Search

Nogo-66 receptors (NgR1-3) are glycosylphosphatidyl inositol-linked proteins that belong to the leucine-rich repeat superfamily. Through binding to myelin-associated inhibitors, NgRs contribute to the inhibition of axonal regeneration after spinal cord injury. Their role in limiting synaptic plasticity and axonal outgrowth in the adult CNS has been described previously, but not much is known about their role during the development of the nervous system. Here, we show that NgR1 and NgR3 mRNAs are expressed during spinal cord development of the chicken embryo. In particular, they are expressed in the dI1 subpopulation of commissural neurons during the time when their axons navigate toward and across the floorplate, the ventral midline of the spinal cord. To assess a potential role of NgR1 and NgR3 in axon guidance, we downregulated them usingin ovoRNAi and analyzed the trajectory of commissural axons by tracing them in open-book preparations of spinal cords. Our results show that loss of either NgR1 or NgR3 causes axons to stall in the midline area and to interfere with the rostral turn of postcrossing axons. In addition, we also show that NgR1, but not NgR3, requires neuronal PlexinA2 for the regulation of commissural axon guidance.SIGNIFICANCE STATEMENTOver the last decades, many studies have focused on the role of NgRs, particularly NgR1, in axonal regeneration in the injured adult CNS. Here, we show a physiological role of NgRs in guiding commissural axons during early development of the chicken spinal cordin vivo. Both NgR1 and NgR3 are required for midline crossing and subsequent turning of postcrossing axons into the longitudinal axis of the spinal cord. NgR1, but not NgR3, forms a receptor complex with PlexinA2 during axon guidance. Overall, these findings provide a link between neural regenerative mechanisms and developmental processes.

Primary sensory afferents of the dorsal root ganglion (DRG) that innervate the skin detect a wide range of stimuli, such as touch, temperature, pain, and itch. Different functional classes of nociceptors project their axons to distinct target zones within the developing skin, but the molecular mechanisms that regulate target innervation are less clear. Here we report that the Nogo66 receptor homolog NgR2 is essential for proper cutaneous innervation. NgR2−/−mice display increased density of nonpeptidergic nociceptors in the footpad and exhibit enhanced sensitivity to mechanical force and innocuous cold temperatures. These sensory deficits are not associated with any abnormality in morphology or density of DRG neurons. However, deletion of NgR2 renders nociceptive nonpeptidergic sensory neurons insensitive to the outgrowth repulsive activity of skin-derived Versican. Biochemical evidence shows that NgR2 specifically interacts with the G3 domain of Versican. The data suggest that Versican/NgR2 signaling at the dermo-epidermal junction actsin vivoas a local suppressor of axonal plasticity to control proper density of epidermal sensory fiber innervation. Our findings not only reveal the existence of a novel and unsuspected mechanism regulating epidermal target innervation, but also provide the first evidence for a physiological role of NgR2 in the peripheral nervous system.

Flavonoids target a variety of pathophysiological mechanisms and are therefore increasingly considered as compounds encompassed with therapeutic potentials in diseases such as cancer, diabetes, arteriosclerosis, and neurodegenerative diseases and mood disorders. Hops (Humulus lupulus L.) is rich in flavonoids such as the flavanone 8-prenylnaringenin, which is the most potent phytoestrogen identified so far, and the prenylchalcone xanthohumol, which has potent tumor-preventive, anti-inflammatory and antiviral activities. In the present study, we questioned whether hops-derived prenylflavonoids and synthetic derivatives thereof act on neuronal precursor cells and neuronal cell lines to induce neuronal differentiation, neurite outgrowth and neuroprotection. Therefore, mouse embryonic forebrain-derived neural precursors and Neuro2a neuroblastoma-derived cells were stimulated with the prenylflavonoids of interest, and their potential to activate the promoter of the neuronal fate-specific doublecortin gene and to stimulate neuronal differentiation and neurite outgrowth was analyzed. In this screening, we identified highly "neuroactive" compounds, which we termed "enhancement of neuronal differentiation factors" (ENDFs). The most potent molecule, ENDF1, was demonstrated to promote neuronal differentiation of neural stem cells and neurite outgrowth of cultured dorsal root ganglion neurons and protected neuronal PC12 cells from cobalt chloride-induced as well as cholinergic neurons of the nucleus basalis of Meynert from deafferentation-induced cell death. The results indicate that hops-derived prenylflavonoids such as ENDFs might be powerful molecules to promote neurogenesis, neuroregeneration and neuroprotection in cases of chronic neurodegenerative diseases, acute brain and spinal cord lesion and age-associated cognitive impairments.

After injury to the central nervous system intrinsic factors such as myelin associated inhibitory factors inhibit cellular and axonal regeneration resulting in permanent disability. Three of these factors (Nogo-A, oligodendrocyte myelin glycoprotein, myelin-associated glycoprotein) bind to a common receptor: the Nogo-66 receptor (NgR1). NgR1 is expressed mainly on neurons and is usually associated in a trimolecular complex. The second member of the complex, LINGO-1, is often connected to NgR1 function and is further found to function independently as a negative regulator of oligodendrocyte proliferation and differentiation. The third member of the NgR complex is either the p75 neurotrophin receptor, TROY, or an as yet unidentified co-receptor. Targeting of factors contained in this complex has been described to lead to the promotion of neurite outgrowth, oligodendrocyte proliferation and differentiation and inhibition of cell death. In the current review, we aim to describe the mechanisms of action of the chemical and biological compounds used in targeting NgR1 and LINGO-1. This will be achieved using three examples: blocking of ligand binding to NgR1 in treatment of spinal cord injury, antibody-mediated inhibition of LINGO-1 to promote oligodendrocyte differentiation in multiple sclerosis, and the use of soluble NgR1 to sequester Abeta peptide in the periphery in Alzheimer's disease.

