Search

Osteosarcoma (OS) is the most frequent bone malignancy in humans. Previous evidence suggest that circ_0032463 is an oncogenic circular RNA (circRNA) in various cancers, including OS. However, the molecular mechanism of circ_0032463 involved in OS is still unclear. Circ_0032463, microRNA-145-5p (miR-145-5p), GDNF receptor alpha 1 (GFRA1), and Wilms tumor 1-associated protein (WTAP) levels were determined using real-time quantitative polymerase chain reaction (RT-qPCR). Cell proliferation, apoptosis, migration, invasion, and angiogenesis were analyzed using 5-ethynyl-2'-deoxyuridine (EdU), flow cytometry, transwell, and tube formation assays. Western blot analysis was performed to measure matrix metalloproteinase 2 (MMP2), MMP9, GFRA1, and WTAP protein levels. Binding between miR-145-5p and circ_0032463 or GFRA1 was confirmed using a dual-luciferase reporter and pull-down assay. The biological role of circ_0032463 on OS cell growth was also analyzed using a xenograft tumor model in vivo. Methylated RNA immunoprecipitation assay validated the interaction between WTAP and circ_0032463. Circ_0032463, GFRA1, and WTAP levels were increased, and miR-145-5p was decreased in OS tissues and cells. Circ_0032463 deficiency might hinder OS cell proliferation, migration, invasion, angiogenesis, and promote apoptosis in vitro. Mechanically, circ_0032463 worked as a miR-145-5p sponge to increase GFRA1 expression. Repression of circ_0032463 knockdown on tumor cell growth was proved in vivo. Besides, N6-methyladenosine (m6A) modification facilitates the biogenesis of circ_0032463. Taken together, m6A-mediated biogenesis of circ_0032463 facilitates OS cell malignant biological behavior partly via regulating the miR-145-5p/GFRA1 axis, suggesting a promising molecular marker for OS treatment., (© 2024 Wiley Periodicals LLC.)

The increase in presynaptic striatal dopamine is the main dopaminergic abnormality in schizophrenia (SCZ). SCZ is primarily treated by modulating the activity of monoamine systems, with a focus on dopamine and serotonin receptors. Glial cell line-derived neurotrophic factor (GDNF) is a strong dopaminergic factor, that recently was shown to correlate with SCZ in human CSF and in striatal tissue. A 2-3-fold increase in GDNF in the brain was sufficient to induce SCZ-like dopaminergic and behavioural changes in mice. Here, we analysed the effect of acute, chronic, and embryonic methamphetamine, a drug known to enhance the risk of psychosis, on Gdnf and its receptors, Gfra1 and Ret , as well as on monoamine metabolism-related gene expression in the mouse brain. We found that acute methamphetamine application increases Gdnf expression in the striatum and chronic methamphetamine decreases the striatal expression of GDNF receptors Gfra1 and Ret . Both chronic and acute methamphetamine treatment upregulated the expression of genes related to dopamine and serotonin metabolism in the striatum, prefrontal cortex, and substantia nigra. Our results suggest a potential mechanism as to how methamphetamine elicits individual psychosis risk in young adults-variation in initial striatal GDNF induction and subsequent GFRα1 and RET downregulation may determine individual susceptibility to psychosis. Our results may guide future experiments and precision medicine development for methamphetamine-induced psychosis using GDNF/GFRa1/RET antagonists.

The spermatogonial compartment maintains spermatogenesis throughout the reproductive lifespan. Single-cell RNA sequencing (scRNA-seq) has revealed the presence of several spermatogonial clusters characterized by specific molecular signatures. However, it is unknown whether the presence of such clusters can be confirmed in terms of protein expression and whether protein expression in the subsets overlaps. To investigate this, we analyzed the expression profile of spermatogonial markers during the seminiferous epithelial cycle in cynomolgus monkeys and compared the results with human data. We found that in cynomolgus monkeys, as in humans, undifferentiated spermatogonia are largely quiescent, and the few engaged in the cell cycle were immunoreactive to GFRA1 antibodies. Moreover, we showed that PIWIL4+ spermatogonia, considered the most primitive undifferentiated spermatogonia in scRNA-seq studies, are quiescent in primates. We also described a novel subset of early differentiating spermatogonia, detectable from stage III to stage VII of the seminiferous epithelial cycle, that were transitioning from undifferentiated to differentiating spermatogonia, suggesting that the first generation of differentiating spermatogonia arises early during the epithelial cycle. Our study makes key advances in the current understanding of male germline premeiotic expansion in primates., Competing Interests: Competing interests The authors declare no competing or financial interests., (© 2023. Published by The Company of Biologists Ltd.)

Cocaine addiction is a serious condition with potentially lethal complications and no current pharmacological approaches towards treatment. Perturbations of the mesolimbic dopamine system are crucial to the establishment of cocaine-induced conditioned place preference and reward. As a potent neurotrophic factor modulating the function of dopamine neurons, glial cell line-derived neurotrophic factor (GDNF) acting through its receptor RET on dopamine neurons may provide a novel therapeutic avenue towards psychostimulant addiction. However, current knowledge on endogenous GDNF and RET function after the onset of addiction is scarce. Here, we utilized a conditional knockout approach to reduce the expression of the GDNF receptor tyrosine kinase RET from dopamine neurons in the ventral tegmental area (VTA) after the onset of cocaine-induced conditioned place preference. Similarly, after establishing cocaine-induced conditioned place preference, we studied the effect of conditionally reducing GDNF in the ventral striatum nucleus accumbens (NAc), the target of mesolimbic dopaminergic innervation. We find that the reduction of RET within the VTA hastens cocaine-induced conditioned place preference extinction and reduces reinstatement, while the reduction of GDNF within the NAc does the opposite: prolongs cocaine-induced conditioned place preference and increases preference during reinstatement. In addition, the brain-derived neurotrophic factor (BDNF) was increased and key dopamine-related genes were reduced in the GDNF cKO mutant animals after cocaine administration. Thus, RET antagonism in the VTA coupled with intact or enhanced accumbal GDNF function may provide a new approach towards cocaine addiction treatment.

