A two-phase growth strategy in cultured neuronal networks as reflected by the distribution of neurite branching angles

Neurite outgrowth and branching patterns are instrumental in dictating the wiring diagram of developing neuronal networks. We study the self-organization of single cultured neurons into complex networks focusing on factors governing the branching of a neurite into its daughter branches. Neurite branching angles of insect ganglion neurons in vitro were comparatively measured in two neuronal categories: neurons in dense cultures that bifurcated under the presence of extrinsic (cellular environment) cues versus neurons in practical isolation that developed their neurites following predominantly intrinsic cues. Our experimental results were complemented by theoretical modeling and computer simulations. A preferred regime of branching angles was found in isolated neurons. A model based on biophysical constraints predicted a preferred bifurcation angle that was consistent with this range shown by our real neurons. In order to examine the origin of the preferred regime of angles we constructed simulations of neurite outgrowth in a developing network and compared the simulated developing neurons with our experimental results. We tested cost functions for neuronal growth that would be optimized at a specific regime of angles. Our results suggest two phases in the process of neuronal development. In the first, reflected by our isolated neurons, neurons are tuned to make first contact with a target cell as soon as possible, to minimize the time of growth. After contact is made, that is, after neuronal interconnections are formed, a second branching strategy is adopted, favoring higher efficiency in neurite length and volume. The two-phase development theory is discussed in relation to previous results.     

Dl-3-n-butylphthalide promotes neurite outgrowth of primary cortical neurons by Sonic Hedgehog signaling via upregulating Gap43

Neurite outgrowth is the basis for wiring during the development of the nervous system. Dl-3-n-butylphthalide (NBP) has been recognized as a promising treatment to improve behavioral, neurological and cognitive outcomes in ischemic stroke. However, little is known about the effect and mechanism of NBP on the neurite outgrowth. In this study, we used different methods to investigate the potential effects of NBP on the neurite extension and plasticity of immature and mature primary cortical neurons and explored the underlying mechanisms. Our results demonstrated that in immature and mature cortical neurons, NBP promoted the neurite length and intersections, increased neuritic arborization, elevated numbers of neurite branch and terminal points and improved neurite complexity and plasticity of neuronal development processes. Besides, our data revealed that NBP promoted neurite extension and branching partly by activating Shh signaling pathway via increasing Gap43 expression both in immature and mature primary cortical neurons. The present study provided new insights into the contribution of NBP in neuronal plasticity and unveiled a novel pathway to induce Gap43 expression in primary cortical neurons.     

Distinct molecular interactions mediate neuronal process outgrowth on non-neuronal cell surfaces and extracellular matrices

We have compared neurite outgrowth on extracellular matrix (ECM) constituents to outgrowth on glial and muscle cell surfaces. Embryonic chick ciliary ganglion (CG) neurons regenerate neurites rapidly on surfaces coated with laminin (LN), fibronectin (FN), conditioned media (CM) from several non-neuronal cell types that secrete LN, and on intact extracellular matrices. Neurite outgrowth on all of these substrates is blocked by two monoclonal antibodies, CSAT and JG22, that prevent the adhesion of many cells, including neurons, to the ECM constituents LN, FN, and collagen. Neurite outgrowth is inhibited even on mixed LN/poly-D-lysine substrates where neuronal attachment is independent of LN. Therefore, neuronal process outgrowth on extracellular matrices requires the function of neuronal cell surface molecules recognized by these antibodies. The surfaces of cultured astrocytes, Schwann cells, and skeletal myotubes also promote rapid process outgrowth from CG neurons. Neurite outgrowth on these surfaces, though, is not prevented by CSAT or JG22 antibodies. In addition, antibodies to a LN/proteoglycan complex that block neurite outgrowth on several LN-containing CM factors and on an ECM extract failed to inhibit cell surface-stimulated neurite outgrowth. After extraction with a nonionic detergent, Schwann cells and myotubes continue to support rapid neurite outgrowth. However, the activity associated with the detergent insoluble residue is blocked by CSAT and JG22 antibodies. Detergent extraction of astrocytes, in contrast, removes all neurite- promoting activity. These results provide evidence for at least two types of neuronal interactions with cells that promote neurite outgrowth. One involves adhesive proteins present in the ECM and ECM receptors on neurons. The second is mediated through detergent- extractable macromolecules present on non-neuronal cell surfaces and different, uncharacterized receptor(s) on neurons. Schwann cells and skeletal myotubes appear to promote neurite outgrowth by both mechanisms.     