Mesial temporal lobe epilepsy (TLE) is associated with pronounced anatomical and biochemical changes in the hippocampal formation including extensive neurodegeneration, reorganization of mossy fibres and sprouting of interneurons. Although the anatomical features and some of the physiological consequences of hippocampal remodeling have been well documented, the molecular mechanisms underlying the profound and orientated outgrowth of hippocampal neurons in TLE are not yet understood. The reticulon protein Nogo-A has been associated with an inhibitory action on axon growth and plasticity. Using immunohistochemistry and in situ hybridization, we investigated the expression of Nogo-A in specimens obtained at surgery from patients with TLE compared with those obtained from autopsy controls. In control specimens, Nogo-A immunoreactivity and mRNA were mainly confined to oligodendrocytes. Only approximately 40% of the specimens revealed low expression of Nogo-A mRNA in neurons. In contrast, in TLE patients with and without Ammon's horn sclerosis, Nogo-A mRNA and immunoreactivity were markedly up-regulated in most neurons (3.6- and 4.4-fold increases in Nogo-A mRNA in granule cells of sclerotic and nonsclerotic specimens) and their processes throughout the hippocampal formation. Similar elevations in Nogo-A mRNA and protein levels were determined by quantitative RT-PCR and Western blotting. Since Nogo-A expression was also up-regulated in specimens without hippocampal sclerosis, it may be induced by seizures prior to progressing neurodegeneration.

Nogo-A is a protein associated with central nervous system (CNS) myelin thought to impair regenerative responses and to suppress sprouting and plastic changes of synaptic terminals. In this study, we report that serum IgM autoantibodies to the recombinant large N-terminal inhibitory domain of Nogo-A are a frequent finding in multiple sclerosis (MS) and acute inflammatory (IND) and non-inflammatory neurological diseases (OND), but not in neurodegenerative diseases (ND), systemic inflammatory disease and healthy controls. Furthermore, we demonstrate intrathecal production of anti-Nogo-A antibodies measured by increased IgG indices. Intrathecal anti-Nogo antibodies were significantly more frequent in patients with relapsing-remitting as compared to chronic progressive (CP) MS. We also found a highly significant negative correlation of these antibody responses with age indicating that they are more frequent in younger patients. We finally demonstrate that human anti-Nogo-A antibodies recognize native Nogo-A in brain extracts, oligodendrocytes and cells expressing human Nogo-A.

Nogo-A is a potent neurite growth inhibitorin vitroand plays a role both in the restriction of axonal regeneration after injury and in structural plasticity in the CNS of higher vertebrates. The regions that mediate inhibition and the topology of the molecule in the plasma membrane have to be defined. Here we demonstrate the presence of three different active sites: (1) an N-terminal region involved in the inhibition of fibroblast spreading, (2) a stretch encoded by the Nogo-A-specific exon that restricts neurite outgrowth and cell spreading and induces growth cone collapse, and (3) a C-terminal region (Nogo-66) with growth cone collapsing function. We show that Nogo-A-specific active fragments bind to the cell surface of responsive cells and to rat brain cortical membranes, suggesting the existence of specific binding partners or receptors. Several antibodies against different epitopes on the Nogo-A-specific part of the protein as well as antisera against the 66 aa loop in the C-terminus stain the cell surface of living cultured oligodendrocytes. Nogo-A is also labeled by nonmembrane-permeable biotin derivatives applied to living oligodendrocyte cultures. Immunofluorescent staining of intracellular, endoplasmic reticulum-associated Nogo-A in cells after selective permeabilization of the plasma membrane reveals that the epitopes of Nogo-A, shown to be accessible at the cell surface, are exposed to the cytoplasm. This suggests that Nogo-A could have a second membrane topology. The two proposed topological variants may have different intracellular as well as extracellular functions.

Unlike neonatal axons, mammalian adult axons of the CNS do not regenerate after injury. This developmental loss of regenerative capacity, is correlated with the onset of myelination. Likewise, myelin, or myelin-associated components such as Nogo-A and myelin-associated glycoprotein (MAG) inhibit regeneration from older but not younger neurons. Identification of the molecular events responsible for this developmental loss of regenerative capacity is central to devise strategies to encourage regeneration in adults after injury. Endogenous levels of the cyclic nucleotides cAMP and cGMP have been suggested to determine the neuronal responsiveness to various axonal guidance factors. Elevating cAMP concentrations block Nogo-A or MAG induced inhibition of neurite outgrowth in older neurons, whereas suppressing cAMP levels in young neurons renders them susceptible to Nogo-A and MAG. Interestingly, elevated cAMP levels abrogated the Nogo-A and MAG mediated activation of RhoA and down regulation of Rac1 in adult neurons. In contrast, elevation of cAMP leads to the inactivation of RhoA and prevents activation of downstream effector proteins, while Rac is activated. We therefore conclude that the endogenous neuronal cAMP levels determine the neuronal responsiveness to myelin-associated neurite growth inhibitors by regulating rho GTPase activities.

The adult mammalian CNS has a limited capacity for nerve regeneration and structural plasticity. The presence of glia-derived inhibitory factors myelin-associated glycoprotein (MAG) and Nogo-A have been suggested to provide a nonpermissive environment for elongating nerve fibers. In particular, Nogo-A, an integral membrane protein predominantly expressed by oligodendrocytes, has been demonstrated to impair neurite growth in vitro and in vivo . Structure function analysis revealed that Nogo-A protein contains at least two active domains, NiG and Nogo-66, with diverse effects on neurite outgrowth and cell spreading. We now provide evidence that these inhibitory domains mediate their effects via an antagonistic regulation of the small GTPases RhoA and Rac1, resulting in activation of RhoA and suppression of Rac1. By inactivating RhoA with C3 transferase or the downstream effector Rho-kinase ROCK with[Y27632][1], the inhibitory effects of both Nogo-A fragments and MAG on neurite outgrowth and oligodendrocyte-mediated growth cone collapse were abolished. Furthermore, we show that the recently cloned receptor for Nogo-66 and MAG, NgR, is not necessary for either NiG- or MAG-induced RhoA activation. [1]: /lookup/external-ref?link_type=GEN&access_num=Y27632&atom=%2Fjneuro%2F22%2F23%2F10368.atom

After nerve injury, adult sensory neurons can regenerate peripheral axons and reconnect with their target tissue. Initiation of outgrowth, as well as elongation of neurites over long distances, depends on the signaling of receptors for neurotrophic growth factors. Here, we investigated the importance of gp130, the signaling subunit of neuropoietic cytokine receptors in peripheral nerve regeneration.After sciatic nerve crush, functional recoveryin vivowas retarded inSNS-gp130−/−mice, which specifically lackgp130in sensory neurons. Correspondingly, a significantly reduced number of free nerve endings was detected in glabrous skin fromSNS-gp130−/−compared with control mice after nerve crush. Neurite outgrowth and STAT3 activationin vitrowere severely reduced in cultures in gp130-deficient cultured neurons. Surprisingly, in neurons obtained fromSNS-gp130−/−mice the increase in neurite length was reduced not only in response to neuropoietic cytokine ligands of gp130 but also to nerve growth factor (NGF), which does not bind to gp130-containing receptors. Neurite outgrowth in the absence of neurotrophic factors was partially rescued in gp130-deficient neurons by leptin, which activates STAT3 downstream of leptic receptor and independent of gp130. The neurite outgrowth response of gp130-deficient neurons to NGF was fully restored in the presence of leptin.Based on these findings, gp130 signaling via STAT3 activation is suggested not only to be an important regulator of peripheral nerve regenerationin vitroandin vivo, but as determining factor for the growth promoting action of NGF in adult sensory neurons.

