bluuebunny/crossref_metadata_2025 huggingface dataset:
Movements of N‐terminus of LeuT during the Substrate Transport Cycle
Authors: Khan Jawad Akbar , Sohail Azmat , Sandtner Walter , Sitte Harald H. , Stockner Thomas
Published: June 2021

LeuT from Aquifex aeolicus aeolicus is a bacterial homologue to mammalian solute carrier family 6 (SLC6) of neurotransmitter transporters. SLC6 transporters are of great pharmacological interests because of their crucial role in neurotransmitter clearance. These proteins are also targets of many clinically relevant drugs, including antidepressant drugs and drugs of abuse such as amphetamines. The amino terminus of SLC6 transporters plays an important regulatory role for function.

High resolution X‐ray crystal structures of LeuT are available in different conformations. However, these structures are not able to fully explain the dynamic nature of LeuT function during the substrate transport cycle. The aim of this project is to look at the movements of the N terminus of LeuT in the proteoliposomes using spectroscopic based technique called lanthanide based resonance energy transfer (LRET).

We developed a LeuT variant in which we inserted a Lanthanide Binding Tag (LBT) at position 335, positioned at the end of extracellular loop 4. Three LeuT mutants (K4C, H7C and A9C) were created in the background of the LeuT‐LBT construct. Using LRET based distance measurements we determined the distance between the LBT bound lanthanide and the fluorophore TMR attached to the cysteines in the N‐terminal of LeuT. The results did not show the expected increase in distance towards the N‐terminus, as the distance to H7C was shorter than to A9C. In contrast, iodate quenching experiments of TMR fluorescence showed no difference in accessibility to KI for all three residues.

Intramolecular distances determined by LRET suggest that the fluorophore attached to H7 could be oriented towards the inner vestibule, while the KI based accessibility experiments displays a contrasting picture. In order to elucidate the dynamics of the N‐terminus and support one of the two findings, I am further planning to stabilize the transporter in an outward open conformation using tryptophan, and as an alternative strategy, stabilize LeuT‐LBT in the inward‐facing and an outward‐facing conformation using the R5A or the R30A mutants.

Support or Funding Information

This work is jointly supported by the Austrian Science Fund (FWF) Austria and Higher Education Commission (HEC) Pakistan

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Co‐expression of Cdr1as and miR‐7a is associated with hypoxia‐affected fetal brain development
Authors: Lv Yehui , Li Shiying
Published: June 2021

Hypoxia influences different cellular biological processes and hypoxia during pregnancy can adversely affect brain development. MicroRNA (miRNA) and emerging circular RNA (circRNA) are critical regulators of multiple cell processes including hypoxia. Recently, one human circRNA, antisense to the cerebellar degeneration‐related protein 1 transcript (Cdr1as) has been identified to share expression domains with miR‐7a. This study aimed to investigate the expression pattern of Cdr1as/miR‐7a and further to understand the impact of prenatal hypoxia on brain development. For this purpose, pregnant SD rats were exposed to either hypoxia or normoxia and fetal brains were further collected at day 20. Fetal cortex weight and ratio of brain‐body weight were reduced. Subsequently, real‐time PCR was performed to quantify the expression of Cdr1as and miR‐7a in both normoxic and hypoxic neonatal cortex samples. The expression levels of both Cdr1as and miR‐7a were both upregulated in the cortex tissues of the offspring exposed to hypoxia. Moreover, miR‐7a's target SP1 (Specificity Protein 1), which had been confirmed in present study (Geng Hai‐Hua, 2016), were found to be down‐regulated in both mRNA and protein levels following prenatal hypoxia. These results indicate that the co‐expression of Cdr1as and miR‐7a play roles in the hypoxia response and these observed molecular alterations may provide insights into prenatal‐hypoxia‐impaired brain development with further study.

Support or Funding Information

Ethical permission was granted through the Science and Ethics Committee of Shanghai University of Medicine & Health Sciences. This study was funded by the Seed Fund of Shanghai University of Medicine & Health Sciences and Open Foundation of Shanghai Key Laboratory of Forensic 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.
Red Blood Cells Specifically Bind a C‐peptide/Albumin Complex
Authors: Geiger Morgan , Kauffman Nathan , Zinn Kurt , Spence Dana
Published: June 2021

People with Type 1 diabetes (T1D) require exogenous insulin due to the destruction of insulin‐secreting pancreatic β‐cells. However, even with insulin treatments, people with T1D develop complications such as retinopathy, neuropathy, and nephropathy that are often associated with poor microvascular blood flow. C‐peptide, a 31‐amino acid length peptide co‐secreted with insulin from the pancreatic β‐cells in a 1:1 mole ratio has been shown to improve microvascular blood flow in T1D rats and small‐scale human studies. C‐peptide indirectly enhances endothelium‐derived nitric oxide (NO) by stimulating the release of red blood cell (RBC) derived ATP, a recognized stimulus of endothelial NO synthase. Unfortunately, a receptor for C‐peptide has never been reported. Previously, using ELISA, we reported C‐peptide uptake by RBCs was approximately 1800 C‐peptide molecules/cell, but only when in the presence of albumin. In the absence of albumin, there was no measurable C‐peptide binding to the RBCs. We also report C‐peptide binding to albumin with a Kd of 5.7 ± (2.1) × 10−6 M; therefore, we hypothesize that C‐peptide binds to RBCs via a receptor that is specific for an albumin/Cpeptide complex. The binding of 99mTc labeled bovine serum albumin (BSA) to isolated RBCs, in the presence and absence of C‐peptide, was quantitatively determined by automatic gamma counting after repeated washing with a physiological salt solution. Sample counts per minute (CPM) were compared to a standard curve to determine the 99mTc‐BSA molecules per RBC. Results suggest BSA total binding to the RBCs saturates at approximately 1600 BSA molecules/RBC, whether or not C‐peptide is present, but that the BSA‐99mTc is specifically binding to RBCs in the presence of C‐peptide. Collectively, our data strongly suggest that the Cpeptide receptor has remained elusive because the receptor specifically binds an albumin/Cpeptide complex, as opposed to C‐peptide alone.

