The neurotrophic factor neurturin contributes toward an aggressive cancer cell phenotype, neuropathic pain and neuronal plasticity in pancreatic cancer.

UNLABELLED: Neurotrophic factors possess an emerging role in the pathophysiology of several gastrointestinal disorders, regulating innervation, pain sensation and disease-associated neuroplasticity. Here, we aimed at characterizing the role of the neurotrophic factor neurturin (NRTN) and its receptor glial-cell-line-derived neurotrophic factor receptor alpha-2 (GFRα-2) in pancreatic cancer (PCa) and pancreatic neuropathy. For this purpose, NRTN and GFRα-2 were studied in normal human pancreas and PCa tissues via immunohistochemistry, quantitative reverse transcription-polymerase chain reaction, immunoblotting and correlated to abdominal pain. The impact of NRTN/GFRα-2 on PCa cell (PCC) biology was investigated via exposure to hypoxia, 3-(4,5-dimethylthiazole-2-yl)-2,5-diphenyl tetrazolium bromide viability and matrigel invasion assays in native and specific small interfering RNA-silenced PCCs. To assess the influence of NRTN on pancreatic neuroplasticity and neural invasion (NI), its impact was explored via an in vitro 'neuroplasticity assay' and a 3D neural migration assay. NRTN and GFRα-2 demonstrated a site-specific upregulation in PCa, predominantly in nerves, PCCs and extracellular matrix. Patients with severe pain demonstrated higher intraneural GFRα-2 immunoreactivity than patients with no pain. PCa tissue and PCCs contained increased amounts of NRTN, which was suppressed under hypoxia. NRTN promoted PCC invasiveness, and silencing of NRTN limited both PCC proliferation and invasion. Depletion of NRTN from PCa tissue extracts and PCC supernatants decreased axonal sprouting in neuronal cultures but did not influence glial density. Silencing of NRTN in PCCs boosted NI. We conclude that increased NRTN/GFRα-2 in PCa seems to promote an aggressive PCC phenotype and neuroplasticity in PCa. Accelerated NI following NRTN suppression constitutes a novel explanation for the attraction of PCC to nerves in the hypoxic PCa tumor microenvironment. SUMMARY: PCa is characterized by intrapancreatic neuroplasticity and NI. Here, we show that PCC produce the neurotrophic factor NRTN, which reinforces their biological properties, triggers neuroplastic alterations, NI and influences pain sensation via the GFRα-2 receptor.

GDNF family ligand dependent STAT3 activation is mediated by specific alternatively spliced isoforms of GFRα2 and RET.

Neurturin (NRTN), a member of the GDNF family of ligands (GFL), is currently investigated in a series of clinical trials for Parkinson's disease. NRTN signals through its cognate receptor GFRα2 and co-receptor RET to induce neurite outgrowth, but the underlying mechanism remains to be better understood. STAT3 was previously shown to be activated by oncogenic RET, independent of ligand and GFRα. In this study, we demonstrated that NRTN induced serine(727) but not tyrosine(705) phosphorylation of STAT3 in primary cortical neuron and neuronal cell lines. Remarkably, STAT3 phosphorylation was found to be mediated specifically by GFRα2c and RET9 isoforms. Furthermore, serine but not tyrosine dominant negative mutant of STAT3 impaired NRTN induced neurite outgrowth, indicative of the role of STAT3 as a downstream mediator of NRTN function. Similar to NGF, the NRTN induced P-Ser-STAT3 was localized to the mitochondria but not to the nucleus. Mitochondrial STAT3 was further found to be intimately involved in NRTN induced neurite outgrowth. Collectively, these findings demonstrated the hitherto unrecognized and novel role of specific GFRα2 and RET isoforms in mediating NRTN activation of STAT3 and the transcription independent mechanism whereby the mitochondria localized P-Ser-STAT3 mediated NRTN induced neurite outgrowth.

The effects of induced type I diabetes on developmental regulation of GDNF, NRTN, and NCAM proteins in the dentate gyrus of male rat offspring.