Molecular mechanisms which stabilize dendrites and dendritic spines are essential for regulation of neuronal plasticity in development and adulthood. The class of Nogo receptor proteins, which are critical for restricting neurite outgrowth inhibition signaling, have been shown to have roles in developmental, experience and activity induced plasticity. Here we investigated the role of the Nogo receptor homolog NgR2 in structural plasticity in a transgenic null mutant for NgR2. Using Golgi-Cox staining to analyze morphology, we show that loss of NgR2 alters spine morphology in adult CA1 pyramidal neurons of the hippocampus, significantly increasing mushroom-type spines, without altering dendritic tree complexity. Furthermore, this shift is specific to apical dendrites in distal CA1 stratum radiatum (SR). Behavioral alterations in NgR2(-/-) mice were investigated using a battery of standardized tests and showed that whilst there were no alterations in learning and memory in NgR2(-/-) mice compared to littermate controls, NgR2(-/-) displayed reduced fear expression in the contextual conditioned fear test, and exhibited reduced anxiety- and depression-related behaviors. This suggests that the loss of NgR2 results in a specific phenotype of reduced emotionality. We conclude that NgR2 has role in maintenance of mature spines and may also regulate fear and anxiety-like behaviors.

The plasma membrane is the outer limit of the animal cell. As such, it is both a border separating inside from outside and a signaling platform for interactions with the surroundings. Among these interactions are extracellular matrix contacts and adhesion sites. The membrane and its contact sites together with the underlying cytoskeleton undergo constant remodeling, which leads to changes of the cell shape. In addition to spatial information micro-topographical maps provide, information about the z-dimension and describe the position of the plasma membrane with respect to the distance to a given substrate. Here we address how to measure height differences in the plasma membrane and how to create topographical maps of the plasma membrane with nanometer resolution. We address the currently used methodologies along with their advantages and drawbacks. Moreover, we delineate a label-free method to obtain topographic maps of the plasma membrane that are corrected for differences in the refractive index of the membrane utilizing an interferometric approach with multiple wavelengths and a normalization procedure to account for changes in the refractive index in the membrane. Copyright © 2014 SCITEPRESS - Science and Technology Publications. All rights reserved. ispartof: pages:31-40 ispartof: Proceedings of the International Conference on Bioimaging pages:31-40 ispartof: BIOIMAGING location:Angers date:3 Mar - 6 Mar 2014 status: published

In this paper, we identify the chondroitin sulfate proteoglycan versican V2 as a major inhibitor of axonal growth in the extracellular matrix of the mature central nervous system. In immunohistochemical and in situ hybridization experiments we show that this tissue-specific splice variant of versican is predominantly present in myelinated fiber tracts of the brain and in the optic nerve, most likely being expressed by oligodendrocytes. We demonstrate that isolated versican V2 strongly inhibits neurite outgrowth of central and peripheral neurons in stripe-choice assays using laminin-1 as permissive substrate. The inhibitory character of versican V2 is maintained after removal of chondroitin sulfate and N- and O-linked oligosaccharide side chains, but it is abolished after core protein digestion with proteinase-K. Our data support the notion, that intact versican V2 prevents excessive axonal growth during late phases of development and hereby participates in the structural stabilization of the mature central nervous system.

Proteoglycans are a heterogeneous class of proteins bearing sulfated glycosaminoglycans. Some of the proteoglycans have distinct core protein structures, and others display similarities and thus may be grouped into families such as the syndecans, the glypicans, or the hyalectans (or lecticans). Proteoglycans can be found in almost all tissues being present in the extracellular matrix, on cellular surfaces, or in intracellular granules. In recent years, brain proteoglycans have attracted growing interest due to their highly regulated spatiotemporal expression during nervous system development and maturation. There is increasing evidence that different proteoglycans act as regulators of cell migration, axonal pathfinding, synaptogenesis, and structural plasticity. This review summarizes the most recent data on structures and functions of brain proteoglycans and focuses on new physiological concepts for their potential roles in the developing central nervous system.

Retinal ganglion cell (RGC) axons in lizards (reptiles) were found to regenerate after optic nerve injury. To determine whether regeneration occurs because the visual pathway has growth-supporting glia cells or whether RGC axons regrow despite the presence of neurite growth-inhibitory components, the substrate properties of lizard optic nerve myelin and of oligodendrocytes were analyzed in vitro, using rat dorsal root ganglion (DRG) neurons. In addition, the response of lizard RGC axons upon contact with rat and reptilian oligodendrocytes or with myelin proteins from the mammalian central nervous system (CNS) was monitored. Lizard optic nerve myelin inhibited extension of rat DRG neurites, and lizard oligodendrocytes elicited DRG growth cone collapse. Both effects were partially reversed by antibody IN-1 against mammalian 35/250 kD neurite growth inhibitors, and IN-1 stained myelinated fiber tracts in the lizard CNS. However, lizard RGC growth cones grew freely across oligodendrocytes from the rat and the reptilian CNS. Mammalian CNS myelin proteins reconstituted into liposomes and added to elongating lizard RGC axons caused at most a transient collapse reaction. Growth cones always recovered within an hour and regrew. Thus, lizard CNS myelin and oligodendrocytes possess nonpermissive substrate properties for DRG neurons--like corresponding structures and cells in the mammalian CNS, including mammalian-like neurite growth inhibitors. Lizard RGC axons, however, appear to be far less sensitive to these inhibitory substrate components and therefore may be able to regenerate through the visual pathway despite the presence of myelin and oligodendrocytes that block growth of DRG neurites.

