bluuebunny/crossref_metadata_2025 huggingface dataset:
SIRT3: a potential target for CHF?
Authors: Liu Peiqing
Published: June 2021

Pathological cardiac hypertrophy is a maladaptive response in a variety of organic heart disease (OHD), which is characterized by mitochondrial dysfunction that results from disturbed energy metabolism. SIRT3, a mitochondria‐localized sirtuin, regulates global mitochondrial lysine acetylation and preserves mitochondrial function. However, the mechanisms by which SIRT3 regulates cardiac hypertrophy remains to be further elucidated. In this study, we firstly demonstrated that expression of SIRT3 was decreased in Angiotension II (Ang II)‐treated cardiomyocytes and in hearts of Ang II‐induced cardiac hypertrophic mice. In addition, SIRT3 overexpression protected myocytes from hypertrophy, whereas SIRT3 silencing exacerbated Ang II‐induced cardiomyocyte hypertrophy. In particular, SIRT3‐KO mice exhibited significant cardiac hypertrophy. Mechanistically, we identified NMNAT3 (nicotinamide mononucleotide adenylyltransferase 3), the rate‐limiting enzyme for mitochondrial NAD biosynthesis, as a new target and binding partner of SIRT3. Specifically, SIRT3 physically interacts with and deacetylates NMNAT3, thereby enhancing the enzyme activity of NMNAT3 and contributing to SIRT3‐mediated anti‐hypertrophic effects. Moreover, NMNAT3 regulates the activity of SIRT3 via synthesis of mitochondria NAD. Taken together, these findings provide mechanistic insights into the negative regulatory role of SIRT3 in cardiac hypertrophy. Sirtuin 3 (SIRT3), a mitochondrial deacetylase that may play an important role in regulating cardiac function and a potential target for CHF.

Support or Funding Information

This work was supported by grants from the National Natural Science Foundation of China (No 81673433, No 81026548 and No 81273499), Team item of the Natural Science Foundation of Guangdong Province (NoS2011030003190)

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
miR‐141 inhibition protects cardiomyocytes from anoxia/reoxygenation‐induced mitochondrial dysfunction
Authors: Zhang Hao , Yan Jun , Wang Hao , Tao Aibin , Rui Tao
Published: June 2021

Background

Mitochondrial dysfunction plays an important role in ischemia/reperfusion (I/R) ‐induced myocardial injury. MicroRNAs are involved in myocardial injury after I/R. It has been reported that miR‐141 contributes to mitochondrial dysfunction in mice with diabetes. The aim of the present study is to dertmine whether the miR‐141 contributes to I/R‐induced myocardial injury and the underlying mechanism.

Methods

HL‐1 cells were challenged with an anoxia/reoxygenation (A/R). miR‐141 expression in HL‐1 cells was detected by qRT‐PCR. Mitofusin‐2 (Mfn2) was assessed with Western blot. HL‐1 cellular oxygen consumption rate (OCR) was assessed with Seahorse XF24 analyzer.

Results

A/R challenge to HL‐1 cells increased miR‐141 expression and peaked at 12 hours after the reoxygenation. A/R challenge resulted in decreased mitochondrial membrane potential, OCR and ATP production of the cardiomyocytes, as well as increased LDH activity in the supernatant of the cardiomyocytes. The above changes were attenuated by transfection of the cardiomyocytes with miR‐141 inhibitor before the A/R. Overexpression of miR‐141 in the HL‐1 cell exaggerated the A/R‐induced mitochondrial dysfunction and cardiomyocyte injury. The A/R challenge to HL‐1 cells led to a decreased Mfn2 expression. Transfection of HL‐1 cells with miR‐141 mimics suppressed Mfn2 expression.

Conclusion

Our data indicate that miR‐141 is involved in A/R‐induced mitochondrial dysfunction by modulating Mfn2 protein expression.

Support or Funding Information

The study was supported by Natural Science Foundation of Jiangsu Province (BK20151332) and Natural Science Foundation of China (81370333).

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Human Stem Cell Derived Cardiomyocyte Maturation is Regulated by Glucose Levels and Metabolic Hormone Supplementation
Authors: Klug Heather May , Karbassi Elaheh , Yang Xiulan , Pabon Lil , Murry Charles
Published: June 2021

Methods to differentiate stem cells into cardiomyocytes have been well established. However, a limitation for the successful application of these cells for research and medicine has been their fetal‐like phenotype with respect to cell size, contractility, calcium handling, metabolism, and electrophysiology. We sought to increase the maturity of human pluripotent stem cell derived cardiomyocytes (hPSC‐CMs) through metabolic pathway regulation. We hypothesized that switching the main metabolic substrates from glucose to fatty acids, mimicking the switch from placental to breast milk nutrient consumption that occurs during development, will increase hPSC‐CM maturation. RUES2 embryonic stem cells were differentiated into cardiomyocytes and then treated with base media with varying glucose and calcium concentrations and fatty acid supplementation. Using qRT‐PCR, we measured an inverse relationship between glucose levels and markers of cardiomyocyte maturation: increased expression of cardiac troponin I relative to skeletal troponin I isoforms (TNNI3:TNNI1), increased expression of metabolic (CPT1B, PPARGC1A) and electrical maturity markers (KCNJ2, RYR2). These maturation markers were not influenced with fatty acid supplementation but were enhanced upon the addition of thyroid hormone and dexamethasone. To understand the mechanisms by which media nutrients and signaling molecules cause phenotypic changes we investigated the relationship between maturation and global epigenetic state. Western blot revealed increases in global acetylation levels, measured by histone H3 acetylation, linked to maturation signaling. These findings depict a direct relationship between glucose metabolism and the development of a mature phenotype in hPSC‐CMs, mediated by epigenetic mechanisms.

