bluuebunny/crossref_metadata_2025_split huggingface dataset:
Analysis of the Relationship Between Learning and Synaptophysin in Obese Rats Treated With DPP4 Inhibitor
Authors: Korkmaz Murat , Balci Sibel Oguzkan , Demirel Can , Yilmaz Ibrahim , Akarsu Ersin 
Published: May 2021

Abstract
In this study, it is aimed to investigate possible changes in cognitive functions in obesity by using targeted treatment hypothesis. Accordingly, the effects of DPP4 inhibitor, which is actively used in the clinic in the treatment of diabetes, and the effect of exercise, which has been proven to be effective in the treatment of obesity, on the change of learning performance and the relationship of these effects with the synaptophysin molecule were investigated. In our study, 42 Wistar albino rats were used. The animals were randomly divided into seven groups as obese, control, obese+DPP4i, control+DPP4i, obese+exercise, control+exercise, control+NaCl. To create experimental obesity, the animals that are targeted to be obese were separated and fed on a high fat diet for 8 weeks. After the obese model was created, sitagliptin was applied to the DPP4i groups and swimming exercise was applied to the exercise groups for obesity treatment. The last week of the study was performed reference memory learning test to the whole group with Morris water maze. Then, the hippocampus tissues were removed from the animals under anesthesia. mRNA and protein isolations were performed from the extracted tissues. Synaptophysin gene expressions were determined from mRNA samples by Real-Time PCR method. Synaptophysin protein levels were determined from protein lysates by Western Blot method. In the learning test, in the obese groups, there was a statistically significant difference between the average escape time of the DPP4i and exercise groups and the groups that did not (p&lt;0.05). As a result, in groups where obesity is treated with DPP4i and exercise; It was concluded that cognitive performance was better than obese groups. There was a evident decrease in synaptophysin gene expression levels in obese groups compared to the control group. In the treatment groups, an increase was observed in synaptophysin gene expression levels in the DPP4 inhibitor and especially in the exercise groups compared to the control groups (P&gt; 0.05). Gene expression results were similar in analyzes performed at the protein level. According to these results, in terms of performance in cognitive function due to obesity and synaptophysin gene relationship; DPP4 inhibitor showed as effective a result as exercise. This provides a resource for advanced molecular and metabolic research.
Acknowledgement: This study was supported by The Scientific And Technological Research Council Of Turkey (TÜBİTAK) Project No. 219S063.
Chronic Excess of Non-Aromatizable Androgens From Puberty Does Not Drive a Neuroendocrine Phenotype Observed in Other Preclinical Rodent Models of PCOS
Authors: Coyle Chris , Ruddenklau Amy , Prescott Mel , Walters Kirsty A , Campbell Rebecca Elaine 
Published: May 2021

Abstract
Polycystic ovary syndrome (PCOS) is the most common form of anovulatory infertility in women of reproductive age, characterised by androgen excess, polycystic appearance of the ovary and irregular menstruation. PCOS is also frequently associated with metabolic abnormalities, including increased adiposity and insulin resistance. The origins of PCOS are unknown, however recent findings in animal models strongly implicate androgen signalling in the brain in the development of PCOS pathophysiology. Exposure to androgen excess, either acutely during prenatal development or chronically from a peripubertal timepoint, can drive the development of PCOS-like features in adulthood. Prenatally androgenized (PNA) mice exhibit the cardinal reproductive features of PCOS and increased luteinizing hormone (LH) pulse frequency. This phenotype is associated with increased GABAergic innervation of gonadotropin-releasing hormone (GnRH) neurons, postulated to drive elevated GnRH/LH release and downstream effects. Chronic exposure to di-hydrotestosterone (DHT) from 3 weeks of age drives both reproductive and metabolic PCOS-like features that are ameliorated by selective AR loss from the brain. Here, we aimed to determine whether chronic exposure to DHT drives a similar increase in LH pulsatility and elevated GABAergic innervation to GnRH neurons as seen following prenatal exposure to androgen excess. GnRH-green fluorescent protein (GFP) female mice received either DHT or blank capsules for 90 days from postnatal day (PND) 21 (N = 6-7/group). Serial tail tip blood sampling was used to measure pulsatile LH and fixed brains were collected and immunolabelled for vesicular GABA transporter (VGAT) to assess putative GABAergic terminals associated with GFP-labelled GnRH neurons. Chronic androgen excess from the peripubertal period resulted in acyclicity and increased body weight as expected. However, LH pulsatility was not different between DHT-treated females and controls. Similarly, the density of VGAT appositions to GnRH neurons was not different between groups. Therefore, the programmed changes in the GnRH neuronal network and hyperactive LH secretion that result from prenatal androgen excess are not affected by chronic DHT exposure initiated at 3 weeks of age. These findings suggest that unique central mechanisms are involved in the reproductive impairments driven by exposure to androgen excess at different developmental stages.
Estrogen Differentially Regulates Protein Abundance in Exosomes Released From Immortalized Kisspeptin Neurons in Vitro
Authors: James Teagan , Chappell Patrick Everett 
Published: May 2021