Numerous studies have shown that the developing tip of a neurite, the growth cone, can respond to environmental cues with behaviors such as guidance or collapse. To assess whether a given cell type can use more than one second-messenger pathway for a single behavior, we compared the influence of two well-characterized guidance cues on growth cones of chick temporal retinal ganglion cells. The first cue was the repulsive activity derived from the posterior optic tectum (p-membranes), and the second was the collapse-inducing activity derived from oligodendrocytes known as NI35/NI250. p-Membranes caused permanent growth cone collapse with no recovery after several hours, while NI35 caused transient collapse followed by recovery after about 10 min. The p-membrane-induced collapse was found to be Ca2+ independent, as shown using the Ca2+-sensitive dye Fura-2 and by the persistence of collapse in Ca2+-free medium. Dantrolene, a blocker of the ryanodine receptor, had only a minor effect on the collapse frequency caused by p-membranes. In contrast, the NI35-induced collapse was clearly Ca2+ dependent. [Ca2+]i increased sevenfold preceding collapse, and both dantrolene and antibodies against NI35 significantly reduced both the Ca2+ increase and the collapse frequency. Thus, even in a single cell type, growth cone collapse induced by two different signals can be mediated by two different second-messenger systems.

A recombinant Fab fragment was prepared from the monoclonal IgM/kappa antibody IN-1, which neutralizes central nervous system myelin-associated neurite growth inhibitors both in vitro and in vivo. The variable domain gene sequences were amplified and cloned after cDNA synthesis from the hybridoma RNA. After insertion into the tet promoter vector pASK85, which provided the constant domains of class IgG1/kappa, equipped with a His6 tag, large amounts of the Fab fragment were produced in Escherichia coli by medium cell density fermentation. The Fab fragment was purified to homogeneity by immobilized metal-affinity chromatography and its biochemical activity was compared with the original IN-1 antibody. In an assay for neurite outgrowth and fibroblast spreading, the Fab fragment showed a similar neutralizing effect on inhibitory substrate properties of central nervous system myelin as the unpurified IgM, although an approximately tenfold higher concentration was necessary. Immunoprecipitation experiments revealed a more selective antigen-binding behaviour for the Fab fragment. The Fab fragment was also successfully applied for antigen detection in immunohistochemical analyses. Therefore, the recombinant Fab fragment of IN-1 shows full functionality in vitro and appears to be well suited for replacing the monoclonal IgM in investigations on fiber tract regeneration in vivo.

Adhesion and neurite outgrowth of PC12 cells, as well as the spreading of 3T3 fibroblasts, were inhibited in a dose dependent manner by detergent solubilized mouse central nervous system myelin proteins as a tissue culture substrate. These inhibitory effects could be neutralized by the monoclonal antibody IN-1 directed against the neurite growth inhibiting proteins NI-35 and NI-250. Separation of the detergent soluble proteins of bovine spinal cord by an anion exchange column showed that the peaks of inhibitory activity for the two cell lines overlapped, such that the PC12 cells were inhibited by a larger number of fractions comprising those inhibitory for 3T3 cells. Neurite outgrowth of PC12 cells was not influenced by the myelin associated glycoprotein, MAG.

Peripheral nerve injury triggers the activation of RhoA in spinal motor and peripheral sensory neurons. RhoA activates a number of effector proteins including the Rho-associated kinase, ROCK, which targets the cytoskeleton and leads to inhibition of neurite outgrowth. Blockade of the Rho/ROCK pathway by pharmacological means improves axon regeneration after experimental injury. C3(bot) transferase, an exoenzyme produced by Clostridium botulinum, inactivates RhoA by ADP-ribosylation. It has been successfully applied in experimental CNS lesions to facilitate axon regeneration. Up to now it was not investigated thoroughly whether C3(bot) exerts positive effects on peripheral axon regeneration as well. In the present study, recombinant membrane permeable C3(bot) produced a small, but significant, axon outgrowth effect on peripheral sensory neurons dissociated from adult dorsal root ganglia (DRG) of the rat. Neuronal overexpression of C3, however, did not enhance axonal growth. Moreover, transfection of plasmids encoding dominant negative RhoA or RhoA specific shRNAs failed to increase axonal growth. Furthermore, we show that the C3(bot) mutant, C3(E174Q), which lacks RhoA inhibitory activity, still stimulates axonal growth. When analyzing possible signaling mechanisms we found that extracellular signal-regulated kinase (ERK) and Akt are activated by C3(bot) and ERK is induced by the C3(E174Q) mutant. Upregulation of kinase activities by C3(bot) occurs significantly faster than inactivation of RhoA indicating a RhoA-independent pathway of action by C3(bot). The induction of ERK signaling by C3(bot) was detected in embryonic hippocampal neurons, too. Taken together, although RhoA plays a central role for inhibition of axon outgrowth by myelin-derived inhibitors, it does not interfere with axonal growth of sensory neurons on a permissive substrate in vitro. C3(bot) blocks neuronal RhoA activity, but its positive effects on axon elongation and branching appear to be mediated by Rho independent mechanisms involving activation of axon growth promoting ERK and Akt kinases.

The Nogo-66 receptor family (NgR) consists in three glycophosphatidylinositol (GPI)-anchored receptors (NgR1, NgR2 and NgR3), which are primarily expressed by neurons in the central and peripheral mammalian nervous system. NgR1 was identified as serving as a high affinity binding protein for the three classical myelin-associated inhibitors (MAIs) Nogo-A, myelin-associated glycoprotein (MAG) and oligodendrocyte myelin glycoprotein (OMgp), which limit axon regeneration and sprouting in the injured brain. Recent studies suggest that NgR signaling may also play an essential role in the intact adult CNS in restricting axonal and synaptic plasticity and are involved in neurodegenerative diseases, particularly in Alzheimer's disease pathology through modulation of β-secretase cleavage. Here, we outline the biochemical properties of NgRs and their functional roles in the intact and diseased CNS.

Cerebral malaria (CM) is associated with a high rate of transient or persistent neurological sequelae. Nogo-A, a protein that is highly expressed in the endoplasmic reticulum (ER) of the mammalian central nervous system (CNS), is involved in neuronal regeneration and synaptic plasticity in the injured CNS. The current study investigates the role of Nogo-A in the course of experimental CM. C57BL/6J mice were infected with Plasmodium berghei ANKA blood stages. Brain homogenates of mice with different clinical severity levels of CM, infected animals without CM and control animals were analyzed for Nogo-A up-regulation by Western blotting and immunohistochemistry. Brain regions with Nogo-A upregulation were evaluated by transmission electron microscopy. Densitometric analysis of Western blots yielded a statistically significant upregulation of Nogo-A in mice showing moderate to severe CM. The number of neurons and oligodendrocytes positive for Nogo-A did not differ significantly between the studied groups. However, mice with severe CM showed a significantly higher number of cells with intense Nogo-A staining in the brain stem. In this region ultrastructural alterations of the ER were regularly observed. Nogo-A is upregulated during the early course of experimental CM. In the brain stem of severely affected animals increased Nogo-A expression and ultrastructural changes of the ER were observed. These data indicate a role of Nogo-A in neuronal stress response during experimental CM.