Support or Funding Information

Washington Research Foundation Undergraduate Fellowship

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Toxoplasma gondii alters NMDAR signaling and induces signs of Alzheimer's disease in wild‐type, C57BL/6 mice
Authors: Torres Luisa , Bynoe Margaret S
Published: June 2021

Alzheimer's disease (AD) is a progressive neurodegenerative disease associated with cognitive decline, resulting in a complete loss of basic functions. The ubiquitous apicomplexan parasite Toxoplasma gondii (T. gondii) infects up to one third of the world's population and is implicated in AD. We infected C57BL/6 wild‐type male and female mice with 10 T. gondii ME49 cysts and assessed whether infection led to behavioral and anatomical effects using immunohistochemistry, immunofluorescence, Western blotting, cell culture assays, as well as an array of mouse behavior tests. We previously reported that T. gondii infection induced two major hallmarks of AD in the brains of C57BL/6 male and female mice: beta‐amyloid (Aβ) immunoreactivity and hyperphosphorylated Tau. Infected mice showed significant neuronal death, loss of N‐methyl‐D‐aspartate receptor (NMDAR) expression, and loss of olfactory sensory neurons. T. gondii infection also caused anxiety‐like behavior, altered recognition of social novelty, altered spatial memory, and reduced olfactory sensitivity. We have new data that suggests that T. gondii might be recruiting the NMDA receptor to the parasitophorous vacuole, a structure that protects T. gondii from the phagolysosomes of its host. This indicates that T. gondii might use the NMDA receptor of the host to survive and might explain the reduction of NMDAR that we observed with T. gondii infection. Since reduced NMDAR expression coincides with Aβ immunoreactivity, it is possible that T. gondii infection leads to reduced NMDAR expression which then leads to Aβ immunoreactivity.

Support or Funding Information

This work was supported by NIH grant R01 NS063011 to M.S Bynoe

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Protease‐Activated Receptor 2 Drives Colonic Epithelial Wound Healing via EGFR Transactivation
Authors: Bandara Mahesha N.S. , MacNaughton Wallace Keith
Published: June 2021

Background

Compromised intestinal barrier function is a prominent feature of inflammatory bowel diseases. Epithelial restitution is required in the resolution of inflammation. For this to occur epithelial cells need to transition to migratory phenotype from its normal barrier phenotype. The inflammatory milieu of the intestinal epithelium contains a variety proteases which are upregulated in IBD patients and which are agonists of protease activated receptors (PARs). PAR2 is membrane‐spanning cell surface receptor which is widely present in the gastrointestinal tract. PAR2 activation can induce expression of cyclooxygenase‐2 (COX‐2) and transactivation of epidermal growth factor receptor (EGFR) in the intestinal epithelium. However, the specific roles of PAR2 activation in IBD remain unclear.

Hypothesis

We hypothesized that PAR2 activation drives COX‐2 and EGFR dependent epithelial wound healing.

Methods

CMT93 mouse intestinal epithelial cells were grown to confluence. PAR2 mRNA expression was detected by RT‐PCR and confirmed by cDNA sequencing. PAR2 A5 antibody was used to detect total PAR2 by immunocytochemistry and the tight junction protein, ZO‐1, demarcated apical and basolateral surfaces. PAR2 was activated by selective activating peptide 2‐furoyl‐LIGRLO‐NH2 (2fLI). Functionality of PAR2 was tested by measuring intracellular Ca2+, since it has been shown that PAR2 activation elicits Ca2+‐dependent signaling. Scratch wounds were made in post‐confluent CMT93 monolayers and imaged using the IncuCyte™ live‐cell imaging system over 24 hours. COX‐2 protein was measured by western blotting and inhibited in wound healing assays with NS‐398 (10 μM). EGFR was inhibited by the EGFR tyrosine kinase inhibitor PD153035 (10 nM) and activated using human EGF (10 ng/ml and 5 ng/ml).

Results

Immunocytochemistry revealed that PAR2 is expressed on mostly basolateral membranes with some expression on apical membrane and punctate immunoreactivity in the cytoplasm in polarized cells. Concentration‐response studies revealed that 2.5 μM 2fLI elicited the highest intracellular Ca2+ response (EC50 = 0.5 μM). PAR2 activation at 10 μM 2fLI significantly increased wound healing (P≤ 0.0001). However, PAR2 activation by 2fLI did not induce COX‐2 expression, nor did COX‐2 inhibition alter PAR2‐induced wound healing. PAR2‐induced wound healing was inhibited by the EGFR tyrosine kinase inhibitor (P ≤ 0.001). Moreover, 2fLI induced wound healing is comparable to the that of EGF (5 ng/ml).

Conclusion

PAR2 activation drives wound healing in CMT93 epithelial cells through the transactivation of EGFR and not through induction of COX‐2. This study enhances our understanding of how proteases in the inflammatory milieu may drive epithelial restitution and mucosal healing.

