Tau inhibits anterograde axonal transport and perturbs stability in growing axonal neurites in part by displacing kinesin cargo: neurofilaments attenuate tau-mediated neurite instability

Overexpression of tau compromises axonal transport and induces retraction of growing neurites. We tested the hypothesis that increased stability provided by neurofilaments (NFs) may prevent axonal retraction. NB2a/d1 cells were differentiated for 3 days, at which time phosphorylated NFs appear and for 14 days, which induces continued neurite elongation and further phospho-NF accumulation. Cultures were transfected with a construct that expresses full-length, 4-repeat tau. Consistent with prior studies, overexpression of tau induced retraction of day three axonal neurites even following treatment with the microtubule-stabilizing drug taxol. Axonal neurites of day 14 cells were more resistant to tau-mediated retraction. To test whether or not this resistance was derived from their additional NF content, day 3 cultures were co-transfected with constructs expressing tau and NF-M (which increases overall axonal NFs). Overexpression of NF-M attenuated tau-mediated retraction of day 3 axonal neurites. By contrast, co-transfection with constructs expressing tau and vimentin (which increases axonal neurites length) did not attenuate tau-mediated neurite retraction. Co-precipitation experiments indicate that tau is a cargo of kinesin, and that tau overexpression may displace other kinesin-based cargo, including both critical cytoskeletal proteins and organelles. However, cultures simultaneously transfected with constructs expressing NF-M and tau, the level of examined vesicles was maintained. These collectively indicate that NFs stabilize developing axonal neurites and can counteract the destabilizing force resulting from overexpression of tau, and underscore that the development and stabilization of axonal neurites is dependent upon a balance of cytoskeletal elements.     

Lysophosphatidic Acid-Induced Neurite Retraction in PC12 Cells: Control by Phosphoinositide-Ca2+ Signaling and Rho

Abstract: The endogenous phospholipid mediator lysophosphatidic acid (LPA) caused growth cone collapse, neurite retraction, and cell flattening in differentiated PC12 cells. Neurite retraction was blocked by cytochalasin B and ADP-ribosylation of the small-molecular-weight G protein Rho by the Clostridium botulinum C-3 toxin. LPA induced a transient rise in the level of inositol 1,4,5-trisphosphate, and retraction was blocked by inhibitors of phospholipase β. Repeated application of LPA elicited homologous desensitization of the Ca²⁺ mobilization response. The activation of the phosphoinositide (PIP)-Ca²⁺ second messenger system played a permissive role in the morphoregulatory response. Blockers of protein kinase C—chelerythrine, a myristoylated pseudosubstrate peptide, staurosporine, and depletion of protein kinase C from the cells by long-term phorbol ester treatment—all diminished neurite retraction by interfering with LPA-induced Ca²⁺ mobilization, which was required for the withdrawal of neurites. A brief 15-min treatment with 4β-phorbol 12-myristate 13-acetate also blocked retraction and Ca²⁺ mobilization, by inactivating the LPA receptor. Inhibition of protein tyrosine phosphorylation by herbimycin diminished retraction. Although activation of the PIP-Ca²⁺ second messenger system appears necessary for the Rho-mediated rearrangements of the actin cytoskeleton, bradykinin, which activates similar signaling events, failed to cause retraction, indicating that a yet unidentified novel mechanism is also involved in the LPA-induced morphoregulatory response.     

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.     

Regulation of SNAREs by tomosyn and ROCK: implication in extension and retraction of neurites

Extension of neurites requires the SNARE-dependent fusion of plasmalemmal precursor vesicles with the plasma membrane of growth cones. Here, we show that tomosyn localizes at the palm of growth cones and inhibits the fusion of the vesicles there, thus promoting transport of the vesicles to the plasma membrane of the leading edges of growth cones. Tomosyn localizes because ROCK activated by Rho small G protein phosphorylates syntaxin-1, which increases the affinity of syntaxin-1 for tomosyn and forms a stable complex with tomosyn, resulting in inhibition of the formation of the SNARE complex. In retraction of neurites, tomosyn localizes all over the edges of the neurites and inhibits fusion of the vesicles with the plasma membrane. Thus, tomosyn demarcates the plasma membrane by binding to syntaxin-1 phosphorylated by ROCK, and thereby regulates extension and retraction of neurites.     