The mammalian CNS exhibits very limited capacity for spontaneous regrowth of fiber tracts upon injury. Recent evidence suggests that the failure of successful axonal regeneration in the CNS is not simply a property of the neurone, but mainly dependent on the local environment encountered by re-extending axons. Glial scar tissue which forms around lesion sites seems to be able to inhibit growth of axons. Different molecular properties of the cells within the scar seem to contribute to a non-conducive environment, hindering successful regeneration. In addition to the role of scars, the presence of neurite growth inhibitory molecules associated with CNS myelin and oligodendrocyte membranes role for the lack of successful fiber regeneration. Antibodies raised against these proteins were shown to neutralize their growth inhibitory effect in vitro and in vivo, resulting in long-distance fiber growth within the adult mammalian CNS. In the PNS, where regeneration of lesioned axons occurs spontaneously, these inhibitory proteins are absent.

Although the role of myelin-derived Nogo-A as an inhibitor of axonal regeneration after CNS injury has been thoroughly described, its physiological function in the adult, uninjured CNS is less well known. We address this question in the hippocampus, where Nogo-A is expressed by neurons as well as oligodendrocytes. We used 21 din vitroslice cultures of neonatal hippocampus where we applied different approaches to interfere with Nogo-A signaling and expression and analyze their effects on the dendritic and axonal architecture of pyramidal cells. Neutralization of Nogo-A by function-blocking antibodies induced a major alteration in the dendrite structure of hippocampal pyramidal neurons. Although spine density was not influenced by Nogo-A neutralization, spine type distribution was shifted toward a more immature phenotype. Axonal complexity and length were greatly increased. Nogo-A KO mice revealed a weak dendritic phenotype resembling the effect of the antibody treatment. To discriminate a possible cell-autonomous role of Nogo-A from an environmental, receptor-mediated function, we studied the effects of short hairpin RNA-induced knockdown of Nogo-A or NgR1, a prominent Nogo-A receptor, within individual neurons. Knockdown of Nogo-A reproduced part of the dendritic and none of the spine or axon alterations. However, downregulation of NgR1 replicated the dendritic, the axonal, and the spine alterations observed after Nogo-A neutralization. Together, our results demonstrate that Nogo-A plays a major role in stabilizing and maintaining the architecture of hippocampal pyramidal neurons. Mechanistically, although the majority of the activity of Nogo-A relies on a receptor-mediated mechanism involving NgR1, its cell-autonomous function plays a minor role.

Background The reticulon Nogo-B participates in cellular and immunological processes in murine macrophages. Since leukocytes are an essential part of the immune system in health and disease, we decided to investigate the expression of Nogo-A, Nogo-B and Nogo-C in different human immune cell subpopulations. Furthermore, we analyzed the localization of Nogo-B in human monocyte-derived macrophages by indirect immunofluorescence stainings to gain further insight into its possible function. Findings We describe an association of Nogo-B with cytoskeletal structures and the base of filopodia, but not with focal or podosomal adhesion sites of monocyte-derived macrophages. Nogo-B positive structures are partially co-localized with RhoA staining and Rac1 positive membrane ruffles. Furthermore, Nogo-B is associated with the tubulin network, but not accumulated in the Golgi region. Although Nogo-B is present in the endoplasmic reticulum, it can also be translocated to large cell protrusions or the trailing end of migratory cells, where it is homogenously distributed. Conclusions Two different Nogo-B staining patterns can be distinguished in macrophages: firstly we observed ER-independent Nogo-B localization in cell protrusions and at the trailing end of migrating cells. Secondly, the localization of Nogo-B in actin/RhoA/Rac1 positive regions supports an influence on cytoskeletal organization. To our knowledge this is the first report on Nogo-B expression at the base of filopodia, thus providing further insight into the distribution of this protein.

We have found a regional distribution of IL 1 beta mRNA and IL 1 activity in the normal adult rat brain, which reveals at least partially a colocalization with nerve growth factor (NGF). The predominantly neuronal signal patterns were found over the granule cells of the dentate gyrus, the pyramidal cells of the hippocampus, the granule cells of the cerebellum, the granule and periglomerular cells of the olfactory bulb, and over dispersed cells of the ventromedial hypothalamus and of the frontal cortex. In these areas also the highest levels of IL 1 activity were observed. In the striatum and septum much lower levels of IL 1 beta mRNA and IL 1 activity (shown for the striatum), most likely synthesized by glial cells, could be determined. IL 1 beta-expressing cells were mainly found in brain regions that also synthesize NGF mRNA as shown by in situ hybridization. NGF mRNA could be demonstrated over pyramidal cells of the hippocampus, granule cells of the dentate gyrus, periglomerular cells of the olfactory bulb and over prefrontal cortex neurons. These data indicate that IL 1 beta, among other factors, might also play a regulatory role in the synthesis of NGF in the CNS, as has been demonstrated in the peripheral nervous system (Lindholm, D., R. Heumann, M. Meyer, and H. Thoenen. 1987. Nature (Lond.). 330:658-659).

Reticulons (RTNs) are a large family of transmembrane proteins present throughout the eukaryotic domain in virtually every cell type. Despite their wide distribution, their function is still mostly unknown. RTN4, also termed Nogo, comes in three isoforms, Nogo-A, -B, and -C. While Nogo-A has been described as potent inhibitor of nerve growth, Nogo-B has been implicated in vascular remodeling and regulation of apoptosis. We show here that Nogo-B gets cleaved by caspase-7, but not caspase-3, during apoptosis at a caspase nonconsensus site. By a combination of MS and site-directed mutagenesis we demonstrate that proteolytic processing of Nogo-B is regulated by phosphorylation of Ser(16) within the cleavage site. We present cyclin-dependent kinase (Cdk)1 and Cdk2 as kinases that phosphorylate Nogo-B at Ser(16) in vitro. In vivo, cleavage of Nogo-B is markedly increased in Schwann cells in a lesion model of the rat sciatic nerve. Taken together, we identified an RTN protein as one out of a selected number of caspase targets during apoptosis and as a novel substrate for Cdk1 and 2. Furthermore, our data support a functionality of caspase-7 that is distinct from closely related caspase-3.

