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Spinal cord injury induces significant cellular stress responses. The Heat Shock Protein 90 (HSP90) plays a pivotal role as a molecular chaperone and is crucial for protein folding, stabilization, and cellular signaling pathways. Despite its important function in stress adaptation, the specific expression patterns and functional roles of HSP90 after nerve injury remain unclear. This study aimed to elucidate the expression dynamics and functional implications of HSP90 following central nervous system (CNS) injury. Using western blotting and immunohistochemical analyses, we observed upregulation of HSP90 expression in spinal cord tissues and within injured neurons in a spinal cord contusion injury model. Additionally, HSP90 was found to enhance neurite outgrowth in primary cortical neurons cultured in vitro. Furthermore, in a glutamate-induced neuronal injury model, the expression of HSP90 was up-regulated, and overexpression of HSP90 promoted neurite re-growth in damaged neurons. Overall, our findings highlight the critical involvement of HSP90 in the neural response to injury and offer valuable insights into potential therapeutic strategies for CNS repair., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024. Published by Elsevier B.V.)

Axonal regeneration is restricted in adults and causes irreversible motor dysfunction following spinal cord injury (SCI). In contrast, neonates have prominent regenerative potential and can restore their neural function. Although the distinct cellular responses in neonates have been studied, how they contribute to neural recovery remains unclear. To assess whether the secreted molecules in neonatal SCI can enhance neural regeneration, we re-analyzed the previously performed single-nucleus RNA-seq (snRNA-seq) and focused on Asporin and Cd109, the highly expressed genes in the injured neonatal spinal cord. In the present study, we showed that both these molecules were expressed in the injured spinal cords of adults and neonates. We treated the cortical neurons with recombinant Asporin or CD109 to observe their direct effects on neurons in vitro. We demonstrated that these molecules enhance neurite outgrowth in neurons. However, these molecules did not enhance re-growth of severed axons. Our results suggest that Asporin and CD109 influence neurites at the lesion site, rather than promoting axon regeneration, to restore neural function in neonates after SCI., (Copyright © 2024 The Authors. Published by Elsevier B.V. All rights reserved.)

Recent studies have found that people with diabetes are more vulnerable to cognitive dysfunction, particularly Alzheimer's disease (AD). Previous studies revealed that Reticulon 3 (RTN3) oligomers could induce cortical neuritic dystrophy (CND) in the brains of diabetic rats. However, it is not clear how diabetes induces RTN3 aggregation. In this study, we examined in vivo and in vitro diabetes models to explore the underlying effects of RTN3-mediated neurite dystrophy. The results showed that the binding ability of ASC and RTN3 was significantly increased during diabetes- or high glucose-induced neuritic dystrophy, and ASC siRNA or an anti-inflammatory drug (CP 424174) could inhibit neuritic dystrophy in vitro. These results suggest that the ASC and RTN3 interaction is involved in diabetes-induced CND, and anti-inflammatory therapy might be an effective way to prevent and inhibit diabetes-induced CND., (Copyright © 2021 Elsevier B.V. All rights reserved.)

Background: Dementia with Lewy bodies (DLB) is one of the major neurodegenerative diseases and a clinical diagnosis is made based on the fourth consensus report on DLB. However, clinicopathological features of DLB are variable among cases., Methods: We analyzed three autopsy-proven cases of DLB (patients 1-3). Their clinical features were variable in spite of their pathological commonality. The entire hemispheric sections were stained for phosphorylated alpha-synuclein (aS), digitized, and each aS positive lesion was mapped using a virtual slide system., Results: The three patients were clinically diagnosed as having DLB. However, patient 1 exhibited amnesia and misrecognition, while patient 3 exhibited abnormal behavior in addition to dementia. Therefore, both patients 1 and 3 did not fulfill the clinical criteria of DLB, in contrast to patient 2. In spite of the clinical heterogeneity, Lewy pathology was similar in patients 1, 2, and 3. Additionally, patient 1 exhibited less frequent Lewy neurites of the amygdala and entorhinal cortex, and less frequent neurofibrillary tangles and senile plaque as compared to patient 2. On the other hand, the Lewy pathology of patient 3 extended far beyond those of patients 1 and 2, wherein the superior to middle frontal cortices, insular cortex, and lentiform nucleus were severely affected by Lewy pathology., Conclusions: Clinical features could be variable among autopsy-proven cases of DLB. Furthermore, we show that the accent of Lewy pathology differs according to the variability of the clinical symptoms. This method will provide a comprehensive strategy to analyze wide-spread aS lesions scattered throughout the entire cerebral hemisphere and help determine the corresponding pathological lesions responsible for the clinical variability of neurodegenerative disorders, including DLB., (Copyright © 2021 Elsevier B.V. All rights reserved.)

Two billion people are chronically infected with Toxoplasma gondii worldwide with unknown consequences. Important neurological diseases have been associated to the brain infection, making essential to understand the neurophysiological changes associated with the neuronal encystment. T. gondii may subvert neuronal functions modifying neurotransmitter concentration in chronically infected mice but the molecular mechanisms involved are still unclear. Parasites were observed inside neuronal cells in cultures from 24-192 hs. The rate of infection increased with time. Neurite density decreased affecting network functionality. Neuronal survival was affected and we detected the presence of cysts inside neuronal bodies and dilated portions of neurites in association with a relative increase of TH-positive neuritic area without noticeable changes in DA immunofluorescence pattern. These results advance our knowledge of the interaction between T. gondii and the neuronal network of the host., (Copyright © 2019 Elsevier B.V. All rights reserved.)

Granulovacuolar degeneration (GVD) was originally reported in Alzheimer's disease (AD) and later found in aging brains. Pathologically, GVD is thought to be associated with the development of tauopathy, but the precise mechanism remains unknown. Previous studies have suggested that GVD contains proteins associated with an inflammatory signal. In this study, we examined phosphorylated p65 (pp65), which is the activated form of a subunit of nuclear factor-kappa B (NF-κB), in the hippocampus of 21 autopsied cases, including AD, amyotrophic lateral sclerosis cases with optineurin mutation (ALS-OPTN), and a variety of other neurodegenerative disorder cases and normal controls. In all cases, GVDs were immunopositive for pp65. The density of pp65-positive GVDs statistically correlated with that of casein kinase 1 delta (CK1δ), which is known as GVD marker. pp65 was also detected in neurites in AD and ALS-OPTN. The number of neurons with pp65-immunoreactive GVD was significantly higher in the AD group than in the non-AD group. Double immunostaining showed the colocalization of CK1δ and pp65. pp65-positive GVD was found in a neuron with AT8-positive neurofibrillary tangles. Moreover, pp65 was also found in neurites that were immunostained with phosphorylated tau, phosphorylated α-synuclein, or TDP-43 (transactivation response element DNA-binding protein 43 kDa). Therefore, the activation of the NF-κB pathway may be related to the pathology of GVD formation and dementia with tauopathy, including AD and ALS-OPTN. We propose that pp65 is useful as a GVD marker, and that the NF-κB pathway could be a therapeutic target not only for AD, but for age-related neurodegenerative diseases in general., (Copyright © 2019 Elsevier B.V. All rights reserved.)