Cytochalasin separates microtubule disassembly from loss of asymmetric morphology

When neuroblastoma cells bearing neurites are incubated with colchicine or Nocodazole, the cytoplasmic microtubules are depolymerized and concomitantly the neurites retract. We report here that cytochalasin separates the two effects of these drugs: it quantitatively inhibits neurite retraction but does not inhibit microtubule assembly. The neurites that remain contain intermediate filaments and actin but are devoid of microtubules. Depletion of cellular ATP also blocks neurite retraction induced by colchicine or Nocodazole, but some assembled microtubules persist under these conditions. The results suggest that neurite retraction is an active cell process.     

Rapid retraction of neurites by sensory neurons in response to increased concentrations of nerve growth factor

The phenomenon of growth cone (GC) and neurite retraction resulting from a rapid incrase in concentration of the trophic molecule NGF was studied. Neurite outgrowth from explants of 8-d chick embryo dorsal root ganglia was achieved at very low NGF concentrations with heart conditioned medium during overnight culture. Quickly incrasing the NGF concentration in the growth medium dramatically affected GC and neurite morphology: the majority of GCs and neurites collapsed and retracted towards the cell body over a course of approximately 2-5 min. Retraction was elicited by increasing NGF levels from 0 to 0.05 ng/ml to as little as 0.5 ng/ml but did not occur if the NGF concentration during the initial overnight culture period exceeded 0.8 ng/ml, regardless of how much the concentration was elevated. Similar concentration changes of cytochrome c or insulin did nt result in retraction. Neurites that had been separated from their cell bodies by cutting close to their exit from the explant still retracted when NGF levels were raised. Cytochalasin B reversible inhibits retraction, whereas colchicine allows retraction to occur. Observation of cell- substratum adhesion during retraction revealed that some adhesion points remain during retraction and that they correspond to the ends of NGF leels and that it may involve microfilaments in the neurite cytoskeleton. The NGF concentration changes that elicit neurite retraction suggest that a primary event in retraction may be increased occupancy of a high-affinity NGF receptor on neurites.     

Spinal neurite reabsorption and regrowth in vitro depend on the polarity of an applied electric field

Retraction and regrowth of frog neural tube neurites have been studied in vitro in control cultures and in the presence of a small, continuously applied electrical field. In control cultures, some degree of retraction was seen in 39% of neurites while 7% were reabsorbed completely. Reabsorption of anodal-facing neurites was at least twice as common, with 67% showing some retraction and 17% almost totally reabsorbed. Cathodal-facing neurites were spared from retraction. Following extreme reabsorption of anodal-facing neurites, reversal of the electric field promoted regeneration in 47% (9/19) of cases studied. growth cone morphology also was determined by the polarity of the applied field. Anodal-facing growth cones had fewer filopodia than cathodal-facing growth cones sharing the same cell body. Field reversal induced a polarity-specific change in filopodia number on individual growth cones: a shift from anodal to cathodal increased filopodia numbers and vice versa. Some possible mechanisms involved and the significance of these results are discussed.     

Respective roles of neurofilaments, microtubules, MAP1B, and tau in neurite outgrowth and stabilization.

The respective roles of neurofilaments (NFs), microtubules (MTs), and the microtubule-associated proteins (MAPs) MAP 1B and tau on neurite outgrowth and stabilization were probed by the intracellular delivery of specific antisera into transiently permeabilized NB2a/d1 cells during treatment with dbcAMP. Intracellular delivery of antisera specific for the low (NF-L), middle (NF-M), or extensively phosphorylated high (NF-H) molecular weight subunits did not prevent initial neurite elaboration, nor did it induce retraction of existing neurites elaborated by cells that had been previously treated for 1 d with dbcAMP. By contrast, intracellular delivery of antisera directed against tubulin reduced the percentage of cells with neurites at both these time points. Intracellular delivery of anti-NF-L and anti-NF-M antisera did not induce retraction in cells treated with dbcAMP for 3 d. However, intracellular delivery of antisera directed against extensively phosphorylated NF-H, MAP1B, tau, or tubulin induced similar levels of neurite retraction at this time. Intracellular delivery of monoclonal antibodies (RT97 or SMI-31) directed against phosphorylated NF-H induced neurite retraction in cell treated with dbcAMP for 3 d; a monoclonal antibody (SMI-32) directed against nonphosphorylated NF-H did not induce neurite retraction at this time. By contrast, none of the above antisera induced retraction of neurites in cells treated with dbcAMP for 7 d. Neurites develop resistance to retraction by colchicine, first detectable in some neurites after 3 d and in the majority of neurites after 7 d of dbcAMP treatment. We therefore examined whether or not colchicine resistance was compromised by intracellular delivery of the above antisera. Colchicine treatment resulted in rapid neurite retraction after intracellular delivery of antisera directed against extensively phosphorylated NF-H, MAP1B, or tau into cells that had previously been treated with dbcAMP for 7 d. By contrast, colchicine resistance was not compromised by the intracellular delivery of antisera directed against NF-L, NF-M, or tubulin. These findings support previous studies indicating that MT polymerization mediates certain aspects of axonal neurite outgrowth and suggest that NFs do not directly participate in these events. These findings further suggest that NFs function in stabilization of the axonal cytoskeleton, apparently by interactions among NFs and MTs that are mediated by NF-H and MAPs.     

