Characterization of cellular phenotypes in neurons derived from induced pluripotent stem cells of male patients with Fabry disease.

Fabry disease (FD) is an X-linked inherited lysosomal metabolism disorder in which globotriaosylceramide (Gb3) accumulates in various organs resulting from a deficiency in alpha-galactosidase A. The clinical features of FD include progressive impairments of the renal, cardiac, and peripheral nervous systems. In addition, patients with FD often develop neuropsychiatric symptoms, such as depression and dementia, which are believed to be induced by the cellular injury of cerebrovascular and partially neuronal cells due to Gb3 accumulation. Although the analysis of autopsy brain tissue from patients with FD showed no accumulation of Gb3, abnormal deposits of Gb3 were found in the neurons of several brain areas, including the hippocampus. Therefore, in this study, we generated induced pluripotent stem cells (iPSCs) from patients with FD and differentiated them into neuronal cells to investigate pathological and biological changes in the neurons of FD. Neural stem cells (NSCs) and neurons were successfully differentiated from the iPSCs we generated; however, cellular damage and morphological changes were not found in these cells. Immunostaining revealed no Gb3 accumulation in NSCs and neurons. Transmission electron microscopy did not reveal any zebra body-like structures or inclusion bodies, which are characteristic of FD. These results indicated that neuronal cells derived from FD-iPSCs exhibited normal morphology and no Gb3 accumulation. It is likely that more in vivo environment-like cultures are needed for iPSC-derived neurons to reproduce disease-specific features.

Dysregulated DNA methylation of GLA gene was associated with dysfunction of autophagy.

Lysosomes are an essential organ for cellular metabolism and play an important role in autophagy. We examined the association between methylation and autophagy in a severely affected female patient with Fabry disease, which is caused by mutation of the GLA gene on the X chromosome, and her two sisters, who had few symptoms. We confirmed autophagic flux by LC3 turnover assay using fibroblasts from each sister. In the severe female patient, autophagic flux showed abnormal while her two sisters with few symptoms had normal autophagic flux, revealing the direct relationship between symptoms and autophagic flux. Furthermore, we observed the levels of p62, which is a substrate for autophagy, and lysosome morphology. In the severe patient of this family, lysosomes were enlarged and p62 was accumulated. The methylated allele of the GLA gene in the severe patient had a high proportion of wild alleles; conversely, the sisters' methylated allele had a high proportion of mutant alleles. Therefore, we examined the mRNA expression level of the mutant allele by allele-specific PCR. It was high in the severe patient and low in the siblings with few symptoms. That is, the correlation between the mRNA expression level of the mutant allele and disease severity was confirmed. We showed a correlation between severe symptoms, dysfunction of autophagy and methylation of wild alleles in Fabry disease. It was suggested that allele-specific PCR may lead to a diagnosis and help to determine the prognosis of female patients with Fabry disease.

Characteristics of PPT1 and TPP1 enzymes in neuronal ceroid lipofuscinosis (NCL) 1 and 2 by dried blood spots (DBS) and leukocytes and their application to newborn screening.

We first characterized PPT1 and TPP1 enzymes in dried blood spots (DBS), plasma/serum, and leukocytes/lymphocytes using neuronal ceroid lipofuscinosis (NCL) 1 and 2 patients and control subjects. PPT1 enzyme had only one acid form in control DBS, plasma/serum, and leukocytes/lymphocytes and showed deficient activities in these samples from NCL 1 patients. Conversely, TPP1 enzymes in control DBS and leukocytes/lymphocytes consisted of two forms, an acidic form and a neutral form, whereas serum TPP1 enzyme had only a neutral form. In control subjects, the optimal pH of PPT1 enzyme in DBS, plasma/serum, and leukocytes/lymphocytes was 4.5 to 5.0 in the acidic form, whereas TPP1 enzyme in control DBS and leukocytes/lymphocytes was pH 4.5 and 6.5, respectively. In NCL 1 and 2, both PPT1 and TPP1 enzyme activities in DBS, plasma, and leukocytes/lymphocytes were markedly reduced in acidic pH, whereas heterozygotes of NCL 1 and 2 in the acidic form showed intermediate activities between patients and control subjects. In neutral conditions, pH 6.0, the PPT1 enzyme activities in NCL 1 patients showed rather higher residual activities and intermediate activities in heterozygotes in NCL 1, which was probably caused by mutated proteins in three cases with NCL 1 patients. TPP1 enzyme activities at neutral pH 6.5 to 7.0 in DBS and leukocytes/lymphocytes showed higher enzyme activities in NCL 2 patients and heterozygotes. The reason for the increases of neutral TPP1 enzyme activities at pH 6.5 to 7.0 in NCL 2 DBS and leukocytes/lymphocytes, is obscure, but possibly caused by secondary activation of neutral TPP1 enzyme due to the absence of the acidic form. Interestingly, TPP1 activity in serum only consisted of a neutral form, no acidic form, and was not deficient in any NCL 2 patient. Therefore, we can diagnose NCL 1 patients by plasma/serum enzyme assay of PPT1, but not diagnose NCL 2 by serum TPP1 enzyme assay. A pilot study of newborn screening of NCL 1 and 2 has been established by more than 1000 newborn DBS assays. Using this assay system, we will be able to perform newborn screening of NCL 1 and 2 by DBS.