Abstract
Estrogen (E2) is essential for multiple physiological effects in females, ensuring maximum reproductive fitness and maintaining skeletal homeostasis. E2 has been shown to stimulate cancellous bone formation via activation of estrogen receptor alpha (ERα), an effect widely accepted to be mediated directly at bone. A recent landmark study (Herber et al., Nat Commun 2019) demonstrated bone density increases in female mice harboring ERα-deletions specifically in arcuate Kiss-1 neurons. In this study, bone from transgenic females showed higher osteoblast functioning and increases in the expression of sp7 and runx2, positing a direct neural-bone regulatory axis altered by circulating E2 acting in brain. Our laboratory has used two immortalized Kisspeptin (Kiss1)-expressing and -secreting cell lines, KTaR-1 (representative of female arcuate Kiss-1 neurons) and KTaV-3 cells (representative of female AVPV Kiss-1 neurons) as models to explore the role of Kiss-1 in multiple physiological regulatory contexts. We recently determined that factors in the media of female ARC-derived KTaR-1 cells can affect parameters of osteoblast function in vitro, including increases in sp7 and runx2 expression, and formation of bone matrix (evaluated by Alizarin Red assay). Exposure of canine osteosarcoma cells to conditioned media from KTaR-1 cells led to increases in sp7 expression in an E2-dependent manner, and 24h E2-deprivation of these neurons stimulated secretion of osteogenic factors. In this current study, we have used LCMS-MS proteomic analysis to determine the contents of exosomes isolated from Kisspeptin neurons under varying E2 exposure conditions in vitro. Preliminary results reveal ~150-170 proteins up-regulated by E2 exposure and ~200-220 proteins downregulated by E2 exposure in exosomes of both KTaR-1 and KTaV-3 Kisspeptin neurons. Estrogen-regulated Kiss-1 exosomal proteins include several candidates involved in bone remodeling (pentraxin, osteonectin, osteoclast-stimulating factor-1) and neuronal synaptic plasticity and signaling (annexins, semaphorins, connexins). Current work is exploring the effects of exposure of purified exosomes on morphology and gene expression in immortalized GnRH neurons and osteoblasts. While further study is required, initial results suggest that exosomes may represent additional cellular communication pathways utilized by Kisspeptin neurons to elicit changes in brain and bone.
Hippocampal Circuits for the Hunger-Dependent Control of Feeding Behaviour
Authors: Shien Wee Ryan Wei , MacAskill Andrew 
Published: May 2021

Abstract
Background: Feeding behavior is a complex motivated behavior that requires organisms to integrate features of the environment, such as food availability and value, and internal states, such as hunger, in deliberating over the decision to eat. The hippocampus - a brain region classically thought to support spatial cognition and episodic memory - is increasingly recognised to contribute to such decision-making processes. This function makes the hippocampus a likely candidate in supporting the higher-order decisions that underpin motivated behaviors such as feeding. However, the role of the hippocampus during free-feeding behavior has not been examined.
Methods and Results: To address this question, we used in vivo calcium imaging during feeding behavior in mice to monitor the neural activity of the ventral subiculum (vS) - one of the main output structures of the ventral hippocampus. In a free-feeding task, we found that the vS encoded the investigative approach phase of feeding behavior and that activity during this period correlated with the probability of transitioning from food investigation to consumption. Calcium imaging during an operant task confirmed the specific encoding of preparatory behaviour preceding food consumption. Furthermore, the sensitivity of vS to the hunger state could be mapped to vS neurons projecting to the nucleus accumbens (vS-NAc). Ghrelin - a hormone signalling the hunger state - altered synaptic transmission specifically in vS-NAc neurons, and molecular knockdown of the ghrelin receptor was required for the hunger sensitivity of vS-NAc. Consequently, both reducing ghrelin signalling in vS-NAc neurons through molecular knockdown and artificially elevating vS-NAc activity through optogenetics were sufficient to shift the feeding strategy of animals, effectively curtailing overall food consumption.
Conclusion: In summary, these results provide evidence for a hippocampal circuit that integrates hunger state signals to regulate the decision to eat.
Memory Improving Effects in Social-Isolated Aged Rats by Seaweed Glycoproteins
Authors: Oh Jeong Hwan , Nam Taek-Jeong , Choi Youn Hee 
Published: May 2021

