Influence of Folic Acid on Neural Connectivity during Dorsal Root Ganglion Neurogenesis.

The vitamin folic acid (FA) is essential for DNA synthesis, repair and methylation, and for methionine synthesis. Although it is necessary for neural development, recent studies suggest a possible link between excess maternal supplemental FA intake and adverse interferences with single-carbon metabolism and neural development. Insufficient FA early in brain development can lead to failure of the neural tube closure, but the consequences of too much intake have not been fully investigated. Plasma FA concentrations can increase greatly with dietary supplementation. To model the development of neural connectivity, we cultured dorsal root ganglia (DRGs) taken from 8-day-old chick embryos in a range of pteroylmonoglutamate (PteGlu, synthetic supplemental FA) concentrations. DRGs were cultured for 36 h, fixed and immunostained to reveal the locations of neural networks with synaptic vesicles. We found a concentration-dependent relationship with significant reduction in neurite length in PteGlu concentrations from 0.25 to 20 µM. The average total of stained synaptic areas surrounding each cultured DRG was significantly reduced as well. To further characterize the effects, we carried out time-lapse imaging of growth cones at terminals of extending neurites. We found that PteGlu reduced the area-changing activity of the growth cone, hindering its exploratory capabilities, along with a tendency to inhibit overall advancement, thus altering the ability to extend and form synapses. Our results show that PteGlu at 250 nM and higher reduces neurite extension and synapse formation in a dose-dependent manner during neurogenesis, and that its effect is mediated through inhibition of growth cone motility.

Polyphenols from green tea prevent antineuritogenic action of Nogo-A via 67-kDa laminin receptor and hydrogen peroxide.

Axonal regeneration after injury to the CNS is hampered by myelin-derived inhibitors, such as Nogo-A. Natural products, such as green tea, which are neuroprotective and safe for long-term therapy, would complement ongoing various pharmacological approaches. In this study, using nerve growth factor-differentiated neuronal-like Neuroscreen-1 cells, we show that extremely low concentrations of unfractionated green tea polyphenol mixture (GTPP) and its active ingredient, epigallocatechin-3-gallate (EGCG), prevent both the neurite outgrowth-inhibiting activity and growth cone-collapsing activity of Nogo-66 (C-terminal domain of Nogo-A). Furthermore, a synergistic interaction was observed among GTPP constituents. This preventive effect was dependent on 67-kDa laminin receptor (67LR) to which EGCG binds with high affinity. The antioxidants N-acetylcysteine and cell-permeable catalase abolished this preventive effect of GTPP and EGCG, suggesting the involvement of sublethal levels of H2 O2 in this process. Accordingly, exogenous sublethal concentrations of H2 O2 , added as a bolus dose (5 µM) or more effectively through a steady-state generation (1-2 µM), mimicked GTPP in counteracting the action of Nogo-66. Exogenous H2 O2 mediated this action by bypassing the requirement of 67LR. Taken together, these results show for the first time that GTPP and EGCG, acting through 67LR and elevating intracellular sublethal levels of H2 O2 , inhibit the antineuritogenic action of Nogo-A. Currently, several agents are being evaluated for overcoming axonal growth inhibitors to promote functional recovery after stroke and spinal cord injury. Epigallocatechin-3-gallate (EGCG), present in green tea polyphenol mixture (GTPP), prevents antineuritogenic activity of Nogo-A, a myelin-derived axonal growth inhibitor. The preventive action of EGCG involves the cell-surface-associated 67-kDa laminin receptor and H2 O2 . GTPP may complement ongoing efforts to treat neuronal injuries.>

Accelerating axon growth to overcome limitations in functional recovery after peripheral nerve injury.

