Nerve Growth Factor-treated PC12 Cells Research Articles

Cocaine- and amphetamine-regulated transcript promoter regulated by nicotine in nerve growth factor-treated PC12 cells.

Nicotine and cocaine- and amphetamine-regulated transcripts (CART) have several overlapping functions, such as the regulation of reward, feeding behavior, stress response, and anxiety. Previous studies showed that nicotine regulates CART expression in various brain regions. However, the molecular mechanisms underlying this regulation are not known. This study investigated the regulatory effect of nicotine on promoter activity of the CART gene in PC12 cells, which were differentiated into a neuronal phenotype by nerve growth factor (NGF) treatment. Two vectors containing reporter genes (Gaussia luciferase or mCherry) and the 1,140-bp upstream of the transcriptional start site of the mouse CART gene are used to analyze the CART promoter activity. Transient transfection of PC12 cells with either vector displayed strong promoter activity in both undifferentiated and differentiated PC12 cells. CART promoter activity in the PC12 cell line is increased by forskolin or NGF treatment. In differentiated PC12 cells, exposure to 50nM nicotine for 6h increased CART promoter activity. However, treatment with higher nicotine doses for 6h and treatment with all nicotine doses for 24h showed no effect. A nicotine concentration of 50nM is comparable to brain nicotine levels experienced by chronic smokers over long periods of time. Taken together, these data indicate that nicotine may exert some of its actions through the regulation of CART transcription in the brain.

P-body-induced inactivation of let-7a miRNP prevents the death of growth factor-deprived neuronal cells.

RNA processing bodies (P-bodies) are cytoplasmic RNA granules in eukaryotic cells that regulate gene expression by executing the translation suppression and degradation of mRNAs that are targeted to these bodies. P-bodies can also serve as storage sites for translationally repressed mRNAs both in mammalian cells and yeast cells. In this report, a unique role of mammalian P-bodies is documented. Depletion of P-body components dedifferentiate nerve growth factor-treated PC12 cells, whereas ectopic expression of P-body components induces the neuronal differentiation of precursor cells. Trophic factor withdrawal from differentiated cells induces a decrease in cellular P-body size and numbers that are coupled with dedifferentiation and cell death. Here, we report how the expression of P-body proteins-by ensuring the phosphorylation of argonaute protein 2 and the subsequent inactivation let-7a miRNPs-prevents the apoptotic death of growth factor-depleted neuronal cells.-Patranabis, S., Bhattacharyya, S. N. P-body-induced inactivation of let-7a miRNP prevents the death of growth factor-deprived neuronal cells.

Comparison of neurons derived from mouse P19, rat PC12 and human SH-SY5Y cells in the assessment of chemical- and toxin-induced neurotoxicity

BackgroundExposure to chemicals might be toxic to the developing brain. There is a need for simple and robust in vitro cellular models for evaluation of chemical-induced neurotoxicity as a complement to traditional studies on animals. In this study, neuronally differentiated mouse embryonal carcinoma P19 cells (P19 neurons) were compared with human neuroblastoma SH-SY5Y cells and rat adrenal pheochromocytoma PC12 cells for their ability to detect toxicity of methylmercury (MeHg), okadaic acid and acrylamide.MethodsRetinoic acid-treated P19 and SH-SY5Y cells and nerve growth factor-stimulated PC12 cells, allowed to differentiate for 6 days, were exposed to MeHg, okadaic acid and acrylamide for 48 h. Cell survival and neurite outgrowth were assessed with the calcein-AM assay and fluorescence detection of antibodies against the cytoskeletal neuron-specific protein βIII-tubulin, respectively. The effects of glutathione (GSH) and the potent inhibitor of GSH synthesis buthionine sulfoximine (BSO) on the MeHg induced-toxicity were assessed using the PrestoBlue™ cell viability assay and the TMRE mitochondrial membrane potential assay.ResultsDifferentiated P19 cells developed the most extensive neuronal network among the three cell models and were the most sensitive neuronal model to detect neurotoxic effects of the test compounds. MeHg produced a concentration-dependent toxicity in differentiated P19 cells and SH-SY5Y cells, with statistically significant effects at concentrations from 0.1 μM in the P19 neurons and 1 μM in the SH-SY5Y cells. MeHg induced a decrease in the cellular metabolic activity and mitochondrial membrane potential (ΔΨm) in the differentiated P19 cells and SH-SY5Y cells, that were attenuated by GSH. Okadaic acid and acrylamide also showed statistically significant toxicity in the P19 neurons, but not in the SH-SY5Y cells or the P12 cells.ConclusionsP19 neurons are more sensitive to detect cytotoxicity of MeHg, okadaic acid and acrylamide than retinoic acid-differentiated SH-SY5Y cells and nerve growth factor-treated PC12 cells. P19 neurons are at least as sensitive as differentiated SH-SY5Y cells to detect the loss of mitochondrial membrane potential produced by MeHg and the protective effects of extracellular GSH on MeHg toxicity. P19 neurons may be a useful model to study neurotoxic effects of chemicals.