Support or Funding Information

NIH

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Tubastatin A attenuates chronic angiotensin‐II‐induced autophagy
Authors: Rajasingh Sheeja , Czirok Andras , Isai Dona Greta , Rajasingh Johnson
Published: June 2021

Introduction

It has been shown that autophagy is playing one of the crucial cellular functions, which maintain the cellular homeostasis as well as cardiac hypertrophy, but the mechanism is poorly understood. Studies have shown that over activation of autophagy is not always protective but may cause autophagic cell death. Recent reports have demonstrated that STAT3 participates in the process of autophagy. Bcl2 is an anti‐apoptotic and anti‐autophagic protein that inhibits autophagy through direct interaction with the BH3 domain of pro‐autophagic protein beclin1. Here we have examined the protective role of HDAC6 inhibitor Tubastatin A (TBSA) during chronic angiotensin II‐induced autophagy in human iPSC‐derived cardiomyocytes (hiCMCs).

Hypothesis

We hypothesize that TBSA treatment mediated activation of STAT3‐Bcl2 is a critical regulator of angiotensin II‐induced hiCMCs autophagy and the over expression of HDAC6 in hiCMCs leads to exaggerate angiotensin II‐induced autophagy.

Methods and Results

We have evaluated the TBSA treatment mediated effects in the contractility of hiCMCs, cell size and mitochondrial membrane potential in response to angiotensin II‐induced autophagy. Our data shows that the functional effect of hiCMCs stimulated with angiotensin II has significantly reduced the beating frequency, whereas treatment with TBSA rescued the beating frequency (Fig. A, B). Furthermore, the angiotensin II‐induced hypertrophic effect on hiCMCs is significantly reduced by TBSA treatment (Fig. C). Moreover, the immunostaining with JC1 (marker for Autophagy) shows that the mitochondrial membrane potential significantly reduced in angiotensin II‐induced hiCMCs, whereas TBSA treatment maintains the mitochondrial membrane potential (Fig D). Moreover, our electron microscopic analysis revealed a dramatic difference was observed between angiotensin II induced hiCMCs and Angiotensin II‐induced hiCMCs treated with TBSA in actin cytoskeletal protein and disorganized sarcomeres.

Conclusions

We report for the first time that the role of HDAC6 in regulation of pathological autophagy, thus can acts as a potential therapeutic target for treatment of chronic heart diseases.

Support or Funding Information

American Heart Association Grant‐in‐Aid Grant 16GRNT30950010

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 Myosin Chaperon, UNC‐45A, Is a Key Regulator of Intestinal Epithelial Apical Junctions
Authors: Lechuga Susana , Ivanov Andrei I
Published: June 2021

Epithelial adherens junctions (AJs) and tight junctions (TJs) play critical roles in regulating the integrity and permeability of epithelial barriers. Proper assembly and functioning of AJs and TJs depend on their associations with underlying actin filaments and the actin motor protein, nonmuscle myosin II (NM II). Our previous studies demonstrated the roles of NM II in controlling assembly and permeability of intestinal epithelial apical junctions in vitro and in vivo. However, the molecular mechanisms that regulate NM IIA functions in intestinal epithelium remain poorly understood. UNC‐45A is a highly conserved member of the UNC‐45/CRO1/She4p family proteins, which acts as a NM II chaperone and controls a proper folding and activity of myosin heavy chains. In the present study, we investigated the roles of UNC‐45A in the regulation of apical junctions in model intestinal epithelial cell monolayers.

UNC‐45A protein was abundantly expressed in several commonly‐used human intestinal epithelial cell lines. Furthermore, its expression in T84 and HT‐29 cells was significantly downregulated by proinflammatory cytokines, IFN‐γ and TNF‐α, in parallel to cytokine induced TJ disassembly and barrier disruption. Consistently, UNC‐45A and NM IIA expression was downregulated in colonic epithelial cells isolated from tissue samples of ulcerative colitis patients. CRISPR/Cas9 mediated knock‐out of UNC‐45A in SK‐CO15 and HT‐29 human colonic epithelial cells resulted in multiple defects of apical junctions. These defects include the decreased transepithelial electrical resistance, increased transepithelial dextran flux, impaired steady‐state structure of AJs and TJs, and attenuated junctional reassembly during calcium switch. Together, our findings revealed a novel cytoskeleton‐dependent mechanism that regulates structure and functions of normal intestinal epithelial barrier and may contribute to barrier disruption during mucosal inflammation.

Support or Funding Information

This work was supported by NIH grant R01DK108278.