The severe clinical phenotype for a heterozygous Fabry female patient correlates to the methylation of non-mutated allele associated with chromosome 10q26 deletion syndrome.

Heterozygous Fabry females usually have an attenuated form of Fabry disease, causing them to be symptomatic; however, in rare cases, they can present with a severe phenotype. In this study, we report on a 37-year-old woman with acroparesthesia, a dysmorphic face, left ventricular hypertrophy, and intellectual disability. Her father had Fabry disease and died due to chronic renal and congestive cardiac failure. Her paternal uncle had chronic renal failure and intellectual disability, and her paternal aunt was affected with congestive cardiac failure. The patient has two sisters with no significant medical illness. However, her nephew has acroparesthesia, anhidrosis, and school phobia, and her niece shows mild phenotypes. The patient's enzyme analysis showed very low α-galactosidase A (α-gal A) activity in dried blood spot (DBS), lymphocytes, and skin fibroblasts with massive excretion of Gb3 and Gb2 in urine and lyso-Gb3 in DBS and plasma. Electron microscopic examination showed a large accumulation of sphingolipids in vascular endothelial cells and keratinocytes. Chromosomal analysis and comparative genomic hybridization microarray showed 10q26 terminal deletion. Molecular data showed a novel heterozygous stop codon mutation in exon 1 of the GLA gene in her sisters and niece, and a hemizygous state in her nephew. When we checked the methylation status, we found her non-mutated allele in the GLA gene was methylated. However, the non-mutated alleles of her sisters were non-methylated, and those of her niece were partially methylated. The chromosomal and methylation study may speculate the severity of her clinical phenotypes.

Involvement of oxidative stress and impaired lysosomal degradation in amiodarone-induced schwannopathy.

Amiodarone hydrochloride (AMD), an anti-arrhythmic agent, has been shown to cause peripheral neuropathy; however, its pathogenesis remains unknown. We examined the toxic effects of AMD on an immortalized adult rat Schwann cell line, IFRS1, and cocultures of IFRS1 cells and adult rat dorsal root ganglion neurons or nerve growth factor-primed PC12 cells. Treatment with AMD (1, 5, and 10 µm) induced time- and dose-dependent cell death, accumulation of phospholipids and neutral lipids, upregulation of the expression of gangliosides, and oxidative stress (increased nuclear factor E2-related factor in nuclear extracts and reduced GSH/GSSG ratios) in IFRS1 cells. It also induced the upregulation of LC3-II and p62 expression, with phosphorylation of p62, suggesting that deficient autolysosomal degradation is involved in AMD-induced IFRS1 cell death. Furthermore, treatment of the cocultures with AMD induced detachment of IFRS1 cells from neurite networks in a time- and dose-dependent manner. These findings suggest that AMD-induced lysosomal storage accompanied by enhanced oxidative stress and impaired lysosomal degradation in Schwann cells might be a cause of demyelination in the peripheral nervous system.

Adenoviral expression of TDP-43 and FUS genes and shRNAs for protein degradation pathways in rodent motoneurons in vitro and in vivo.