Evidence that calcium may control neurite outgrowth by regulating the stability of actin filaments

We investigated the effects of calcium removal and calcium ionophores on the behavior and ultrastructure of cultured chick dorsal root ganglia (DRG) neurons to identify possible mechanisms by which calcium might regulate neurite outgrowth. Both calcium removal and the addition of calcium ionophores A23187 or ionomycin blocked outgrowth in previously elongating neurites, although in the case of calcium ionophores, changes in growth cone shape and retraction of neurites were also observed. Treatment with calcium ionophores significantly increased growth cone calcium. The ability of the microtubule stabilizing agent taxol to block A23187-induced neurite retraction and the ability of the actin stabilizing agent phalloidin to reverse both A23187-induced growth cone collapse and neurite retraction suggested that calcium acted on the cytoskeleton. Whole mount electron micrographs revealed an apparent disruption of actin filaments in the periphery (but not filopodia) of growth cones that were exposed to calcium ionophores in medium with normal calcium concentrations. This effect was not seen in cells treated with calcium ionophores in calcium-free medium or cells treated with the monovalent cation ionophore monensin, indicating that these effects were calcium specific. Ultrastructure of Triton X-100 extracted whole mounts further indicated that both microtubules and microfilaments may be more stable or extraction resistant after treatments which lower intracellular calcium. Taken together, the data suggest that calcium may control neurite elongation at least in part by regulating actin filament stability, and support a model for neurite outgrowth involving a balance between assembly and disassembly of the cytoskeleton.     

Proteolytic regulation of neurite outgrowth from neuroblastoma cells by thrombin and protease nexin-1

This review summarizes studies on the reciprocal regulation of neuroblastoma neurite outgrowth by thrombin and protease nexin-1 (PN-1). PN-1 recently was shown to possess the same deduced amino acid sequence as the glial-derived neurite-promoting factor. The neurite outgrowth activity of PN-1 depends on its ability to inhibit thrombin. Thrombin not only blocks the neurite outgrowth activity of PN-1, but it also brings about neurite retraction in the presence of PN-1. Thrombin also produces neurite retraction in the absence of PN-1 and other regulatory factors. This suggests that its activity is due to a direct action on cells. The neurite retraction by thrombin depends on its proteolytic activity. It does not occur with the other serine proteases that have been tested, indicating that it is a specific effect and is not due to a general proteolytic effect that could detach neurites from the culture dish. Serum brings about neurite retraction in certain neuroblastoma cells and primary neuronal cultures; most of this activity is due to residual thrombin in the serum. Together, these results suggest that PN-1 and thrombin (or a thrombin-like protease) play a role in regulation of neurite outgrowth.     

Alterations in dynamics of microtubule assembly during axonal neuritogenesis in NB2a/d1 cells

During dibutyryl cyclic AMP (dbcAMP)-mediated differentiation, axonal neurites elaborated by mouse NB2a/d1 neuroblastoma cells are initially colchicine-labile but attain colchicine-stability after 7 days. To examine whether or not differences in tubulin subunit turnover could account for the development of colchicine-stability, anti-tubulin antibodies were delivered into NB2a/d1 cells at various times during dbcAMP-mediated neurite outgrowth. These antibodies prevented initial neurite elaboration, and induced neurite retraction in cells treated with dbcAMP for up to 3 days, but did not induce neurite retraction for cells treated for 7 days. We conclude that a less dynamic, more slowly-turning over population of microtubules develops within neurites of cells treated with dbcAMP for 7 days.     

