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Background: Research into the pathophysiology of bipolar disorder (BD) is limited by the inability to examine brain cellular processes in subjects with the illness., Methods: Endoscopic biopsy was performed in subjects with bipolar I disorder to establish olfactory neural progenitor (ONP) cell lines. Olfactory function was assessed prebiopsy and postbiopsy using the University of Pennsylvania Smell Identification Test (UPSIT). Cells were characterized to determine their lineage., Results: There were no significant complications associated with the biopsy procedure, including olfaction. Outpatient olfactory neuroepithelial biopsy yielded ONP cells in three out of 13 biopsy attempts (23.1%). ONPs were positive for neuron-specific proteins (β-tubulin III, nestin, hexaribonucleotide binding protein-3, and peripherin) and glia-specific proteins (glial fibrillary acidic protein and myelin basic protein)., Conclusions: ONP cells can be obtained safely from awake outpatients and are potentially useful for pathophysiological studies of bipolar illness and perhaps other neuropsychiatric conditions. Such cells allow for the investigation of potential pathological cellular processes without the confounding factors of genetic manipulation, which is required for induced pluripotent cells., Competing Interests: DECLARATION OF CONFLICTING INTERESTS: The author(s) declared the following potential conflicts of interest with respect to the research, authorship, and/or publication of this article: Drs. CL and FJR have a financial interest in RhinoCyte™ Inc., which owns the cells. However, mechanisms have been put into places that prevent them from directly benefiting from this work. Dr. RSE receives research funding from the National Institutes of Health, the state of Kentucky, Merck, Alexza, Janssen, and Psychnostics; and he is also a speaker for Allergan, Lundbeck, Merck, Otsuka, Sunovion, and Takeda. None of the other others have relevant conflicts of interest.

Objectives: Disturbed ion homeostasis and apoptosis have been implicated in the pathophysiology of bipolar disorder (BD). TRPM2, a nonselective cation channel, is involved in apoptosis and is possibly linked with BD. In this study, monensin, a sodium ionophore, was used to model the increase [Na(+)](in) and [Ca(2+)](in) seen in BD patients., Methods: Human olfactory neuroepithelial-derived progenitors (ONP), which possess neuronal markers, were utilized to investigate the effects of monensin on apoptosis and the response of TRPM2, and the effects of lithium on the cellular response to monensin. Monensin treatment for 6 h activated caspase-3, -7 and poly(ADP-ribose) polymerase (PARP), inducing apoptosis., Results: [Na(+)](in) increased to twice the basal level and reached steady state after 2 h of 10(-6) M monensin treatment, while [Ca(2+)](in) rose after 6 h of the treatment. Monensin treatment for 24 h decreased expression of the long form of TRPM2, and increased expression of the short form. Lithium (1 mM) pretreatment reduced the [Na(+)](in) and [Ca(2+)](in) elevation caused by monensin, down-regulated the levels of caspase-3, -7 and PARP, and reduced expression of TRPM2., Conclusions: Our findings suggest that the elevation of [Na(+)](in) and [Ca(2+)](in) induced ONP apoptosis and altered the expression of TRPM2. Lithium pretreatment attenuated the apoptosis induced by ionic stress.

Low levels of brain-derived neurotrophic factor (BDNF) peptide are linked to the pathophysiology of mood disorders. Several single-nucleotide polymorphisms (SNPs) across the BDNF gene (BDNF) have been associated with bipolar illness. Since both elevated intracellular sodium and apoptosis are believed to contribute to cellular dysfunction in bipolar disorder, it is important to determine the effect of exogenous BDNF on apoptosis induced by the high levels of intracellular sodium seen in ill bipolar patients. Human olfactory neuroepithelial progenitor cells were treated with monensin, a sodium ionophore that increases intracellular sodium and leads to apoptosis. Apoptosis was quantified with enzyme-linked immunosorbent assay (ELISA) for mono- and oligonucleosomes. Elevation of intracellular sodium concentration by monensin induced apoptosis. BDNF 100ng/mL pretreatment or co-treatment attenuated the monensin-induced apoptosis. Pretreatment with BDNF for 24h reduced monensin-induced apoptosis by 93%. Co-treatment of BDNF and monensin increased intracellular sodium concentration and reduced apoptosis by 66%. Monensin for 24h models a process that is believed to occur during ill phases of bipolar illness. Treatment with BDNF greatly attenuates or prevents monensin-induced apoptosis. The functional consequences of BDNF SNPs, known to be associated with bipolar illness, need to be examined., (Copyright 2009 Elsevier Ltd. All rights reserved.)

We investigated the effects of engrafted human adult olfactory neuroepithelial neurosphere forming cells (NSFCs) on regeneration and reinnervation of rubrospinal tract (RST) axons and locomotor recovery following partial cervical hemisection that completely ablated the RST. Weekly behavioral testing showed greater functional recovery of forelimb use during the 12 weeks after NSFCs engraftment than in the control rats. Anterograde tracing with biotinylated dextran amine (BDA) confirmed the presence of RST axons within the white matter 4-8 segments caudal to the grafts. Both immunofluorescence and immunoelectron microscopy revealed the BDA-labeled RST axonal terminals reestablished synaptic connections with motoneurons in the ventral horn of the distal cervical spinal cord. Further study of forelimb functional recovery in NSFCs-engrafted subgroups considered the effects of a second dorsolateral funiculotomy, irreversibly destroying the recovery, and the injection of muscimol, blocking RST neuronal activity reversibly. These results highlight the unique potential of human olfactory neuroepithelial-derived progenitors as an autologous cell source for spinal cord repair.

Stem cells from fetal and adult central nervous system have been isolated and characterized, providing populations for potential replacement therapy for traumatic injury repair and neurodegenerative diseases. The regenerative capacity of the olfactory system has attracted scientific interest. Studies focusing on animal and human olfactory bulb ensheathing cells (OECs) have heightened the expectations that OECs can enhance axonal regeneration and repair demyelinating diseases. Harvest of OECs from the olfactory bulb requires highly invasive surgery, which is a major obstacle. In contrast, olfactory epithelium (OE) has a unique regenerative capacity and is readily accessible from its location in the nasal cavity, allowing for harvest without lasting damage to the donor. Adult OE contains progenitors responsible for the normal life-long continuous replacement of neurons and supporting cells. Culture techniques have been established for human OE that generate populations of mitotically active neural progenitors that form neurospheres (Roisen et al., 2001; Winstead et al., 2005). The potential application of this technology includes autologous transplantation where minimal donor material can be isolated, expanded ex vivo, and lineage restricted to a desired phenotype prior to/or after re-implantation. Furthermore, these strategies circumvent the ethical issues that arise with embryonic or fetal tissues. The long term goal is to develop procedures through which a victim of a spinal cord injury or neurodegenerative condition would serve as a source of progenitors for his/her own regenerative grafts, avoiding the need for immunosuppression and ethical controversy. In addition, these cells can provide populations for pharmacological and/or diagnostic evaluation.

