Glial Cell Line-derived Neurotrophic Factor Family Research Articles

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.

Preventing obesity, insulin resistance and type 2 diabetes by targeting MT1-MMP

Obesity is one of the predominant risk factors for type 2 diabetes. Despite all the modern advances in medicine, an effective drug treatment for obesity without overt side effects has not yet been found. The discovery of growth and differentiation factor 15 (GDF15), an appetite-regulating hormone, created hopes for the treatment of obesity. However, an insufficient understanding of the physiological regulation of GDF15 has been a major obstacle to mitigating GDF15-centric treatment of obesity. Our recent studies revealed how a series of proteolytic events predominantly mediated by membrane-type 1 matrix metalloproteinase (MT1-MMP/MMP14), a key cell-surface metalloproteinase involved in extracellular remodeling, contribute to the pathogenesis of metabolic disorders, including obesity and diabetes. The MT1-MMP-mediated cleavage of the GDNF family receptor-α-like (GFRAL), a key neuronal receptor of GDF15, controls the satiety center in the hindbrain, thereby regulating non-homeostatic appetite and bodyweight changes. Furthermore, increased activation of MT1-MMP does not only lead to increased risk of obesity, but also causes age-associated insulin resistance by cleaving Insulin Receptor in major metabolic tissues. Importantly, inhibition of MT1-MMP effectively protects against obesity and diabetes, revealing the therapeutic potential of targeting MT1-MMP for the management of metabolic disorders.

Artesunate treats obesity in male mice and non-human primates through GDF15/GFRAL signalling axis

Obesity, a global health challenge, is a major risk factor for multiple life-threatening diseases, including diabetes, fatty liver, and cancer. There is an ongoing need to identify safe and tolerable therapeutics for obesity management. Herein, we show that treatment with artesunate, an artemisinin derivative approved by the FDA for the treatment of severe malaria, effectively reduces body weight and improves metabolic profiles in preclinical models of obesity, including male mice with overnutrition-induced obesity and male cynomolgus macaques with spontaneous obesity, without inducing nausea and malaise. Artesunate promotes weight loss and reduces food intake in obese mice and cynomolgus macaques by increasing circulating levels of Growth Differentiation Factor 15 (GDF15), an appetite-regulating hormone with a brainstem-restricted receptor, the GDNF family receptor α-like (GFRAL). Mechanistically, artesunate induces the expression of GDF15 in multiple organs, especially the liver, in mice through a C/EBP homologous protein (CHOP)-directed integrated stress response. Inhibition of GDF15/GFRAL signalling by genetic ablation of GFRAL or tissue-specific knockdown of GDF15 abrogates the anti-obesity effect of artesunate in mice with diet-induced obesity, suggesting that artesunate controls bodyweight and appetite in a GDF15/GFRAL signalling-dependent manner. These data highlight the therapeutic benefits of artesunate in the treatment of obesity and related comorbidities.

Endogenous GDNF Is Unable to Halt Dopaminergic Injury Triggered by Microglial Activation.

Overactivation of microglial cells seems to play a crucial role in the degeneration of dopaminergic neurons occurring in Parkinson's disease. We have previously demonstrated that glial cell line-derived neurotrophic factor (GDNF) present in astrocytes secretome modulates microglial responses induced by an inflammatory insult. Therefore, astrocyte-derived soluble factors may include relevant molecular players of therapeutic interest in the control of excessive neuroinflammatory responses. However, in vivo, the control of neuroinflammation is more complex as it depends on the interaction between different types of cells other than microglia and astrocytes. Whether neurons may interfere in the astrocyte-microglia crosstalk, affecting the control of microglial reactivity exerted by astrocytes, is unclear. Therefore, the present work aimed to disclose if the control of microglial responses mediated by astrocyte-derived factors, including GDNF, could be affected by the crosstalk with neurons, impacting GDNF's ability to protect dopaminergic neurons exposed to a pro-inflammatory environment. Also, we aimed to disclose if the protection of dopaminergic neurons by GDNF involves the modulation of microglial cells. Our results show that the neuroprotective effect of GDNF was mediated, at least in part, by a direct action on microglial cells through the GDNF family receptor α-1. However, this protective effect seems to be impaired by other mediators released in response to the neuron-astrocyte crosstalk since neuron-astrocyte secretome, in contrast to astrocytes secretome, was unable to protect dopaminergic neurons from the injury triggered by lipopolysaccharide-activated microglia. Supplementation with exogenous GDNF was needed to afford protection of dopaminergic neurons exposed to the inflammatory environment. In conclusion, our results revealed that dopaminergic protective effects promoted by GDNF involve the control of microglial reactivity. However, endogenous GDNF is insufficient to confer dopaminergic neuron protection against an inflammatory insult. This reinforces the importance of further developing new therapeutic strategies aiming at providing GDNF or enhancing its expression in the brain regions affected by Parkinson's disease.