The use of next-generation sequencing (NGS) has helped in identifying many genes that cause congenital anomalies of the kidney and urinary tract (CAKUT). Bilateral renal agenesis (BRA) is the most severe presentation of CAKUT, and its association with autosomal recessively inherited genes is expanding. Highly consanguineous populations can impact the detection of recessively inherited genes. Here, we report two families harboring homozygous missense variants in recently described genes, NPNT and GFRA1. Two consanguineous families with neonatal death due to CAKUT were investigated. Fetal ultrasound of probands identified BRA in the first family and severe renal cystic dysplasia in the second family. Exome sequencing coupled with homozygosity mapping was performed, and Sanger sequencing was used to confirm segregation of alleles in both families. In the first family with BRA, we identified a homozygous missense variant in GFRA1: c.362A>G; p.(Tyr121Cys), which is predicted to damage the protein structure. In the second family with renal cystic dysplasia, we identified a homozygous missense variant in NPNT: c.56C>G; p.(Ala19Gly), which is predicted to disrupt the signal peptide site. We report two Saudi Arabian consanguineous families with CAKUT phenotypes that included renal agenesis caused by missense variants in GFRA1 and NPNT, confirming the role of these two genes in human kidney development.

Complete surgical resection, accessible therapeutic targets and effective tyrosine kinase inhibitors (TKIs) have not completely cured gastrointestinal stromal tumours (GISTs), with most patients suffering from residual tumours and recurrence. The existence of nerve infiltration in GIST provides a way for tumour cells to escape local resection and systemic targeted therapy, which may challenge the previous understanding of its behaviour patterns and inspire the development of more radical excision and more precise targeted therapy. Moreover, tumour dormancy has emerged as a major cause of drug resistance and tumour relapse. Among these pathways, the nerve-tumour regulatory axis GDNF-GFRA1 is activated in GISTs, assists tumour cells in achieving dormancy and protects them from apoptosis under environmental stress by enhancing autophagic flux. The concrete mechanism is that the GDNF-regulating interaction between GFRA1 and the lysosomal calcium channel MCOLN1 activates Ca 2+ -dependent TFEB signalling. Activated TFEB transcriptionally regulates intracellular lysosome levels, which could achieve feedback upregulation of cellular autophagy flux during TKI treatment. This dormancy-transition axis fills parts of the mechanistic vacancy before the onset of secondary mutations, and strategies for TKIs combined with targeting GFRA1-dependent autophagy have distinct promise as prospective clinical therapies., (Copyright © 2022 The Authors. Published by Elsevier B.V. All rights reserved.)

The ability of an animal to regenerate lost tissue and body parts has obviously life-saving implications. Understanding how this ability became restricted or active in specific animal lineages will help us understand our own regeneration. According to phylogenic analysis, the glial cell line-derived neurotrophic factor (GDNF) signaling pathway, but not other family members, is conserved in axolotls, a salamander with remarkable regenerative capacity. Furthermore, comparing the pro-regenerative Spiny mouse to its less regenerative descendant, the House mouse, revealed that the GDNF signaling pathway, but not other family members, was induced in regenerating Spiny mice. According to GDNF receptor expression analysis, GDNF may promote hair follicle neogenesis - an important feature of skin regeneration - by determining the fate of dermal fibroblasts as part of new hair follicles. These findings support the idea that GDNF treatment will promote skin regeneration in humans by demonstrating the GDNF signaling pathway's ancestral and cellular nature., Competing Interests: The authors have no conflict interests to declare., (© 2022 The Japanese Society for Regenerative Medicine. Production and hosting by Elsevier B.V.)

We propose that GDNF, a glial cell line-derived neurotrophic factor, can promote hair follicle neogenesis and skin regeneration after wounding by directing the fate of dermal fibroblasts. Our hypothesis is largely based on detailed GDNF and receptor analysis during skin regenerative stages, as well as the induction of GDNF receptors after wounding between the pro-regenerative spiny mouse (genus Acomys) and its less-regenerative descendant, the house mouse (Mus musculus). To characterize the GDNF-target cells, we will conduct a series of lineage-tracing experiments in conjunction with single-cell RNA and assay for transposase-accessible chromatin sequencing experiments. The heterogenetic dynamics of skin regeneration have yet to be fully defined, and this research will help to advance the fields of regenerative medicine and biology. Finally, we believe that stimulating the GDNF signalling pathway in fibroblasts from less-regenerative animals, such as humans, will promote skin regeneration, morphogenesis and scarless wound healing., (© 2022 The Authors. Experimental Dermatology published by John Wiley & Sons Ltd.)