Neurosphere forming cells (NSFCs) have been established from cultures of adult olfactory neuroepithelium obtained from patients and cadavers as described previously. They remained undifferentiated in serum or defined media with or without neurotrophic factors. Many factors affect the differentiation of stem cells along a neuronal pathway. Retinoic acid (RA), forskolin (FN), and sonic hedgehog (Shh) have been reported to act as growth promoters during neurogenesis of embryonic CNS in vivo. The effect of RA, FN, and Shh on NSFCs' neuronal lineage restriction has not been described. The application of RA, FN, and Shh to NSFCs induced the expression of motoneuronal transcription factors, tyrosine hydroxylase, an indicator of dopamine production, and neurite formation. These studies further heighten the potential for using olfactory neuroepithelial progenitors for future autologous cell replacement strategies in neurodegenerative conditions and trauma as well as for use in diagnostic evaluation.

Neurosphereforming cell (NSFC) lines have been established from cultures of human adult olfactory neuroepithelium. Few of these cells ever express mature neuronal or glial markers in minimal essential medium supplemented with 10% fetal bovine serum or defined medium. However, these neural progenitors have the potential to differentiate along glial or neuronal lineages. To evaluate the potential of NSFCs to form motoneurons, transcription factors Olig2, Ngn2, and HB9 were introduced into NSFCs to determine if their expression is sufficient for motoneuron specification and differentiation, as has been shown in the early development of the avian and murine central nervous systems in vivo. NSFCs transfected with Olig2, Ngn2, and HB9 alone exhibited no phenotypic lineage restriction. In contrast, simultaneous transfection of Ngn2 and HB9 cDNA increased the expression of Isl1/2, a motoneuron marker, when the cells were maintained in medium supplemented with retinoic acid, forskolin, and sonic hedgehog. Furthermore, a population of Olig2-expressing NSFCs also expressed Ngn2. Cotransfection of NSFCs with Olig2 and HB9, but not Olig2 and Ngn2, increased Isl1/2 expression. Coculture of NSFCs trans-fected with Ngn2-HB92 or Olig2 and HB9 with purified chicken skeletal muscle demonstrated frequent contacts that resembled neuromuscular junctions. These studies demonstrate that transcription factors governing the early development of chick and mouse motoneuron formation are able to drive human adult olfactory neuroepithelial progenitors to differentiate into motoneurons in vitro. Our long-term goal is to develop cell populations for future studies of the therapeutic utility of these olfactory-derived NSFCs for autologous cell replacement strategies for central nervous system trauma and neurodegenerative diseases.

Previously, our lab reported the isolation of patient-specific neurosphere-forming progenitor lines from human adult olfactory epithelium from cadavers as well as patients undergoing nasal sinus surgery. RT-PCR and ELISA demonstrated that the neurosphere-forming cells (NSFCs) produced BDNF. Since rubrospinal tract (RST) neurons have been shown to respond to exogenous BNDF, it was hypothesized that if the NSFCs remained viable following engraftment into traumatized spinal cord, they would rescue axotomized RS neurons from retrograde cell atrophy and promote functional recovery. One week after a partial cervical hemisection, GFP-labeled NSFCs suspended in Matrigel matrix or Matrigel matrix alone was injected into the lesion site. GFP-labeled cells survived up to 12 weeks in the lesion cavity or migrated within the ipsilateral white matter; the apparent number and mean somal area of fluorogold (FG)-labeled axotomized RST neurons were greater in the NSFC-engrafted rats than in lesion controls. Twelve weeks after engraftment, retrograde tracing with FG revealed that some RST neurons regenerated axons 4-5 segments caudal to the engraftment site; anterograde tracing with biotinylated dextran amine confirmed regeneration of RST axons through the transplants within the white matter for 3-6 segments caudal to the grafts. A few RST axons terminated in gray matter close to motoneurons. Matrix alone did not elicit regeneration. Behavioral analysis revealed that NSFC-engrafted rats displayed better performance during spontaneous vertical exploration and horizontal rope walking than lesion Matrigel only controls 11 weeks post transplantation. These results emphasize the unique potential of human olfactory neuroepithelial-derived progenitors as an autologous source of stem cells for spinal cord repair.

Olfactory epithelium (OE) contains a population of progenitors responsible for its life-long regenerative capacity. Procedures for the isolation of these progenitors have been established [F.J. Roisen, K.M. Klueber, C.L. Lu, L.M. Hatcher, A. Dozier, C.B. Shields, Adult human olfactory stem cells, Brain Res., 890 (2001) 11-12.] and over 40 patient-specific cell lines from adult postmortem OE and endoscopic biopsy from patients undergoing nasal sinus surgery have been obtained. As these cells emerged in primary cultures, they formed neurospheres (NSFCs). The purpose of the present study was to further characterize these adult human olfactory-derived progenitors. Subcultures of the NSFCs have been passaged nearly 200 times, with a mitotic cycle of 18-20 h. Telomerase activity remains in stem cells; therefore, ELISA was employed to determine the telomerase activity of different lines and passages. Since progenitors undergo low levels of apoptosis, the levels of apoptosis were also examined in these populations. The levels of telomerase and apoptotic activity in 12 NSFC lines remained relatively constant irrespective of donor age, culture duration, or sex. To further study the apoptotic characteristics of the NSFCs, nine different caspases (cysteine proteases) known to be critical in apoptosis were evaluated using gene-microarrays comparing cells from a single line at passages 14, 88, and 183. No increases were found in caspase activity in all passages studied. ELISA confirmed the absence of caspase activity over the entire range of passages. This study further suggests that NSFCs can be obtained and used from patients, irrespective of age, sex, or time in culture without altered viability expanding the potential utility of these cells for autologous transplantation and possible diagnostic testing.

Neurosphere-forming cell (NSFC) lines have been derived from cultures of adult olfactory neuroepithelium obtained from patients and cadavers. These progenitors remain undifferentiated when maintained in minimal essential medium with 10% fetal bovine serum, but have the potential to differentiate along glial or neuronal lineages. However, few of these cells ever express mature neuronal or glial markers in defined medium. To evaluate the potential of NSFCs to form oligodendrocytes, two transcription factors, Olig2 and Nkx2.2, were introduced into NSFCs to determine whether their expression is sufficient for oligodendrocyte differentiation, as has been shown in the embryonic avian and murine central nervous systems in vivo. NSFCs transfected with Olig2 or Nkx2.2 alone exhibited no phenotypic lineage restriction. In contrast, simultaneous transfection of Olig2 and Nkx2.2 cDNA produced characteristic oligodendrocyte morphology and antigenicity, including myelin basic protein (MBP). Furthermore, a population of Olig2-expressing NSFCs also expressed Sox10. Cotransfection of NSFCs with Nkx2.2 and Sox10, but not Olig2 and Sox10, produced a MBP(+) oligodendrocytic phenotype. Coculture of NSFCs transfected with Olig2 and Nkx2.2 or Nkx2.2 and Sox10 with purified sensory neurons, demonstrated frequent contacts between NSFC processes and axons, including the early stages of ensheathment. These studies demonstrate transcription factors governing early development of chick and mouse oligodendrocyte formation, also apply to human progenitors isolated from adult olfactory neuroepithelium. Our long-term goal is to develop cell populations for future studies used to determine the therapeutic utility of these olfactory-derived NSFCs for autologous transplantation into donors with central nervous system trauma or neurodegenerative diseases.