We investigated the role of the p38 mitogen-activated protein kinase (MAPK) pathway in electrical stimulation-induced neurite outgrowth of PC12 mutant cells in which nerve growth factor (NGF)-induced neurite outgrowth is impaired. When cultures of the PC12 mutant (PC12m3) cells were exposed to electrical stimulation at 100 mA for 30 min, activity of p38 MAPK increased and neurite outgrowth was greatly enhanced. The frequency of neurite outgrowth induced by electrical stimulation was approximately 10-fold greater than that of neurite outgrowth induced by NGF alone. The neurite outgrowth induced by electrical stimulation was inhibited by a specific p38 MAPK inhibitor, SB203580. The activation of p38 MAPK induces activation of the transcription factor CREB. The activation of CREB induced by electrical stimulation was inhibited by SB203580. Longer electrical stimulation of PC12m3 cells provoked cell death, which was enhanced by SB203580. These findings suggest that electrical stimulation-induced activation of the p38 MAPK/CREB pathway is responsible for the neurite outgrowth and survival of PC12m3 cells., (Copyright © 2019 Elsevier B.V. All rights reserved.)

Proteins containing an expanded polyglutamine tract tend to aggregate, leading to the neuronal damage observed in polyglutamine diseases. We recently reported that free electron laser (FEL) irradiation markedly dissociates naked polyglutamine aggregates as well as the aggregate in the 293 T cells. In the present study, we investigated whether FEL irradiation of neuron-like cells with polyglutamine aggregates would restore the cellular damage and dysfunction. The aggregated polyglutamine peptides induced neurite retraction of differentiated SH-SY5Y cells. Upon FEL irradiation, the polyglutamine aggregates in the SH-SY5Y cells were dissociated, and the shorter length of individual neurite, fewer number of neurites per cell and shorter total length of neurite by polyglutamine were inhibited. Same results were essentially obtained in PC12 cells. Moreover, when FEL irradiation was applied to undifferentiated SH-SY5Y cells, the deficits in neuron-like differentiation seen in expanded polyglutamine peptide-containing cells were also rescued. Thus, FEL irradiation restored both the damage and differentiation caused by polyglutamine in neuron-like cells., (Copyright © 2018 Elsevier B.V. All rights reserved.)

α-Synuclein shares structural homology with 14-3-3 proteins. Seven 14-3-3 protein isoforms have been identified in mammals. Among them, the 14-3-3 sigma isoform was initially considered absent in the mammalian brain. However, we previously identified immunohistochemical association of 14-3-3 sigma with Pick bodies. Because 14-3-3 isoforms other than sigma isoform have been identified in Lewy bodies, we were prompted to look for this 14-3-3 sigma-like immunoreactivity (IR) in Lewy bodies in the brainstem, cerebral cortex, and Lewy neurites in seven patients with Lewy body disease. Unexpectedly, 14-3-3 sigma-like IR was consistently found in various types of Lewy pathologies in all cases examined. Double labeling studies confirmed its colocalization with alpha-synuclein. In general, 14-3-3 proteins can trap and hold some phosphorylated proteins in the cytoplasm and they can prevent or mediate apoptosis and survival of some cells. More precisely, the 14-3-3 sigma isoform is unique in its multiple cellular functions such as facilitating cell cycle arrest in the G2 phase, positively regulating p53, and suppressing tumor growth. Although the precise role of 14-3-3 sigma in the development of Lewy pathology remains elusive, its consistent association to Lewy pathology may expand our understanding of Lewy pathogenesis., (Copyright © 2018 Elsevier B.V. All rights reserved.)

Long-term repeated application of morphine induces plasticity changes in sensitive neurons, which represents the neurobiological basis of morphine addiction. Caveolin-1 (Cav-1) is an important scaffolding protein that plays a key role in the regulation of neurostructural plasticity. However, in-depth studies focused on the role of Cav-1 in morphine-induced changes in neurostructural plasticity are lacking. The present study explored the effects of Cav-1 on the expression levels of 2 markers of neurite outgrowth, growth association protein 43 (GAP-43) and microtubule-associated protein 2 (MAP-2), during the process of morphine-induced changes in the structural plasticity. A primary mouse cerebral cortical neuron culture was established, and Cav-1 knockdown was achieved. The results showed that morphine at a concentration of 10.0μmol/L had no adverse effect on neuronal viability, but enhanced the Cav-1 and GAP-43 levels and induced the outgrowth of MAP-2-labeled neurites. The qRT-PCR data showed that 10.0μmol/L of morphine increased the Cav-1 mRNA level and promoted Cav-1 expression at the transcriptional level. Moreover, Cav-1 knockdown inhibited the morphine-induced upregulation of GAP-43 expression and the prolongation of MAP-2-labeled neurites. In conclusion, morphine was capable of inducing changes in the structural plasticity of primary cultured mouse cortical neurons, and Cav-1 played an important role in this process. Inhibition of Cav-1 expression reduced the morphine-induced increase in the neuronal growth markers GAP-43 and MAP-2. Thus, Cav-1 may be a potential molecular target for inhibiting morphine-induced neuroplasticity., (Copyright © 2017 Elsevier B.V. All rights reserved.)

The Rab family small GTPases are key players in the membrane traffic that underlies various cellular phenomena. Neurite outgrowth, which is a prerequisite for neuronal network formation, also requires membrane traffic from the cell body to the tips of neurites. Although several Rabs have been shown to promote neurite outgrowth, very little is known about Rab involvement in the negative regulation of neurite outgrowth. Here we used nerve growth factor-stimulated PC12 cells to perform siRNA-based comprehensive knockdown screenings for Rabs that negatively regulate neurite outgrowth and succeeded in identifying Rab20 as a novel negative regulator of neurite outgrowth. Our findings showed that knockdown of endogenous Rab20 in PC12 cells promoted neurite outgrowth, whereas overexpression of active Rab20 inhibited it. We also found that the presence of Gly-64 and Cys-70, both of which are conserved only in the switch II region, a putative effector binding domain, of Rab20 is required for the inhibitory effect of Rab20 on neurite outgrowth. These findings indicated that active Rab20 suppresses neurite outgrowth of PC12 cells, possibly through interaction with an unidentified effector molecule that specifically recognizes certain amino acids in the switch II region of Rab20., (Copyright © 2017 Elsevier B.V. All rights reserved.)