Bilateral renal agenesis belongs to a group of perinatal lethal renal diseases. To date, pathogenic variants in three genes (ITGA8, GREB1L, and FGF20) have been shown to cause renal agenesis in humans. Recently GFRA1 has been linked to a phenotype consistent with a nonsyndromic form of bilateral renal agenesis. GFRA1 encodes a member of the glial cell line-derived neurotrophic factor receptor family of proteins. The receptor on the Wolffian duct regulates ureteric bud outgrowth in developing a functional renal system. We report on four additional affected neonates from a consanguineous family who presented with a similar lethal phenotype whereby whole exome sequencing identified a homozygous deleterious sequence variant in GFRA1 (NM_005264.8:c.628G > T:p.[Gly210Ter]). The current study represents a second confirmation report on the causal association of GFRA1 pathogenic variants with lethal nonsyndromic bilateral renal agenesis in humans., (Copyright © 2021 Elsevier Masson SAS. All rights reserved.)

Background: Osteosarcoma is a common primary malignant bone cancer. Long noncoding RNA small nucleolar RNA host gene 15 (SNHG15) has been reported to play an oncogenic role in many cancers. Nevertheless, the role of SNHG15 in the doxorubicin (DXR) resistance of osteosarcoma cells has not been fully addressed., Methods: Cell Counting Kit-8 assay was conducted to measure the half-maximal inhibitory concentration value of DXR in osteosarcoma cells. Western blotting was carried out to examine the levels of autophagy-related proteins and GDNF family receptor alpha-1 (GFRA1). Quantitative reverse transcription-polymerase chain reaction was performed to determine the levels of SNHG15, miR-381-3p, and GFRA1. The proliferation of osteosarcoma cells was measured by MTT assay. The binding sites between miR-381-3p and SNHG15 or GFRA1 were predicted by Starbase bioinformatics software, and the interaction was confirmed by dual-luciferase reporter assay. Murine xenograft model was established to validate the function of SNHG15 in vivo ., Results: Autophagy inhibitor 3-methyladenine sensitized DXR-resistant osteosarcoma cell lines to DXR. SNHG15 was upregulated in DXR-resistant osteosarcoma tissues and cell lines. SNHG15 knockdown inhibited the proliferation, DXR resistance, and autophagy of osteosarcoma cells. MiR-381-3p was a direct target of SNHG15, and GFRA1 bound to miR-381-3p in osteosarcoma cells. SNHG15 contributed to DXR resistance through the miR-381-3p/GFRA1 axis in vitro . SNHG15 depletion contributed to the inhibitory effect of DXR on osteosarcoma tumor growth through the miR-381-3p/GFRA1 axis in vivo ., Conclusions: SNHG15 enhanced the DXR resistance of osteosarcoma cells through elevating the autophagy via targeting the miR-381-3p/GFRA1 axis. Restoration of miR-381-3p expression might be an underlying therapeutic strategy to overcome the DXR resistance of osteosarcoma., Competing Interests: Conflict of interest: The authors state no conflict of interest., (© 2020 Jinshan Zhang et al., published by De Gruyter.)

Background: Radiotherapy is an effective strategy for preventing cancer metastasis, including osteosarcoma. However, cancer radioresistance limits the efficiency of radiotherapy. Therefore, it is essential to investigate the mechanism of osteosarcoma radioresistance., Methods: The osteosarcoma tissues and adjacent healthy tissues were collected from 53 osteosarcoma patients. The expression of LINC00210, miR-342-3p, and GFRA1 mRNA were determined using qRT-PCR. Cell viability, cell apoptosis, and cell surviving fraction were determined by MTT assay, flow cytometry, and colony formation assay, respectively. Western blot was performed to detect the protein levels. Luciferase assay was conducted to verify the relationship between LINC00210, miR-342-3p, and GFRA1., Results: LINC00210 and GFRA1 were up-regulated, and miR-342-3p was down-regulated in osteosarcoma tissues and cells. The expression of LINC00210 in osteosarcoma was negatively related to miR-342-3p expression and positively associated with GFRA1. Besides, there was a negative correlation between LINC00210 and GFRA1 expression in osteosarcoma. Also, LINC00210 and GFRA1 were up-regulated, and miR-342-3p was down-regulated in osteosarcoma cells exposed to 4 Gy irradiation treatment. Furthermore, either LINC00210 knockdown or miR-342-3p overexpression enhanced the radiosensitivity of osteosarcoma cells. Moreover, LINC00210 increased GFRA1 expression via sponging miR-342-3p. Additionally, LINC00210 knockdown improved the radiosensitivity of osteosarcoma cells by regulating GFRA1 expression via sponging miR-342-3p., Conclusion: LINC00210 modulated the radiosensitivity of osteosarcoma cells via the miR-342-3p/GFRA1 axis, making LINC00210 a novel target for improving radiotherapy efficiency in osteosarcoma., (© 2020 The Authors. Journal of Clinical Laboratory Analysis Published by Wiley Periodicals LLC.)

