Days Of Nerve Growth Factor Treatment Research Articles

Nerve growth factor for the treatment of spinocerebellar ataxia type 3: an open-label study.

Background:Spinocerebellar ataxia type 3 (SCA3) is the most common subtype of SCA worldwide, and runs a slowly progressive and unremitting disease course. There is currently no curable treatment available. Growing evidence has suggested that nerve growth factor (NGF) may have therapeutic effects in neurodegenerative diseases, and possibly also in SCA3. The objective of this study was to test the efficacy of NGF in SCA3 patients.Methods:We performed an open-label prospective study in genetically confirmed adult (>18 years old) SCA3 patients. NGF was administered by intramuscular injection (18 μg once daily) for 28 days consecutively. All the patients were evaluated at baseline and 2 and 4 weeks after treatment using the Chinese version of the scale for assessment and rating of ataxia (SARA).Results:Twenty-one SCA3 patients (10 men and 11 women, mean age 39.14 ± 7.81 years, mean disease duration 4.14 ± 1.90 years, mean CAG repeats number 77.57 ± 2.27) were enrolled. After 28 days of NGF treatment, the mean total SARA score decreased significantly from a baseline of 8.48 ± 2.40 to 6.30 ± 1.87 (P < 0.001). Subsections SARA scores also showed significant improvements in stance (P = 0.003), speech (P = 0.023), finger chase (P = 0.015), fast alternating hand movements (P = 0.009), and heel-shin slide (P = 0.001).Conclusions:Our preliminary data suggest that NGF may be effective in treating patients with SCA3.

The Ubiquitin Ligase Praja1 Reduces NRAGE Expression and Inhibits Neuronal Differentiation of PC12 Cells

Evidence suggests that regulated ubiquitination of proteins plays a critical role in the development and plasticity of the central nervous system. We have previously identified the ubiquitin ligase Praja1 as a gene product induced during fear memory consolidation. However, the neuronal function of this enzyme still needs to be clarified. Here, we investigate its involvement in the nerve growth factor (NGF)-induced differentiation of rat pheochromocytoma (PC12) cells. Praja1 co-localizes with cytoskeleton components and the neurotrophin receptor interacting MAGE homologue (NRAGE). We observed an enhanced expression of Praja1 after 3 days of NGF treatment and a suppression of neurite formation upon Praja1 overexpression in stably transfected PC12 cell lines, which was associated with a proteasome-dependent reduction of NRAGE levels. Our data suggest that Praja1, through ubiquitination and degradation of NRAGE, inhibits neuronal differentiation. The two murine isoforms, Praja1.1 and Praja1.2, appear to be functionally homologous in this respect.

Regulation of Neurite Outgrowth by Interactions between the Scaffolding Protein, JNK-associated Leucine Zipper Protein, and Neuronal Growth-associated Protein Superior Cervical Ganglia Clone 10

JLP (JNK-associated leucine zipper protein) is a novel scaffolding protein involved in JNK signaling. Although it is known that JLP is highly expressed in brain, the biological function of JLP in neuronal systems remains unknown. Here, we report a novel interaction between JLP and SCG10 (superior cervical ganglia clone 10), which is a microtubule-destabilizing factor that is essential for neurite outgrowth. Inhibition of endogenous JLP expression using small interference RNA methodology strongly enhanced nerve growth factor (NGF)-induced neurite outgrowth in PC12 cells. Our results show that JLP negatively regulates NGF-induced neurite outgrowth by decreasing the level of phosphorylated SCG10. Furthermore, inhibition of JNK phosphorylation by a small molecule inhibitor, SP600125, resulted in inhibition of SCG10 phosphorylation and inhibition of neurite growth. Taken together, our results suggest that JLP negatively regulates NGF-induced neurite outgrowth through a sequestering mechanism that results in an attenuation of NGF-induced SCG10 phosphorylation.