Background: The adult central nervous system contains progenitor cells; however, invasive surgery is required for their harvest. Olfactory neuroepithelium (ONe) has attracted attention because it is extracranial and contains progenitor cells that account for its regenerative capacity. Olfactory progenitor cells have been cultured from postmortem ONe. Our aim was to determine if olfactory progenitors could be obtained via biopsy from patients in a feasible, effective, and safe manner., Methods: Endoscopic biopsy was performed on individuals undergoing sinus surgery (n = 42). Olfactory function was assessed pre- and postoperatively. Specimens were cultured under conditions for olfactory progenitor cell development., Results: Progenitor cells emerged in cultures from 50% of our patients. The superior turbinate, biopsied with cutting punch forceps, gave the highest yield. No adverse impact on olfaction or complications with the biopsy were observed., Conclusion: Endoscopic biopsy of ONe for obtaining olfactory progenitor cells from living donors is feasible, effective, and safe.

Neurosphere-forming cells (NSFCs) derived from primary cultures of adult human olfactory epithelium were established in minimum essential medium (MEM) with Hanks balanced salts and 10% heat-inactivated fetal bovine serum (FBS). A totally defined medium (DM) was employed to examine their proliferation, lineage restriction and differentiation. DMEM/F12 (DF) was found to support NSFCs and served as the base medium for this study. NSFCs were adapted to the DM through serial serum reductions at successive feedings. NSFCs in DF supplemented with N2, B27 or insulin attained saturation density and formed extensive processes. Immunolocalization of lineage specific markers [i.e., nestin, beta-tubulin III, peripherin, neural cell adhesion molecule, A2B5, O4, microtubule-associated-protein-2 (MAP2) and glial fibrillary acidic protein], as well as 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromide and ornithine decarboxylase assays were employed to characterize the NSFCs. The effects of trophic factors including epidermal growth factor (EGF), nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF), ciliary neurotrophic factor (CNTF), neurotrophic factors (NT-3), and basic fibroblast growth factor (bFGF) were evaluated. With the reduction of serum and the addition N2, B27, and other nutrients, there was a change in lineage restriction including an increase the expression of A2B5 and other glial markers as well as the expression of mature neuronal markers with a simultaneous reduction of nestin reactivity. NSFCs proliferated and maintained their pluripotency for over a year in the DM. Further studies will determine the utility of NSFCs for cell replacement therapy.

Microglia are thought to play a key role in the development and regeneration of the central nervous system although the mechanisms regulating their presence and activity are not fully understood. Substantial evidence suggests that members of the neurotrophin family such as nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF), and neurotrophin-3 and -4 (NT-3/4) have a dramatic effect on both neurons and perineuronal cells. This study employed two murine microglial lines, BV-2 and N9, to examine the action of these neurotrophins on the mitotic activity and survival of microglia in vitro. Neurotrophins were incorporated into the media at the time of plating and cell number and levels of mitochondrial dehydrogenase activity (MTT) were determined at various time points in vitro. NGF increased cell number and MTT levels of both cell lines in a dose-dependent manner. BV-2 was more sensitive to NGF than N9. Similar responses were elicited by BDNF, although the sensitivity of each cell line was different than that found for NGF. NT-3 and NT-4 had no effect on cell proliferation. However, NT-4 had an effect on the survival of BV-2 and N9 cells. The response of these cells to neurotrophins was blocked by K252a, a tyrosine kinase inhibitor, suggesting that actions of neurotrophins were mediated by high-affinity tyrosine kinase receptors (Trk). Immunolocalization studies revealed positive Trk (pan) reactivity in the above cell lines and in primary microglia, but an absence of the low-affinity p75 neurotrophin receptor. Western blot analysis supported the above observations. These studies suggest that in addition to their neurotrophic actions, NGF and BDNF may also regulate microglial dynamics, thereby influencing the surrounding milieu during neuronal regeneration.

The location of stem cells within the adult CNS makes them impractical for surgical removal and autologous transplantation. Their limited availability and histocompatibility issues further restrict their use. In contrast, olfactory neuroepithelium (ONe) located in the nasal passageways has a continuous regenerative capability and can be biopsied readily. To investigate the potential of human ONe to provide viable populations of pluripotent cells, ONe was harvested from cadavers 6-18 h postmortem, dissociated, plated and fed every 3-4 days. Heterogeneous populations of neurons, glia, and epithelia were identified with lineage-specific markers. After several weeks, 5-10% of the cultures produced a population of rapidly dividing cells, which in turn, produced neurospheres containing at least two subpopulations based on neuronal and glial specific antigens. Most contained one or more neuronal markers; a few were positive for A2B5 and/or GFAP. To determine if growth modulators would affect the neurosphere forming cells, they were exposed to dibutyryl-cAMP. The nucleotide reduced cell division and increased process formation. Although the cells had been passaged more than 70 times, their viability remained constant as shown by the MTT viability index. Donor age or sex were not limiting factors, because neurospheres have been established from cadavers of both sexes from 50 to 95 years old at time of death. The ex vivo expansion of these cells will provide a patient-specific population of cells for immunological, genetic and pharmacological evaluation. Our long-term goal is to determine the utility of these cells to facilitate CNS repair.

To investigate mechanisms of neurite outgrowth, murine Neuro-2a neuroblastoma cells were exposed to ganglioside GM1 in the presence or absence of specific protein kinase inhibitors. Isoquinolinesulfonamide (H-89), an inhibitor of cyclic AMP dependent protein kinase A (PKA), and bisindolylmaleimide I (BIM), which inhibits protein kinase C, each stimulated neurite outgrowth in a dose-dependent manner in the absence of exogenous GM1. Minimally effective (threshold) concentrations of H-89 or BIM potentiated outgrowth when they were used in combination with GM1. To search for a shared component in the mechanisms of GM1, H-89 and BIM, phosphorylation of ERK1/2 was examined. Inhibition of the activation of extracellular signal regulated kinases (ERK1/2) by U0126, prevented neuritogenesis of Neuro-2a by all the three agents. Pretreatment of serum-depleted Neuro-2a cultures with GM1 or BIM enhanced ERK1/2 phosphorylation when the serum level was restored to 10%. In contrast, H-89 did not alter the serum-mediated response. In cells exposed to GM1 or BIM without additional serum, a transitory decrease in ERK phosphorylation occurred. These data suggest that GM1 influences two neuritogenic pathways, one modulated by PKC and the other regulated by PKA. Therefore, GM1 may have the potential to stimulate alternate pathways resulting in outgrowth.

