WTAP-mediated m6A modification of circ_0032463 promotes osteosarcoma progression by sponging miR-145-5p and regulating GFRA1 expression.

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.

PTN from Leydig cells activates SDC2 and modulates human spermatogonial stem cell proliferation and survival via GFRA1.

BACKGROUND: Spermatogonial stem cells (SSCs) are essential for the maintenance and initiation of male spermatogenesis. Despite the advances in understanding SSC biology in mouse models, the mechanisms underlying human SSC development remain elusive. RESULTS: Here, we analyzed the signaling pathways involved in SSC regulation by testicular somatic cells using single-cell sequencing data (GEO datasets: GSE149512 and GSE112013) and identified that Leydig cells communicate with SSCs through pleiotrophin (PTN) and its receptor syndecan-2 (SDC2). Immunofluorescence, STRING prediction, and protein immunoprecipitation assays confirmed the interaction between PTN and SDC2 in spermatogonia, but their co-localization was observed only in approximately 50% of the cells. The knockdown of SDC2 in human SSC lines impaired cell proliferation, DNA synthesis, and the expression of PLZF, a key marker for SSC self-renewal. Transcriptome analysis revealed that SDC2 knockdown downregulated the expression of GFRA1, a crucial factor for SSC proliferation and self-renewal, and inhibited the HIF-1 signaling pathway. Exogenous PTN rescued the proliferation and GFRA1 expression in SDC2 knockdown SSC lines. In addition, we found downregulation of PTN and SDC2 as well as altered localization in non-obstructive azoospermia (NOA) patients, suggesting that downregulation of PTN and SDC2 may be associated with impaired spermatogenesis. CONCLUSIONS: Our results uncover a novel mechanism of human SSC regulation by the testicular microenvironment and suggest a potential therapeutic target for male infertility.

Neutralizing antibody against GDF15 for treatment of cancer-associated cachexia.

GDF15 (growth differentiation factor 15), also known as macrophage inhibitory cytokine 1 (MIC-1), is a circulating protein involved in the regulation of energy balance and weight control. Elevated levels of GDF15 have been associated with cachexia and reduced survival rates in cancer patients. Through the activation of the GFRAL (GDNF-family receptor α-like)-RET (Rearranged during Transfection) signaling pathway, GDF15 can induce weight loss, making it a potential target for treating cachexia. Currently, there are no approved antibody drugs specifically targeting GDF15 for cancer cachexia treatment. However, efforts have been made to develop antibody-based therapeutics against this emerging target. In this study, we generated a monoclonal antibody KY-NAb-GDF15 against GDF15 that effectively blocks downstream signaling mediated by GFRAL upon stimulation by GDF15. This antibody demonstrates robust neutralizing activity and exhibits high binding specificity. Importantly, our findings indicate that this antibody holds promise in alleviating cancer-induced cachexia and mitigating chemotherapy-induced weight loss, thereby offering significant therapeutic potential for managing cancer cachexia.

Discovery of the first selective, small-molecule GFRα2/3 inhibitors through DNA-encoded library technology.

Studies have shown that disrupting the formation of the ligand-RET-GFRα complex could be an effective way of treating pain and itch. Compared to traditional high-throughput screens, DNA encoded libraries (DELs) have distinguished themselves as a powerful technology for hit identification in recent years. The present work demonstrates the use of DEL technology identifying compound 16 as the first GFRa2/GFRa3 small molecule inhibitor (0.1/0.2 µM respectively) selective over RET. This molecule represents an opportunity to advance the development of small-molecule inhibitors targeting the GFRα-RET interface for the treatment of pain and itch.

GDNF/GFRA1 signaling contributes to chemo- and radioresistance in glioblastoma.