DNA Methyltransferase 3b Regulates Nerve Growth Factor-Induced Differentiation of PC12 Cells by Recruiting Histone Deacetylase 2

To elucidate the role of epigenetic reprogramming in cell- or tissue-specific differentiation, we explored the role of DNA methyltransferases (Dnmts) in the nerve growth factor (NGF)-induced differentiation of PC12 (pheochromocytoma) cells into neuronal cells. The mRNA and protein levels of de novo methyltransferase Dnmt3b increased, whereas those of Dnmt3a and Dnmt1 decreased, during NGF-induced neurite outgrowth. Dnmt3b localized in the nucleus, as well as in the growing neurites. When the expression of Dnmt3b was inhibited by antisense or small interfering RNA, PC12 cells continued to proliferate and failed to generate neurites. Cells depleted of Dnmt3b were unable to exit the cell cycle even after 6 days of NGF treatment. Furthermore, this failure in differentiation correlated with significant attenuation in tyrosine phosphorylation of TrkA (a marker for NGF-induced differentiation) and reduced the expression of neuronal markers, Hu antigen, and MAP2. The methyl-CpG content of the PC12 genome or the methylation status of repetitive elements was not significantly altered after differentiation and was not affected by Dnmt3b depletion. This was consistent with the ability of the catalytic-site mutant of Dnmt3b to induce differentiation in Dnmt3b-depleted cells after NGF treatment. The Dnmt3b-mediated differentiation was attributed to its N-terminal domain, which recruits histone deacetylase 2 (Hdac2), as demonstrated by (i) impeding of differentiation by the Hdac inhibitors, (ii) facilitation of the differentiation process by overexpression of the N-terminal domain of Dnmt3b, (iii) higher Hdac activity associated with Dnmt3b after NGF treatment, and (iv) coimmunoprecipitation and cosedimentation of Dnmt3b specifically with Hdac2 in a glycerol density gradient. These data indicate a novel role of Dnmt3b in neuronal differentiation.

Nerve Growth Factor Signals through TrkA, Phosphatidylinositol 3-Kinase, and Rac1 to Inactivate RhoA during the Initiation of Neuronal Differentiation of PC12 Cells

In PC12 rat pheochromocytoma cells, nerve growth factor (NGF)-induced neuronal differentiation is blocked by constitutively active dominant mutants of RhoA but augmented by negative ones, suggesting a not yet elucidated inhibitory signaling link between NGF receptors and RhoA. Here we show that NGF treatment rapidly translocates RhoA from the plasma membrane to the cytosol and simultaneously decreases RhoA affinity to its target Rho-associated kinase (ROK), a key mediator of neurite outgrowth. This effect was transient, because after 2 days of NGF treatment, RhoA relocated from the cytosol to the plasma membrane, and its GTP loading returned to a level found in undifferentiated cells. Inhibition of RhoA is mediated by activation of the TrkA receptor, because NGF failed to induce RhoA translocation and inhibition of ROK binding in nnr5 cells that lack TrkA, whereas the inhibition was reconstituted in receptor add-back B5 cells. In MM17-26 cells, which due to expression of dominant negative Ras do not differentiate, NGF-stimulated transient RhoA inhibition was unaffected. The inhibitory pathway from TrkA to RhoA involves phosphatidylinositol-3-kinase (PI3K), because the inhibitors LY294002 or wortmannin prevented NGF-induced RhoA translocation and increased RhoA association with ROK. Furthermore, inhibition of PI3K significantly reduced NGF- mediated Rac1 activation, whereas dominant negative Rac1 abolished the inhibitory signaling to RhoA. Taken together, these data indicate that NGF-mediated activation of TrkA receptor stimulates PI3K, which in turn increases Rac1 activity to induce transient RhoA inactivation during the initial phase of neurite outgrowth.

Collaboration of JNKs and ERKs in nerve growth factor regulation of the neurofilament light chain promoter in PC12 cells.

