Neuritogenesis: the prion protein controls β1 integrin signaling activity

Cytoskeleton modifications are required for neuronal stem cells to acquire neuronal polarization. Little is known, however, about mechanisms that orchestrate cytoskeleton remodeling along neuritogenesis. Here, we show that the silencing of the cellular prion protein (PrP(C)) impairs the initial sprouting of neurites upon induction of differentiation of the 1C11 neuroectodermal cell line, indicating that PrP(C) is necessary to neuritogenesis. Such PrP(C) function relies on its capacity to negatively regulate the clustering, activation, and signaling activity of β1 integrins at the plasma membrane. β1 Integrin aggregation caused by PrP(C) depletion triggers overactivation of the RhoA-Rho kinase-LIMK-cofilin pathway, which, in turn, alters the turnover of focal adhesions, increases the stability of actin microfilaments, and in fine impairs neurite formation. Inhibition of Rho kinases is sufficient to compensate for the lack of PrP(C) and to restore neurite sprouting. We also observe an increased secretion of fibronectin in the surrounding milieu of PrP(C)-depleted 1C11 cells, which likely self-sustains β1 integrin signaling overactivation and contributes to neuritogenesis defect. Our overall data reveal that PrP(C) contributes to the acquisition of neuronal polarization by modulating β1 integrin activity, cell interaction with fibronectin, and cytoskeleton dynamics.     

RhoA/ROCK regulation of neuritogenesis via profilin IIa-mediated control of actin stability

Neuritogenesis, the first step of neuronal differentiation, takes place as nascent neurites bud from the immediate postmitotic neuronal soma. Little is known about the mechanisms underlying the dramatic morphological changes that characterize this event. Here, we show that RhoA activity plays a decisive role during neuritogenesis of cultured hippocampal neurons by recruiting and activating its specific kinase ROCK, which, in turn, complexes with profilin IIa. We establish that this previously uncharacterized brain-specific actin-binding protein controls neurite sprouting by modifying actin stability, a function regulated by ROCK-mediated phosphorylation. Furthermore, we determine that this novel cascade is switched on or off by physiological stimuli. We propose that RhoA/ROCK/PIIa-mediated regulation of actin stability, shown to be essential for neuritogenesis, may constitute a central mechanism throughout neuronal differentiation.     

Ena/VASP Is Required for neuritogenesis in the developing cortex

Mammalian cortical development involves neuronal migration and neuritogenesis; this latter process forms the structural precursors to axons and dendrites. Elucidating the pathways that regulate the cytoskeleton to drive these processes is fundamental to our understanding of cortical development. Here we show that loss of all three murine Ena/VASP proteins, a family of actin regulatory proteins, causes neuronal ectopias, alters intralayer positioning in the cortical plate, and, surprisingly, blocks axon fiber tract formation during corticogenesis. Cortical fiber tract defects in the absence of Ena/VASP arise from a failure in neurite initiation, a prerequisite for axon formation. Neurite initiation defects in Ena/VASP-deficient neurons are preceded by a failure to form bundled actin filaments and filopodia. These findings provide insight into the regulation of neurite formation and the role of the actin cytoskeleton during cortical development.     

Cytoskeletal dynamics underlying collateral membrane protrusions induced by neurotrophins in cultured Xenopus embryonic neurons

The establishment and refinement of neuronal connections depend on dynamic modification of the morphology and physiology of developing axons in response to extrinsic factors. In embryonic cultures of Xenopus spinal neurons, acute application of brain-derived neurotrophic factor (BDNF) induced rapid collateral protrusion of filopodium-like microspikes and lamellipodia along the neurite processes, leading to a morphologic alternation of the neuron. Both types of membrane protrusions contained high concentrations of actin filaments and depended on the polymerization of the actin cytoskeleton. Immunofluorescent staining, however, revealed the presence of microtubules (MTs) in lamellipodia induced by BDNF. These MTs appeared to have arisen from debundling of MTs in the neurite shaft at the protrusion sites, splaying and extending in the rapidly protruding lamellipodia. Inhibition of microtubule polymerization by nocodazole largely abolished the formation of lamellipodia but not of microspikes. Taken together, our results suggest that collateral sprouting of microspikes and lamellipodia involve distinctly different cytoskeletal mechanisms. Although the actin cytoskeleton is solely responsible for microspike formation, cooperative efforts by microtubules and actin filaments are essential for lamellipodial protrusion in response to extrinsic factors.     

