Phenolic and Steroidal Metabolites from the Cultivated Edible Inonotus hispidus Mushroom and Their Bioactivities.

A chemical study on the fruiting bodies of cultivated edible mushroom Inonotus hispidus resulted in 14 metabolites including three new hispolon congeners, named inonophenols A-B and one new lanostane triterpenoid, named inonoterpene A. These structures were identified by NMR, high-resolution electrospray ionization mass spectrometry (HRESIMS), and electronic circular dichroism (ECD) data analysis. All metabolites were assessed for neurotrophic, anti-inflammatory, and antioxidative activities. Among them, inonophenols B and C were the most active in promoting PC-12 cell neurite outgrowth at a concentration of 10 µM. The phenolic derivatives reduced NO generation by lipopolysaccharide (LPS)-induced BV-2 microglial cells by suppressing the expression of toll-like receptor-4 (TLR-4) and the nuclear factor-kappa-B (NF-κB) signaling pathway as well as the inflammatory mediators including inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2). Moreover, the phenolics showed antioxidant effects in DPPH scavenging assay with the IC50 values of 9.82-21.43 µM. These findings showed that I. hispidus may be a new source of neurotrophic and protective agents against neurodegenerative disorders.

Specific Akt Family Members Impair Stress-Mediated Transactivation of Viral Promoters and Enhance Neuronal Differentiation: Important Functions for Maintaining Latency.

Neurotropic Alphaherpesvirinae subfamily members such as bovine herpesvirus 1 (BoHV-1) and herpes simplex virus 1 (HSV-1) establish and maintain lifelong latent infections in neurons. Following infection of ocular, oral, or nasal cavities, sensory neurons within trigeminal ganglia (TG) are an important site for latency. Certain external stressors can trigger reactivation from latency, in part because activation of the glucocorticoid receptor (GR) stimulates productive infection and promoters that drive expression of key viral transcriptional regulators. The Akt serine/threonine protein kinase family is linked to maintaining latency. For example, Akt3 is detected in more TG neurons during BoHV-1 latency than in reactivation and uninfected calves. Furthermore, Akt signaling correlates with maintaining HSV-1 latency in certain neuronal models of latency. Finally, an active Akt protein kinase is crucial for the ability of the HSV-1 latency-associated transcript (LAT) to inhibit apoptosis in neuronal cell lines. Consequently, we hypothesized that viral and/or cellular factors impair stress-induced transcription and reduce the incidence of reactivation triggered by low levels of stress. New studies demonstrate that Akt1 and Akt2, but not Akt3, significantly reduced GR-mediated transactivation of the BoHV-1 immediate early transcription unit 1 (IEtu1) promoter, the HSV-1 infected cell protein 0 (ICP0) promoter, and the mouse mammary tumor virus long terminal repeat (MMTV-LTR). Akt3, but not Akt1 or Akt2, significantly enhanced neurite formation in mouse neuroblastoma cells, which correlates with repairing damaged neurons. These studies suggest that unique biological properties of the three Akt family members promote the maintenance of latency in differentiated neurons.IMPORTANCE External stressful stimuli are known to increase the incidence of reactivation of Alphaherpesvirinae subfamily members. Activation of the glucocorticoid receptor (GR) by the synthetic corticosteroid dexamethasone (DEX) stimulates bovine herpesvirus 1 (BoHV-1) and herpes simplex virus 1 (HSV-1) reactivation. Furthermore, GR and dexamethasone stimulate productive infection and promoters that drive expression of viral transcriptional regulators. These observations lead us to predict that stress-induced transcription is impaired by factors abundantly expressed during latency. Interestingly, activation of the Akt family of serine/threonine protein kinases is linked to maintenance of latency. New studies reveal that Akt1 and Ak2, but not Akt3, impaired GR- and dexamethasone-mediated transactivation of the BoHV-1 immediate early transcription unit 1 and HSV-1 ICP0 promoters. Strikingly, Akt3, but not Akt1 or Akt2, stimulated neurite formation in mouse neuroblastoma cells, a requirement for neurogenesis. These studies provide insight into how Akt family members may promote the maintenance of lifelong latency.

