Tumor-Induced Generation of Splenic Erythroblast-like Ter-Cells Promotes Tumor Progression.

Identifying tumor-induced leukocyte subsets and their derived circulating factors has been instrumental in understanding cancer as a systemic disease. Nevertheless, how primary tumor-induced non-leukocyte populations in distal organs contribute to systemic spread remains poorly defined. Here, we report one population of tumor-inducible, erythroblast-like cells (Ter-cells) deriving from megakaryocyte-erythroid progenitor cells with a unique Ter-119+CD45-CD71+ phenotype. Ter-cells are enriched in the enlarged spleen of hosts bearing advanced tumors and facilitate tumor progression by secreting neurotrophic factor artemin into the blood. Transforming growth factor ß (TGF-ß) and Smad3 activation are important in Ter-cell generation. In vivo blockade of Ter-cell-derived artemin inhibits hepatocellular carcinoma (HCC) growth, and artemin deficiency abolishes Ter-cells' tumor-promoting ability. We confirm the presence of splenic artemin-positive Ter-cells in human HCC patients and show that significantly elevated serum artemin correlates with poor prognosis. We propose that Ter-cells and the secreted artemin play important roles in cancer progression with prognostic and therapeutic implications.

Ret is a multifunctional coreceptor that integrates diffusible- and contact-axon guidance signals.

Growing axons encounter multiple guidance cues, but it is unclear how separate signals are resolved and integrated into coherent instructions for growth cone navigation. We report that glycosylphosphatidylinositol (GPI)-anchored ephrin-As function as "reverse" signaling receptors for motor axons when contacted by transmembrane EphAs present in the dorsal limb. Ephrin-A receptors are thought to depend on transmembrane coreceptors for transmitting signals intracellularly. We show that the receptor tyrosine kinase Ret is required for motor axon attraction mediated by ephrin-A reverse signaling. Ret also mediates GPI-anchored GFRα1 signaling in response to GDNF, a diffusible chemoattractant in the limb, indicating that Ret is a multifunctional coreceptor for guidance molecules. Axons respond synergistically to coactivation by GDNF and EphA ligands, and these cooperative interactions are gated by GFRα1 levels. Our studies uncover a hierarchical GPI-receptor signaling network that is constructed from combinatorial components and integrated through Ret using ligand coincidence detection.

Camptothecin effectively treats obesity in mice through GDF15 induction.

Elevated circulating levels of growth differentiation factor 15 (GDF15) have been shown to reduce food intake and lower body weight through activation of hindbrain receptor glial-derived neurotrophic factor (GDNF) receptor alpha-like (GFRAL) in rodents and nonhuman primates, thus endogenous induction of this peptide holds promise for obesity treatment. Here, through in silico drug-screening methods, we found that small molecule Camptothecin (CPT), a previously identified drug with potential antitumor activity, is a GDF15 inducer. Oral CPT administration increases circulating GDF15 levels in diet-induced obese (DIO) mice and genetic ob/ob mice, with elevated Gdf15 expression predominantly in the liver through activation of integrated stress response. In line with GDF15's anorectic effect, CPT suppresses food intake, thereby reducing body weight, blood glucose, and hepatic fat content in obese mice. Conversely, CPT loses these beneficial effects when Gdf15 is inhibited by a neutralizing antibody or AAV8-mediated liver-specific knockdown. Similarly, CPT failed to reduce food intake and body weight in GDF15's specific receptor GFRAL-deficient mice despite high levels of GDF15. Together, these results indicate that CPT is a promising anti-obesity agent through activation of GDF15-GFRAL pathway.

Plzf regulates germline progenitor self-renewal by opposing mTORC1.