Factors that trigger emotional expression may be divided into two patterns according to the type of motivation, acquiring reward (pleasure) and avoiding aversion (punishment). Repeated exposure to certain external stimuli accompanied by aberrant motivation may produce psychiatric diseases such as bipolar disorder and addiction via dysregulation of the central nervous system. However, neurobiological underpinnings of such diseases have not been clarified, especially at the neuronal level. In the present study, plasma from rats undergoing intracranial self-stimulation (ICSS) produced neurite outgrowth in PC12-variant cells (PC12m3). Stimulated PC12m3 cells also exhibited heightened activity of the p38 MAPK pathway. These findings indicate that reward states lead to not only morphological changes but also increases in p38 MAPK activity at the neuronal level in the central nervous system., (Copyright © 2017 Elsevier B.V. All rights reserved.)

Bone morphogenetic protein 2 (BMP2) is a neurotrophic factor which induces the growth of midbrain dopaminergic (DA) neurons in vitro and in vivo, and its neurotrophic effects have been shown to be dependent on activation of BMP receptors (BMPRs) and Smad 1/5/8 signalling. However, the precise intracellular cascades that regulate BMP2-BMPR-Smad-signalling-induced neurite growth remain unknown. Endocytosis has been shown to regulate Smad 1/5/8 signalling and differentiation induced by BMPs. However, these studies were carried out in non-neural cells. Indeed, there are scant reports regarding the role of endocytosis in BMP-Smad signalling in neurons. To address this, and to further characterise the mechanisms regulating the neurotrophic effects of BMP2, the present study examined the role of dynamin-dependent endocytosis in BMP2-induced Smad signalling and neurite growth in the SH-SY5Y neuronal cell line. The activation, temporal kinetics and magnitude of Smad 1/5/8 signalling induced by BMP2 were significantly attenuated by dynasore-mediated inhibition of endocytosis in SH-SY5Y cells. Furthermore, BMP2-induced increases in neurite length and neurite branching in SH-SY5Y cells were significantly reduced following inhibition of dynamin-dependent endocytosis using dynasore. This study demonstrates that BMP2-induced Smad signalling and neurite growth is regulated by dynamin-dependent endocytosis in a model of human midbrain dopaminergic neurons., (Copyright © 2017 Elsevier B.V. All rights reserved.)

Immunotherapeutic approaches reducing α-synuclein deposits may provide therapeutic benefit for Dementia with Lewy Bodies (DLB). Immunization with full-length human α-synuclein (hα-Syn) protein in a Parkinson's disease mouse model decreased the accumulation of the aggregated forms of this protein in neurons and reduced neurodegeneration. To enhance the immunogenicity of candidate vaccines and to avoid the risk of autoreactive anti-hα-Syn T-helper (Th) cell responses, we generated three peptide-based epitope vaccines composed of different B-cell epitopes of hα-Syn fused with a "non-self" Th epitope from tetanus toxin (P30). Immunization of mice with these epitope vaccines produced high titers of anti-hα-Syn antibodies that bound to Lewy bodies (LBs) and Lewy neurites (LNs) in brain tissue from DLB cases and induced robust Th cell responses to P30, but not to hα-Syn. Further development of these first generation epitope vaccines may facilitate induction of anti-hα-Syn immunotherapy without producing potentially harmful autoreactive Th cell responses., (Copyright © 2013 Elsevier Ireland Ltd. All rights reserved.)

Curcumin is well known as an antioxidant and anti-inflammatory agent. In this study, we provided convincing evidence for the function of curcumin in neurite outgrowth of cortical neurons. We discovered that curcumin treatment could promote the number of processes, mean process length, and maximum process length of primary neurons, which were inhibited by reggie-1 siRNAs or extracellular signal-regulated kinase (ERK) 1/2 antagonist. Furthermore, curcumin-induced neurite growth was related to the ERK1/2 phosphorylation, which was blocked by reggie-1 knockdown. Overall, our results implied that curcumin could mediate neurite outgrowth through reggie-1 and ERK1/2 pathway., (Copyright © 2013 Elsevier Ireland Ltd. All rights reserved.)

Injuries in the mammalian central nervous system induce a variety of factors which promote or inhibit neuronal axon regeneration/sprouting. However, the inhibitory activities are much stronger, and indeed are the major obstacle to functional recovery. Chondroitin sulfate proteoglycans (CSPGs) are produced by activated glial cells, and are among the strongest inhibitors. Here, we investigated the role of the growth factor midkine (MK), which binds to CSPGs, in neuronal injury. MK expression was induced by spinal cord injury, and was mainly produced by activated astrocytes. A prolonged culture of neurons also produced MK. MK not only enhanced neurite outgrowth on the substratum coated with poly-l-lysine, but also overcame the neurite growth inhibition by the CSPG substratum. Moreover, we found that MK activated neither astrocytes nor microglia as evaluated by morphological changes and cell proliferation or nitric oxide production. These properties would be advantageous for the treatment of neuronal injuries in vivo. Therefore, we next explored the therapeutic effect of MK in a rat spinal cord injury model. MK or vehicle was administered intrathecally for 2 weeks using an osmotic pump after spinal cord contusion injury. Rats treated with MK showed significantly better functional recovery after 5 weeks. These results suggest that MK may offer a potent alternative for the treatment of neuronal injuries without activating glial cells., (Copyright © 2013 Elsevier Ireland Ltd. All rights reserved.)