Formation of cytoplasmic aggregates in neuronal and glial cells is one of the pathological hallmarks of amyotrophic lateral sclerosis (ALS). Mutations in two genes encoding transactivation response (TAR) DNA-binding protein 43 (TDP-43) and fused in sarcoma (FUS), both of which are main constituents of cytoplasmic aggregates, have been identified in patients with familial and sporadic ALS. Impairment of protein degradation machineries has also been recognized to participate in motoneuron degeneration in ALS. In the present study, we produced recombinant adenovirus vectors encoding wild type and mutant TDP-43 and FUS, and those encoding short hairpin RNAs (shRNAs) for proteasome (PSMC1), autophagy (ATG5), and endosome (VPS24) systems to investigate whether the coupled gene transductions in motoneurons by these adenoviruses elicit ALS pathology. Cultured neurons, astrocytes and oligodendrocytes differentiated from adult rat neural stem cells and motoneurons derived from mouse embryonic stem cells were successfully infected with these adenoviruses showing cytoplasmic aggregate formation. When these adenoviruses were injected into the facial nerves of adult rats, exogenous TDP-43 and FUS proteins were strongly expressed in facial motoneurons by a retrograde axonal transport of the adenoviruses. Co-infections of adenovirus encoding shRNA for PSMC1, ATG5 or VPS24 with TDP-43 or FUS adenovirus enhanced cytoplasmic aggregate formation in facial motoneurons, suggesting that impairment of protein degradation pathways accelerates formation of TDP-43 and FUS-positive aggregates in ALS.

Myelination in coculture of established neuronal and Schwann cell lines.

Establishing stable coculture systems with neuronal and Schwann cell lines has been considered difficult, presumably because of their high proliferative activity and phenotypic differences from primary cultured cells. The present study is aimed at developing methods for myelin formation under coculture of the neural crest-derived pheochromocytoma cell line PC12 and the immortalized adult rat Schwann cell line IFRS1. Prior to coculture, PC12 cells were seeded at low density (3 × 10(2)/cm(2)) and maintained in serum-free medium with N2 supplement, ascorbic acid (50 µg/ml), and nerve growth factor (NGF) (50 ng/ml) for a week. Exposure to such a NGF-rich environment with minimum nutrients accelerated differentiation and neurite extension, but not proliferation, of PC12 cells. When IFRS1 cells were added to NGF-primed PC12 cells, the cell density ratio of PC12 cells to IFRS1 cells was adjusted from 1:50 to 1:100. The cocultured cells were then maintained in serum-free medium with B27 supplement, ascorbic acid (50 µg/ml), NGF (10 ng/ml), and recombinant soluble neuregulin-1 type III (25 ng/ml). Myelin formation was illustrated by light and electron microscopy performed at day 28 of coculture. The stable PC12-IFRS1 coculture system is free of technical and ethical problems arising from the primary culture and can be a valuable tool to study peripheral nerve degeneration and regeneration.

Spontaneously immortalized Schwann cells from adult Fischer rat as a valuable tool for exploring neuron-Schwann cell interactions.

We established spontaneously immortalized Schwann cell lines from long-term cultures of adult Fischer 344 rat dorsal root ganglia (DRG) and peripheral nerves. One of these cell lines, designated immortalized Fischer rat Schwann cells 1 (IFRS1), showed spindle-shaped morphology; immunoreactivity for S100, p75 neurotrophin receptor (p75(NTR) ), glial fibrillary acidic protein (GFAP), laminin, and vimentin; and mRNA expression of neurotrophic factors (NGF, GDNF, and CNTF), neurotrophin receptors (p75(NTR) , truncated TrkB, and TrkC), cell adhesion molecules (L1, NCAM, and N-cadherin), myelin proteins [P0, PMP22, and myelin-associated glycoprotein (MAG)], transcription factors (Krox20, Sox10, and Oct6), neuregulin-1 receptors (ErbB2 and ErbB3), and an orphan G protein-coupled receptor (Gpr126). Conditioned medium (CM) obtained from IFRS1 cells exhibited potent biological activity for the promotion of neuronal survival and neurite outgrowth of cultured adult rat DRG neurons. Furthermore, light and electron microscopic analyses revealed that IFRS1 cells were capable of myelinating neurites while in coculture with adult rat DRG neurons. These findings indicate that IFRS1 cells possess some biological properties of mature Schwann cells and that the coculture system with adult DRG neurons and IFRS1 cells can be a useful tool for the study of peripheral nerve degeneration and regeneration.

Expression of transforming growth factor-beta receptors in meningeal fibroblasts of the injured mouse brain.