Background: The glial cell-derived neurotrophic factor (GDNF) family plays essential roles in the maintenance, growth, regulatory and signalling pathways of spermatogonial stem cells (SSCs). In this study, we analysed the expression of anti-GDNF family receptor alpha 1 antibody (GFRa1) by immunohistochemistry (IHC), immunocytochemistry (ICC), Fluidigm real-time polymerase chain reaction (RT-PCR) and flow cytometry analyses., Materials and Methods: In this experiment study, ICC, IHC, Fluidigm RT-PCR and flow cytometry were used to analyse the expression of the germ cell marker GFRa1 in testis tissue and SSC culture., Results: IHC analysis showed that there were two groups of GFRa1 positive cells in the seminiferous tubules based on their location and expression shape - a small round punctuated shape on the basal compartment donut shape and a C-shaped expression located between the basal and the luminal compartments of the seminiferous tubules. OCT4 and PLZF positive cells may have similar patterns of expression as the first group. Assessment of the seminiferous tubule sections demonstrated that about 27% of the SSCs were positive for GFRa1. Fluidigm RT-PCR confirmed the significant expression (P<0.001) of GFRa1 in the SSCs compared to testicular stromal cells (TSCs). Flow cytometry analysis demonstrated that about 75% of the isolated SSCs colonies were positive for GFRa1., Conclusion: The results indicated that GFRa1 had a specific expression pattern both in vivo and in vitro. This finding could be helpful for understanding the proliferation, maintenance and signalling pathways of SSCs, and differentiation of meiotic and haploid germ cells., Competing Interests: The authors declare no conflicts of interest., (Copyright© by Royan Institute. All rights reserved.)

Background: The expression of the membrane receptor protein GFRA1 is frequently upregulated in many cancers, which can promote cancer development by activating the classic RET-RAS-ERK and RET-RAS-PI3K-AKT pathways. Several therapeutic anti-GFRA1 antibody-drug conjugates are under development. Demethylation (or hypomethylation) of GFRA1 CpG islands (dmGFRA1) is associated with increased gene expression and metastasis risk of gastric cancer. However, it is unknown whether dmGFRA1 affects the metastasis of other cancers, including colon cancer (CC)., Aim: To study whether dmGFRA1 is a driver for CC metastasis and GFRA1 is a potential therapeutic target., Methods: CC and paired surgical margin tissue samples from 144 inpatients and normal colon mucosal biopsies from 21 noncancer patients were included in this study. The methylation status of GFRA1 islands was determined by MethyLight and denaturing high-performance liquid chromatography and bisulfite-sequencing. Kaplan-Meier analysis was used to explore the effect of dmGFRA1 on the survival of CC patients. Impacts of GFRA1 on CC cell proliferation and migration were evaluated by a battery of biological assays in vitro and in vivo . The phosphorylation of AKT and ERK proteins was examined by Western blot analysis., Results: The proportion of dmGFRA1 in CC, surgical margin, and normal colon tissues by MethyLight was 68.4%, 73.4%, and 35.9% (median; nonparametric test, P = 0.001 and < 0.001), respectively. Using the median value of dmGFRA1 peak area proportion as the cutoff, the proportion of dmGFRA1-high samples was much higher in poorly differentiated CC samples than in moderately or well-differentiated samples (92.3%% vs 55.8%, Chi-square test, P = 0.002) and significantly higher in CC samples with distant metastasis than in samples without (77.8% vs 46.0%, P = 0.021). The overall survival of patients with dmGFRA1-low CC was significantly longer than that of patients with dmGFRA1-high CC (adjusted hazard ratio = 0.49, 95% confidence interval: 0.24-0.98), especially for 89 CC patients with metastatic CC (adjusted hazard ratio = 0.41, 95% confidence interval: 0.18-0.91). These data were confirmed by the mining results from TCGA datasets. Furthermore, GFRA1 overexpression significantly promoted the proliferation/invasion of RKO and HCT116 cells and the growth of RKO cells in nude mice but did not affect their migration. GFRA1 overexpression markedly increased the phosphorylation levels of AKT and ERK proteins, two key molecules in two classic GFRA1 downstream pathways., Conclusion: GFRA1 expression is frequently reactivated by DNA demethylation in CC tissues and is significantly associated with a poor prognosis in patients with CC, especially those with metastatic CC. GFRA1 can promote the proliferation/growth of CC cells, probably by the activation of AKT and ERK pathways. GFRA1 might be a therapeutic target for CC patients, especially those with metastatic potential., (©The Author(s) 2020. Published by Baishideng Publishing Group Inc. All rights reserved.)

The glial cell line-derived neurotrophic factor (GDNF) is required for the survival and differentiation of diverse neuronal populations during nervous system development. Despite the high expression of GDNF and its receptor GFRα1 in the adult hippocampus, the functional role of this system remains unknown. Here, we show that GDNF, acting through its GFRα1 receptor, controls dendritic structure and spine density of adult-born granule cells, which reveals that GFRα1 is required for their integration into preexisting circuits. Moreover, conditional mutant mice for GFRα1 show deficits in behavioral pattern separation, a task in which adult neurogenesis is known to play a critical role. We also find that running increases GDNF in the dentate gyrus and promotes GFRα1-dependent CREB (cAMP response element-binding protein) activation and dendrite maturation. Together, these findings indicate that GDNF/GFRα1 signaling plays an essential role in the plasticity of adult circuits, controlling the integration of newly generated neurons., (Copyright © 2019 The Authors. Published by Elsevier Inc. All rights reserved.)