Abstract
Social isolation and loneliness that could induce cognitive decline are serious public health problems in elderly. The trafficking of α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptors (AMPARs) is a critical process in long-term potentiation of synapses that is necessary for memory formation. We previously found that glycoproteins of an edible seaweed Capsosiphon fulvescens (C. fulvescens) prevent aging-induced cognitive impairment via regulation of brain-derived neurotrophic factor in hippocampus. This study was to investigate whether chronic administration of hydrophilic glycoproteins of C. fulvescens (Cf-hGP) prevent cognitive dysfunctions caused by social isolation in aged rats and this effect is regulated by post synaptic density protein 95 (PSD-95)-mediated AMPAR trafficking and the glycosylation of Cf-hGP. Social isolation for four weeks decreased phosphorylation of extracellular signal-regulated protein kinase 1/2 (ERK1/2), PSD-95 (Ser295) and AMPAR GluR1 (Ser845) and increased expression of metabotropic glutamate receptor 5 (mGluR5) in synaptosome of the dorsal hippocampus, which was attenuated by chronic Cf-hGP treatment (15 mg/kg/day, 4 weeks). Blockade of mGluR5 abolished decrease in ERK1/2-mediated phosphorylation of PSD-95 (Ser295) and GluR1 (Ser845) in the socially isolated rats. In particular, chronic Cf-hGP treatment enhanced binding of ERK1/2 to PSD-95 and upregulated the surface movement of AMPAR GluR1 in the dorsal hippocampus. In addition, Cf-hGP treatment prevented spatial memory impairment caused by the social isolation, which was attenuated by inhibition of ERK1/2 or deglycosylation of Cf-hGP. These findings suggest that Cf-hGP-induced clustering of ERK1/2-mediated PSD-95 in the dorsal hippocampus improves memory formation in socially isolated aged rats and protein glycosylation contributes to enhancing the Cf-hGP effect.
Neuromedin B Receptor Antagonist Suppresses Pomc Expression in AtT-20 Cells and Human Corticotroph Adenoma Cells
Authors: Sekizaki Tomonori , Kameda Hiraku , Nakamura Akinobu , Nomoto Hiroshi , Cho Kyu Yong , Ishi Yukitomo , Motegi Hiroaki , Miyoshi Hideaki , Atsumi Tatsuya 
Published: May 2021

Abstract
Objective: We previously reported that Neuromedin B (NMB) is expressed in murine pituitary corticotrophs under adrenal insufficiency (1). Because NMB is also expressed in several cancer cells and stimulates ACTH secretion, we hypothesized that NMB is related to corticotroph adenoma cell proliferation and hormone secretion. To examine this hypothesis, we investigated the expression of NMB and its receptor NMBR in human corticotroph adenoma and the effects of a NMBR antagonist on AtT-20 cells, a mouse corticotroph adenoma cell line, and patient-derived corticotroph adenoma cells. Methods: 1. NMB and NMBR expression in human pituitary adenoma: We performed real-time qPCR and immunostaining on human pathological specimens of corticotrophs, somatotrophs, and non-functioning pituitary adenoma to investigate NMB and NMBR expression. 2. Experiments in AtT-20 cells: We extracted mRNAs and proteins from AtT-20 cells after incubation with 100nM NMBR antagonist PD168368, and performed real-time qPCR and western blotting analyses to investigate Pomc expression. 3. Experiments in patient-derived corticotroph adenoma cells: We isolated surgically resected human corticotroph adenoma cells from patients who underwent trans-sphenoidal surgery and investigated POMC mRNA expression by real-time qPCR after incubation with PD168368. Statistical analysis: One-way ANOVA was employed to compare values among multiple groups. If the ANOVA revealed significant differences, the Tukey-Kramer post-hoc test was employed to compare values between two specific groups. Dunnett’s post-hoc test was employed to compare values with the control group. Statistical significance was defined as p &lt; 0.05. Results: 1. NMB and NMBR expression levels were significantly higher in human corticotroph adenoma (13 and 33 times higher than non-functioning adenoma, respectively) than in somatotroph adenoma (2 and 3 times higher than non-functioning adenoma, respectively) and non-functioning adenoma in the qPCR analyses. Immunostaining confirmed higher expression of NMB and NMBR in corticotroph adenoma than in somatotroph and non-functioning adenoma. 2. Treatment with 100 nM PD168368 significantly suppressed Pomc mRNA and protein expression in AtT-20 cells by 22%±3% and 25%±10%, respectively. 3. Treatment with 1 µM PD168368 significantly suppressed POMC mRNA expression in human corticotroph adenoma cells by 18%±1%. Conclusions: NMB and NMBR were both expressed in human corticotroph adenoma, suggesting that NMB may stimulate adenoma cell proliferation and hormone secretion in autocrine or paracrine manners. Because the NMBR antagonist suppressed Pomc expression in both AtT-20 cells and human corticotroph adenoma cells, it may represent a potential treatment for Cushing disease. Reference: (1) Kameda H et al., Endocrinology 2014;155(7):2492-9.
Overactive Reproductive Axis Due to Fragile X Gene Mutation
Authors: Villa Pedro , Lainez Nancy , Ethell Iryna , Coss Djurdjica 
Published: May 2021