Hyperactivity of mTORC1, a key mediator of cell growth, leads to stem cell depletion, although the underlying mechanisms are poorly defined. Using spermatogonial progenitor cells (SPCs) as a model system, we show that mTORC1 impairs stem cell maintenance by a negative feedback from mTORC1 to receptors required to transduce niche-derived signals. We find that SPCs lacking Plzf, a transcription factor essential for SPC maintenance, have enhanced mTORC1 activity. Aberrant mTORC1 activation in Plzf(-/-) SPCs inhibits their response to GDNF, a growth factor critical for SPC self-renewal, via negative feedback at the level of the GDNF receptor. Plzf opposes mTORC1 activity by inducing expression of the mTORC1 inhibitor Redd1. Thus, we identify the mTORC1-Plzf functional interaction as a critical rheostat for maintenance of the spermatogonial pool and propose a model whereby negative feedback from mTORC1 to the GDNF receptor balances SPC growth with self-renewal.

The GDF15-GFRAL Pathway in Health and Metabolic Disease: Friend or Foe?

GDF15 is a cell activation and stress response cytokine of the glial cell line-derived neurotrophic factor family within the TGF-ß superfamily. It acts through a recently identified orphan member of the GFRα family called GFRAL and signals through the Ret coreceptor. Cell stress and disease lead to elevated GDF15 serum levels, causing anorexia, weight loss, and alterations to metabolism, largely by actions on regions of the hindbrain. These changes restore homeostasis and, in the case of obesity, cause a reduction in adiposity. In some diseases, such as advanced cancer, serum GDF15 levels can rise by as much as 10-100-fold, leading to an anorexia-cachexia syndrome, which is often fatal. This review discusses how GDF15 regulates appetite and metabolism, the role it plays in resistance to obesity, and how this impacts diseases such as diabetes, nonalcoholic fatty liver disease, and anorexia-cachexia syndrome. It also discusses potential therapeutic applications of targeting the GDF15-GFRAL pathway and lastly suggests some potential unifying hypotheses for its biological role.

Adult medial habenula neurons require GDNF receptor GFRα1 for synaptic stability and function.

The medial habenula (mHb) is an understudied small brain nucleus linking forebrain and midbrain structures controlling anxiety and fear behaviors. The mechanisms that maintain the structural and functional integrity of mHb neurons and their synapses remain unknown. Using spatiotemporally controlled Cre-mediated recombination in adult mice, we found that the glial cell-derived neurotrophic factor receptor alpha 1 (GFRα1) is required in adult mHb neurons for synaptic stability and function. mHb neurons express some of the highest levels of GFRα1 in the mouse brain, and acute ablation of GFRα1 results in loss of septohabenular and habenulointerpeduncular glutamatergic synapses, with the remaining synapses displaying reduced numbers of presynaptic vesicles. Chemo- and optogenetic studies in mice lacking GFRα1 revealed impaired circuit connectivity, reduced AMPA receptor postsynaptic currents, and abnormally low rectification index (R.I.) of AMPARs, suggesting reduced Ca2+ permeability. Further biochemical and proximity ligation assay (PLA) studies defined the presence of GluA1/GluA2 (Ca2+ impermeable) as well as GluA1/GluA4 (Ca2+ permeable) AMPAR complexes in mHb neurons, as well as clear differences in the levels and association of AMPAR subunits with mHb neurons lacking GFRα1. Finally, acute loss of GFRα1 in adult mHb neurons reduced anxiety-like behavior and potentiated context-based fear responses, phenocopying the effects of lesions to septal projections to the mHb. These results uncover an unexpected function for GFRα1 in the maintenance and function of adult glutamatergic synapses and reveal a potential new mechanism for regulating synaptic plasticity in the septohabenulointerpeduncular pathway and attuning of anxiety and fear behaviors.

Activation of the hypothalamic-pituitary-adrenal axis by exogenous and endogenous GDF15.

