Glial cell line-derived neurotrophic factor (GDNF) promotes the survival of axotomized retinal ganglion cells in adult rats: comparison to and combination with brain-derived neurotrophic factor (BDNF)

Adult rat retinal ganglion cells (RGC) undergo degeneration after optic nerve transection. Studies have shown that exogenously applied neurotrophic factors such as brain-derived neurotrophic factor (BDNF) can attenuate axotomy-induced as well as developmental RGC death. Here, we examined whether glial cell line-derived neurotrophic factor (GDNF), a known neurotrophic factor for dopaminergic neurons and motor neurons, could provide neurotrophic support to RGC in adult rats. We determined whether RGC could retrogradely transport GDNF from their target tissue. After injection into the superior colliculus of adult rats, 125I-GDNF was retrogradely transported to contralateral eyes but not to ipsilateral eyes. The transport of 125I-GDNF could be blocked by coinjection of excess unlabeled GDNF, indicating that it was receptor mediated. We tested whether intravitreally applied GDNF could prevent axotomy-induced RGC degeneration. The RGC were prelabeled with Fluorogold (FG) and axotomized by intraorbital optic nerve transection. GDNF, BDNF (positive control), cytochrome c (negative control), or a GDNF/BDNF combination was injected intravitreally on days 0 and 7. On day 14, FG-labeled RGC were counted from whole-mount retinas. We found that, similar to BDNF, GDNF could significantly attenuate the degeneration of RGC in a dose-dependent fashion. Furthermore, the combination treatment of GDNF and BDNF showed better protection than either factor used individually. Our data indicate that GDNF is a neurotrophic factor for the adult rat RGC. GDNF, like BDNF, may be useful for the treatment of human RGC degenerative diseases.     

Glial cell line-derived neurotrophic factor (GDNF) is a neurotrophic factor for sensory neurons: Comparison with the effects of the neurotrophins

We compared the effects of glial cell line-derived neurotrophic factor (GDNF) on dorsal root ganglion (DRG) sensory neurons to that of nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF), and neurotrophin 3 (NT-3). All of these factors were retrogradely transported to sub-populations of sensory neuron cell bodies in the L4/L5 DRG of neonatal rats. The size distribution of ¹²⁵I-GDNF-labeled neurons was variable and consisted of both small and large DRG neurons (mean of 506.60 μm²). ¹²⁵I-NGF was preferentially taken up by small neurons with a mean cross-sectional area of 383.03 μm². Iodinated BDNF and NT-3 were transported by medium to large neurons with mean sizes of 501.48 and 529.27 μm², respectively. A neonatal, sciatic nerve axotomy-induced cell death model was used to determine whether any of these factors could influence DRG neuron survival in vivo. GDNF and NGF rescued nearly 100% of the sensory neurons. BDNF and NT-3 did not promote any detectable level of neuronal survival despite the fact that they underwent retrograde transport. We examined the in vitro survival-promoting ability of these factors on neonatal DRG neuronal cultures derived from neonatal rats. GDNF, NGF, and NT-3 were effective in vitro, while BDNF was not. The range of effects seen in the models described here underscores the importance of testing neuronal responsiveness in more than one model. The biological responsiveness of DRG neurons to GDNF in multiple models suggests that this factor may play a role in the development and maintenance of sensory neurons. © 1997 John Wiley & Sons, Inc. J Neurobiol 32: 22–32, 1997.     