Glioblastoma is the most common primary brain tumor in adults, characterized by an inherent aggressivity and resistance to treatment leading to poor prognoses. While some resistance mechanisms have been elucidated, a deeper understanding of these mechanisms is needed to increase therapeutic efficacy. In this study we first discovered glial-cell derived neurotrophic factor (GDNF) to be upregulated in patient-derived glioblastoma spheroid cultures after chemotherapeutic temozolomide treatment, through RNA-Seq experiments. Therefore, we investigated the role of the GDNF/GDNF receptor alpha 1 (GFRA1) signaling pathway as a resistance mechanism to chemotherapy with temozolomide and lomustine, as well as irradiation using patient-derived glioblastoma spheroid cultures. With qPCR experiments we showed a consistent upregulation of GDNF and its primary receptor GFRA1 following all three lines of treatment. Moreover, CRISPR/Cas9 knock-outs of GDNF in two patient-derived models sensitized these cells to chemotherapy treatment, but not radiotherapy. The increased sensitivity was completely reversed by the addition of exogeneous GDNF, confirming the key role of this factor in chemoresistance. Finally, a CRISPR KO of GFRA1 demonstrated a similar increased sensitivity to temozolomide and lomustine treatment, as well as radiotherapy. Together, our findings support the role of the GDNF/GFRA1 signaling pathway in glioblastoma chemo and radioresistance.

ARTN-GFRA3 axis induces epithelial-mesenchymal transition phenotypes, migration, and invasion of gastric cancer cells via KRAS signaling.

Neural invasion underlies the local spread of gastric cancer and is associated with poor prognosis. This process has been receiving increasing attention in recent years. However, the relationship between neural invasion and the malignant phenotypes of gastric cancer cells, as well as the molecular mechanism involved in this process, remain unclear. In this study, bioinformatics analysis was performed using a dataset obtained from The Cancer Genome Atlas-Stomach Adenocarcinoma. The results revealed that high expression of GDNF family receptor alpha 3 (GFRA3) was associated with a poor prognosis of patients with gastric cancer. GFRA3 is a receptor for artemin (ARTN), a glial cell line-derived neurotrophic factor (GDNF). This association was indicated by short overall/disease-free survival, as well as the presence of high-stage and high-grade disease. Gene set enrichment analysis showed that two cancer-associated pathways, namely KRAS signaling and epithelial-mesenchymal transition (EMT), were activated when GFRA3 was highly expressed in gastric cancer. Further studies confirmed that GFRA3 activated KRAS downstream signaling phosphatidylinositol 3 kinase/protein kinase B (PI3K/AKT) or extracellular signal-regulated kinase (ERK) and induced EMT markers, as well as promoted the migration and invasion of gastric cancer cells. As a ligand of GFRA3, ARTN induced the EMT, migration, and invasion of gastric cancer cells via GFRA3. Notably, the effects of the ARTN-GFRA3 axis were attenuated by treatment with a KRAS inhibitor. The present findings indicated that, during the neural invasion of gastric cancer, ARTN-mediated activation of GFRA3 induces EMT phenotypes, migration, and invasion of gastric cancer cells via KRAS signaling.

GDF15 controls primary cilia morphology and function thereby affecting progenitor proliferation.

We recently reported that growth/differentiation factor 15 (GDF15) and its receptor GDNF family receptor alpha-like (GFRAL) are expressed in the periventricular germinal epithelium thereby regulating apical progenitor proliferation. However, the mechanisms are unknown. We now found GFRAL in primary cilia and altered cilia morphology upon GDF15 ablation. Mutant progenitors also displayed increased histone deacetylase 6 (Hdac6) and ciliary adenylate cyclase 3 (Adcy3) transcript levels. Consistently, microtubule acetylation, endogenous sonic hedgehog (SHH) activation and ciliary ADCY3 were all affected in this group. Application of exogenous GDF15 or pharmacological antagonists of either HDAC6 or ADCY3 similarly normalized ciliary morphology, proliferation and SHH signalling. Notably, Gdf15 ablation affected Hdac6 expression and cilia length only in the mutant periventricular niche, in concomitance with ciliary localization of GFRAL. In contrast, in the hippocampus, where GFRAL was not expressed in the cilium, progenitors displayed altered Adcy3 expression and SHH signalling, but Hdac6 expression, cilia morphology and ciliary ADCY3 levels remained unchanged. Thus, ciliary signalling underlies the effect of GDF15 on primary cilia elongation and proliferation in apical progenitors.