2 Water-Based Exercise Improves Cognition and Reduces Neurological-Related Plasma Proteins in Older Adults.

Objective:In the United States, Alzheimer’s disease (AD) is the most common cause of dementia and the seventh leading cause of death. Exercise has demonstrated health benefits in older adults and reduces the risk of developing AD. Exploring underlying biological mechanisms of exercise could aid in identifying therapeutic targets to prevent AD progression, especially for high-risk individuals such as those with Mild Cognitive Impairment (MCI). Many studies of dementia focus on memory; however, executive function and processing speed are also vulnerable to the neuropathology that causes AD. This exploratory study aimed to identify potential mechanisms by which physical activity can facilitate change in cognitive functioning in older adults. This was accomplished by investigating correlations between changes in neurology-related plasma proteins and changes in measures of executive function and processing speed after participation in a water-based exercise intervention.Participants and Methods:The sample included 20 older adults with amnestic MCI, ages 55-82 years (mean 68.15 ±7.75). Participants were predominately male (90%), White (70%), and non-Hispanic (85%), with more than high school education (95%). Participants engaged in supervised high-intensity water-based exercise three times per week for six months. Neuropsychological assessments and blood samples were assessed at baseline and after completion of the exercise intervention. Cognitive measures included: the Digit Span subtests from the Wechsler Adult Intelligence Scale, 4th Edition, Trail-Making Test (TMT), Stroop Color Word Test (SCWT), and the Symbol Digit Modalities Test (SDMT). Plasma protein levels were analyzed using the Olink Target 96 Neurology assay (Uppsala, Sweden), selected a priori for the established markers linked to neurobiological processes and diseases. Changes in cognitive measures and protein levels were assessed using paired-sample t-tests, and Pearson’s correlations were calculated for significant findings.Results:Participants’ cognitive performance significantly improved on the SCWT color trial (t = -2.19, p = 0.042) and SDMT (t = -2.17, p = .043). Significant decreases in plasma proteins levels were found for GDNF family receptor alpha-1 ([GFRA1]: t =2.05, p = 0.055), neuroblastoma suppressor tumorigenicity-1 ([NBL1]: t = 2.13, p= .046), and neuropilin-2 ([NRP2]: t = 2.61 p= 0.017). Correlational analyses showed reductions in NBL1 were significantly associated with changes in both SDMT (r = -.61, p = 0.006) and the color trial of SCWT (r = .48, p = .038), and NRP2 was significantly associated with improvement on the SDMT (r = -.46, p = 0.045). GFRA1 was not significantly associated with change on any cognitive measure.Conclusions:In a sample of older adults with MCI, participation in high-intensity water-based exercise led to significant improvements in cognitive function as well as changes in neurological plasma proteome. Improved outcomes in processing speed, attention, visuospatial scanning, and working memory were associated with changes in specific plasma protein concentrations. This highlights potential activity-dependent neurobiological mechanisms that may underlie the cognitive benefits derived from physical activity. Future studies should explore these findings in Randomized Control Trials with a comparative condition and larger sample size.