Regulation of RhoA activity by the cellular prion protein

The cellular prion protein (PrPC) is a highly conserved glycosylphosphatidylinositol (GPI)-anchored membrane protein that is involved in the signal transduction during the initial phase of neurite outgrowth. The Ras homolog gene family member A (RhoA) is a small GTPase that is known to have an essential role in regulating the development, differentiation, survival, and death of neurons in the central nervous system. Although recent studies have shown the dysregulation of RhoA in a variety of neurodegenerative diseases, the role of RhoA in prion pathogenesis remains unclear. Here, we investigated the regulation of RhoA-mediated signaling by PrPC using both in vitro and in vivo models and found that overexpression of PrPC significantly induced RhoA inactivation and RhoA phosphorylation in hippocampal neuronal cells and in the brains of transgenic mice. Using siRNA-mediated depletion of endogenous PrPC and overexpression of disease-associated mutants of PrPC, we confirmed that PrPC induced RhoA inactivation, which accompanied RhoA phosphorylation but reduced the phosphorylation levels of LIM kinase (LIMK), leading to cofilin activation. In addition, PrPC colocalized with RhoA, and the overexpression of PrPC significantly increased neurite outgrowth in nerve growth factor-treated PC12 cells through RhoA inactivation. However, the disease-associated mutants of PrPC decreased neurite outgrowth compared with wild-type PrPC. Moreover, inhibition of Rho-associated kinase (ROCK) substantially facilitated neurite outgrowth in NGF-treated PC12 cells, similar to the effect induced by PrPC. Interestingly, we found that the induction of RhoA inactivation occurred through the interaction of PrPC with RhoA and that PrPC enhanced the interaction between RhoA and p190RhoGAP (a GTPase-activating protein). These findings suggest that the interactions of PrPC with RhoA and p190RhoGAP contribute to neurite outgrowth by controlling RhoA inactivation and RhoA-mediated signaling and that disease-associated mutations of PrPC impair RhoA inactivation, which in turn leads to prion-related neurodegeneration.

Varying butyric acid amounts induce different stress- and cell death-related signals in nerve growth factor-treated PC12 cells: implications in neuropathic pain absence during periodontal disease progression.

Neuropathic pain is absent from the early stages of periodontal disease possibly due to neurite retraction. Butyric acid (BA) is a periodontopathic metabolite that activates several stress-related signals and, likewise, induce neurite retraction. Neuronal cell death is associated to neurite retraction which would suggest that BA-induced neurite retraction is ascribable to neuronal cell death. However, the underlying mechanism of BA-related cell death signaling remains unknown. In this study, we exposed NGF-treated PC12 cells to varying BA concentrations [0 (control), 0.5, 1.0, 5.0mM] and determined selected stress-related (H2O2, glutathione reductase, calcium (Ca(2+)), plasma membrane Ca(2+) ATPase (PMCA), and GADD153/CHOPS) and cell death-associated (extrinsic: FasL, TNF-α, TWEAK, and TRAIL; intrinsic: cytochrome C (CytC), NF-kB, CASP8, CASP9, CASP10, and CASP3) signals. Similarly, we confirmed cell death execution by chromatin condensation. Our results showed that low (0.5mM) and high (1.0 and 5.0mM) BA levels differ in stress and cell death signaling. Moreover, at periodontal disease-level BA concentration (5mM), we observed that only FasL amounts were affected and occurred concurrently with chromatin condensation insinuating that cells have fully committed to neurodegeneration. Thus, we believe that both stress and cell death signaling in NGF-treated PC12 cells are affected differently depending on BA concentration. In a periodontal disease scenario, we hypothesize that during the early stages, low BA amounts accumulate resulting to both stress- and cell death-related signals that favor neurite non-proliferation, whereas, during the later stages, high BA amounts accumulate resulting to both stress- and cell death-related signals that favor neurodegeneration. More importantly, we propose that neuropathic pain absence at any stage of periodontal disease progression is ascribable to BA accumulation regardless of amount.