An acute increase in the circulating concentration of glucocorticoid hormones is essential for the survival of severe somatic stresses. Circulating concentrations of GDF15, a hormone that acts in the brain to reduce food intake, are frequently elevated in stressful states. We now report that GDF15 potently activates the hypothalamic-pituitary-adrenal (HPA) axis in mice and rats. A blocking antibody to the GDNF-family receptor α-like receptor completely prevented the corticosterone response to GDF15 administration. In wild-type mice exposed to a range of stressful stimuli, circulating levels of both corticosterone and GDF15 rose acutely. In the case of Escherichia coli or lipopolysaccharide injections, the vigorous proinflammatory cytokine response elicited was sufficient to produce a near-maximal HPA response, regardless of the presence or absence of GDF15. In contrast, the activation of the HPA axis seen in wild-type mice in response to the administration of genotoxic or endoplasmic reticulum toxins, which do not provoke a marked rise in cytokines, was absent in Gdf15-/- mice. In conclusion, consistent with its proposed role as a sentinel hormone, endogenous GDF15 is required for the activation of the protective HPA response to toxins that do not induce a substantial cytokine response. In the context of efforts to develop GDF15 as an antiobesity therapeutic, these findings identify a biomarker of target engagement and a previously unrecognized pharmacodynamic effect, which will require monitoring in human studies.

GFRAL-expressing neurons suppress food intake via aversive pathways.

The TGFß cytokine family member, GDF-15, reduces food intake and body weight and represents a potential treatment for obesity. Because the brainstem-restricted expression pattern of its receptor, GDNF Family Receptor α-like (GFRAL), presents an exciting opportunity to understand mechanisms of action for area postrema neurons in food intake; we generated GfralCre and conditional GfralCreERT mice to visualize and manipulate GFRAL neurons. We found infection or pathophysiologic states (rather than meal ingestion) stimulate GFRAL neurons. TRAP-Seq analysis of GFRAL neurons revealed their expression of a wide range of neurotransmitters and neuropeptides. Artificially activating GfralCre -expressing neurons inhibited feeding, decreased gastric emptying, and promoted a conditioned taste aversion (CTA). GFRAL neurons most strongly innervate the parabrachial nucleus (PBN), where they target CGRP-expressing (CGRPPBN) neurons. Silencing CGRPPBN neurons abrogated the aversive and anorexic effects of GDF-15. These findings suggest that GFRAL neurons link non-meal-associated pathophysiologic signals to suppress nutrient uptake and absorption.

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.

Non-homeostatic body weight regulation through a brainstem-restricted receptor for GDF15.

Under homeostatic conditions, animals use well-defined hypothalamic neural circuits to help maintain stable body weight, by integrating metabolic and hormonal signals from the periphery to balance food consumption and energy expenditure. In stressed or disease conditions, however, animals use alternative neuronal pathways to adapt to the metabolic challenges of altered energy demand. Recent studies have identified brain areas outside the hypothalamus that are activated under these 'non-homeostatic' conditions, but the molecular nature of the peripheral signals and brain-localized receptors that activate these circuits remains elusive. Here we identify glial cell-derived neurotrophic factor (GDNF) receptor alpha-like (GFRAL) as a brainstem-restricted receptor for growth and differentiation factor 15 (GDF15). GDF15 regulates food intake, energy expenditure and body weight in response to metabolic and toxin-induced stresses; we show that Gfral knockout mice are hyperphagic under stressed conditions and are resistant to chemotherapy-induced anorexia and body weight loss. GDF15 activates GFRAL-expressing neurons localized exclusively in the area postrema and nucleus tractus solitarius of the mouse brainstem. It then triggers the activation of neurons localized within the parabrachial nucleus and central amygdala, which constitute part of the 'emergency circuit' that shapes feeding responses to stressful conditions. GDF15 levels increase in response to tissue stress and injury, and elevated levels are associated with body weight loss in numerous chronic human diseases. By isolating GFRAL as the receptor for GDF15-induced anorexia and weight loss, we identify a mechanistic basis for the non-homeostatic regulation of neural circuitry by a peripheral signal associated with tissue damage and stress. These findings provide opportunities to develop therapeutic agents for the treatment of disorders with altered energy demand.

A dual role for peripheral GDNF signaling in nociception and cardiovascular reflexes in the mouse.