Support or Funding Information

CIHR

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 derived reactive oxygen species activates NLRP3 inflammasome in cardiac fibroblasts in sepsis
Authors: Yan Jun , Zhang Hao , Wang Hao , Tao Aibin , Rui Tao
Published: June 2021

Background

Sepsis is a common disease in intensive care unit in hospital. It is caused by exaggerated host response to infection. Septic myocardial dysfunction plays an important role in pathogenesis of multiple organ failure and subsequent high mortality of patients with sepsis. Our previous studies indicate that activation of NLRP3 inflammasome in cardiac fibroblasts (CF) contributes to sepsis‐induced myocardial dysfunction. The aim of the present study is to identify the role of mitochondrial ROS (mtROS) in the activation of the NLRP3 inflammasome in CF.

Methods

Cultured CF was primed with LPS (1 ng/mL) followed by ATP (3 mmol/L) treatment for activation of NLRP3 inflammasome. CF mtROS was detected with MitoSOX reagent, a probe specifically detects mitochondrial ROS. NLRP3 inflammasome activation was assessed with Western blot, ELISA and co‐immunoprecipitation.

Results

ATP challenge of CF primed with LPS increased CF mtROS production which was attenuated by pretreatment of the LPS primed CF with mito‐TEMPO (25 mmol/L), an ROS scavenger targeted at mtROS. ATP challenge of the LPS primed CF activated the NLRP3 inflammasome as indicated by increase in intracellular levels of NLRP3, pro‐IL‐1β and released IL‐1β. Mito‐TEMPO pretreatment of the CF with LPS+ATP prevented the increase of released IL‐1β in CF. Finally, ATP challenge of the LPS primed CF promoted NLRP3 interaction with the ASC. The Mito‐TEMPO pretreatment of the CF diminished the interaction between the NLRP3 and ASC.

Conclusion

Our data suggest that mitochondrial derived ROS plays a pivotal role in the activation of the CF NLRP3 inflammasome in sepsis.

Support or Funding Information

The study was supported by Natural Science Foundation of Jiangsu Province (BK20151332) and Natural Science Foundation of China (81370333).

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Actions of Angeli's salt, a nitroxyl (HNO) donor, on Trypanosoma cruzi infection
Authors: Pinge‐Filho Phileno , Pereira Rito Santo , Malvezi Aparecida Donizette , Lucchetti Bruno Fernando Cruz , Cecchini Rubens , Yamauchi Lucy Megumi , Yamada‐Ogatta Sueli Fumie , Verri Waldiceu Aparecido , Martins‐Pinge Marli Cardoso , Tatakihara Vera Lucia Hideko
Published: June 2021

Chagas disease, caused by Trypanosoma cruzi infection, is endemic to much of Latin America, but also present in the United States (U.S.) The goal of treatment is to eliminate the parasite and decrease the probability of cardiomyopathy and interrupt the cycle of disease transmission. Currently, only benznidazole (BZN) and nifurtimox are recognized by the World Health Organization as effective drugs for treatment of Chagas disease, but both present low cure rates in the chronic phase and often have serious side effects. T. cruzi infection produces an intense inflammatory response in several tissues which is critical for controlling parasites proliferation and evolution of Chagas disease. Compounds liberating nitric oxide (NO donors) have been used as therapeutic agents against T. cruzi. Here we test HNO donor, Angeli's salt (AS) in the outcome of T. cruzi infection. C57BL/6 mice were infected with T. cruzi (Y strain, 5 × 103 trypomastigotes) intraperitoneally (ip). After 15 minutes of infection and the subsequent 12 days, the animals were treated with different concentrations of AS ip. The control group received 100 uL of PBS by the same route. Treatment with AS showed decreased parasitemia and in the number of nests in the cardiac tissue, decreased oxidative stress in erythrocytes and improved leukopenia and thrombocytopenia resulting in reduced disease severity. The in vitro treatment was able to reduce the parasite internalization rate and release of infective trypomastigotes by these cells. Our results suggest for the first time the therapeutic potential of AS in a mouse model of Chagas disease.

Support or Funding Information

CAPES, Fundação Araucária, CNPQ, UniZambeze and MCTESTP Mozambique.

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 of Factors Involved in the Pathogenesis of Chagas Disease
Authors: Hagström Luciana , Wesley Moisés , Fereira Tatiana Shiroma Borges , de Souza Alves Jehny Pereira , de Cássia Rosa Ana , Dallago Bruno , Nitz Nadjar , Hecht Mariana Machado
Published: June 2021

Chagas disease, caused by the protozoan Trypanosoma cruzi, represents a serious health public problem with around seven million infected people worldwide. Approximately 30% of infected individuals will develop cardiac disorders during the chronic phase. However, the pathogenesis of Chagas disease remain incompletely elucidated with fundamental questions partially unanswered as the factors that lead to the development of clinical manifestations. Thereby, this study aims to investigate the importance of the association of T. cruzi strain, host sex, host immunological factors and changes in heart tissue in the progression of cardiac lesions in experimental Chagas disease. For this, 60 BALB/c mice were divided into three groups according to the T. cruzi strain (discrete typing unit TcI, TcII and TcVI). They were analyzed in acute phase (30 days pos‐infection ‐ 30dpi) and chronic phase (90 dpi). Ten uninfected mice formed the control group. Each group were formed by five males and five females. After the experimental period, mice were euthanized and blood and heart were collected. Anti‐T cruzi antibody and anti‐cardiac tissue antibody production were evaluated by ELISA. Parasite load in the heart was determined by qPCR. Histopathological analysis of heart was also performed. Most of the animals produced anti‐T. cruzi IgM antibody in the acute phase, whereas all mice were seropositive for IgG in chronic phase. TcI was characterized by elevated antibody levels with autoantibodies titers against the heart in chronic phase significantly higher than the other groups. Besides that TcI presented a greater number of parasites both in acute and chronic phase as well as more damage in heart tissue. No difference was observed between males and females. Amastigote nests were seen in mice analyzed in acute phase in TcI and TcVI, but not in TcII. The results confirm the participation of autoimmune response in chronic Chagas disease, which varies according to T. cruzi strain. It seems that the TcI was the most aggressive. However, the relationship between the studied factors and the severity of disease has not been established.