Abstract
Women carrying a pre-mutation or mutation of the Fragile X mental retardation gene (FMR1) comprise the largest portion of premature ovarian failure (POF) cases due to known genetic factors. FMR1 mutation causes Fragile X syndrome, the most common cause of inherited mental impairment. The mutation inhibits the expression of the fragile X mental retardation protein (FMRP), a ubiquitously expressed mRNA binding protein. The specific molecular mechanism(s) leading to premature ovarian failure in Fragile X carriers are not known. Here, we utilize the complete KO mouse model, to mimic the lack of FMRP in Fragile X mutations and analyze the hypothalamic-pituitary-gonadal axis to uncover causes of POF due to FMR1 mutation. Consistent with mutations in human population, KO females experience early cessation of reproductive function and stop having litters at 150 days of age, compared to controls that stop reproducing at 250 days of age. Since POF can be caused by either insufficient pool of primordial follicles or by increased recruitment in each cycle and early depletion, we analyzed ovaries at 3 weeks of age and determined that the FMR1 KO mice had the same number of primordial follicles when compared to the controls, suggesting that POF is not due to a deficit in primordial follicles. However, at 8 weeks of age, FMR1 KO ovaries had higher number of corpora lutea, and KO females had larger litters, indicating that FMR1 KO mice have more follicles recruited in each estrous cycle. FMR1 KO mice have higher FSH, which corresponds to the high FSH in women. Serum estradiol levels and inhibin b expression levels were unaffected by FMR1 mutation suggesting normally functioning negative feedback signals from the ovaries. Analyses of hypothalamic gene expression demonstrated elevated GnRH mRNA in KO mice. To further investigate alterations is hypothalamic protein levels, western blot analyses determined that FMR1 KO mice have higher levels of NMDAR1 and higher levels of GABAA receptor G2 subunit. Dual label immunofluorescence analyses revealed higher number of NMDAR1 and GABAA receptors specifically in GnRH neurons of FMR1 KO mice when compared to control, suggesting that GnRH neurons themselves are affected by FMR1 mutation. Given that both glutamate and GABA can activate GnRH neurons, alterations in the number of these receptors can potentially cause hyperactivity in the HPG axis at the hypothalamic level leading to elevated FSH and the subsequent POF. In summary, our results reveal a potential mechanism of premature ovarian failure in Fragile X mutation carriers.
PI3K Inhibition by BKM120 Results in Antiproliferative Effects on Corticotroph Tumor Cells
Authors: Oliveira Helen A , Bueno Ana C , Pugliesi Renata S , Silva-Júnior Rui M P , de Castro Margaret , Martins Clarissa S 
Published: May 2021