High butyric acid amounts induce oxidative stress, alter calcium homeostasis, and cause neurite retraction in nerve growth factor-treated PC12 cells.

Butyric acid (BA) is a common secondary metabolite by-product produced by oral pathogenic bacteria and is detected in high amounts in the gingival tissue of patients with periodontal disease. Previous works have demonstrated that BA can cause oxidative stress in various cell types; however, this was never explored using neuronal cells. Here, we exposed nerve growth factor (NGF)-treated PC1(2) cells to varying BA concentrations (0.5, 1.0, 5.0 mM). We measured total heme, H(2)O(2), catalase, and calcium levels through biochemical assays and visualized the neurite outgrowth after BA treatment. Similarly, we determined the effects of other common periodontal short-chain fatty acids (SCFAs) on neurite outgrowth for comparison. We found that high (1.0 and 5.0 mM) BA concentrations induced oxidative stress and altered calcium homeostasis, whereas low (0.5 mM) BA concentration had no significant effect. Moreover, compared to other SCFAs, we established that only BA was able to induce neurite retraction.

Retraction Notice

Microarray and Synchronization of Neuronal Differentiation with Pathway Changes in the Kyoto Encyclopedia of Genes and Genomes (KEGG) Databank in Nerve Growth Factor-Treated PC12 Cells; Current Neurovascular Research, Vol. 9, No. 1; Pp. 222-229. Chih-Ming Lin and Wayne Feng This article has been withdrawn at the request of the authors. The Publisher apologizes for any inconvenience this may have caused.

Retraction Notice: Microarray and synchronization of neuronal differentiation with pathway changes in the Kyoto Encyclopedia of Genes and Genomes (KEGG) databank in nerve growth factor-treated PC12 cells

The Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway database creates networks from interrelations between molecular biology and underlying chemical elements. This allows for analysis of biologic networks, genomic information, and higher-order functional information at a systems level. We performed microarray experiments and used the KEGG database, systems biology analysis, and annotation of pathway function to study nerve growth factor (NGF)-induced differentiation of PC12 cells. Cells were cultured to 70%-80% confluence, treated with NGF for 1 or 3 hours (h), and RNA was extracted. Stage 1 data analysis involved analysis of variance (ANOVA), and stage 2 involved cluster analysis and heat map generation. We identified 2020 NGF-induced PC12 genes (1038 at 1 h and 1554 at 3 h). Results showed changes in gene expression over time. We compared these genes with 6035 genes from the KEGG database. Cross-matching resulted in 830 genes. Among these, we identified 395 altered genes (155 at 1 h and 301 at 3 h; 2-fold increase from 1 h to 3 h). We identified 191 biologic pathways in the KEGG database; the top 15 showed correlations with neuronal differentiation (mitogen-activated protein kinase [MAPK] pathway: 35 genes at 1 h, 54 genes at 3 h; genes associated with axonal guidance: 12 at 1 h, 26 at 3 h; Wnt pathway: 16 at 1 h, 25 at 3 h; neurotrophin pathway: 4 at 1 h, 14 at 3 h). Thus, we identified changes in neuronal differentiation pathways with the KEGG database, which were synchronized with NGF-induced differentiation.

