morphine and N‐palmitoylethanolamide in rats with chronic constriction of the sciatic nerve
Authors: Hernández Yisel Blanco , Valdés Licet Caridad Mena , Muñoz Francisco Javier López
Published: June 2021

Neuropathic pain (NP) is a chronic disorder with a complex and multifactorial etiology. The NP prevalence constitute a significant health problem worldwide. Among the first choice drugs used for NP treatment is gabapentin (GBP), an antiepileptic drug and morphine (MOR), a prototype of analgesic opioid drug. Moreover, it has recently described the antinociceptive effects of N‐palmitoylethanolamide (PEA) on inflammatory pain.

Objective

To evaluate and compare the anti‐allodynic effects of GBP, MOR and PEA in rats with chronic constriction of the sciatic nerve (CCI model).

Methods

The anti‐allodynic effects of GBP (3.16–177.83 mg/Kg, p.o.), MOR (0.010–3.16 mg/Kg, s.c.) and PEA (3.16–10.00 mg/Kg, p.o.) were determined after single‐doses, using could allodynia test in the CCI model. Evaluations were done until 10 days post‐surgery at 30, 60, 90, 120 and 180 minutes after drugs administration.

Result

It was found that GBP, MOR and PEA showed anti‐allodynic effects in rats with NP induced by CCI. PEA achieved the same anti‐allodynic efficacy as the control group (GBP) and similar to the maximum efficacy achieved by MOR (p>0.05). The analysis of pharmacological potency demonstrated that: MOR (ED50 = 0.047 mg/Kg) was 28 time more potent than PEA (ED50 (1.319 mg/Kg), but PEA was 78 time more potent than GBP (ED50 = 103.3 mg/Kg).

Conclusions

These results demonstrate that PEA have anti‐allodynic effect similar to MOR and GBP, and also that PEA is more potent than GBP in rats with NP induced by CCI.

Support or Funding Information

Department of Pharmacobiology, Cinvestav‐Unidad Coapa, C.P. 14330, Tlálpan, Mexico City, Mexico.

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Differences in Depressive‐ and Anxiety‐like effects in S426A/S430A Mutant and Wild‐type Mice Following the Induction of Neuropathic Pain
Authors: Sepulveda Diana E , Oleksyuk Veronika , Nolan Bradley J , Morgan Daniel J , Henderson‐Redmond Angela N
Published: June 2021

Clinically, chronic pain has been associated with increases in anxiety‐ and depressive‐like symptoms, concomitant with the severity and duration of pain. Pursuant to the on‐going opioid crisis, novel therapeutic approaches, including the use of cannabinoids, have been investigated for the management of chronic pain. We have previously demonstrated that mice expressing a desensitization form of the cannabinoid type‐1 (CB1) receptor (S426A/S430A) show delayed tolerance and heightened sensitivity to the anti‐nociceptive effects of Δ9‐tetrahydrocannabinol (Δ9‐THC). In addition to analgesia, cannabinoids have been implicated in mediating affect in select mood disorders. Therefore, the purpose of the present study was to examine whether the induction of chronic neuropathic pain would alter anxiety and/or depressive‐like phenotypes in either wild‐type and/or S426A/S430A mutant mice, and whether the severity of these symptoms correlated with either basal allodynia or Δ9‐THC‐induced anti‐allodynic responses. Male and female wild‐type and S426A/S430A mice were assessed for alterations in locomotor behavior, anxiety‐like (elevated plus maze; open field) and depressive‐like (forced swim test) behaviors before and after the induction of cisplatin‐evoked neuropathic pain. Mice were also assessed to see whether basal pain levels correlated with either anxiety‐ and/or depressive‐like behaviors, and whether increased anxiety‐ and/or depressive‐like symptoms correlated with the degree of anti‐allodynia produced following pretreatment with Δ9‐THC. All mice showed evidence of neuropathic pain following repeated cisplatin‐treatment. Interestingly, the S426A/S430A mutation caused female (but not male) mice to exhibit reduced anxiety‐like behavior prior to the induction of cisplatin pain but caused them to be more susceptible to anxiety and depressive‐like symptoms once they exhibited cisplatin‐evoked pain. Pretreatment with Δ9‐THC was also able to completely reverse allodynia in male and female mutant, but not wild‐type, mice. Taken together, these results highlight the importance of understanding the role that sex may have in the potential clinical use of cannabinoids to modulate both affective and sensory aspects of chronic pain.

Support or Funding Information

Penn State University College of Medicine Department of Anesthesiology & Perioperative Medicine

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.