The cytomechanics of axonal elongation and retraction

Neurites of PC12 and chick dorsal root ganglion neurons behave as viscoelastic solids in response to applied forces. This passive behavior can be modeled with three mechanical elements; a relatively stiff, undamped spring in series with a Voight element composed of a less stiff spring in parallel with a dashpot. In response to applied tensions greater than 100 microdynes, PC12 cells show lengthening behavior distinct from and in addition to the passive viscoelastic response. We interpret this as "towed growth" (Bray, D. 1984. Dev. Biol. 102:379-389) because the neurites can become twice as long without obvious thinning of the neurite and because in two cases neurite tensions fell below original rest tensions, a result that cannot be obtained with passive viscoelastic elements. The rate of towed growth showed a linear dependence of growth rate with applied tensions in 8 of 12 PC12 neurites exposed to applied tension greater than 100 microdynes. Both PC12 and chick sensory neurons showed evidence of retraction when neurite tensions were suddenly diminished. This response was measured as tension recovery after slackening in chick sensory neurites. In 62% of the cases, tension recovery exceeded and sometimes doubled the preexperimental steady-state tension. Our data indicate that this response is active tension generation by the neurite shaft. We conclude that neurite length is regulated by axial tension in both elongation and retraction. Our data suggest a three-way controller: above some tension set point, the neurite is stimulated to elongate. Below some different, lower tension threshold the neurite is stimulated to retract. Between these two tension thresholds, the neurite responds passively as a viscoelastic solid.     

Tension and compression in the cytoskeleton of PC 12 neurites

We report in this article that the retraction of PC 12 neurites, unlike that of other cultured neurons, is due to tension within the neurite. Retraction is rapid and independent of metabolic energy. Transection of one arm of a branched neurite immediately causes the remaining arm to take up a new equilibrium position between attachment points. Similarly, detachment of one growth cone of a cell causes the cell body to move to a new equilibrium position between the remaining neurites. These observations provide direct evidence for the suspension of the cell soma among a network of tensioned neurites. We used retraction as an assay for neurite tension to examine the role of actin filaments and microtubules in neurite support and elongation. Our data suggest that microtubules (MTs) within PC 12 neurites are under compression, supporting tension within the actin network. Treatment of cells with drugs that disrupt actin networks, cytochalasin D or erythro-9-[3-(2-hydroxynonyl)]adenosine eliminates retraction regardless of the absence of MTs, lack of adhesion to the substratum, or integrity of the neurite. Conversely, stimulation of actin polymerization by injection of phalloidin causes retraction of neurites. Treatments that depolymerize MTs, nocodazole or cold, cause retraction of neurites, which suggests that microtubules support this tension, i.e., are under compression. Stabilization of MTs with taxol stabilizes neurites to retraction and under appropriate circumstances can drive neurite extension. Taxol-stimulated neurite extension is augmented by combined treatment with anti-actin drugs. This is consistent with the actin network's normally exerting a force opposite that of MT assembly. Cytochalasin and erythro-9-[3-(2-hydroxynonyl)] adenosine were found to increase slightly the dose of nocodazole required for MT depolymerization. This is consistent with the postulated balance of forces and also suggests that alteration of the compression borne by the microtubules could serve as a local regulator for MT polymerization during neurite outgrowth.     

The role of the cytoskeleton in volume regulation and beading transitions in PC12 neurites

We present investigations on volume regulation and beading shape transitions in PC12 neurites, conducted using a flow-chamber technique. By disrupting the cell cytoskeleton with specific drugs, we investigate the role of its individual components in the volume regulation response. We find that microtubule disruption increases both swelling rate and maximum volume attained, but does not affect the ability of the neurite to recover its initial volume. In addition, investigation of axonal beading—also known as pearling instability—provides additional clues on the mechanical state of the neurite. We conclude that volume recovery is driven by passive diffusion of osmolites, and propose that the initial swelling phase is mechanically slowed down by microtubules. Our experiments provide a framework to investigate the role of cytoskeletal mechanics in volume homeostasis.     