Neuronal calcium sensor-1 modulation of optimal calcium level for neurite outgrowth

Neurite extension and branching are affected by activity-dependent modulation of intracellular Ca2+, such that an optimal window of [Ca2+] is required for outgrowth. Our understanding of the molecular mechanisms regulating this optimal [Ca2+]i remains unclear. Taking advantage of the large growth cone size of cultured primary neurons from pond snail Lymnaea stagnalis combined with dsRNA knockdown, we show that neuronal calcium sensor-1 (NCS-1) regulates neurite extension and branching, and activity-dependent Ca2+ signals in growth cones. An NCS-1 C-terminal peptide enhances only neurite branching and moderately reduces the Ca2+ signal in growth cones compared with dsRNA knockdown. Our findings suggest that at least two separate structural domains in NCS-1 independently regulate Ca2+ influx and neurite outgrowth, with the C-terminus specifically affecting branching. We describe a model in which NCS-1 regulates cytosolic Ca2+ around the optimal window level to differentially control neurite extension and branching.     

Phosphatidylcholine biosynthesis via CTP:phosphocholine cytidylyltransferase 2 facilitates neurite outgrowth and branching

Hallmarks of neuronal differentiation are neurite sprouting, extension, and branching. We previously showed that increased expression of CTP:phosphocholine cytidylyltransferase beta2 (CTbeta2), an isoform of a key phosphatidylcholine (PC) biosynthetic enzyme, accompanies neurite outgrowth (Carter, J. M., Waite, K. A., Campenot, R. B., Vance, J. E., and Vance, D. E. (2003) J. Biol. Chem. 278, 44988-44994). CTbeta2 mRNA is highly expressed in the brain. We show that CTbeta2 is abundant in axons of rat sympathetic neurons and retinal ganglion cells. We used RNA silencing to decrease CTbeta2 expression in PC12 cells differentiated by nerve growth factor. In CTbeta2-silenced cells, numbers of primary and secondary neurites were markedly reduced, suggesting that CTbeta2 facilitates neurite outgrowth and branching. However, the length of individual neurites was significantly increased, and the total amount of neuronal membrane was unchanged. Neurite branching of PC12 cells is known to be inhibited by activation of Akt and promoted by the Akt inhibitor LY294002. Our experiments showed that LY294002 increases neurite sprouting and branching in control PC12 cells but not in CTbeta2-deficient cells. CTbeta2 was not phosphorylated in vitro by Akt. However, inhibition of Cdk5 by roscovitine blocked CTbeta2 phosphorylation and reduced neurite outgrowth and branching. These results highlight the importance of CTbeta2 in neurons for promoting neurite outgrowth and branching and represent the first identification of a lipid biosynthetic enzyme that facilitates these functions.     

Effects of apolipoprotein E, beta-very low density lipoproteins, and cholesterol on the extension of neurites by rabbit dorsal root ganglion neurons in vitro.

Previous studies suggest that during nerve regeneration apoE acts as a lipid transport protein that assists in the rapid initial extension of axons and then in their myelination. To determine whether apoE and/or apoE-containing lipoproteins can modulate axon growth, we assessed their effect on the out-growth of neurites from neurons in mixed cultures of fetal rabbit dorsal root ganglion cells in vitro. Incubation with beta-very low density lipoprotein (beta-VLDL) particles, which are rich in apoE and cholesterol, increased neurite outgrowth and branching. Unesterified cholesterol added to the cultures had a similar, but less pronounced, effect. These data suggest that cholesterol might be the component responsible for the enhanced neurite growth. In contrast, purified, lipid-free apoE added to the cultures reduced neurite branching. Neurite branching was also reduced when purified apoE was added along with beta-VLDL or cholesterol; however, the striking finding was that under these conditions the neurites extended farther from the neuronal cell body. Dorsal root ganglion cells were examined for the presence of receptors for native and apoE-enriched beta-VLDL. Immunocytochemistry, ligand blots, 45Ca2+ blots, and studies of the interaction of the cells with fluorescent lipoproteins provided evidence of two types of receptors for apoE-containing lipoproteins on neurons: the low density lipoprotein (LDL) receptor, which binds native beta-VLDL, and the LDL receptor-related protein, which binds apoE-enriched beta-VLDL. These findings indicate that apoE may play two complementary roles in neurite outgrowth. When complexed with lipoproteins, apoE stimulates neurite growth by the receptor-mediated delivery of cholesterol and perhaps other components necessary for neurite outgrowth. When apoE as a free protein is added together with apoE-containing lipoproteins, apoE decreases neurite branching and promotes neurite extension away from the cell body. These actions, which would be complementary in promoting target-directed nerve growth in vivo, provide the first direct evidence that apoE and apoE-containing lipoproteins can modulate the outgrowth of neuronal processes.     