Support or Funding Information

This study was financed by Fundação de Apoio a Pesquisa do Distrito Federal (FAPDF), Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) and Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES).

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Different Roles of Epigenetic Regulators and Inflammasome in Hepatocellular Carcinoma Tumorigenesis in Patients with ASH vs. NASH
Authors: Jia Yue , Ji Ping , Nguyen Luan , French Barbara , Tillman Brittany , French Sameul W
Published: June 2021

As the fifth most common cancer and the second leading cause of cancer related deaths worldwide, hepatocellular carcinoma (HCC) is an aggressive tumor type with poor prognosis causing 250,000 to one million deaths annually. Alcoholic steatohepatitis (ASH) and non‐alcoholic steatohepatitis (NASH) are the two major growing risk factors and both may develop liver fibrosis or even HCC. However, compared with the rate of patients with ASH progressing to HCC annually, it is much lower in NASH patients. The present study is to clarify the protein expression of epigenetic regulators and Inflammasome components in the oncogenesis pathway between ASH and NASH. By using the immunofluorescence method and morphometrically quantitating the fluorescence intensity in liver biopsied specimens from NASH and ASH patients, we studied the protein expression within hepatocytes cytoplasm of candidate epigenetic regulators G9A and LHPP and inflammasome component ASC. Compared with the control group patients, the expression levels of all three proteins were upregulated in the ASH and NASH group of patients (p < 0.001 in all molecules). While compared with the ASH group of patients, only the expression levels of G9A were significantly lower in the NASH group of patients (p < 0.01). The other two molecules, LHPP and ASC did not change. These results are consistent with our previous work that there are significant differences of many molecules including epigenetic modulators and the inflammasome pathway in both NASH and ASH compared to the control group. Thus, we conclude that there are significantly different molecules and pathways involved during the pathogenesis of HCC development in NASH compared to ASH which may focus the light on developing prevention methods and targeted treatments in NASH and ASH patients.

Support or Funding Information

This study was funded by NIH/AAA grant # UO‐21898‐05.

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Combined effects of phenmetrazine and nor‐binaltorphimine on dopamine function and motivation to self‐administer cocaine
Authors: Estave Paige , Jones Sara
Published: June 2021

Despite numerous clinical trials, the FDA has not approved any medication assisted treatment for cocaine addiction. Our laboratory has shown that chronic cocaine self‐administration reduced overall dopamine (DA) neurotransmission, resulting in a hypodopaminergic state. Other studies have shown that repeated, non‐contingent cocaine exposure resulted in a hyper‐functioning kappa opioid receptor (KOR) system. Thus, this study aims to examine the effectiveness of a combination therapy, targeting the dopamine transporter and the KOR, in reducing cocaine‐seeking behaviors in rats chronically exposed to cocaine.

In order to understand the impacts of cocaine self‐administration on the DA and KOR systems, we used ex vivo fast scan cyclic voltammetry in the nucleus accumbens core to examine DA dynamics after chronic cocaine exposure (40 infusions of cocaine for 5 consecutive days). We found that cocaine exposure reduced stimulated DA release and attenuated cocaine potency at the dopamine transporter. Additionally, we examined KOR function through activation of KORs using the agonist U50,488, which inhibited DA release. We observed that responses to U50,488 were augmented post‐cocaine exposure.

Male Sprague Dawley rats were used to determine the individual and combined behavioral effects of the DA releaser phenmetrazine and the KOR antagonist nor‐binaltorphimine (nor‐BNI). After rats self‐administered forty infusions of cocaine (1.5 mg/kg/infusion) on a fixed‐ratio 1 schedule for five consecutive days, a progressive ratio schedule was used to determine cocaine breakpoints (0.1875 mg/kg/infusion) at baseline, and following phenmetrazine (25 mg/kg/day; osmotic mini‐pump), nor‐BNI (10 mg/kg; i.p.), or a combination of the two drugs. Administration of phenmetrazine alone significantly reduced breakpoints (60% of baseline), while nor‐BNI had no effect as a monotherapy. Interestingly, cocaine breakpoints were further attenuated by the combination (20% of baseline), indicating augmented effects of targeting both the DA and KOR systems.

In this study, voltammetry results showed that chronic cocaine exposure reduced DA transmission and augmented KOR function. Dual targeting of the DA and KOR systems through a combination therapy approach revealed a greater decrease in motivation to take cocaine compared to either monotherapy. Taken together, these data support the potential beneficial effects of the dopamine transporter and KOR as dual‐cellular targets to treat multiple aspects of the withdrawal syndrome, ultimately decreasing the risk of relapse in individuals with cocaine dependence.

