Neural cells in the esophagus respond to glial cell line-derived neurotrophic factor and neurturin, and are RET-dependent

Glial cell line-derived neurotrophic factor (GDNF) is expressed in the gastrointestinal tract of the developing mouse and appears to play an important role in the migration of enteric neuron precursors into and along the small and large intestines. Two other GDNF family members, neurturin and artemin, are also expressed in the developing gut although artemin is only expressed in the esophagus. We examined the effects of GDNF, neurturin, and artemin on neural crest cell migration and neurite outgrowth in explants of mouse esophagus, midgut, and hindgut. Both GDNF and neurturin induced neural crest cell migration and neurite outgrowth in all regions examined. In the esophagus, the effect of GDNF on migration and neurite outgrowth declined with age between E11.5 and E14.5, but neurturin still had a strong neurite outgrowth effect at E14.5. Artemin did not promote neural migration or neurite outgrowth in any region investigated. The effects of GDNF family ligands are mediated by the Ret tyrosine kinase. We examined the density of neurons in the esophagus of Ret-/- mice, which lack neurons in the small and large intestines. The density of esophageal neurons in Ret-/- mice was only about 4% of the density of esophageal neurons in Ret+/- and Ret+/+ mice. These results show that GDNF and neurturin promote migration and neurite outgrowth of crest-derived cells in the esophagus as well as the intestine. Moreover, like intestinal neurons, the development of esophageal neurons is largely Ret-dependent.     

Reprint of "on the size of the active site in proteases. I. Papain"

α-Synuclein is the major component of Lewy bodies and Lewy neurites, the pathological hallmarks of surviving neuronal cells in Parkinson's disease patients. However, the physiological role played by α-synuclein remains unclear. In this study, spectrin beta non-erythrocyte 1 (SPTBN1) interacted with α-synuclein in phage display assays using a normalized human brain cDNA library. A direct interaction between α-synuclein and SPTBN1 was confirmed by GST pull-down and co-immunoprecipitation assays. SPTBN1 and α-synuclein proteins colocalized in N2a neuronal cells. Transfection of SPTBN1 caused human SH-SY5Y dopaminergic neuron cells to inappropriately induce neurites, which extended from cell bodies. Cotransfection with α-synuclein reversed SPTBN1-induced excessive neurite branching in SH-SY5Y cells, and only a single neurite extended from each neuron. These results suggest that α-synuclein modulates neurite outgrowth by interacting with cytoskeletal proteins such as SPTBN1.     

Calcium: A novel and efficient inducer of differentiation of adipose‐derived stem cells into neuron‐like cells

This study comparatively investigated the effectiveness of calcium and other well‐known inducers such as isobutylmethylxanthine (IBMX) and insulin in differentiating human adipose‐derived stem cells (ADSCs) into neuronal‐like cells. ADSCs were immunophenotyped and differentiated into neuron‐like cells with different combinations of calcium, IBMX, and insulin. Calcium mobilization across the membrane was determined. Differentiated cells were characterized by cell cycle profiling, staining of Nissl bodies, detecting the gene expression level of markers such as neuronal nuclear antigen (NeuN), microtubule associated protein 2 (MAP2), neuron‐specific enolase (NSE), doublecortin, synapsin I, glial fibrillary acidic protein (GFAP), and myelin basic protein (MBP) by quantitative real‐time polymerase chain reaction (quantitative real‐time polymerase chain reaction (qRT‐PCR) and protein level by the immunofluorescence technique. Treatment with Ca + IBMX + Ins induced neuronal appearance and projection of neurite‐like processes in the cells, accompanied with inhibition of proliferation and halt in the cell cycle. A significantly higher expression of MBP, GFAP, NeuN, NSE, synapsin 1, doublecortin, and MAP2 was detected in differentiated cells, confirming the advantages of Ca + IBMX + Ins to the other combinations of inducers. Here, we showed an efficient protocol for neuronal differentiation of ADSCs, and calcium fostered differentiation by augmenting the number of neuron‐like cells and instantaneous increase in the expression of neuronal markers.     