BDNF, NT-3 and Trk receptor agonist monoclonal antibodies promote neuron survival, neurite extension, and synapse restoration in rat cochlea ex vivo models relevant for hidden hearing loss.

Neurotrophins and their mimetics are potential treatments for hearing disorders because of their trophic effects on spiral ganglion neurons (SGNs) whose connections to hair cells may be compromised in many forms of hearing loss. Studies in noise or ototoxin-exposed animals have shown that local delivery of NT-3 or BDNF has beneficial effects on SGNs and hearing. We evaluated several TrkB or TrkC monoclonal antibody agonists and small molecules, along with BDNF and NT-3, in rat cochlea ex vivo models. The TrkB agonists BDNF and a monoclonal antibody, M3, had the greatest effects on SGN survival, neurite outgrowth and branching. In organotypic cochlear explants, BDNF and M3 enhanced synapse formation between SGNs and inner hair cells and restored these connections after excitotoxin-induced synaptopathy. Loss of these synapses has recently been implicated in hidden hearing loss, a condition characterized by difficulty hearing speech in the presence of background noise. The unique profile of M3 revealed here warrants further investigation, and the broad activity profile of BDNF observed underpins its continued development as a hearing loss therapeutic.

A fully human inhibitory monoclonal antibody to the Wnt receptor RYK.

RYK is an unusual member of the receptor tyrosine kinase (RTK) family that is classified as a putative pseudokinase. RYK regulates fundamental biological processes including cell differentiation, migration and target selection, axon outgrowth and pathfinding by transducing signals across the plasma membrane in response to the high affinity binding of Wnt family ligands to its extracellular Wnt inhibitory factor (WIF) domain. Here we report the generation and initial characterization of a fully human inhibitory monoclonal antibody to the human RYK WIF domain. From a naïve human single chain fragment variable (scFv) phage display library, we identified anti-RYK WIF domain-specific scFvs then screened for those that could compete with Wnt3a for binding. Production of a fully human IgG1κ from an inhibitory scFv yielded a monoclonal antibody that inhibits Wnt5a-responsive RYK function in a neurite outgrowth assay. This antibody will have immediate applications for modulating RYK function in a range of settings including development and adult homeostasis, with significant potential for therapeutic use in human pathologies.

Cell adhesion molecule 1 (CADM1) on mast cells promotes interaction with dorsal root ganglion neurites by heterophilic binding to nectin-3.

Cell adhesion molecule 1 (CADM1) on mast cells promotes attachment and communication with neurons by homophilic binding. However, we found that mast cell CADM1 was responsible for both the attachment of mast cells to dorsal root ganglia (DRG) neurites and their calcium responses to activated DRG neurites, despite the low expression of CADM1 in DRG. Instead, nectin-3 was expressed on DRG neurons and localized to regions of cell-cell contact. A neutralizing antibody to nectin-3 inhibited both mast cell attachment and subsequent calcium responses. This suggests that heterophilic binding between CADM1 and nectin-3 mediates functional DRG-mast cell interactions in vitro.

Myeloid lineage cells inhibit neurite outgrowth through a myosin II-dependent mechanism.

The molecular mechanisms that underlie the axonal damage that accompanies CNS inflammation are largely unknown. Here, we investigate the effects of immune cells on neuronal viability and axonal growth and show that conditioned media from myeloid lineage cells inhibit neurite outgrowth without causing apoptosis. Treatment with monocyte conditioned medium enhances myosin light chain phosphorylation in neurons and the neurite outgrowth inhibitory effect of myeloid lineage cells can be attenuated with the myosin II inhibitor blebbistatin. Our results suggest that in the context of CNS inflammation myeloid cells may limit axonal repair in the CNS via a myosin II-dependent mechanism.

