Anemarrhenae asphodeloides rhizoma Extract Enriched in Mangiferin Protects PC12 Cells against a Neurotoxic Agent-3-Nitropropionic Acid.

The rhizome of Anemarrhena asphodeloides Bunge, used in Traditional Chinese Medicine as a brain function-improving herb, is a promising source of neuroprotective substances. The aim of this study was to evaluate the protective action of xanthones from A. asphodeloides rhizomes on the PC12 cell line exposed to the neurotoxic agent-3-nitropropionic acid (3-NP). The xanthone-enriched fraction of the ethanolic extract of A. asphodeloides (abbreviated from now on as XF, for the Xanthone Fraction), rich in polyphenolic xanthone glycosides, in concentrations from 5 to 100 µg/mL, and 3-NP in concentrations from 2.5 to 15 mM, were examined. After 8, 16, 24, 48, and 72 h of exposure of cells to various combinations of 3-NP and XF, the MTT viability assay was performed and morphological changes were estimated by confocal fluorescence microscopy. The obtained results showed a significant increase in the number of cells surviving after treatment with XF with exposure to neurotoxic 3-NP and decreased morphological changes in PC12 cells in a dose and time dependent manner. The most effective protective action was observed when PC12 cells were pre-incubated with the XF. This effect may contribute to the traditional indications of this herb for neurological and cognitive complaints. However, a significant cytotoxicity observed at higher XF concentrations (over 10 µg/mL) and longer incubation time (48 h) requires caution in future research and thorough investigation into potential adverse effects.

A Chemical Investigation of the Leaves of Morus alba L.

The leaves of Morus alba L. are an important herbal medicine in Asia. The systematic isolation of the metabolites of the leaves of Morus alba L. was achieved using a combination of liquid chromatography techniques. The structures were elucidated by spectroscopic data analysis and the absolute configuration was determined based on electronic circular dichroism (ECD) spectroscopic data and hydrolysis experiments. Their biological activity was evaluated using different biological assays, such as the assessment of their capacity to inhibit the aldose reductase enzyme; the determination of their cytotoxic activity and the evaluation of their neuroprotective effects against the deprivation of serum or against the presence of nicouline. Chemical investigation of the leaves of Morus alba L. resulted in four new structures 1⁻4 and a known molecule 5. Compounds 2 and 5 inhibited aldose reductase with IC50 values of 4.33 µM and 6.0 µM compared with the potent AR inhibitor epalrestat (IC50 1.88 × 10−3 µM). Pretreatment with compound 3 decreased PC12 cell apoptosis subsequent serum deprivation condition and pretreatment with compound 5 decreased nicouline-induced PC12 cell apoptosis as compared with control cells (p < 0.001).

Phospholipase Cß-TRAX Association Is Required for PC12 Cell Differentiation.

When treated with nerve growth factor, PC12 cells will differentiate over the course of several days. Here, we have followed changes during differentiation in the cellular levels of phosphoinositide-specific phospholipase Cß (PLCß) and its activator, Gαq, which together mediate Ca2+ release. We also followed changes in the level of the novel PLCß binding partner TRAX (translin-associated factor X), which promotes RNA-induced gene silencing. We find that the level of PLCß increases 4-fold within 24 h, whereas Gαq increases only 1.4-fold, and this increase occurs ∼24 h later than PLCß. Alternately, the level of TRAX remains constant over the 72 h tested. When PLCß1 or TRAX is down-regulated, differentiation does not occur. The impact of PLCß on differentiation appears independent of Gαq as down-regulating Gαq at constant PLCß does not affect differentiation. Förster resonance energy transfer studies after PLCß association with its partners indicate that PLCß induced soon after nerve growth factor treatment associates with TRAX rather than Gαq Functional measurements of Ca2+ signals to assess the activity of PLCß-Gαq complexes and measurements of the reversal of siRNA(GAPDH) to assess the activity of PLCß-TRAX complexes additionally suggest that the newly synthesized PLCß associates with TRAX to impact RNA-induced silencing. Taken together, our studies show that PLCß, through its ability to bind TRAX and reverse RNA silencing of specific genes, plays a key role in switching PC12 cells to their differentiated state.