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Light/Ultrasound Responsive 750 nm‐Emitted Non‐toxic Indium Phosphide Quantum Dots Hybrid Nanobubble for Brain Tumor Imaging
Authors: Chan Ming‐Hsien , Liu Ru‐Shi , Hsiao Michael
Published: June 2021

Background

The blood‐brain barrier (BBB) hardly allows all substances to pass. Except for oxygen, carbon dioxide, and blood sugar, most dyes and proteins are generally unable to pass due to their molecular structure. Therefore, bioimaging in brain tumor is very challenging. Moreover, different diagnostic methods have their own advantages and disadvantages. Composite imaging is considered to be a more efficient method of bioimaging.

Design

The study initially constructed an ultrasonic imaging material that can be used for quantum dot bonding, which allows the nanomaterial to have both supersonic and optical diagnosis. We intend to synthesize lipid‐phase nanobubbles to combine with 750 nm emitted non‐toxic indium phosphide (InP) quantum dots, providing multiple imaging functions. Choose for high biocompatibility and for in vivo and in situ diagnosis. Moreover, recent researches have demonstrated that cavitated nanobubbles can introduce a small opening in the BBB, allowing materials extravasation. It makes InP Quantum dots can enter in brain tissue to generate the near‐infrared signal for the signal tracking.

Results

Nanobubble subjected to a shock wave that disrupts the BBB. The cavitation effect makes an opening space, allowing small particles extravasation. InP quantum dots‐loaded nanobubbles activated by ultrasound can temporarily permeabilize into the BBB, causing quantum dots accumulation in the brain tumor tissue, as evidenced by ultrasound and fluorescence imaging.

Conclusions

Nanobubbles cavitation bioimaging displays the mean intensity of acoustic cavitation over time and was correlated with areas of acoustic cavitation‐induced BBB opening. After combining the InP quantum dots to the lipid bubbles, the nanocomposite is expected to have the functions of ultrasonic and optical imaging. We chose the best near‐infrared light to make the signal of brain tumor can be detected.

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Analysis of the Effects of Toll‐like Receptor 3 (TLR3) on the Pathology of Chlamydia muridarum in the Genital Tract of Wild‐Type and TLR3 Knockout C57BL/6N Mice
Authors: Billingsley Caylin , Derbigny Wilbert , Sandusky George
Published: June 2021

In 2017, a total 1,708,569 chlamydial infections were reported to Centers for Disease Control and Prevention; the most prevalent sexually transmitted disease (STD). Chlamydia trachomatis primarily replicates in epithelial cells lining the reproductive tract. Epithelial cells recognize chlamydia through cell surface and cytosolic receptors, and/or endosomal innate receptors such as Toll‐like receptors (TLRs). The activation of these receptors triggers immune mechanisms that are required for chlamydial clearance. It was previously shown that Chlamydia muridarum (Cm) induces IFN‐β in oviduct epithelial cells in a TLR3‐dependent manner. TLR3's regulation of inflammatory chemicals involved in host defense against Cm infection led to the hypothesis that TLR3 plays a protective role against Cm‐induced genital tract pathology in congenic C57BL/6N mice. To test this hypothesis, wild‐type and TLR3‐deficient mice were treated with vehicle and Cm by swabbing the vaginal vault and cervix. Tissues were extracted from mice sacrificed on day 7, 21, or 42 post‐infection and fixed in 4% paraformaldehyde for 48 hrs. Paraffin‐embedded, 4‐μm sections containing cervix and both uterine horns and oviducts were stained with H&E. Genital tract sections (cervix, uterine body and horns, and oviducts) were microscopically examined and graded for the presence of acute inflammation (neutrophilic infiltrates), chronic inflammation (lymphocytic infiltrates), epithelial erosion and disruption, and luminal distension of uterine horns and oviducts using a semi qualitative pathology assessment. Inflammatory cell infiltrates, epithelial erosion and disruption were scored as follows: 0, normal; 1, mild; 2, moderate; 3, severe. All scoring was performed in a double‐blinded fashion with regards to the treatment groups. The results revealed an inflammatory immune response showing minute differences in both groups at day 7 (acute inflammation) in the oviducts, cervix and uterus. By day 21 (chronic inflammation), there was more inflammation seen in the WT group compared to the knock out TLR3 group. By day 42, there was mild chronic inflammation in the uterus in both groups; however, the knockout group had more cystic endometrial change in the uterus compared to the wild type group. In conclusion, the data suggest that TLR3 may promote the clearance of Cm.

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
mTOR Inhibition Delays Hepatoblastoma Growth in a Relevant Mouse Model
Authors: Monga Satdarshan Pal , Molina Laura Maria , Yang Hong , Adebayo Michael Adeola O. , Tao Junyan , Bell Aaron
Published: June 2021