Support or Funding Information

Support for this work was provided by R01 DA014030, P50 DA006634, and T32 DA041329

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Gut‐Brain Actions Underlying Comorbid Neuropsychiatric Disturb Associated with Inflammatory Bowel Disease
Authors: Cordaro Marika , Siracusa Rosalba , D'Amico Ramona , Gugliandolo Enrico , Peritore Alessio Filippo , Crupi Rosalia , Impellizzeri Daniela , Cuzzocrea Salvatore , Di Paola Rosanna
Published: June 2021

Inflammatory bowel disease (IBD) is a chronic relapsing and remitting disorder characterized by inflammation of the gastrointestinal tract and its associated with different neurological and psychiatric disorders, which are integrated among the extra‐intestinal manifestation of this disease.

In fact, epidemiological studies have shown an increased incidence of depression in patients with colitis, in which strong inflammation in the gut occurs. The possible mechanisms connecting gut inflammation and neuroinflammation in the brain could be via an increase in peripheral cytokines or via damaged nerve terminals in the gut.

Recent evidence demonstrated that the gut‐brain‐axis has a central function in the perpetuation of IBS, and, for this reason it can be considered as a possible therapeutic target.

N‐Palmitoylethanolamine‐oxazoline (PEA‐OXA) possesses anti‐inflammatory and potent neuroprotective effects. Although, recent studies have explained the neuroprotective properties of PEA‐OXA, nothing is known about its effects on gut‐brain axis during colitis.

The aim of this study is to explore the mechanism and the effect of PEA‐OXA on gut brain axis in mice subjected to experimental colitis induced by intracolonic administration of Dextran sulfate sodium. Daily orally administration of PEA‐OXA 10 mg/kg daily o.s. was able to decrease body weight loss, macroscopic score and colon length and histology alteration after DSS induction. On the other hand was able to reduce histology modification and c‐FOS expression in the hippocampus.

Additionally, we demonstrate that PEA‐OXA administration significantly increase neurogenesis and promoting neurotrophic grow factor release and decrease astroglial and microglial activation DSS‐induced. Moreover, PEA‐OXA, was able to decrease inflammatory marker in both hippocampus and colon.

In conclusion, in our work, we demonstrate for the fort time the action of PEAOXA on gut‐brain axis during in a model of DSS induced colitis. induced. Moreover, PEA‐OXA, was able to decrease inflammatory marker in both hippocampus and colon. In conclusion, in our work, we demonstrate for the fort time the action of PEAOXA on gut‐brain axis during in a model of DSS induced colitis.

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Psychedelics Improve the Mental Health of Rats
Authors: Hibicke Meghan , Landry Alexus N , Talman Zoe K , Nichols Charles
Published: June 2021

Introduction

Psilocybin has recently demonstrated profound efficacy to alleviate depression and anxiety in several clinical trials and has received Breakthrough Status by the FDA. Symptomatic relief after only one or two therapeutic treatments last for at least several months in the vast majority of individuals. It is unclear whether the persistent antidepressant and anxiolytic effects are a psychological integration of the subject's personal experience while under the influence of the drug, or are a consequence of neurochemical changes induced by psilocybin that correct abnormalities leading to a healthier brain state. To distinguish between these possibilities, we investigated the antidepressant and anxiolytic effects of psilocybin in a rat model of treatment resistant depression, Wistar‐Kyoto (WKY) rats. Rats do not, as we currently believe, have a sense of self to reflect upon during a psychedelic experience. Therefore, if the effects are purely psychological, psilocybin should not occasion long lasting therapeutic effects as are seen in humans.

Objective

To evaluate the long‐term effects of psychedelics on depressive‐like and anxiety‐like behaviors in a rat model of depression.

Methods

Two experiments were performed in which rats were administered a single bolus dose of saline or treatment in saline vehicle: psilocybin (1 mg/kg), lysergic acid diethylamide (LSD, 0.15 mg/kg), or ketamine (5.0 mg/kg) intraperitoneally at a volume of 1 mL/kg. Saline (experiment 1, n=8) and repeat measures psilocybin (experiment 1, n=8) groups were evaluated in the Forced Swim Test (FST) weekly for five weeks, and then the Evaluated Plus Maze (EPM) on the sixth week. Interval psilocybin (experiment 1, n=8), saline (experiment 2, n=6), LSD (experiment 2, n=6), and ketamine (experiment 2, n=6) were evaluated in the FST a single time on the fifth week, and then in the EPM on the sixth week. All measures were evaluated with one‐way ANOVA using Holm‐Sidak post hoc, or with t‐test as appropriate.

Results

Remarkably, we have found that a single treatment with psilocybin produces context‐dependent, long lasting antidepressant‐like (p=0.046, p=0.026, and p<0.0001) and anxiolytic effects (p=0.039) in male WKY rats as measured by the FST and EPM. We have also found that LSD, a related psychedelic, produces a long lasting antidepressant‐like effect in male WKY rats (p=0.003), while ketamine does not.

Conclusions

These results indicate that at least a substantial portion of the ability of psychedelics to produce long lasting therapeutic effects has a biological basis and can be studied in animal models.

Support or Funding Information

This work was supported by Eleusis, PBC.