Backgroud: Increasing studies show that circular RNAs (circRNAs) play important roles in tumor progression. However, the function of circRNAs in ovarian cancer is mostly unclear. Methods: We detected the expression of circGFRA1 by quantitative real-time PCR (qRT-PCR) in 50 pairs of ovarian cancer tissues and adjacent normal tissues. Then, we explored the function of circGFRA1 in ovarian cancer progression, such as cell proliferation, apoptosis and invasion. Moreover, we performed luciferase reporter and RNA immunoprecipitation (RIP) assay to study the competing endogenous RNA (ceRNA) function of circGFRA1 in ovarian cancer progression. Results: qRT-PCR showed that circGFRA1 was overexpressed in ovarian cancer tissues. Inhibition of circGFRA1 suppressed cell proliferation and invasion, but induced cell apoptosis in ovarian cancer. Luciferase reporter and RIP assay revealed that circGFRA1 could regulate the expression of GFRA1 by sponging miR-449a. Conclusions: In summary, circGFRA1 regulated GFRA1 expression and ovarian cancer progression by sponging miR-449a. circGFRA1 could be a potential diagnostic biomarker and therapeutic target for ovarian cancer., Competing Interests: Competing Interests: The authors have declared that no competing interest exists.

Undifferentiated spermatogonia (A und ) or spermatogonial stem cells (SSCs) are committed to the establishment and maintenance of spermatogenesis and fertility throughout a male's life and are located in a highly specialized microenvironment called niche that regulates their fate. Although several studies have been developed on SSCs in mammalian testis, little is known about other vertebrate classes. The present study is the first to perform a more detailed investigation on the spermatogonial cells and their niche in a reptilian species. Thus, we characterized A und /SSCs and evaluated the existence of SSCs niche in the Kinosternon scorpioides, a freshwater turtle found from Mexico to northern and central South America. Our results showed that, in this species, A und /SSCs exhibited a nuclear morphological pattern similar to those described for other mammalian species already investigated. However, in comparison to other spermatogonial cell types, A und /SSCs presented the largest nuclear volume in this turtle. Similar to some mammalian and fish species investigated, both GFRA1 and CSF1 receptors were expressed in A und /SSCs in K. scorpioides. Also, as K. scorpioides A und /SSCs were preferentially located near blood vessels, it can be suggested that this niche characteristic is a well conserved feature during evolution. Besides being valuable for comparative reproductive biology, our findings represent an important step towards the understanding of SSCs biology and the development of valuable systems/tools for SSCs culture and cryopreservation in turtles. Moreover, we expect that the above-mentioned results will be useful for reproductive biotechnologies as well as for governmental programs aiming at reptilian species conservation., (Copyright © 2018 Elsevier Inc. All rights reserved.)

The present study aimed to investigate whether the Gfra1/Gdnf and/or Kit/Kitlg regulatory pathways could be involved in the regulation of spermatogonial cell proliferation and/or differentiation in fish. Homologs of the mammalian gfra1, gdnf, kitr, and kitlg genes were identified in gnathostomes and reliable orthologous relationships were established using phylogenetic reconstructions and analyses of syntenic chromosomal fragments. Gene duplications and losses occurred specifically in teleost fish and members of the Salmoninae family including rainbow trout (Oncorhynchus mykiss) and Atlantic salmon (Salmo salar). Some duplicated genes exhibited distinct spatiotemporal expression profiles and were differently regulated by hormones in rainbow trout. Undifferentiated type A spermatogonia expressed higher levels of kitrb and kitra2 making them possible target cells for the gonadal kitlgb and somatic kitlga before the onset of spermatogenesis. Interestingly, gdnfa and gdnfb ohnologous genes were poorly expressed before the onset of spermatogenesis. The expression level of gdnfb was correlated with that of the vasa gene suggesting that the late increased abundance of gdnfb during spermatogenesis onset was due to the increased number of spermatogonial cells. gfra1a2 was expressed in undifferentiated type A spermatogonia whereas gfra1a1 was mainly detected in somatic cells. These observations indicate that the germinal gdnfb ligand could exert autocrine and paracrine functions on spermatogonia and on testicular somatic cells, respectively. Fsh and androgens inhibited gfra1a2 and gdnfb whereas gfra1a1 was stimulated by Fsh, androgens, and 17α, 20β progesterone. Finally, our data provide evidences that the molecular identity of the male germ stem cells changes during ontogenesis prior to spermatogenesis onset.

GFRA1 and RET are overexpressed in estrogen receptor (ER)-positive breast cancers. Binding of GDNF to GFRA1 triggers RET signaling leading to ER phosphorylation and estrogen-independent transcriptional activation of ER-dependent genes. Both GFRA1 and RET are membrane proteins which are N-glycosylated but no O-linked sialylation site on GFRA1 or RET has been reported. We found GFRA1 to be a substrate of ST3GAL1-mediated O-linked sialylation, which is crucial to GDNF-induced signaling in ER-positive breast cancer cells. Silencing ST3GAL1 in breast cancer cells reduced GDNF-induced phosphorylation of RET, AKT and ERα, as well as GDNF-mediated cell proliferation. Moreover, GDNF induced transcription of ST3GAL1, revealing a positive feedback loop regulating ST3GAL1 and GDNF/GFRA1/RET signaling in breast cancers. Finally, we demonstrated ST3GAL1 knockdown augments anti-cancer efficacy of inhibitors of RET and/or ER. Moreover, high expression of ST3GAL1 was associated with poor clinical outcome in patients with late stage breast cancer and high expression of both ST3GAL1 and GFRA1 adversely impacted outcome in those with high grade tumors., (Copyright © 2018 Elsevier B.V. All rights reserved.)