Regulation of early neurite morphogenesis by the Na+/H+ exchanger NHE1.

The ubiquitously expressed Na(+)/H(+) exchanger NHE1 plays an important role in regulating polarized membrane protrusion and directional motility in non-neuronal cells. Using NGF-differentiated PC12 cells and murine neocortical neurons in vitro, we now show that NHE1 plays a role in regulating early neurite morphogenesis. NHE1 was expressed in growth cones in which it gave rise to an elevated intracellular pH in actively extending neurites. The NHE1 inhibitor cariporide reversibly reduced growth cone filopodia number and the formation and elongation of neurites, especially branches, whereas the transient overexpression of full-length NHE1, but not NHE1 mutants deficient in either ion translocation activity or actin cytoskeletal anchoring, elicited opposite effects. In addition, compared with neocortical neurons obtained from wild-type littermates, neurons isolated from NHE1-null mice exhibited reductions in early neurite outgrowth, an effect that was rescued by overexpression of full-length NHE1 but not NHE1 mutants. Finally, the growth-promoting effects of netrin-1, but not BDNF or IGF-1, were markedly reduced by cariporide in wild-type neocortical neurons and were not observed in NHE1-null neurons. Although netrin-1 failed to increase growth cone intracellular pH or Na(+)/H(+) exchange activity, netrin-1-induced increases in early neurite outgrowth were restored in NHE1-null neurons transfected with full-length NHE1 but not an ion translocation-deficient mutant. Collectively, the results indicate that NHE1 participates in the regulation of early neurite morphogenesis and identify a novel role for NHE1 in the promotion of early neurite outgrowth by netrin-1.

RhoE stimulates neurite-like outgrowth in PC12 cells through inhibition of the RhoA/ROCK-I signalling.

Neurite formation involves coordinated changes between the actin cytoskeleton and the microtubule network. Rho GTPases are clearly implicated in several aspects of neuronal development and function. Indeed, RhoA is a negative regulator of neurite outgrowth and its effector Rho-kinase mediates the Rho-driven neurite retraction. Considering that RhoE/round protein (Rnd3) acts antagonistically to RhoA and it is also able to bind and inhibit rho kinase-I (p160ROCK) - ROCK-I, it is tempting to speculate a role of RhoE in neurite formation. We show for the first time that, in the absence of nerve growth factor (NGF), RhoE induces neurite-like outgrowth. Our results demonstrate that over-expression of RhoE decreases the activity of RhoA and reduces the expression of both ROCK-I and the phosphorylated myosin light chain phosphatase (MLCPp). Conversely, over-expression of either active RhoA or ROCK-I abolishes the RhoE-promoted neurite outgrowth, suggesting that RhoE induces neurite-like formation through inhibition of the RhoA/ROCK-I signalling. We also show that Rac and Cdc42 have a role in RhoE-induced neurite outgrowth. Finally, the present data further indicate that RhoE may be involved in the NGF-induced neurite outgrowth in PC12 cells, as depletion of RhoE by siRNA reduces the neurite formation induced by NGF. These findings provide new insights into the molecular mechanism implicated in neuronal development and may provide novel therapeutic targets in neurodegenerative disorders.

NRAGE is a negative regulator of nerve growth factor-stimulated neurite outgrowth in PC12 cells mediated through TrkA-ERK signaling.

NRAGE, also denominated as MAGE-D1 or Dlxin-1, is firstly identified as a molecule interacting with NGF low affinity receptor p75NTR. It facilitates cell cycle arrest and NGF-dependent neuronal apoptosis. Here we report that NRAGE is downregulated while p75NTR is upregulated during the process of NGF-induced neuronal differentiation of PC12 cells. Knockdown of NRAGE by RNA interference accelerates NGF-mediated neurite outgrowth. In addition, in the NRAGE-suppressed cells, NGF-induced ERK activation is increased and this activation is MEK-dependent. Conversely, NRAGE overexpression significantly represses NGF-induced ERK activation. Further studies revealed that NRAGE downregulates TrkA expression through a post-transcriptional manner and thereby blocks NGF-induced TrkA phosphrylation at tyrosine-490. Altogether, these data indicate for the first time that NRAGE is an endogenous inhibitor for NGF-induced neuronal differentiation of PC12 cells by regulating TrkA-ERK signaling.