OBJECTIVE: Injured peripheral nerves regenerate at very slow rates. Therefore, proximal injury sites such as the brachial plexus still present major challenges, and the outcomes of conventional treatments remain poor. This is in part attributable to a progressive decline in the Schwann cells' ability to provide a supportive milieu for the growth cone to extend and to find the appropriate target. These challenges are compounded by the often considerable delay of regeneration across the site of nerve laceration. Recently, low-frequency electrical stimulation (as brief as an hour) has shown promise, as it significantly accelerated regeneration in animal models through speeding of axon growth across the injury site. METHODS: To test whether this might be a useful clinical tool, we carried out a randomized controlled trial in patients who had experienced substantial axonal loss in the median nerve owing to severe compression in the carpal tunnel. To further elucidate the potential mechanisms, we applied rolipram, a cyclic adenosine monophosphate agonist, to rats after axotomy of the femoral nerve. RESULTS: We demonstrated that effects similar to those observed in animal studies could also be attained in humans. The mechanisms of action of electrical stimulation likely operate through up-regulation of neurotrophic factors and cyclic adenosine monophosphate. Indeed, the application of rolipram significantly accelerated nerve regeneration. CONCLUSION: With new mechanistic insights into the influencing factors of peripheral nerve regeneration, the novel treatments described above could form part of an armament of synergistic therapies that could make a meaningful difference to patients with peripheral nerve injuries.

Cocultures of rat sensorimotor cortex and spinal cord slices to investigate corticospinal tract sprouting.

STUDY DESIGN: Experimental study of corticospinal axonal sprouting in an organotypic slice culture model. OBJECTIVE: To develop an in vitro model that simplifies the study of various factors regulating neuronal regeneration. SUMMARY OF BACKGROUND DATA: Spinal cord injury leads to permanent neurologic damage, mainly due to the inability of the adult central nervous system to regenerate. Much attention has been focused on promoting axonal regeneration and sprouting, either by exogenous administration of various neurotrophic factors or by the antagonization of factors inhibiting regeneration. METHODS: An in vitro system that allows coculture of slices from rat sensorimotor cortex and spinal cord (p4) was established. Two groups of cultures were investigated: In the first group, intact spinal cord slices were cultured adjacent to sensorimotor cortex slices, while in the second group the spinal cord slices were sagittally cut into halves, with the sectioned interface placed directly adjacent to the sensorimotor cortex, to prevent the spinal white matter from interference. Each group was further divided into 2 subgroups: The neurotrophin-3 (NT-3) group, where the culture medium contained 50 ng/mL NT-3 and the control group treated with normal culture medium. Sensorimotor cortex pyramidal neurons were anterogradely labeled with Mini-Ruby, a 10 kD biotinylated dextran amine. RESULTS: Cocultures of cortical and spinal cord tissue were propagated in vitro, and axonal sprouting occurred. The group of cocultures treated with NT-3 showed an improved cortical cytoarchitecture, and sprouting axons were more frequently observed. In NT-3-treated cocultures where spinal cord gray matter was directly opposed to cortical slices sprouting axons entered the adjacent spinal cord tissue. This phenomenon was not observed if spinal cord pia mater and white matter were opposed to the cortical slices, or if NT-3 was absent. CONCLUSION: Our data suggest that the absence of repellent factors such as white matter and the presence of neurotrophic factors promote axonal sprouting. Cocultures of sensorimotor cortex and spinal cord slices combined with anterograde axonal labeling could provide a valuable in vitro model for the simplified screening of factors influencing corticospinal tract regeneration.

Prolyl oligopeptidase binds to GAP-43 and functions without its peptidase activity.

Inhibitors of the enzyme prolyl oligopeptidase (PO) improve performance in rodent learning and memory tasks. PO inhibitors are also implicated in the action of drugs used to treat bipolar disorder: they reverse the effects of three mood stabilizers on the dynamic behaviour of neuronal growth cones. PO cleaves prolyl bonds in short peptides, suggesting that neuropeptides might be its brain substrates. PO is located in the cytosol, however, where it would not contact neuropeptides. Here, we show that mice with a targeted PO null-mutation have altered growth cone dynamics. The wild-type phenotype is restored by PO cDNAs encoding either native or a catalytically-dead enzyme. In addition, we show that PO binds to the growth-associated protein GAP-43, which is a key regulator of synaptic plasticity. Taken together, our results show that peptidase activity is not required for PO function in neurons and suggest that PO instead acts by binding to cytosolic proteins that control growth cone and synaptic function.