Hepatoblastoma (HB) is the most common pediatric liver malignancy, affecting children in their first few years of life. Around 80% of HB demonstrate simultaneous activation of β‐catenin and Yes‐associated protein 1 (YAP1), which are transcriptional coactivators downstream of the Wnt and Hippo signaling pathways, respectively. Recently, mTORC1 activation was reported in human HB cell lines as well as HB tumors in a mouse model driven by β‐catenin and YAP1. In the current study we investigated directly the impact of mTOR inhibition on HB development in a YAP1‐β‐catenin HB mouse model. Hydrodynamic tail vein injection (HTVI) was used to co‐deliver Sleeping Beauty transposase along with plasmids coding for ΔN90‐β‐catenin and S127A‐YAP1. Five weeks post‐HTVI, when HB tumors are evident, we treated half of the mice with rapamycin via diet, and both control and treated groups were monitored with ultrasound imaging to assess tumor growth over time. All mice were sacrificed at 10 weeks post‐HTVI. Our results show that mice treated with rapamycin for 5 weeks developed significantly less HB tumor burden than control mice, with almost normal liver weight to body weight ratio and fewer, smaller tumors observed histologically. We show by immunohistochemistry that rapamycin treatment reduces activation of downstream targets of mTORC1 signaling such as phosphorylation of 4EBP1 and S6‐ribosomal protein. Importantly, we show by ultrasound imaging that control mice exhibit a dramatic rise in tumor number and volume starting at ~5 weeks post‐HTVI, while rapamycin‐treated mice begin to show a similar increase only at 8–9 weeks post‐HTVI (after 3–4 weeks of rapamycin treatment). Interestingly, while a majority of HB tumors in the control group exhibit embryonal histology, the tumors that developed in rapamycin‐treated mice exhibit more well differentiated, fetal histology. Thus we show that while rapamycin does not completely block HB tumor formation, it significantly delays the development of tumors driven by β‐catenin and YAP1. Our study also demonstrates the utility of standard and 3D ultrasound imaging for the monitoring of liver tumor formation in mice and supports the use of mTORC1 inhibitors for HB treatment.

Support or Funding Information

This work was supported by NIH grants 1R01DK62277, 1R01DK100287, 1R01CA204586, and the Endowed Chair for Experimental Pathology (S.P.M.), as well as CATER T32 grant 5T32EB001026‐14 (L.M.).

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Isoflurane‐Induced Hypothermia Does Not Aggravate Memory Impairment in A Sporadic Alzheimer's Disease Rat Model Produced by Intracerebroventricular Streptozotocin
Authors: Vizin Robson Cristiano Lillo , Harris Grant , Kunstetter Ana Cançado , Almeida Maria Camila , Carrettiero Daniel Carneiro , Romanovsky Andrej A.
Published: June 2021

Several studies have shown that hypothermia and anesthesia are risk factors for Alzheimer's disease. Most of them were performed on healthy elderly rodents. However, the effect of anesthesia‐induced hypothermia in memory in a sporadic Alzheimer's disease animal model has never been studied. In this study, Wistar rats (3‐month‐old) were induced to anesthesia with 3% isoflurane and maintained with 1.5–2% isoflurane in 100% oxygen for 3 hours. In the first 30 min of anesthesia, rats were subjected to a single bilateral intracerebroventricular injection of streptozotocin (STZ, 1 mg/kg) to produce the sporadic Alzheimer's disease model, or its vehicle (0.05 mol/L citrate buffer, pH 4.5, 2 μL/ventricle). The rats were either subjected to hypothermia or not, induced by isoflurane (hypothermic and normothermic groups, respectively). In this sense, during anesthesia, normothermic rats were kept on a heating pad, and their rectal temperature was maintained at 37.5 ± 0.5°C. Hypothermic rats were kept without a heating pad. Spatial working memory was evaluated using Morris Water Maze from the 3rd to 28th‐day postsurgery, and by Y‐maze test on the 30th‐day postsurgery. Recognition memory was evaluated on the 31st‐day postsurgery using the Novel Object Recognition Test. All the procedures were approved by and are in accordance with the local ethics committee from St Joseph's Hospital and Medical Center (protocol n° 485). In the Morris water maze test, both hypothermic and normothermic STZ‐injected rats shown similar impairment in spatial working memory compared to their respective controls, i.e., hypothermic and normothermic vehicle‐injected rats, respectively. In the Y‐maze test, normothermic STZ‐injected rats shown a deficit in spatial working memory compared to normothermic vehicle‐injected rats (p = 0.028). However, this effect was missed in hypothermic groups with no difference between STZ‐ and vehicleinjected groups (p = 0.073). Similarly, in the Novel Object Recognition Test, recognition memory was impaired in normothermic STZ‐injected rats compared to normothermic vehicleinjected rats (p = 0.012) and no effect was observed between hypothermic STZ‐ and vehicleinject rats (p = 0.118). In conclusion, isoflurane‐induced hypothermia does not aggravate the STZ‐deleterious effect in recognition memory and spacial work memory in rats.

Support or Funding Information

This work was supported by the Brazilian National Council for Scientific and Technological Development (grant 449102/2014‐9 to D.C.C.) and the São Paulo Research Foundation (grants 2015/02991‐0 to M.C.A., 2015/23426‐9 to D.C.C., and 2016/01836‐3 to R.C.L.V.).