Tension and compression in the cytoskeleton of PC-12 neurites. II: Quantitative measurements

We assessed the mechanical properties of PC-12 neurites by applying a force with calibrated glass needles and measured resulting changes in neurite length and deflection of the needle. We observed a linear relationship between force and length change that was not affected by multiple distensions and were thus able to determine neurite spring constants and initial, nondistended, rest tensions. 81 out of 82 neurites showed positive rest tensions ranging over three orders of magnitude with most values clustering around 30-40 mu dynes. Treatment with cytochalasin D significantly reduced neurite rest tensions to an average compression equal to 14% of the former tension and spring constants to an average of 17% of resting values. Treatment with nocodazole increased neurite rest tensions to an average of 282% of resting values but produced no change in spring constant. These observations suggest a particular type of complementary force interaction underlying axonal shape; the neurite actin network under tension and neurite microtubules under compression. Thermodynamics suggests that microtubule (MT) assembly may be regulated by changes in compressive load. We tested this effect by releasing neurite attachment to a polylysine-coated surface with polyaspartate, thus shifting external compressive support onto internal elements, and measuring the relative change in MT polymerization using quantitative Western blotting. Neurons grown on polylysine or collagen without further treatment had a 1:2 ratio of soluble to polymerized tubulin. When neurites grown on polylysine were treated with 1% polyaspartate for 15-30 min, 80% of neurites retracted, shifting the soluble: polymerized tubulin ratio to 1:1. Polyaspartate treatment of cells grown on collagen, or grown on polylysine but treated with cytochalasin to reduce tension, caused neither retraction nor a change in the soluble:polymerized tubulin ratio. We suggest that the release of adhesion to the dish shifted the compressive load formerly borne by the dish onto Mts causing their partial depolymerization. Our observations are consistent with the possibility that alterations in MT compression during growth cone advance integrates MT assembly with the advance.     

Influence of pulsed electromagnetic field with different pulse duty cycles on neurite outgrowth in PC12 rat pheochromocytoma cells

The influence of low frequency (50 Hz repetition rate) pulsed electromagnetic field (EMF) on PC12 cell neurite outgrowth in vitro was investigated in this study. We studied the percentage of neurite bearing cells, average length of neurites, and directivity of neurite outgrowth in PC12 cells cultured for 96 h in the presence of nerve growth factor (NGF). PC12 cells were exposed in one incubator to pulsed EMF at 1.36 mT (peak value) generated by a pair of Helmholtz coils, and the control samples were placed in another identical incubator. We found that the pulse duty cycle had significant effect on neurite outgrowth. Low (10%) pulse on-time significantly inhibited the percentage of neurite bearing cells, but at the same time increased the average length of neurites, while 100% on-time (DC) had exactly the opposite effects. Furthermore, we found that neurites were prone to extend along the direction of pulsed EMF with 10% pulse on-time. Our studies show that neurite outgrowth in PC12 cells is sensitive to the pulse duty and this sensitivity was associated with NGF concentration.     

Okadaic Acid, a Protein Phosphatase Inhibitor, Inhibits Nerve Growth Factor-Directed Neurite Outgrowth in PC 12 Cells

The biochemical mechanisms involved in neurite outgrowth in response to nerve growth factor (NGF) have yet to be completely resolved. Several recent studies have demonstrated that protein kinase activity plays a critical role in neurite outgrowth. However, little information exists about the role of protein phosphatases in the process. In the present study, okadaic acid, a phosphatase inhibitor (specific for types 2A and 1) and tumor promoter, was used to investigate the role of protein phosphatases in neurite outgrowth in PC12 cells. PC12 cells cultured in the presence of 50 ng/ml of NGF started to extend neurites after 1 day. After 3 days, 20-25% of the cells had neurites. Okadaic acid inhibited the rate of neurite outgrowth elicited by NGF with an IC50 of approximately 7 nM. This inhibition was rapidly reversed after washout of okadaic acid. Okadaic acid also enhanced the neurite degeneration of NGF-primed PC12 cells, indicating that continual phosphatase activity is required to maintain neurites. Taken together, these results reveal the presence of an okadaic acid-sensitive pathway in neurite outgrowth and imply that protein phosphatase plays a positive role in regulating the neuritogenic effects of NGE.     