GFRAL Is Widely Distributed in the Brain and Peripheral Tissues of Mice.

In 2017, four independent publications described the glial cell-derived neurotrophic factor (GDNF) receptor alpha-like (GFRAL) as receptor for the growth differentiation factor 15 (GDF15, also MIC-1, NAG-1) with an expression exclusively in the mice brainstem area postrema (AP) and nucleus tractus solitarii (NTS) where it mediates effects of GDF15 on reduction of food intake and body weight. GDF15 is a cell stress cytokine with a widespread expression and pleiotropic effects, which both seem to be in contrast to the reported highly specialized localization of its receptor. This discrepancy prompts us to re-evaluate the expression pattern of GFRAL in the brain and peripheral tissues of mice. In this detailed immunohistochemical study, we provide evidence for a more widespread distribution of this receptor. Apart from the AP/NTS region, GFRAL-immunoreactivity was found in the prefrontal cortex, hippocampus, nucleus arcuatus and peripheral tissues including liver, small intestine, fat, kidney and muscle tissues. This widespread receptor expression, not taken into consideration so far, may explain the multiple effects of GDF-15 that are not yet assigned to GFRAL. Furthermore, our results could be relevant for the development of novel pharmacological therapies for physical and mental disorders related to body image and food intake, such as eating disorders, cachexia and obesity.

Expression of Glial-Cell-Line-Derived Neurotrophic Factor Family Ligands in Human Intervertebral Discs.

Glial-cell-line-derived neurotrophic factor (GDNF) family ligands (GFLs) contribute to the sensitization of primary afferents and are involved in the pathogenesis of inflammatory pain. The purpose of this preliminary study was to examine the expression of other GFLs (neurturin (NRTN), artemin (ARTN), persephin (PSPN)) and receptors in human IVD cells and tissues exhibiting early and advanced stages of degeneration. Human IVD cells were cultured as a monolayer after isolation from the nucleus pulposus (NP) and anulus fibrosus (AF) tissues. The mRNA expression of NRTN, ARTN, PSPN, and their receptors (GFRA2-GFRA4) was quantified using real-time PCR. Protein expression was evaluated using immunohistochemistry and Western blotting. The expression of NRTN, ARTN, PSPN, and their co-receptors (GFRA2-GFRA4) was identified in human IVD cells at both mRNA and protein levels. A trend was noted wherein the mRNA expression of ARTN, PSPN, and GFRA2 was upregulated by IL-1ß treatment in a dose-dependent manner. The percentages of immunopositive cells in the advanced degenerate stage of ARTN, PSPN, and GFRA2 were significantly higher than those in the early degenerate stage. Their expression was enhanced in advanced tissue degeneration, which suggests that GFLs (ARTN and PSPN) may be involved in the pathogenesis of discogenic pain.

Overview of growth differentiation factor 15 in metabolic syndrome.

Growth and differentiation factor 15 (GDF15) is a member of the transforming growth factor-ß (TGF-ß) superfamily. GDF15 has been linked with several metabolic syndrome pathologies such as obesity and cardiovascular diseases. GDF15 is considered to be a metabolic regulator, although its precise mechanisms of action remain to be determined. Glial cell-derived neurotrophic factor family receptor alpha-like (GRAL), located in the hindbrain, has been identified as the receptor for GDF15 and signals through the coreceptor receptor tyrosine kinase (RET). Administration of GDF15 analogues in preclinical studies using various animal models has consistently been shown to induce weight loss through a reduction in food intake. GDF15, therefore, represents an attractive target to combat the current global obesity epidemic. In this article, we review current knowledge on GDF15 and its involvement in metabolic syndrome.

Tumor-associated macrophage-derived GDNF promotes gastric cancer liver metastasis via a GFRA1-modulated autophagy flux.