A critical importance of polyamine site in NMDA receptors for neurite outgrowth and fasciculation at early stages of P19 neuronal differentiation.

We have investigated the role of N-methyl-d-aspartate receptors (NMDARs) and gamma-aminobutyric acid receptors type A (GABA(A)Rs) at an early stage of P19 neuronal differentiation. The subunit expression was profiled in 24-hour intervals with RT-PCR and functionality of the receptors was verified via fluo-3 imaging of Ca(2+) dynamics in the immature P19 neurons showing that both NMDA and GABA excite neuronal bodies, but only polyamine-site sensitive NMDAR stimulation leads to enhanced Ca(2+) signaling in the growth cones. Inhibition of NR1/NR2B NMDARs by 1 muM ifenprodil severely impaired P19 neurite extension and fasciculation, and this negative effect was fully reversible by polyamine addition. In contrast, GABA(A)R antagonism by a high dose of 200 microM bicuculline had no observable effect on P19 neuronal differentiation and fasciculation. Except for the differential NMDAR and GABA(A)R profiles of Ca(2+) signaling within the immature P19 neurons, we have also shown that inhibition of NR1/NR2B NMDARs strongly decreased mRNA level of NCAM-180, which has been previously implicated as a regulator of neuronal growth cone protrusion and neurite extension. Our data thus suggest a critical role of NR1/NR2B NMDARs during the process of neuritogenesis and fasciculation of P19 neurons via differential control of local growth cone Ca(2+) surges and NCAM-180 signaling.

A high-throughput screen to identify novel compounds to promote neurite outgrowth.

Following spinal cord injury, a variety of inhibitory molecules hinder the success of axon regeneration. The motile tip of the axon, the growth cone, shares a similar cytoskeletal array as a migrating cell, and in general the cytoskeleton is regulated by a conserved set of signaling pathways that act downstream of guidance cue and growth factor receptors. We exploit these similarities by using migrating cells as a model system to screen for extracts that promote axon outgrowth. The screen is a high-throughput wound-healing assay performed by a 96-pin tool Biogrid robot where positive candidates are identified as extracts that stimulate complete wound healing. Testing of positive candidates on chick DRG explants has lead to the identification of extracts that promote neurite outgrowth on permissive and inhibitory substrates. Extracts can be fractionated to purity, identifying novel compounds that promote neurite outgrowth on inhibitory substrates.

Acetylcholine influences growth cone motility and morphology of developing thalamic axons.

The neurotransmitter acetylcholine (ACh) is expressed in the developing telencephalon at the time when thalamic axons project to the cortex, long before synapses are being formed. Since previous studies demonstrated an influence of ACh on neurite extension we used different in vitro assays to examine possible effects of ACh on the growth of thalamic axons. In explant cultures, application of ACh reduced the length of thalamic axons in a dose dependent manner, an effect that could also be evoked by selective muscarinic and nicotinic agonists. Time-lapse imaging of thalamic axons exposed to microscopic gradients of ACh revealed that growth cones no longer advanced, but maintained high filopodial activity. This growth cone pausing was not accompanied by axon retraction or growth cone collapse. It could at least partially be blocked by muscarinic and nicotinic antagonists, indicating that both types of ACh receptors contribute to mediate these effects on thalamic axons. Finally, we also found that ACh changed the morphology of growth cones; they became larger and extended more filopodia. Since such changes in the structure and motility of growth cones are observed at decision regions along the path of many fiber populations including thalamic axons, we suggest that ACh plays a role during the elaboration of thalamocortical projections.

Olfactory ensheathing cells exert a trophic effect on the hypothalamic neurons in vitro.