BACKGROUND: Maternal diabetes during pregnancy can affect the neurological development of offspring. Glial cell-derived neurotrophic factor (GDNF), neurturin (NRTN), and neural cell adhesion molecules (NCAM) are three important proteins for brain development. Therefore, this study aimed to investigate the impacts of the mentioned neurotrophic factors in the hippocampal dentate gyrus (DG) of rat offspring born to diabetic mothers. METHODS: Wistar female rats were randomly allocated into diabetic (STZ-D) [(45 mg/kg BW, STZ (Streptozotocin), i.p)], diabetic + NPH insulin (STZ-INS) [(4-6 unit/kg/day SC)], and control groups. The animals in all groups were mated by non-diabetic male rats. Two weeks after birth, male pups from each group were sacrificed and then protein contents of GDNF, NRTN, and NCAM were evaluated using immunohistochemistry. RESULTS: The study found that the expression of GDNF and NRTN in the hippocampus of diabetic rat offspring was significantly higher compared to the diabetic+ insulin and control groups, respectively (P < 0.01, P < 0.001). Additionally, the expression of NCAM was significantly higher in the diabetic group the diabetic+ insulin and control groups (P < 0.01, P < 0.001). CONCLUSIONS: The results of the study revealed that diabetes during pregnancy significantly impacts the distribution pattern of GDNF, NRTN, and NCAM in the hippocampus of rat neonates.

Muscle-secreted neurturin couples myofiber oxidative metabolism and slow motor neuron identity.

Endurance exercise promotes skeletal muscle vascularization, oxidative metabolism, fiber-type switching, and neuromuscular junction integrity. Importantly, the metabolic and contractile properties of the muscle fiber must be coupled to the identity of the innervating motor neuron (MN). Here, we show that muscle-derived neurturin (NRTN) acts on muscle fibers and MNs to couple their characteristics. Using a muscle-specific NRTN transgenic mouse (HSA-NRTN) and RNA sequencing of MN somas, we observed that retrograde NRTN signaling promotes a shift toward a slow MN identity. In muscle, NRTN increased capillary density and oxidative capacity and induced a transcriptional reprograming favoring fatty acid metabolism over glycolysis. This combination of effects on muscle and MNs makes HSA-NRTN mice lean with remarkable exercise performance and motor coordination. Interestingly, HSA-NRTN mice largely recapitulate the phenotype of mice with muscle-specific expression of its upstream regulator PGC-1ɑ1. This work identifies NRTN as a myokine that couples muscle oxidative capacity to slow MN identity.

Protection of dopamine neurons by CDNF and neurturin variant N4 against MPP+ in dissociated cultures from rat mesencephalon.

Parkinson's disease is associated with the loss of dopamine (DA) neurons in ventral mesencephalon. We have previously reported that no single neurotrophic factor we tested protected DA neurons from the dopaminergic toxin 1-methyl-4-phenylpyridinium (MPP+) in dissociated cultures isolated from the P0 rat substantia nigra, but that a combination of five neurotrophic factors was protective. We now report that cerebral DA neurotrophic factor (CDNF) and a variant of neurturin (NRTN), N4, were also not protective when provided alone but were protective when added together. In cultures isolated from the substantia nigra, MPP+ (10 µM) decreased tyrosine hydroxylase-positive cells to 41.7 ± 5.4% of vehicle control. Although treatment of cultures with 100 ng/ml of either CDNF or N4 individually before and after toxin exposure did not significantly increase survival in MPP+-treated cultures, when the two trophic factors were added together at 100 ng/ml each, survival of cells was increased 28.2 ± 6.1% above the effect of MPP+ alone. In cultures isolated from the ventral tegmental area, another DA rich area, a higher dose of MPP+ (1 mM) was required to produce an EC50 in TH-positive cells but, as in the substantia nigra, only the combination of CDNF and N4 (100 ng/ml each) was successful at increasing the survival of these cells compared to MPP+ alone (by 22.5 ± 3.5%). These data support previous findings that CDNF and N4 may be of therapeutic value for treatment of PD, but suggest that they may need to be administered together.

Neurturin effects on nigrostriatal dopamine release and content: comparison with GDNF.

