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Since spermatogonial stem cells (SSCs) are capable of both self-renewal and differentiation to daughter cells for subsequent spermatogenesis, the development of an efficient in vitro culture system is essential for studies related to spermatogenesis. Although the currently available system is serum-free and contains only chemically-defined components, it highly relies upon bovine serum albumin (BSA), a component with batch-to-batch quality variations similar to those of fetal bovine serum. Thus, we searched for an alternative BSA-free culture system that preserved the properties of SSCs. In this study, we utilized Knockout Serum Replacement (KSR) in the SSC culture medium, as a substitute for BSA. The results demonstrated that KSR supported the continuous growth of SSCs in vitro and the SSC activity in vivo without BSA, in a feeder-cell combination with mouse embryonic fibroblasts. The addition of BSA to KSR further facilitated cell cycle progression, whereas a transplantation assay revealed that the addition of BSA did not affect the number of SSCs in vivo. The combination of KSR with BSA also allowed the elimination of GFRA1 and FGF2, and the reduction of the GDNF concentration from 20 ng/ml to 5 ng/ml, while maintaining the growth rate and the expression of SSC markers. Furthermore, KSR was also useful with SSCs from non-DBA/2 strains, such as C57BL/6 and ICR. These results suggested that KSR is an effective substitute for BSA for long-term in vitro cultures of SSCs. Therefore, this method is practical for various studies related to SSCs, including spermatogenesis and germ stem cell biology.

Metformin, the world’s most prescribed anti-diabetic drug, is also effective in preventing type 2 diabetes in people at high risk 1 , 2 . More than 60% of this effect is attributable to the ability of metformin to lower body weight in a sustained manner 3 . The molecular mechanisms by which metformin lowers body weight are unknown. Here we show—in two independent randomized controlled clinical trials—that metformin increases circulating levels of the peptide hormone growth/differentiation factor 15 (GDF15), which has been shown to reduce food intake and lower body weight through a brain-stem-restricted receptor. In wild-type mice, oral metformin increased circulating GDF15, with GDF15 expression increasing predominantly in the distal intestine and the kidney. Metformin prevented weight gain in response to a high-fat diet in wild-type mice but not in mice lacking GDF15 or its receptor GDNF family receptor α-like (GFRAL). In obese mice on a high-fat diet, the effects of metformin to reduce body weight were reversed by a GFRAL-antagonist antibody. Metformin had effects on both energy intake and energy expenditure that were dependent on GDF15, but retained its ability to lower circulating glucose levels in the absence of GDF15 activity. In summary, metformin elevates circulating levels of GDF15, which is necessary to obtain its beneficial effects on energy balance and body weight, major contributors to its action as a chemopreventive agent. In mouse studies, metformin treatment results in increased secretion of growth/differentiation factor 15 (GDF15), which prevents weight gain in response to high-fat diet, and GDF15-independent lowering of circulating blood glucose.

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.

Parkinson’s disease is the second most common neurodegenerative condition with its prevalence projected to 8.9 million individuals globally in the year 2019. Parkinson’s disease affects both motor and certain non-motor functions of an individual. Numerous research has focused on the neuroprotective effect of the glial cell line–derived neurotrophic factor (GDNF) in Parkinson’s disease. Discovered in 1993, GDNF is a neurotrophic factor identified from the glial cells which was found to have selective effects on promoting survival and regeneration of certain populations of neurons including the dopaminergic nigrostriatal pathway. Given this property, recent studies have focused on the exogenous administration of GDNF for relieving Parkinson’s disease–related symptoms both at a pre-clinical and a clinical level. This review will focus on enumerating the molecular connection between Parkinson’s disease and GDNF and shed light on all the available drug delivery approaches to facilitate the selective delivery of GDNF into the brain paving the way as a potential therapeutic candidate for Parkinson’s disease in the future.

In this study, the authors show that, during the development of the enteric nervous system, a subset of enteric neural crest cells migrate from midgut across the mesentery to colonize caudal hindgut. This trans-mesenteric migration requires GDNF and GFRα1 signaling. Cell migration is fundamental to organogenesis. During development, the enteric neural crest cells (ENCCs) that give rise to the enteric nervous system (ENS) migrate and colonize the entire length of the gut, which undergoes substantial growth and morphological rearrangement. How ENCCs adapt to such changes during migration, however, is not fully understood. Using time-lapse imaging analyses of mouse ENCCs, we show that a population of ENCCs crosses from the midgut to the hindgut via the mesentery during a developmental time period in which these gut regions are transiently juxtaposed, and that such 'trans-mesenteric' ENCCs constitute a large part of the hindgut ENS. This migratory process requires GDNF signaling, and evidence suggests that impaired trans-mesenteric migration of ENCCs may underlie the pathogenesis of Hirschsprung disease (intestinal aganglionosis). The discovery of this trans-mesenteric ENCC population provides a basis for improving our understanding of ENS development and pathogenesis.

Fibro-adipogenic progenitors (FAPs) are muscle-resident mesenchymal progenitors that can contribute to muscle tissue homeostasis and regeneration, as well as postnatal maturation and lifelong maintenance of the neuromuscular system. Recently, traumatic injury to the peripheral nerve was shown to activate FAPs, suggesting that FAPs can respond to nerve injury. However, questions of how FAPs can sense the anatomically distant peripheral nerve injury and whether FAPs can directly contribute to nerve regeneration remained unanswered. Here, utilizing single-cell transcriptomics and mouse models, we discovered that a subset of FAPs expressing GDNF receptors Ret and Gfra1 can respond to peripheral nerve injury by sensing GDNF secreted by Schwann cells. Upon GDNF sensing, this subset becomes activated and expresses Bdnf . FAP-specific inactivation of Bdnf ( Prrx1 Cre ; Bdnf fl/fl ) resulted in delayed nerve regeneration owing to defective remyelination, indicating that GDNF-sensing FAPs play an important role in the remyelination process during peripheral nerve regeneration. In aged mice, significantly reduced Bdnf expression in FAPs was observed upon nerve injury, suggesting the clinical relevance of FAP-derived BDNF in the age-related delays in nerve regeneration. Collectively, our study revealed the previously unidentified role of FAPs in peripheral nerve regeneration, and the molecular mechanism behind FAPs’ response to peripheral nerve injury.