Publisher Summary This chapter discusses many studies that provide insights into the origins of central (CNS) and peripheral nervous system (PNS) myelination and into the reasons that CNS regeneration in adult vertebrates is restricted to fish and amphibians. As in other fields, studying biological processes through their evolutionary history, in their own right, provides important and novel insights into human diseases in which myelin sheaths develop abnormally and/or following normal development they disassemble and are degraded. The chapter studies mechanisms for the adaptation of sophisticated regulation that underlies myelination for animals living under conditions far different from example, without thermoregulation or different osmotic environments. Among the animal models with a rapidly growing following is the zebra, which is being used more frequently by ‘‘myelin ’’ scientists. As more and more sequences from genome-sequencing projects relevant to vertebrate and invertebrate evolution become available, appreciation of evolutionary adaptations made by the plethora of molecules associated with differing facets of the myelination process is realized.

In the adult mammalian central nervous system (CNS), growth of neuronal fibers is actively inhibited by myelin. The proteins myelin-associated glycoprotein (MAG), oligodendrocyte myelin glycoprotein (OMgP), and Nogo-66 have been identified as inhibitory components present in CNS myelin. All three proteins exert their inhibitory activity by binding to a neuronal receptor complex containing the Nogo-66 receptor (NgR) and the neurotrophin (NT) receptor p75 NTR . In their recent publication, Mi et al . identify the novel protein Lingo-1 as an interactor of p75 NTR and NgR. The Lingo-1-NgR-p75 NTR complex is shown to confer the inhibitory effects on nerve cell regeneration of Nogo-66, OMgP, and MAG by activating the small guanosine triphosphatase (GTPase) RhoA. Together with the recent finding that p75 NTR interacts with the transmembrane protein sortilin to form a different receptor complex with cell death-promoting activity, the results of Mi et al . indicate that p75 NTR exerts its diverse cellular functions by associating with function-specific co-receptors.

Mammalian neurons require a constant supply of oxygen to maintain adequate cellular functions and survival. Following sustained hypoxia during ischemic events in brain, the energy status of neurons and glia is compromised, which may subsequently lead to cell death by apoptosis and necrosis. Concomitant with energy depletion is the formation of the purine nucleoside adenosine, a powerful endogenous neuroprotectant. In this paper the effect of chemical hypoxia on cell survival and neurite outgrowth of primary cerebellar granule cells was investigated. Rotenone, a mitochondrial complex I inhibitor, induced a 30.4 +/- 3.6% loss of viable cells and a 35.0 +/- 4.4% loss of neurite formation of cerebellar granule cells, which was partially restored by the addition of purine nucleosides adenosine, inosine and guanosine. Inosine had the most striking effect of 37.7 +/- 2.9% rescue of viability and 71.2 +/- 18.4% rescue of neurite outgrowth. Data confirm the suggested role of purine nucleosides for the neuronal regeneration of primary brain cells following hypoxic insult.

Myelin is a major obstacle for regenerating nerve fibers of the adult mammalian central nervous system (CNS). Several proteins including Nogo-A, myelin-associated glycoprotein (MAG), oligodendrocyte myelin glycoprotein (OMgp) and the chondroitin-sulfate proteoglycan (CSPG) Versican V2 have been identified as inhibitory components present in CNS myelin. MAG, OMgp as well as the Nogo specific domain Nogo-66 exert their inhibitory activity by binding to a neuronal receptor complex containing the Nogo-66 receptor NgR and the neurotrophin receptor p75(NTR). While this suggests a converging role of the p75(NTR)/NgR receptor complex for myelin-derived neurite growth inhibitors, we show here that NgR/p75(NTR) is not required for mediating the inhibitory activity of the two myelin components NiG, unlike Nogo-66 a distinct domain of Nogo-A, and Versican V2. Primary neurons derived from a complete null mutant of p75(NTR) are still sensitive to NiG and Versican V2. In line with this result, neurite growth of p75(NTR) deficient neurons is still significantly blocked on total bovine CNS myelin. Furthermore, modulation of RhoA and Rac1 in p75(NTR)-/- neurons persists with NiG and Versican V2. Finally, we demonstrate that neither NiG nor Versican V2 interact with the p75(NTR)/NgR receptor complex and provide evidence that the binding sites of NiG and Nogo-66 are physically distinct from each other on neural tissue. These results indicate not only the existence of neuronal receptors for myelin inhibitors independent from the p75(NTR)/NgR receptor complex but also establish Rho GTPases as a common point of signal convergence of diverse myelin-induced regeneration inhibitory pathways.

Growth cones are known as the site of action of many factors that influence neurite growth behavior. To assess how different collapsing agents influence the growth cone cytoskeleton, we used recombinant human Semaphorin III (hSema III) and the serine protease thrombin. Embryonic chick dorsal root ganglion neurons showed a dramatic depolymerization of actin filaments within 5 min upon hSema III exposure and virtually no influence on microtubules (MT). Only at later time points (20–30 min) was the polymerization/depolymerization rate of MT significantly affected. Thrombin induced a morphologically and kinetically similar growth cone collapse. Moreover, thrombin induced an early and selective depolymerization of dynamic MT, accompanied by the formation of loops of stable MT bundles. Selective changes in the phosphorylation pattern of tau were associated with microtubule assembly in thrombin-induced responses. Our data provide evidence that different signal transduction pathways lead to distinct changes of the growth cone cytoskeleton.