Nerve growth factor (NGF) induces transcription-dependent neural differentiation of PC12 cells, and the ERK family of MAPKs has been implicated as the dominant signal pathway that mediates this response. We employed a neurofilament light chain (NFLC) promoter-luciferase (NFLC-Luc) reporter to define the role of the ERKs as well as additional MAPK pathways in NGF induction of this neural specific gene. Constitutive active forms of c-Raf-1, MEKK1 and MKK6, proximal regulators of the ERKs, JNKs, and p38 MAPKs, respectively, all stimulated NFLC-Luc activity. NFLC-Luc activity stimulated by NGF, however, was partially (approximately 50%) inhibited by the MEK inhibitor, PD098059, or by co-transfection of kinase-inactive MEK1 but not by the p38 MAPK inhibitor, SB203580, indicating a role for the ERKs, but not the p38 MAPKs, in NGF regulation of the NFLC promoter. Importantly, a gain-of-function MKK7-JNK3 fusion protein stimulated NFLC-Luc and synergized with gain-of-function c-Raf-1 to activate the NFLC promoter. In addition, transfection of kinase-inactive forms of MEK1 and MKK7 produced an additive inhibition of NGF-stimulated NFLC-Luc relative to either inhibitor alone. These findings indicate that the ERK and JNK pathways collaborate downstream of the NGF receptor for regulation of the NFLC promoter. Truncation analysis and electromobility shift assays established the requirement for a cAMP-response element/activating transcription factor-like site in the NFLC promoter that minimally interacts with constitutively expressed cAMP-response element-binding protein and JunD as well as c-Jun which is induced by NGF in an ERK-dependent manner. Cumulatively, these findings indicate that the ERK pathway requires collaboration with the JNK pathway for maximal activation of the NFLC gene in PC12 cells through the integrated control of c-Jun function.

Modifications of intracellular Ca2+ signalling during nerve growth factor-induced neuronal differentiation of rat adrenal chromaffin cells.

Postnatal sympathetic neurons (SNs) and chromaffin cells (CCs) derive from neural crest precursors. CCs can differentiate in vitro into SN-like cells after nerve growth factor (NGF) exposure. This study examines changes of intracellular Ca2+ homeostasis and dynamics of CCs under conditions that promote a neuronal phenotype. Spontaneous Ca2+ fluctuations, a frequent observation in early cultures of CCs, diminished after > 10 days in vitro in control cells and ceased in NGF-treated ones. At the same time, Ca2+ rises resulting from entry upon membrane depolarization, gradually increased both their size and peak d[Ca2+]i/dt, resembling those recorded in SNs. Concomitantly, caffeine-induced Ca2+ rises, resulting from Ca2+ release from intracellular stores, increased their size and their peak d[Ca2+]i/dt by > 1000%, and developed transient and sustained release components, similar to those of SNs. The transient component, linked to regenerative Ca2+ release, appeared after > 10 days of NGF treatment, suggesting a delayed steep enhancement of Ca2+-induced Ca2+ release (CICR). Immunostaining showed that proteins coded by the three known isoforms of ryanodine receptors (RyRs) are present in CCs, but that only RyR2 increased significantly after NGF treatment. Since the transient release component increased more steeply than RyR2 immunostaining, we suggest that the development of robust CICR requires both an increased expression of RyRs and more efficient functional coupling among them. NGF-induced transdifferentiation of chromaffin cells involves the enhancement of both voltage-gated Ca2+ influx and Ca2+ release from intracellular stores. These modifications are likely to complement the extensive morphological and functional reorganization required for the replacement of the endocrine phenotype with the neuronal one.

Estrogen and NGF synergistically protect terminally differentiated, ERalpha-transfected PC12 cells from apoptosis.