Oligomeric forms of insulin amyloid aggregation disrupt outgrowth and complexity of neuron-like PC12 cells

Formation of protein amyloid fibrils consists of a series of intermediates including oligomeric aggregates, proto-fibrillar structures, and finally mature fibrils. Recent studies show higher toxicity for oligomeric and proto-fibrillar intermediates of protein relative to their mature fibrils. Here the kinetic of the insulin amyloid fibrillation was evaluated using a variety of techniques including ThT fluorescence, Congo red absorbance, circular dichroism, and atomic force microscopy (AFM). The solution surface tension changes were attributed to hydrophobic changes in insulin structure and were detected by Du Noüy Ring method. Determination of the surface tension of insulin oligomeric, proto-fibrillar and fibrillar forms indicated that the hydrophobicity of solution is enhanced by the formation of the oligomeric forms of insulin compared to other forms. In order to investigate the toxicity of the different forms of insulin we monitored morphological alterations of the differentiated neuron-like PC12 cells following incubation with native, oligomeric aggregates, proto-fibrillar, and fibrillar forms of insulin. The cell body area, average neurite length, neurite width, number of primary neurites, and percent of bipolar cells and node/primary neurite ratios were used to assess the growth and complexity of PC12 cells exposed to different forms of insulin. We observed that the oligomeric form of insulin impaired the growth and complexity of PC12 cells compared to other forms. Together our data suggest that the lower surface tension of oligomers and their perturbation affects the morphology of PC12 cells, mainly due to their enhanced hydrophobicity and detergent-like structures.     

Neurite development in PC12 cells on flexible micro-textured substrates under cyclic stretch

We investigated the combined effect of micro-texture and mechanical strain on neuronal cell development such as neurite length and neurite density in a rat pheochromocytoma cell line (PC12 cells). Cells were seeded on flexible silicone substrates with micro-texture or no texture (smooth) and cultured under static and dynamic conditions. In the static condition substrates were not stretched and in the dynamic conditions substrates were subjected to cyclic uniaxial stretching at three different strain levels of 4%, 8%, and 16% with each at three different strain rates at 0.1, 0.5, and 1.0 Hz. Results showed that of all cell cultures there was no significant difference in neurite development between cells on smooth and textured substrates, except in the static and 4% at 0.1 Hz conditions, where micro-texture induced significantly longer neurites. With both types of substrates, a lower mechanical condition (4% at 1.0 Hz or 16% at 0.1 Hz) resulted in more and longer neurites and lower cell density, and a higher mechanical condition (16% at 1.0 Hz) resulted in fewer and shorter neurites and lower cell density as compared to the static condition. These findings suggest that the effect of the micro-texture on neurite development is more prominent in low mechanical conditions than in high mechanical conditions and that the strain level and strain rate have an interrelated effect on neurite development: a higher strain level at a lower strain rate has a similar effect as a lower strain level at a higher strain rate in terms of promoting neurite development.     

Synergistic effects of cyclic AMP and nerve growth factor on neurite outgrowth and microtubule stability of PC12 cells

The outgrowth of neurites from rat PC12 cells stimulated by combined treatment of nerve growth factor (NGF) with cAMP is significantly more rapid and extensive than the outgrowth induced by either factor alone. We have compared the responses of PC12 cells under three different growth conditions, NGF alone, cAMP alone, and combined treatment, with respect to surface morphology, rapidity of neurite outgrowth, and stability of neurite microtubules, to understand the synergistic action of NGF and cAMP on PC12. Surface events at early times in these growth conditions varied, suggesting divergent pathways of action of NGF and cAMP. This suggestion is strongly supported by the finding that cells exposed to saturating levels of dibutyryl cAMP without substantial neurite outgrowth initiated neurites within 5 min of NGF. This response has been adopted as a convenient assay for NGF. Neurites that regenerated in the three growth conditions showed marked differences in stability to treatments that depolymerize microtubules. The results indicate that microtubules in cells treated with both NGF and cAMP are significantly more stable than in either growth factor alone. We suggest that a shift of the assembly equilibrium favoring tubulin assembly is a necessary prerequisite for the initiation of neurites by PC12.     