Recently, 3-hydroxy-3-methyl glutaryl coenzyme A (HMG-CoA) reductase inhibitors were reported to induce neurite outgrowth in vitro. However, the mechanism underlying this effect remains unclear. Cellular prion protein (PrP(C)) is a ubiquitous glycoprotein present on the surfaces of various cells, including neurons, and is suggested to be involved in neurite outgrowth. Therefore, the present study aimed to determine whether PrP(C) mediates neurite outgrowth induced by HMG-CoA reductase inhibitors. Atorvastatin, a strong HMG-CoA reductase inhibitor, induced neurite outgrowth and increased PrP(C) levels in Neuro2a cells in a time- and dose-dependent manner. PrP(C) mRNA expression was also increased by atorvastatin. Farnesol, a non-sterol mevalonate derivative, attenuated the atorvastatin-induced neurite outgrowth and increase in PrP(C). Neuro2a cells overexpressing PrP(C) showed a remarkable enhancement of atorvastatin-induced neurite outgrowth compared with mock cells transfected with empty pCI-neo vector. These findings suggest that PrP(C) contributes, at least in part, to atorvastatin-induced neurite outgrowth. This phenomenon may be included among the mechanisms underlying decreased risk of Alzheimer's disease in patients treated with HMG-CoA reductase inhibitors., (Copyright © 2012 Elsevier Ireland Ltd. All rights reserved.)

Mutations in presenilins are the major cause of early onset familial Alzheimer disease. It has recently been argued that clinical presenilin mutations work as loss-of-function but not toxic gain-of-function. To investigate whether presenilins are involved in the regulation of the distribution of neuronal membrane lipids, we treated neuronally differentiated PC12 cells with DAPT, an inhibitor of presenilin-dependent γ-secretase, and performed lipid analyses of neuritic terminals, which is an initial site of Aβ deposition in brains, using liquid chromatography/electrospray ionization tandem mass spectrometry (LC/ESI-MS/MS) in combination with multiple reaction monitoring (MRM). With DAPT treatment, levels of sphingomyelin, phosphatidylcholine, and cholesterol remained unchanged. However, DAPT treatment increased the ganglioside levels in PC12 neuritic terminals. Together with a previous finding that accumulation of gangliosides at neuritic terminals facilitates Aβ assembly and deposition, the present data suggest that the loss-of-function of presenilins, i.e., a decrease in γ-secretase activity, has an impact on neuronal membrane architecture in a way that eventually exacerbates Alzheimer pathology., (Copyright © 2012 Elsevier Ireland Ltd. All rights reserved.)

The axons of the adult mammalian brain and spinal cord fail to regenerate after injury, and it has been suggested that Nogo-66 could prevent CNS axon repair. However, the mechanism of Nogo-66 inhibiting neurite outgrowth remains unknown. Our previous results indicated that protein kinase B (PKB) is involved in the inhibition of the neurite outgrowth by Nogo-66. Glycogen synthase kinase-3β (GSK-3β) is implicated in many processes in the nervous system, including differentiation, specification, polarity, plasticity and axon growth. In addition, GSK-3β is one of the most important molecules downstream of PKB. In the present study, we report on the role of GSK-3β signaling on Nogo-66-treated mouse neuroblastoma N2a cells. Nogo-66 reduced the phosphorylation of GSK-3β at Ser9 in N2a cells. In contrast, pretreatment with SB216763, a specific inhibitor of GSK-3β, resulted in an amelioration of neurite outgrowth by Nogo-66, compared with the Nogo-66 alone group (P<0.05). Moreover, we performed RNA interference experiments to knock down GSK-3β expression levels in N2a cells via transient transfection of shRNA plasmids. The inhibition of neurite outgrowth by Nogo-66 was subdued in shRNA cells, compared to the non-RNAi cells (P<0.05). Taken together, these data suggest that GSK-3β is involved in the inhibition by Nogo-66 of neurite outgrowth in N2a cells., (Copyright © 2011 Elsevier Ireland Ltd. All rights reserved.)

Staurosporine as an inhibitor of protein kinases can induce neuronal differentiation in PC12 cells. We investigated the role of extracellular Ca(2+) on staurosporine inducing neurite outgrowth in PC12 cells. The cells were cultured during cell differentiation in the presence of 214 nM staurosporine with 0.0-0.7 Ca(2+)mM as treatment media. We obtained the fraction of neurite-bearing cells, total neurite length and the percentage of cytotoxiciy. The results showed that decrease or increase of extracellular calcium ions resulted in correspondingly significant decrease or increase in total neurite length and cell differentiation in treated cells. With an increase of extracellular calcium concentration from 0.0 to 0.7 mM, the percentage of cytotoxicity of the PC12 cells decreased (p<0.05). Our data suggest that staurosporine uses the extracellular calcium ions to affect on neurite outgrowth., (Copyright © 2011 Elsevier Ireland Ltd. All rights reserved.)

Thyroid hormone (TH) plays an essential role in growth and differentiation of the central nervous system. Deficiency of TH during perinatal period results in abnormal brain development known as cretinism in human. We recently reported that an environmental chemical 1,2,5,6,9,10-α-hexabromocyclododecane (HBCD) suppressed TH receptor (TR)-mediated transcription. To examine the effect of HBCD on cerebellar granule cells, we used purified rat cerebellar granule cells in reaggregate culture. Low dose HBCD (10(-10)M) significantly suppressed TH-induced neurite extension of granule cell aggregate. To clarify further the mechanisms of such suppression, we added brain-derived neurotrophic factor (BDNF) into culture medium, since BDNF plays a critical role in promoting granule cell development and is regulated by TH. BDNF completely rescued HBCD-induced suppression of granule cell neurite extension in the presence of T3. These results indicate that HBCD may disrupt TH-mediated brain development at least in part due to a disruption of the T3 stimulated increase in BDNF and BDNF may possess ability to ameliorate the effect of HBCD in granule cells., (Copyright © 2011 Elsevier Ireland Ltd. All rights reserved.)

Neurite elongation is a critical process in the formation of nerve systems from neural cells. During metamorphosis, the holometabolous insect Drosophila melanogaster reorganizes its central nervous system (CNS) under the influence of the steroid molting hormone 20-hydroxyecdysone (20E). A neural cell line that responds to 20E treatment is therefore desired in order to analyze its signal transduction process. Here, we show that cells of the Drosophila neural cell line BG2-c6 extended long projections of over 30 microm in length after being stimulated with 20E. Most of these projections contained both actin filaments and microtubules. Since microtubules are structural markers of neurites, the projections were considered to be neurites. Live imaging of cells expressing GFP tagged alpha-tubulin showed that the neurites did not have a lamellipodial structure at their tips. Under an electron microscope, microtubules were found to run alongside the actin filaments in the neurite shaft but did not reach the tip, where the actin filaments were loosely bundled rather than being arranged into a meshwork as in lamellipodia. These results indicate that BG2-c6 cells project neurites without the typical growth-corn structure at their tips after 20E stimulation., ((c) 2010 Elsevier Ireland Ltd. All rights reserved.)