Spirocyclic Nortriterpenoids with NGF-Potentiating Activity from the Fruits of Leonurus heterophyllus

Four new spirocyclic nortriterpenoids, leonurusoleanolide A (1), leonurusoleanolide B (2), leonurusoleanolide C (3), and leonurusoleanolide D (4), were isolated from the MeOH extract of the fruits of Leonurus heterophyllus. Compounds 1 and 2, and compounds 3 and 4, were found to exist as equilibrium mixtures of trans and cis isomers. Mixtures of 1 and 2, and 3 and 4, significantly enhanced the neurite outgrowth of nerve growth factor-treated PC12 cells at concentrations ranging from 1 to 30 μM. Compound 8 was also found to have a neurite outgrowth-promoting effect at concentrations of 1 and 10 μM. The structure-activity relationship of these compounds is discussed.

Proteases Inhibition Assessment on PC12 and NGF Treated Cells after Oxygen and Glucose Deprivation Reveals a Distinct Role for Aspartyl Proteases

Hypoxia is a severe stressful condition and induces cell death leading to neuronal loss both to the developing and adult nervous system. Central theme to cellular death is the activation of different classes of proteases such as caspases calpains and cathepsins. In the present study we investigated the involvement of these proteases, in the hypoxia-induced PC12 cell death. Rat PC12 is a model cell line for experimentation relevant to the nervous system and several protocols have been developed for either lethal hypoxia (oxygen and glucose deprivation OGD) or ischemic preconditioning (IPS). Nerve Growth Factor (NGF) treated PC12 differentiate to a sympathetic phenotype, expressing neurites and excitability. Lethal hypoxia was established by exposing undifferentiated and NGF-treated PC12 cells to a mixture of N2/CO2 (93:5%) in DMEM depleted of glucose and sodium pyruvate for 16 h. The involvement of caspases, calpains and lysosomal cathepsins D and E to the cell death induced by lethal OGD was investigated employing protease specific inhibitors such as z-VAD-fmk for the caspases, MDL28170 for the calpains and pepstatin A for the cathepsins D and E. Our findings show that pepstatin A provides statistically significant protection from cell death of both naive and NGF treated PC12 cells exposed to lethal OGD. We propose that apart from the established processes of apoptosis and necrosis that are integral components of lethal OGD, the activation of cathepsins D and E launches additional cell death pathways in which these proteases are key partners.

Neurite Outgrowth of Mature Retinal Ganglion Cells and PC12 Cells Requires Activity of CK1δ and CK1ε

Mature retinal ganglion cells (RGCs) do not normally regenerate severed axons after optic nerve injury and show only little neurite outgrowth in culture. However, RGCs can be transformed into an active regenerative state after lens injury (LI) enabling these neurons to regrow axons in vitro and in vivo. In the current study we investigated the role of CK1δ and CK1ε activity in neurite outgrowth of LI stimulated RGCs and nerve growth factor (NGF) stimulated PC12 cells, respectively. In both cell types CK1δ and ε were localized in granular particles aligned at microtubules in neurites and growth cones. Although LI treatment did not measurably affect the expression of CK1δ and ε, it significantly elevated the specific kinase activity in the retina. Similarly, CK1δ/ε specific kinase activity was also elevated in NGF treated PC12 cells compared with untreated controls. Neurite extension in PC12 cells was associated with a change in the activity of CK1δ C-terminal targeting kinases, suggesting that activity of these kinases might be necessary for neurite outgrowth. Pharmacological inactivation of CK1δ and ε markedly compromised neurite outgrowth of both, PC12 cells and LI stimulated RGCs in a concentration dependent manner. These data provide evidence for a so far unknown, but essential role of CK1 isoforms in neurite growth.

Ankyrin repeat-rich membrane spanning/Kidins220 protein interacts with mammalian Septin 5.