Differential effects of the trophic factors BDNF,NT‐4, GDNF,and IGF‐I on the isthmo‐optic nucleus in chick embryos

The isthmo‐optic nucleus (ION) of chick embryos is a model system for the study of retrograde trophic signaling in developing CNS neurons. The role of brain‐derived neurotrophic factor (BDNF) is well established in this system. Recent work has implicated neurotrophin‐4 (NT‐4), glial cell line–derived neurotrophic factor (GDNF), and insulin‐like growth factor I (IGF‐I) as additional trophic factors for ION neurons. Here it was examined in vitro and in vivo whether these factors are target‐derived trophic factors for the ION in 13‐ to 16‐day‐old chick embryos. Unlike BDNF, neither GDNF, NT‐4, nor IGF‐I increased the survival of ION neurons in dissociated cultures identified by retrograde labeling with the fluorescent tracer DiI. BDNF and IGF‐I promoted neurite outgrowth from ION explants, whereas GDNF and NT‐4 had no effect. Injections of NT‐4, but not GDNF, in the retina decreased the survival of ION neurons and accelerated cell death in the ION. NT‐4–like immunoreactivity was present in the retina and the ION. Exogenous, radiolabeled NT‐4, but not GDNF or IGF‐I, was retrogradely transported from the retina to the ION. NT‐4 transport was significantly reduced by coinjection of excess cold nerve growth factor (NGF), indicating that the majority of NT‐4 bound to p75 neurotrophin receptors during axonal transport. Binding of NT‐4 to chick p75 receptors was confirmed in L‐cells, which express chick p75 receptors. These data indicate that GDNF has no direct trophic effects on ION neurons. IGF‐I may be an afferent trophic factor for the ION, and NT‐4 may act as an antagonist to BDNF, either by competing with BDNF for p75 and/or trkB binding or by signaling cell death via p75. © 2000 John Wiley & Sons, Inc. J Neurobiol 43: 289–303, 2000     

Target‐derived and locally derived neurotrophins support retinal ganglion cell survival in the neonatal rat retina

Brain‐derived neurotrophic factor (BDNF) and neurotrophin‐4/5 (NT‐4/5) protein and mRNA are found in the neonatal rat retina and also in target sites such as the superficial layers of the superior colliculus. Both neurotrophins support neonatal retinal ganglion cell survival in vitro. In vivo, injections of recombinant BDNF and NT‐4/5 reduce naturally occurring cell death as well as death induced by removal of the contralateral superior colliculus. In the latter case, the peak of retinal ganglion cell death occurs about 24 h postlesion. We wished to determine: whether a similar time‐course of degeneration occurs after selective removal of target cells or depletion of target‐derived trophic factors, and whether ganglion cell viability also depends on intraretinally derived neurotrophins. Retinal ganglion cell death was measured 24 and 48 h following injections of kainic acid or a mixture of BDNF and NT‐4/5 blocking antibodies into the superior colliculus and 24 h after intraocular injection of the same antibodies. Retinotectally projecting ganglion cells were identified by retrograde labeling with the nucleophilic dye diamidino yellow. We show that collicular injections of either kainic acid or BDNF and NT‐4/5 blocking antibodies significantly increased retinal ganglion cell death in the neonatal rat 24 h postinjection, death rates returning to normal by 48 h. This increase in death was greatest following collicular injections; however, death was also significantly increased 24 h following intravitreal antibody injection. Thus retinal ganglion cell survival during postnatal development is not only dependent upon trophic factors produced by central targets but may also be influenced by local intraretinal neurotrophin release. © 2004 Wiley Periodicals, Inc. J Neurobiol 60: 319–327, 2004     

Serotonin (5-HT) induces glial cell line-derived neurotrophic factor (GDNF) mRNA expression via the transactivation of fibroblast growth factor receptor 2 (FGFR2) in rat C6 glioma cells