PURPOSE: Liver metastasis, a lethal malignancy of gastric cancer (GC) patients, execrably impairs their prognosis. As yet, however, few studies have been designed to identify the driving molecules during its formation, except screening evidence pausing before their functions or mechanisms. Here, we aimed to survey a key driving event within the invasive margin of liver metastases. METHODS: A metastatic GC tissue microarray was used for exploring malignant events during liver-metastasis formation, followed by assessing the expression patterns of glial cell-derived neurotrophic factor (GDNF) and GDNF family receptor alpha 1 (GFRA1). Their oncogenic functions were determined by both loss- and gain-of-function studies in vitro and in vivo, and validated by rescue experiments. Multiple cell biological studies were performed to identify the underlying mechanisms. RESULTS: In the invasive margin, GFRA1 was identified as a pivotal molecule involved in cellular survival during liver metastasis formation, and we found that its oncogenic role depends on tumor associated macrophage (TAM)-derived GDNF. In addition, we found that the GDNF-GFRA1 axis protects tumor cells from apoptosis under metabolic stress via regulating lysosomal functions and autophagy flux, and participates in the regulation of cytosolic calcium ion signalling in a RET-independent and non-canonical way. CONCLUSION: From our data we conclude that TAMs, homing around metastatic nests, induce the autophagy flux of GC cells and promote the development of liver metastasis via GDNF-GFRA1 signalling. This is expected to improve the comprehension of metastatic pathogenesis and to provide a novel direction of research and translational strategies for the treatment of metastatic GC patients.

The Role of Promyelocytic Leukemia Zinc Finger (PLZF) and Glial-Derived Neurotrophic Factor Family Receptor Alpha 1 (GFRα1) in the Cryopreservation of Spermatogonia Stem Cells.

The cryopreservation of spermatogonia stem cells (SSCs) has been widely used as an alternative treatment for infertility. However, cryopreservation itself induces cryoinjury due to oxidative and osmotic stress, leading to reduction in the survival rate and functionality of SSCs. Glial-derived neurotrophic factor family receptor alpha 1 (GFRα1) and promyelocytic leukemia zinc finger (PLZF) are expressed during the self-renewal and differentiation of SSCs, making them key tools for identifying the functionality of SSCs. To the best of our knowledge, the involvement of GFRα1 and PLZF in determining the functionality of SSCs after cryopreservation with therapeutic intervention is limited. Therefore, the purpose of this review is to determine the role of GFRα1 and PLZF as biomarkers for evaluating the functionality of SSCs in cryopreservation with therapeutic intervention. Therapeutic intervention, such as the use of antioxidants, and enhancement in cryopreservation protocols, such as cell encapsulation, cryoprotectant agents (CPA), and equilibrium of time and temperature increase the expression of GFRα1 and PLZF, resulting in maintaining the functionality of SSCs. In conclusion, GFRα1 and PLZF have the potential as biomarkers in cryopreservation with therapeutic intervention of SSCs to ensure the functionality of the stem cells.

The GDF15-GFRAL axis mediates chemotherapy-induced fatigue in mice.

Cancer-related fatigue is defined as a distressing persistent subjective sense of physical, emotional, and/or cognitive tiredness or exhaustion related to cancer or cancer treatment that is not proportional to recent activity and that interferes with usual functioning. This form of fatigue is highly prevalent during cancer treatment and in some patients, it can persist for years after treatment has ended. An understanding of the mechanisms that drive cancer-related fatigue is still lacking, which hampers the identification of effective treatment options. Various chemotherapeutic agents including cisplatin are known to induce mitochondrial dysfunction and this effect is known to mediate chemotherapy-induced peripheral neuropathy and cognitive dysfunction. Mitochondrial dysfunction results in the release of mitokines that act locally and at distance to promote metabolic and behavioral adjustments to this form of cellular stress. One of these mitokines, growth differentiation factor 15 (GDF15) and its receptor, glial cell line-derived neurotrophic factor family receptor α-like (GFRAL), have received special attention in oncology as activation of GFRAL mediates the anorexic response that is responsible for cancer anorexia. The present study was initiated to determine whether GDF15 and GFRAL are involved in cisplatin-induced fatigue. We first tested the ability of cisplatin to increase circulating GDF15 in mice before assessing whether GDF15 can induce behavioral fatigue measured by decreased wheel running in healthy mice and increase behavioral fatigue induced by cisplatin. Mice administered a long acting form of GDF15, mGDF15-fc, decreased their voluntary wheel running activity. When the same treatment was administered to mice receiving cisplatin, it increased the amplitude and duration of cisplatin-induced decrease in wheel running. To determine whether endogenous GDF15 mediates the behavioral fatigue induced by cisplatin, we then administered a neutralizing monoclonal antibody to GFRAL to mice injected with cisplatin. The GFRAL neutralizing antibody mostly prevented cisplatin-induced decrease in wheel running and accelerated recovery. Taken together these findings demonstrate for the first time the role of the GDF15/GFRAL axis in cisplatin-induced behaviors and indicate that this axis could be a promising therapeutic target for the treatment of cancer-related fatigue.