Targeting of pseudorabies virus structural proteins to axons requires association of the viral Us9 protein with lipid rafts.

The pseudorabies virus (PRV) Us9 protein plays a central role in targeting viral capsids and glycoproteins to axons of dissociated sympathetic neurons. As a result, Us9 null mutants are defective in anterograde transmission of infection in vivo. However, it is unclear how Us9 promotes axonal sorting of so many viral proteins. It is known that the glycoproteins gB, gC, gD and gE are associated with lipid raft microdomains on the surface of infected swine kidney cells and monocytes, and are directed into the axon in a Us9-dependent manner. In this report, we determined that Us9 is associated with lipid rafts, and that this association is critical to Us9-mediated sorting of viral structural proteins. We used infected non-polarized and polarized PC12 cells, a rat pheochromocytoma cell line that acquires many of the characteristics of sympathetic neurons in the presence of nerve growth factor (NGF). In these cells, Us9 is highly enriched in detergent-resistant membranes (DRMs). Moreover, reducing the affinity of Us9 for lipid rafts inhibited anterograde transmission of infection from sympathetic neurons to epithelial cells in vitro. We conclude that association of Us9 with lipid rafts is key for efficient targeting of structural proteins to axons and, as a consequence, for directional spread of PRV from pre-synaptic to post-synaptic neurons and cells of the mammalian nervous system.

Mechanisms involved in suppression of NGF-induced neuronal differentiation of PC12 cells by hyaluronic acid.

In the present study, we found that hyaluronic acid (HA) suppressed the neuronal differentiation mediated by nerve growth factor (NGF). In addition, we examined the mechanism by which HA inhibits the NGF-induced neurite outgrowth of PC12 cells. We elucidated the direct interaction between NGF and HA, and found that HA did not bind to NGF directly using a quartz-crystal microbalance. Western blot analysis revealed that HA suppressed NGF-induced phosphorylation of p38 MAPK, ERKs, and transcriptional factor CREB in PC12 cells. Furthermore, HA inhibited the luciferase activity of pCRE-Luc transfected PC12 cells in the presence of NGF. We confirmed that the p38 MAPK inhibitor SB203580 and ERK inhibitor U0126 suppressed NGF-induced neurite outgrowth of PC12 cells, and found that the inhibitory effects of HA on phosphorylation of ERKs, but not of p38 MAPK, were restored by the anti-RHAMM antibody. The number of PC12 cells with neurites increased remarkably when pre-cultured with the anti-RHAMM antibody, then treated with NGF and HA. Our findings indicate that HA inhibits NGF-induced neuronal differentiation of PC12 cells partially by inhibiting ERK phosphorylation through RHAMM, and suggest that the binding of HA to RHAMM modifies the signaling pathways in PC12 cells treated with NGF.

Evaluation of PRGD/FK506/NGF conduits for peripheral nerve regeneration in rats.