The absence of fiber regrowth in the injured mammalian CNS is influenced by several different factors and mechanisms. Besides the nonconducive properties of the glial scar tissue that forms around the lesion site, individual molecules present in CNS myelin and expressed by oligodendrocytes, such as NI-35/NI-250, bNI-220, and myelin-associated glycoprotein (MAG), have been isolated and shown to inhibit axonal growth. Here, we report an additional neurite growth-inhibitory activity purified from bovine spinal cord myelin that is not related to bNI-220 or MAG. This activity can be ascribed to the presence of two chondroitin sulfate proteoglycans (CSPGs), brevican and the brain-specific versican V2 splice variant. Neurite outgrowth of neonatal cerebellar granule cells and of dorsal root ganglion neuronsin vitrowas strongly inhibited by this myelin fraction enriched in CSPGs. Immunohistochemical staining revealed that brevican and versican V2 are present on the surfaces of differentiated oligodendrocytes. We provide evidence that treatment of oligodendrocytes with the proteoglycan synthesis inhibitors β-xylosides can strongly influence the growth permissiveness of oligodendrocytes. β-Xylosides abolished cell surface presentation of brevican and versican V2 and reversed growth cone collapse in encounters with oligodendrocytes as demonstrated by time-lapse video microscopy. Instead, growth cones were able to grow along or even into the processes of oligodendrocytes. Our results strongly suggest that brevican and versican V2 are additional components of CNS myelin that contribute to its nonpermissive substrate properties for axonal growth. Expression of these CSPGs on oligodendrocytes may indicate that they participate in the restriction of structural plasticity and regeneration in the adult CNS.

We investigated whether neuron-specific enolase (NSE) in serum or cerebrospinal fluid (CSF) reflects subtle or manifest brain injury in children undergoing cardiac surgery using cardiopulmonary bypass (CPB). NSE was measured in serum (s-NSE) before, and up to, 102 h after surgery in 27 children undergoing cardiac surgery with CPB. In 11 children, CSF-NSE was also measured 48 or 66 h post-surgery. As erythrocytes contain NSE, hemoglobin concentration in the samples was determined spectrophotometrically at 550 nm (cut-off limit: absorbance 0.4=560 mg/l) in 14 children and in a further 13 children by spectroscopic multicomponent analysis (cut-off limit 5 μ mol/l=80 mg/l). One hundred and one of 214 post-operative serum samples (47%) had to be discarded because of hemolysis (18% spectrophotometrically at 550 nm and 88% with spectroscopic multicomponent analysis). On the first and second post-operative day, the median s-NSE values were significantly higher when compared with samples taken after 54 h or longer ( P =0.008 and P =0.002). All CSF-NSE levels were within the normal range and below the s-NSE measured in the same patient. Although in our study elevated s-NSE seems to indicate brain injury in CPB-surgery, the low concentration of NSE in the post-operative CSF of 11 children puts the neuronal origin of s-NSE in question. NSE from other non-neuronal tissues probably contributes to the elevated s-NSE. Additionally, normal post-operative CSF-NSE values in two children with post-operative neurological sequelae might question the predictive value of CSF-NSE with regard to brain injury.

All cranial nerves, as well as the VIIIth nerve which invades the cochlea, have a proximal end in which myelin is formed by Schwann cells and a distal end which is surrounded by oligodendrocytes. The question which arises in this context is whether peripheral and central parts of these nerves myelinate simultaneously or subsequently and whether the myelination of either of the parts occurs simultaneously at the onset of the cochlea function and under the control of neuronal activity. In the present paper, we examined the relative time course of the myelinogenesis of the distal part of the VIIIth nerve by analyzing the expression of peripheral protein P0, proteolipid protein and myelin basic protein. To our surprise, we observed that the expression of myelin markers in the peripheral and central part of the intradural part of the VIIIth nerve started simultaneously, from postnatal day 2 onwards, long before the onset of cochlea function. The expression rapidly achieved saturation levels on the approach to postnatal day 12, the day on which the cochlea function commenced. Because of its importance for the neuronal and morphological maturation of the cochlea during this time, an additional role of thyroid hormone in cochlear myelinogenesis was considered. Indeed, it transpires that this hormone ensures the rapid accomplishment of glial gene expression, not only in the central but also in the peripheral part of the cochlea. Furthermore, an analysis of the thyroid hormone receptors, TRα and TRβ, indicates that TRβ is necessary for myelinogenesis of the VIIIth nerve. Rapid thyroid hormone-dependent saturation of myelin marker gene expression in Schwann cells and oligodendrocytes of the VIIIth nerve may guarantee nerve conduction and synchronized impulse transmission at the onset of hearing. The thyroid hormone-dependent commencement of nerve conduction is discussed in connection with the patterning refinement of central auditory pathways and the acquisition of deafness.

The poor axonal regeneration that follows lesions of the central nervous system (CNS) is crucially influenced by the local CNS tissue environment through which neurites have to grow. In addition to an inhibitory role of the glial scar, inhibitory substrate effects of CNS myelin and oligodendrocytes have been demonstrated. Several proteins including NI-35/250, myelin-associated glycoprotein, tenascin-R, and NG-2 have been described to have neurite outgrowth inhibitory or repulsive properties in vitro. Antibodies raised against NI-35/250 (monoclonal antibody IN-1) were shown to partially neutralize the growth inhibitory effect of CNS myelin and oligodendrocytes, and to result in long distance fiber regeneration in the lesioned adult mammalian CNS in vivo. We report here the purification of a myelin protein to apparent homogeneity from bovine spinal cord which exerts a potent neurite outgrowth inhibitory effect on PC12 cells and chick dorsal root ganglion cells, induces collapse of growth cones of chick dorsal root ganglion cells, and also inhibits the spreading of 3T3 fibroblasts. These activities could be neutralized by the monoclonal antibody IN-1. The purification procedure includes detergent solubilization, anion exchange chromatography, gel filtration, and elution from high resolution SDS-polyacrylamide gel electrophoresis. The active protein has a molecular mass of 220 kDa and an isoelectric point between 5.9 and 6.2. Its inhibitory activity is sensitive to protease treatment and resists harsh treatments like 9 M urea or short heating. Glycosylation is, if present at all, not detectable. Microsequencing resulted in six peptides and strongly suggests that this proteins is novel.

The extent of fibre regeneration in the adult injured vertebrate nervous system appears to be primarily determined by the local environment. Thus, the failure of axon regrowth in the central nervous system (CNS) is crucially influenced by the presence of the myelin-associated neurite growth inhibitor NI-35/250 and possibly also by molecules such as the myelin-associated glycoprotein and the proteoglycans. Developmental time course studies have shown that the capacity for regeneration declines sharply with the appearance of mature oligodendrocytes and myelin, which indicates a role of NI-35/250 in restricting CNS regeneration and plasticity. However, recent in vitro and in vivo studies showed that embryonic neurons are capable of extending fibres on and in adult CNS tissue apparently unaffected by myelinated areas. A possible explanation is that very immature neurons have yet to express the appropriate receptors and response mechanisms for factors that normally induce growth inhibition at a later stage of development. Here we report that embryonic rat dorsal root ganglion and chick retinal ganglion cells display different sensitivity to bovine NI-35/250 compared with mature neurons. In older neurons NI-35/250 could evoke long-lasting collapse responses accompanied by a large increase in the intracellular calcium level, persisting for several minutes. In contrast, their embryonic counterparts collapsed only transiently when exposed to NI-35/250, and increases in intracellular calcium concentration were small and transient. Calcium influx induced experimentally by the calcium ionophore A23187 revealed that it was not the maximal size of the calcium increase but rather the duration of elevated calcium concentration that was the most important determinant for subsequent morphological alterations of the growth cone. Our data further suggest that developing neurons acquire their complete sensitivity for NI-35/250 around the time of myelination.