Inhibition of lysophosphatidate- and thrombin-induced neurite retraction and neuronal cell rounding by ADP ribosylation of the small GTP-binding protein Rho

Addition of the bioactive phospholipid lysophosphatidic acid (LPA) or a thrombin receptor-activating peptide (TRP) to serum-starved N1E-115 or NG108-15 neuronal cells causes rapid growth cone collapse, neurite retraction, and transient rounding of the cell body. These shape changes appear to be driven by receptor-mediated contraction of the cortical actomyosin system independent of classic second messengers. Treatment of the cells with Clostridium botulinum C3 exoenzyme, which ADP-ribosylates and thereby inactivates the Rho small GTP-binding protein, inhibits LPA- and TRP-induced force generation and subsequent shape changes. C3 also inhibits LPA-induced neurite retraction in PC12 cells. Biochemical analysis reveals that the ADP-ribosylated substrate is RhoA. Prolonged C3 treatment of cells maintained in 10% serum induces the phenotype of serum-starved cells, with initial cell flattening being followed by neurite outgrowth; such C3-differentiated cells fail to retract their neurites in response to agonists. We conclude that RhoA is essential for receptor-mediated force generation and ensuing neurite retraction in N1E-115 and PC12 cells, and that inactivation of RhoA by ADP-ribosylation abolishes actomyosin contractility and promotes neurite outgrowth.     

Neuroserpin regulates neurite outgrowth in nerve growth factor-treated PC12 cells

Neuroserpin is a serine protease inhibitor widely expressed in the developing and adult nervous systems and implicated in the regulation of proteases involved in processes such as synaptic plasticity, neuronal migration and axogenesis. We have analysed the effect of neuroserpin on growth factor-induced neurite outgrowth in PC12 cells. We show that small changes in neuroserpin expression result in changes to the number of cells extending neurites and total neurite length following NGF treatment. Increased expression of neuroserpin resulted in a decrease in the number of cells extending neurites and a reduction in total free neurite length whereas reduced levels of neuroserpin led to a small increase in the number of neurite extending cells and a significant increase in total free neurite length compared to the parent cell line. Neuroserpin also altered the response of PC12 cells to bFGF and EGF treatment. Neuroserpin was localised to dense cored secretory vesicles in PC12 cells but was unable to complex with its likely enzyme target, tissue plasminogen activator at the acidic pH found in these vesicles. These data suggest that modulation of neuroserpin levels at the extending neurite growth cone may play an important role in regulating axonal growth.     

Extracellular matrix allows PC12 neurite elongation in the absence of microtubules

Several groups have shown that PC12 will extend microtubule-containing neurites on extracellular matrix (ECM) with no lag period in the absence of nerve growth factor. This is in contrast to nerve growth factor (NGF)-induced neurite outgrowth that occurs with a lag period of several days. During this lag period, increased synthesis or activation of assembly-promoting microtubule-associated proteins (MAPs) occurs and is apparently required for neurite extension. We investigated the growth and microtubule (MT) content of PC12 neurites grown on ECM in the presence or absence of inhibitors of neurite outgrowth. On ECM, neurites of cells with or without prior exposure to NGF contain a normal density of MTs, but frequently contain unusual loops of MTs in their termini that may indicate increased MT assembly. On ECM, neurites extend from PC12 cells in the presence of 10 microM LiCl at significantly higher frequency than on polylysine. On other substrates, LiCl inhibits neurite outgrowth, apparently by inhibiting phosphorylation of particular MAPs (Burstein, D. E., P. J. Seeley, and L. A. Greene. 1985. J. Cell Biol. 101:862-870). Although 35-45% of 60 Li(+)-neurites examined were found to contain a normal array of MTs, 25-30% were found to have a MT density approximately 15% of normal. The remaining 30% of these neurites were found to be nearly devoid of MTs, containing only occasional, ambiguous, short tubular elements. We also found that neurites would extend on ECM in the presence of the microtubule depolymerizing drug, nocodazole. At 0.1 micrograms/ml nocodazole, cells on ECM produce neurites that contain a normal density of MTs. This is in contrast to the lack of neurite outgrowth and retraction of extant neurites that this dose produces in cells grown on polylysine. At 0.2 microgram/ml nocodazole, neurites again grew out in substantial number and four of five neurites examined ultrastructurally were found to be completely devoid of microtubules. We interpret these results by postulating that growth on ECM relieves the need for MTs to serve as compressive supports for neurite tension (Dennerll, T. J., H. C. Joshi, U. L. Steel, R. E. Buxbaum, and S. R. Heidemann. 1988. J. Cell Biol. 107:665). Because compression destabilizes MTs and favors disassembly, this would tend to increase MT assembly relative to other conditions, as we found. Additionally, if MTs are not needed as compressive supports, neurites could grow out in their absence, as we also observed.     