CONTEXT: Both tacrolimus (FK506) and nerve growth factor (NGF) enhance peripheral nerve regeneration, and in vitro experimental results demonstrate that the combination of FK506 and NGF increased neurite outgrowth compared with either treatment alone. AIM: To determine if the combination of FK506 and NGF benefits peripheral nerve regeneration compared with either treatment alone in vivo. SETTINGS AND DESIGN: Rat sciatic nerves were cut off to form a 10 mm defect and repaired with the nerve conduits. All of the 32 Wistar rats were randomly divided into 4 groups: Group A: RGD peptide modification of poly{(lactic acid)-co-[(glycolic acid)-alt-(L-lysine)]} (PRGD)/FK506/NGF; Group B: PRGD/FK506; Group C: PRGD/NGF; and Group D: autologous nerves. MATERIALS AND METHODS: At 3 months after surgery, the regenerated rat sciatic nerve was evaluated by electrophysiology, calf triceps wet weight recovery rate, and histologic assessment. STATISTICAL ANALYSIS USED: The SPSS 10.0 software (Bizinsight, Beijing China) was used for statistical analysis. RESULTS: The compound muscle action potentials (CMAPs) of groups A and D were significantly stronger than those of groups B and C. The calf triceps wet weight recovery rate of groups A and D were higher than those of groups B and C. The regenerated nerves of groups A and D were more mature than those of groups B and C. There was no significant difference between groups A and D. CONCLUSIONS: PRGD/FK506/NGF sustained-release nerve conduits are more effective in regenerating nerves than both PRGD/FK506 sustained-release nerve conduits and PRGD/NGF sustained-release nerve conduits. The effect is as good as that of an autograft.

Different outcomes of unliganded and liganded estrogen receptor-alpha on neurite outgrowth in PC12 cells.

A precise description of the mechanisms by which estrogen receptor-alpha (ERalpha) exerts its influences on cellular growth and differentiation is still pending. Here, we report that the differentiation of PC12 cells is profoundly affected by ERalpha. Importantly, depending upon its binding to 17beta-estradiol (17betaE2), ERalpha is found to exert different effects on pathways involved in nerve growth factor (NGF) signaling. Indeed, upon its stable expression in PC12 cells, unliganded ERalpha is able to partially inhibit the neurite outgrowth induced by NGF. This process involves a repression of MAPK and phosphatidylinositol 3-kinase/Akt signaling pathways, which leads to a negative regulation of markers of neuronal differentiation such as VGF and NFLc. This repressive action of unliganded ERalpha is mediated by its D domain and does not involve its transactivation and DNA-binding domains, thereby suggesting that direct transcriptional activity of ERalpha is not required. In contrast with this repressive action occurring in the absence of 17betaE2, the expression of ERalpha in PC12 cells allows 17betaE2 to potentiate the NGF-induced neurite outgrowth. Importantly, 17betaE2 has no impact on NGF-induced activity of MAPK and Akt signaling pathways. The mechanisms engaged by liganded ERalpha are thus unlikely to rely on an antagonism of the inhibition mediated by the unliganded ERalpha. Furthermore, 17betaE2 enhances NGF-induced response of VGF and NFLc neuronal markers in PC12 clones expressing ERalpha. This stimulatory effect of 17betaE2 requires the transactivation functions of ERalpha and its D domain, suggesting that an estrogen-responsive element-independent transcriptional mechanism is potentially relevant for the neuritogenic properties of 17betaE2 in ERalpha-expressing PC12 cells.

Structure-based discovery of low molecular weight compounds that stimulate neurite outgrowth and substitute for nerve growth factor.

Olanzapine, an atypical antipsychotic drug, was previously shown to protect neuronal cells against nutrient deprivation and to enhance neurite outgrowth. In an effort to identify small molecules with greater potency, the structure of olanzapine was used as a template to search commercially available chemical inventories for compounds with similar features. These compounds were evaluated for their ability to protect cells against glutamine deprivation and low-serum conditions. Positive compounds, 'hits' from initial screening, were then tested for stimulation of neurite outgrowth, alone and in combination with suboptimum concentrations of nerve growth factor (NGF). Numerous neuroprotective compounds (mw < 550 Da) were identified that significantly stimulated neurite outgrowth in PC12 cells. These included 4', 6'-diamidino-2-phenylindole, a nuclear stain; staurosporine, an antibiotic and kinase inhibitor; and 2-phenylamino-adenosine, an adenosine analog. The small molecules were comparable with NGF, and in fact, replaced NGF in outgrowth assays. Pharmacophore analysis of the hits led to the design and synthesis of an active compound, LSU-D84, which represented an initial lead for drug discovery efforts.

Late endocytic dysfunction as a putative cause of amyloid fibril formation in Alzheimer's disease.