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 opioid receptor positive allosteric modulator BMS‐986187 enhances some, but not all, delta‐opioid receptor mediated behaviors
Authors: Jutkiewicz Emily M , Palleiko Benjamin A , Dripps Isaac J , Rice Kenner C
Published: June 2021

Activation of the delta opioid receptor (DOR) by orthosteric agonists produces antinociception, antihyperalgesia, antidepressant‐like effects, and convulsions in animal models. Positive allosteric modulators (PAMs) bind to a receptor site distinct from the orthosteric binding site and can augment the activity of the endogenous system. Therefore, a DOR PAM could be an effective strategy for eliciting potential therapeutic effects without convulsions. To evaluate this hypothesis, we investigated the ability of the opioid receptor PAM BMS‐986187 to elicit DOR‐mediated behaviors in mice alone or in combination with the DOR orthosteric agonist SNC80 and evaluated the potential contribution of multiple opioid receptor subtypes to the observed behavioral effects. Antidepressant‐like effects were evaluated in the mouse forced swim test and convulsions were evaluated by continuous observation. The contribution of DORs and mu‐opioid receptors (MORs) was evaluated through pharmacological and genetic manipulation of the receptor subtypes. BMS‐986187 alone produced antidepressant‐like effects without convulsions. These antidepressant‐like effects were blocked by the DOR antagonist naltrindole and absent in DOR knockout mice, indicating a DOR‐mediated effect. The antidepressant‐like effects of BMS‐986187 were enhanced in MOR knockout mice and following betaFNA pretreatment, suggesting that activation of MORs functionally inhibited DOR‐mediated antidepressant‐like effects. BMS‐986187 increased the potency of the orthosteric DOR agonist SNC80 to decrease immobility in the forced swim test but not to elicit convulsions. In addition, SNC80, but not BMS‐986187, enhanced the convulsive effects of pentylenetetrazol. Overall, these findings suggest that BMS‐986187 has activity at DORs and MORs in vivo and enhances some, but not all, behavioral effects of DOR agonists.

Support or Funding Information

This work was funded in part by a Research Starter Grant from the PhRMA Foundation.

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
MDMA and Pavlovian Fear Memory: Dose‐Effect Analysis
Authors: Pantoni Madeline M , Anagnostaras Stephan G
Published: June 2021

±3,4‐methylenedioxymethamphetamine (MDMA) is a recreational drug that is also being pursued as a therapeutic for PTSD and other mood and anxiety disorders. Despite strong evidence of its therapeutic potential, these pursuits are hindered by evidence that MDMA produces robust neurotoxicity and cognitive deficits at high doses. These findings, however, may not generalize to typical recreational or therapeutic use of low‐dose MDMA. To date, there is little research on the cognitive effects of low/moderate doses of MDMA (less than 3 mg/kg) – the doses that users typically take. In the present study, we examined the effects of MDMA on learning and memory across a range of doses using a Pavlovian fear conditioning paradigm. Hybrid C57BL/6Jx129S1/SvImJ mice were randomly assigned to groups by MDMA dose. Mice were trained on‐drug with a single tone‐shock pairing and then tested off‐drug one week later for long‐term context and tone fear memory. We assessed the effects of doses of 0.1 to 8 mg/kg MDMA relative to vehicle control on immediate memory and long‐term contextual and cued memory. With increasing interest in therapeutic uses of MDMA, this research will help determine if low doses are therapeutically viable in terms of adverse effects on learning and memory.

Support or Funding Information

NIH/NIDA Grant DA020041 (Anagnostaras)

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Mice with Gnao1 G203R Gain‐of‐Function (GOF) Mutation Phenocopy Combined Movement Disorder and Seizures of Patients
Authors: Larrivee Casandra L , Feng Huijie , Leipprandt Jefferey , Dimireva Elena , Xie Huirong , Neubig Richard
Published: June 2021

Patients with de novo mutations in the gene for Gao (GNAO1) exhibit either early infantile epileptic encephalopathy (EIEE17) or neurodevelopmental delay with involuntary movements (NEDIM). Gao is the a subunit of the most abundant heterotrimeric G protein in brain. As with other Gi/o family G proteins, it can signal directly to effectors or by release of free Gbg resulting in inhibition of adenylyl cyclase or modulation of Ca++ and K+ ion channels. Loss‐of‐Function (LOF) alleles in GNAO1 result in epilepsy while GOF mutations result in movement disorder with or without seizures. Here we report the first mouse model of a human GNAO1 encephalopathy. Heterozygous GOF mutant mice (Gnao1+/G203R) were created using CRISPR/Cas9 to determine whether they replicate the clinical pattern of children with de novo GNAO1+/G203R mutations who display epilepsy as well as severe choreoathetosis and dystonia. There was a striking sex difference in the observed phenotypes. Male Gnao1+/G203R mice displayed significant deficits in open field, Rotarod®, Digigait®, and grip strength assessments. Also, they had enhanced seizure susceptibility in pentylenetrazol (PTZ) kindling studies. None of these differences were seen in female mice. Unlike children with the GNAO1 G203R mutation, there were no obvious dystonic or choreoathetotic movements or postures at baseline. After administration of oxotremorine, however, male Gnao1+/G203R mice displayed more forelimb raising and hindlimb splaying than was observed in WT mice. This novel model phenocopies the combined epilepsy and movement disorder phenotype of children with GNAO1 G203R mutations. These mice should facilitate studies of neural mechanisms and permit preclinical drug repurposing.

Support or Funding Information

Supported by grants from the MSU Clinical and Translational Science Institute and The Bow Foundation.