Roles of 5-HT on phase transition of neurite outgrowth in the identified serotoninergic neuron C1, <Emphasis Type="Italic">Helisoma trivolvis</Emphasis>

Neurite outgrowth is a morphological marker of neuronal differentiation and neuroregeneration, and the process includes four essential phases, namely initiation, elongation, guidance and cessation. Intrinsic and extrinsic signaling molecules seem to involve morphological changes of neurite outgrowth via various cellular signaling cascades phase transition. Although mechanisms associated with neurite outgrowth have been studied extensively, little is known about how phase transition is regulated during neurite outgrowth. 5-HT has long been studied with regard to its relationship to neurite outgrowth in invertebrate and vertebrate culture systems, and many studies have suggested 5-HT inhibits neurite elongation and growth cone motility, in particular, at the growing parts of neurite such as growth cones and filopodia. However, the underlying mechanisms need to be investigated. In this study, we investigated roles of 5-HT on neurite outgrowth using single serotonergic neurons C1 isolated from Helisoma trivolvis. We observed that 5-HT delayed phase transitions from initiation to elongation of neurite outgrowth. This study for the first time demonstrated that 5-HT has a critical role in phase-controlling mechanisms of neurite outgrowth in neuronal cell cultures.     

Modulation of Synapsin I Gene Expression in Rat Cortical Neurons by Extracellular Matrix

1. Neuronal differentiation depends on crosstalk between genetic program and environmental cues. In this study we tried to dissect this complex interplay by culturing neurons from fetal rat brain cortices in a chemically defined, neuron-specific, medium and on different substrata, either artificial (poly-D-lysine) or natural.2. Among the extracellular matrix compounds used in this study, two (collagen I and fibronectin) allowed only a weak attachment of cortical neurons to the substratum, while the others (collagen IV, laminin, and basal lamina from Engelbreth-Holm-Swarm sarcoma) allowed both firm attachment and moderate to extensive neurite outgrowth from neuronal cell bodies.3. By using synapsin I gene expression as a parameter of neuronal differentiation, we found that neurite outgrowth and neuronal differentiation are not linearly linked. Synapsin I gene expression, in fact, was maximal in neurons cultured on laminin, while the fastest neuritic outgrowth was recorded in cultures on poly-D-lysine.4. The data presented in this paper are consistent with the hypothesis that the extracellular matrix plays an active role in modulating the differentiative program of neurons.     

Target cell contact suppresses neurite outgrowth from soma-soma paired Lymnaea neurons

Neurite extension from developing and/or regenerating neurons is terminated on contact with their specific synaptic partner cells. However, a direct relationship between the effects of target cell contact on neurite outgrowth suppression and synapse formation has not yet been demonstrated. To determine whether physical/synaptic contacts affect neurite extension from cultured cells, we utilized soma-soma synapses between the identified Lymnaea neurons. A presynaptic cell (right pedal dorsal 1, RPeD1) was paired either with its postsynaptic partner cells (visceral dorsal 4, VD4, and Visceral dorsal 2, VD2) or with a non-target cell (visceral dorsal 1, VD1), and the interactions between their neurite outgrowth patterns and synapse formation were examined. Specifically, when cultured in brain conditioned medium (CM, contains growth-promoting factors), RPeD1, VD4, and VD2 exhibited robust neurite outgrowth within 12-24 h of their isolation. Synapses, similar to those seen in vivo, developed between the neurites of these cells. RPeD1 did not, however, synapse with its non-target cell VD1, despite extensive neuritic overlap between the cells. When placed in a soma-soma configuration (somata juxtaposed against each other), appropriate synapses developed between the somata of RPeD1 and VD4 (inhibitory) and between RPeD1 and VD2 (excitatory). Interestingly, pairing RPeD1 with either of its synaptic partner (VD4 or VD2) resulted in a complete suppression of neurite outgrowth from both pre- and postsynaptic neurons, even though the cells were cultured in CM. A single cell in the same dish, however, extended elaborate neurites. Similarly, a postsynaptic cell (VD4) contact suppressed the rate of neurite extension from a previously sprouted RPeD1. This suppression of the presynaptic growth cone motility was also target cell contact specific. The neurite suppression from soma-soma paired cells was transient, and neuronal sprouting began after a delay of 48-72 h. In contrast, when paired with VD1, both RPeD1 and this non-target cell exhibited robust neurite outgrowth. We demonstrate that this neurite suppression from soma-soma paired cells was target cell contact/synapse specific and Ca(2+) dependent. Specifically, soma-soma pairing in CM containing either lower external Ca(2+) concentration (50% of its control level) or Cd(2+) resulted in robust neurite outgrowth from both cells; however, the incidence of synapse formation between the paired cells was significantly reduced. Taken together, our data show that contact (physical and/or synaptic) between synaptic partners strongly influence neurite outgrowth patterns of both pre- and postsynaptic neurons in a time-dependent and cell-specific manner. Moreover, our data also suggest that neurite outgrowth and synapse formation are differentially regulated by external Ca(2+) concentration.     