Neurturin (NTN) is a member of the glial cell line-derived neurotrophic factor (GDNF) family; and, while GDNF has been shown to increase dopamine (DA) release in normal animals, the ability of NTN to alter DA release has not been previously reported. The purpose of the present study was to determine if NTN could alter striatal DA release, and to compare the effects of NTN to GDNF. Male Fischer-344 rats were given a single injection of vehicle or 5 microg NTN or GDNF into the right substantia nigra. Three weeks later microdialysis experiments were conducted to assess striatal DA release. Basal extracellular levels of striatal DA were not affected by either NTN or GDNF. However, both NTN and GDNF led to increases in amphetamine-evoked overflow of DA from the ipsilateral striatum, and there was a trend for potassium-evoked overflow to be augmented. Postmortem tissue levels of DA were decreased by approximately 20% in the striatum, and increased by approximately 100% in the substantia nigra, on the ipsilateral side of the brain compared to the contralateral side following both NTN and GDNF injection. Thus, NTN, like GDNF, can augment striatal DA release, and the magnitude of the NTN effects are similar to those of GDNF.

GDNF-induced cell signaling and neurite outgrowths are differentially mediated by GFRalpha1 isoforms.

Glial cell line-derived neurotrophic factor (GDNF) transduces signal and promotes neurite outgrowths in diverse neurons through the interactions of GDNF family receptor alpha 1 (GFRalpha1) and other co-receptors including Ret receptor tyrosine kinase and NCAM. GFRalpha1 is alternatively spliced into two isoforms, GFRalpha1a and GFRalpha1b, with five amino acids difference. In this study, we found that both GFRalpha1a and GFRalpha1b were expressed in various human tissues. Interestingly, when stimulated with GDNF, GFRalpha1a but not GFRalpha1b promoted neurite outgrowth in neuroblastoma cells through the activations of ERK1/2, Rac1 and Cdc42. Remarkably, in cells co-expressing GFRalpha1a and GFRalpha1b, GDNF inhibited neurite outgrowths. The inhibitory activity of GFRalpha1b was dependent on RhoA and ROCK activation. Furthermore, GFRalpha1b but not GFRalpha1a activated Rho and various ROCK downstream effectors LIMK1/2, cofilin and MLC2. This study demonstrates the hitherto unrecognized roles of GFRalpha1 isoforms in the activation of distinct signaling pathways and in neurite outgrowths.

Neurturin regulates the lung-resident macrophage inflammatory response to viral infection.

Lung-resident macrophages are crucial to the maintenance of health and in the defence against lower respiratory tract infections. Macrophages adapt to local environmental cues that drive their appropriate function; however, this is often dysregulated in many inflammatory lung pathologies. In mucosal tissues, neuro-immune interactions enable quick and efficient inflammatory responses to pathogenic threats. Although a number of factors that influence the antimicrobial response of lung macrophages are known, the role of neuronal factors is less well understood. Here, we show an intricate circuit involving the neurotrophic factor, neurturin (NRTN) on human lung macrophages that dampens pro-inflammatory cytokine release and modulates the type of matrix metalloproteinases produced in response to viral stimuli. This circuit involves type 1 interferon-induced up-regulation of RET that when combined with the glial cell line-derived neurotrophic factor (GDNF) receptor α2 (GFRα2) allows binding to epithelial-derived NRTN. Our research highlights a non-neuronal immunomodulatory role for NRTN and a novel process leading to a specific antimicrobial immune response by human lung-resident macrophages.

The upregulation of GLAST-1 is an indirect antiapoptotic mechanism of GDNF and neurturin in the adult CNS.

Glial cell-line-derived neurotrophic factor (GDNF) and neurturin (NTN) protect retinal ganglion cells (RGCs) from axotomy-induced apoptosis. It is likely that neuroprotection by GDNF or NTN in the adult central nervous system (CNS) involves indirect mechanisms and independent signal transduction events. Extracellular glutamate is a trigger of apoptosis in injured RGCs, and glutamate transporter levels can be upregulated by GDNF. Therefore, GDNF may indirectly protect RGCs by enhancing glutamate uptake in the retina. We studied the upregulation of the glutamate transporters GLAST-1 and GLT-1 by GDNF and NTN, and the intracellular pathways required for GDNF/NTN neuroprotection. GDNF required phosphoinositide-3 kinase (PI3K) and Src activity to upregulate GLAST-1 and GLT-1. NTN required PI3K activity to upregulate GLAST-1 and did not affect GLT-1 levels. PI3K activity was also important for GDNF and NTN neuroprotection following optic nerve transection. However, GDNF also required Src and mitogen-activated protein kinase activity to prevent RGC apoptosis. RNA interference demonstrated that the upregulation of GLAST-1 by GDNF and NTN is required to rescue RGCs. Thus, additional independent signal transduction events, together with the upregulation of GLT-1 by GDNF, differentiate the biological activity of GDNF from NTN. Furthermore, the upregulation of the glial glutamate transporter GLAST-1 by both factors is an indirect neuroprotective mechanism in the CNS.