Group III/IV muscle afferents transduce nociceptive signals and modulate exercise pressor reflexes (EPRs). However, the mechanisms governing afferent responsiveness to dually modulate these processes are not well characterized. We and others have shown that ischemic injury can induce both nociception-related behaviors and exacerbated EPRs in the same mice. This correlated with primary muscle afferent sensitization and increased expression of glial cell line-derived neurotrophic factor (GDNF) in injured muscle and increased expression of GDNF family receptor α1 (GFRα1) in dorsal root ganglia (DRG). Here, we report that increased GDNF/GFRα1 signaling to sensory neurons from ischemia/reperfusion-affected muscle directly modulated nociceptive-like behaviors and increased exercise-mediated reflexes and group III/IV muscle afferent sensitization. This appeared to have taken effect through increased cyclic adenosine monophosphate (cAMP) response element binding (CREB)/CREB binding protein-mediated expression of the purinergic receptor P2X5 in the DRGs. Muscle GDNF signaling to neurons may, therefore, play an important dual role in nociception and sympathetic reflexes and could provide a therapeutic target for treating complications from ischemic injuries.

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.

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.

Small-molecule agonists of the RET receptor tyrosine kinase activate biased trophic signals that are influenced by the presence of GFRa1 co-receptors.

Glial cell line-derived neurotrophic factor (GDNF) is a growth factor that regulates the health and function of neurons and other cells. GDNF binds to GDNF family receptor α1 (GFRa1), and the resulting complex activates the RET receptor tyrosine kinase and subsequent downstream signals. This feature restricts GDNF activity to systems in which GFRa1 and RET are both present, a scenario that may constrain GDNF breadth of action. Furthermore, this co-dependence precludes the use of GDNF as a tool to study a putative functional cross-talk between GFRa1 and RET. Here, using biochemical techniques, terminal deoxynucleotidyl transferase dUTP nick end labeling staining, and immunohistochemistry in murine cells, tissues, or retinal organotypic cultures, we report that a naphthoquinone/quinolinedione family of small molecules (Q compounds) acts as RET agonists. We found that, like GDNF, signaling through the parental compound Q121 is GFRa1-dependent. Structural modifications of Q121 generated analogs that activated RET irrespective of GFRa1 expression. We used these analogs to examine RET-GFRa1 interactions and show that GFRa1 can influence RET-mediated signaling and enhance or diminish AKT Ser/Thr kinase or extracellular signal-regulated kinase signaling in a biased manner. In a genetic mutant model of retinitis pigmentosa, a lead compound, Q525, afforded sustained RET activation and prevented photoreceptor neuron loss in the retina. This work uncovers key components of the dynamic relationships between RET and its GFRa co-receptor and provides RET agonist scaffolds for drug development.

Glial-derived neurotrophic factor in human airway smooth muscle.

Airway smooth muscle (ASM) cells modulate the local airway milieu via production of inflammatory mediators and growth factors including classical neurotrophins, such as brain-derived neurotrophic factor (BDNF). The glial cell-derived neurotrophic factor (GDNF) family of ligands (GFLs) are nonclassical neurotrophins and their role in the airway is barely understood. The major GFLs, GDNF and Neurturin (NRTN) bind to GDNF family receptor (GFR) α1 and α2 respectively that pair with Ret receptor to accomplish signaling. In this study, we found GDNF is expressed in human lung and increased in adult asthma, while human ASM expresses GDNF and its receptors. Accordingly, we used human ASM cells to test the hypothesis that ASM expression and autocrine signaling by GFLs regulate [Ca2+ ]i . Serum-deprived ASM cells from non-asthmatics were exposed to 10 ng/ml GDNF or NRTN for 15 min (acute) or 24 h (chronic). In fura-2 loaded cells, acute GDNF or NRTN alone induced [Ca2+ ]i responses, and further enhanced responses to 1 µM ACh or 10 µM histamine. Ret inhibitor (SPP86; 10 µM) or specific GDNF chelator GFRα1-Fc (1 µg/ml) showed roles of these receptors in GDNF effects. In contrast, NRTN did not enhance [Ca2+ ]i response to histamine. Furthermore, conditioned media of nonasthmatic and asthmatic ASM cells showed GDNF secretion. SPP86, Ret inhibitor and GFRα1-Fc chelator markedly decreased [Ca2+ ]i response compared with vehicle, highlighting autocrine effects of secreted GDNF. Chronic GDNF treatment increased histamine-induced myosin light chain phosphorylation. These novel data demonstrate GFLs particularly GDNF/GFRα1 influence ASM [Ca2+ ]i and raise the possibility that GFLs are potential targets of airway hyperresponsiveness.