Inhibition of cyclin-dependent kinase 5 but not of glycogen synthase kinase 3-ß prevents neurite retraction and tau hyperphosphorylation caused by secretable products of human T-cell leukemia virus type I-infected lymphocytes.

Human T-cell leukemia virus type I (HTLV-I)-associated myelopathy/tropical spastic paraparesis (HAM/TSP) is a neurodegenerative disease characterized by selective loss of axons and myelin in the corticospinal tracts. This central axonopathy may originate from the impairment of anterograde axoplasmic transport. Previous work showed tau hyperphosphorylation at T(181) in cerebrospinal fluid of HAM/TSP patients. Similar hyperphosphorylation occurs in SH-SY5Y cells incubated with supernatant from MT-2 cells (HTLV-I-infected lymphocytes secreting viral proteins, including Tax) that produce neurite shortening. Tau phosphorylation at T(181) is attributable to glycogen synthase kinase 3-ß (GSK3-ß) and cyclin-dependent kinase 5 (CDK5) activation. Here we investigate whether neurite retraction in the SH-SY5Y model associates with concurrent changes in other tau hyperphosphorylable residues. Threonine 181 turned out to be the only tau hyperphosphorylated residue. We also evaluate the role of GSK3-ß and CDK5 in this process by using specific kinase inhibitors (LiCl, TDZD-8, and roscovitine). Changes in both GSK3-ß active and inactive forms were followed by measuring the regulatory phosphorylable sites (S(9) and Y(216) , inactivating and activating phosphorylation, respectively) together with changes in ß-catenin protein levels. Our results showed that LiCl and TDZD-8 were unable to prevent MT-2 supernatant-mediated neurite retraction and also that neither Y(216) nor S(9) phosphorylations were changed in GSK3-ß. Thus, GSK3-ß seems not to play a role in T(181) hyperphosphorylation. On the other hand, the CDK5 involvement in tau phosphorylation was confirmed by both the increase in its enzymatic activity and the absence of MT-2 neurite retraction in the presence of roscovitine or CDK5 siRNA transfection.

Neurons preferentially respond to self-MHC class I allele products regardless of peptide presented.

Studies of mice lacking MHC class I (MHC I)-associated proteins have demonstrated a role for MHC I in neurodevelopment. A central question arising from these observations is whether neuronal recognition of MHC I has specificity for the MHC I allele product and the peptide presented. Using a well-established embryonic retina explant system, we observed that picomolar levels of a recombinant self-MHC I molecule inhibited neurite outgrowth. We then assessed the neurobiological activity of a panel of recombinant soluble MHC Is, consisting of different MHC I heavy chains with a defined self- or nonself-peptide presented, on cultured embryonic retinas from mice with different MHC I haplotypes. We observed that self-MHC I allele products had greater inhibitory neuroactivity than nonself-MHC I molecules, regardless of the nature of the peptide presented, a pattern akin to MHC I recognition by some innate immune system receptors. However, self-MHC I molecules had no effect on retinas from MHC I-deficient mice. These observations suggest that neuronal recognition of MHC I may be coordinated with the inherited MHC I alleles, as occurs in the innate immune system. Consistent with this notion, we show that MHC I and MHC I receptors are coexpressed by precursor cells at the earliest stages of retina development, which could enable such coordination.

Neuroprotective and axon growth-promoting effects following inflammatory stimulation on mature retinal ganglion cells in mice depend on ciliary neurotrophic factor and leukemia inhibitory factor.