Dietary medium-chain fatty acids reduce food intake via the GDF15-GFRAL axis in mice

ObjectiveMedium chain fatty acids (MCFAs), which are fatty acids with chain lengths of 8–12 carbon atoms, have been shown to reduce food intake in rodents and humans, but the underlying mechanisms are unknown. Unlike most other fatty acids, MCFAs are absorbed from the intestine into the portal vein and enter first the liver. We thus hypothesized that MCFAs trigger the release of hepatic factors that reduce appetite. MethodsThe liver transcriptome in mice that were orally administered MCFAs as C8:0 triacylglycerol (TG) was analyzed. Circulating growth/differentiation factor 15 (GDF15), tissue Gdf15 mRNA and food intake were investigated after acute oral gavage of MCFAs as C8:0 or C10:0 TG in mice. Effects of acute and subchronic administration of MCFAs as C8:0 TG on food intake and body weight were determined in mice lacking either the receptor for GDF15, GDNF Family Receptor Alpha Like (GFRAL), or GDF15. ResultsHepatic and small intestinal expression of Gdf15 and circulating GDF15 increased after ingestion of MCFAs, while intake of typical dietary long-chain fatty acids (LCFAs) had no effect. Plasma GDF15 levels also increased in the portal vein with MCFA intake, indicating that in addition to the liver, the small intestine contributes to the rise in circulating GDF15. Acute oral provision of MCFAs decreased food intake over 24 h compared with a LCFA-containing bolus, and this anorectic effect required the GDF15 receptor, GFRAL. Moreover, subchronic oral administration of MCFAs reduced body weight over 7 days, an effect that was blunted in mice lacking either GDF15 or GFRAL. ConclusionsWe have identified ingestion of MCFAs as a novel nutritional approach that increases circulating GDF15 in mice and have revealed that the GDF15-GFRAL axis is required for the full anorectic effect of MCFAs.

Differential expression of RET and GDNF family receptor, GFR-α1, between striatum and substantia nigra following nigrostriatal lesion: A case for diminished GDNF-signaling

Although glial cell line-derived neurotrophic factor (GDNF) showed efficacy in preclinical and early clinical studies to alleviate parkinsonian signs in Parkinson's disease (PD), later trials did not meet primary endpoints, giving pause to consider further investigation. While GDNF dose and delivery methods may have contributed to diminished efficacy, one crucial aspect of these clinical studies is that GDNF treatment began ∼8 years after PD diagnosis; a time point representing several years after near 100% depletion of nigrostriatal dopamine markers in striatum and at least 50% in substantia nigra (SN), which represents a time point of initiating GDNF treatment later than reported in some preclinical studies. With nigrostriatal terminal loss exceeding 70% at PD diagnosis, we utilized hemiparkinsonian rats to determine if expression of GDNF family receptor, GFR-α1, and receptor tyrosine kinase, RET, differed between striatum and SN at 1 and 4 weeks following a 6-hydroxydopamine (6-OHDA) hemilesion. Whereas GDNF expression changed minimally, GFR-α1 expression decreased progressively in striatum and in tyrosine hydroxylase positive (TH+) cells in SN, correlating with reduced TH cell number. However, in nigral astrocytes, GFR-α1 expression increased. RET expression decreased maximally in striatum by 1 week, whereas in the SN, a transient bilateral increase occurred, returning to control levels by 4 weeks. Expression of brain-derived neurotrophic factor (BDNF) or its receptor, TrkB, were unchanged throughout lesion progression. Together, these results reveal that differential GFR-α1 and RET expression between the striatum and SN, and cell-specific differences in GFR-α1 expression in SN, occur during nigrostriatal neuron loss. Targeting loss of GDNF receptors thus appears critical to enhance GDNF therapeutic efficacy against nigrostriatal neuron loss. Significance statementAlthough preclinical evidence supports that GDNF provides neuroprotection and improves locomotor function in preclinical studies, there is uncertainty if it can alleviate motor impairment in Parkinson's disease patients. Using the established 6-OHDA hemiparkinsonian rat model, we determined whether expression of its cognate receptors, GFR-α1 and RET, were differentially affected between striatum and substantia nigra in a timeline study. In striatum, there was early and significant loss of RET, but a gradual, progressive loss of GFR-α1. In contrast, RET transiently increased in lesioned substantia nigra, but GFR-α1 progressively decreased only in nigrostriatal neurons and correlated with TH cell loss. Our results indicate that direct availability of GFR-α1 may be a critical element that determines GDNF efficacy following striatal delivery.