Olfactory epithelium (OE) is unique in the adult nervous system for it produces new neurons throughout life. The cultured cells of adult mouse OE in vitro were studied by histochemistry and immunocytochemistry techniques, and the results showed that they were neurons as up to 50% of the bipolar cells were immunopositive to NF, NSE, MAP2, OMP and tau. The effects of basic fibroblast growth factor (bFGF) and brain-derived neurotrophic factor (BDNF) in OE medium with different concentration serum on OE cell number and development of cell processes were also observed. The results showed that the combination of bFGF and BDNF added to 10% and 5% serum OE medium increased the bipolar cell number and percentage, stimulated the process outgrowth of bipolar cells, and decreased the numbers of giant cells and fusiform cells. The effects of the combination of bFGF and BDNF in the 10% serum OE medium were more significant than those of bFGF and BDNF applied alone; and the effects of neurotrophic factors in the 10% serum OE medium were more significant than those observed in the 5% serum OE medium.

The ganglioside GM1 is a glycosphingolipid which enhances process formation of several neuronal lines and potentiates some growth factor-mediated responses. Previously we have shown that 24 h exposure of Neuro 2a cells to GM1 mobilized the neuron-specific microtubule-associated protein, MAP2, away from microtubule-rich areas to areas of neurite sprouting where MAP2 was more closely associated with the subcortical actin network. To examine the role of GM1 in fostering the shift of the association of MAP2 from tubulin to actin, NIH 3T3 cells were co-transfected with pHook-1, which expresses a surface antigen, and a construct expressing MAP2. Transfected cells were selected with magnetic beads coated with a hapten that binds to the expressed surface antigen and treated with 150 microg/ml GM1 for 18-24 h. Actin and MAP2 or tubulin and MAP2 were immunolocalized and examined with confocal microscopy. MAP2 was found throughout the cytoplasm as well as associated with actin filaments. As observed previously with Neuro 2a, GM1 treatment of transfected fibroblasts redistributed the MAP2 away from direct association with microtubules to peripheral areas where the association of MAP2 with actin was enhanced. GM1 did not induce neurite-like processes in MAP2-transfected cells. Treatment with cytochalasin B, which is reported to result in process formation, also did not induce neurite-like processes. These studies suggest that GM1's ability to mobilize MAP2 and promote its association with actin is not restricted to neurons.

Objective: Filamentous actin (F-actin) plays a central role in maintaining endothelial barrier function. Thrombin and histamine, two inflammatory mediators that increase endothelial permeability, can alter F-actin production and distribution. In this study, we use a newly developed image analysis technique to show that these two inflammatory mediators differentially alter F-actin structure., Methods: Human umbilical vein endothelial cells were grown to confluence and treated with either histamine (1 microM), thrombin (1 microM) or the agonist's vehicle. The endothelium was stained with BODIPY-phallodin, and digitized images were taken of the treated cells. The digitized images of individual human umbilical vein endothelial cells (HUVEC) were imported into a F-actin image analysis program (FAAP) and converted to layers, each one pixel thick. The program then determined the mean gray level (which corresponded to the amount of F-actin) in each layer starting from the outside of the cell (layer 1) and progressing in one pixel layer increments towards the center of the cell (layer 32)., Results: Both inflammatory mediators increased endothelial F-actin production, however, the distribution of the actin was different. Thrombin increased the presence of stress fibers, while also decreasing peripheral banding actin. In contrast, histamine had no effect on peripheral actin compared to control, but did increase the presence of F-actin stress fibers., Conclusions: These results establish that thrombin and histamine alter endothelial F-actin production in different locations within the cell, which can be quantified using an image analysis program.

Olfactory receptor neurons (ORNs) are unique because they can be replaced by stem cells throughout life. Previous studies have demonstrated that adult mouse olfactory epithelium (OE) injured by exposure to ZnSO4 through nasal irrigation can stimulate stem cell mitotic activity in situ, which continues when placed in culture. We report on an improved ZnSO4 delivery method, mist inhalation, which produces more consistent and greater yields of OE cells. Cultures established following this method contained bipolar, nest, fusiform, and giant cells. The bipolar cells usually underwent asymmetric process development. Some bipolar cells reacted positively to neuron-specific antibodies and were immunonegative for keratin and glia-specific proteins, suggesting that they were ORNs. Those that were negative for the neuron-specific proteins may represent either neuron progenitors or olfactory ensheathing cells. The fusiform cells were relatively small and undifferentiated, exposure to brain-derived neurotrophic factor resulted in their decrease and an increase in bipolar cells. Therefore, they might be the stem cells. The nest cells had morphological characteristics of epithelia and bound keratin antibodies. The giant cells had the morphology of epithelial cells but were negative for keratin; they may represent a unique cell population induced by the ZnSO4. These results indicate that the major cell types of intact OE are present in our cultures, and each retains characteristics found in situ. The mist inhalation method provides an in vitro population of adult mitotically active neurons for study., (Copyright 1998 Academic Press.)

In previous studies, we demonstrated that the exogenous ganglioside GM1 increased the complexity of the microtubular network and level of tubulin, selectively changed the distribution of microtubule associated protein-2 (MAP2) immunoreactivity from the perikarya to distal neuritic processes and increased immunogold label of MAP2 in the subplasmalemmal cytoplasm, neuritic filopodia and growth cones of Neuro-2a neuroblastoma cells. Since these areas are rich in actin filaments, our data suggested that MAP2 may be associated with microfilaments in the early stages of ganglioside-induced neuritogenesis. To determine if GM1 alters neuronal morphology by facilitating the interaction of actin and MAP2, we examined the immunolocalization of these two proteins with confocal and electron microscopy. We found that along with the redistribution of MAP2 from perikaryal to neuritic regions, there was parallel redistribution of actin. The uniform subplasmalemmal actin meshwork was disrupted in areas of processes and filopodia with a redistribution of actin to these areas in close association with MAP2. Our present results suggest that gangliosides enhance neuritogenesis by redistributing actin as well as MAP2 to processes and filopodia thereby facilitating their interaction. The association of MAP2 with actin filaments is likely to be an early step in ganglioside-mediated filopodia formation.