Macrophage inhibitory cytokine-1 promotes angiogenesis by eliciting the GFRAL-mediated endothelial cell signaling.

Macrophage inhibitory cytokine-1 (MIC-1) is a cytokine with pleotropic actions and its expression is markedly increased by inflammation and cardiac injury and in cancers. In particular, MIC-1 production after cardiac ischemia injury is associated with enhanced cardiac angiogenesis as well as myocardial protection. However, it remains uncertain whether MIC-1 itself has proangiogenic activity. In this study, we tried to determine the precise role of MIC-1 in physiological and pathological angiogenesis. Human microvessel endothelial cells responded to MIC-1 with enhanced angiogenic behaviors. Employing various angiogenesis assays, MIC-1 was found to promote vessel formation and development with a potency similar to that of vascular endothelial growth factor (VEGF). MIC-1 transgenic (Tg) mice also displayed enhanced neovascularization in both developing embryos and neonatal mouse retinas, compared with wild-type mice. Furthermore, endothelial cells (ECs) isolated from MIC-1 Tg mouse lung exhibited higher angiogenic potential than ECs from wild-type lung. MIC-1-induced angiogenesis was also observed in the recovery or healing processes of injuries such as hindlimb ischemia and skin wounds in mice. However, unlike VEGF, MIC-1 induced neither endothelial inflammation nor increased vascular permeability. In ECs, the MIC-1 signal exerted proangiogenic actions via the MEK/extracellular signal-regulated kinase- and phosphatidylinositol 3-kinase/Akt-dependent pathways. Notably, these MIC-1 signaling events in ECs were abrogated by small interfering RNA-mediated knockdown of GFRAL, suggesting that GFRAL is an EC receptor for MIC-1. In summary, we here show a novel role of MIC-1 as a potent EC activator, which promotes both normal and injury-related angiogenesis.

Human splenic TER cells: A relevant prognostic factor acting via the artemin-GFRα3-ERK pathway in pancreatic ductal adenocarcinoma.

Splenectomy is routinely performed during distal or total pancreatectomy (DP or TP) for pancreatic ductal adenocarcinoma (PDAC), but information about its oncological value is limited. TER cells, nonimmune cells discovered in the spleens of tumour-bearing mice, are elicited by tumours and promote tumour progression, while their role in the clinical outcomes of patients with PDAC remains unclear. In our study, postoperative specimens from 622 patients who underwent DP or TP with splenectomy were analysed by flow cytometry or immunofluorescence, and the relationship between splenic TER cell count and clinical parameters was calculated. We also purified human TER cells for functional experiments and mechanistic studies. We found that TER cell numbers were increased only in the spleens of patients with PDAC but not in PDAC tissue and adjacent pancreatic tissue. High splenic TER cell counts independently predicted poor prognosis (P < .001) and indicated large tumour size, lymph node metastasis, advanced 8th AJCC/mAJCC stage and high CA19-9 classification (all P < .050) in patients with PDAC. Mechanistic analysis showed that TER cells express artemin, which facilitates the proliferation and invasion of PDAC cells by activating GFRα3-ERK signalling. Our study reveals that TER cell count is an indicator of poor prognosis of PDAC, while splenectomy during pancreatic surgery might provide oncological benefits in addition to ensuring the radical resection of PDAC.

GILZ-dependent modulation of mTORC1 regulates spermatogonial maintenance.