GDF15 has potent anti-obesity effects, but its receptor was unknown. GFRAL has now been identified as the receptor that mediates GDF15's effects via central actions in the hindbrain. Growth differentiation factor 15 (GDF15; also known as MIC-1) is a divergent member of the TGF-β superfamily and is associated with body-weight regulation in humans and rodents. However, the cognate receptor of GDF15 is unknown. Here we show that GDF15 binds specifically to GDNF family receptor α-like (GFRAL) with high affinity, and that GFRAL requires association with the coreceptor RET to elicit intracellular signaling in response to GDF15 stimulation. We also found that GDF15-mediated reductions in food intake and body weight of mice with obesity were abolished in GFRAL-knockout mice. We further found that GFRAL expression was limited to hindbrain neurons and not present in peripheral tissues, which suggests that GDF15–GFRAL-mediated regulation of food intake is by a central mechanism. Lastly, given that GDF15 did not increase energy expenditure in treated mice with obesity, the anti-obesity actions of the cytokine are likely driven primarily by a reduction in food intake.

Absence of glial cell line-derived neurotrophic factor (GDNF) leads to intestinal aganglionosis. We recently demonstrated that patients with diverticular disease (DD) exhibit hypoganglionosis suggesting neurotrophic factor deprivation. Thus, we screened mRNA expression pattern of the GDNF system in DD and examined the effects of GDNF on cultured enteric neurons. Colonic specimens obtained from patients with DD (n = 21) and controls (n = 20) were assessed for mRNA expression levels of the GDNF system (GDNF, GDNF receptors GFRα1 and RET). To identify the tissue source of GDNF and its receptors, laser-microdissected (LMD) samples of human myenteric ganglia and intestinal muscle layers were analyzed separately by qPCR. Furthermore, the effects of GDNF treatment on cultured enteric neurons (receptor expression, neuronal differentiation and plasticity) were monitored. mRNA expression of GDNF and its receptors was significantly down-regulated in the muscularis propria of patients with DD. LMD samples revealed high expression of GDNF in circular and longitudinal muscle layers, whereas GDNF receptors were also expressed in myenteric ganglia. GDNF treatment of cultured enteric neurons increased mRNA expression of its receptors and promoted neuronal differentiation and plasticity revealed by synaptophysin mRNA and protein expression. Our results suggest that the GDNF system is compromised in DD. In vitro studies demonstrate that GDNF enhances expression of its receptors and promotes enteric neuronal differentiation and plasticity. Since patients with DD exhibit hypoganglionosis, we propose that the observed enteric neuronal loss in DD may be due to lacking neurotrophic support mediated by the GDNF system.

Stem cells support tissue maintenance by balancing self-renewal and differentiation. In mice, it is believed that a homogeneous stem cell population of single spermatogonia supports spermatogenesis, and that differentiation, which is accompanied by the formation of connected cells (cysts) of increasing length, is linear and nonreversible. We evaluated this model with the use of lineage analysis and live imaging, and found that this putative stem cell population is not homogeneous. Instead, the stem cell pool that supports steady-state spermatogenesis is contained within a subpopulation of single spermatogonia. We also found that cysts are not committed to differentiation and appear to recover stem cell potential by fragmentation, and that the fate of individual spermatogonial populations was markedly altered during regeneration after damage. Thus, there are multiple and reversible paths from stem cells to differentiation, and these may also occur in other systems.

Two of the glial-cell-line-derived neurotrophic factor (GDNF) family ligands (GFLs), namely GDNF and neurturin (NRTN), are essential neurotropic factors for enteric nerve cells. Signal transduction is mediated by a receptor complex composed of GDNF family receptor alpha 1 (GFRα1) for GDNF or GFRα2 for NRTN, together with the tyrosine kinase receptor RET (rearranged during transfection). As both factors and their receptors are crucial for enteric neuron survival, we assess the site-specific gene expression of these GFLs and their corresponding receptors in human adult colon. Full-thickness colonic specimens were obtained after partial colectomy for non-obstructing colorectal carcinoma. Samples were processed for immunohistochemistry and co-localization studies. Site-specific gene expression was determined by real-time quantitative polymerase chain reaction in enteric ganglia and in circular and longitudinal muscle harvested by microdissection. Protein expression of the receptors was mainly localized in the myenteric and submucosal plexus. Dual-label immunohistochemistry with PGP 9.5 as a pan-neuronal marker detected immunoreactivity of the receptors in neuronal somata and ganglionic neuropil. RET immunoreactivity co-localized with neuronal GFRα1 and GFRα2 signals. The dominant source of receptor mRNA expression was in myenteric ganglia, whereas both GFLs showed higher expression in smooth muscle layers. The distribution and expression pattern of GDNF and NRTN and their corresponding receptors in the human adult enteric nervous system indicate a role of both GFLs not only in development but also in the maintenance of neurons in adulthood. The data also provide a basis for the assessment of disturbed signaling components of the GDNF and NRTN system in enteric neuropathies underlying disorders of gastrointestinal motility.