Immunohistochemical expression of two members of the GDNF family of growth factors and their receptors in the olfactory system

The glial cell line-derived (GDNF) family of trophic factors, GDNF, neurturin, persephin and artemin, are known to support the survival and regulate differentiation of many neuronal populations, including peripheral autonomic, enteric and sensory neurons. Members of this family of related ligands bind to specific GDNF family receptor (GFR) proteins, which complex and signal through the Ret receptor tyrosine kinase. We showed previously that GDNF protein was detectable in olfactory sensory neurons (OSNs) in the olfactory neuroepithelium (ON). In this immunohistochemical study, we localized GDNF, neurturin, GFRα1, GFRα2 and Ret in the adult rat ON and olfactory bulb. We found that GDNF and Ret were widely expressed by immature and mature OSNs, while neurturin was selectively expressed in a subpopulation of OSNs zonally restricted in the ON. The GFRs had differential expression, with mature OSNs and their axons preferentially expressing GFRα1, whereas progenitors and immature neurons more avidly expressed GFRα2. In the bulb, GDNF was highly expressed by the mitral and tufted cells, and by periglomerular cells, and its distribution generally resembled that of Ret, with the exception that Ret was far more predominant on fibers than cell bodies. Neurturin, in contrast, was present at lower levels and was more restricted in its expression to the axonal compartment. GFRα2 appeared to be the dominant accessory protein in the bulb. These data are supportive of two members of this neurotrophic family, GDNF and neurturin, playing different physiological roles in the olfactory neuronal system.     

Coordinated expression of cytoskeleton regulating genes in the accelerated neurite outgrowth of P19 embryonic carcinoma cells

The embryonal carcinoma P19 cells provide a model to study neuronal differentiation. Cells that are exposed to retinoic acid become mature neurons within a few days with a pronounced axonal and dendritic polarity. Notably, an accelerated rate of neurite extension characterizes densely but not sparsely plated cells. DNA microarray experiments show maximal differences in gene expression of the dense compared to sparse plated cultures at 18 h after plating. The differentially expressed genes are enriched by functions of cell adhesion and cytoskeletal regulation. Doublecortin, Lis1, Reelin, Map2 and dozens of proteins that regulate cytoskeleton dynamics increase in concordance with a rapid neurite extension. A brief elevation in intracellular cAMP via PKA is sufficient to instigate the phenotype of accelerated neurite extension with no effect on P19 cell fate. Furthermore, we show that the cAMP dependent changes in the expression of cytoskeleton regulators such as doublecortin are restricted to a short time window prior to the establishment of functional neurons. We propose that the wave of gene expression of cytoskeletal regulators that is accompanied by accelerated neurite extension acts in remodeling young developing neurons in the CNS.     

Ret-dependent and Ret-independent mechanisms of Gfl-induced sensitization

Background

The GDNF family ligands (GFLs) are regulators of neurogenic inflammation and pain. We have previously shown that GFLs increase the release of the sensory neuron neuropeptide, calcitonin gene-related peptide (CGRP) from isolated mouse DRG.

Results

Inhibitors of the mitogen-activated protein kinase (MAPK) pathway abolished the enhancement of CGRP release by GDNF. Neurturin-induced enhancement in the stimulated release of CGRP, used as an indication of sensory neuronal sensitization, was abolished by inhibition of the phosphatidylinositol-3 kinase (PI-3K) pathway. Reduction in Ret expression abolished the GDNF-induced sensitization, but did not fully inhibit the increase in stimulus-evoked release of CGRP caused by neurturin or artemin, indicating the presence of Ret-independent GFL-induced signaling in sensory neurons. Integrin β-1 and NCAM are involved in a component of Ret-independent GFL signaling in sensory neurons.

Conclusions

These data demonstrate the distinct and variable Ret-dependent and Ret-independent signaling mechanisms by which GFLs induce sensitization of sensory neurons. Additionally, there is a clear disconnect between intracellular signaling pathway activation and changes in sensory neuronal function.     

Effect of carbamate esters on neurite outgrowth in differentiating human SK-N-SH neuroblastoma cells

Carbamate esters are widely used as pesticides and can cause neurotoxicity in humans and animals; the exact mechanism is still unclear. In the present investigation, the effects of carbamates at sublethal concentration on neurite outgrowth and cytoskeleton as well as activities of acetylcholinesterase (AChE) and neuropathy target esterase (NTE) in differentiating human SK-N-SH neuroblastoma cells were studied. The results showed that 50 microM of either aldicarb or carbaryl significantly decreased neurite length in the retinoic acid-induced differentiation of the neuroblastoma cells, compared to cells treated with vehicle. Western blot analyses revealed that neither carbamate had significant effects on the levels of actin, or total neurofilament high molecular proteins (NF-H). However, increased NF-H phosphorylation was observed following carbamate treatment. These changes may represent a useful in vitro marker of carbamate neurotoxicity within a simple model of neuronal cell differentiation. Furthermore, activity of AChE, but not NTE, was significantly inhibited by aldicarb and carbaryl in differentiating cells, which suggested that cytoskeletal protein changes induced by carbamate esters in differentiating cells was associated with inhibition of AChE but not NTE.     