The fibrotic scar which is formed after traumatic damage of the central nervous system (CNS) is considered as a major impediment for axonal regeneration. In the process of the fibrotic scar formation, meningeal fibroblasts invade and proliferate in the lesion site to secrete extracellular matrix proteins, such as collagen and laminin. Thereafter, end feet of reactive astrocytes elaborate a glia limitans surrounding the fibrotic scar. Transforming growth factor-beta1 (TGF-beta1), a potent scar-inducing factor, which is upregulated after CNS injury, has been implicated in the formation of the fibrotic scar and glia limitans. In the present study, expression of receptors to TGF-beta1 was examined by in situ hybridization histochemistry in transcortical knife lesions of the striatum in the mouse brain in combination with immunofluorescent staining for fibroblasts and astrocytes. Type I and type II TGF-beta receptor mRNAs were barely detected in the intact brain and first found in meningeal cells near the lesion 1 day postinjury. Many cells expressing TGF-beta receptors were found around the lesion site 3 days postinjury, and some of them were immunoreactive for fibronectin. After 5 days postinjury, many fibroblasts migrated from the meninges to the lesion site formed the fibrotic scar, and most of them expressed TGF-beta receptors. In contrast, few of reactive astrocytes expressed the receptors throughout the postinjury period examined. These results indicate that meningeal fibroblasts not reactive astrocytes are a major target of TGF-beta1 that is upregulated after CNS injury.

Future clinical and biochemical predictions of Fabry disease in females by methylation studies of the GLA gene.

Fabry disease is an X-linked lysosomal storage disorder caused by a deficiency of α-galactosidase A (α-gal A). The clinical variability of the phenotypes of Fabry disease in females is still poorly understood. The degree of aberrant methylation of non-mutated alleles is thought to have significant effects on X-chromosome inactivation (XCI). We previously reported that one heterozygous Fabry female showing classical phenotypes had complete methylation of the non-mutated allele of the GLA gene. In this report, we summarized 36 heterozygous females with a clinical severity score based on the FAbry STabilization indEX (FASTEX). We measured their α-gal A activity and plasma/ serum globotriaosylsphingosine (lyso-Gb3) accumulation and performed electron microscopy of skin biopsies. We analyzed the methylation-sensitive restriction enzyme sites throughout the GLA gene, including the 5'UTR, and found a single SacII site and multiple HhaI and HpaII sites aggregated in exon 1 and the 5'UTR. One HpaII sequence in exon 7 was also detected as a methylation-sensitive site. With methylation-sensitive restriction enzymes, methylated and non-methylated alleles could be separated, and the ratio of the methylation was quantified. We found a clear correlation between the severity of the phenotype and lyso-Gb3 accumulation for heterozygous Fabry disease in females. Methylation of the non-mutated allele was also proportionately correlated to the clinical severity score measured by FASTEX.

Ten-year-long enzyme replacement therapy shows a poor effect in alleviating giant leg ulcers in a male with Fabry disease.

Fabry disease is an X-linked lysosomal storage disorder caused by a deficiency of α-galactosidase A (α-gal A), leading to the progressive accumulation of glycosphingolipids. Classical hemizygous males usually present symptoms, including pain and paresthesia in the extremities, angiokeratoma, hypo- or anhidrosis, abdominal pain, cornea verticillata, early stroke, tinnitus, and/or hearing loss, during early childhood or adolescence. Moreover, proteinuria, renal impairment, and cardiac hypertrophy can appear with age. Enzyme replacement is the most common therapy for Fabry disease at present which has been approved in Japan since 2004. We report a case involving a 27-year-old male with extreme terminal pain, anhidrosis, abdominal pain, tinnitus, hearing impairment, cornea verticillata, and recurrent huge ulcers in the lower extremities. At the age of 16 years, he was diagnosed with Fabry disease with a positive family history and very low α-gal A activity. He then received enzyme replacement therapy (ERT) with recombinant human agalsidase beta at 1 mg/kg every 2 weeks for 10 years. Throughout the course of ERT, his leg ulcers recurred, and massive excretion of urinary globotriaosylceramide and plasma globotriaosylsphingosine was observed. Electron microscopy of the venous tissue in the regions of the ulcer showed massive typical zebra bodies in the vascular wall smooth muscle cells.

Application of a diagnostic methodology by quantification of 26:0 lysophosphatidylcholine in dried blood spots for Japanese newborn screening of X-linked adrenoleukodystrophy.