We previously reported that serotonin (5-HT) increased glial cell line-derived neurotrophic factor (GDNF) release in a 5-HT₂ receptor (5-HT₂R) and mitogen-activated protein kinase kinase/extracellular signal-related kinase (MEK/ERK)-dependent manner in rat C6 glioma cells (C6 cells), a model of astrocytes. We herein found that 5-HT-induced rapid ERK phosphorylation was blocked by 5-HT₂R antagonists in C6 cells. We therefore examined 5-HT-induced ERK phosphorylation to reveal the mechanism of 5-HT-induced GDNF mRNA expression. As 5-HT-induced ERK phosphorylation was blocked by inhibitors for Gα_q/11 and fibroblast growth factor receptor (FGFR), but not for second messengers downstream of Gα_q/11, 5-HT₂R-mediated FGFR transactivation was suggested to be involved in the ERK phosphorylation. Although FGFR1 and 2 were functionally expressed in C6 cells, 5-HT selectively phosphorylated FGFR2. Indeed, small interfering RNA for FGFR2, but not for FGFR1, blocked 5-HT-induced ERK phosphorylation. As Src family tyrosine kinase inhibitors and microtubule depolymerizing agents blocked 5-HT-induced FGFR2 phosphorylation, Src family tyrosine kinase and stabilized microtubules were suggested to act upstream of FGFR2. Finally, 5-HT-induced GDNF mRNA expression was also inhibited by the blockade of 5-HT₂R, FGFR, and Src family tyrosine kinase. In conclusion, our findings suggest that 5-HT induces GDNF mRNA expression via 5-HT₂R-mediated FGFR2 transactivation in C6 cells.     

A cell‐survival factor (N‐acetyl‐L‐cysteine) alters the in vivo fate of constitutively proliferating subependymal cells in the adult forebrain

The adult mouse brain contains a population of constitutively proliferating subependymal cells that surround the lateral ventricle and are the direct progeny of the neural stem cell. Constitutively proliferating cells divide rapidly; 6 days after labeling, 60% of their progeny undergo cell death, 25% migrate to the olfactory bulbs, and 15% continue to proliferate within the subependyma. We have intraventricularly infused a cell survival factor N‐acetyl‐L ‐cysteine (NAC), which is known to have survival effects without concomitant proliferative effects on cells in vitro, and examined the resulting fate of cells spared from the normally occurring cell death. NAC infusion for 5 days results in a five‐fold increase in the number of retrovirally labeled subependymal cells compared to saline‐infused controls. The increase in the number of subependymal cells is directly proportional to the amount of time during which NAC is present and is not due to increased proliferation. While NAC is able to keep all the normally dying progeny alive, the cells spared from death remain confined to the subependyma lining the lateral ventricles and do not migrate to the olfactory bulbs (one normal fate of constitutively proliferating progeny) or into the surrounding brain parenchyma. When animals survive for an additional 6 days following NAC infusion, the number of retrovirally labeled subependymal cells returns to control values, indicating that the continued presence of NAC is necessary for cell survival. These data suggest that preventing cell death is not sufficient to keep all of the progeny of these cells in a proliferative mode. © 2000 John Wiley & Sons, Inc. J Neurobiol 42: 338–346, 2000     

Targeting amphiregulin (AREG) derived from senescent stromal cells diminishes cancer resistance and averts programmed cell death 1 ligand (PD‐L1)‐mediated immunosuppression

Aging is characterized by a progressive loss of physiological integrity, while cancer represents one of the primary pathological factors that severely threaten human lifespan and healthspan. In clinical oncology, drug resistance limits the efficacy of most anticancer treatments, and identification of major mechanisms remains a key to solve this challenging issue. Here, we highlight the multifaceted senescence‐associated secretory phenotype (SASP), which comprises numerous soluble factors including amphiregulin (AREG). Production of AREG is triggered by DNA damage to stromal cells, which passively enter senescence in the tumor microenvironment (TME), a process that remarkably enhances cancer malignancy including acquired resistance mediated by EGFR. Furthermore, paracrine AREG induces programmed cell death 1 ligand (PD‐L1) expression in recipient cancer cells and creates an immunosuppressive TME via immune checkpoint activation against cytotoxic lymphocytes. Targeting AREG not only minimized chemoresistance of cancer cells, but also restored immunocompetency when combined with classical chemotherapy in humanized animals. Our study underscores the potential of in vivo SASP in driving the TME‐mediated drug resistance and shaping an immunosuppressive niche, and provides the proof of principle of targeting major SASP factors to improve therapeutic outcome in cancer medicine, the success of which can substantially reduce aging‐related morbidity and mortality.