Glial cell line-derived neurotrophic factor alters lipid composition and protein distribution in MPP+-injured differentiated SH-SY5Y cells

Parkinson's disease (PD) is a neurodegenerative disease characterized by progressive loss of dopaminergic neurons in the substantia nigra and striatum. Glial cell line-derived neurotrophic factor (GDNF) can effectively promote the differentiation and survival of many types of neurons, especially dopaminergic neurons, suggesting it could be a treatment for PD. Lipid rafts are highly dynamic cell membrane domains that contain numerous signal protein receptors, providing an important platform for signal transduction. Compelling evidence indicates that alterations in lipid rafts are associated with PD, and some studies have reported that GDNF can regulate the expression of caveolin-1, a lipid raft-marker protein. However, the precise effects of GDNF on lipid rafts remain unknown. We developed a cellular PD model, purified detergent-resistant membranes (membrane rafts), and performed proteomic and lipid metabolomics analyses to examine changes in lipid rafts after GDNF treatment. The results showed considerable protein and lipid alterations in response to GDNF, especially altered levels of dopamine-β-hydroxylase, heat shock 70 kDa protein, neural cell adhesion molecule, cytoskeletal proteins, and long-chain polysaturated/unsaturated fatty acids. These findings reveal a new avenue to explore the relationships between GDNF, lipid rafts, and PD and support the hypothesis that GDNF may be a useful treatment for PD.     

Small GTPase Rin induces neurite outgrowth through Rac/Cdc42 and calmodulin in PC12 cells

The novel Ras-like small GTPase Rin is expressed prominently in adult neurons, and binds calmodulin (CaM) through its COOH-terminal-binding motif. It might be involved in calcium/CaM-mediated neuronal signaling, but Rin-mediated signal transduction pathways have not yet been elucidated. Here, we show that expression of Rin induces neurite outgrowth without nerve growth factor or mitogen-activated protein kinase activation in rat pheochromocytoma PC12 cells. Rin-induced neurite outgrowth was markedly inhibited by coexpression with dominant negative Rac/Cdc42 protein or CaM inhibitor treatment. We also found that expression of Rin elevated the endogenous Rac/Cdc42 activity. Rin mutant proteins, in which the mutation disrupted association with CaM, failed to induce neurite outgrowth irrespective of Rac/Cdc42 activation. Disruption of endogenous Rin function inhibited the neurite outgrowth stimulated by forskolin and extracellular calcium entry through voltage-dependent calcium channel evoked by KCl. These findings suggest that Rin-mediated neurite outgrowth signaling requires not only endogenous Rac/Cdc42 activation but also Rin-CaM association, and that endogenous Rin is involved in calcium/CaM-mediated neuronal signaling pathways.     

Coordinated interactions between actin and microtubules through crosslinkers in neurite retraction induced by lysophosphatidic acid

Neurite development requires rearrangement of cytoskeletal elements, which are mechanically and functionally integrated with each other. Although the process of how an extracellular signal induces rearrangement of a single element has been closely examined, the mechanisms by which the signal regulates cytoskeletal integration during cell shape changes are poorly understood. We previously reported that lysophosphatidic acid (LPA) induces actin polymerization-dependent microtubule (MT) rearrangement, leading to neurite retraction in cultured neurons. Here we examined whether the crosslinker proteins were involved in LPA-induced neurite retraction using immortalized mouse neuroblast TR cells. When the MT-binding domains of MACF (MT actin-crosslinking factor) were exogenously expressed in TR cells, MTs were found to be stabilized and become resistant to exposure to LPA. On the other hand, expression of MT-associated protein 2c showed no effect on LPA-induced neurite retraction. These findings suggest that MACF is involved in actin-dependent MT rearrangement during LPA-induced neurite retraction.     