CLP36, a member of the alpha-actinin-associated LIM protein (ALP)/enigma protein family, plays a role in neurite outgrowth in the peripheral nervous system. However, the underlying molecular mechanisms are not known. In this study, we performed yeast two-hybrid screening of an E18 mouse whole-body cDNA library with CLP36 as the bait and isolated palladin as a CLP36-binding protein. Palladin is an actin-binding protein and it was shown to have a role in the extension of cortical neurons. A coimmunoprecipitation study showed that CLP36 and palladin formed a complex in the dorsal root ganglion (DRG). In addition, CLP36 and palladin were colocalized in the neurites and cell bodies of primary DRG neurons. Furthermore, sciatic nerve transection increased the expression of both CLP36 and palladin mRNAs in DRG neurons, with the increase in CLP36 mRNA being more prominent. This implies that CLP36 has a more specific role in nerve regeneration than palladin. Our results suggest that CLP36 may interact with palladin to influence neurite outgrowth during sciatic nerve regeneration., (Copyright 2010 Elsevier Ireland Ltd. All rights reserved.)

The effects of radiofrequency electromagnetic field (RF-EMF) exposure on neuronal phenotype maturation have been studied in two different in vitro models: murine SN56 cholinergic cell line and rat primary cortical neurons. The samples were exposed at a dose of 1W/kg at 900 MHz GSM modulated. The phenotype analysis was carried out at 48 and 72 h (24 and 48 h of SN56 cell line differentiation) or at 24, 72, 120 h (2, 4 and 6 days in vitro for cortical neurons) of exposure, on live and immunolabeled neurons, and included the morphological study of neurite emission, outgrowth and branching. Moreover, cortical neurons were studied to detect alterations in the expression pattern of cytoskeleton regulating factors, e.g. beta-thymosin, and of early genes, e.g. c-Fos and c-Jun through real-time PCR on mRNA extracted after 24h exposure to EMF. We found that RF-EMF exposure reduced the number of neurites generated by both cell systems, and this alteration correlates to increased expression of beta-thymosin mRNA.

Huntington's disease (HD) is an autosomal dominant inheritable neurodegenerative disorder caused by expansion of a polyglutamine repeat in the amino-terminal region of huntingtin. Polyglutamine expansion causes mutant huntingtin to aggregate and accumulate in the nuclei and cytoplasm of neurons. The aggregated amino-terminal fragments of mutant huntingtin are toxic to neuronal cells and may be involved in the neurodegeneration in HD patient brains. Although nuclear mutant huntingtin has been found to affect gene expression, the effect of cytoplasmic mutant huntingtin remains to be investigated. We established stably transfected mouse neuroblastoma (N2a) cells that express soluble amino-terminal fragments of huntingtin containing 20 (20Q) or 150 (150Q) glutamine repeats. In these stable cell lines, both 20Q and 150Q are diffusely distributed in the cytoplasm without aggregate formation. However, the stable 150Q cells are deficient in neurite outgrowth. Compared with wild-type N2a cells and cells stably expressing 20Q, stable 150Q cells also have decreased viability and are more susceptible to apoptotic stimulation. These findings suggest that the cytoplasmic soluble mutant huntingtin is also toxic to cells.

As recent evidence has revealed a pro-survival role for the anti-obesity hormone leptin in the nervous system, we investigated the generality of this finding on cerebellar Purkinje and granule neurons in vitro. We found that whilst leptin promoted cerebellar Purkinje neuron survival, it had no affect on cerebellar granule cells. In addition, we discovered that leptin promoted both the outgrowth of neurites from cerebellar Purkinje neurons and increased the complexity of the neurite arbor. Thus, leptin has different effects on two neighbouring populations of neurons within the cerebellum implying specificity of its actions in the central nervous system.

Ret is a receptor tyrosine kinase for the GDNF family of ligands and plays important roles during nervous system development for cell proliferation, cell migration and neurite growth. Signaling initiated from intracellular tyrosine 1062, by recruitment of several different phosphotyrosine binding (PTB) proteins (i.e. Shc, Frs2 and Dok), is important for these biological effects. By a single amino acid substitution in the PTB domain binding sequence of Ret, we have rewired the receptor such that it preferentially recruits Dok (Ret(Dok+)) with little or no remaining interactions with Shc and Frs2. Ret(Dok+) displays a sustained MAP kinase activation and a loss of Akt signaling compared to Ret(WT). We show that early events after ligand stimulation of Ret(Dok+) include massive formation of fine microspikes that are believed to be priming structures for neurite growth from the cell soma. The Ret(Dok+) receptors relocated in the membrane compartment into focal clusters at the tip of the microspikes, which was associated with Cdc42 activation. These results suggest that engagement of different adaptor proteins by Ret results in very different downstream signaling and functions within neurons and that Dok recruitment leads to a rapid receptor relocation and formation of microspikes.

Chondroitin sulfate proteoglycans elicit a selective inhibition to neurite growth from ventrotemporal (VT) but not dorsonasal (DN) retina, potentiating the bilateral routing of axons in the mouse optic chiasm. We examined whether this selective response is mediated by a difference in protein kinase C (PKC) expression. Effects of suppressing PKC activity in explant preparations of embryonic day 14 retinae with inhibitor Gö6976 or Ro-32-0432 abolished the chondroitin sulfate inhibition to the VT neurites but had no effect to the DN neurites. Whether these responses rely on a difference in expression of PKC in the growth cones was examined using antibodies against six isozymes of PKC. Among these the alpha, betaI and epsilon isozymes were expressed prominently in the retinal growth cones; whilst the betaII, delta and gamma isozymes were barely detected. Moreover, while the alpha and epsilon isozymes were abundant in the filopodial and lamellipodial processes, the betaI isozyme was restricted largely in the core region of the growth cones. Despite these subtype specific localization, there was no significant difference in expression of any of these PKC isozymes between growth cones from VT and DN retina, indicating that the selective response to chondroitin sulfates is not likely generated by a regulation of PKC expression, but by expression of surface molecules that interact with chondroitin sulfate proteoglycans.