Neurotrophin receptors utilize specific adaptor proteins to activate signaling pathways involved in various neuronal functions, such as neurite outgrowth and cytoskeletal remodeling. The Ankyrin-Repeat Rich Membrane Spanning (ARMS)/kinase D-interacting substrate-220 kDa (Kidins220) serves as a unique downstream adaptor protein of Trk receptor tyrosine kinases. To gain insight into the role of ARMS/Kidins220, a yeast two-hybrid screen of a rat dorsal root ganglion library was performed using the C-terminal region of ARMS/Kidins220 as bait. The screen identified a mammalian septin, Septin 5 (Sept5), as an interacting protein. Co-immunoprecipitation using lysates from transiently transfected HEK-293 cells revealed the specific interaction between ARMS/Kidins220 and Sept5. Endogenous ARMS/Kidins220 and Sept5 proteins were colocalized in primary hippocampal neurons and were also predominantly expressed at the plasma membrane and in the tips of growing neurites in nerve growth factor-treated PC12 cells. Mapping of Sept5 domains important for ARMS/Kidins220 binding revealed a highly conserved N-terminal region of Sept5. The direct interaction between ARMS/Kidins220 and Sept5 suggests a possible role of ARMS/Kidins220 as a functional link between neurotrophin receptors and septins to mediate neurotrophin-induced intracellular signaling events, such as neurite outgrowth and cytoskeletal remodeling.

Mutation-, Aging-, and Gene Dosage-dependent Accumulation of Neuroserpin (G392E) in Endoplasmic Reticula and Lysosomes of Neurons in Transgenic Mice

Mutations in human neuroserpin gene cause an autosomal dementia, familial encephalopathy with neuroserpin inclusion bodies (FENIB). We generated and analyzed transgenic mice expressing high levels of either FENIB-type (G392E) or wild-type human neuroserpin in neurons of the central nervous system. G392E neuroserpin accumulated age-dependently in neurons of the neocortex, thalamus, amygdala, pons, and spinal cord of homozygous transgenic mice. Such accumulations were not observed in hemizygous transgenic mice nor in transgenic mice for wild-type neuroserpin. In differential centrifugation of brain homogenates, G392E neuroserpin recovered in the nucleus-rich fraction dramatically increased along with aging, suggesting that the aggregations gradually increase their densities presumably by their conversion into heavier and more compact configurations. In immunoelectron microscopical analyses, immunopositivities for G392E neuroserpin were found not only in endoplasmic reticulum but also in lysosomes. G392E neuroserpin transgenic mice were much more susceptible to seizures induced by kainate administration than nontransgenic mice. Overall, G392E neuroserpin accumulated in the central nervous system neurons of transgenic mice in mutation-, aging-, and gene dosage-dependent manners. The established transgenic mice will be valuable to elucidate not only mechanisms for the formation of G392E neuroserpin aggregations but also pathways for the degradation and/or clearance of the already formed aggregations in neurons.

Iron loading inhibits ferroportin1 expression in PC12 cells

Ferroportin1 (FP1 or MTP1/IREG1), the product of the SLC40A1 gene, is a main iron export protein in mammals. Its mRNA contains an iron response element (IRE) in its 5′ untranslated region, but the way this gene is regulated by iron is still unclear. The existence of FP1 in the brain has been recently confirmed. To better understand the role of this important transmembrane iron exporter in brain iron homeostasis, we investigated the effects of iron and nitric oxide (NO) on FP1 expression and that of a FP1 antibody on iron release in nerve growth factor-treated rat PC12 cells. We found that FP1 expression was down-regulated by iron loading but stimulated by iron chelation and treatment with a NO donor, S-nitroso- N-acetylpenicillamine (SNAP). In addition, a significant decrease in iron release was found in cells treated with a FP1 antibody. Our findings imply that regulation of FP1 by iron in the cells is at the transcriptional level, rather than by an IRE/IRP-mediated pathway. Based on our results and published data, it is suggested that the transcriptional and translational (IRP/IRE pathway) mechanisms of FP1 expression might both operate in a tissue-specific manner and that FP1 might have a role in iron export from PC12 cells.

Magnesium inhibits norepinephrine release by blocking N-type calcium channels at peripheral sympathetic nerve endings.