Background: Previous studies using systematically administered lathyrogens to inhibit wound contractures have produced inconsistent results. The purpose of this study was to investigate the effects of lathyrogenic drugs on wound contraction when injected locally., Methods: Two symmetrical full-thickness wounds were made on the dorsum of either side of hairless (hr/hr) mice; thus, each animal served as its own control. Animals were divided into groups receiving daily local injections of beta-aminopropionitrile or D-penicillamine, or both beta-aminopropionitrile and D-penicillamine and normal saline vehicle (control side) for 5 or 10 days. The rate of contraction was determined by serial measurements of the surface area of each wound during the treatment period. At the end of the treatment period, the wounds were excised en bloc with the chest wall and prepared for blinded histological analysis. Granulation tissue thickness, number of fibroblasts in granulation tissue per unit area, number of inflammatory cells (neutrophils, lymphocytes, macrophages and mast cells) in subjacent muscle per unit area, and collagen deposition in subjacent muscle were determined., Results: Wound contraction, granulation tissue thickness, and collagen deposition in subjacent muscle were decreased only in wounds treated with beta-aminopropionitrile plus D-penicillamine. Collagen deposition in subjacent muscle was also decreased in wounds treated with D-penicillamine alone. Neither drug alone nor the combination affected the number of inflammatory cells in subjacent muscle. Body weight was not affected by the experimental procedures., Conclusions: The combination of beta-aminopropionitrile and D-penicillamine is potentially useful for inhibiting contracture formation when injected locally.

The effect of ganglioside GM1 on components of the neuronal cytoskeleton was studied in Neuro-2a neuroblastoma cells using immunofluorescent, immunogold-labeled, and Western-blot analysis. Exposure of cells to GM1 for 24 h resulted in an increased microtubular network and level of tubulin, a redistribution of MAP2 immunoreactivity from perikarya to distal neuritic processes, and an increased MAP2 gold label in the subplasmalemmal cytoplasm, neuritic spines, and growth cones. A similar change in the distribution of actin-positive fluorescent immunoreactivity was observed. In contrast to the redistribution of MAP2, immunolocalization of MAP5 and tau did not change following 24 h GM1 exposure. Our results suggest that gangliosides enhance neuritogenesis by selectively altering the distribution of MAP2 from perikaryon to neuritic spines. Furthermore, the enhanced presence of MAP2 in regions known to be rich in microfilaments following GM1 treatment suggests that an interaction of MAP2 with microfilaments may be necessary for early neurite formation.

The ability of PC12 cells to regenerate processes is substantially enhanced in vitro if they have been previously exposed to nerve growth factor (NGF-primed), compared to cells that have not been exposed (NGF-naive). These studies were carried out to determine if the enhanced neuritogenic ability of NGF-primed cells is retained following transplantation. NGF-naive or NGF-primed PC12 cells were transplanted into the striatum of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-treated mice and allowed to survive for 2 weeks. Mice were given daily injections of cyclosporin A (CyA) to prevent anti-species graft rejection. The transplanted PC12 cells were visualized by tyrosine hydroxylase immunoreactivity. The NGF-naive transplanted cells formed dense clusters and large tumor masses in more than half the animals. Only a few of the naive PC12 cells had short processes. In contrast, many of the transplanted NGF-primed PC12 cells had processes. Furthermore, fewer of the animals transplanted with primed cells produced tumor masses in the striatum compared to animals that received NGF-naive cells. Transplantation of NGF-naive PC12 cells leads to a significant increase in the number of dopaminergic neurons in the host substantia nigra (SN) compared to MPTP-treated animals. The increase of host dopaminergic neurons was not statistically significant when NGF-primed PC12 cells were used. Following MPTP treatment and PC12 cell transplantation, injection of CyA did not affect the dopaminergic neurons in the host SN. These data suggest that exposure of cells to trophic factors, prior to transplantation, can enhance their level of differentiation and integration into the host brain.

The effects of locally injected combined colchicine and D-penicillamine on wound contraction were investigated in a murine model. Two full-thickness excisional wounds were made on either side of the back of hairless (hr/hr) mice. A volume of 0.15 ml of colchicine, D-penicillamine, or combined colchicine and D-penicillamine in normal saline vehicle were injected daily into the wound on one side of the animal and 0.15 ml of vehicle alone was injected into the wound on the other side for 5 or 10 days; thus, each animal served as its own control. The surface area of each wound was measured on Days 0, 5, and 10 to determine an index of the rate of wound contraction. At the end of the experimental period (Day 5 or 10), wounds were excised en bloc from euthanized animals for histological studies. The following histological parameters were determined: the thickness of the granulation tissue, the number of fibroblasts in granulation tissue per unit area, and the number of inflammatory cells (neutrophils, lymphocytes, macrophages, mast cells) in subjacent muscle per unit area. Our data showed that after 5 days of treatment, wound contraction was significantly inhibited only in wounds treated with combined colchicine and D-penicillamine. Wound contraction was significantly inhibited even after 10 days of treatment with the combination. Histological studies revealed that although the thickness of the granulation tissue and the number of inflammatory cells in subjacent muscle were decreased by D-penicillamine alone, only combined colchicine and D-penicillamine decreased the thickness of the granulation tissue, fibroblasts in granulation tissue, and inflammatory cells in subjacent muscle. Our data suggests that very low concentrations of colchicine and D-penicillamine when combined and injected locally may be potentially useful in controlling surface scar formation.

This study demonstrates that ZnSO4-induced chemical trauma results in an in situ regeneration of the olfactory epithelium which, when maintained in vitro, provides an enriched population of olfactory neurons. Therefore, the ability of the olfactory epithelium to respond to chemical trauma with increased mitotic activity can be used to increase growth of neurons in culture. Tissue obtained from normal or vehicle-treated adult mice produced few olfactory neurons, when maintained in culture, compared to cultures established from tissue following an in situ ZnSO4 trauma. Maximal neuronal yields were obtained in cultures established from tissue that was removed 4-6 days following chemical trauma. The morphological appearance and the presence of cell specific intermediate filament proteins were used to classify the cell types in these olfactory epithelial cultures. Single cells and aggregates of cells which were immunopositive for keratin, but immunonegative for neurofilament protein and GFAP, were identified as epithelioid. Flattened polygonal cells immunopositive for GFAP were identified as glia. A small population of flattened cells was immunonegative for all of the antibodies used in this study. Cells that had processes were immunonegative for GFAP and keratin. Some were immunopositive for 200 kDa and 160 kDa neurofilament proteins but immunonegative for the 68 kDa neurofilament protein. A few of these cells showed positive immunoreactivity with the olfactory marker protein (OMP) antibody and most likely represented the most mature olfactory neurons in the cultures. This trauma-induced culture model using olfactory tissue from adult mice can serve as a source of CNS neurons for comparison with cultured embryonic neurons.