Glial cell line-derived neurotrophic factor family ligands and their therapeutic potential

Four glial cell line-derived neurotrophic factor (GDNF) family ligands (GFLs) have been characterized: GDNF, neurturin (NRTN), artemin (ARTN) and persephin (PSPN). These proteins support and restore multiple neuronal populations such as dopaminergic, sensory, motor, hippocampal, basal forebrain, enteric, sympathetic and parasympathetic neurons. Therefore, GFLs attracted significant attention as a potential cure for the diseases caused by neuronal injury and degeneration. Results of multiple experiments indicate that GFLs can alleviate behavioral symptoms and restore affected neurons in animal models of several neurological disorders including, among others, Parkinson’s disease (PD). During the last decade, GDNF protein and NRTN gene therapy have been tested in several clinical trials in patients with PD. Although the results of phase I clinical trials were positive, phase II clinical trials failed to reach primary end-points. Poor pharmacokinetic properties of GFLs (inability to penetrate tissues barriers, high affinity for extracellular matrix, etc.) could contribute to the absence of clear clinical benefits of these proteins for the patients. The purpose of this paper was to review therapeutic potential of GFLs and discuss possibilities to overcome difficulties associated with pharmacokinetic properties and delivery of GFLs to target neurons.     

GDNF promotes neurite outgrowth and upregulates galectin-1 through the RET/PI3K signaling in cultured adult rat dorsal root ganglion neurons

Galectin-1 (GAL-1), a member of a family of β-galactoside binding animal lectins, is predominantly expressed in isolectin B4 (IB4)-binding small non-peptidergic (glial cell line-derived neurotrophic factor (GDNF)-responsive) sensory neurons in the sections of adult rat dorsal root ganglia (DRG), but its functional role and the regulatory mechanisms of its expression in the peripheral nervous system remain unclear. In the present study, both recombinant nerve growth factor (NGF) and GDNF (50 ng/ml) promoted neurite outgrowth from cultured adult rat DRG neurons, whereas GDNF, but not NGF, significantly increased the number of IB4-binding neurons and the relative protein expression of GAL-1 in the neuron-enriched culture of DRG. The GAL-1 expression in immortalized adult rat Schwann cells IFRS1 and DRG neuron-IFRS1 cocultures was unaltered by treatment with GDNF, which suggests that GDNF/GAL-1 signaling axis is more related to neurite outgrowth, rather than neuron-Schwann cell interactions. The GDNF-induced neurite outgrowth and GAL-1 upregulation were attenuated by anti-GDNF family receptor (RET) antibody and phosphatidyl inositol-3′-phosphate-kinase (PI3K) inhibitor LY294002, suggesting that the neurite-outgrowth promoting activity of GDNF may be attributable, at least partially, to the upregulation of GAL-1 through RET-PI3K pathway. On the contrary, no significant differences were observed between GAL-1 knockout and wild-type mice in DRG neurite outgrowth in the presence or absence of GDNF. Considerable immunohistochemical colocalization of GAL-3 with GAL-1 in DRG sections and GDNF-induced upregulation of GAL-3 in cultured DRG neurons imply the functional redundancy between these galectins.     

N1E-115 mouse neuroblastoma cells express MT1 melatonin receptors and produce neurites in response to melatonin

Melatonin, a pineal hormone that induces sleep, has become a popular over-the-counter drug. The cellular effects of melatonin, however, are only beginning to be studied. We have recently shown that stimulation of the MT1 melatonin receptor induces rapid and dramatic cytoskeletal rearrangements in transformed non-neuronal cells (Witt-Enderby et al., Cell. Motil. Cytoskel. 46 (2000) 28). These cytoskeletal changes result in the formation of structures that closely resemble neurites. In this work, we show that the N1E-115 mouse neuroblastoma cell line rapidly responds to melatonin stimulation and forms neurites within 24 h. We also demonstrate that these cells readily bind 2-[125I]iodomelatonin at levels consistent with what is noted for native tissues (B(max)=3.43+/-1.56 fmol/mg protein; K(d)=240 pM). Western analysis shows that these cells possess and express melatonin receptors of the MT1 subtype. Treatment with pertussis toxin eliminates neurite formation whereas treatment with the MT2 subtype-specific activator, BMNEP, does not induce neurite formation. We have previously shown that increases in MEK 1/2 and ERK 1/2 phosphorylation are correlated with the shape changes in transformed CHO cells. Western analysis of the MEK/ERK signaling pathway in N1E-115 cells shows that this pathway is most likely maximally and constitutively stimulated. This may account for the spontaneous production of neurites noted for this cell line after long culture periods. The results of this work show that melatonin receptor stimulation in a neuronal cell type results in the formation of neurites and that the receptors responsible for melatonin-induced neurite formation in N1E-115 cells are most likely of the MT1 subtype.     