Metformin triggers a kidney GDF15-dependent area postrema axis to regulate food intake and body weight

Metformin, the most widely prescribed medication for obesity-associated type 2 diabetes (T2D), lowers plasma glucose levels, food intake, and body weight in rodents and humans, but the mechanistic site(s) of action remain elusive. Metformin increases plasma growth/differentiation factor 15 (GDF15) levels to regulate energy balance, while GDF15 administration activates GDNF family receptor α-like (GFRAL) that is highly expressed in the area postrema (AP) and the nucleus of the solitary tract (NTS) of the hindbrain to lower food intake and body weight. However, the tissue-specific contribution of plasma GDF15 levels after metformin treatment is still under debate. Here, we found that metformin increased plasma GDF15 levels in high-fat (HF) fed male rats through the upregulation of GDF15 synthesis in the kidney. Importantly, the kidney-specific knockdown of GDF15 expression as well as the AP-specific knockdown of GFRAL expression negated the ability of metformin to lower food intake and body weight gain. Taken together, we unveil the kidney as a target of metformin to regulate energy homeostasis through a kidney GDF15-dependent AP axis.

GDNF family receptor alpha-like antagonist antibody alleviates chemotherapy-induced cachexia in melanoma-bearing mice.

Patients with cancer undergoing chemotherapy experience cachexia with anorexia, body weight loss, and the depletion of skeletal muscles and adipose tissues. Effective treatment strategies for chemotherapy-induced cachexia are scarce. The growth differentiation factor 15 (GDF15)/GDNF family receptor alpha-like (GFRAL)/rearranged during transfection (RET) axis is a critical signalling pathway in chemotherapy-induced cachexia. In this study, we developed a fully human GFRAL antagonist antibody and investigated whether it inhibits the GDF15/GFRAL/RET axis, thereby alleviating chemotherapy-induced cachexia in tumour-bearing mice. Anti-GFRAL antibodies were selected via biopanning, using a human combinatorial antibody phage library. The potent GFRAL antagonist antibody A11 was selected via a reporter cell assay and its inhibitory activity of GDF15-induced signalling was evaluated using western blotting. To investigate the in vivo function of A11, a tumour-bearing mouse model was established by inoculating 8-week-old male C57BL/6 mice with B16F10 cells (n=10-16 mice per group). A11 was administered subcutaneously (10mg/kg) 1day before intraperitoneal treatment with cisplatin (10mg/kg). Animals were assessed for changes in food intake, body weight, and tumour volume. Plasma and key metabolic tissues such as skeletal muscles and adipose tissues were collected for protein and mRNA expression analysis. A11 reduced serum response element-luciferase reporter activity up to 74% (P<0.005) in a dose-dependent manner and blocked RET phosphorylation up to 87% (P=0.0593), AKT phosphorylation up to 28% (P=0.0593) and extracellular signal regulatory kinase phosphorylation up to 75% (P=0.0636). A11 inhibited the action of cisplatin-induced GDF15 on the brainstem and decreased GFRAL-positive neuron population expressing c-Fos in the area postrema and nucleus of the solitary tract by 62% in vivo (P<0.05). In a melanoma mouse model treated with cisplatin, A11 recovered anorexia by 21% (P<0.05) and tumour-free body weight loss by 13% (P<0.05). A11 significantly improved the cisplatin-induced loss of skeletal muscles (quadriceps: 21%, gastrocnemius: 9%, soleus: 13%, P<0.05) and adipose tissues (epididymal white adipose tissue: 37%, inguinal white adipose tissue: 51%, P<0.05). Our study suggests that GFRAL antagonist antibody may alleviate chemotherapy-induced cachexia, providing a novel therapeutic approach for patients with cancer experiencing chemotherapy-induced cachexia.