X-linked adrenoleukodystrophy (X-ALD) is a rare inherited metabolic disease that results in the accumulation of very long chain fatty acids (VLCFA) in plasma and all tissues. Recent studies regarding cerebral X-ALD (CALD) treatment emphasize the importance of its early diagnosis. 26:0 lysophosphatidylcholine (LysoPC) is a sensitive biomarker for newborn screening of X-ALD, while its application for Japanese DBS is unclear. Therefore, we evaluated the feasibility of 20:0 LysoPC and 24:0 LysoPC along with 26:0 LysoPC for diagnosing X-ALD in a cohort of newborns (n = 604), healthy adults (n = 50) and patients (n = 4). Results indicated that 26:0 LysoPC had strong significance for discrimination of patients by the amounts of 2.0 to 4.0 and 0.1 to 1.9 pmol/punch for patients and newborns/healthy adults, respectively. Based on these values, we recommend that further diagnostic confirmation is essential if the amount of 26:0 LysoPC in DBS is above 1.7 pmol/punch.

L-leucine and SPNS1 coordinately ameliorate dysfunction of autophagy in mouse and human Niemann-Pick type C disease.

Lysosomal storage disorders are characterized by progressive accumulation of undigested macromolecules within the cell due to lysosomal dysfunction. 573C10 is a Schwann cell line derived from a mouse model of Niemann-Pick type C disease-1, NPC (-/-). Under serum-starved conditions, NPC (-/-) cells manifested impaired autophagy accompanied by an increase in the amount of p62 and lysosome enlargement. Addition of L-leucine to serum-starved NPC (-/-) cells ameliorated the enlargement of lysosomes and the p62 accumulation. Similar autophagy defects were observed in NPC (-/-) cells even without serum starvation upon the knockdown of Spinster-like 1 (SPNS1), a putative transporter protein thought to function in lysosomal recycling. Conversely, SPNS1 overexpression impeded the enlargement of lysosomes, p62 accumulation and mislocalization of the phosphorylated form of the mechanistic Target of rapamycin in NPC (-/-) cells. In addition, we found a reduction in endogenous SPNS1 expression in fibroblasts derived from NPC-1 patients compared with normal fibroblasts. We propose that SPNS1-dependent L-leucine export across the lysosomal membrane is a key step for triggering autophagy, and that this mechanism is impaired in NPC-1.

Neonicotinoid Insecticides Alter the Gene Expression Profile of Neuron-Enriched Cultures from Neonatal Rat Cerebellum.

Neonicotinoids are considered safe because of their low affinities to mammalian nicotinic acetylcholine receptors (nAChRs) relative to insect nAChRs. However, because of importance of nAChRs in mammalian brain development, there remains a need to establish the safety of chronic neonicotinoid exposures with regards to children's health. Here we examined the effects of longterm (14 days) and low dose (1 µM) exposure of neuron-enriched cultures from neonatal rat cerebellum to nicotine and two neonicotinoids: acetamiprid and imidacloprid. Immunocytochemistry revealed no differences in the number or morphology of immature neurons or glial cells in any group versus untreated control cultures. However, a slight disturbance in Purkinje cell dendritic arborization was observed in the exposed cultures. Next we performed transcriptome analysis on total RNAs using microarrays, and identified significant differential expression (p < 0.05, q < 0.05, ≥1.5 fold) between control cultures versus nicotine-, acetamiprid-, or imidacloprid-exposed cultures in 34, 48, and 67 genes, respectively. Common to all exposed groups were nine genes essential for neurodevelopment, suggesting that chronic neonicotinoid exposure alters the transcriptome of the developing mammalian brain in a similar way to nicotine exposure. Our results highlight the need for further careful investigations into the effects of neonicotinoids in the developing mammalian brain.

Upregulation of galectin-3 in immortalized Schwann cells IFRS1 under diabetic conditions.

A spontaneously immortalized adult Fischer rat Schwann cell line IFRS1 retains the characteristic features of normal Schwann cells, and can be a useful tool for the study of diabetic neuropathy. In the present study, we examined the effects of high glucose and 3-deoxyglucosone (3-DG) on the viability and the protein expression of advanced glycation endproducts (AGE)-binding proteins, such as galectin-3 (GAL-3) and receptor for AGE (RAGE) in IFRS1 cells. Exposure to 30mM of glucose or 0.2mM of 3-DG for 7 days failed to impair the IFRS1 cell viability, but significantly upregulated the expression of GAL-3. The same exposure tended to increase the expression of RAGE, but the changes were not significant. The high glucose-induced upregulation of GAL-3 was attenuated by cotreatment with 0.2mM of an anti-glycated agent aminoguanidine or 20nM of an anti-oxidant trans-resveratrol. In addition, treatment of IFRS1 cells with 1µg/ml of recombinant GAL-3 for 48h resulted in the upregulation of B-cell lymphoma 2 (Bcl-2) and the downregulation of 4-hydroxynonenal (4HNE). These findings suggest the involvement of GAL-3 in the glycation and oxidative stress under diabetic conditions and its cytoprotective role in Schwann cells.