Although Mg2+ contributes to blood pressure regulation partly in terms of vasodilator action, its sympatholytic effect may also play an important role to control blood pressure. Thus, in the present study, we investigated the effect of Mg2+ on sympathetic tone and blood pressure. We studied its actions on the blood pressure response to hydralazine, a direct vasodilator, in conscious spontaneously hypertensive rats (SHRs), and to electrical stimulation in the pithed Sprague-Dawley rat; catecholamine release by peripheral sympathetic nerve endings; and the N-type Ca2+ channels of cultured neural cells. Intravenous Mg2+ infusion (MgSO4: 3x10(-6) mol/kg body weight/min) induced the greater hypotensive response to hydralazine with attenuated reflex tachycardia in SHRs. In pithed rats, Mg2+ infusion significantly attenuated the blood pressure elevation (2+/-2 mm Hg versus 27+/-6 mm Hg, P<0.01) in response to spinal electrical stimulation. In the perfused mesenteric arteries system, norepinephrine release was significantly attenuated (51+/-2%, P<0.01) by high Mg2+ concentration solution (4.8 mmol/L) compared with normal Mg2+ solution (1.2 mmol/L). When we applied the perforated whole-cell patch clamp method to nerve growth factor-treated PC12 cells, Mg2+ blocked voltage-gated Ca2+ currents in a concentration-dependent manner. The majority of the voltage-gated Ca2+ currents were carried through N-type channels, followed by L-type channels. Mg2+ blocked both of these channels. These findings suggest that Mg2+ blocks mainly N-type Ca2+ channels at nerve endings, and thus inhibits norepinephrine release, which decreases blood pressure independent of its direct vasodilating action.

Botulinum toxin type A targets RhoB to inhibit lysophosphatidic acid-stimulated actin reorganization and acetylcholine release in nerve growth factor-treated PC12 cells.

Botulinum toxin type A (BoNT/A) produced by Clostridium botulinum inhibits Ca2+-dependent acetylcholine (ACh) release (neuroexocytosis) at peripheral neuromuscular junctions, sometimes causing neuromuscular paralysis. This inhibitory effect is attributed to its metalloprotease activity to cleave the 25-kDa synaptosomal-associated protein, which is essential for the exocytotic machinery. However, deletion of this protein does not result in a complete block of neuroexocytosis, suggesting that botulinum-mediated inhibition may occur via another mechanism. Rho GTPases, a class of small GTP-binding proteins (G proteins), control actin cytoskeletal organization, thereby regulating a variety of cellular functions in various cells, including neuronal cells. We have shown that the G protein activator lysophosphatidic acid (LPA) triggered actin reorganization followed by Ca2+-dependent ACh release in nerve growth factor-treated PC12 cells and that BoNT/A blocked both events through degradation of RhoB by the proteasome. Overexpression of wild-type RhoB caused actin reorganization and enhanced the release of ACh by LPA to overcome toxin's inhibitory effect on actin reorganization/exocytosis stimulated by LPA, whereas overexpression of a dominant negative RhoB inhibited ACh release, regardless of LPA and/or toxin treatment. Finally, a knockdown of the RhoB gene via sequence-specific, post-transcriptional gene silencing reduced RhoB expression in PC12 cells, resulting in total inhibition of both actin reorganization and ACh release induced by LPA. We conclude that the RhoB signaling pathway regulates ACh release via actin cytoskeletal reorganization and that botulinum toxin inhibits neuroexocytosis by targeting RhoB pathway.

HMG-CoA reductase inhibition causes neurite loss by interfering with geranylgeranylpyrophosphate synthesis.

To determine whether neurite outgrowth depends upon the mevalonate pathway, we blocked mevalonate synthesis in nerve growth factor-treated PC12 cells or primary cortical neurones with atorvastatin, a 3-hydroxymethylglutaryl coenzyme A (HMG-CoA) reductase inhibitor, and substituted different intermediates of the mevalonate pathway. We show that HMG-CoA reductase inhibition causes a profound reduction of neurite length, neurite loss and ultimatively cell death in undifferentiated and pre-differentiated PC12 cells and also in rat primary cortical neurones. Geranylgeranylpyrophosphate, but not farnesylpyrophosphate, squalene or cholesterol, completely compensated for the lack of mevalonate. Our data indicate that, under HMG-CoA reductase inhibition, geranylgeranylpyrophosphate rather than farnesylpyrophosphate or cholesterol is critical for neurite outgrowth and/or maintenance. Loss of neurites is an early manifestation of various neurodegenerative disorders, and dysfunction of isoprenylation might play a role in their pathogenesis.