GDNF revisited: A novel mammalian cell-derived variant form of GDNF increases dopamine turnover and improves brain biodistribution.

Parkinson's disease (PD) is a disorder affecting dopamine neurons for which there is no cure. Glial cell line-derived neurotrophic factor (GDNF) and the closely related protein neurturin are two trophic factors with demonstrated neuroprotective and neurorestorative properties on dopamine neurons in multiple animal species. However, GDNF and neurturin Phase-2 clinical trials have failed to demonstrate a significant level of improvement over placebo controls. Insufficient drug distribution in the brain parenchyma has been proposed as a major contributing factor for the lack of clinical efficacy in the Phase-2 trial patients. To address this issue, a novel mammalian cell-derived variant form of GDNF (GDNFv) was designed to promote better tissue distribution by reducing its heparin binding to the extracellular matrix and key amino acids were substituted to enhance its chemical stability. Administration of this fully glycosylated GDNFv in the normal rat striatum increased dopamine turnover and produced significantly greater brain distribution than E. coli-produced wildtype GDNF (GDNFwt). Intrastriatal GDNFv also protected midbrain dopamine neuron function in 6-hydroxydopamine-lesioned rats. Studies conducted in normal adult rhesus macaques support that GDNFv was well tolerated in all animals and demonstrated a greater volume of distribution than GDNFwt in the brain following intrastriatal infusion. Importantly, favorable physiological activity of potential therapeutic value was maintained in this variant trophic factor with significant target activation in GDNFv recipients as indicated by dopamine turnover modulation. These data suggest that GDNFv may be a promising drug candidate for the treatment of PD. Additional studies are needed in non-human primates with dopamine depletion. This article is part of the Special Issue entitled 'Drug Repurposing: old molecules, new ways to fast track drug discovery and development for CNS disorders'.

Intraputamenal infusion of exogenous neurturin protein restores motor and dopaminergic function in the globus pallidus of MPTP-lesioned rhesus monkeys.

The neurorestorative effects of exogenous neurturin (NTN) delivered directly into the putamen via multiport catheters were studied in 10 MPTP-lesioned rhesus monkeys expressing stable parkinsonism. The parkinsonian animals were blindly assigned to receive coded solutions containing either vehicle (n = 5) or NTN (n = 5, 30 microg/day). Both solutions were coinfused with heparin using convection-enhanced delivery for 3 months. The NTN recipients showed a significant and sustained behavioral improvement in their parkinsonian features during the treatment period, an effect not seen in the vehicle-treated animals. At study termination, locomotor activity levels were increased by 50% in the NTN versus vehicle recipients. Also, DOPAC levels were significantly increased by 150% ipsilateral (right) to NTN infusion in the globus pallidus, while HVA levels were elevated bilaterally in the NTN-treated animals by 10% on the left and 67% on the right hemisphere. No significant changes in DA function were seen in the putamen. Volumetric analysis of putamenal NTN labeling showed between-subject variation, with tissue distribution ranging from 214 to 744 mm3, approximately equivalent to 27-93% of area coverage. Our results support the concept that intraparenchymal delivery of NTN protein may be effective for the treatment of PD. More studies are needed to determine strategies that would enhance tissue distribution of exogenous NTN protein, which could contribute to optimize its trophic effects in the parkinsonian brain.

Specificity in the crosstalk of TGFbeta/GDNF family members is determined by distinct GFR alpha receptors.