Predominant release of lysosomal enzymes by newborn rat microglia after LPS treatment revealed by proteomic studies

Growing evidence suggest that microglia may play an important role in the pathogenesis of neurodegenerative disease including Parkinson's disease, Alzheimer's disease, and so forth. The activation of microglia may cause neuronal damage through the release of reactive oxygen species and proinflammatory cytokines. However, the early response of microglial cells remains unclear before cells can secrete the proinflammatory cytokines. Here, a time course analysis showed the earliest expression of inducible nitric oxide synthase and cyclooxygenase-2 at 3 and 24 h following lipopolysaccharide (LPS) treatment. To further define initial response proteins of microglia after LPS treatment, we utilized a novel mass spectrometry-based quantitative proteomic technique termed SILAC (for stable isotope labeling by amino acids in cell culture) to compare the protein profiles of the cell culture-conditioned media of 1 h LPS-treated microglia as compared with controls. The proteomic analysis identified 77 secreted proteins using SignalP; of these, 28 proteins were associated with lysosome of cells and 13 lysosome-related proteins displayed significant changes in the relative abundance after 1 h LPS treatment. Four proteins were further evaluated with Western blot, demonstrating good agreement with quantitative proteomic data. These results suggested that microglia first released some lysosomal enzymes which may be involved in neuronal damage process. Furthermore, ammonium chloride, which inhibits microglia lysosomal enzyme activity, could prevent microglia from causing neuronal injury. Hence, in addition to the numerous novel proteins that are potentially important in microglial activation-mediated neurodegeneration revealed by the search, the study has indicated that the early release of lysosomal enzymes in microglial cells would contribute to LPS-activated inflammatory response.     

Direct activation of second messenger pathways mimics cell adhesion molecule-dependent neurite outgrowth

We present evidence that direct activation of neuronal second messenger pathways in PC12 cells by opening voltage-dependent calcium channels mimics cell adhesion molecule (CAM)-induced differentiation of these cells. PC12 cells were cultured on monolayers of control 3T3 cells or 3T3 cells expressing transfected N-cadherin in the presence of KCl or a calcium channel agonist Bay K 8644. Both potassium depolarization and agonist-induced activation of calcium channels promoted substantial neurite outgrowth from PC12 cells cultured on control 3T3 monolayers and increased neurite outgrowth from those cultured on N-cadherin-expressing 3T3 monolayers. The potassium-induced response could be inhibited by L- and N-type calcium channel antagonists and by kinase inhibitor K-252b but was unaffected by pertussis toxin. In contrast activators of protein kinase C did not stimulate neurite outgrowth, and the neurite outgrowth response induced by activation of protein kinase A was not inhibited by calcium channel antagonists or pertussis toxin. These studies support the postulate that CAM-induced neuronal differentiation involves a specific transmembrane signaling pathway and suggest that activation of this pathway after CAM binding may be more important for the neurite outgrowth response than CAM-dependent adhesion per se.     

Temporal Analysis of Changes in Neuronal c-fos mRNA Levels Induced by Depletion of Endoplasmic Reticulum Calcium Stores: Effect of Clamping Cytoplasmic Calcium Activity at Resting Levels

Abstract: Activation of immediate early gene expression is a key event in stress-induced neuronal cell injury. To study whether changes in cytoplasmic calcium activity are necessary to activate neuronal immediate early gene expression, endoplasmic reticulum (ER) calcium stores of primary neurons were depleted by exposing cells to thapsigargin (Tg), an irreversible inhibitor of ER Ca²⁺-ATPase. Tg-induced rise in [Ca²⁺]_i and the effect of loading neurons with the cell-permeable calcium chelator BAPTA-AM on this increase in [Ca²⁺]_i were measured in fura-2-loaded cells by fluorescence microscopy. Changes in c- fos mRNA levels were evaluated by quantitative PCR. Tg treatment of neurons produced a pronounced rise in c- fos mRNA levels (∼10-fold more than DMSO) which peaked at 1 h after exposure. The Tg-induced rise in c- fos mRNA content was unchanged (hippocampal neurons) or even increased further (cortical neurons) by preloading cells with BAPTA before incubation with Tg. It is concluded that in neuronal cells an increase in cytoplasmic calcium activity is not a prerequisite for a rise in mRNA levels of c- fos . Thus, stress-induced changes in mRNA levels of immediate early genes of neurons may also result from disturbances in ER calcium homeostasis and not necessarily by an overload of cells with calcium ions. The results of the present series of experiments cast further doubt on the widely accepted hypothesis that the stress-induced cytoplasmic overload of neurons with calcium ions is the primary event triggering cell injury.     