A comparison was made of neurite growth across spinal cord lesions in the isolated central nervous system (CNS) of newborn opossums (Monodelphis domestica) at various stages of development. The aim was to define the critical period at which growth after injury ceases to occur, with emphasis on growth-inhibitory proteins, myelin and glial cells. In postnatal opossums 3-6 days old (P3-6), repair was observed 5 days after lesions were made in culture at the cervical level (C7) by crushing with forceps. Through-conduction of action potentials was re-established and axons stained by Dil grew into and beyond the crush. In a series of 66 animals 29 showed repair. In 28 animals at P11-12 with comparable lesions repair was observed in five preparations. At P13-14, the CNS was still viable in culture, but none of the 25 preparations examined showed any axonal growth into the crush or conduction through it. The rostro-caudal gradient of development permitted lesions to be made in mature cervical and immature lumbar regions of P11-12 spinal cord. Growth across crushes occurred in lumbar but not in cervical segments of the same preparation. The development of glial cells and myelin was assessed by electron microscopy and by staining with specific antibodies (Rip-1 and myelin-associated glycoprotein) in cervical segments of neonatal P6-14 opossums. At P8, oligodendrocytes and thin myelin sheaths started to appear followed at P9 by astrocytes stained with antibody against glial fibrillary acidic protein. By P14, astrocytes, oligodendrocytes and well-developed myelin sheaths were abundant. The cervical crush sites of P12 cords contained occasional astrocytes but no oligodendrocytes. Specific antibodies (IN-1) to neurite growth-inhibiting proteins (NI-35/250) associated with oligodendrocytes and myelin in the rat CNS cross-reacted with opossum proteins. Assays using the spreading of 3T3 fibroblasts and IN-1 showed that by P7 inhibitory proteins became apparent, particularly in the hindbrain and cervical spinal cord. The concentrations of NI-35/250 thereafter increased and became abundant in the adult opossum. Our finding of a well-defined critical period, encompassing only 5 days, in CNS preparations that can be maintained in culture offers advantages for analysing mechanisms that promote or prevent CNS repair.

In the central nervous system (CNS) the ability of nerve fibres to regenerate upon injury is very limited. In contrast, peripheral nerve fibres have been observed to regrow over long distances. Transplantation experiments with CNS and PNS nerve explants have shown that the local microenvironment is crucial for regenerating nerve fibers: CNS neurons can extend their lesioned neurites into the peripheral nerve environment, but cease growth at the transition between PNS and CNS (Aguayo et al 1990; David and Aguayo 1981; Ramon y Cajal 1928; Tello 1911). To test if a possible lack of trophic factors in the adult CNS might be responsible for this observation, perinatal rat sensory, sympathetic, or retinal neurons were cocultured with explants of adult rat sciatic (PNS) or optic nerves (CNS) in the presence of nerve growth factor (NGF) or brain-derived neurotrophic factor (BDNF) (Schwab & Thoenen 1985). The pattern of fiber growth corresponded exactly to that found in vivo: many axons grew into the sciatic nerve explants, but none grew into the optic nerves. These results suggested that there is a neurite growth inhibitory activity present in adult CNS tissue that cannot be overcome by the stimulatory effects of neurotrophic factors (Schwab & Thoenen, 1985).

Myelin-associated inhibition of axonal regrowth after injury is considered one important factor that contributes to regeneration failure in the adult central nervous system (CNS). Blocking strategies targeting this pathway have been successfully applied in several nerve injury models, including experimental autoimmune encephalomyelitis (EAE), suggesting myelin-associated inhibitors (MAIs) and functionally related molecules as targets to enhance regeneration in multiple sclerosis. NgR1 and NgR2 were identified as interaction partners for the myelin proteins Nogo-A, MAG and OMgp and are probably mediating their growth-inhibitory effects on axons, although the in vivo relevance of this pathway is currently under debate. Recently, alternative functions of MAIs and NgRs in the regulation of immune cell migration and T cell differentiation have been described. Whether and to what extent NgR1 and NgR2 are contributing to Nogo and MAG-related inhibition of neuroregeneration or immunomodulation during EAE is currently unknown. Here we show that genetic deletion of both receptors does not promote functional recovery during EAE and that NgR1 and NgR2-mediated signals play a minor role in the development of CNS inflammation. Induction of EAE in Ngr1/2-double mutant mice resulted in indifferent disease course and tissue damage when compared to WT controls. Further, the development of encephalitogenic CD4(+) Th1 and Th17 responses was unchanged. However, we observed a slightly increased leukocyte infiltration into the CNS in the absence of NgR1 and NgR2, indicating that NgRs might be involved in the regulation of immune cell migration in the CNS. Our study demonstrates the urgent need for a more detailed knowledge on the multifunctional roles of ligands and receptors involved in CNS regeneration failure.

A myelin-associated protein from the central nervous system, the neurite growth inhibitor NI-35, inhibits regeneration of lesioned neuronal fiber tracts in vivo and growth of neurites in vitro. Growth cones of cultured rat dorsal root ganglion neurons arrested their growth and collapsed when exposed to liposomes containing NI-35. Before morphological changes, the concentration of free intracellular calcium ([Ca2+]i) showed a rapid and large increase in growth cones exposed to liposomes containing NI-35. Neither an increase in [Ca2+]i nor collapse of growth cones was detected in the presence of antibodies to NI-35. Dantrolene, an inhibitor of calcium release from caffeine-sensitive intracellular calcium stores, protected growth cones from collapse evoked by NI-35. Depletion of these caffeine-sensitive intracellular calcium stores prevented the increase in [Ca2+]i evoked by NI-35. The NI-35-evoked cascade of intracellular messengers that mediates collapse of growth cones includes the crucial step of calcium release from intracellular stores.