Glial cell line-derived neurotrophic factor (GDNF) and neurturin (NRTN) are neurotrophic factors for parasympathetic neurons including ciliary ganglion (CG) neurons. Recently, we have shown that survival and signaling mediated by GDNF in CG neurons essentially requires transforming growth factor beta (TGFbeta). We have provided evidence that TGFbeta regulates the availability of the glycosyl phosphatidylinositol (GPI)-anchored GDNF receptor alpha 1 (GFRalpha1) by promoting the recruitment of the receptor to the plasma membrane. We report now that in addition to GDNF, NRTN, but not persephin (PSPN) or artemin (ARTN), is able to promote survival of CG neurons. Interestingly, in contrast to GDNF, NRTN is not dependent on cooperation with TGFbeta, but efficiently promotes neuronal survival and intracellular signaling in the absence of TGFbeta. Additional treatment with TGFbeta does not further increase the NRTN response. Both NRTN and GDNF exclusively bind to and activate their cognate receptors, GFRalpha2 and GFRalpha1, respectively, as shown by the use of receptor-specific neutralizing antibodies. Immunocytochemical staining for the two receptors on the surface of CG neurons reveals that, in contrast to the effect on GFRalpha1, TGFbeta is not required for recruitment of GFRalpha2 to the plasma membrane. Moreover, binding of radioactively labeled GDNF but not NRTN is increased upon treatment of CG neurons with TGFbeta. Disruption of TGFbeta signaling does interfere with GDNF-, but not NRTN-mediated signaling and survival. We propose a model taking into account data from GFRalpha1 crystallization and ontogenetic development of the CG that may explain the differences in TGFbeta-dependence of GDNF and NRTN.

Neurturin and a GLP-1 Analogue Act Synergistically to Alleviate Diabetes in Zucker Diabetic Fatty Rats.

Neurturin (NRTN), a member of the glial-derived neurotrophic factor family, was identified from an embryonic chicken pancreatic cDNA library in a screen for secreted factors. In this study, we assessed the potential antidiabetic activities of NRTN relative to liraglutide, a glucagon-like peptide 1 receptor agonist, in Zucker diabetic fatty (ZDF) rats. Subcutaneous administration of NRTN to 8-week-old male ZDF rats prevented the development of hyperglycemia and improved metabolic parameters similar to liraglutide. NRTN treatment increased pancreatic insulin content and ß-cell mass and prevented deterioration of islet organization. However, unlike liraglutide-treated rats, NRTN-mediated improvements were not associated with reduced body weight or food intake. Acute NRTN treatment did not activate c-Fos expression in key feeding behavior and metabolic centers in ZDF rat brain or directly enhance glucose-stimulated insulin secretion from pancreatic ß-cells. Treating 10-week-old ZDF rats with sustained hyperglycemia with liraglutide resulted in some alleviation of hyperglycemia, whereas NRTN was not as effective despite improving plasma lipids and fasting glucose levels. Interestingly, coadministration of NRTN and liraglutide normalized hyperglycemia and other metabolic parameters, demonstrating that combining therapies with distinct mechanism(s) can alleviate advanced diabetes. This emphasizes that therapeutic combinations can be more effective to manage diabetes in individuals with uncontrolled hyperglycemia.

Nerve growth factor, glial cell line-derived neurotrophic factor and neurturin prevent semaphorin 3A-mediated growth cone collapse in adult sensory neurons.

Developmentally, semaphorin 3A (sema3A) is an important chemorepellent that guides centrally projecting axons of dorsal root ganglion (DRG) neurons. Sema3A-mediated growth cone collapse can be prevented by cyclic GMP (cGMP) and nerve growth factor (NGF) in embryonic neurons. Sema3A may also play a role in directing regrowth of injured axons in adults, and interactions with neurotrophic factors near the injury site may determine the extent and targeting of both regenerative and aberrant growth. The aim of this study was to determine whether NGF, glial cell line-derived neurotrophic factor (GDNF) and neurturin (NTN) modulate sema3A-mediated growth cone collapse in cultured adult rat DRG neurons. Sema3A caused a significant increase in growth cone collapse, which was completely prevented by prior treatment with NGF, GDNF or NTN. Immunocytochemical experiments showed that sema3A-sensitive neurons were heterogeneous in their expression of neurotrophic factor receptors and responses to neurotrophic factors, raising the possibility of novel, convergent signaling mechanisms between these substances. Increasing cGMP levels caused growth cone collapse, whereas sema3A-mediated collapse was prevented by inhibition of guanylate cyclase or by increasing cyclic AMP levels. In conclusion, sema3A signaling pathways in adult neurons differ to those described in embryonic neurons. Three different neurotrophic factors each completely prevent sema3A-mediated collapse, raising the possibility of novel converging signaling pathways. These studies also show that there is considerable potential for neurotrophic factors to regulate sema3A actions in the adult nervous system. This may provide insights into the mechanisms underling misdirected growth and targeting of sensory fibers within the spinal cord after injury, that is thought to contribute to development of autonomic dysreflexia and neuropathic pain.