Orexin A Inhibits Propofol-Induced Neurite Retraction by a Phospholipase D/Protein Kinase Cε-Dependent Mechanism in Neurons

Background

The intravenous anaesthetic propofol retracts neurites and reverses the transport of vesicles in rat cortical neurons. Orexin A (OA) is an endogenous neuropeptide regulating wakefulness and may counterbalance anaesthesia. We aim to investigate if OA interacts with anaesthetics by inhibition of the propofol-induced neurite retraction.

Methods

In primary cortical cell cultures from newborn rats’ brains, live cell light microscopy was used to measure neurite retraction after propofol (2 µM) treatment with or without OA (10 nM) application. The intracellular signalling involved was tested using a protein kinase C (PKC) activator [phorbol 12-myristate 13-acetate (PMA)] and inhibitors of Rho-kinase (HA-1077), phospholipase D (PLD) [5-fluoro-2-indolyl des-chlorohalopemide (FIPI)], PKC (staurosporine), and a PKCε translocation inhibitor peptide. Changes in PKCε Ser⁷²⁹ phosphorylation were detected with Western blot.

Results

The neurite retraction induced by propofol is blocked by Rho-kinase and PMA. OA blocks neurite retraction induced by propofol, and this inhibitory effect could be prevented by FIPI, staurosporine and PKCε translocation inhibitor peptide. OA increases via PLD and propofol decreases PKCε Ser⁷²⁹ phosphorylation, a crucial step in the activation of PKCε.

Conclusions

Rho-kinase is essential for propofol-induced neurite retraction in cortical neuronal cells. Activation of PKC inhibits neurite retraction caused by propofol. OA blocks propofol-induced neurite retraction by a PLD/PKCε-mediated pathway, and PKCε maybe the key enzyme where the wakefulness and anaesthesia signal pathways converge.     

Autonomous and non-autonomous regulation of mammalian neurite development by Notch1 and Delta1

BACKGROUND: On the basis of experiments suggesting that Notch and Delta have a role in axonal development in Drosophila neurons, we studied the ability of components of the Notch signaling pathway to modulate neurite formation in mammalian neuroblastoma cells in vitro. RESULTS: We observed that N2a neuroblastoma cells expressing an activated form of Notch, Notch1(IC), produced shorter neurites compared with controls, whereas N2a cell lines expressing a dominant-negative Notch1 or a dominant-negative Delta1 construct extended longer neurites with a greater number of primary neurites. We then compared the effects on neurites of contacting Delta1 on another cell and of overexpression of Delta1 in the neurite-extending cell itself. We found that N2a cells co-cultured with Delta1-expressing quail cells produced fewer and shorter neuritic processes. On the other hand, high levels of Delta1 expressed in the N2a cells themselves stimulated neurite extension, increased numbers of primary neurites and induced expression of Jagged1 and Notch1. CONCLUSIONS: These studies show that Notch signals can antagonize neurite outgrowth and that repressing endogenous Notch signals enhances neurite outgrowth in neuroblastoma cells. Notch signals therefore act as regulators of neuritic extension in neuroblastoma cells. The response of neuritic processes to Delta1 expressed in the neurite was opposite to that to Delta1 contacted on another cell, however. These results suggest a model in which developing neurons determine their extent of process outgrowth on the basis of the opposing influences on Notch signals of ligands contacted on another cell and ligands expressed in the same cell.     

Steps in the formation of a bipolar outgrowth pattern by cultured neurons, and their substrate dependence.

We studied the steps in the formation of the bipolar outgrowth pattern of cultured adult Anterior Pagoda (AP) neurons of the leech growing on a central nervous system (CNS) homogenate as substrate. This pattern, which consists of two primary neurites directed in opposite directions plus some bifurcations, resembles their embryonic pattern but is different from the patterns they develop in culture on leech laminin or Concanavalin A as substrates. In eight neurons that were studied, one primary neurite formed and branched several hours before the second one. Time-lapse video analysis showed that between 12 and 36 h of growth, the more proximal branch of the early neurite migrated retrogradely, rotated, and formed the second primary branch. Both neurites elongated until the total neurite length reached 130-160 microm, when the elongation of primary neurites became synchronous with the retraction of secondary processes, suggesting competition. The substrate dependence of these events was tested by plating AP neurons on leech laminin. On this substrate AP neurons produced multiple independent primary neurites with branches. Retraction of some large branches was followed by their regrowth, and did not correlate with the changes in other neurites. We propose that the dynamics in the formation of the bipolar outgrowth pattern of AP neurons arise from inhibitory extracellular matrix molecules, which reduce the synthesis of precursors for neurite formation.     