Neuro-2a murine neuroblastomal cells exposed to exogenous ganglioside undergo increased neuritogenesis in vitro. To determine if the distribution of exogenous ganglioside (GM1) in neuronal membranes is related to neuritogenesis, the surface topography of exogenous ganglioside in these cells was examined by localization with cholera toxin B-FITC. Following exposure to exogenous ganglioside, levels of fluorescent label appeared similar on perikaryal and neuritic surfaces. Scanning electron microscopic studies using protein G-gold to label antibody against exogenous ganglioside confirmed these observations at higher magnification. Within the general labelling pattern, occasionally labelled material was observed which seemed to form short linear arrays. This suggested that elements of the cytoskeleton might be influencing the surface distribution of exogenous ganglioside. To examine this possibility, Neuro-2a cells were exposed to agents known to alter the stability of specific cytoskeletal components, after which the general distribution of exogenous ganglioside was determined. Treatment with Colcemid, which disrupted microtubules, resulted in restriction of most exogenous ganglioside-positive label to the perikaryal surfaces. In contrast, exposure to taxol which enhanced microtubule stability diminished perikaryal fluorescence and increased neuritic labelling. The disruption of cytochalasin D-sensitive microfilaments did not influence the topographic distribution of exogenous ganglioside. Under the experimental conditions employed, mean neuritic lengths for Colcemid- and taxol-treated cells were nearly equal, indicating that altered neuritic length resulting from treatment with cytoskeletal agents was not a major factor in the redistribution of exogenous ganglioside. These studies suggest that microtubules play a role in determining the distribution of recently incorporated ganglioside in neuronal plasma membranes.

Nerve growth factor receptor (NGFR)-like immunoreactivity (IR) was studied in PC12 cells treated for 96 hr with NGF (40 ng/ml), using immunogold labeling and electron microscopic morphometric analysis. The cells were exposed to the anti-NGFR antibody 192-IgG, followed by immunoglobulin (IgG) conjugated with colloidal gold. PC12 cells exhibited occasional gold label (positive NGFR-IR) on all surfaces. Cells treated with colcemid (0.05 micrograms/ml) or cytochalasin D (2 micrograms/ml), which limit microtubule (MT) and microfilament (MF) formation, respectively, displayed an increased NGFR-IR in terms of gold labeling. NGFR-IR was also seen on taxol (0.85 micrograms/ml)-exposed cells, an agent that promotes MT assembly. Cells treated simultaneously with cytochalasin D and taxol had a dramatically augmented NGFR-IR on their surfaces, which exceeded levels obtained with either agent alone. Prominent NGFR-IR was localized frequently in coated endocytotic vesicles, in smooth endoplasmic reticulum, and in secondary multivesicular lysosomes, in both treated and untreated cells. The results suggest that a large number of NGFRs (positive NGFR-IR) in PC12 cells are cryptic and not available for ligand binding. Changes in cytoskeletal organization that may affect mobility of integral membrane proteins can modulate the distribution of NGFR-IR on neuronal surfaces.

The effects of nerve growth factor (NGF) and dibutyryl cyclic AMP (DBC) on the density of cytoskeletal structures in cultured dorsal root ganglia were examined using morphometric techniques. After 24 hr in culture, NGF-treated neurites were longer than either DBC-treated or control neurites. At 48 hr, neurites produced in response to NGF and DBC were of equivalent length, while controls were considerably shorter. Comparison of electron micrographs of neuritic profiles revealed some differences of area and cytoskeletal density between treatment groups. Morphometric analysis was used to determine these differences under several growth conditions, at various rates of elongation and at different neurite lengths. As shown by analysis of variance, both NGF-treated and control neurites tapered in diameter at 48 hr in vitro, while DBC-induced neurites increased in area. An increase in cytoskeletal density for all treatment groups indicated that density was not always correlated with changes in area. An increased density of microtubules as compared to neurofilaments was seen at 24 hr, with equal densities of both cytoskeletal elements present after 48 hr in vitro. Comparisons between individual groups of data indicated that NGF-treated neurites relied primarily on microtubular density at 24 hr in vitro, when NGF induced longer, faster growing neurites. At 48 hr, there was an increase in neurofilaments proximal to the explant in the presence of DBC, implying that DBC may cause increased synthesis and/or transport of these structures. A comparison of microtubule to neurofilament ratios indicated that at 24 hr, there was always a greater density of microtubules. However, after 48 hr, neurofilament density increased such that there were equivalent densities of both cytoskeletal elements, possibly due to the overall increase in length observed in each treatment group. These data imply that 1) neurites with different rates of elongation may exhibit differences in cytoskeletal density; 2) neurites of equivalent lengths may be of differing stabilities; 3) NGF and DBC produce neurites with different cytoskeletal densities, implying divergent mechanisms of neurite induction; 4) the presence or absence of NGF may be partially responsible for variations in cytoskeletal densities observed between peripheral and central processes of DRG during development.

Cultures of mouse Neuro-2a neuroblastoma cells treated with 3-6 mM extracellular Ca2+ exhibited enhanced neurite extension characterized by increased neurite numbers and lengths. The ganglioside GM1 potentiated the effect of extracellular Ca2+ by increasing further the number and length of the neurites formed in response to exogenous Ca2+. Maximal neuritic numbers were achieved with 4 mM Ca2+ while the longest neurites were observed in medium containing 4-6 mM Ca2+. Stimulation of the Ca2+ influx with the ionophore A23187 or the amino acid taurine also enhanced neurite formation and GM1 potentiated these actions. Transmission electron microscopy revealed numerous microtubules and neurofilaments in neurites and microfilaments with the spine-like processes along fine neuritic branches and in the filopodia of growth cones. Neuritic varicosities and growth cones contained a variety of vesicles. All of these structures were increased in the presence of GM1 and were increased further by extracellular Ca2+ or A23187. The ability of GM1 to enhance neuritogenesis was diminished by EGTA or Ruthenium red. Similarly, the effect of GM1 was diminished or abolished by Ca2+ channel blockers such as CdCl2 or LaCl3. X-ray microprobe analysis revealed that GM1 alone enhanced intracellular levels of total ionic and membrane bound Ca2+, perhaps accounting for the increased neuritogenesis observed under conditions in which Ca2+ was manipulated. The present study suggest that the neuritogenic action of GM1 is Ca2+ dependent.

The role of gangliosides in preventing neuronal degeneration was examined in a 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-induced mouse parkinsonian model. Intraventricular injections of a ganglioside mixture prior to MPTP treatment reduced MPTP's toxicity on tyrosine hydroxylase-positive neurons in the substantia nigra. This raises the interesting possibility that early ganglioside administration may be beneficial in the treatment of neurodegenerative disorders.