Clustering of the neural cell adhesion molecule (NCAM) at the neuronal cell surface induces caspase-8- and -3-dependent changes of the spectrin meshwork required for NCAM-mediated neurite outgrowth

Changes in neuronal morphology underlying neuronal differentiation depend on rapid and sustained cytoskeleton rearrangements in the growing neurites. Whereas cell adhesion molecules are well established as regulators of neuronal differentiation, less is known about the signaling mechanisms by which they influence the cytoskeleton. Here we show that the neural cell adhesion molecule (NCAM) associates with the active form of caspase-8 and that clustering of NCAM at the neuronal cell surface leads to activation of caspase-8 and -3 followed by the cleavage of the sub-membranous brain spectrin meshwork, but not of the actin or tubulin cytoskeleton. Inhibitors of caspase-8 and -3 specifically block the NCAM-dependent spectrin cleavage and abolish NCAM-dependent neurite outgrowth. NCAM-dependent rearrangements of the membrane associated spectrin meshwork via caspase-8 dependent caspase-3 activation are thus indispensable for NCAM-mediated neurite outgrowth.     

AlphaII-spectrin participates in the surface expression of cell adhesion molecule L1 and neurite outgrowth

AlphaII-spectrin, a basic component of the spectrin-based scaffold which organizes and stabilizes membrane microdomains in most animal cells, has been recently implicated in cell adherence and actin dynamics. Here we investigated the contribution of αΙΙ-spectrin to neuritogenesis, a highly complex cellular process which requires continuous actin cytoskeleton remodeling and cross-talk between extracellular cues and their cell surface receptors, including cell adhesion molecules. Using RNA interference-mediated gene silencing to down-regulate αΙΙ-spectrin expression in human neuroblastoma SH-SY5Y cells, we observed major changes in neurite morphology and cell shape: (1) reduced mean length and a higher number of neurites per cell; occasional long neurites were thinner and displayed abnormal adhesiveness during cell migration resulting in frequent breaks; similar persisting adhesiveness and breaks were also observed in trailing edges of cell bodies; (2) irregular polygonal cell shape in parallel with loss of cortical F-actin from neuronal cell bodies; (3) reduction in protein levels of αΙ- and βΙ-spectrins, but not βΙΙ-spectrin (4) decreased global expression of adhesion molecule L1 and spectrin-binding adapter ankyrin-B, which links L1 to the plasma membrane. Remarkably, αΙΙ-spectrin depletion affected L1 – but not NCAM – cell surface expression, and L1 clustering at growth cones. This study demonstrates that αΙΙ-spectrin is implicated in normal morphology and adhesive properties of neuron cell bodies and neurites, and in cell surface expression and organization of adhesion molecule L1.     

Spatial phosphoprotein profiling reveals a compartmentalized extracellular signal-regulated kinase switch governing neurite growth and retraction

Brain development and spinal cord regeneration require neurite sprouting and growth cone navigation in response to extension and collapsing factors present in the extracellular environment. These external guidance cues control neurite growth cone extension and retraction processes through intracellular protein phosphorylation of numerous cytoskeletal, adhesion, and polarity complex signaling proteins. However, the complex kinase/substrate signaling networks that mediate neuritogenesis have not been investigated. Here, we compare the neurite phosphoproteome under growth and retraction conditions using neurite purification methodology combined with mass spectrometry. More than 4000 non-redundant phosphorylation sites from 1883 proteins have been annotated and mapped to signaling pathways that control kinase/phosphatase networks, cytoskeleton remodeling, and axon/dendrite specification. Comprehensive informatics and functional studies revealed a compartmentalized ERK activation/deactivation cytoskeletal switch that governs neurite growth and retraction, respectively. Our findings provide the first system-wide analysis of the phosphoprotein signaling networks that enable neurite growth and retraction and reveal an important molecular switch that governs neuritogenesis.     