Olfactory ensheathing cells (OECs) constitute an usual population of glial cells sharing properties with both Schwann cells (SC) of peripheral nervous system (PNS) and astrocytes of the central nervous system (CNS). They express a high level of growth factors which play a very important role as neuronal support. Recent evidence in literature suggests that OECs may facilitate axonal regeneration in the injured nervous system. In this study, we developed an in vitro model to evaluate the neurotrophic effect of OECs on the survival and axonal outgrowth of hypothalamic neurons. Co-cultures of OECs and hypothalamus neuronal cells of postnatal rats were successfully established and cells were immunocytochemically characterized. Furthermore, some neuronal cultures were added with NGF, bFGF and GDNF to compare with the co-cultures. Our results indicate that in co-cultures of hypothalamic neurons and OECs, the number of neurons was significantly increased compared to control cultures exhibiting a dense axonal outgrowth. Moreover, we show that NGF promoted a major neuronal survival than bFGF and GDNF, while bFGF and GDNF exerted an evidence axonal and dendritic outgrowth compared to NGF. In conclusion, these data suggest that OECs have the capacity to promote the survival and axonal outgrowth of hypothalamic neurons in vitro and that bFGF, NGF and GDNF differentially support hypothalamic neurons promoting and enhancing the neuronal survival and outgrowth. Therefore, the OECs are a source of growth factors and might be considered a better approach for functional recovery and growth factors might exert a neuroprotective effect in neurodegenerative disorders.

Regulation of neurite growth in immortalized mouse hypothalamic neurons and rat hippocampal primary cultures by teneurin C-terminal-associated peptide-1.

Teneurins are a highly conserved family of four type II transmembrane proteins that are expressed in the CNS. The protein possesses several functional domains including a unique bioactive 40-41 amino acid sequence at the extracellular terminus. Synthetic versions of this teneurin C-terminal-associated peptide (TCAP) can modulate cyclic AMP accumulation, cell proliferation and teneurin mRNA levels in vitro. Furthermore, i.c.v. injections of TCAP-1 into rat brain induce major changes in acoustic startle response behavior 3 weeks after administration, suggesting that the peptide may act to alter interneuron communication via changes in neurite and axon outgrowth. Synthetic mouse/rat TCAP-1 was used to treat cultured immortalized mouse hypothalamic cells, to determine if TCAP-1 could directly regulate neurite and axon growth. TCAP-1-treated cells showed a significant increase in the length of neurites accompanied by a marked increase in beta-tubulin transcription and translation as determined by real-time PCR and Western blot analysis, respectively. Changes in alpha-actinin-4 transcription and beta-actin protein expression were also noted. Immunofluorescence confocal microscopy using beta-tubulin antiserum showed enhanced resolution of beta-tubulin cytoskeletal elements throughout the cell. In order to determine if the effects of TCAP-1 could be reproduced in primary neuronal cultures, primary cultures of E18 rat hippocampal cells were treated with 100 nM TCAP-1. The TCAP-1-treated hippocampal cultures showed a significant increase in both the number of cells, dendritic branching and the presence of large and fasciculated beta-tubulin immunoreactive axons. These data suggest that TCAP acts, in part, as a functional region of the teneurins to regulate neurite and axonal growth of neurons.

Axonal plasticity is associated with motor recovery following amphetamine treatment combined with rehabilitation after brain injury in the adult rat.

Clinical and laboratory studies have suggested that amphetamine treatment when paired with rehabilitation results in improved recovery of function after stroke or traumatic brain injury. In the present study, we investigated whether new anatomical pathways developed in association with improved motor function after brain damage and amphetamine treatment linked with rehabilitation. Following a unilateral sensorimotor cortex lesion in the adult rat, amphetamine (2 mg/kg) was administered in conjunction with physiotherapy sessions on postoperative days two and five. Physiotherapy was continued twice daily for the first 3 weeks after injury, and then once daily until week six. Performance on skilled forelimb reaching and ladder rung walking was used to assess motor improvement. Our results show that animals with sensorimotor cortical lesions receiving amphetamine treatment linked with rehabilitation had significant improvement in both tasks. Neuroanatomical tracing of efferent pathways from the opposite, non-damaged cortex resulted in the novel finding that amphetamine treatment linked with rehabilitation, significantly increased axonal growth in the deafferented basilar pontine nuclei. These results support the notion that pharmacological interventions paired with rehabilitation can enhance neuronal plasticity and thereby improve functional recovery after CNS injury.