The assembly of amyloid beta-protein to amyloid fibrils is a critical event in Alzheimer's disease. Evidence exists that endocytic pathway abnormalities, including the enlargement of early endosomes, precede the extraneuronal amyloid fibril deposition in the brain. We determined whether endocytic dysfunction potently promotes the assembly of amyloid beta-protein on the surface of cultured cells. Blocking the early endocytic pathway by clathrin suppression, inactivation of small GTPases, removal of membrane cholesterol, and Rab5 knockdown did not result in amyloid fibril formation on the cell surface from exogenously added soluble amyloid beta-protein. In contrast, blocking the late endocytic pathway by Rab7 suppression markedly induced the amyloid fibril formation in addition to the enlargement of early endosomes. Notably, a monoclonal antibody specific to GM1-ganglioside-bound amyloid beta-protein, an endogenous seed for Alzheimer amyloid, completely blocks the amyloid fibril formation. Our results suggest that late but not early endocytic dysfunction contributes to the amyloid fibril formation by facilitating the generation of amyloid seed in the Alzheimer's brain.

Characterization of CLP36/Elfin/PDLIM1 in the nervous system.

CLP36, one of the alpha-Actinin Associated LIM Protein (ALP)/Enigma family proteins, has a wide tissue distribution, but little is known about its expression and role in the nervous system. We show here that CLP36 is expressed in sensory ganglia but not in the CNS of adult rats. In primary dorsal root ganglion (DRG) neurons, CLP36 is distributed in the soma and neurites with enrichment in the growth cones. CLP36 forms a complex with alpha-actinin and is localized to actin cytoskeleton. To examine the role of CLP36 in neuronal cells, we transfected PC12 cells with a series of CLP36 deletion mutants and found that over-expression of CLP36 PDZ domain affects neurite outgrowth. Reduction of CLP36 function in PC12 cells by RNA interference (RNAi) induced lamellipodial protrusions around cell periphery and activated growth-cone movements, resulting in an increase in the length and number of neurites. Similarly, inhibition of CLP36 in primary DRG neurons increased the rate of neurite-bearing cells. We also found that CLP36 is up-regulated in DRG neurons and facial motoneurons after nerve injury. These findings suggest that CLP36 serves as a scaffold to form a multiprotein complex that regulates actin cytoskeleton dynamics and plays a role in controlling neurite outgrowth.

1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one inhibits neurite outgrowth and causes neurite retraction in PC12 cells independently of soluble guanylyl cyclase.

The effect of the potent soluble guanylyl cyclase (sGC) inhibitor 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ) on neurite outgrowth and retraction was investigated in PC12 cells and SH-SY5Y human neuroblastoma cells. ODQ inhibited neurite outgrowth and triggered neurite retraction in the cells stimulated with nerve growth factor (NGF), staurosporine, or Y-27632. The nitric oxide (NO) scavenger 2-(4-carboxyphenyl)-4,4,5,5-tetramethyl-imidazoline-1-oxyl-3-oxide (PTIO) had little effect on neurite outgrowth induced by Y-27632 or staurosporine. In the presence of ODQ, treatment of the cells with the cell-permeable cGMP analogue 8-bromo-cGMP failed to retrigger Y-27632- and staurosporine-induced neurite outgrowth. Furthermore, the depletion of sGC by RNA interference failed to prevent Y-27632- and staurosporine-induced neurite outgrowth. These results indicate that the NO/sGC/cGMP signaling cascade is not critically involved in ODQ-induced neurite remodeling. The MEK inhibitor PD98059 did not inhibit neurite outgrowth, and Y-27632 and staurosporine did not induce ERK phosphorylation, suggesting that the inhibitory effect of ODQ on neurite outgrowth is independent of the ERK signaling pathway. In contrast, pretreatment with dithionite or a hemin-glutathione mixture reversed the inhibitory effect of ODQ on Y-27632- and staurosporine-induced neurite outgrowth, indicating that ODQ might act on an intracellular redox-sensitive molecule. We conclude that ODQ inhibits Y-27632- and staurosporine-induced neurite outgrowth and triggers neurite retraction in an sGC-independent manner in neuronal cells and suggest that oxidation of unidentified redox-sensitive protein could be responsible for these effects.