Glial cell line-derived neurotrophic factor (GDNF) family ligands reduce the sensitivity of neuroblastoma cells to pharmacologically induced cell death, growth arrest and differentiation.

The glial cell line-derived neurotrophic factor (GDNF) family of ligands play essential roles in promoting normal neural crest differentiation during embryogenesis, and, may have a therapeutic role in malignancies of neural crest origin, such as neuroblastoma. However, we report here that GDNF and neurturin blocked the growth inhibitory and neuritogenic effects of all-trans-retinoic acid in neuroblastoma cells in vitro. GDNF caused neuroblastoma cells to proliferate in the presence of a range of cytotoxic chemotherapeutic agents at low concentrations. Thus, our findings suggest a role for GDNF signaling in promoting resistance to differentiation or cytotoxic therapy of neuroblastoma, and, preclude their use in this neural crest tumor.

The GDNF family members neurturin, artemin and persephin promote the morphological differentiation of cultured ventral mesencephalic dopaminergic neurons.

Neurturin (NRTN), artemin (ARTN), persephin (PSPN) and glial cell line-derived neurotrophic factor (GDNF) form a group of neurotrophic factors, also known as the GDNF family ligands (GFLs). They signal through a receptor complex composed of a high-affinity ligand binding subunit, postulated ligand specific, and a common membrane-bound tyrosine kinase RET. Recently, also NCAM has been identified as an alternative signaling receptor. GFLs have been reported to promote survival of cultured dopaminergic neurons. In addition, GDNF treatments have been shown to increase morphological differentiation of tyrosine hydroxylase immunoreactive (TH-ir) neurons. The present comparative study investigated the dose-dependent effects of GFLs on survival and morphological differentiation of TH-ir neurons in primary cultures of E14 rat ventral mesencephalon. Both NRTN and ARTN chronically administered for 5 days significantly increased survival and morphological differentiation of TH-ir cells at all doses investigated [0.1-100 ng/ml], whereas PSPN was found to be slightly less potent with effects on TH-ir cell numbers and morphology at 1.6-100 ng/ml and 6.3-100 ng/ml, respectively. In conclusion, our findings identify NRTN, ARTN and PSPN as potent neurotrophic factors that may play an important role in the structural development and plasticity of ventral mesencephalic dopaminergic neurons.

Enhanced delivery and bioactivity of the neurturin neurotrophic factor through focused ultrasound-mediated blood--brain barrier opening in vivo.

The blood-brain barrier (BBB) constitutes a major obstacle in brain drug delivery. Focused ultrasound (FUS) in conjunction with microbubbles has been shown to open the BBB noninvasively, locally, and transiently to allow large molecules diffusion. Neurturin (NTN), a member of the glial-derived neurotrophic factor (GDNF) family, has been demonstrated to have neuroprotective and regenerative effects on dopaminergic neurons in vivo using invasive drug delivery methods. The brain's ascending nigrostriatal pathway is severely damaged in Parkinson's disease (PD), and therefore the substantia nigra (SN) and striatal caudoputamen (CP) were selected as the target areas. The objective of the study was to investigate whether safe and efficient NTN delivery can be achieved through FUS-induced BBB opening via intravenous administration, and thus trigger the neuroregeneration cascade in the nigrostriatal pathway. After the optimization of FUS parameters and target locations in the murine brain, NTN bioavailability and downstream signaling were detected and characterized through immunostaining. FUS significantly enhanced the delivery of NTN compared with the direct injection technique, whereas triggering of the signaling cascade was detected downstream to the neuronal nuclei. These findings thus indicate the potential of the FUS method to mediate transport of proteins through the blood-brain barrier in a PD animal model.