The potential cytoprotective effects of the gonadal steroid estrogen, acting via its receptor (ER) on target neurons, are of considerable interest in aging, disease, and trauma. In this study, we examined the effects of estrogen on a prototypical neuronal-like cell, namely, nerve growth factor (NGF) differentiated PC12 cells when these cells are placed into an apoptosis-inducing environment. A clonal line of PC12 cells stably transfected to express full-length rat ERalpha mRNA and protein (PCER cells), as well as control cells (vector DNA transfected: PCCON cells), were differentiated into neurite bearing cells by 14 days of NGF treatment. Reverse transcription polymerase chain reaction (RT-PCR) and immunocytochemistry for ERalpha revealed that terminally differentiated PCER cells continue to show robust ER mRNA and protein expression after differentiation. PCCON cells do not express ERalpha either before or after NGF-induced differentiation. After differentiation, serum and NGF withdrawal from wild type PC12 cells is well known to induce cell death via apoptotic mechanisms. We challenged differentiated PCER and PCCON cells with serum-free media and assessed the effects of various treatments on cell survival and apoptosis using trypan blue staining and quantitative TUNEL staining. When differentiated PCER cells were treated with 17beta-estradiol (E2) plus NGF during a 2-day serum-free media challenge, cell survival was significantly greater, and the apoptotic index was significantly lower, than when PCER cells were treated with NGF only during the challenge. No additional improvement in cell survival, and no further reduction in apoptosis, was observed in non-ER expressing cells (PCCON) by E2 plus NGF treatment over that obtained by NGF alone. The effects of E2 on the expression of several apoptosis-related genes in this system were also examined. We found that E2 treatment of differentiated PCER cells in serum-free media increased the levels of Bcl-XL mRNA and reduced the levels of BAD mRNA relative to those in vehicle-treated PCER cells, and also relative to those in PCCON cells. Thus, estrogen's protective effects on PCER cells appear to, at least partly, involve a modulation of apoptosis-regulating genes.

NGF-induction of the expression of ChAT mRNA in PC12 cells and primary cultures of embryonic rat basal forebrain

The objective of this study was to examine the role of nerve growth factor (NGF) in regulation of expression of the cholinergic phenotype. NGF was administered to PC12 cells or primary cultures of embryonic (E17) rat basal forebrain for 2 days, then steady-state levels of choline acetyltransferase (ChAT) mRNA was monitored. Expression of ChAT mRNA isoforms was investigated using reverse transcription-polymerase chain reaction (RT-PCR) to amplify different upstream regions of the ChAT transcripts, and Southern blot analysis was used to verify identity of the PCR products. An NGF-induced increase of 1.8- and 1.5-fold in steady-state level of the ChAT transcript containing the M-exon (M-ChAT) was observed in PC12 cells and embryonic rat basal forebrain neurons, respectively. Also, a 2-fold increase in ChAT protein as determined by western blot analysis was associated with an NGF-mediated increase of 1.7-fold in ChAT activity in rat basal forebrain neurons within the same cultures following 4 days of NGF treatment.

Nerve growth factor (NGF) induces increase in multi-ubiquitin chains and concomitant decrease in free ubiquitin in nuclei of PC 12h.

Changes in Ubiquitin-immunoreactivity after nerve growth factor (NGF) treatment were investigated in PC12h cells. Ubiquitin-immunoreacitivity was increased in the nucleus of NGF-treated cells. The quantitative analysis revealed that, after 7 days of NGF treatment, almost 20% of cells had ubiquitin-immunoreactive nuclei and the frequency was increased thereafter. Levels of free ubiquitin and multi-ubiquitin chains were measured by radioimmunoassay (RIA) and enzyme-linked immunosorbent assay (ELISA), respectively. Measurements were carried out for four subcellular fractions: urea- and water-soluble extracts of nuclei and cytoplasm. Decrease in free ubiquitin was observed in water-soluble cytoplasmic extracts of NGF-treated cells, though increase in multi-ubiquitin chains in the same fraction was not observed. As for nuclei, increase in multi-ubiquitin chains and concomitant decrease in free ubiquitin were found in the water-soluble extracts after NGF treatment. Levels of multi-ubiquitin chains did not change in urea-soluble cytoplasmic extracts as well as nuclear urea-soluble ones after NGF treatment. These results indicated that multi-ubiquitination of nuclear proteins is increased during NGF-induced neuronal differentiation of PC 12h cells.