Male fertility is dependent on spermatogonial stem cells (SSCs) that self-renew and produce differentiating germ cells. Growth factors produced within the testis are essential for SSC maintenance but intrinsic factors that dictate the SSC response to these stimuli are poorly characterised. Here, we have studied the role of GILZ, a TSC22D family protein and spermatogenesis regulator, in spermatogonial function and signalling. Although broadly expressed in the germline, GILZ was prominent in undifferentiated spermatogonia and Gilz deletion in adults resulted in exhaustion of the GFRα1+ SSC-containing population and germline degeneration. GILZ loss was associated with mTORC1 activation, suggesting enhanced growth factor signalling. Expression of deubiquitylase USP9X, an mTORC1 modulator required for spermatogenesis, was disrupted in Gilz mutants. Treatment with an mTOR inhibitor rescued GFRα1+ spermatogonial failure, indicating that GILZ-dependent mTORC1 inhibition is crucial for SSC maintenance. Analysis of cultured undifferentiated spermatogonia lacking GILZ confirmed aberrant activation of ERK MAPK upstream mTORC1 plus USP9X downregulation and interaction of GILZ with TSC22D proteins. Our data indicate an essential role for GILZ-TSC22D complexes in ensuring the appropriate response of undifferentiated spermatogonia to growth factors via distinct inputs to mTORC1.

Glial cell line-derived neurotrophic factor increases matrix metallopeptidase 9 and 14 expression in microglia and promotes microglia-mediated glioma progression.

Glial cell line-derived neurotrophic factor (GDNF) is released by glioma cells and promotes tumor growth. We have previously found that GDNF released from the tumor cells is a chemoattractant for microglial cells, the immune cells of the central nervous system. Here we show that GDNF increases matrix metalloproteinase (MMP) 9 and MMP14 expression in cultured microglial cells from mixed sexes of neonatal mice. The GDNF-induced microglial MMP9 and MMP14 upregulation is mediated by GDNF family receptor alpha 1 receptors and dependent on p38 mitogen-activated protein kinase signaling. In organotypic brain slices, GDNF promotes the growth of glioma and this effect depends on the presence of microglia. We also previously found that MMP9 and MMP14 upregulation can be mediated by Toll-like receptor (TLR) 2 signaling and here we demonstrate that GDNF increases the expression of TLR1 and TLR2. In conclusion, GDNF promotes the pro-tumorigenic phenotype of microglia.

Corneal nonmyelinating Schwann cells illuminated by single-cell transcriptomics and visualized by protein biomarkers.

The cornea is the most innervated tissue in the human body. Myelinated axons upon inserting into the peripheral corneal stroma lose their myelin sheaths and continue into the central cornea wrapped by only nonmyelinating corneal Schwann cells (nm-cSCs). This anatomical organization is believed to be important for central vision. Here we employed single-cell RNA sequencing (scRNA-seq), microscopy, and transgenics to characterize these nm-cSCs of the central cornea. Using principal component analysis, uniform manifold approximation and projection, and unsupervised hierarchal cell clustering of scRNA-seq data derived from central corneal cells of male rabbits, we successfully identified several clusters representing different corneal cell types, including a unique cell cluster representing nm-cSCs. To confirm protein expression of cSC genes, we performed cross-species validation, employing corneal whole-mount immunostaining with confocal microscopy in mouse corneas. The expression of several representative proteins of nm-cSCs were validated. As the proteolipid protein 1 (PLP1) gene was also expressed in nm-cSCs, we explored the Plp1-eGFP transgenic reporter mouse line to visualize cSCs. Specific and efficient eGFP expression was observed in cSCs in adult mice of different ages. Of several putative cornea-specific SC genes identified, Dickkopf-related protein 1 was shown to be present in nm-cSCs. Taken together, our findings, for the first time, identify important insights and tools toward the study nm-cSCs in isolated tissue and adult animals. We expect that our results will advance the future study of nm-cSCs in applications of nerve repair, and provide a resource for the study of corneal sensory function.