Paracrine factors such as glial cell line-derived neurotrophic factor (GDNF), which was originally derived from the supernatants of a rat glioma cell line, play pivotal roles in oocyte maturation and early embryo development in mammals, such as mice, rats, pigs, sheep, and even humans. However, whether GDNF facilitates in vitro oocyte maturation or early embryo development in bovines is not yet known. We show for the first time that GDNF and its receptor, GDNF family receptor alpha-1 (GFRA1), are presented in ovarian follicles at different stages as well as during oocyte maturation and early embryo development. Immunostaining results revealed the subcellular localizations of GDNF and GFRA1 in oocytes throughout follicle development, first in germinal vesicles and during blastocyst embryo stages. The ability of exogenously applied GDNF to promote oocyte maturation and early embryo development was evaluated in culture, where we found that an optimal concentration of 50 ng/mL promotes the maturation of cumulus-oocyte complexes and the nuclei of denuded oocytes as well as the development of embryos after IVF. To further investigate the potential mechanism by which GDNF promotes oocyte maturation, bovine oocytes were treated with morpholinos targeting Gfra1. The suppression of GFRA1 presence blocked endogenous and exogenous GDNF functions, indicating that the effects of GDNF that are essential and beneficial for bovine oocyte maturation and early embryo development occur through this receptor. Furthermore, we show that supplementation with GDNF improves the efficiency of bovine IVF embryo production., (Copyright © 2018 Elsevier Inc. All rights reserved.)

Despite recent advances in treatment, breast cancer remains the second-most common cause of cancer death among American women. A greater understanding of the molecular characteristics of breast tumors could ultimately lead to improved tumor-targeted treatment options, particularly for subsets of breast cancer patients with unmet needs. Using an unbiased genomics approach to uncover membrane-localized tumor-associated antigens (TAAs), we have identified glial cell line derived neurotrophic factor (GDNF) family receptor α 1 (GFRA1) as a breast cancer TAA. Immunohistochemistry (IHC) revealed that GFRA1 displays a limited normal tissue expression profile coupled with overexpression in specific breast cancer subsets. The cell surface localization as determined by fluorescence-activated cell sorting (FACS) and the rapid internalization kinetics of GFRA1 makes it an ideal target for therapeutic exploitation as an antibody-drug conjugate (ADC). Here, we describe the development of a pyrrolobenzodiazepine (PBD)-armed, GFRA1-targeted ADC that demonstrates cytotoxicity in GFRA1-positive cell lines and patient-derived xenograft (PDX) models. The safety profile of the rat cross-reactive GFRA1-PBD was assessed in a rat toxicology study to find transient cellularity reductions in the bone marrow and peripheral blood, consistent with known off-target effects of PBD ADC's. These studies reveal no evidence of on-target toxicity and support further evaluation of GFRA1-PBD in GFRA1-positive tumors., Competing Interests: CONFLICTS OF INTEREST Jiping Zha is currently an employee of NGM Pharmaceuticals, Joanne Ayriss is currently an employee of Pfizer, Partha Chowdhury is currently an employee of Sanofi Genzyme, Binyam Bezabeh is currently an employee of Salubris Biotherapeutics. All additional authors are employees of MedImmune, LLC.

The glycosylphosphatidylinositol-linked GDNF (glial cell derived neurotrophic factor) receptor alpha (GFRA), a coreceptor that recognizes the GDNF family of ligands, has a crucial role in the development and maintenance of the nervous system. Of the four identified GFRA isoforms, GFRA1 specifically recognizes GDNF and is involved in the regulation of proliferation, differentiation, and migration of neuronal cells. GFRA1 has also been implicated in cancer cell progression and metastasis. Recent findings show that GFRA1 can contribute to the development of chemoresistance in osteosarcoma. GFRA1 expression was induced following treatment of osteosarcoma cells with the popular anticancer drug, cisplatin and induction of GFRA1 expression significantly suppressed apoptosis mediated by cisplatin in osteosarcoma cells. GFRA1 expression promotes autophagy by activating the SRC-AMPK signaling axis following cisplatin treatment, resulting in enhanced osteosarcoma cell survival. GFRA1-induced autophagy promoted tumor growth in mouse xenograft models, suggesting a novel function of GFRA1 in osteosarcoma chemoresistance.

Background Susceptibility genes for migraine, despite it being a highly prevalent and disabling neurological disorder, have not been analyzed in Asians by genome-wide association study (GWAS). Methods We conducted a two-stage case-control GWAS to identify susceptibility genes for migraine without aura in Han Chinese residing in Taiwan. In the discovery stage, we genotyped 1005 clinic-based Taiwanese migraine patients and 1053 population-based sex-matched controls using Axiom Genome-Wide CHB Array. In the replication stage, we genotyped 27 single-nucleotide polymorphisms with p < 10 -4 in 1120 clinic-based migraine patients and 604 sex-matched normal controls by using Sequenom. Variants at LRP1, TRPM8, and PRDM, which have been replicated in Caucasians, were also genotyped. Results We identified a novel susceptibility locus (rs655484 in DLG2) that reached GWAS significance level for migraine risk in Han Chinese ( p = 1.45 × 10 -12 , odds ratio [OR] = 2.42), and also another locus (rs3781545in GFRA1) with suggestive significance ( p = 1.27 × 10 -7 , OR = 1.38). In addition, we observed positive association signals with a similar trend to the associations identified in Caucasian GWASs for rs10166942 in TRPM8 (OR = 1.33, 95% confidence interval [CI] = 1.14-1.54, P permutation  = 9.99 × 10 -5 ; risk allele: T) and rs1172113 in LRP1 (OR = 1.23, 95% CI = 1.04-1.45, P permutation  = 2.9 × 10 -2 ; risk allele: T). Conclusion The present study is the first migraine GWAS conducted in Han-Chinese and Asians. The newly identified susceptibility genes have potential implications in migraine pathogenesis. DLG2 is involved in glutamatergic neurotransmission, and GFRA1 encodes GDNF receptors that are abundant in CGRP-containing trigeminal neurons. Furthermore, positive association signals for TRPM8 and LRP1 suggest the possibility for common genetic contributions across ethnicities.