Selective Inhibition of the Expression of Signal Transduction Proteins by Lithium in Nerve Growth Factor‐Differentiated PC12 Cells

This investigation examined if lithium, the primary therapeutic treatment for bipolar affective disorder, modulated the levels of selected signal transduction proteins in PC12 cells. Nerve growth factor (NGF) induced differentiation of PC12 cells, and after 12 days of NGF treatment there were large increases in the levels of the heterotrimeric G protein subunits alpha o1, alpha i1, beta, and alpha s, small increases in those of alpha i2 and alpha q, and a slight decrease in that of alpha o2. Lithium (1 mM, equivalent to the therapeutic concentration) selectively reduced NGF-induced increases in levels of G protein subunits, generally having the greatest inhibition on those that were increased the most by NGF. Lithium at 5 mM had greater inhibitory effects than 1 mM lithium on NGF-induced increases in levels of G proteins, but neither concentration of lithium affected the induction of the cytoskeletal protein beta-tubulin. Examination of other proteins involved in signal transduction revealed that 12 days of NGF treatment increased the level of protein kinase C-alpha, but not those of the beta, epsilon, or zeta subtypes, and did not alter the levels of beta, gamma, or delta phospholipase C. Pretreatment with lithium inhibited the increase in content of protein kinase C-alpha induced by NGF but had little effect on the proteins not responsive to NGF except for decreasing the levels of protein kinase C-epsilon. The inhibitory effect of lithium was found not to be due to inhibition of NGF-induced tyrosine phosphorylation, which was unaffected by 5 mM lithium, or to inositol depletion. In summary, use of the dynamic system of NGF-induced PC12 cell differentiation provided a sensitive model in which to identify signal transduction proteins that were influenced by lithium treatment. The large changes caused by a therapeutically equivalent concentration of lithium lend support to the proposal that the selective inhibitory effects of lithium on subtypes of G proteins and protein kinase C may be important therapeutic targets.

Selective translocation of protein kinase C-delta in PC12 cells during nerve growth factor-induced neuritogenesis.

The specific intracellular signals initiated by nerve growth factor (NGF) that lead to neurite formation in PC12 rat pheochromocytoma cells are as of yet unclear. Protein kinase C-delta (PKC delta) is translocated from the soluble to the particulate subcellular fraction during NGF-induced-neuritogenesis; however, this does not occur after treatment with the epidermal growth factor, which is mitogenic but does not induce neurite formation. PC12 cells also contain both Ca(2+)-sensitive and Ca(2+)-independent PKC enzymatic activities, and express mRNA and immunoreactive proteins corresponding to the PKC isoforms alpha, beta, delta, epsilon, and zeta. There are transient decreases in the levels of immunoreactive PKCs alpha, beta, and epsilon after 1-3 days of NGF treatment, and after 7 days there is a 2.5-fold increase in the level of PKC alpha, and a 1.8-fold increase in total cellular PKC activity. NGF-induced PC12 cell neuritogenesis is enhanced by 12-O-tetradecanoyl phorbol-13-acetate (TPA) in a TPA dose- and time-dependent manner, and this differentiation coincides with abrogation of the down-regulation of PKC delta and other PKC isoforms, when the cells are treated with TPA. Thus a selective activation of PKC delta may play a role in neuritogenic signals in PC12 cells.

Lectin binding distinguishes between neuroendocrine and neuronal derivatives of the sympathoadrenal neural crest.