After optic nerve injury retinal ganglion cells (RGCs) normally fail to regenerate axons in the optic nerve and undergo apoptosis. However, lens injury (LI) or intravitreal application of zymosan switch RGCs into an active regenerative state, enabling these neurons to survive axotomy and to regenerate axons into the injured optic nerve. Several factors have been proposed to mediate the beneficial effects of LI. Here, we investigated the contribution of glial-derived ciliary neurotrophic factor (CNTF) to LI-mediated regeneration and neuroprotection using wild-type and CNTF-deficient mice. In wild-type mice, CNTF expression was strongly upregulated in retinal astrocytes, the JAK/STAT3 pathway was activated in RGCs, and RGCs were transformed into an active regenerative state after LI. Interestingly, retinal LIF expression was correlated with CNTF expression after LI. In CNTF-deficient mice, the neuroprotective and axon growth-promoting effects of LI were significantly reduced compared with wild-type animals, despite an observed compensatory upregulation of LIF expression in CNTF-deficient mice. The positive effects of LI and also zymosan were completely abolished in CNTF/LIF double knock-out mice, whereas LI-induced glial and macrophage activation was not compromised. In culture CNTF and LIF markedly stimulated neurite outgrowth of mature RGCs. These data confirm a key role for CNTF in directly mediating the neuroprotective and axon regenerative effects of inflammatory stimulation in the eye and identify LIF as an additional contributing factor.

CD40 ligation mediates plaque-associated tau phosphorylation in beta-amyloid overproducing mice.

Neuritic dystrophy with amyloid burden and neurofibrillary tangles are pathological hallmarks of Alzheimer's disease. Genetic disruption of CD40 or CD40L alleviates amyloid burden, astrocytosis, and microgliosis in transgenic animal models of Alzheimer's disease. It has been reported that phosphorylated tau-positive dystrophic neurites are observed in transgenic mice over-expressing human mutant beta-amyloid precursor protein (Tg2576). Here, we studied the pattern of phosphorylated tau (labeled with AT8, CP13, PG5, and PHF1 antibodies) and plaques using immunohistochemical techniques. Phosphorylated tau-positive dystrophic neurites were exclusively associated with Congo red-positive plaques as previously reported. Further, we show that CD40L or CD40 deficiency reduces the mean ratio of dystrophic neurite area to congophilic plaque area and the level of expression of cdk5 and p35/p25 in mice. In addition, we show that in a human neuroblastoma cell line treated with CD40L, cdk5 and p35/p25 are increased. Together, our data suggest that CD40-CD40L interaction has an effect on tau phosphorylation independent of beta-amyloid pathology, and that this effect may occur through a decrease of cdk5 and p35/p25.

Anti-Thy-1 antibody-induced neurite outgrowth in cultured dorsal root ganglionic neurons is mediated by the c-Src-MEK signaling pathway.

Our previous study has shown that anti-Thy-1 antibody promotes neurite outgrowth of cultured dorsal root ganglion (DRG) neurons in a protein kinase A (PKA)-dependent manner. The present study provided another intracellular signaling pathway for the neurotrophic effect of anti-Thy-1 antibody. In DMSO-treated control cells, Thy-1 was enriched in microdomain-like structures on cell membranes by immunofluorescence observation. Treatment of DRG neurons with anti-Thy-1 antibody not only stimulated neurite outgrowth, but also increased the branching complexity of the neurites in both small and large neurons. We have previously shown that anti-Thy-1 antibody causes a time-dependent activation of mitogen-activated protein kinase (MEK) and of cyclic AMP response-element binding protein (CREB). Here, anti-Thy-1 antibody elicited a transient activation of c-Src kinase, and the activation of c-Src kinase appeared occurring upstream of the activation of MEK and CREB, since pretreatment with the Src kinase inhibitor, PP2, effectively abolished the anti-Thy-1 antibody-induced neurite outgrowth and the phosphorylation of MEK and CREB. CREB phosphorylation might result in upregulation of certain neurite outgrowth-related proteins. We therefore conclude that anti-Thy-1 antibody activates the c-Src kinase-MEK-CREB cascade and overcomes the inhibitory effect of Thy-1 on neurite outgrowth in DRG neurons.

Galanin plays a role in the conditioning lesion effect in sensory neurons.