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 ß-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.

Immortalized adult rodent Schwann cells as in vitro models to study diabetic neuropathy.

We have established spontaneously immortalized Schwann cell lines from normal adult mice and rats and murine disease models. One of the normal mouse cell lines, IMS32, possesses some biological properties of mature Schwann cells and high proliferative activities. The IMS32 cells under hyperglycemic and/or hyperlipidemic conditions have been utilized to investigate the pathogenesis of diabetic neuropathy, especially the polyol pathway hyperactivity, glycation, increased oxidative stress, and reduced synthesis of neurotrophic factors. In addition to the mouse cell lines, our current study focuses on the characterization of a normal rat cell line, IFRS1, under normal and high glucose conditions. These Schwann cell lines can be valuable tools for exploring the detailed mechanisms leading to diabetic neuropathy and novel therapeutic approaches against that condition.

Pleiotrophin induces neurite outgrowth and up-regulates growth-associated protein (GAP)-43 mRNA through the ALK/GSK3beta/beta-catenin signaling in developing mouse neurons.

Pleiotrophin (PTN) is highly expressed in the nervous system during embryogenesis; however, little is known about its functional role in neural development. By using whole mount in situ hybridization, we observed that the expression pattern of PTN was similar to that of Wnt3a; PTN mRNA was abundant in the nervous tissue along the dorsal midline and in the forelimb and hindlimb buds of embryonic mice (E8.5-E12.5). Treatment with recombinant PTN (100ng/ml) induced phosphorylation of glycogen synthase kinase 3beta (GSK3beta), nuclear localization of beta-catenin and up-regulation of growth-associated protein (GAP)-43 mRNA in cultured embryonic mouse (E14.5) neurons. Furthermore, recombinant PTN enhanced neurite outgrowth from cortical explants embedded in Matrigel. These PTN-induced biochemical changes and neurite outgrowth were attenuated by the co-treatment with anti-anaplastic lymphoma kinase (ALK) antibodies, but not with anti-protein tyrosine phosphatase (PTP)zeta antibodies. These findings imply that ALK is involved in the PTN signaling on neural development.

Neuroprotective properties of ciliary neurotrophic factor for cultured adult rat dorsal root ganglion neurons.

We observed that recombinant ciliary neurotrophic factor (CNTF) enhanced survival and neurite outgrowth of cultured adult rat dorsal root ganglion (DRG) neurons. Among other neurotrophic factors (NGF and GDNF) and interleukin (IL)-6 cytokine members [IL-6, LIF, cardiotrophin-1, and oncostatin M (OSM)] at the same concentration (50 ng/ml), CNTF, as well as LIF and OSM, displayed high efficacy for the promotion of the number of viable neurons and neurite-bearing cells. CNTF enhanced the number of neurite-bearing cells in both small neurons (soma diameter <30 microm) and large neurons (soma diameter > or =30 microm), whereas NGF and GDNF promoted that in only small neurons. Western blot analysis revealed that CNTF induced phosphorylation of STAT3, Akt, and ERK1/2 in the neurons. Furthermore, the neurite outgrowth-promoting activity of CNTF was diminished by co-treatment with Janus kinase (JAK) 2 inhibitor, AG490; STAT3 inhibitor, STA-21; phosphatidyl inositol-3'-phosphate-kinase (PI3K) inhibitor, LY294002; and mitogen-activated protein kinase kinase (MEK) inhibitor, PD98059, in a concentration-dependent manner. Its survival-promoting activity was also affected by AG490, STA-21, and LY294002 at higher concentrations, but not by PD98059. These findings suggest the involvement of JAK2/STAT3, PI3K/Akt, and MEK/ERK signaling pathways in CNTF-induced neurite outgrowth, where the former two pathways are thought to play major roles in mediating the survival response of neurons to CNTF.