Induction of neurites by the regulatory domains of PKCdelta and epsilon is counteracted by PKC catalytic activity and by the RhoA pathway

We have shown that protein kinase C (PKC) epsilon, independently of its kinase activity, via its regulatory domain (RD), induces neurites in neuroblastoma cells. This study was designed to evaluate whether the same effect is obtained in nonmalignant neural cells and to dissect mechanisms mediating the effect. Overexpression of PKCepsilon resulted in neurite induction in two immortalised neural cell lines (HiB5 and RN33B). Phorbol ester potentiated neurite outgrowth from PKCepsilon-overexpressing cells and led to neurite induction in cells overexpressing PKCdelta. The effects were potentiated by blocking the PKC catalytic activity with GF109203X. Furthermore, kinase-inactive PKCdelta induced more neurites than the wild-type isoform. The isolated regulatory domains of novel PKC isoforms also induced neurites. Experiments with PKCdelta-overexpressing HiB5 cells demonstrated that phorbol ester, even in the presence of a PKC inhibitor, led to a decrease in stress fibres, indicating an inactivation of RhoA. Active RhoA blocked PKC-induced neurite outgrowth, and inhibition of the RhoA effector ROCK led to neurite outgrowth. This demonstrates that neurite induction by the regulatory domain of PKCdelta can be counteracted by PKCdelta kinase activity, that PKC-induced neurite outgrowth is accompanied by stress fibre dismantling indicating an inactivation of RhoA, and that the RhoA pathway suppresses PKC-mediated neurite outgrowth.     

Force generation by cytoskeletal motor proteins as a regulator of axonal elongation and retraction

Axons elongate and retract in response to environmental signals during the development of the nervous system. There is broad agreement that these signals must affect the cytoskeleton to elicit bouts of elongation or retraction. Most contemporary studies have speculated that bouts of elongation involve polymerization of the cytoskeleton whereas bouts of retraction involve depolymerization of the cytoskeleton. Here we present an alternative view, namely that molecular motor proteins generate forces on the cytoskeletal polymers that can affect their distribution and configuration. In this view, bouts of axonal elongation involve net forward movement of cytoskeletal elements whereas bouts of retraction involve net backward movements. We propose that environmental cues elicit bouts of elongation or retraction via biochemical pathways that modulate the activities of relevant motors.     

Lysophosphatidic Acid-Induced Neurite Retraction in PC12 Cells: Neurite-Protective Effects of Cyclic AMP Signaling

Abstract: Effects of the cyclic AMP second messenger system were studied on the retraction of neurites elicited by the phospholipid mediator lysophosphatidic acid (LPA) in PC12 cells. LPA stimulation inhibited adenylyl cyclase, indicating that the LPA receptor couples to the heterotrimeric G_i proteins. However, pertussis toxin or expression of dominant negative Ras did not prevent neurite retraction. In contrast, cholera toxin, forskolin, and application of dibutyryl-cyclic AMP prevented neurite retraction. The neurite-protective effect of forskolin was blocked by Rp -adenosine 3',5'-phosphorothioate. Forskolin and dibutyryl-cyclic AMP both failed to protect neurites in A126-1B2 and 123.7 cells, which lack cyclic AMP-activated protein kinase. Data indicate that elevation of cyclic AMP levels triggers a cyclic AMP-activated protein kinase-dependent mechanism that opposes the functioning of the morphoregulatory signaling activated by LPA. ADP-ribosylation of Rho by the Clostridium botulinum C-3 toxin in 123.7 cells caused neuronal differentiation, indicated by neurite extension, and blocked LPA-induced neurite retraction. LPA activates G_q- and G_i-linked signaling in parallel; therefore, a morphoregulatory signaling network hypothesis is proposed versus the simplistic approach of a signaling pathway. The signaling network integrates the receptor-activated individual, sequential, and parallel signaling events into an interactive network whose individual components may fulfill required and permissive functions encoding the cellular response.     

Neurite Extension by Mouse Neuroblastoma: Evidence for Two Modes of Induction

The induction of neurite extension by mouse neuroblastoma cells in vitro can be reversed by adding colchicine or cytochalasin B (CB) to the culture medium. The relative sensitivity of the neurites to retraction is a function of the method used to induce extension. Cells incubated in the absence of serum or in the presence of serum and dibutyryl cyclic-AMP are sensitive to both colchicine and CB; cells incubated in serum-free medium containing either cycloheximide or dimethylsulphoxide are sensitive only to colchicine.