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Upregulation of Mitofusin2 in neurons slows down skeletal muscles atrophy in ALS disease and aging model
Authors: Wang Luwen , Gao Ju , Wang Xinglong
Published: June 2021

Skeletal muscles undergo atrophy in response to diseases and aging. Mitochondria, the source of cellular energy, were found to be fragmented in experimental models of ALS disease and aging. However, whether and how mitochondrial fragmentation plays a role in the progression of disease and aging is not clear. Mitofusin2 (Mfn2) is a key regulator of mitochondrial fusion which is crucial for the maintenance of mitochondrial morphology. To test whether Mfn2 reduction in motor neurons contributed to the disease progression in SOD1G93A mice or aged mice, we generated transgenic mice overexpressing Mfn2 specifically in neurons under the control of the Thy1.2 promoter (TMFN mice). Mfn2 overexpression was confirmed in spinal cords but not skeletal muscles. By crossing Mfn2 overexpressed mice with SOD1G93A mice, we obtained double transgenic Mfn2/SOD1G93A mice. Interestingly, mitochondrial fragmentation was abolished in motor neurons of Mfn2/SOD1G93A mice even in the disease endstage. Strikingly, characteristic neuromuscular junction (NMJ) denervation, collapse, and dysfunction in SOD1G93A mice were also greatly alleviated in Mfn2/SOD1G93A mice. The dramatic protective effect of neuronal Mfn2 in SOD1G93A mice was further indicated by the greatly delayed symptom onset of more than 60 days and the complete prevention of behavioral deficits at 120 days old, a very late stage immediately before endstage onset.

A similar protective effect was observed in aged mice. The Mfn2 neuronal overexpression mice suffered less neuromuscular synaptic dysfunction and skeletal muscle atrophy than their non‐transgenic litter mates even at 22 months old, which in human years is equivalent to the 70s. Taken together, our study provides evidence suggesting that Mfn2 deficiency induced mitochondrial fragmentation in motor neurons is likely an important mechanism leading to NMJ dysfunction and skeletal muscle atrophy in ALS and aging, and that Mfn2 could be a common therapeutic target for skeletal muscle atrophy.

Support or Funding Information

This study is supported by grants from the US NIH (1R01NS097679 and 1R01NS089604), US Alzheimer's Association (AARG‐17‐499682), and US Association for Frontotemporal Degeneration and Alzheimer's Drug Discovery Foundation (20161206).

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Pharmacological Characterization of Kappa Opioid Receptor Agonists
Authors: Kaska Sophia , Kornberger Lindsay , Biggerstaff Andrew , Kivell Bronwyn , Prisinzano Thomas
Published: June 2021

Agonists of the kappa opioid receptor (KOR) demonstrate promise for treating pain without the typical side effects of mu opioid receptor agonists, such as respiratory depression and tolerance. Unfortunately, KOR agonists have their own side effects such as sedation and dysphoria, which necessitates further development of new KOR agonists that will maintain analgesic effects while eliminating these negative side effects. Through structure‐activity relationship studies, our laboratory has identified a series of potent and selective KOR agonists that demonstrate functional selectivity for the G‐protein pathway, increasing the propensity to display analgesic effects with fewer side effects that are mediated through beta‐arrestin. Furthermore, in vivo evaluation of the analgesic effects of these compounds demonstrated an increase in potency, compared to U50,488, consistent with our in vitro data. To further demonstrate the analgesic effects of our compounds, a time course experiment was performed using the hot water tail flick assay. Over this time course, these analogs at lower doses demonstrated the same efficacy compared to U50,488, indicating an increase in analgesic effect of these compounds. Together, these data demonstrate that our series of KOR agonists have improved in vitro and in vivo activity compared to U50,488 and can potentially be used for treating pain.

Support or Funding Information

This work was supported by NIH R01 DA018151 and the Health Research Council of New Zealand.

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Buprenorphine C7‐Esters with Improved Nociceptin Receptor Agonist Potency as Analgesics
Authors: Olson Keith Mathew , Cami‐Kobeci Gerta , Husbands Stephen M. , Traynor John R.
Published: June 2021

There is an urgent need for analgesics with reduced abuse liability, withdrawal, respiratory depression and constipation to treat chronic pain patients. While clinical opiates produce analgesia and associated side effects through Mu Opioid Receptor (MOR) agonist activity, preclinical evidence indicates activity at the Kappa‐ (KOR), Delta‐ (DOR), and Nociception Receptors (NOP) subtypes can minimize side effects. Compounds displaying NOP agonist and weak MOR partial agonist activity produce antinociception with minimal abuse liability in primates and therefore are desirable therapeutic target. In the present study we evaluated buprenorphine analogues with C7 ester substituents for opioid receptor affinity by competition binding assay and for function using the [35S]‐GTPgS assay at MOR, NOP, DOR and KOR. Compounds showed a nearly 1000‐fold range in binding and [35S]‐GTPgS activity at NOP, ranging from low nanomolar to micromolar. One compound, containing a phenyl ester at C7, and lacking a keto group at C6, showed the highest potency NOP activity (EC50= 0.97 +/− 0.22 nM, EMAX= 47% +/− 11) with weak partial agonist potency at MOR (EC50= 3.9 +/− 1.3 nM, EMAX= 23% +/− 7). Since this compound has less MOR agonist efficacy than buprenorphine, it would be predicted to have minimal abuse liability. The series also showed a synergistic relationship in NOP activity between the C7 functional group and the C6 oxidation state. For example, the presence of a keto group at C6 decreased NOP potency significantly (EC50= 164 +/− 51 nM, EMAX= 47% +/− 9). This pattern was observed with numerous C7 functional groups. All compounds in the series were putative antagonists at DOR and KOR as they bound with high affinity but did stimulate significant activity in the [35S]‐GTPgS assay. This series of compounds provides different degrees of partial agonism at MOR and NOP and should help to determine the minimal MOR activity necessary for effective antinociception while minimizing potential abuse liability.