Embryonic neural stem cells in a 3D bioassay for trophic stimulation studies.

Progenitors were discovered in the corpus striatum several years ago, but little is known about their proliferation and differentiation. The aim of this study was to analyze embryonic progenitor cells from the corpus striatum using a bioassay with trophic stimulation. Primary cells obtained from brains of rat embryos at E13-14 were dissected from striatum niches and cultured in stem cell media. These floating dispersed cells clumped together to forming floating bodies like irregular spheres (spheroids), which were placed in type I collagen gel and cultured under basal conditions or with the addition of NGF, NT-3, or NTN. Optimum growth of neurites was obtained, and after 24 and 48 h, they were measured for number and length. The expression of proliferation markers such as PCNA and Ki67, and of neural progenitor markers such as GFAP, nestin, vimentin, O4, A2B5, Pax6, S100, TubIII, and NeuN, was then analyzed. The initial behavior in cell cultures showed distinguishable spheroids that, when placed in 3D gels and with trophic support, generated neurites. A similar effect was observed in glial cell outgrowth from the spheroids. Our assay showed high reproducibility, short culture time, and high resolution for tracing neuron-neurite outgrowth or visualizing glial outgrowth in a few hours.

Parasympathetic stimulation improves epithelial organ regeneration.

Parasympathetic nerves are a vital component of the progenitor cell niche during development, maintaining a pool of progenitors for organogenesis. Injured adult organs do not regenerate after parasympathectomy, and there are few treatments to improve organ regeneration, particularly after damage by therapeutic irradiation. Here we show that restoring parasympathetic function with the neurotrophic factor neurturin increases epithelial organ regeneration after damage. We use mouse salivary gland explant culture containing fluorescently labelled progenitors, and injure the tissue with irradiation. The progenitors survive, parasympathetic function is diminished and epithelial apoptosis reduces the expression of neurturin, which increases neuronal apoptosis. Treatment with neurturin reduces neuronal apoptosis, restores parasympathetic function and increases epithelial regeneration. Furthermore, adult human salivary glands damaged by irradiation also have reduced parasympathetic innervation. We propose that neurturin will protect the parasympathetic nerves from damage and improve organ regeneration. This concept may be applicable for other organs where parasympathetic innervation influences their function.

Protection effect of GDNF and neurturin on photosensitized crayfish neurons and glial cells.

Neurons and glial cells can protect each other from stress and following death by mutual exchange with neurotrophins. In order to examine involvement of different neurotrophic factors in neuroglial interactions in a photosensitized crayfish stretch receptor, a simple model object consisting of only two sensory neurons enveloped by glial cells, we studied the influence of glial cell line-derived neurotrophic factor (GDNF), neurturin, and ciliary neurotrophic factor (CNTF) on its photodynamic injury. Photodynamic treatment, which causes strong oxidative stress, induced firing abolition and necrosis of neurons, necrosis, and apoptosis of glial cells. GDNF significantly reduced photoinduced neuronal necrosis and neurturin but not CNTF showed a similar tendency. Both of them significantly reduced necrosis and apoptosis of glial cells. At the ultrastructural level, neurons and glial cells treated with GDNF in the darkness contained large mitochondria with well-developed cristae, numerous ribosomes, polysomes, rough endoplasmic reticulum (ER), and dictyosomes. This indicated the high level of bioenergetic, biosynthetic, and transport processes. Photodynamic treatment caused swelling and vacuolization of mitochondria, dictyosomes, and ER. It also impaired formation of glial protrusions and double membrane vesicles that transfer glial material into the neuron. GDNF prevented photoinduced mitochondria swelling that disturbed the cellular bioenergetics and cytoplasm vacuolization associated with injury of intracellular organelles. It also preserved the structures involved in protein synthesis and transport: rough ER, dictyosomes, polysomes, microtubule bundles, submembrane cisterns, and double membrane vesicles. GDNF-mediated maintenance of metabolism and ultrastructure of photosensitized neurons and glial cells may be the basis of its neuro- and glia protective effects.