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Bimolecular modeling and simulation of antagonist binding to the alpha‐1A adrenergic receptor
Authors: O'Connell Timothy D , Aragon Ingrid R , Healy Chastity L , Muthyala Ramaiah , Sham Yuk
Published: June 2021

Benign prostatic hyperplasia (BPH), which is a noncancerous enlargement of the prostate, currently affects 70% of men 60–69 years old, and 80% of men 70 years and older. By 2030, BPH incidence is predicted to increase based on the aging US population, and estimates suggest 20 million men 65 years and older will be diagnosed with BPH. BPH symptoms include frequent urination, difficulty initiating, maintaining, or a weak stream during urination, as well as inability to urinate or loss of bladder control. α1‐Adrenergic receptors (α1‐ARs), and particularly the α1A‐subtype, are highly expressed in prostate smooth muscle cells and mediate contraction. Clinically, α1‐AR antagonists, or α1‐blockers, are the primary treatment BPH with lower urinary tract symptoms (LUTS), including tamsulosin, touted as an α1A‐subtype selective antagonist. Here, our goal was to understand the molecular forces governing α1‐blocker binding to α1‐ARs to better understand α1‐blocker pharmacology. We have developed a homology model of α1‐antagonist binding to the α1A‐AR to identify essential molecular parameters that can be exploited for structure‐based drug design. Through homology modeling, docking, and molecular dynamics simulation, our preliminary model complex suggests for the first time 1) the burial of the terminal aromatic core within the hydrophobic pocket α1A‐AR and 2) the salt bridge interaction between its charged ammonium group with ionized aspartate are essential for ligand binding. This finding provides invaluable insight into design of the next generation, subtype‐specific of novel a1‐blockers.

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 Docking‐Based Virtual Screen for Bifunctional μ‐Opioid Agonist/δ‐Opioid Antagonist Compounds
Authors: Chan Wallace , Zhang Yang , Traynor John
Published: June 2021

The opioid receptors are family of G protein‐coupled receptors responsible for the modulation of pain, motor control, and mood. Opioid drugs, such as morphine, typically target the mu opioid receptor to elicit analgesia. However, a plethora of side effects, such as respiratory depression and addiction, manifest alongside their use, limiting their effectiveness in the long term. With the opioid crisis in our midst, the need for the development of a safer opioid with fewer or no side effects for pain management is of utmost importance. Recently, “mixed‐efficacy” bifunctional compounds acting as mu opioid receptor (MOR) agonists and delta opioid receptor (DOR) antagonists have been developed and shown in animal studies to produce analgesia with less potential for the development of tolerance and dependence. Consequently, the discovery of novel scaffolds for such compounds could lead to the development of safer opioid analgesics. Here, we report the development and validation of a sequential ligand‐ and structure‐based virtual screening pipeline. A ligand‐based virtual screen was used to establish an opioid‐enriched, targeted database for subsequent docking with AutoDock Vina. Retrospective virtual screening against a docking‐based benchmark dataset resulted in enrichment factor values from 7.8‐fold to over 10‐fold over random for both MOR and DOR, respectively, validating the efficacy of the model. In a prospective virtual screen, over 13 million compounds from the drug‐like subset of compounds in the ZINC database were screened, after which 360 unique compounds chemically dissimilar to currently‐known opioids were selected by manual inspection or clustering. Experimental validation of high‐scoring candidate compounds is underway to determine affinity and efficacy at MOR and DOR, and selectivity over other receptors.

Support or Funding Information

Discovery of New Treatments for Drug Abuse. Husbands, Stephen. Traynor, John. National Institute of Health (NIH). National Institute on Drug Abuse (NIDA). Project #: 5R01DA007315‐24

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
In Vitro Evaluation of Cimetidine Reno‐protective Effects on Cisplatin‐Induced Kidney Injury
Authors: Mody Hardik , Vaidya Tanaya , Lesko Lawrence , Ait‐Oudhia Sihem
Published: June 2021

Objectives

Cisplatin (CIS), a platinum‐based drug, is one of the most efficacious anti‐cancer agents prescribed for a variety of solid tumors. However, CIS‐induced kidney injury (CIKI) remains a clinical challenge. The Organic Cation Transporter 2 (OCT2) is an uptake transporter predominantly expressed in the kidney proximal tubule cells. OCT2 is responsible for the cellular uptake of CIS by kidney cells. Our study aims to evaluate and quantify the in vitro renotoxicity effect of CIS and the reno‐protective effect of cimetidine (CIM), an OCT2 inhibitor, against CIKI.

Methods

Human renal proximal tubular epithelial cells, SA7K, were exposed for 72 hours to a range of concentrations of CIS (1–100 μM), CIM (0.1–2 mM), or to at least 12 different combinations of the two drugs. The inhibition of cellular viability was measured at 0, 12, 24, 48, and 72 hours using a CCK‐8 cellular viability kit. CIKI was captured with CIS stimulation of apoptosis in SA7K cells. CIM action on SA7K cells was fixed to the control (no treatment) baseline value. The combination of CIM with CIS was characterized with a competitive antagonism model using the Gaddum equation. Mathematical modeling was performed using Monolix (2016R1). All estimated parameters were determined as mean±SE.

Results

A dose‐dependent pattern in CIS toxicity on SA7K cells and CIM protection from CIKI is observed. The first‐order rate constant for SA7K cells death was estimated at 0.0047±0.00024 h−1. The maximal effect of CIS on CIKI (Smax) was determined at 0.07±0.0064 h−1, while CIS concentration inducing 50% of Smax was estimated at 70.3±10.5 μM. The concentration of CIM leading to 50% of Smax was determined at 486.3±69.8 μM. All time course profiles of cell viability responses to the single agents and combinations of CIS with CIM were described with our developed models.