A network map of GDNF/RET signaling pathway in physiological and pathological conditions.

Glial cell line-derived neurotrophic factor (GDNF) signals through a multi-component receptor system predominantly consisting of glycosyl-phosphatidylinositol-anchored GDNF family receptor alpha-1 (GFRα1) and the Rearranged during transfection (RET) receptor tyrosine kinase. GDNF/RET signaling is vital to the central and peripheral nervous system, kidney morphogenesis, and spermatogenesis. In addition, the dysregulation of the GDNF/RET signaling has been implicated in the pathogenesis of cancers. Despite the extensive research on GDNF/RET signaling, a molecular network of reactions induced by GDNF reported across the published literature. However, a comprehensive GDNF/RET pathway resource is currently unavailable. We describe an integrated signaling pathway reaction map of GDNF/RET consisting of 1151 molecular reactions. These include information pertaining to 52 molecular association events, 70 enzyme catalysis events, 36 activation/inhibition events, 22 translocation events, 856 gene regulation events, and 115 protein-level expression events induced by GDNF in diverse cell types. We developed a comprehensive GDNF/RET signaling network map based on these molecular reactions. The pathway map was made accessible through WikiPathways database ( https://www.wikipathways.org/index.php/Pathway:WP5143 ). Biocuration and development of gene regulatory network map of GDNF/RET signaling pathway.

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.

P10.06.A Expression of GDNF and its receptors in glioblastoma

Abstract Background Neurotrophic factors are important for brain development, but their role in glioblastoma, the most aggressive primary brain cancer, is not fully understood. We focused on glial cell line-derived neurotrophic factor (GDNF), which has been shown to play a role in growth and migration in glioblastoma. The aim of this study was to investigate the expression of GDNF and its primary receptor GFRA1 as well as GDNF family receptors GFRA2-4 and RET. Material and Methods Tissue sections from 10 glioblastoma patients were stained with antibodies against GDNF (frozen tissue) and each of the receptors (formalin-fixed paraffin-embedded tissue). Moreover, the sections were stained with an RNA Scope assay for GDNF mRNA. The area fractions of positive receptor staining were quantified with a software-based classifier. Double immunofluorescence stainings were performed for GFRA1 and the astrocytic tumor cell marker GFAP, the microglia and macrophage marker IBA1, and the tumor stem cell markers OLIG2 and SOX2, respectively, for assessment of co-expression. Results GDNF staining in a varying number of cells was seen in 5 of the 10 samples. GDNF mRNA stainings were observed in a varying number of cells in all stained tissues, including in hypoxic areas and in microvascular proliferations. Mean area fractions of the receptors were 31 % (range 7-56) for GFRA1, 3 % (0,2-7) for GFRA2, 0,4 % (0,01-2) for GFRA3, 0,1 % (0,02-0,3) for GFRA4 and 1 % (0,1-2) for RET. Co-expression of GFRA1 and GFAP was widely present. Co-expression of GFRA1 and either OLIG2 or SOX2 was partially present. Co-expression of GFRA1 and IBA1 was limited. Conclusion Our results suggest that GDNF and GFRA1 are widely expressed in glioblastoma tissue, but GFRA2-4 and RET are also expressed and may play a role in glioblastoma tumor biology. Future experimental studies are needed to clarify the functional role of GDNF and its receptors in glioblastoma biology.

Evaluation of Nrf2/ARE Signaling Pathway in the Presence of Pentoxifylline as a Cryoprotectant in Mouse Spermatogonial Stem Cells.