Sensory neurons show enhanced neurite outgrowth in vivo and in vitro following a conditioning lesion. Previous studies have shown that these effects are dependent on two members of the gp130 family of cytokines, leukemia inhibitory factor and interleukin-6. Here, we asked whether galanin, a neuropeptide induced by these cytokines, plays a role in the conditioning lesion response. Following a conditioning lesion, neurite outgrowth in culture was reduced in sensory neurons from galanin -/- mice compared with those from wild type controls. In neurons from wild type mice, the length of the longest neurite was increased 2.4-fold after a conditioning lesion, compared with 1.8-fold in neurons from knockout animals. The results indicate that the induction of galanin plays an important role in triggering the conditioning lesion response.

[Confocal laser scanning microscopy to study molecular mechanism of mast cell activation].

In the immune system, mast cells are a key cell type in the pathogenesis of immunoglobulin E (IgE)-dependent hypersensitivity reactions. Engagement of the high-affinity IgE receptors by multivalent antigens initiates the downstream activation of signal-transducing enzymes and evokes degranulation and cytokine production via an increase in the intracellular Ca2+ concentration. In addition, mast cells also play a prominent role in non-IgE-mediated hypersensitivity reactions. Mast cells are closely apposed to nerves in vivo and are likely to be regulated functionally by nerves. However, the molecular mechanisms for mast cell activation in an IgE-dependent and -independent manner have not been fully clarified. Confocal laser scanning microscopy has played an essential role in cell biology by allowing visualization of specific intracellular signaling molecules with high spatiotemporal resolution in living cells. We have studied intracellular movements of Ca2+ using a specific fluorescent probe and several types of signaling molecules using derivatives of green fluorescent protein in a living single mast cell using a microscopic strategy. We here describe our imaging analysis of the calcium signals to the nucleus, the movement of secretory granules in the degranulation process, and the nucleocytoplasmic shuttling of mitogen-activated protein kinase in mast cells. Further, we demonstrate that direct communication between mast cells and nerves occurs. These findings provide useful information from a new perspective to understand the molecular mechanisms of allergic reaction and inflammation.

Direct neurite-mast cell communication in vitro occurs via the neuropeptide substance P.

Communication between nerves and mast cells is a prototypic demonstration of neuroimmune interaction. However, whether mast cell activation occurs as a direct response to neuronal activation or requires an intermediary cell is unclear. Addressing this issue, we used an in vitro coculture approach comprising cultured murine superior cervical ganglia and rat leukemia basophilic cells (RBLs; possesses properties of mucosal-type mast cells). Following loading with the calcium fluorophore, Fluo-3, neurite-RBL units (separated by <50 nm) were examined by confocal laser scanning microscopy. Addition of bradykinin, or scorpion venom, dose-dependently elicited neurite activation (i.e., Ca2+ mobilization) and, after a lag period, RBL Ca2+ mobilization. Neither bradykinin nor scorpion venom had any direct effect on the RBLs in the absence of neurites. Addition of a neutralizing substance P Ab or a neurokinin (NK)-1 receptor antagonist, but not an NK-2 receptor antagonist, dose-dependently prevented the RBL activation that resulted as a consequence of neural activation by either bradykinin or scorpion venom. These data illustrate that nerve-mast cell cross-talk can occur in the absence of an intermediary transducing cell and that the neuropeptide substance P, operating via NK-1 receptors, is an important mediator of this communication. Our findings have implications for the neuroimmune signaling cascades that are likely to occur during airways inflammation, intestinal hypersensitivity, and other conditions in which mast cells feature.

The Escherichia coli-derived Fab fragment of the IgM/kappa antibody IN-1 recognizes and neutralizes myelin-associated inhibitors of neurite growth.