Ral GTPases regulate neurite branching through GAP-43 and the exocyst complex

Neurite branching is essential for the establishment of appropriate neuronal connections during development and regeneration. We identify the small GTPase Ral as a mediator of neurite branching. Active Ral promotes neurite branching in cortical and sympathetic neurons, whereas Ral inhibition decreases laminin-induced branching. In addition, depletion of endogenous Ral by RNA interference decreases branching in cortical neurons. The two Ral isoforms, RalA and -B, promote branching through distinct pathways, involving the exocyst complex and phospholipase D, respectively. Finally, Ral-dependent branching is mediated by protein kinase C-dependent phosphorylation of 43-kD growth-associated protein, a crucial molecule involved in pathfinding, plasticity, and regeneration. These findings highlight an important role for Ral in the regulation of neuronal morphology.     

Metalloprotease-induced ectodomain shedding of neural cell adhesion molecule (NCAM)

Transmembrane forms of neural cell adhesion molecule (NCAM140, NCAM180(1)) are key regulators of neuronal development. The extracellular domain of NCAM can occur as a soluble protein in normal brain, and its levels are elevated in neuropsychiatric disorders, such as schizophrenia; however the mechanism of ectodomain release is obscure. Ectodomain shedding of NCAM140, releasing a fragment of 115 kD, was found to be induced in NCAM-transfected L-fibroblasts by the tyrosine phosphatase inhibitor pervanadate, but not phorbol esters. Pervanadate-induced shedding was mediated by a disintegrin metalloprotease (ADAM), regulated by ERK1/2 MAP kinase. In primary cortical neurons, NCAM was shed at high levels, and the metalloprotease inhibitor GM6001 significantly increased NCAM-dependent neurite branching and outgrowth. Moreover, NCAM-dependent neurite outgrowth and branching were inhibited in neurons isolated from a transgenic mouse model of NCAM shedding. These results suggest that regulated metalloprotease-induced ectodomain shedding of NCAM down-regulates neurite branching and neurite outgrowth. Thus, increased levels of soluble NCAM in schizophrenic brain have the potential to impair neuronal connectivity.     

Essential role of ERK activation in neurite outgrowth induced by α-lipoic acid

Background

Neurite outgrowth is an important aspect of neuronal plasticity and regeneration after neuronal injury. Alpha-lipoic acid (LA) has been used as a therapeutic approach for a variety of neural disorders. We recently reported that LA prevents local anesthetics-induced neurite loss. In this study, we hypothesized that LA administration promotes neurite outgrowth.

Methods

To test our hypothesis, we treated mouse neuroblastoma N2a cells and primary neurons with LA. Neurite outgrowth was evaluated by examination of morphological changes and by immunocytochemistry for β-tubulin-3. ROS production was examined, as were the phosphorylation levels of ERK and Akt. In separate experiments, we determined the effects of the inhibition of ERK or PI3K/Akt as well as ROS production on LA-induced neurite outgrowth.

Results

LA promoted significantly neurite outgrowth in a time- and concentration-dependent manner. LA stimulation significantly increased the phosphorylation levels of both Akt and ERK and transiently induced ROS production. PI3K/Akt inhibition did not affect LA-induced neurite outgrowth. However, the inhibition of ERK activation completely abolished LA-induced neurite outgrowth. Importantly, the prevention of ROS production by antioxidants attenuated LA-stimulated ERK activation and completely abolished LA-promoted neurite outgrowth.

Conclusion

Our data suggest that LA stimulates neurite outgrowth through the activation of ERK signaling, an effect mediated through a ROS-dependent mechanism.     