Gial derived neurotrophic factor (GDNF) modulates neuronal cell differentiation during development and protects against neurodegeneration by preventing apoptosis at maturity. GDNF's role in tissue maintenance has generated interest in the therapeutic potential of GDNF in treating neurological disorders such as Parkinson's disease. Heparan sulfate has been shown to be essential for GDNF signaling and altering the levels of heparan sulfate promotes or inhibits GDNF functional activity. To search for other oligosaccharides capable of modulating GDNF activity as potential therapeutic molecules, we investigated the effect of acidic oligosaccharide sugar chain (AOSC) and its sulfated derivative on GDNF induced neurotrophic events by using Western-blotting, immunofluorescence cell staining, and immunoprecipitation techniques in PC12 cells expressing the GDNF receptors GFR alpha 1-Ret. AOSC significantly improved the neurite outgrowth and activated c-Ret phosphorylation in PC12-GFR alpha 1-Ret cells, but its sulfated derivative inhibited GDNF activity. Studies to understand the opposing biological effects of AOSC and its sulfated derivative on GDNF activity demonstrated that reduced GDNF binding to PC12-GFR alpha 1-Ret cell surface in the presence of the sulfated derivative likely suppressed GDNF activity as both AOSC and its sulfated derivatives had similar binding affinities to GDNF. This study illustrates the importance of oligosaccharide structure and charge on influencing GDNF activity and the potential use of oligosaccharides in modulating GDNF activity for therapeutic purposes.

Among the myriad of cellular functions played by nitric oxide in the brain, there is increasing evidence that nitric oxide might be a primary player in the program of neurogenesis and neuronal differentiation. We have recently reported that tyrosine nitration of proteins is implicated in the signaling pathway triggered by nitric oxide during NGF-induced neuronal differentiation in PC12 cells. The cytoskeleton becomes the main cellular fraction containing nitrotyrosinated proteins, and the cytoskeletal proteins alpha-tubulin and tau are two of the targets. Here, we have studied the association of nitrated proteins with the cytoskeletal fraction in differentiating PC12 cells following exposure to microtubule depolymerising treatments and found that nitration of the cytoskeleton correlates with the increased microtubule stability underlying the progression of neuronal differentiation. These results suggest a novel functional role for nitrated cytoskeletal proteins in the stabilisation of neurites occurring in differentiated neuronal cells.

Nogo-A mRNA and protein are present in the perikarya of neurons in both the intact and injured CNS. The present study focused primarily on Nogo-A protein expression in the primary somatosensory cortex of the adult rat. Coronal brain sections were probed with double-immunofluorescent labeling against Nogo-A together with NeuN, RBPC, or MAP-2 for confocal imaging. The sizes of the cell somata in pyramidal neurons and the thicknesses of neurites were measured on the captured confocal images. Nogo-A was expressed in larger pyramidal neurons and thicker neurites in layer V, but not in smaller pyramidal neurons and thinner neurites. Considering the morphological properties and the cell soma size reported in previous studies together with the present data, Nogo-A-positive neurons of layer V appear to be intrinsically bursting neurons that project axons to the subcortical regions. This suggests that intraneuronal Nogo-A may play roles in neurite growth and axonal regeneration of the corticofugal neurons, but not of columnar intrinsic neurons, in layer V of the S1 barrel cortex. Additionally, this study demonstrates a novel result, which is that layer V pyramidal neurons of the S1 barrel cortex exhibit a pattern of cell size-dependent intraneuronal Nogo-A expression.

This study was performed to determine whether early diabetes accelerates retinal neuronal cell death and inhibits neurite regeneration. Five of ten rats were rendered diabetic with streptozotocin injection. After 3 weeks of diabetes, retinas were isolated and retinal explants were cultured in serum-free media. On day 6, the number of neurites was counted in retinal explants obtained from control and diabetic rat retinas. To identify neuronal cells undergoing apoptosis, retinas were fixed, cryosections prepared, and TdT-dUTP terminal nick-end labeling (TUNEL) was performed. Furthermore, the expression of Bax was determined in the retinas by immunohistochemistry and Western blot analysis. The number of TUNEL-positive cells in the ganglion cell layer of diabetic retinas was significantly increased (144.8+/-33.6% of control, p<0.01) compared to those in non-diabetic control rats. The number of regenerating neurites in the retinal explants of diabetic rats was significantly reduced (63.2+/-25.1% of control, p<0.05). In retinal neuronal cells of the diabetic retinas, proapoptotic Bax expression determined by immunohistochemistry and Western blot analysis showed a significant increase compared to the non-diabetic control retinas (158.1+/-55.2% of control, p<0.01; 161.6+/-48.8% of control, p<0.01, respectively). The findings indicate that diabetes upregulates Bax expression, accelerates retinal neuronal cell death, and inhibits neurite regeneration in rat retinas. Thus, it is likely that Bax dependent pathways may be activated in injured neuronal cells of the diabetic retinas.

Ndrg2 is a member of the N-myc downstream-regulated genes. Thus far, two different isoforms of rat Ndrg2 protein, Ndrg2S and Ndrg2L, have been identified. Recently, we have identified rat Ndrg2 as a novel target molecule of antidepressants and ECT. The functional role of Ndrg2 in the central nervous system, however, remains unclear. In the present study, we examined the expression of endogenous Ndrg2, cellular localization of transfected Ndrg2 protein, and morphological changes resulting from overexpression of Ndrg2 in NGF-differentiated PC12 cells. Neurites began to sprout 1-2 days after exposure to NGF; subsequent neurite growth continued for 5 days. During this time, we evaluated Ndrg2 mRNA expression by real-time quantitative PCR and found that expression significantly increased in a time-dependent manner. Interestingly, V5-conjugated Ndrg2S and Ndrg2L proteins expressed in NGF-differentiated PC12 specifically localized to cell surface membranes and growth cones. Moreover, Ndrg2S and Ndrg2L overexpression promoted neurite elongation in NGF-differentiated PC12 cells. In conclusion, our findings offer novel insights into the physiological roles of Ndrg2 in the central nervous system.

Lesch-Nyhan disease (LND), caused by complete deficiency of hypoxanthine guanine phosphoribosyltransferase (HPRT), is characterized by a neurological deficit, the etiology of which is unknown. Evidence has accumulated indicating that it might be related to dysfunction of the basal ganglia with a prominent loss of striatal dopamine fibers. Guanine nucleotide depletion has been shown to occur in cells from Lesch-Nyhan patients. In this study we demonstrate that chronic guanine nucleotide depletion induced by inhibition of inosine monophosphate dehydrogenase with low levels (50 nM) of mycophenolic acid (MPA) lead human neuroblastoma cell lines to differentiate toward the neuronal phenotype. The MPA-induced morphological changes were more evident in the dopaminergic line LAN5, than in the cholinergic line IMR32. MPA-induced differentiation, unlike that induced by retinoic acid, caused a less extensive neurite outgrowth and branching (similar to that observed in cultured HPRT-deficient dopaminergic neurons) and involved up-regulation of p53, p21 and bax, and bcl-2 down-regulation without p27 protein accumulation. These results suggest that guanine nucleotide depletion following HPRT deficiency, might lead to earlier and abnormal brain development mainly affecting the basal ganglia, displaying the highest HPRT activity, and could be responsible for the specific neurobehavioral features of LND.