Taurine-induced differentiation was examined in the murine neuroblastoma Neuro-2a cell line in the presence or absence of the monosialoganglioside GM1 and under conditions in which Ca2+ levels were manipulated. Taurine (4 mM), GM1 (200 micrograms/ml), or taurine with GM1 were applied to culture media that contained either various concentrations of Ca2+ or the Ca2+ ionophore A23187. Taurine or GM1 and taurine with GM1 increased the number of cells emitting neurites above that found for controls. A significant interaction was found between treatment (taurine, GM1 or taurine + GM1) and the manipulations of Ca2+ levels, affecting the number of neurites and producing changes on the neuritic and perikaryal surfaces. Treatment with both taurine and taurine + GM1 and the various concentrations of Ca2+ resulted in a significant increase in neurite elongation. The Ca2+ ionophore A23187 in the presence of taurine or taurine + GM1 caused neurites to grow longer than observed in media containing Ca2+, either in a low concentration (about 125 microM) or at 1-2 mM. Taurine-treated cultures in the presence of extracellular Ca2+ or A23187 were characterized by surfaces with numerous microvillar, spine-like projections. This effect was enhanced with GM1 and was less pronounced in the medium containing low levels of Ca2+. Transmission electron microscopy of the taurine-stimulated neurons revealed an excessive number of clear-core vesicles (40-200 nm in diameter) in perikarya, neurites and neuritic varicosities and growth cones. In addition, numerous aggregates of intermediate filaments were seen. They were most abundant in the taurine + GM1 treated cultures. The taurine + A23187 cultures also exhibited numerous microtubules within the elongated processes. The different neuritic patterns induced by taurine under conditions in which Ca2+ levels were manipulated and/or when cells were exposed to exogenous GM1 suggest that taurine's actions depend in part on Ca2+ flux.

We have examined the role of microtubules and microfilaments in neurite outgrowth by chemically modifying their interaction in Neuro-2a neuroblastoma cells. Cells exposed to taxol (1 microM), an agent that promotes microtubule polymerization and stabilization, did not form neurites over a 24 h period. Similarly, cells exposed to cytochalasin D (4 microM), an agent which promotes microfilament depolymerization, did not develop neurites. However, cells treated simultaneously with taxol (1 microM) and cytochalasin D (4 microM) produced long (50 microns) thin, unbranched neurites. Neurites formed during this simultaneous treatment grew in a circular pattern, lacked typical growth cones, were packed densely with microtubules and were deficient in microfilaments. Untreated cells maintained in control medium for 24 h formed short (15 microns), thick, highly branched neurites containing a dense meshwork of microtubules, microfilaments and neurofilaments. These results demonstrate that taxol does not block neurite outgrowth from Neuro-2a cells maintained under microfilament-limiting conditions. They suggest further that microtubules may provide the major cytoskeletal framework for neurite elongation.

We have modified the Lowicryl K4M low-temperature dehydration and embedding procedure for immunoelectron microscopy to provide improved ultrastructural detail and facilitate the localization of actin and tubulin in isolated rat adrenocortical cells, chick spinal cord with attached dorsal root ganglia (SC-DRG), and cultured dorsal root ganglia (DRG). Cells and tissues were fixed for immunocytochemistry either in a mixture of 2% paraformaldehyde and 0.25% glutaraldehyde (0.1 M PIPES buffer, pH 7.3) or in a mixture of 0.3% glutaraldehyde and 1.0% ethyldimethylaminopropylcarbodiimide (0.1 M phosphate buffered saline, pH 7.3). Dehydration was in ethanol at progressively lower temperatures to -35 degrees C. Infiltration at -35 degrees C was followed by ultraviolet polymerization at -20 degrees C. Comparable samples were fixed in glutaraldehyde and osmium tetroxide and embedded in Epon 812 or Epon-Araldite. Post-embedding immunostaining of thin sections utilized commercially available monoclonal antibodies to tubulin and actin followed by the protein A-gold technique (Roth et al., Endocrinology 108:247, 1981). Actin immunoreactivity was observed at the periphery of mitochondria and between mitochondria and lipid droplets in rat adrenocortical cells and at the periphery of neuronal cell processes of SC-DRG. Tubulin immunoreactivity was associated with microtubules throughout neurites of cultured DRG. Our modified technique allows preservation of ultrastructural details as well as localization of antigens by immunoelectron microscopy.

A method utilizing polyethylene glycol (PEG) as an extractable embedment for electron microscopy is described. Tissues are fixed according to conventional protocols, embedded in PEG, and sectioned. Sections (ranging from 100 to 500 nm in thickness) are mounted on grids, divested of their PEG matrix, critical-point-dried, and examined stereoscopically. This method greatly facilitates studies on the three-dimensional organization of cytoskeletal and cytoplasmic contractile systems in both muscle and nonmuscle cells.

The 4 major ganglioside species, GM1, GD1a, GD1b and GT1b (200 micrograms/ml), were tested individually for the ability to stimulate neuronal trophic responses. The growth parameters measured were: morphologic changes, quantitated by computer-assisted morphometry of neurite length and number per soma, and metabolic changes, indicated by alterations in ornithine decarboxylase activity (ODC). In addition, the interaction of each ganglioside with nerve growth factor (NGF) was investigated with an NGF-responsive pheochromocytoma PC12 cell line and NGF-insensitive neuroblastoma Neuro-2a cultures. PC12 cells responded to gangliosides only in the presence of NGF (20 micrograms/ml): GM1 produced the greatest morphologic response, but did not alter metabolic levels; GT1b increased both parameters. The presence (5 micrograms/ml) or absence of NGF did not have an effect on the ganglioside-mediated morphologic responses of Neuro-2a cells to each species: GD1b elicited the greatest increase in neurite length, while GD1a and GT1b stimulated both length and number. In contrast, while GT1b alone was able to elevate ODC activity independently of NGF, the simultaneous exposure of Neuro-2a cultures to NGF and GM1 or GD1a resulted in a stimulation of cellular metabolism. These results indicate that each ganglioside species has a specific target action in the stimulation of different trophic responses and that performance in one category is not a predictor of the result in another. In addition, it is possible to confer a sensitivity to NGF by simultaneous treatment with specific gangliosides. This indicates that membrane gangliosides may modulate the actions of neurotrophic factors.

Diazepam (Valium/Roche) inhibited the morphogenesis of explanted stage 8 chick embryos in a dose-related manner. Diazepam, at concn of 400-500 micrograms/ml, preferentially inhibited closure of the neural tube. This inhibition was accompanied by a significant reduction in myosin content of the developing neuroepithelium. Diazepam can be used as a probe to study the contributory role of myosin in cellular and morphogenetic movements.

In these studies, DMSO at concentration above 1% was shown to be a potent inhibitor of neurite maturation in vitro. The inhibition was not reversible, and occurred in both newly formed and established neurites. In the presence of DMSO, the addition of NGF resulted in a general shift of the dose-response curve. The data indicate that at levels of DMSO below 5%, DRG neurons are capable of responding to NGF stimulation. It has been suggested that DMSO blocks a specific process associated with neurite extension. These studies suggest that the inhibitory action of membranous organelles in the developing neurite, a finding consistent with previous speculation that DMSO is responsible for an increased intracellular disorganization.