GDF-15 Inhibits ADP-Induced Human Platelet Aggregation through the GFRAL/RET Signaling Complex.

Growth differentiation factor-15 (GDF-15) is proposed to be strongly associated with several cardiovascular diseases, such as heart failure and atherosclerosis. Moreover, some recent studies have reported an association between GDF-15 and platelet activation. In this study, we isolated peripheral blood platelets from healthy volunteers and evaluated the effect of GDF-15 on adenosine diphosphate (ADP)-induced platelet activation using the platelet aggregation assay. Subsequently, we detected the expression of GDF-15-related receptors on platelets, including the epidermal growth factor receptor (EGFR), human epidermal growth factor receptor 2 (HER2), human epidermal growth factor receptor 3 (HER3), transforming growth factor-beta receptor I (TGF-ßRI), transforming growth factor-beta receptor II (TGF-ßRII), glial-cell-line-derived neurotrophic factor family receptor α-like (GFRAL), and those rearranged during transfection (RET). Then, we screened for GDF-15 receptors using the GDF-15-related receptor microarray comprising these recombinant proteins. We also performed the immunoprecipitation assay to investigate the interaction between GDF-15 and the receptors on platelets. For the further exploration of signaling pathways, we investigated the effects of GDF-15 on the extracellular signal-regulated kinase (ERK), protein kinase B (AKT), and Janus kinase 2 (JAK2) pathways. We also investigated the effects of GDF-15 on the ERK and AKT pathways and platelet aggregation in the presence or absence of RET agonists or inhibition. Our study revealed that GDF-15 can dose-independently inhibit ADP-induced human platelet aggregation and that the binding partner of GDF-15 on platelets is GFRAL. We also found that GDF-15 inhibits ADP-induced AKT and ERK activation in platelets. Meanwhile, our results revealed that the inhibitory effects of GDF-15 can be mediated by the GFRAL/RET complex. These findings reveal the novel inhibitory mechanism of ADP-induced platelet activation by GDF-15.

GDNF Increases Inhibitory Synaptic Drive on Principal Neurons in the Hippocampus via Activation of the Ret Pathway.

Glial cell line-derived neurotrophic factor (GDNF) has been shown to counteract seizures when overexpressed or delivered into the brain in various animal models of epileptogenesis or chronic epilepsy. The mechanisms underlying this effect have not been investigated. We here demonstrate for the first time that GDNF enhances GABAergic inhibitory drive onto mouse pyramidal neurons by modulating postsynaptic GABAA receptors, particularly in perisomatic inhibitory synapses, by GFRα1 mediated activation of the Ret receptor pathway. Other GDNF receptors, such as NCAM or Syndecan3, are not contributing to this effect. We observed similar alterations by GDNF in human hippocampal slices resected from epilepsy patients. These data indicate that GDNF may exert its seizure-suppressant action by enhancing GABAergic inhibitory transmission in the hippocampal network, thus counteracting the increased excitability of the epileptic brain. This new knowledge can contribute to the development of novel, more precise treatment strategies based on a GDNF gene therapy approach.

Missense Variants in GFRA1 and NPNT Are Associated with Congenital Anomalies of the Kidney and Urinary Tract.

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.

Mitochondrial stress-induced GFRAL signaling controls diurnal food intake and anxiety-like behavior.