Sox21, a high-mobility group box transcription factor, is expressed throughout the immature neural stem/progenitor population in ventricular zone but not in the cortical plate in embryonic mouse brain and its expression is restricted to the subventricular zone in the adult brain. In undifferentiated PC12 cells, endogenous Sox21 expression is detected but its expression ceases during the nerve growth factor (NGF)-induced neuronal differentiation. Overexpression of Sox21 results in a substantial repression of NGF-induced neurite outgrowth in PC12 cells. Further, we biochemically identified a Sox21-associating protein, Y-box binding protein 1 which not only binds to Sox21 but also partially restores NGF-induced neurite outgrowth of PC12 cells inhibited by Sox21. These results suggest that Sox21 is a repressor of neuronal differentiation in the developing nervous system.

We examined neuronal and oligodendroglial nuclear inclusions and neurites in the pontine base of multiple system atrophy brains using an antibody to alpha-synuclein. Immunohistochemistry showed alpha-synuclein positive inclusions in the nuclei of some neurons and oligodendroglia. Immunoelectron microscopy showed that the labeled inclusions were composed of bundles of tightly packed straight filaments with a diameter of 10-20 nm. The filaments were similar, if not identical, in morphology and immunoreactivity, to those found in the soma of neurons and oligodendrocytes with glial cytoplasmic inclusions. In addition, similar immuno-positive filaments were found in dendrites or unmyelinated axons, but not in myelinated axons. The functional significance of these inclusions in terms of transcriptional and axonal dysfunction is unknown.

Glypican-1, a heparan sulfate proteoglycan, is expressed in various tissues including developing and postnatal central nervous system. It serves as a receptor for heparin-binding molecules such as fibroblast growth factors (FGFs). We investigated whether glypican-1 was expressed after brain injury in adult mice. In situ hybridization study showed that glypican-1 mRNA was expressed in the region surrounding necrotic tissue, and that the signal intensity peaked 7 days after the cryo-injury. In addition, both FGF-2 and amyloid precursor protein (APP) were concurrently upregulated and colocalized with glypican-1 mRNA. Since FGF-2 and APP can bind to glypican-1 in vitro, the present study suggested that their autocrine/paracrine interactions with glypican-1 may be involved in neuronal regeneration and/or neurite-outgrowth inhibition after brain injury.

In the present study, the effect of KCl-induced calcium influx on PC12 cell differentiation was examined. We show that KCl depolarization potentiates the effect of forskolin-induced neurite outgrowth of PC12 cells. The effects of KCl and forskolin were mediated via the protein kinase A (PKA) and the extracellular signal-regulated kinase (ERK) signaling pathways, since addition of the ERK kinase (MEK1) inhibitor PD98059 and the PKA inhibitor H89 inhibits neurite outgrowth. KCl depolarization and forskolin synergistically activate the ERK signaling pathway, but whereas KCl-mediated ERK activation depends on both PKA and MEK1, forskolin activates ERK in a PKA-independent manner. Finally, we find that KCl depolarization and forskolin both induce nuclear ERK2 translocation via a PKA- and MEK1-dependent pathway. The results demonstrate that PKA and ERK play a key role in KCl- and forskolin-induced neuronal differentiation by integration of signals from both pathways.

There is an accumulation of evidence implicating a role for vitamin D(3) in the developing brain. The receptor for this seco-steroid is expressed in both neurons and glial cells, it induces nerve growth factor (NGF) and it is a potent inhibitor of mitosis and promoter of differentiation in numerous cells. We have therefore assessed the direct effect of vitamin D(3) on mitosis, neurite outgrowth, as well as NGF production as a possible mediator of those effects, in developing neurons. Using cultured embryonic hippocampal cells and explants we found the addition of vitamin D(3) significantly decreases the percentage of cultured hippocampal cells undergoing mitosis in conjunction with increases in both neurite outgrowth and NGF production. The role of vitamin D(3) during brain development warrants closer scrutiny.

Organotypic hippocampal slice cultures prepared from newborn rats were maintained in vitro for 9 days. Cultures were then exposed to 30 min of combined oxygen-glucose deprivation (OGD). After OGD, the area covered by neurites was decreased. The dead cells of hippocampal slices in the ischemia group were 40.4% at day 3 and 41.6% at day 7 after OGD. The ultrastructure of the CA1 region of the slices was seriously damaged. While hippocampal slices were cultured in combination with bone marrow stromal cells (MSCs), the average area covered by neurites was comparatively increased. The dead cells were only 25.2% at day 3 and 27.1% at day 7 after coculture. The damage of the ultrastructure of the CA1 region in the coculture group was reduced significantly. Thus, in an in vitro model of simulated ischemia, MSCs can promote the outgrowth of neurites from hippocampal slices and alleviate cell damage. The neuroprotective effect might be mediated through diffusible neurotrophic factors secreted from MSCs.

In traumatic brain injury, the brain is subjected to mechanical shear and varying degrees of hypoxia/ischemia. To compare effects of stretch injury, hypoxia, and the combination of both insults on neurons, mixed neuronal and astrocytic cultures were established from day 17 fetal rat brains. On days 17-19 in vitro, cultures were subjected to stretch injury or hypoxia of varying degrees, alone and in combination. Cultures were assayed for lactate dehydrogenase release and Trypan Blue uptake. Hypoxia or Stretch injury alone induced a graded response (P<0.05) on both assays. Stretch+Hypoxia (4 or 6 h) resulted in significantly greater injury as compared with controls (P<0.05), and as compared with either isolated Stretch or Hypoxia (1, 2, 4 or 6 h) alone (P<0.05).

The synapsin family consists of three neuronal-specific phosphoproteins associated with dynamic reorganization of the neuronal cytoskeleton. Synapsin I and II are implicated in axonal and synaptic differentiation, formation and maintenance, whereas the function of synapsin III is not as well defined. We report a significant transcriptional upregulation of all three synapsins (synapsin I, 2.1-fold; synapsin II, 2.6-fold; and synapsin III, 5.5-fold) by retinoic acid-induced differentiation of NTera-2cl.D1 cells, a human paradigm for neuronal differentiation. The observed stronger regulation of synapsin III might be due to still active neurite elongation and a rather early state of presynaptic maturation at the time-point investigated, as synapsin III was previously found to be highly enriched in growth cones and during early synaptic development.