A recombinant Fab fragment was prepared from the monoclonal IgM/kappa antibody IN-1, which neutralizes central nervous system myelin-associated neurite growth inhibitors both in vitro and in vivo. The variable domain gene sequences were amplified and cloned after cDNA synthesis from the hybridoma RNA. After insertion into the tet promoter vector pASK85, which provided the constant domains of class IgG1/kappa, equipped with a His6 tag, large amounts of the Fab fragment were produced in Escherichia coli by medium cell density fermentation. The Fab fragment was purified to homogeneity by immobilized metal-affinity chromatography and its biochemical activity was compared with the original IN-1 antibody. In an assay for neurite outgrowth and fibroblast spreading, the Fab fragment showed a similar neutralizing effect on inhibitory substrate properties of central nervous system myelin as the unpurified IgM, although an approximately tenfold higher concentration was necessary. Immunoprecipitation experiments revealed a more selective antigen-binding behaviour for the Fab fragment. The Fab fragment was also successfully applied for antigen detection in immunohistochemical analyses. Therefore, the recombinant Fab fragment of IN-1 shows full functionality in vitro and appears to be well suited for replacing the monoclonal IgM in investigations on fiber tract regeneration in vivo.

Accelerated Nerve Regeneration in Mice by upregulated expression of interleukin (IL) 6 and IL-6 receptor after trauma.

In this study we aimed to examine a role for interleukin 6 (IL-6) and its receptor (IL-6R) in peripheral nerve regeneration in vivo. We first observed that cultured mouse embryonic dorsal root ganglia exhibited dramatic neurite extension by simultaneous addition of IL-6 and soluble IL-6R (sIL-6R), a complex that is known to interact with and activate a signal transducing receptor component, gp130. After injury in the hypoglossal nerve in adult mice by ligation, immunoreactivity to IL-6 was upregulated in Schwann cells at the lesional site as well as in the cell bodies of hypoglossal neurons in the brain stem. In the latter, upregulation of the immunoreactivity to IL-6R was also observed. Regeneration of axotomized hypoglossal nerve in vivo was significantly retarded by the administration of anti-IL-6R antibody. Surprisingly, accelerated regeneration of the axotomized nerve was achieved in transgenic mice constitutively expressing both IL-6 and IL-6R, as compared with nontransgenic controls. These results suggest that the IL-6 signal may play an important role in nerve regeneration after trauma in vivo.

Expression of a unique globo-series glycolipid in cultured rat dorsal root ganglion neurons: relationship with neuronal development.

Previous studied from the laboratory demonstrated the presence of a UDP-galactose:Gb3Cer alpha 1-3galactosyltansferase activity responsible for the synthesis of a unique glycosphingolipid (GSL), Gal alpha 1-3Gb3Cer, in cultured PC12 pheochromocytoma cells (21). In this investigation, we examined the presence of this enzyme activity in isolated rat embryonic dorsal root ganglion neurons (DRGN), which, like pheochromocytoma cells, originate from the neural crest cells. DRGN exhibited the alpha-galactosyltransferase activity and the activity was comparable to that of the PC12 cells while several other rat tissues, with the exception of kidney, showed minimal activity. In order to define the spatial and temporal expression of Gal alpha 1-3Gb3Cer in DRGN, we examined the expression of Gal alpha 1-3Gb3Cer in cultured DRGN derived from embryonic day 16 rat embryos. Using a polyclonal antibody raised against Gal alpha 1-3Gb3Cer, we examined the localization of this glycolipid in DRGN cells after 5, 8, 12, and 15 days in culture. Immunostaining was restricted to the neurons while Schwann cells were negative. At day 5, the immunostaining was weak and confined to the cell body of the DRGN, though neurites were present at this stage. The period between days 5 and 15 represented a period of rapid neuritic growth and continued enlargement of the cell bodies. Immunoreactivity in the cell bodies increased dramatically by day 8. By day 12, immunoreactivity was present in neurites, and by day 15, was strong in both cells bodies and neurites. The expression of Gal alpha 1-3Gb3Cer in vivo was confirmed by immunostaining of frozen sections of dorsal root ganglia. Our present studies which demonstrate neuron-specific expression of Gal alpha 1-3Gb3Cer during neurotigenesis combined with previous observations for its expression in PC12 cells, strongly implicates this GSL in neuronal development.

Clustering and co-localization of immunogold double labelled neural cell adhesion molecule isoforms in chick forebrain.