Support or Funding Information

Supported by R01 DA07315 and 035316.

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 of the mGluR2/3 agonist LY379268, alone and in combination with monoamineenhancing drugs, on cocaine self‐administration in rhesus monkeys
Authors: Czoty Paul W , Coller Michael , Odom Jill , Landavazo Antonio , Blough Bruce E , Nader Michael A
Published: June 2021

Cocaine addiction persists as a major public health problem but still lacks any FDA‐approved pharmacotherapies. Wake Forest's Center for the Neurobiology of Drug Abuse Treatment (CNAT) has demonstrated the effectiveness of ‘agonist medications’ such as the monoamine releaser phenmetrazine (PM) and its pro‐drug phendimetrazine (PDM) to decrease cocaine use in animal models. CNAT investigators have hypothesized that combination of a dopamine‐enhancing drug like PM/PDM with a drug with a different pharmacological and/or behavioral mechanism may display enhanced effectiveness and represent a promising pharmacotherapeutic approach to treating cocaine use disorder. The present studies characterized the effects of the mGluR2/3 receptor agonist LY379268, alone and in combination with daily PDM or the dopamine transporter inhibitor methylphenidate (MPD) on cocaine reinforcement in rhesus monkeys. In one experiment (n=4), a multiple schedule was used in which food reinforcers were available in the first component under a fixed‐ratio schedule of reinforcement and cocaine self‐administration was studied under a progressive‐ratio schedule. A second study (n=4) used a food‐cocaine choice procedure in which a complete cocaine dose‐effect curve was generated each day. Effects of acute and chronic LY379268 were studied. Under the multiple schedule, acute LY379268 decreased food‐but not cocaine‐maintained responding; when administered chronically (as an i.v. injection) during chronic oral PDM treatment, a drug combination that persistently decreased cocaine self‐administration was found in only one of four subjects. Under the choice procedure, LY379268 increased the reinforcing strength of cocaine relative to food. After 5 days of treatment, tolerance developed to this effect in a dose‐related manner but at no point were decreases in cocaine choice observed. Finally, when chronic LY379268 was administered in combination with chronic oral PDM or chronic MPD (via subcutaneous osmotic pump), dose combinations were observed that modestly decreased cocaine choice in two of four monkeys. Although some results were positive, taken together these studies, which used highly translational nonhuman primate models of cocaine use disorder, do not support the effectiveness of LY 279638 alone or in combination with agonist medications.

Support or Funding Information

P50 DA06634

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Varying Effects of the G‐Protein Coupled Estrogen Receptor During Development in Embryonic Rat Hippocampal and Cortical Neurons
Authors: Pemberton Kyle , Xu Fenglian
Published: June 2021

Estrogen has been known to play a role in enhancing brain development for over half a century. Most studies of estrogen's effect on the nervous system have focused on the traditional estrogen receptors Estrogen Receptor‐α (ERα) and Estrogen Receptor‐β (ERβ). The recent discovery of the G‐Protein Coupled Estrogen Receptor (GPER) suggests knowledge of estrogen's effects may be incomplete. While evidence has suggested the roles GPER plays in the nervous system include neuroprotection, neuronal recovery from stroke and degeneration, neuron proliferation, as well as many others. A caveat is that most studies have looked only at the effect in mature neurons and not the effect GPER has during early neuronal development. Here we sought to study the role of GPER activity in neurite outgrowth and synapse formation during early development.

Embryonic day 18 rat cortical and hippocampal neurons were cultured in estrogen‐ and phenol red‐ free medium in the presence of a nonselective ER agonist (E2), a selective GPER agonist (G‐1), and a selective GPER antagonist (G‐15). Neurite growth was measured at 20, 48, 72, and 96 hours in culture using the ImageJ plugin NeuronJ. Synapses were visualized at 7 and 14 days in culture by using immunofluorescence of the pre‐ and post‐synaptic markers, synaptophysin and PSD‐95, respectively. Synapse formation was measured by measuring the overlap between these markers in ImageJ. Our data revealed activation of GPER promoted neurite growth in hippocampal neurons, but not in cortical neurons, but blocking the receptor inhibited neurite growth in cortical neurons. In addition, low levels of G1 (1μM) inhibited neurite outgrowth in cortical neurons. Cortical neurons showed no significant difference in synapse formation, while in hippocampal neurons the effect varied based on time period and dosage. This provides the first evidence that GPER activation may play different roles in brain regions, as well as having specific dosage based effects in specific regions. Future experiments will investigate the molecular and cellular mechanisms underlying the pharmacological differences in GPER effects between developing cortical and hippocampal neurons.

Support or Funding Information

This project was supported by the Saint Louis University Start‐up Fund for Dr. Xu lab and the Sigma XI Grant‐In‐Aid of Research awarded to Mr. Kyle Pemberton.