Integration of topographical and biochemical cues by axons during growth on microfabricated 3-D substrates.

During embryonic neural development, axon tips ("growth cones") are guided through a dynamic three-dimensional (3-D) landscape by soluble chemotropic factors and by immobilized, growth-permissive or growth-inhibiting contact cues present in the extracellular matrix and on the surface of surrounding cells. It has been difficult to probe the search algorithms of growth cones in response to multiple contact cues during 3-D navigation using traditional two-dimensional (2-D) substrates. Here, we present an in vitro study in which the axons of murine embryonic cortical neurons are challenged with competing growth options, using 3-D substrates that feature variations in permissiveness and microtopography. As 3-D substrates, we used poly-D-lysine (PDL) coatings on microfabricated steps of polydimethylsiloxane (PDMS) and complementary features of Matrigel. We found that axons display a preference for PDL over Matrigel and for the straightest path within a distance consistent with the exploratory range of the growth cone. When these two preferences are in conflict, axons choose to grow straight into Matrigel; when the straight path is not permissive, the axon turns in the direction that minimizes the turning angle. These results suggest that growth cones make 3-D navigation decisions by integrating permissiveness and topographical cues.

Docosahexaenoic acid promotes neurite growth in hippocampal neurons.

Docosahexanoic acid (22:6n-3; DHA) deficiency during development is associated with impairment in learning and memory, suggesting an important role of DHA in neuronal development. Here we provide evidence that DHA promotes neuronal differentiation in rat embryonic hippocampal primary cultures. DHA deficiency in vitro was spontaneously induced by culturing hippocampal cells in chemically defined medium. DHA supplementation improved DHA levels to values observed in freshly isolated hippocampus. We found that DHA supplementation in culture increased the population of neurons with longer neurite length per neuron and with higher number of branches. However, supplementation with arachidonic, oleic or docosapentaenoic acid did not have any effect, indicating specificity of the DHA action on neurite growth. Furthermore, hippocampal cultures obtained from n-3 fatty acid deficient animals contained a lower DHA level and a neuronal population with shorter neurite length per neuron in comparison to those obtained from animals with adequate n-3 fatty acids. DHA supplementation to the deficient group recovered the neurite length to the level similar to n-3 fatty acid adequate cultures. Our data demonstrates that DHA uniquely promotes neurite growth in hippocampal neurons. Inadequate neurite development due to DHA deficiency may contribute to the cognitive impairment associated with n-3 fatty acid deficiency.

Layer-specific thalamocortical innervation in organotypic cultures is prevented by substances that alter neural activity.

Cortical layer IV is the major target of thalamocortical axons and many previous studies have shown that the development of this layer-specific innervation can be modelled in vitro by organotypic cocultures of thalamus and cortex. The mechanisms causing thalamic axons to terminate in layer IV are unknown. We used these in vitro models to test the possibility that neural activity plays a part in this termination process by adding substances that raise or lower levels of neural activity to the cocultures. We found that addition of tetrodotoxin or 2-amino-5-phosphonovalerate, to block activity, or potassium, to raise it, all interfered with termination in layer IV. These findings suggest that termination in layer IV requires neural activity at an appropriate level in the thalamocortical system. They also add support to recent findings that show that the importance of neural activity in development may extend to an earlier period than thought previously, to include the correct targeting of axons as well as the later refinement of connections.

Arginase I and polyamines act downstream from cyclic AMP in overcoming inhibition of axonal growth MAG and myelin in vitro.

Elevation of cAMP can overcome myelin inhibitors to encourage regeneration of the CNS. We show that a consequence of elevated cAMP is the synthesis of polyamines, resulting from an up-regulation of Arginase I, a key enzyme in their synthesis. Inhibiting polyamine synthesis blocks the cAMP effect on regeneration. Either over-expression of Arginase I or exogenous polyamines can overcome inhibition by MAG and by myelin in general. While MAG/myelin support the growth of young DRG neurons, they become inhibitory as DRGs mature. Endogenous Arginase I levels are high in young DRGs but drop spontaneously at an age that coincides with the switch from promotion to inhibition by MAG/myelin. Over-expressing Arginase I in maturing DRGs blocks that switch. Arginase I and polyamines are more specific targets than cAMP for intervention to encourage regeneration after CNS injury.