We used Northern blot analysis to measure SCG10 mRNA expression in various areas of the monkey cerebral cortex during postnatal development. In all areas, SCG10 mRNA expression was highest immediately after birth, decreased sharply until postnatal day 70 (P70) and then decreased slowly until the adult stage. Expression levels were similar in all areas at all time points during development, including the adult stage. These findings confirm that axonal growth in the monkey cerebral cortex is most vigorous before or during the perinatal period, progresses similarly in all areas until P70, and continues at lower levels during late development.

The nerve growth factor (NGF)-potentiating effect of K vitamins on PC12D cells was investigated. Treatment of PC12D cells with vitamin K(1) or K(2) in the presence of NGF significantly enhanced the proportion of neurite-bearing cells and acetylcholinesterase activity compared with NGF treatment alone. The K vitamins-enhanced neurite outgrowth on PC12D cells was completely blocked by a protein kinase A (PKA) inhibitor or mitogen-activated protein kinase (MAPK) kinase inhibitor PD98059, whereas a protein kinase C inhibitor chelerythrine chloride did not significantly inhibit the enhancing effect of the K vitamins. These results suggest that the K vitamins enhance neurite outgrowth via the activation of PKA and MAPK-mediated signaling pathways in PC12D cells.

The impairment of neuronal differentiation for long-term opioid drug-exposed infants is a serious problem and there are several speculated mechanisms. We have previously reported that altering the endogenous delta opioid receptor by long-term interaction with its antagonist augmented the nerve growth factor (NGF)-induced neuronal differentiation of rat pheochromocytoma PC12 cells. In this study using subtype-specific antagonist, we present data showing further that type 2 delta opioid receptor (delta(2)-DOR) is the receptor on the PC12 cells participating in the progression of neuronal differentiation upon NGF-stimulation. Unlike the delta(2)-DOR, alteration of type 1 DOR (delta(1)-DOR) activity by delta(1)-DOR-specific antagonist appeared to be toxic for the PC12 cells. The different influence of the subtypes of delta opioid receptors in the neuronal differentiation of the PC12 cells suggests that each subtype of opioid receptor may trigger different biological activities in vivo.

Fibronectin appears to be present in Senile Plaques of Alzheimer's disease brains. These senile or neuritic plaques are surrounded by dystrophic neurites, activated microglia and reactive astrocytes. The purpose of this work was to establish if a direct correlation exists between the production of Fibronectin (FN) by astrocytes and the presence of amyloid, analysing the modification of this protein produced after the treatment of cultured astrocytes with amyloid peptide (25-35). Our data showed that the addition of previously polymerised A beta-peptide to cultured astrocytes induced a marked increase in FN immunoreactivity that is in part dependent on phosphatases 2A or phosphatase 1, since was partially inhibited by okadaic acid. The increased amount of FN did not appear to be associated to any specific single isoform of which are mainly present in the rat brain. Our data suggest that in vivo FN accumulated in senile plaques may be the result, at least in part, of the response of reactive astrocyte to the presence of amyloid peptide. The importance of FN up-regulation in vivo, as part of a 'positive' response of the astrocytes to produce molecules that favours neurite outgrowth, is discussed.

The effects of aluminum(III) on microtubular meshwork have been investigated using cultured murine neuroblastoma cells grown in a medium containing aluminum lactate at defined metal concentrations (10-20 microM). A role of aluminum(III) in promoting neuronal plasticity events is suggested. These events including sprouting and neurite outgrowth are associated with an increased tyrosine-tubulin (Tyr-Tub) expression, which can be due to the enhanced needs of recently formed, highly dynamic microtubules typical of neuronal plasticity. After 48 and 72 h aluminum exposure, an upregulation of Tyr-Tub expression is detected and this is concentration-dependent. A high amount of Tyr-Tub is observed also in non-treated cells, although later than in aluminum-exposed cells. Thus, it is possible that aluminum(III) accelerates neuronal plasticity events, for which Tyr-Tub is confirmed to be a useful marker.

Myelin associated glycoprotein (MAG) has growth promoting effect on mouse cerebellar neurons. In the present study, we examined which isoform of MAG has the effect. cDNA for L-MAG and S-MAG was stably transfected into BALB/c 3T3 cells, on which cerebellar neurons were cultured. The neurons were stained with antibody against microtubule-associated protein-2. Neurites of the neurons cultured on cells expressing L-MAG extended significantly further than those cultured on cells expressing S-MAG or on control cells. Therefore, intracellular domain of MAG may have the potential to affect MAG-neurite interaction.

Lumbar spinal cord explants, harvested from neonatal rat pups aged between postnatal day 0 (P0) and P7, were cultured for a period of 48 h in the chemically defined medium R(12) [17] (Romijn, H.J., van-Huijen, F., Wolters, P.S., Neurosci Biobehav Rev, 8 (1984) 301-334), embedded in a collagen matrix. The outgrowth into the surrounding matrix was quantified. Age-matched cortical explants were used as controls. Despite adaptations of the culture protocol, outgrowth remained variable. Statistical analysis demonstrated a clear relation between the age of the explant (at the time of explantation) and the number of neurites in the corona surrounding the explant. The number of outgrowing neurites decreased sharply with age. The average number of neurites per explant obeyed to the expression log(N)= -0.652 A+17 (N: the number of neurites per explant; A: the age expressed in gestational days; A epsilon [G23-G30]; G23 signifying gestational day 23, or P0). The observed age-related decrease of outgrowth could not be explained by progressive myelination of the spinal cord white matter, nor by the absence of trophic support from muscle, but may be related to a progressive inability of the spinal neurites to interact with collagen.

The generation of fully differentiated post-mitotic human neuronal cells from stem cells (human teratocarcinoma (hNT2) cells) might enable the development of a co-culture model of human neurons with human Schwann cells (SCs). This co-culture model is an important tool to study formation of myelin sheaths. However, the thin process of the post-mitotic human neuronal cells formed under known culture conditions do not provide a good substrate for human SCs to start myelination. We optimized the culture conditions of these cells to obtain axons with a larger diameter. Western blotting and immunofluorescence studies were performed to confirm the neuronal status of the cells and diameter of the processes. In this study, we show that addition of cAMP-inducing factors to hNT2 cells resulted in rapid morphological changes including the development of processes with a larger diameter.