Lectin cytochemistry was used to identify surface epitopes selectively expressed by chromaffin cell chemoreceptors (glomus cells) in the rat carotid body. Unexpectedly, these studies revealed that binding sites for peanut agglutinin (PNA; Arachis hypogea) were highly expressed by all neuroendocrine-derivatives of the sympathoadrenal neural crest, including glomus cells, small, intensely fluorescent cells, and adrenal chromaffin cells in situ. In contrast, principal sympathetic neurons did not express PNA receptors. PNA binding was inhibited by 2% galactose. To determine whether expression of PNA receptors was selectively induced by neuroendocrine differentiation of sympathoadrenal precursors, we compared PNA labeling of embryonic sympathoblasts in the presence of either nerve growth factor (NGF) or the synthetic glucocorticoid dexamethasone (DEX). DEX-treated cells, which expressed several neuroendocrine traits, bound PNA, whereas NGF-treated neuronal derivatives did not. In addition, to examine whether expression of existing PNA receptors was down-regulated by neuronal differentiation of chromaffin cells, we compared labeling of PC12 cells, which normally bind PNA, in the presence and absence of NGF. Although PC12 cells acquired characteristic neuronal morphologies in the presence of NGF, they did not lose PNA labeling, even after 8 days of NGF treatment. These findings indicate that neuronal and neuroendocrine derivatives of the sympathoadrenal lineage can be distinguished by differential expression of carbohydrate epitopes and suggest that PNA receptors are induced by neuroendocrine differentiation.

Characterization of the rat light neurofilament (NF-L) gene promoter and identification of NGF and cAMP responsive regions.

We have isolated a genomic DNA clone covering the coding and 14 kb upstream region of the rat light neurofilament (NF-L) gene and sequenced 2.3 kb of its promoter. DNase I hypersensitive sites have been mapped in PC12 cells. For functional analysis of the NF-L promoter, constructs carrying 38, 97, 407, 564, 650, 1,099, 1,660, 2,003 base pairs (bp) upstream region in front of the chloramphenicol acetyltransferase (CAT) reporter gene were tested for their capability to direct CAT expression after transient transfection into various cell lines. Similar CAT activities were recorded both in rat pheochromocytoma (PC12) and mouse neuroblastoma N115 cells and also in several nonneural cell lines (HeLa, C127, NIH 3T3). Regions responsible for the basic promoter activity were located between -407 and +75 bp from the transcription initiation site. The NGF-responsive element was located between -38 and +75 bp, and sequence -97 to -38 was found to contain a functional cAMP-responsive element. In PC12 cells in which nerve growth factor (NGF) induces neurite outgrowth and NF-L transcription, NF-L promoter-driven CAT expression was stimulated up to 12-fold within three days of NGF treatment, whereas epidermal growth factor (EGF) had no effect. Rat NF-L promoter contained Sp1, AP-2 and CGCCCCCGC elements. In PC12 cells, NGF transiently induced the binding of transcription factors to the deoxyoligonucleotide probes containing the binding sites of these elements. The role of these factors in NF-L gene transcriptional induction by NGF in PC12 cells is discussed.

Constitutive upregulation of calcium channel currents in rat phaeochromocytoma cells: role of c‐fos and c‐jun.