Sciatic nerve conditioned medium depleted of pro-NGF modulates sodium currents and neurite outgrowth in PC12 cells.

Excitability and axon/dendrite specification are the most distinctive features in the establishment of neuronal polarization. Conditioned medium from rat sciatic nerve (CM) induced a neuronal-like morphology in PC12 cells. Here we show that CM neuritogenic activity is limited to the induction of dendrites in PC12 cells. However, treatment of these cells with CM in combination with a generic inhibitor for tyrosine kinase receptors (k252a) promoted the enhancement of neurite length, development of axons and induction of sodium currents. On the other hand, specific inhibition of TrkA and p75(NTR) receptors in CM-treated cells reduced the neurite length in comparison with cells treated only with CM, although the effect over the induction of sodium currents was continuously observed. These results suggested that CM had some components that, even though are able to start the morphological cell differentiation and produce short neurites (likely acting through TrkA and p75(NTR)), can restrain further neurite extension. Depletion of pro-NGF isoforms from CM produced a similar effect as the exerted by k252a, TrkA and p75(NTR) receptor inhibitors in CM-treated cells, inducing the elicitation of sodium currents. These results suggested that the effect of CM might be mediated through pro-NGF. The difference between the results obtained with the generic inhibitor for Trk receptors and the specific inhibitors for TrkA and p75(NTR) receptors in CM-treated cells, suggested that alternative pathways could be used to regulate neurite elongation, axon specification and sodium currents in PC12 cells. These findings represent important clues to improve the understanding of the initiation of neuronal polarity.

Quantitative assessment of neuronal differentiation in three-dimensional collagen gels using enhanced green fluorescence protein expressing PC12 pheochromocytoma cells.

There is a paucity of quantitative methods for evaluating the morphological differentiation of neuronal cells in a three-dimensional (3-D) system to assist in quality control of neural tissue engineering constructs for use in reparative medicine. Neuronal cells tend to aggregate in the 3-D scaffolds, hindering the application of two-dimensional (2-D) morphological methods to quantitate neuronal differentiation. To address this problem, we developed a stable transfectant green fluorescence protein (GFP)-PC12 neuronal cell model, in which the differentiation process in 3-D can be monitored with high sensitivity by fluorescence microscopy. Under 2-D conditions, the green cells showed collagen adherence, round morphology, proliferation properties, expression of the nerve growth factor (NGF) receptors TrkA and p75(NTR), stimulation of extracellular signal-regulated kinase phosphorylation by NGF and were able to differentiate in a dose-dependent manner upon NGF treatment, like wild-type (wt)-PC12 cells. When grown within 3-D collagen gels, upon NGF treatment, the GFP-PC12 cells differentiated, expressing long neurite outgrowths. We describe here a new validated method to measure NGF-induced differentiation in 3-D. Having properties similar to those of wt-PC12 and an ability to grow and differentiate in 3-D structures, these highly visualized GFP-expressing PC12 cells may serve as an ideal model for investigating various aspects of differentiation to serve in neural engineering.

A novel method for analyzing mitochondrial movement: inhibition by paclitaxel in a pheochromocytoma cell model.

A method was developed to assess mitochondrial movement in the living cell that is dependent, in part, on microtubule and/or associating protein interactions. The leader sequence from cytochrome-c was used to drive DsRed2 fluorescent proteins to accumulate in the mitochondria, thus enabling to follow mitochondrial (cytochrome-c's) movement. For calculating the percentage of mitochondrial movement, an image-processing program was used (ImageJ). Paclitaxel, an antitumor agent, is a potent microtubule-stabilizing agent that increases the stability of tubulin polymers, inhibiting mitosis and mitochondrial activity in dividing cells. Here, we tested whether paclitaxel inhibits mitochondrial movement in pheochromocytoma cells (a neuronal model, when tested in a differentiated state). While a 2-day exposure to paclitaxel resulted in cellular toxicity (measured as inhibition of mitochondrial activity), 2-3 h exposure to paclitaxel were sufficient to inhibit mitochondrial movement as assessed in 10-20-s imaging sessions in living cells. Mitotracker deep-red staining validated the staining obtained with DsRed2-cytochrome-c and identified intact mitochondria. Results showed a significant paclitaxel dose-dependent inhibition of mitochondrial movement. This new method should enable further assessment of microtubule-interacting drugs and other cytoskeletal components for their potential influence of mitochondrial movement as a test for activity and side effects.