Nongenomic mechanism mediates estradiol stimulation of axon growth in male rat hypothalamic neurons in vitro.

The purpose of the present work was to investigate the participation of estradiol receptors (ER) in estrogen-induced axon growth in vitro. Hypothalamic neurons from 16 day (E16) male rat fetuses were cultured with or without 17-beta-estradiol at 1 x 10(-7) M in basal medium or medium conditioned by astroglia derived from ventral mesencephalon (CM). After 48 hr in vitro, neurite outgrowth was quantified by morphometric analysis. An axogenic effect could be demonstrated for estradiol added to CM. With RT-PCR, the mRNA transcript for ERalpha was found in the donor tissues as well as in the neuron cultures. In this model two specific nuclear ER blockers (tamoxifen and ICI 182,780) were ineffective in blocking the neuritogenic effect, and a membrane-impermeable estrogen-albumin construct (E2-BSA) was as effective as estradiol. These results indicate that the axogenic effect of estradiol at E16 is not exerted through the classical intracellular receptor signal transduction system and suggest the possibility of a membrane-mediated mechanism. The data are discussed in light of our previous findings pointing to the interdependent activation of the estrogenic and the trophic factor signaling pathways that mediate stimulated axon growth.

Effects of Cerebrolysin on the outgrowth and protection of processes of cultured brain neurons.

Cerebrolysin (Cere, EBEWE Arzneimittel, Austria), a peptidergic drug produced by a standardised enzymatic breakdown of porcine brain proteins, consists of a mixture of 75% free amino acids and 25% low molecular weight peptides (<10 k DA). Cerebrolysin was shown to protect against MAP2 loss in primary embryonic chick neuronal cultures after brief histotoxic hypoxia and in a rat model of acute brain ischemia. Since MAP2 is involved in processes like neuronal growth, plasticity and dendritic branching, we address the question whether Cere is protecting processes against degeneration in a chronic low serum (2% FCS) cell stress model and whether the spontaneous outgrowth of axon-like processes is influenced. This was accomplished by quantification of the neurite lengths of embryonic chicken telencephalon neurons after 4 and 8 days. Additionally, time-lapse video microscopy was performed to study a possible influence of Cere on the growth cone behaviour of axon-like processes. To distinguish between effects caused by the peptide fraction and the effects related to free amino acids, we used an artificial amino acid solution (AA-mix). Results demonstrate a process outgrowth promoting effect of the AA-mix and Cere after 4 DIV. After 8 days neuronal network degeneration occurred in the AA-mix treated cultures, whereas Cere treated cultures still presented a well differentiated neuronal network. Dying neurons could release factors possibly impeding neurite outgrowth and Cere was shown to increase the viability of chicken cortical neurons. Neither the addition of BDNF nor serum supplementation (5% and 10% FCS) could protect the neuronal network against degeneration after 8 DIV, although these treatments were shown to ameliorate the viability of chicken telencephalon neurons. This result together with the finding obtained using the artificial amino acid solution points to the peptide fraction of Cere to be responsible for the protection of processes against degeneration. Time-lapse studies of Cere treated cultures revealed a significant decrease of the velocities characterising random growth cone movements, which is thought to be responsible for an increase in the length of axon-like processes after 4 DIV.

Axonal surface molecules act in combination with semaphorin 3a during the establishment of corticothalamic projections.

Interactions between growing axons are considered to play important roles for the establishment of precise neuronal connections during the development of the nervous system. Here we used time-lapse imaging techniques to examine the behavior of neocortical and thalamic axons when they encounter each other in vitro. Results indicate that axonal growth cones are able to respond to specific cues expressed on the surface of fibers. Thalamic growth cones often extended along the surface of other thalamic axons and, likewise, cortical growth cones formed fascicles with cortical axons. In contrast, after contacts between cortical and thalamic fibers, in most cases growth cones collapsed and retracted from the axons. Collapse assays using membrane preparations from cortical or thalamic explants demonstrated the existence of cell-type specific collapsing factors whose activity was enhanced by a member of the semaphorin protein family, Sema3A (expressed in the thalamocortical pathway), as it increased the rate of homotypic fasciculations and at the same time amplified the segregation between cortical and thalamic axons. The interaction between axonal surface molecules and environmental cues might mediate the segregation of afferent and efferent fiber tracts in the neocortical white matter.