1. Northern blot analysis and cell transfection were used in conjunction with whole-cell current recordings to examine the involvement of the immediate early genes, c-fos and c-jun, in the expression of calcium channel currents. 2. Phaeochromocytoma cells (PC12 clone) were exposed to nerve growth factor (NGF) and to depolarizing concentrations of KCl for 60 min every day. Cells challenged with NGF developed extensive networks of neurites within 3 days. Cells depolarized periodically retained their undifferentiated morphology even after 5 days of treatment. 3. The maximal amplitude of high-voltage-activated calcium currents (ICa) increased from the control level of 117.8 +/- 48.3 (mean +/- S.D.) to 387.2 +/- 90.1 pA within 3 days of NGF treatment. omega-Conotoxin (5-10 microM) inhibited 24.6 +/- 8.5% of ICa in undifferentiated cells and 57.8 +/- 6.9% in NGF-treated cells. 4. The levels of c-fos and c-jun mRNAs increased transiently during each daily exposure to NGF. The level of c-fos mRNA also increased transiently during repeated KCl-induced depolarizations but c-jun mRNA remained low or absent. 5. Naive PC12 cells were transiently co-transfected with expression plasmids that contained the full length of c-fos and c-jun cDNA. After 2 days following transfection, the PC12 cells could be grouped according to the size of ICa. In 56% of cells, ICa was similar to control currents (106.1 +/- 37.4 pA). In the remaining 44% of cells, ICa showed a 2.2-fold enhancement with respect to control cells. Transfection of only c-fos had no effect on ICa but, in 24% of cells transfected with c-jun, ICa was 176.6 +/- 124.6 pA. Since periodic membrane depolarization induced c-fos but not c-jun mRNA, c-jun transfection was combined with a high-K+ treatment over 3 days. In 18% of treated cells, ICa was 3.7 times larger than control currents. Morphological differentiation was not observed in transfected cells. 6. In PC12 cells co-transfected with c-fos and c-jun or treated with high K+ after transfection of c-jun, omega-conotoxin (5-10 microM) inhibited 68.7 +/- 11.9% of ICa when the current amplitude was in the range of 200-600 pA. since similar concentrations of omega-conotoxin blocked 19.2 +/- 5.4% of ICa in control cells, the current increase induced by c-fos and c-jun was supported by up to 11-fold enhancement of the omega-conotoxin-sensitive component of ICa.(ABSTRACT TRUNCATED AT 400 WORDS)

Ubiquitin and ubiquitin-protein conjugates in PC12h cells: changes during neuronal differentiation.

Ubiquitin and ubiquitin-protein conjugates in PC12h cells were detected with in vitro [125I]ubiquitination, and quantified by immunoblotting. These levels were altered by nerve growth factor (NGF), which promotes neuronal differentiation. (i) Levels of high molecular weight (HMW) ubiquitin-protein conjugates ranging from 40 to 1,000 kDa were increased by 2 days of NGF treatment, and remained high up to 10 days of NGF treatment. (ii) Ubiquitin and a 23-kDa conjugate tended to be decreased from days 2 to 10 of NGF treatment. 10-Day culture with 10 nM staurosporine, n protein kinase inhibitor, that blocks NGF-induced neurite outgrowth suppressed the NGF-induced increases in levels of HMW conjugates. Cyclic AMP and forskolin, both of which promote neurite outgrowth, mimicked the NGF-induced changes in ubiquitin and HMW conjugates, but phorbol ester and epidermal growth factor had little effect. These findings suggest that changes in ubiquitin-protein conjugates are closely coupled with neuronal differentiation.

Regulation of microtubule associated protein 2 (MAP2) expression by nerve growth factor in PC12 cells

In the presence of nerve growth factor (NGF), PC12 cells cease to divide and differentiate, extending long microtubule-containing neurites. We showed by immunoblot analysis that MAP2 was detectable in PC12 after 4 days of NGF treatment and that its levels increased five- to sevenfold after 12 days of NGF treatment. The apparent molecular weight of MAP2 in PC12 cells was similar to that of rat brain MAP2 (280,000), with a doublet representing the MAP2 isoforms. However, the relative levels of MAP2 in differentiated PC12 cells were 5–10% of those found in rat brain. Immunofluorescence analysis of NGF-treated PC12 cells revealed that MAP2 co-localized with tubulin and was present in cell bodies and neurites. Northern blot analysis showed that the levels of MAP2 mRNA increased in PC12 cells during NGF-treatment in a pattern that paralleled the protein levels, suggesting that MAP2 expression is transcriptionally regulated.

Isolation of the chicken middle-molecular weight neurofilament (NF-M) gene and characterization of its promoter

We have isolated and sequenced genomic DNA clones covering the coding region of the chicken mid-size neurofilament (NF-M) gene and greater than 1 kb of its 5' upstream region. The NF-M gene contains two introns which both are located within the highly conserved C-terminal region of the rod domain. The 5' end of the corresponding mRNA was assigned to a G residue 40 nucleotides upstream of the translation start site and in appropriate distance from a potential TATA box. To functionally analyze the NF-M promoter, constructs carrying 112, 222, and 1026 nucleotides of the 5' upstream region in front of a luciferase reporter gene were tested for their capability to direct luciferase expression after transient transfection into various cell lines. Significant luciferase activity was recorded both in rat phaeochromocytoma (PC12) cells and murine fibroblasts. In PC12 cells, in which neurite outgrowth is induced by nerve growth factor (NGF), expression was stimulated up to 13-fold within 3 days of NGF treatment. This closely resembles expression of the endogenous NF-M gene in response to this hormone.