The mood stabilizer valproic acid stimulates GABA neurogenesis from rat forebrain stem cells

Valproate, an anticonvulsant drug used to treat bipolar disorder, was studied for its ability to promote neurogenesis from embryonic rat cortical or striatal primordial stem cells. Six days of valproate exposure increased by up to fivefold the number and percentage of tubulin beta III-immunopositive neurons, increased neurite outgrowth, and decreased by fivefold the number of astrocytes without changing the number of cells. Valproate also promoted neuronal differentiation in human fetal forebrain stem cell cultures. The neurogenic effects of valproate on rat stem cells exceeded those obtained with the neurotrophins brain-derived growth factor (BDNF) or NT-3, and slightly exceeded the effects obtained with another mood stabilizer, lithium. No effect was observed with carbamazepine. Most of the newly formed neurons were GABAergic, as shown by 10-fold increases in neurons that immunostained for GABA and the GABA-synthesizing enzyme GAD65/67. Double immunostaining for bromodeoxyuridine and tubulin beta III showed that valproate increased by four- to fivefold the proliferation of neuronal progenitors derived from rat stem cells and increased cyclin D2 expression. Valproate also regulated the expression of survival genes, Bad and Bcl-2, at different times of treatment. The expression of prostaglandin E synthase, analyzed by quantitative RT-PCR, was increased by ninefold as early as 6 h into treatment by valproate. The enhancement of GABAergic neuron numbers, neurite outgrowth, and phenotypic expression via increases in the neuronal differentiation of neural stem cell may contribute to the therapeutic effects of valproate in the treatment of bipolar disorder.     

胶质源性神经营养因子对不同组织细胞的影响

GDNF来自于小胶质神经元,首先作为中脑多巴胺能神经元的复活因子被发现,可促进细胞存活,并有增加多巴胺神经元细胞大小及轴突长度的作用。GDNF通过与锚定蛋白细胞表面受体糖基磷脂酰肌醇的相互作用来调节细胞活性。GDNF家族a-1受体,通过跨膜酪氨酸受体或者神经元细胞黏附分子,来促进细胞存活,神经突生长,以及突触发育。后续的研究提示,无论未成年还是成体大脑,GDNF对多种神经细胞都有复活的作用,并与一些周围神经复活、迁移、分化相关。不同的脑缺血实验模型均证实了外源性GDNF对于病灶部位及全脑的神经保护作用,包括局部应用营养因子,利用病毒载体运载GDNF基因以及移植表达GDNF的细胞。近来研究还证实,GDNF不仅对多巴胺能神经元,中枢和周围神经系统的运动、感觉神经元,以及自主神经元有营养和保护作用,对于非神经系统也有不同调节作用。本文将重点讨论这些GDNF作用的不同策略以及机制。     

Ribosomal protein L10 interacts with the SH3 domain and regulates GDNF-induced neurite growth in SH-SY-5y cells

The 24.5 kDa ribosomal protein L10 (RP-L10), which was encoded by QM gene, was known to interact with the SH3 domain of Yes kinase. Herein, we demonstrate that RP-L10 interacts with the SH3 domain of Src and activates the binding of the Nck1 adaptor protein with skeletal proteins such as the Wiskott-Aldrich Syndrome Protein (WASP) and WASP interacting protein (WIP) in neuroblastoma cell line, SH-SY-5y. The RP-L10 was associated with the SH3 domains of Src and Yes. It is shown that two different regions of RP-L10 are associated with the Src-SH3. The effect of ectopic RP-L10 expression on neuronal cell scaffolding was explored in cells transiently transfected with QM. SH-SY-5y human neuroblastoma cells transfected with QM were considerably more susceptible to neurite outgrowth induced by glial cell line-derived neurotrophic factor (GDNF). However, RP-L10 did not directly interact with actin assembly. Taken together, these results suggest that the RP-L10 may positively regulate the GDNF/Ret-mediated signaling of neurite outgrowth in the neuroblastoma cell line, SH-SY-5y.