Conclusions

The combination of CIM with CIS was successfully evaluated in vitro. The competitive antagonism model adequately captured the reno‐protective effects of CIM against CIKI. Further studies are in progress to investigate the molecular mechanisms underlying the beneficial effects of CIM on CIKI.

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 Pharmacological Properties of Artemisia monosperma (Del.)
Authors: Abu‐Niaaj Lubna F. , Katampe Ibrahim , Abdulla Shtaywy
Published: June 2021

Plants are rich in secondary metabolites reputed for their pharmacological properties. With demands for bioactive compounds of natural origin, the attention is on commercializing the use of plants for pharmaceutical benefits. Our focus in on defining the biological activities of plants reputed in folk medicine to treat illnesses. We have been focusing on studying Artemisia monosperma to evaluate its potential usage for therapeutic uses. A. monosperma is a desert plant commonly known as an antispasmodic herb. We report that the boiled aqueous extract showed a concentration‐dependent antispasmodic effect of rat smooth muscle of ileum, pulmonary artery and uterus. The effect induced on the tracheal muscle was minimal while the contractile activity of the urinary bladder muscle was not affected. The activity of the cardiac muscle was inhibited upon addition of the aqueous extract which caused a decreased the rat heart rate accompanied by an increased in the contractile activity. This effect was reversible upon removal of the extract. Among compounds identified from A. monosperma, three flavonoids (arcapillin, eupatilin,7‐O‐Methyleriodictyol) showed a concentration‐dependent (10−7M −3×10−4 M) antispasmodic effect on rat smooth muscle of ileum, uterus and pulmonary artery. Only eupatilin and 7‐O‐Methyleriodictyol inhibited the contraction of trachea and urinary bladder. Further evaluation of the physiological effect of eupatilin on cardiac muscle showed that low concentrations (10−6 M and 10−5 M) caused an increase in the magnitude of cardiac muscle contraction accompanied by a decrease in heart rate which was followed by a partial heart blockage. At a concentration of 10−4M, the heart rate was reduced significantly leading to irreversible death of the infused heart. Eupatilin toxicity was determined by the median lethal dose (LD50) over 48hours on albino mice which came up to 31.8mg/kg. These finding indicate the importance of A. monosperma as a source for bioactive compounds of therapeutic significance.

Support or Funding Information

Special thanks to Central State University and the University of Jordan for the technical support.

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Identification of Covalent Modifications Derived from the GSTZ1‐catalyzed Metabolism of Dichloroacetate
Authors: Squirewell Edwin , Zhong Guo , James Margaret
Published: June 2021

Glutathione transferase zeta 1 (GSTZ1) is a cytosolic and mitochondrial enzyme with a critical role in tyrosine catabolism via its trans‐isomerization of maleylacetoacetate (MAA) and maleylacetone (MA). Dichloroacetate (DCA) is an investigational drug utilized in the treatment of acquired and congenital mitochondrial diseases. Dechlorination of DCA to its inactive metabolite, glyoxylate, is catalyzed by GSTZ1, and allelic variants within GSTZ1 affect the kinetics of DCA conversion. DCA is a mechanism‐based inactivator of GSTZ1, and chronic DCA dosing results in the auto‐inhibition of DCA metabolism. The tissue accumulation of DCA, MAA, and MA due to GSTZ1 degradation may contribute to the observed side effects of DCA, as chronic dosing can lead to the development of reversible peripheral neuropathy in some patient populations. The mechanism for DCA‐induced inactivation of GSTZ1 is postulated to proceed via the formation of a carbonium‐sulfonium intermediate derived from the GSTZ1‐catalyzed reaction of glutathione and DCA. The intermediate may react with nucleophilic sites on GSTZ1 to yield covalently‐modified enzyme or undergo hydrolysis to form glyoxylate. Modification to Cys‐16 within the 14SSCSWR19 glutathione binding motif of GSTZ1 has been described as a mechanism of DCA inactivation, although the mechanism for haplotype differences remain unclear. Therefore, we examined multiple GSTZ1 isoforms for variations in covalent modifications derived from DCA metabolism. GSTZ1 haplotypes 1B, 1C, and 1D (0.5 mg, each) were incubated for 10 hours at 37C in the presence of 2 mM DCA, 5 mM GSH, and 1 mg/mL BSA as stabilizer. The precipitates were collected by centrifugation and separated via SDS‐PAGE to isolate bands for GSTZ1, which were then excised and subjected to tryptic digestion for mass spectral characterization. We confirmed the addition of glyoxylate (+74 m/z) to multiple serine residues within GSTZ1 and present the following serine modifications: Ser‐47 (1C), Ser‐187 (1B and 1D), Ser‐188 (1C and 1D), and Ser‐202 (1B, 1C, and 1D). Each haplotype demonstrated a single adduct of +364 m/z to Cys‐205 within the peptide 193LLVLEAFQVSHPCR206. The change in mass is consistent with an adduct that contains a molecule of glutathione bound to the backbone of DCA, which we describe as S‐carboxy‐methylglutathione. This is a newly characterized modification for the DCA‐induced inactivation of GSTZ1. The incidence of S‐carboxy‐methylglutathione was low and its role in GSTZ1 inactivation has yet to be explored. DCA‐induced inactivation of GSTZ1 could be due, in part, to the aggregation of proteins covalently modified by glyoxylate, and the number of sites accessible to covalent linkages may vary between GSTZ1 haplotypes.

Support or Funding Information

Supported in part by the US Public Health Service GM 099871

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