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Dimethyl fumarate alleviates the nitroglycerin (NTG)‐induced migraine in mice
Authors: Esposito Emanuela , Casili Giovanna , Lanza Marika , Filippone Alessia , Campolo Michela , Paterniti Irene , Cuzzocrea Salvatore
Published: June 2021

Oxidative stress and inflammatory pathways are involved in nitroglycerin (NTG)‐induced migraine and endogenous antioxidant defense system has a role in the prevention of hyperalgesia in migraine. In this study, we aimed to evaluate the role of the most pharmacologically effective molecules among the Fumaric acid esters (FAEs), dimethyl fumarate (DMF), nuclear factor E2‐related factor 2/antioxidant response element (Nrf‐2/ARE) pathway‐mediated, in regulating the hypersensitivity in a mouse model of NTG‐induced migraine. Mice were orally administered with DMF at the doses of 10, 30 and 100 mg/kg, 5 minutes after NTG intraperitoneal injections. We performed histological and molecular analysis on the whole brainand behavioral tests after 4 h by NTG‐migraine induction. The expression of nuclear factor kappa‐light‐chain‐enhancer of activated B cells (NF‐κB) subunit p65, nuclear factor of kappa light polypeptide gene enhancer in B‐cells inhibitor alpha (IκBα), inducible nitrite oxide synthase (iNOS), cyclooxygenase 2 (COX‐2), Nrf‐2, manganese superoxide dismutase (Mn‐SOD) and heme‐oxygenase‐1 (HO‐1) were detected by Western blot. Tail flick, hot plate, formalin and photophobia tests were used to evaluate neuropathic pain and migraine‐related light sensitivity. DMF treatment notably reduced histological damage as showed by cresyl violet staining; also, regulating both NF‐κB and Nrf‐2 pathway, DMF treatment decreased the severity of inflammation and increased the protective antioxidant action. Moreover, the headache‐associated neuropathic pain was significantly reduced. These results provide the evidence that DMF has a modulating effect on central sensitization induced by NTG, suggesting a new insight into the potential application of DMF as novel candidates in drug development for migraine.

Support or Funding Information

No funding

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 Role of Integrin β3 in Nucleus Accumbens Function
Authors: Jameson Alexis , Carneiro Ana , Grueter Brad
Published: June 2021

Stress disorders, depression, anxiety, and substance use disorder, are regulated by serotonin (5HT). The dorsal raphe nucleus (DRN), the major site for neuronal 5HT synthesis, sends projections to the nucleus accumbens (NAc). NAc glutamatergic synapse adaptations are consistent with changes in mood and reward, key components of stress disorders. A subpopulation of DRN projection neurons express the β3 subunit of the integrin αvβ3 receptor. The integrin αvβ3 receptor regulates extracellular 5HT concentration by modulation of 5HT transporters. Previous work has implicated this cell adhesion receptor in modulating stress, as mice lacking the β3 subunit have increased vulnerability to chronic stress. However, how NAc function is regulated by the DRN β3 subunit and contributes to maladaptive reward behaviors as seen in stress disorders is unknown.

Because the β3 subunit of the integrin avβ3 receptor is only expressed in a subpopulation of 5HT neurons, this suggests that it confers a specific function to this neuronal population. Using a mouse line with selective deletion of the β3 subunit within 5HT neurons, we are able to characterize its role in 5HT synaptic transmission. Using acute brain slice whole‐cell patch‐clamp electrophysiology, we characterize how the β3 subunit of 5HT inputs contribute to glutamatergic NAc synaptic transmission. Our data suggests that selective deletion of the β3 subunit in 5HT neurons blunts excitatory synaptic strength in the NAc. This suggests that the β3 subunit in 5HT neurons modulates excitatory input to the NAc, which may relate to stress response.

Support or Funding Information

NIH T32 5T32MH064913‐15

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 Involvement of GLT‐1 in The Drug Seeking Like Effects of Pregabalin
Authors: Althobaiti Yusuf , Nasr Abdulrahman , Alzahrani Omar , Almalki Hussam
Published: June 2021

Pregabalin is a GABA analogue that can bind with high affinity to voltage‐gated calcium channel to minimize excitatory neurotransmitters release. However, several case reports in Middle East and other parts of the world raised the concern of its abuse potential. We have shown in our previous studies that higher doses of pregabalin can cause drug seeking like effect in conditioned place preference (CPP) mouse model. In this study, we tested the involvement of glutamate transporter 1 (GLT‐1), which has a significant role in glutamate homeostasis and in mediating the rewarding and drug seeking effects of several drugs of abuse. Male BALB/c mice were randomly separated into four groups; the control group (C‐C) received saline in home cages and during the conditioning training. The second group (CEF‐C) received ceftriaxone (CEF) in a dose that was consistently shown to upregulate GLT‐1, (200 mg/kg, i.p.), in home cages and received saline during conditioning training. The third group (C‐Preg) received saline in home cages and were administered pregabalin in a dose that previously shown to induce CPP (60 mg/kg) during conditioning training. The last group (CEF‐Preg) received CEF in home cages and injections of pregabalin during the conditioning training. Consistent with our previous studies, the time spent in drug‐paired chamber as compared to the vehicle‐paired chamber was significantly increased in C‐Preg group. Importantly, CEF treatment for eight days blocked pregabalin induced CPP. These results showed for the first time the possible involvement of GLT‐1 in mediating the rewarding effects of pregablin in animal model of drug addiction.