N-cadherin, NCAM, and integrins promote retinal neurite outgrowth on astrocytes in vitro

Retinal ganglion neurons extend axons that grow along astroglial cell surfaces in the developing optic pathway. To identify the molecules that may mediate axon extension in vivo, antibodies to neuronal cell surface proteins were tested for their effects on neurite outgrowth by embryonic chick retinal neurons cultured on astrocyte monolayers. Neurite outgrowth by retinal neurons from embryonic day 7 (E7) and E11 chick embryos depended on the function of a calcium-dependent cell adhesion molecule (N-cadherin) and beta 1-class integrin extracellular matrix receptors. The inhibitory effects of either antibody on process extension could not be accounted for by a reduction in the attachment of neurons to astrocytes. The role of a third cell adhesion molecule, NCAM, changed during development. Anti-NCAM had no detectable inhibitory effects on neurite outgrowth by E7 retinal neurons. In contrast, E11 retinal neurite outgrowth was strongly dependent on NCAM function. Thus, N-cadherin, integrins, and NCAM are likely to regulate axon extension in the optic pathway, and their relative importance varies with developmental age.     

Target cell contact suppresses neurite outgrowth from soma–soma paired Lymnaea neurons

Neurite extension from developing and/or regenerating neurons is terminated on contact with their specific synaptic partner cells. However, a direct relationship between the effects of target cell contact on neurite outgrowth suppression and synapse formation has not yet been demonstrated. To determine whether physical/synaptic contacts affect neurite extension from cultured cells, we utilized soma–soma synapses between the identified Lymnaea neurons. A presynaptic cell (right pedal dorsal 1, RPeD1) was paired either with its postsynaptic partner cells (visceral dorsal 4, VD4, and Visceral dorsal 2, VD2) or with a non‐target cell (visceral dorsal 1, VD1), and the interactions between their neurite outgrowth patterns and synapse formation were examined. Specifically, when cultured in brain conditioned medium (CM, contains growth‐promoting factors), RPeD1, VD4, and VD2 exhibited robust neurite outgrowth within 12–24 h of their isolation. Synapses, similar to those seen in vivo, developed between the neurites of these cells. RPeD1 did not, however, synapse with its non–target cell VD1, despite extensive neuritic overlap between the cells. When placed in a soma–soma configuration (somata juxtaposed against each other), appropriate synapses developed between the somata of RPeD1 and VD4 (inhibitory) and between RPeD1 and VD2 (excitatory). Interestingly, pairing RPeD1 with either of its synaptic partner (VD4 or VD2) resulted in a complete suppression of neurite outgrowth from both pre‐ and postsynaptic neurons, even though the cells were cultured in CM. A single cell in the same dish, however, extended elaborate neurites. Similarly, a postsynaptic cell (VD4) contact suppressed the rate of neurite extension from a previously sprouted RPeD1. This suppression of the presynaptic growth cone motility was also target cell contact specific. The neurite suppression from soma–soma paired cells was transient, and neuronal sprouting began after a delay of 48–72 h. In contrast, when paired with VD1, both RPeD1 and this non‐target cell exhibited robust neurite outgrowth. We demonstrate that this neurite suppression from soma–soma paired cells was target cell contact/synapse specific and Ca²⁺ dependent. Specifically, soma–soma pairing in CM containing either lower external Ca²⁺ concentration (50% of its control level) or Cd²⁺ resulted in robust neurite outgrowth from both cells; however, the incidence of synapse formation between the paired cells was significantly reduced. Taken together, our data show that contact (physical and/or synaptic) between synaptic partners strongly influence neurite outgrowth patterns of both pre‐ and postsynaptic neurons in a time‐dependent and cell‐specific manner. Moreover, our data also suggest that neurite outgrowth and synapse formation are differentially regulated by external Ca²⁺ concentration. © 2000 John Wiley & Sons, Inc. J Neurobiol 42: 357–369, 2000     

Metalloprotease‐induced ectodomain shedding of neural cell adhesion molecule (NCAM)