1,2-dioctanoyl-s,n-glycerol-induced activation of protein kinase C results in striking, but reversible growth cone shape changes and an accumulation of f-actin and serine 41-phosphorylated GAP-43 in the axonal process.

In spinal cord explant cultures from embryonic chicken (E7) we found that both a long-time downregulation of PKC by phorbol-12,13-dibutyrate (PDBu) and an inhibition of PKC by RO-31-8220 strongly reduce neurite outgrowth. Unlike this, in the presence of a high dose of 1,2-dioctanoyl-s,n-glycerol (diC8, 60 microM), PKCalpha,beta isoforms are not downregulated, but neurite outgrowth appeared reduced up to 37 %. A low dose of diC8 (5 microM), however, was found to stimulate neurite outgrowth up to 25 %. Using this tissue culture system as well as neuronal cell culture we then studied the effects of diC8 on the shapes and actin-based motility of distal axonal processes and growth cones as well as on the spatial distribution of f-actin and serine 41-phosphorylated GAP-43 (neuromodulin, B50). High-resolution microscopy showed that addition of 30-60 microM diC8 leads within a few minutes to a retraction of filopodia and to an increased protrusion of lamellipodia followed by the formation of club-shaped dense growing tips, axonal varicosities, and a cessation of any actin dynamics. These striking shape changes are completely reversed after replacement of the medium by drug-free medium. Presence of cytochalasins and a panel of different PKC inhibitors prevent or respectively attenuate the diC8 effects. Immuno- and phalloidin-staining confirmed that in control neurons f-actin and serine 41-phosphorylated GAP-43 are confined to and enriched in the growth cones. In parallel with diC8-induced shape changes there is an accretion of f-actin and serine 41-phosphorylated GAP-43 in the entire axonal processes and the rounded growing tips. With respect to the fundamental role of the actin dynamics in growth cone steering and neuronal pathfinding, the data supports the view that in neurons local PKC-regulated phosphorylation of GAP-43 may represent an important mechanism to transduce guiding signals into actincytoskeletal responses mediating directed axonal growth.

Role of cdk5 and tau phosphorylation in heterotrimeric G protein-mediated retinal growth cone collapse.

During axonal growth, repulsive guidance cues cause growth cone collapse and retraction. In the chick embryo, membranes from the posterior part of the optic tectum containing ephrins are original collapsing factors for axons growing from the temporal retina. We investigated signal transduction pathways in retinal axons underlying this membrane-evoked collapse. Perturbation experiments using pertussis toxin (PTX) showed that membrane-induced collapse is mediated via G(o/i) proteins, as is the case for semaphorin/collapsin-1-induced collapse. Studies with Indo-1 revealed that growth cone collapse by direct activation of G(o/i) proteins with mastoparan did not cause elevation of the intracellular Ca(2+) level, and thus this signal transduction pathway is Ca(2+) independent. Application of the protein phosphatase inhibitor okadaic acid alone induced growth cone collapse in retinal culture, suggesting signals involving protein dephosphorylation. In addition, pretreatment of retinal axons with olomoucine, a specific inhibitor of cdk5 (tau kinase II), prevented mastoparan-evoked collapse. Olomoucine also blocks caudal tectal membrane-mediated collapse. These results suggest that rearrangement of the cytoskeleton is mediated by tau phosphorylation. Immunostaining visualized complementary distributions of tau phospho- and dephosphoisoforms within the growth cone, which also supports the involvement of tau. Taking these findings together, we conclude that cdk5 and tau phosphorylation probably lie downstream of growth cone collapse signaling mediated by PTX-sensitive G proteins.