The process of spermatogonial stem cell cryopreservation (SSCs) in young male cancer survivors is associated with increased reactive oxygen species (ROS), DNA fragmentation, apoptosis, decreased cell activity, and finally reduced fertility of SSCs. Therefore, it is necessary to add cryoprotectants to the freezing medium to minimize the injuries associated with cryopreservation. In addition, the Nrf2/ARE pathway is a main cellular pathway that regulates the antioxidant defense system. The purpose of this study was to evaluate the cryoprotective effect of pentoxifylline (PTX) on SSCs after freezing-thawing through the Nrf2/ARE pathway. SSCs extracted from neonatal mice testes were isolated and their purity was measured by flow cytometry with GDNF family receptor alpha-1 (GFRα1) and inhibitor of differentiation 4 (ID4). After culturing, the cells were frozen in different groups for 1 month. After freezing-thawing, cell viability, colonization rate, and intracellular ROS, malondialdehyde (MDA), superoxide dismutase (SOD), and catalase (CAT) were evaluated. Quantitative real-time polymerase chain reaction and western blotting were done to assess the expression levels of Nrf2, Keap-1, PI3K, and AKT genes and proteins. The survival and colonization rates of SSCs, SOD, and CAT levels, and Nrf2, PI3K, and AKT expression levels were significantly higher in the PTX group compared with the other cryopreservation groups. The Keap-1 expression level and the ROS and MDA production levels also decreased significantly in the PTX group (p-value <0.05). According to our findings, PTX can activate the antioxidant defense through the Nrf2/ARE signaling pathway; therefore, it could be a suitable cryoprotectant candidate for freezing and long-term storage of SSCs in the clinical setting.

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 500ng/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 200ng/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.

Abstract 1524: Expression of GDNF and its receptors in glioblastoma

Abstract Introduction: Glioblastoma is the most aggressive primary brain cancer and one of the most lethal types of cancer overall. Despite multimodal treatment strategies, the prognosis is poor with a median survival of approx. 15 months. Although neurotrophic factors are of major importance in the nervous system, their role in brain cancer is still not fully understood. We focused on the glial cell line-derived neurotrophic factor (GDNF), which has been shown to play a role in growth and migration in glioblastoma. The aim of this study was to investigate the expression of GDNF and it primary receptor GFRA1, as well as the other GDNF family receptors GFRA2-4 and RET. Methods: Tissue sections from 10 glioblastoma patients were stained with antibodies against GDNF and each of the receptors. GDNF stainings were performed on frozen tissue slides, while receptor stainings were performed on formalin-fixed paraffin-embedded tissue sections. The area fractions of positive staining were quantified with a software-based classifier. Double immunofluorescence stainings with the different markers and Iba1 as well as GFAP were performed to assess expression in microglia/macrophages and tumor cells, respectively. Results: GDNF staining was observed in five of ten patient specimens, whereas GFRA1 and GFRA2 staining was observed in all samples. GFRA1 and GFRA2 staining was seen in the membrane and cytoplasm with a mean area fraction of GFRA1 staining at 31% (range: 7-56%), and a mean area fraction of GFRA2 staining at 3% (range: 0.2-7%). GFRA3, GFRA4 and RET were not expressed in glioblastoma tissue. Double immunofluorescence stainings demonstrated co-expression of GFRA1 and GFAP, while co-expression of GFRA1 and IBA1 was limited. Conclusion and perspectives: Our results suggest that GDNF and GFRA1 are widely expressed in glioblastoma tumor tissue, but GFRA2 is also expressed and may play a role in glioblastoma biology. Additional stainings are being established to clarify cellular expression and expression levels of GDNF and its receptors. Citation Format: Jesper D. Ewald, Arnon M. Knudsen, Bjarne W. Kristensen. Expression of GDNF and its receptors in glioblastoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 1524.