Calcium overload is essential for the acceleration of staurosporine-induced cell death following neuronal differentiation in PC12 cells.

Differentiation of neuronal cells has been shown to accelerate stress-induced cell death, but the underlying mechanisms are not completely understood. Here, we find that early and sustained increase in cytosolic ([Ca2(+)]c) and mitochondrial Ca2(+) levels ([Ca2(+)]m) is essential for the increased sensitivity to staurosporine- induced cell death following neuronal differentiation in PC12 cells. Consistently, pretreatment of differentiated PC12 cells with the intracellular Ca2(+)-chelator EGTA-AM diminished staurosporine-induced PARP cleavage and cell death. Furthermore, Ca2(+) overload and enhanced vulnerability to staurosporine in differentiated cells were prevented by Bcl-XL overexpression. Our data reveal a new regulatory role for differentiation-dependent alteration of Ca2(+) signaling in cell death in response to staurosporine.

Small GTPase Rin induces neurite outgrowth through Rac/Cdc42 and calmodulin in PC12 cells.

The novel Ras-like small GTPase Rin is expressed prominently in adult neurons, and binds calmodulin (CaM) through its COOH-terminal-binding motif. It might be involved in calcium/CaM-mediated neuronal signaling, but Rin-mediated signal transduction pathways have not yet been elucidated. Here, we show that expression of Rin induces neurite outgrowth without nerve growth factor or mitogen-activated protein kinase activation in rat pheochromocytoma PC12 cells. Rin-induced neurite outgrowth was markedly inhibited by coexpression with dominant negative Rac/Cdc42 protein or CaM inhibitor treatment. We also found that expression of Rin elevated the endogenous Rac/Cdc42 activity. Rin mutant proteins, in which the mutation disrupted association with CaM, failed to induce neurite outgrowth irrespective of Rac/Cdc42 activation. Disruption of endogenous Rin function inhibited the neurite outgrowth stimulated by forskolin and extracellular calcium entry through voltage-dependent calcium channel evoked by KCl. These findings suggest that Rin-mediated neurite outgrowth signaling requires not only endogenous Rac/Cdc42 activation but also Rin-CaM association, and that endogenous Rin is involved in calcium/CaM-mediated neuronal signaling pathways.

In situ localization of mitochondrial DNA replication in intact mammalian cells.

Nearly all of the known activities required for mitochondrial DNA (mtDNA) replication and expression are nuclear-encoded gene products, necessitating communication between these two physically distinct intracellular compartments. A significant amount of both general and specific biochemical information about mtDNA replication in mammalian cells has been known for almost two decades. Early studies achieved selective incorporation of the thymidine analog 5-Bromo-2-deoxy-Uridine (BrdU) into mtDNA of thymidine kinase-deficient (TK[-]) cells. We have revisited this approach from a cellular perspective to determine whether there exist spatiotemporal constraints on mtDNA replication. Laser-scanning confocal microscopy was used to selectively detect mtDNA synthesis in situ in cultured mammalian cells using an immunocytochemical double-labeling approach to visualize the incorporation of BrdU into mtDNA of dye-labeled mitochondria. In situ detection of BrdU-incorporated mtDNA was feasible after a minimum of 1-2 h treatment with BrdU, consistent with previous biochemical studies that determined the time required for completion of a round of mtDNA replication. Interestingly, the pattern of BrdU incorporation into the mtDNA of cultured mammalian cells consistently radiated outward from a perinuclear position, suggesting that mtDNA replication first occurs in the vicinity of nuclear-provided materials. Newly replicated mtDNA then appears to rapidly distribute throughout the dynamic cellular mitochondrial network.