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Regulation of Mood by Dopamine D1 Receptors on Cerebral Cortical Interneurons
Authors: Stanwood Gregg , Jin Yuanhu , Graham Devon
Published: June 2021

Depression is a multifactorial brain disorder caused by a variety of alterations in neural structure and function. We have used the Cre/loxP system to selectively delete dopamine D1 receptors from GABAergic neurons from the Nkx2.1 lineage within the medial ganglionic eminence (GABA‐D1‐cKO), which include a subset of crucial cerebral cortical interneurons within the frontal cortex. Cell‐type specific loss of D1 receptor expression was validated by in situ hybridization. Neurobehavioral analyses of GABA‐D1‐cKO mice revealed normal patterns of locomotor activity, anxiety, and spatial and nonspatial memory. However, GABA‐D1‐cKO mice exhibit decreased immobility in the forced swim test and decreased latency to consume a palatable liquid in a novel stressful environment; these are both indicative of an antidepressant‐like effect. GABA‐D1‐cKO mice also show reduced basal plasma corticosterone levels and down‐regulation of the neurexin Cntnap4 in cerebral cortical interneurons. Conditional deletion of dopamine D1 receptors from telencephalic glutamatergic neurons (using Emx1‐Cre as a deletor) does not induce these phenotypes. These data suggest a new mechanism by which cerebral cortical interneurons may contribute to mood regulation and identify a new potential mechanism to exploit in the treatment and prevention of this prevalent brain disorder.

Support or Funding Information

Supported by FSU College of Medicine and MH116429.

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 of α7 nicotinic receptor positive allosteric modulator, PNU120596, on BDNF, NKCC1, and KCC2 expressions following LPS‐induced depressive‐like behavior in mice
Authors: Alzarea Sami , Rahman Shafiqur
Published: June 2021

Previous evidence indicates that neuroinflammation plays a critical role in the pathophysiology of major depressive disorder (MDD) by inducing neuronal excitability via dysregulation of microglial brain‐derived neurotrophic factor (BDNF), Na‐K‐Cl cotransporter‐1 (NKCC1), and K‐Cl cotransporter‐2 (KCC2). However, the role of microglial α7 nicotinic acetylcholine receptors (nAChRs), exhibiting anti‐inflammatory function during the activation, on these mediators is unknown. The objective of the present study was to determine the effects of PNU120596 on BDNF, NKCC1, and KCC2 expressions in lipopolysaccharide (LPS)‐induced mouse model of MDD using Western blot analysis, immunofluorescence staining, and real‐time polymerase chain reaction. We also examined the pharmacological interaction between PNU120596 and ANA12, an antagonist of TrkB which is the primary receptor for BDNF, on LPS‐induced depressive‐like behavior using forced swim test (FST), tail suspension test (TST), and Y‐maze test. Systemic administration of LPS (1 mg/kg, i.p.) significantly increased BDNF protein expression and mRNA ratio of NKCC1 to KCC2, and decreased KCC2 mRNA expression in the hippocampus and prefrontal cortex, the brain regions implicated in MDD. The PNU120596 (1 or 4 mg/kg, i.p.) dose‐dependently prevented the LPS‐induced dysregulated BDNF, NKCC1, and KCC2 expressions in these brain regions. Combination treatment with PNU120596 (1 mg/kg, i.p.) and ANA12 (0.25 mg/kg, i.p.) prevented LPS‐induced increased immobility time during FST and TST and LPS‐induced decreased spontaneous alternations (%) during Y‐maze test. Overall, our results suggest that PNU120596 prevented LPS‐induced depressive‐like behavior by decreasing neuronal excitability by targeting microglial α7 nAChR in the hippocampus and prefrontal cortex. (Supported in part by grant from Saudi Arabian Cultural Mission, USA).

Support or Funding Information

Supported in part by grant from Saudi Arabian Cultural Mission, USA.

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Defects of Oligodendrocyte Migration in the Scn1b−/− Mouse Model of Dravet Syndrome
Authors: Clemons Krisyah , O'Malley Heather A. , Isom Lori L.
Published: June 2021

The Scn1b−/− mouse is a model of Dravet Syndrome (DS), a severe pediatric and developmental epileptic encephalopathy that begins in the first year of life and has multiple comorbidities. Scn1b−/− mice have severe seizures beginning at postnatal day (P)10 and die prior to ~P21. DS patients display defects in white matter, which may contribute to cognitive impairment, language issues, and problems with movement. Our previous research demonstrated that there are fewer nodes of Ranvier and that neuronal migration is impaired in Scn1b−/− brain. The purpose of this study is to determine if Scn1b−/− oligodendrocytes (OLs) migrate correctly from birth in the ventricles to destinations in the cortex. We chose to look at migration from the ventricles to cortical layers II/III and layers V/VI. We hypothesized that we would observe impairments in OL migration in Scn1b−/− animals compared to wildtype. We tested this hypothesis using Scn1b wildtype and Scn1b−/− mice in which OL lineage cells have been genetically labeled with green fluorescent protein (GFP). We performed immunocytochemistry on brain slices from mice of both genotypes at P13‐14 and P17‐18, using CDP as a marker for cortical layers II/III and CTIP2 as a marker of cortical layers V/VI, and then counting the number of GFP+ cells which had migrated to each of these regions. Our data demonstrate that, although the numbers of OLs in layers V/VI were similar between genotypes at both ages, there were fewer OLs in layers II/III in Scn1b−/− brain, suggesting that OL migration in these mice is impaired.

Support or Funding Information

Funded by: R37NS076752 to LLI.

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