235-LB: YH34160, a Novel Long-Acting GDF15 Fusion Protein, Exerts Potent and Sustained Body Weight Loss in Rodent Obesity Models

Growth differentiation factor 15 (GDF15) is a multifunctional protein associated with energy homeostasis and body weight regulation. GDF15 has emerged as an attractive therapeutic target to treat obesity-related metabolic disorders. GDF15 reduces food intake with a unique mode of action via its receptor GDNF family receptor α-like (GFRAL) and the co-receptor RET. YH34160 is an engineered GDF15 variant-Fc fusion protein to have extended half-life and potent functional activity by enhancing binding affinity to GDF15 receptors (GFRAL/RET) . Based on rodent and monkey pharmacokinetic (PK) data, YH34160 is expected to have an optimal PK profile for once-weekly dosing in humans. In efficacy studies using obese mouse models, YH34160 demonstrated a potent and sustained weight-lowering effect. After a single subcutaneous injection in diet-induced obese (DIO) mice, YH34160-treated groups showed sustained and dose-dependent body weight (BW) reduction compared to long-acting glucagon like peptide-1 receptor agonist (GLP-1RA) . Following a 6-week multiple-dose study in DIO mice, YH34160-treated groups exhibited a greater and more prolonged BW loss compared to the group of GLP-1RA. Marked BW reduction and improved lipid profile by YH34160 were also proved in leptin-deficient (ob/ob) mice compared to GLP-1RA. In addition, YH34160 caused significantly better anti-obesity effects, and a much more improved metabolic profile than that of albumin-GDF15, and dual GLP-1/Glucagon receptor agonist. Interestingly, YH34160 in combination with GLP-1RA or dual GLP-1/glucose-dependent insulinotropic polypeptide receptor agonist (GIPRA) achieved more potent and greater BW loss compared to each mono-treated group. Overall, these findings indicate that YH34160 would be a promising therapeutic candidate, as well as in combination with GLP-1-based therapeutics, for the treatment of obesity and obesity-related comorbidities. Disclosure S. Lim: None. D. Kim: None. J. Yang: None. M. Ju: None. S. Kim: None. B. Sim: None. J. Kim: None. S. Oh: Employee; Yuhan, Stock/Shareholder; Yuhan.

154-OR: The Site Specific Role of GDF15 in Metformin Action

Metformin lowers glucose levels, appetite and body weight in rodents and humans, but the mechanisms on energy balance regulation have been rarely studied. Metformin increases plasma growth/differentiation factor 15 (GDF15) levels in humans and rodents, while elevating GDF15 via GDF15 infusion in rodents lowers food intake and body weight via its receptor GDNF family receptor α-like (GFRAL) , which is expressed in the hindbrain nucleus area postrema (AP) . However, whether the GDF15-GFRAL axis in the AP mediates metformin effect is unknown. In this study, metformin (200 mg/kg) was infused into the upper small intestine (USI) of rats, while fasting-refeeding study or intravenous glucose tolerance test was performed. USI metformin infusion elevated plasma GDF15 levels (met 138 ± 25 vs. veh 59 ± 7 pg/ml, n = 8, p &amp;lt; 0.01) in parallel to a lowering of food intake (met 30 ± 2 vs. veh 40 ± 2 kcal, n = 16, p &amp;lt; 0.01) and an increase in glucose tolerance (AUC met 5243 ± 75 vs. veh 5674 ± 153, n = 7, p &amp;lt; 0.05) . Further, metformin lowered not only food intake but also body weight gain by 12 h (met 14 ± 1 vs. veh 23 ± 3 g, n = 8, p &amp;lt; 0.01) . Importantly, a knockdown of GFRAL achieved via an injection of lentivirus expressing shRNA GFRAL vs. mismatch into the AP by ∼40% (p &amp;lt; 0.05) negated the ability of metformin to not only lower food intake and body weight, but also increase glucose tolerance independent of body weight. Finally, acute AP GDF15 infusion (2 pmol) decreased food intake (GDF15-25 ± 2 vs. veh-36 ± 3 kcal, n = 10, p &amp;lt; 0.01) and weight gain (as early at 6 h, GDF15 3.6 ± 1.2 vs. vehicle 8.7 ± 1.6 g, n = 10, p &amp;lt; 0.05) , and increased glucose tolerance independently from body weight (AUC, GDF15-5322 ± 94 vs. veh-5722 ± 169, n = 6, p &amp;lt; 0.05) , and these metabolic effects of AP GDF15 infusion were prevented by GFRAL knockdown in the AP. In summary, our findings indicate that the AP GDF15-GFRAL axis is sufficient and necessary for metformin to regulate energy and glucose homeostasis in rodents. Disclosure S.Zhang: None. R.Li: None. Z.Danaei: None. Y.Lim: None. T.K.Lam: None. Funding Canadian Institutes of Health Research (FDN-143204)