Enhanced expression and activation of CTP:phosphocholine cytidylyltransferase beta2 during neurite outgrowth

During differentiation neurons increase phospholipid biosynthesis to provide new membrane for neurite growth. We studied the regulation of phosphatidylcholine (PC) biosynthesis during differentiation of two neuronal cell lines: PC12 cells and Neuro2a cells. We hypothesized that in PC12 cells nerve growth factor (NGF) would up-regulate the activity and expression of the rate-limiting enzyme in PC biosynthesis, CTP:phosphocholine cytidylyltransferase (CT). During neurite outgrowth, NGF doubled the amount of cellular PC and CT activity. CTbeta2 mRNA increased within 1 day of NGF application, prior to the formation of visible neurites, and continued to increase during neurite growth. When neurites retracted in response to NGF withdrawal, CTbeta2 mRNA, protein, and CT activity decreased. NGF specifically activated CTbeta2 by promoting its translocation from cytosol to membranes. In contrast, NGF did not alter CTalpha expression or translocation. The increase in both CTbeta2 mRNA and CT activity was inhibited by U0126, an inhibitor of mitogen-activated kinase/extracellular signal-regulated kinase kinase 1/2 (MEK1/2). In Neuro2a cells, retinoic acid significantly increased CT activity (by 54%) and increased CTbeta2 protein, coincident with neurite outgrowth but did not change CTalpha expression. Together, these data suggest that the CTbeta2 isoform of CT is specifically up-regulated and activated during neuronal differentiation to increase PC biosynthesis for growing neurites.     

Thy-1 antibody-triggered neurite outgrowth requires an influx of calcium into neurons via N- and L-type calcium channels

We present evidence that the neurite out-growth stimulated by the binding of Thy-1 antibodies to PC12 cells is mediated by calcium influx through both N- and L-type calcium channels. PC12 cells cultured on a noncellular substratum in the presence of NGF, or on a cellular substratum in the absence of NGF, responded to soluble Thy-1 antibody by extending longer neurites. The response required bivalent antibody and could be blocked by removing Thy-1 from the surface of PC12 cells with phosphatidylinositol specific phospholipase C. The response could also be blocked by reducing extracellular calcium to 0.25 mM, or by antagonists of L- and N-type calcium channels. Additionally, the response could be fully inhibited by preloading PC12 cells with BAPTA/AM which buffers changes in intracellular calcium. A heterotrimeric G-protein is also implicated in the pathway as the response could be fully inhibited by pertussis toxin. These data suggest that antibody-induced clustering of Thy-1 stimulates neurite outgrowth by activating a second messenger pathway that has previously been shown to underlie cell adhesion molecule (NCAM, N-cadherin, and L1), but not integrin or NGF-dependent neurite outgrowth.     

Changes in the Expression of β and Actins During Differentiation of PC 12 Cells

Cells of the rat pheochromocytoma line PC12 cease proliferation and develop neurites in response to nerve growth factor (NGF). Quantification of beta and gamma isoforms of nonmuscle actin in extracts of these differentiating cells showed that the beta:gamma ratio decreased from 1.30 +/- 0.05 to 0.99 +/- 0.05 after 6 days of NGF treatment. Cells treated with N6,O2-dibutyryl cyclic AMP (dbcAMP) also showed a shift in the ratio of beta:gamma isoforms, although few of these cells extended neurites. Administration of dbcAMP or both NGF and dbcAMP to cells accelerated the decrease in the beta:gamma actin isoform ratio relative to treatment with NGF alone. Those cells treated with both NGF and dbcAMP also showed an accelerated rate of neurite outgrowth. Suspension-grown PC12 cells treated with NGF showed neither an isoform ratio decrease nor neurite development. Our results suggest that either cyclic AMP may be a "second messenger" for NGF or it may effect the isoform ratio change by an independent mechanism. In addition, our data demonstrate an alteration in actin isoform expression, which accompanies the morphological differentiation of PC12 cells.     