Fucosylation of Glycolipids in PC12 Cells Is Dependent on the Sequence of Nerve Growth Factor Treatment and Adenylate Cyclase Activation

Synthesis of complex glycosphingolipids was analyzed in cultures of PC12 cells treated with nerve growth factor (NGF) and forskolin, either sequentially or simultaneously. For the sequential treatment, forskolin was added following 15 days of NGF treatment and cultures were continued for 3 additional days. For simultaneous treatment, cells maintained in medium with no additives received both agents and were then incubated for 3 additional days. Sequential NGF/forskolin treatment induced hypertrophy in PC12 cells and produced cells which had more neuron-like characteristics than cells treated with NGF alone. Simultaneous treatment initially accelerated the outgrowth of neurites and then reduced neurite elongation. Sequential treatment of PC12 cells resulted in a greater than 50% increase in the incorporation of fucose into neutral glycolipids and gangliosides compared to treatment with NGF alone. Galactose incorporation was unchanged, indicating that there was no net increase in glycolipid synthesis. The lack of an increase in total glycolipid accumulation was confirmed using monoclonal antibodies reacting with specific fucosylated and nonfucosylated PC12 cell glycolipids. However, TLC immunostaining analysis revealed that sequential NGF/forskolin treatment selectively altered the expression of some neutral glycolipid species. In contrast to the effects of the sequential treatment, simultaneous NGF and forskolin treatment was accompanied by a decrease in the incorporation of fucose and galactose into neutral glycolipids and gangliosides. These findings suggest that the timing of exposure to neuronotrophic factors and agents which activate adenylate cyclase might in part account for developmentally regulated patterns of glycoconjugate expression and provide further evidence that fucosylation of glycolipids may be associated with maturation of sympathetic neurons.

Nerve growth factor (NGF) reverses axotomy-induced decreases in choline acetyltransferase, NGF receptor and size of medial septum cholinergic neurons

Intraventricular nerve growth factor (NGF) infusion in the adult rat can prevent and also, if delayed, reverse the disappearance of most of the axotomized medial septum cholinergic neurons immunostained for choline acetyltransferase (ChAT). We have utilized the delayed NGF treatment protocol to (i) extend to 3 months the delay time between axotomy and NGF treatment, (ii) define the time course of their recovery, (iii) determine that immunostaining for the (lower affinity) NGF receptor (NGFR) parallels loss and reversal of the ChAT marker, and (iv) evaluate changes in cholinergic somal size following axotomy and subsequent NGF treatment. While NGF treatments starting only 7 days after the fimbria-fornix transection (axotomy) almost entirely restored the number of both ChAT- and NGFR-positive medial septum neurons, longer delayed (2–3 weeks) treatment brought about recovery from the baseline of 20–25% to only about 70% of the control numbers. This limited recoverability, however, persisted even after a 95 day delay period. In all cases examined maximal recoveries were achieved within 3–7 days of NGF treatment. Neuronal size analyses provided evidence for an axotomy-induced atrophy. NGF treatments, started with 1 or 2 week delays, not only reversed fully the average somal size loss but also induced an actual hypertrophy of several of those neurons. These results provide additional evidence that at least half of the apparent loss of cholinergic medial septum neurons upon axotomy is due to a loss of markers such as the transmitter-related enzyme ChAT and NGFR rather than to actual neuronal cell death. These results also show that NGF exerts a genuine trophic influence by regulating the size of its target neurons as well as their content of several proteins.