Growth differentiation factor 15 (GDF15) is a mitochondrial stress-induced cytokine that modulates energy balance in an endocrine manner. However, the importance of its brainstem-restricted receptor GDNF family receptor alpha-like (GFRAL) to mediate endocrine GDF15 signaling to the brain upon mitochondrial dysfunction is still unknown. Using a mouse model with muscle-specific mitochondrial dysfunction, we here show that GFRAL is required for activation of systemic energy metabolism via daytime-restricted anorexia but not responsible for muscle wasting. We further find that muscle mitochondrial stress response involves a GFRAL-dependent induction of hypothalamic corticotropin-releasing hormone, without elevated corticosterone levels. Finally, we identify that GFRAL signaling governs an anxiety-like behavior in male mice with muscle mitochondrial dysfunction, with females showing a less robust GFRAL-dependent anxiety-like phenotype. Together, we here provide novel evidence of a mitochondrial stress-induced muscle-brain crosstalk via the GDF15-GFRAL axis to modulate food intake and anxiogenic behavior.

GDNF Promotes Astrocyte Abnormal Proliferation and Migration Through the GFRα1/RET/MAPK/pCREB/LOXL2 Signaling Axis.

Glial cell-line derived neurotrophic factor (GDNF) is a powerful astroglioma (AG) proliferation and migration factor that is highly expressed in AG cells derived from astrocytes. However, it is still unclear whether high levels of GDNF promote AG occurrence or if they are secondary to AG formation. We previously reported that high concentrations of GDNF (200 and 500 ng/mL) can inhibit DNA damage-induced rat primary astrocytes (RA) apoptosis, suggesting that high concentrations of GDNF may be involved in the malignant transformation of astrocytes to AG cells. Here we show that 200 ng/mL GDNF significantly increased the proliferation and migration ability of RA cells and human primary astrocytes (HA). This treatment also induced RA cells to highly express Pgf, Itgb2, Ibsp, Loxl2, Lif, Cxcl10, Serpine1, and other genes that enhance AG proliferation and migration. LOXL2 is an important AG occurrence and development promotion factor and was highly expressed in AG tissues and cells. High concentrations of GDNF promote LOXL2 expression and secretion in RA cells through GDNF family receptor alpha-1(GFRα1)/rearranged during transfection proto-oncogene (RET)/mitogen-activated protein kinase (MAPK)/phosphorylated cyclic AMP response element binding protein (pCREB) signaling. GDNF-induced LOXL2 significantly promotes RA and HA cell proliferation and migration, and increases the expression of Ccl2, Gbp5, MMP11, TNN, and other genes that regulate the extracellular microenvironment in RA cells. Our results demonstrate that high concentrations of GDNF activate LOXL2 expression and secretion via the GFRα1/RET/MAPK/pCREB signal axis, which leads to remodeling of the astrocyte extracellular microenvironment through molecules such as Ccl2, Gbp5, MMP11, TNN. This ultimately results in abnormal astrocyte proliferation and migration. Collectively, these findings suggest that high GDNF concentrations may promote the malignant transformation of astrocytes to AG cells.

The GDF15-GFRAL pathway is dispensable for the effects of metformin on energy balance.

Metformin is a blood-glucose-lowering medication with physiological effects that extend beyond its anti-diabetic indication. Recently, it was reported that metformin lowers body weight via induction of growth differentiation factor 15 (GDF15), which suppresses food intake by binding to the GDNF family receptor α-like (GFRAL) in the hindbrain. Here, we corroborate that metformin increases circulating GDF15 in mice and humans, but we fail to confirm previous reports that the GDF15-GFRAL pathway is necessary for the weight-lowering effects of metformin. Instead, our studies in wild-type, GDF15 knockout, and GFRAL knockout mice suggest that the GDF15-GFRAL pathway is dispensable for the effects of metformin on energy balance. The data presented here question whether metformin is a sufficiently strong stimulator of GDF15 to drive anorexia and weight loss and emphasize that additional work is needed to untangle the relationship among metformin, GDF15, and energy balance.