cAMP promotes branching of laminin-induced neuronal processes

Laminin is a potent stimulator ofneurite outgrowth. We have examined the signal transduction events involved in the neuronal cell response to laminin. Cyclic nucleotides, calcium, and sodium-proton exchange do not appear to be required for the transduction of the laminin signal during neurite outgrowth. Direct measurement of cAMP and cGMP levels shows no changes in NG108-15 cells when cultured on laminin. Exogenous cAMP alone had no effect on either the rate of process formation or process length, but did alter the morphology of laminin-induced neurites. A four-fold increase in the number of branches per neurite and a two-to-three-fold increase in the number of neurites per cell were observed in both NG108-15 and PC12 cells cultured on laminin when either 8-BrcAMP or forskolin was added. The cAMP-induced branching was also observed when PC12 cells were cultured on a laminin-derived synthetic peptide (PA22-2), which contains the neurite-promoting amino acid sequence IKVAV. By immunofluorescence analysis with axonal or dendritic markers, the PC12 processes on laminin and PA22-2 were axonal, not dendritic, and the cAMP-induced morphological changes were due to axonal branching. These data demonstrate that changes in cAMP are not involved in laminin-mediated neurite outgrowth, but cAMP can modulate the effects of laminin.     

Varicones and Growth Cones: Two Neurite Terminals in PC12 Cells

The rat adrenal pheochromocytoma PC12 cell line is one of the traditional models for the study of neurite outgrowth and growth cone behavior. To clarify to what extent PC12 neurite terminals can be compared to neuronal growth cones, we have analyzed their morphology and protein distribution in fixed PC12 cells by immunocytochemistry. Our results show that that PC12 cells display a special kind of neurite terminal that includes a varicosity in close association with a growth cone. This hybrid terminal, or “varicone”, is characterized by the expression of specific markers not typically present in neuronal growth cones. For example, we show that calpain-2 is a specific marker of varicones and can be detected even before the neurite develops. Our data also shows that a fraction of PC12 neurites end in regular growth cones, which we have compared to hippocampal neurites as a control. We also report the extraordinary incidence of varicones in the literature referred to as “growth cones”. In summary, we provide evidence of two different kinds of neurite terminals in PC12 cells, including a PC12-specific terminal, which implies that care must be taken when using them as a model for neuronal growth cones or neurite outgrowth.     

Nerve Growth Factor Promotes Neurite Regeneration in PC12 Cells by Translational Control

Abstract: When PC12 cells are primed with nerve growth factor (NGF) for periods of ≥1 week, they acquire the ability to regenerate neurites rapidly in response to NGF. It is not known how NGF promotes this regeneration, but it does not require ongoing RNA synthesis. Previous studies have suggested that NGF directs the accumulation of precursor molecules that are rapidly assembled to form the regenerated neurites. To address the nature of these precursor molecules, we have treated PC12 cells with macromolecular synthesis inhibitors during the priming and regeneration phases of neurite growth. Here we show that NGF promotes neurite regeneration by inducing the synthesis of new proteins. These proteins are encoded by short-lived mRNAs that are generated during the NGF priming period. The isolation and identification of these mRNAs will allow a further understanding of how NGF promotes neurite regeneration.     

The neurite-initiating effect of microbial extracellular glycolipids in PC12 cells

The effects of several kinds of microbial extracellular glycolipids on neurite initiation in PC12 cells were examined. Addition of mannosylerythritol lipid-A (MEL-A), MEL-B, and sophorose lipid (SL) to PC12 cells caused significant neurite outgrowth. Other glycolipids, such as polyol lipid (PL), rhamnose lipid (RL), succinoyl trehalose lipid-A (STL-A) and STL-B caused no neurite-initiation. MEL-A increased acetylcholine esterase (AChE) activity to an extent similar to nerve growth factor (NGF). However, MEL-A induced one or two long neurites from the cell body, while NGF induced many neurites. In addition, MEL-A-induced differentiation was transient, and after 48 h, percentage of cells with neurites started to decrease in contrast to neurons induced by NGF, which occurred in a time-dependent manner. MEL-A could induce neurite outgrowth after treatment of PC12 cells with an anti-NGF receptor antibody that obstructed NGF action. These results indicate that MEL-A and NGF induce differentiation of PC12 cells through different mechanisms. This revised version was published online in August 2006 with corrections to the Cover Date.