Spermatogenesis requires retinoic acid (RA) induction of the undifferentiated to differentiating transition in transit amplifying (TA) progenitor spermatogonia, whereas continuity of the spermatogenic lineage relies on the RA response being suppressed in spermatogonial stem cells (SSCs). Here, we discovered that, in mouse testes, both spermatogonial populations possess intrinsic RA-response machinery and exhibit hallmarks of the differentiating transition following direct exposure to RA, including loss of SSC regenerative capacity. We determined that SSCs are only resistant to RA-driven differentiation when situated in the normal topological organization of the testis. Furthermore, we show that the soma is instrumental in "priming" TA progenitors for RA-induced differentiation through elevated RA receptor expression. Collectively, these findings indicate that SSCs and TA progenitor spermatogonia inhabit disparate niche microenvironments within seminiferous tubules that are critical for mediating extrinsic cues that drive fate decisions., (Copyright © 2018 The Authors. Published by Elsevier Inc. All rights reserved.)

Backgroud: Accumulating evidences indicate that circular RNAs (circRNAs), a class of non-coding RNAs, play important roles in tumorigenesis. However, the function of circRNAs in triple negative breast cancer (TNBC) is largely unknown., Methods: We performed circRNA microarrays to identify circRNAs that are aberrantly expressed in TNBC cell lines. Expression levels of a significantly upregulated circRNA, circGFRA1, was detected by quantitative real-time PCR (qRT-PCR) in TNBC cell lines and tissues. Kaplan-Meier survival analysis was used to explore the significance of circGFRA1 in clinical prognosis. Then, we examined the functions of circGFRA1 in TNBC by cell proliferation, apoptosis and mouse xenograft assay. In addition, luciferase assay was used to explore the miRNA sponge function of circGFRA1 in TNBC., Results: Microarray analysis and qRT-PCR verified a circRNA termed circGFRA1 that was upregulated in TNBC. Kaplan-Meier survival analysis showed that upregulated circGFRA1 was correlated with poorer survival. Knockdown of circGFRA1 inhibited proliferation and promoted apoptosis in TNBC. Via luciferase reporter assays, circGFRA1 and GFRA1 was observed to directly bind to miR-34a. Subsequent experiments showed that circGFRA1 and GFRA1 regulated the expression of each other by sponging miR-34a., Conclusions: Taken together, we conclude that circGFRA1 may function as a competing endogenous RNA (ceRNA) to regulate GFRA1 expression through sponging miR-34a to exert regulatory functions in TNBC. circGFRA1 may be a diagnostic biomarker and potential target for TNBC therapy.

The human spermatogonial compartment is essential for daily production of millions of sperm. Despite this crucial role, the molecular signature, kinetic behavior and regulation of human spermatogonia are poorly understood. Using human testis biopsies with normal spermatogenesis and by studying marker protein expression, we have identified for the first time different subpopulations of spermatogonia. MAGE-A4 marks all spermatogonia, KIT marks all B spermatogonia and UCLH1 all Apale-dark (Ap-d) spermatogonia. We suggest that at the start of the spermatogenic lineage there are Ap-d spermatogonia that are GFRA1 High , likely including the spermatogonial stem cells. Next, UTF1 becomes expressed, cells become quiescent and GFRA1 expression decreases. Finally, GFRA1 expression is lost and subsequently cells differentiate into B spermatogonia, losing UTF1 and acquiring KIT expression. Strikingly, most human Ap-d spermatogonia are out of the cell cycle and even differentiating type B spermatogonial proliferation is restricted. A novel scheme for human spermatogonial development is proposed that will facilitate further research in this field, the understanding of cases of infertility and the development of methods to increase sperm output., Competing Interests: Competing interestsThe authors declare no competing or financial interests., (© 2017. Published by The Company of Biologists Ltd.)

Recent progress in chemotherapy has significantly increased its efficacy, yet the development of chemoresistance remains a major drawback. In this study, we show that GFRA1/GFRα1 (GDNF family receptor α 1), contributes to cisplatin-induced chemoresistance by regulating autophagy in osteosarcoma. We demonstrate that cisplatin treatment induced GFRA1 expression in human osteosarcoma cells. Induction of GFRA1 expression reduced cisplatin-induced apoptotic cell death and it significantly increased osteosarcoma cell survival via autophagy. GFRA1 regulates AMPK-dependent autophagy by promoting SRC phosphorylation independent of proto-oncogene RET kinase. Cisplatin-resistant osteosarcoma cells showed NFKB1/NFκB-mediated GFRA1 expression. GFRA1 expression promoted tumor formation and growth in mouse xenograft models and inhibition of autophagy in a GFRA1-expressing xenograft mouse model during cisplatin treatment effectively reduced tumor growth and increased survival. In cisplatin-treated patients, treatment period and metastatic status were associated with GFRA1-mediated autophagy. These findings suggest that GFRA1-mediated autophagy is a promising novel target for overcoming cisplatin resistance in osteosarcoma.