Abstract
Purpose: Cushing’s disease is associated with significant morbidity, thus additional tumor-directed drugs with the potential to exert antineoplastic effects on corticotroph adenoma cells are desired. The PI3K (phosphoinositide-3-kinase)/AKT (protein kinase B) pathway, which plays regulatory roles in cell survival and proliferation, is activated in pituitary adenomas. The present study evaluated the effects of BKM120 (Buparlisib), an oral PI3K inhibitor, in corticotroph tumor cells. Methods: AtT-20/D16v-F2 mouse pituitary corticotroph tumor cells were treated with increasing concentrations of BKM120 or vehicle. Cell viability was measured using MTS-based assay. Apoptosis was evaluated by Annexin V staining. ACTH levels were measured in the culture supernatants by chemiluminescent immunometric assay. Cell cycle analysis was performed by propidium iodide DNA staining and flow cytometry. Gene expression of cell cycle regulators (Cdkn1b, Rb1, Ccnd1, Cdk4, Cdk2, and Myc) was assessed by qPCR. Protein expression of p27, p70 S6 Kinase, p85 S6 Kinase, and phosphorylated AKT was assessed by Western blot. Results: Treatment with BKM120 decreased AtT-20/D16v-F2 cell proliferation and ACTH levels in the cell culture supernatants. Furthermore, BKM120 treatment diminished the phosphorylation of AKT at residue 473, increased p27 expression and induced a G0/G1 cell cycle arrest. Conclusion:In vitro inhibition of PI3K/AKT pathway by BKM120 resulted in antiproliferative effects on corticotroph tumor cells, decreasing cell viability and ACTH production. These encouraging findings shape the path for further experiments with the inhibition of PI3K/AKT pathway in Cushing’s disease.
Potential Role for the RASD1 Glucocorticoid-Responsive Gene in Corticotroph Tumorigenesis
Authors: Hernández-Ramírez Laura C , Pankratz Nathan , Lane John , Hu Mingming , Faucz Fabio R , Chittiboina Prashant , Denise Kay M , Mills James L , Stratakis Constantine A 
Published: May 2021

Abstract
Introduction: Originally identified due to its dexamethasone inducibility in mouse corticotropinoma AtT20 cells, RASD1 is a receptor-independent activator of G-proteins, via guanine nucleotide exchange factor (GEF) activity. It remains unclear, however, whether, and if so, how RASD1 mediates the effects of glucocorticoids on corticotroph cells. We identified a rare germline RASD1 variant and investigated its functional effects in vitro. Methods: We screened 209 CD patients (94.3% pediatric) studied at the at the National Institutes of Health Clinical Research Center between 1997 and 2018 by germline whole-exome sequencing (WES) only (n=157), germline and tumor WES (n=27), and/or RASD1 droplet digital PCR germline copy number variant (CNV) analysis (n=201). Corticotropinoma DNA was available in 72 patients to screen for USP8 hotspot variants by Sanger sequencing. A RASD1 variant was identified and functionally characterized. Results: We studied 119 female (56.9%) and 90 (43.1%) male CD cases, including 197 pediatric (≤18 years at disease onset) and 12 adult patients. USP8 defects were present in 19.4% (14/72) of cases. No RASD1 CNVs were found. A rare (with a minor allele frequency of 0.0022% in gnomAD v3) heterozygous germline missense RASD1 variant, c.580A&gt;C, p.M194L was detected in one male sporadic case. Neither USP8 variants nor loss of heterozygosity at the RASD1 variant position were observed in the patient’s microadenoma. The wild type and p.M194L RASD1 transiently overexpressed proteins displayed similar short half-lives (&lt;1 h) by cycloheximide chase in HEK293 cells, as well as cytoplasmic localization by immunocytofluorescence in AtT20 cells. A CRISPR/Cas9 Rasd1 knockout AtT20 cell line displayed reduced Pomc expression compared with the parental cell line at the mRNA level (Actb-normalized absolute quantification 5.80±0.92 vs 9.62±0.7, P=0.005). Viability of the cell lines did not differ significantly by MTT assay. Overexpression of p.M194L resulted in increased accumulation of phospho-CREB S133 (1.83±0.8 vs 1±0.2 in empty vector control, P=0.0390) as well as a non-significant increase in Pomc expression in wild type, but not in Rasd1 knockout AtT20 cells by immunoblot band densitometry. Conclusions: We found an infrequent RASD1 variant in one CD patient. Rasd1 seems to have a role within the intracellular signaling pathways controlling Pomc expression. Overexpression of the p.M194L variant caused phospho-CREB S133 activation, suggesting increased GEF activity for this variant. Interestingly, another variant at the same position, p.M194I, was found in the COSMIC database (COSS2121715) as a somatic change in cutaneous malignant melanoma. Further studies are required to better define the role of RASD1 in corticotroph physiology and its possible involvement in tumorigenesis.