Post-transcriptional Regulation of Acetylcholinesterase mRNAs in Nerve Growth Factor-treated PC12 Cells by the RNA-binding Protein HuD

Expression of acetylcholinesterase (AChE) is greatly enhanced during neuronal differentiation, but the nature of the molecular mechanisms remains to be fully defined. In this study, we observed that nerve growth factor treatment of PC12 cells leads to a progressive increase in the expression of AChE transcripts, reaching approximately 3.5-fold by 72 h. Given that the AChE 3'-untranslated region (UTR) contains an AU-rich element, we focused on the potential role of the RNA-binding protein HuD in mediating the increase in AChE mRNA seen in differentiating neurons. Using PC12 cells engineered to stably express HuD or an antisense to HuD, our studies indicate that HuD can regulate the abundance of AChE transcripts in neuronal cells. Furthermore, transfection of a reporter construct containing the AChE 3'-UTR showed that this 3'-UTR can increase expression of the reporter gene product in cells expressing HuD but not in cells expressing the antisense. RNA gel shifts and Northwestern blots revealed an increase in the binding of several protein complexes in differentiated neurons. Immunoprecipitation experiments demonstrated that HuD can bind directly AChE transcripts. These results show the importance of post-transcriptional mechanisms in regulating AChE expression in differentiating neurons and implicate HuD as a key trans-acting factor in these events.

Selective in Vivo Inhibition of Mitogen-activated Protein Kinase Activation Using Cell-permeable Peptides

The extracellular signal-regulated kinase (ERK), a member of the mitogen-activated protein kinases (MAPKs), is essential for cellular proliferation and differentiation, and thus there exists great interest to develop specific and selective inhibitors of this enzyme. Whereas small molecule inhibitors PD098095 and U0126 have been used to study MAPK/ERK kinase (MEK), their target selectivity has been questioned recently. The cross-reactivity of ATP-directed inhibitors with other protein kinases prompted us to develop structure-based selective peptide inhibitors of ERK activation. Based on a MEK1-derived peptide, we developed inhibitors of ERK activation in vitro and in vivo. The inclusion of either an alkyl moiety or a membrane-translocating peptide sequence facilitated the cellular uptake of the peptide inhibitor and prevented ERK activation in 4-phorbol 12-myristate 13-acetate-stimulated NIH 3T3 cells or nerve growth factor-treated PC12 cells in a concentration-dependent manner. In addition, cell-permeable peptides inhibited ERK-mediated activation of the transcriptional activity of ELK1. The peptides did not have an inhibitory effect on the activity of two other closely related classes of MAPKs, c-Jun amino-terminal kinase or p38 protein kinase. Thus, these peptides may serve as valuable tools for investigating ERK activation and for selective investigation of ERK-mediated responses. With the knowledge of other kinase interacting domains, it would be possible to design cell-permeable inhibitors for investigating diverse cellular signaling mechanisms and for possible therapeutic applications.

Synuclein-1 is selectively up-regulated in response to nerve growth factor treatment in PC12 cells.

Mutations in the alpha-synuclein gene have recently been identified in families with inherited Parkinson's disease and the protein product of this gene is a component of Lewy bodies, indicating that alpha-synuclein is involved in Parkinson's disease pathogenesis. A role for normal alpha-synuclein in synaptic function, apoptosis or plasticity responses has been suggested. We show here that in rat pheochromocytoma PC12 cells synuclein-1, the rat homolog of human alpha-synuclein, is highly and selectively up-regulated at the mRNA and protein levels after 7 days of nerve growth factor treatment. Synuclein-1 expression appears neither sufficient nor necessary for the neuritic sprouting that occurs within 1-2 days of nerve growth factor treatment. Rather, it likely represents a component of a late neuronal maturational response. Synuclein-1 redistributes diffusely within the cell soma and the neuritic processes in nerve growth factor-treated PC12 cells. Cultured neonatal rat sympathetic neurones express high levels of synuclein-1, with a diffuse intracellular distribution, similar to neuronal PC12 cells. These results suggest that levels of synuclein-1 may be regulated by neurotrophic factors in the nervous system and reinforce a role for alpha-synuclein in plasticity-maturational responses. In contrast, there is no correlation between synuclein expression and apoptotic death following trophic deprivation.