In many mammalian species, surface markers have been used to obtain enriched populations of spermatogonial stem cells (SSCs) for assisted reproduction and other applications; however, little is known about the expression patterns of feline SSCs. In this study, we assessed expression of the SSC surface markers commonly used in other species, KIT, ITGA6, CD9, GFRalpha1, ADGRA3, and THY1, in addition to the less frequently used pluripotent markers TRA-1-60, TRA-1-81, SSEA-1, and SSEA-4 in SSCs of both prepubertal and adult domestic cats (Felis catus). To further characterize cat SSCs, we sorted cells using SSC-specific markers and evaluated the expression of the pluripotent transcription factors NANOG, POU5F1, and SOX2 and the proto-oncogene MYC within these populations. We concluded that SSC surface markers used in other mammalian species were not specific for identifying cat SSCs. However, the pluripotent markers we evaluated were more specific to cat spermatogonia, and the presence of SSEA-1 and SSEA-4 in fewer and primarily individual cells suggests that these two markers may be used for enrichment of cat SSCs. The expression of pluripotent transcription factors at mRNA level by single-stained cells positive for SSEA-4 and by dual-stained cells positive for both GFRalpha1 and SSEA-4 reflects the undifferentiated stage of cat SSCs. The absence of transcription factors in double-stained cells positive for only one marker implies the loss of the stem cell-like identity with the loss of either GFRalpha1 or SSEA-4. Further investigation is warranted to elucidate the biological characteristics of these spermatogonial subpopulations., (© 2016 by the Society for the Study of Reproduction, Inc.)

What makes the spermatogonial stem cells (SSCs) self-renew or differentiate to produce spermatozoa is barely understood, in particular in nonmammalian species. Our research explores possible regulations of the SSC niche in teleost, locally by paracrine factors and peripherally by hormonal regulation. In the present study, we focus on the Gdnf-Gfra1 pathway that plays a major role in the regulation of SSC self-renewal in mammals. We describe a complex evolution of the genes encoding for Gdnf and Gfra1 proteins in trout with the emergence of three gdnf and two gfra1 paralogs. Using quantitative PCR measurements in isolated testicular cell populations, the gdnfb paralog was found expressed in A-spermatogonia and probably in another testicular cell type. In contrast, the transcript of gfra1a, the Gdnf receptor, was preferentially expressed in a population of undifferentiated A-spermatogonia (und A-Spg) separated by centrifugal elutriation. These und A-Spg also demonstrated high stemness potential in transplantation studies and preferentially expressed nanos2, a putative SSC marker in trout (Bellaiche et al., Biol Reprod 2014; 90:79). Flow cytometer experiments demonstrate that only a subfraction of und A-Spg express Gfra1. In trout, spermatogenesis develops along a strict annual cycle, and gdnfb and its receptor were expressed in a spermatogenetic activity-dependent manner. In particular, a dramatic increase of the gdnfb transcript coincided with the progressive cessation of rapid spermatogonial proliferation and of meiosis toward the end of the reproductive cycle. Together these results suggest that, in trout, Gdnfb is involved in the repression of und A-Spg differentiation. Fsh is an endocrine regulator of SSCs self-renewal through the up-regulation of Gdnf in rodents. We demonstrate that in trout, in vitro Fsh treatment stimulated the expression of the gfra1a1 but not of its ligand, gdnfb. Fsh treatment also stimulated the proliferation of und A-Spg cocultured with testicular somatic cells. Based on those results, the Gfra1-positive cells could correspond to the putative SSCs in rainbow trout, and we propose that the balance between SSC self-renewal and differentiation during the trout spermatogenetic cycle is under paracrine regulation by Gdnfb, which represses, and under peripheral regulation by Fsh via the control of gfra1a1 expression., (© 2014 by the Society for the Study of Reproduction, Inc.)

In association with in vitro culture and transplantation, isolation of spermatogonial stem cells (SSCs) is an excellent approach for investigating spermatogonial physiology in vertebrates. However, in fish, the lack of SSC molecular markers represents a great limitation to identify/purify these cells, rendering it difficult to apply several valuable biotechnologies in fish-farming. Herein, we describe potential molecular markers, which served to phenotypically characterize, cultivate and transplant Nile tilapia SSCs. Immunolocalization revealed that Gfra1 is expressed exclusively in single type A undifferentiated spermatogonia (Aund, presumptive SSCs). Likewise, the expression of Nanos2 protein was observed in Aund cells. However, Nanos2-positive spermatogonia have also been identified in cysts with two to eight germ cells that encompass type A differentiated spermatogonia (Adiff). Moreover, we also established effective primary culture conditions that allowed the Nile tilapia spermatogonia to expand their population for at least one month while conserving their original undifferentiated (stemness) characteristics. The maintenance of Aund spermatogonial phenotype was demonstrated by the expression of early germ cell specific markers and, more convincingly, by their ability to colonize and develop in the busulfan-treated adult Nile tilapia recipient testes after germ cell transplantation. In addition to advancing our knowledge on the identity and physiology of fish SSCs, these findings provide the first step in establishing a system that will allow fish SSCs expansion in vitro, representing an important progress towards the development of new biotechnologies in aquaculture, including the possibility of producing transgenic fish., (Copyright © 2013 Elsevier Inc. All rights reserved.)