Transmembrane forms of neural cell adhesion molecule (NCAM140, NCAM180¹) are key regulators of neuronal development. The extracellular domain of NCAM can occur as a soluble protein in normal brain, and its levels are elevated in neuropsychiatric disorders, such as schizophrenia; however the mechanism of ectodomain release is obscure. Ectodomain shedding of NCAM140, releasing a fragment of 115 kD, was found to be induced in NCAM‐transfected L‐fibroblasts by the tyrosine phosphatase inhibitor pervanadate, but not phorbol esters. Pervanadate‐induced shedding was mediated by a disintegrin metalloprotease (ADAM), regulated by ERK1/2 MAP kinase. In primary cortical neurons, NCAM was shed at high levels, and the metalloprotease inhibitor GM6001 significantly increased NCAM‐dependent neurite branching and outgrowth. Moreover, NCAM‐dependent neurite outgrowth and branching were inhibited in neurons isolated from a transgenic mouse model of NCAM shedding. These results suggest that regulated metalloprotease‐induced ectodomain shedding of NCAM down‐regulates neurite branching and neurite outgrowth. Thus, increased levels of soluble NCAM in schizophrenic brain have the potential to impair neuronal connectivity. © 2006 Wiley Periodicals, Inc. J Neurobiol, 2006     

Computer vision profiling of neurite outgrowth dynamics reveals spatiotemporal modularity of Rho GTPase signaling

Rho guanosine triphosphatases (GTPases) control the cytoskeletal dynamics that power neurite outgrowth. This process consists of dynamic neurite initiation, elongation, retraction, and branching cycles that are likely to be regulated by specific spatiotemporal signaling networks, which cannot be resolved with static, steady-state assays. We present NeuriteTracker, a computer-vision approach to automatically segment and track neuronal morphodynamics in time-lapse datasets. Feature extraction then quantifies dynamic neurite outgrowth phenotypes. We identify a set of stereotypic neurite outgrowth morphodynamic behaviors in a cultured neuronal cell system. Systematic RNA interference perturbation of a Rho GTPase interactome consisting of 219 proteins reveals a limited set of morphodynamic phenotypes. As proof of concept, we show that loss of function of two distinct RhoA-specific GTPase-activating proteins (GAPs) leads to opposite neurite outgrowth phenotypes. Imaging of RhoA activation dynamics indicates that both GAPs regulate different spatiotemporal Rho GTPase pools, with distinct functions. Our results provide a starting point to dissect spatiotemporal Rho GTPase signaling networks that regulate neurite outgrowth.     

miR-124-regulated RhoG reduces neuronal process complexity via ELMO/Dock180/Rac1 and Cdc42 signalling

The small GTPase RhoG plays a central role in actin remodelling during diverse biological processes such as neurite outgrowth, cell migration, phagocytosis of apoptotic cells, and the invasion of pathogenic bacteria. Although it is known that RhoG stimulates neurite outgrowth in the rat pheochromocytoma PC12 cell line, neither the physiological function nor the regulation of this GTPase in neuronal differentiation is clear. Here, we identify RhoG as an inhibitor of neuronal process complexity, which is regulated by the microRNA miR-124. We find that RhoG inhibits dendritic branching in hippocampal neurons in vitro and in vivo. RhoG also inhibits axonal branching, acting via an ELMO/Dock180/Rac1 signalling pathway. However, RhoG inhibits dendritic branching dependent on the small GTPase Cdc42. Finally, we show that the expression of RhoG in neurons is suppressed by the CNS-specific microRNA miR-124 and connect the regulation of RhoG expression by miR-124 to the stimulation of neuronal process complexity. Thus, RhoG emerges as a cellular conductor of Rac1 and Cdc42 activity, in turn regulated by miR-124 to control axonal and dendritic branching.     

Rho‐associated kinase (ROCK) inhibitor,Y27632, promotes neurite outgrowth in PC12 cells in the absence of NGF

Neurite extension and retraction are very important processes in the formation of neuronal networks. A strategy for fostering axonal regrowth/regeneration of injured adult neurons is attractive therapeutically for various diseases such as traumatic brain injury, stroke and Alzheimer's disease. The Rho family of small GTPases, including Rac and Cdc42 have been shown to be involved in promoting neurite outgrowth. On the other hand, activation of RhoA induces collapse of growth cone and retraction of neurites. Rho‐associated kinase (ROCK) an effector molecule of RhoA, is downstream of a number of axonal outgrowth and growth cone collapse inhibition mechanisms. In the present study, we sought to identify the role of ROCK in neurite outgrowth in PC12 cells. Y27632, a specific inhibitor of ROCK, induced a robust increase in neurite outgrowth in these cells within 24–48 h as visualized by phase contrast microscopy. Staining with FITC‐tubulin or phalloidin show extended neurites in PC12 cells treated with Y27632, comparable to that with 100 ng/mL of NGF. Assessment of other biochemical markers of neurite outgrowth such as GAP43, neurofilament and tyrosine hydroxylase phosphorylation further indicates that inhibition of ROCK in PC12 cells causes differentiation of these cells to a neuronal phenotype.