Modulation of nigral dopamine signaling mitigates parkinsonian signs of aging: evidence from intervention with calorie restriction or inhibition of dopamine uptake.

Identifying neurobiological mechanisms of aging-related parkinsonism, and lifestyle interventions that mitigate them, remain critical knowledge gaps. No aging study, from rodent to human, has reported loss of any dopamine (DA) signaling marker near the magnitude associated with onset of parkinsonian signs in Parkinson's disease (PD). However, in substantia nigra (SN), similar loss of DA signaling markers in PD or aging coincide with parkinsonian signs. Alleviation of these parkinsonian signs may be possible by interventions such as calorie restriction (CR), which augment DA signaling markers like tyrosine hydroxylase (TH) expression in the SN, but not striatum. Here, we interrogated respective contributions of nigral and striatal DA mechanisms to aging-related parkinsonian signs in aging (18months old) rats in two studies: by the imposition of CR for 6months, and inhibition of DA uptake within the SN or striatum by cannula-directed infusion of nomifensine. Parkinsonian signs were mitigated within 12weeks after CR and maintained until 24months old, commensurate with increased D1 receptor expression in the SN alone, and increased GDNF family receptor, GFR-α1, in the striatum, suggesting increased GDNF signaling. Nomifensine infusion into the SN or striatum selectively increased extracellular DA. However, only nigral infusion increased locomotor activity. These results indicate mechanisms that increase components of DA signaling in the SN alone mitigate parkinsonian signs in aging, and are modifiable by interventions, like CR, to offset parkinsonian signs, even at advanced age. Moreover, these results give evidence that changes in nigral DA signaling may modulate some parameters of locomotor activity autonomously from striatal DA signaling.

Gdnf Acts as a Germ Cell-Derived Growth Factor and Regulates the Zebrafish Germ Stem Cell Niche in Autocrine- and Paracrine-Dependent Manners.

Glial cell line-derived neurotrophic factor (GDNF) and its receptor (GDNF Family Receptor α1-GFRα1) are well known to mediate spermatogonial stem cell (SSC) proliferation and survival in mammalian testes. In nonmammalian species, Gdnf and Gfrα1 orthologs have been found but their functions remain poorly investigated in the testes. Considering this background, this study aimed to understand the roles of the Gdnf-Gfrα1 signaling pathway in zebrafish testes by combining in vivo, in silico and ex vivo approaches. Our analysis showed that zebrafish exhibit two paralogs for Gndf (gdnfa and gdnfb) and its receptor, Gfrα1 (gfrα1a and gfrα1b), in accordance with a teleost-specific third round of whole genome duplication. Expression analysis further revealed that both ligands and receptors were expressed in zebrafish adult testes. Subsequently, we demonstrated that gdnfa is expressed in the germ cells, while Gfrα1a/Gfrα1b was detected in early spermatogonia (mainly in types Aund and Adiff) and Sertoli cells. Functional ex vivo analysis showed that Gdnf promoted the creation of new available niches by stimulating the proliferation of both type Aund spermatogonia and their surrounding Sertoli cells but without changing pou5f3 mRNA levels. Strikingly, Gdnf also inhibited late spermatogonial differentiation, as shown by the decrease in type B spermatogonia and down-regulation of dazl in a co-treatment with Fsh. Altogether, our data revealed that a germ cell-derived factor is involved in maintaining germ cell stemness through the creation of new available niches, supporting the development of spermatogonial cysts and inhibiting late spermatogonial differentiation in autocrine- and paracrine-dependent manners.