A quantitative immunoelectron microscopic study was carried out in the intermediate portion of hyperstriatum ventrale of chick forebrain (a region exhibiting learning-related morphological plasticity) in order to examine the fine distribution of the neural cell adhesion molecule (N-CAM). Antibodies against alpha 2,8 polysialic acid (PSA), characteristic for embryonic N-CAM, and those against the protein backbones of all N-CAM isoforms were sequentially labelled (post-embedding) with 5- and 15-nm gold particles, and binding to their epitopes was analyzed using the statistics of point processes. The results showed that: (1) PSA tends to form clusters (up to 200 nm in size), in contrast to the protein backbones of all N-CAM isoforms, and (2) the two tissue bound antibodies are co-localized spatially in clusters of sizes up to 100 nm. The data suggest that there is a spatial sub-cellular domain enriched in both PSA and protein isoforms of N-CAM.

Distribution of phosphorylated microtubule-associated protein 1B during neurite outgrowth in PC12 cells.

The functional significance of microtubule-associated protein 1B (MAP1B) phosphorylation during neuronal differentiation is unknown. In the present study we examined the hypothesis that the phosphorylation of MAP1B is required for neurite outgrowth. We reasoned that if MAP1B phosphorylation was important for neurite outgrowth then the intracellular distribution of phosphorylated MAP1B might exist as a discrete subset of the pattern for total MAP1B. We utilized a monoclonal antibody (mAb 7-1.1) that specifically recognizes a phosphorylated epitope on MAP1B and a polyclonal antiserum that recognizes all MAP1B protein to compare the distributions of phosphorylated and total MAP1B during neurite outgrowth. Phosphorylated MAP1B progressively accumulated in both the soluble and cytoskeletal fractions of differentiating cells. Similar proportions of total and phosphorylated MAP1B were associated with the cytoskeletons of differentiating PC12 cells. Within individual cells, phosphorylated MAP1B, in comparison with total MAP1B, was not limited to a particular intracellular domain. Phosphorylated MAP1B was present in both neurites and cell bodies. It was associated with fibrillar microtubules in neurites and growth cones, but it appeared nonfibrillar within cell bodies. In some cells that differentiated rapidly, there was little phosphorylated MAP1B in the early neurites despite the presence of extensive microtubules. In addition, although phosphorylated MAP1B increased in populations of mature PC12 cell cultures, increases in phosphorylated MAP1B did not always correlate with neurite outgrowth in individual cells. These results suggest that the phosphorylated isoform of MAP1B recognized by mAb 7-1.1 may not be required for neurite outgrowth.

Microtubule-associated protein tau is required for axonal neurite elaboration by neuroblastoma cells.

NB2a/d1 neuroblastoma cells constitutively express multiple isoforms of the microtubule-associated protein tau and incorporate this protein into the axonal neurites elaborated during serum deprivation. To examine whether or not tau played an essential role in axonal outgrowth, cells cultured in serum-free medium were treated at 24 h intervals with antisense- and sense-oriented cDNA oligonucleotides (25 or 36 mers that span or are upstream of tau initiation codon) and were simultaneously serum deprived. Oligonucleotide uptake was confirmed by determination of intracellular levels of radiolabeled oligonucleotides. Treatment for 48 h with tau antisense oligonucleotides reversibly inhibited the expression of tau and the number of neurite-bearing cells compared with treatment with sense oligonucleotides. By contrast, tubulin expression was not affected. When cells were treated with antisense oligonucleotide simultaneously with serum deprivation, the initial outgrowth of neurites was unaffected, but continued neurite elongation was prevented. By contrast, neurite outgrowth at 4 h was inhibited when cells were pretreated with tau antisense 24 h before serum deprivation. Furthermore, intracellular delivery of anti-tau antiserum prevented neurite outgrowth and, in cells that had previously been deprived of serum for 24 h, induced retraction of existing neurites. These findings indicate that both the initiation and the continued outgrowth of neurites are dependent on tau and that pre-existing cytoplasmic pools of tau can mediate initial neuritogenesis.