Our previous studies have demonstrated that a mixture of bovine brain gangliosides ( BBG ) applied to Neuro-2a neuroblastoma cells markedly increased the degree and rate of neurite formation. In the present study, the cytoskeletal basis for BBG -mediated neurite outgrowth was investigated by comparing cells grown in the presence or absence of BBG (250 micrograms/ml). After 24-48 h, neurite morphology and the distribution of cytoskeletal components were analyzed with correlative whole-cell transmission electron microscopy, thin-section transmission electron microscopy and scanning electron microscopy. BBG treatment enhanced markedly the organization of the microfilamentous system, and had a less pronounced effect on the number and organization of microtubules. The most prominent changes in microfilament organization were in the distal segment of the neurite and the growth cone. BBG -treated cells had a complex cytoskeletal consisting of numerous bundles of microfilaments. These filament bundles were distributed into the secondary and teritary neuritic branches. Cells grown in serum-depleted medium to stimulate neurite outgrowth, lacked these bundles of microfilaments, suggesting that the formation of microfilament bundles was not required for non- BBG -mediated neuritogenesis . The role that the cytoskeletal components play in BBG -induced neurite outgrowth was examined following disruption of microtubules or microfilaments with Colcemid and cytochalasin D, respectively. Simultaneous treatment of cells with BBG and Colcemid (0.25 microgram/ml) at the time of plating resulted in cells with numerous spine-like projections which did not extend neurites. In contrast, the simultaneous treatment of cells with BBG and cytochalasin D (2 micrograms/ml) at the time of plating resulted in cells devoid of spines, but exhibiting anomalous neurite outgrowth consisting of many long, thin, unbranched neurites. These neurites lacked characteristic flattened growth cones and had a tendency to grow in a circular fashion. These results demonstrate that neurite outgrowth under microfilament-limiting conditions results in reduced neuritic branching while growth under microtubule-limiting conditions allows initiation, but prevents significant elongation. The different neuritic growth patterns induced by serum deprivation, ganglioside treatment or the various cytoskeletal disruptive agents reflect changes in the organization of microtubules and microfilaments. Our studies suggest that the organizational state and activity of these cytoskeletal elements determine neurite morphology. Microfilaments appear to be the primary determinants in ganglioside-mediated growth.

The parasympathetic ciliary ganglion (CG) of the embryonic chick in vitro, is unresponsive to Nerve Growth Factor and has been reported to form neurites only in response to neurotrophic factors derived from striated muscle or optic tissues. We investigated a possible role of cyclic adenosine-3',5'-monophosphate (cAMP) in the neurotrophic activity of skeletal muscle-conditioned medium (SCM) on CG explants. We showed that treatment with cAMP or dibutyryl cAMP stimulated neuritogenesis of CG explants in a dose-dependent fashion. SCM and cyclic nucleotide stimulation produced distinctly different types of neuritic growth. The growth cones of SCM-stimulated neurites were observed consistently to contact other cells or processes whereas nucleotide-stimulated neurites were not associated with other cells. These observations suggested that the two neuritogenic agents do not act through identical mechanisms, a conclusion supported by experiments demonstrating that stimulation of the CG with dibutyryl cAMP enhanced ornithine decarboxylase (ODC) activity relative to controls, whereas stimulation with SCM had no effect on ODC activity. We conclude that although cAMP does exhibit neuritogenic activity on the CG in vitro, it does not appear to be involved directly in the neurite formation elicited by SCM. It is feasible that the two types of neuritic growth which we described are representative of the two populations of CG neurons. Because only one of these neuronal populations innervates striated muscle in vivo, SCM and cAMP might act on different neurons.

These studies demonstrate that while microtubules are essential for BBG-mediated neurite initiation and elongation, they are not involved in microfilament-dependent ganglioside-mediated surface activity. Microfilaments may be more directly altered by exogenous gangliosides than microtubules since they are the major structural elements of microvilli and are required for neurite branching. Our studies suggest that normal neuritogenesis requires a delicately balanced interaction between various cytoskeletal elements. Since there is a close relationship between membrane-associated lipid molecules and submembranous cytoskeletal elements, the incorporation of gangliosides into membranes may alter this balance and result in neurite formation. The use of gangliosides to enhance neurite production provides a unique model for the study of nerve development. We have shown that bovine brain gangliosides stimulate an immediate sequence of surface-related changes as well as microtubule and microfilament dependent neurite formation in Neuro-2a cells. However, the precise molecular events by which gangliosides enhance neuritogenesis await further study.

Gangliosides were previously reported to induce neuritogenesis in primary neuronal cultures and in some neurally derived cell lines. Because isolated gangliosides usually contain variable quantities of peptides, we investigated the possibility that neurite-stimulating activity could be caused by these contaminants. Ganglioside preparations from bovine brain and other sources were subjected to a three-step purification procedure that eliminated at least 95% of the contaminating peptides. These purified preparations retained their capacity to induce extensive neurite growth in neuro-2A murine neuroblastoma. Proteolytic digestion and a number of additional procedures were used to reduce residual contamination further without loss of activity. Several crude ganglioside samples had negative effects on neurite development until freed of their inhibitory factors, which were derived from the tissue and/or introduced during laboratory operations. This was particularly evident for bovine white matter gangliosides whose activity increased in proportion to peptide removal. When carefully purified, virtually all of 11 different gangliosides tested were highly active, with the possible exception of GM4, which demonstrated only moderate activity in a limited number of tests. All of the neutral glycolipids tested, as well as sulfatides and free sialic acid, were inactive.

Muscle development is characterized by the fusion of myoblasts to form myotubes and the co-ordinate expression of muscle-specific proteins such as actin, myosin and creatine phosphokinase. Our laboratory has been involved in the study of the role of lysosomal proteinases, namely, cathepsins B, H and L and the endogenous cysteine proteinase inhibitor, cystatin, during muscle differentiation in vitro. Specific activities of the cysteine proteinase in chicken primary cultures and a number of rat myogenic lines increased with the degree of myotube formation. This has suggested that lysosomal proteinases play an important role in myogenesis. We have measured the mRNA levels of two of the lysosomal enzymes, cathepsins B and D. This is advantageous because the presence of the endogenous inhibitor, cystatin, masks the levels of the specific activities of the cysteine proteinases present in the cell. RNA was extracted from developing muscle at three stages of development: proliferating myoblasts, confluent cells, and myotubes. Hybridization of RNA extracted from the L6 myogenic cell line with cathepsin B cDNA showed an increase in the level of cathepsin B mRNA. However, in the L8 rat myogenic line the level decreased after fusion. Cathepsin D mRNA levels remained constant throughout differentiation of the L8 cells. This paper also reports on the characterization of lysosomal proteinases of a newly obtained mouse myogenic line, C2.