GRK5 promotes F-actin bundling and targets bundles to membrane structures to control neuronal morphogenesis

Neuronal morphogenesis requires extensive membrane remodeling and cytoskeleton dynamics. In this paper, we show that GRK5, a G protein-coupled receptor kinase, is critically involved in neurite outgrowth, dendrite branching, and spine morphogenesis through promotion of filopodial protrusion. Interestingly, GRK5 is not acting as a kinase but rather provides a key link between the plasma membrane and the actin cytoskeleton. GRK5 promoted filamentous actin (F-actin) bundling at the membranes of dynamic neuronal structures by interacting with both F-actin and phosphatidylinositol-4,5-bisphosphate. Moreover, separate domains of GRK5 mediated the coupling of actin cytoskeleton dynamics and membrane remodeling and were required for its effects on neuronal morphogenesis. Accordingly, GRK5 knockout mice exhibited immature spine morphology and deficient learning and memory. Our findings identify GRK5 as a critical mediator of dendritic development and suggest that coordinated actin cytoskeleton and membrane remodeling mediated by bifunctional actin-bundling and membrane-targeting molecules, such as GRK5, is crucial for proper neuronal morphogenesis and the establishment of functional neuronal circuitry.     

Tropomodulins are negative regulators of neurite outgrowth

Regulation of the actin cytoskeleton is critical for neurite formation. Tropomodulins (Tmods) regulate polymerization at actin filament pointed ends. Previous experiments using a mouse model deficient for the neuron specific isoform Tmod2 suggested a role for Tmods in neuronal function by impacting processes underlying learning and memory. However, the role of Tmods in neuronal function on the cellular level remains unknown. Immunofluorescence localization of the neuronal isoforms Tmod1 and Tmod2 in cultured rat primary hippocampal neurons revealed that Tmod1 is enriched along the proximal part of F-actin bundles in lamellipodia of spreading cells and in growth cones of extending neurites, while Tmod2 appears largely cytoplasmic. Functional analysis of these Tmod isoforms in a mouse neuroblastoma N2a cell line showed that knockdown of Tmod2 resulted in a significant increase in the number of neurite-forming cells and in neurite length. While N2a cells compensated for Tmod2 knockdown by increasing Tmod1 levels, over-expression of exogenous Tmod1 had no effect on neurite outgrowth. Moreover, knockdown of Tmod1 increased the number of neurites formed per cell, without effect on the number of neurite-forming cells or neurite length. Taken together, these results indicate that Tmod1 and Tmod2 have mechanistically distinct inhibitory roles in neurite formation, likely mediated via different effects on F-actin dynamics and via differential localizations during early neuritogenesis.     

p190 RhoGAP is the principal Src substrate in brain and regulates axon outgrowth, guidance and fasciculation

The Src tyrosine kinases have been implicated in several aspects of neural development and nervous system function; however, their relevant substrates in brain and their mechanism of action in neurons remain to be established clearly. Here we identify the potent Rho regulatory protein, p190 RhoGAP (GTPase-activating protein), as the principal Src substrate detected in the developing and mature nervous system. We also find that mice lacking functional p190 RhoGAP exhibit defects in axon guidance and fasciculation. p190 RhoGAP is co-enriched with F-actin in the distal tips of axons, and overexpressing p190 RhoGAP in neuroblastoma cells promotes extensive neurite outgrowth, indicating that p190 RhoGAP may be an important regulator of Rho-mediated actin reorganization in neuronal growth cones. p190 RhoGAP transduces signals downstream of cell-surface adhesion molecules, and we find that p190-RhoGAP-mediated neurite outgrowth is promoted by the extracellular matrix protein laminin. Together with the fact that mice lacking neural adhesion molecules or Src kinases also exhibit defects in axon outgrowth, guidance and fasciculation, our results suggest that p190 RhoGAP mediates a Src-dependent adhesion signal for neuritogenesis to the actin cytoskeleton through the Rho GTPase.     

N-cadherin specifies first asymmetry in developing neurons

The precise polarization and orientation of developing neurons is essential for the correct wiring of the brain. In pyramidal excitatory neurons, polarization begins with the sprouting of opposite neurites, which later define directed migration and axo-dendritic domains. We here show that endogenous N-cadherin concentrates at one pole of the newborn neuron, from where the first neurite subsequently emerges. Ectopic N-cadherin is sufficient to favour the place of appearance of the first neurite. The Golgi and centrosome move towards this newly formed morphological pole in a second step, which is regulated by PI3K and the actin/microtubule cytoskeleton. Moreover, loss of function experiments in vivo showed that developing neurons with a non-functional N-cadherin misorient their cell axis. These results show that polarization of N-cadherin in the immediate post-mitotic stage is an early and crucial mechanism in neuronal polarity.     

Microtubules and growth cone function

It has been recognized for a long time that the neuronal cytoskeleton plays an important part in neurite growth and growth cone pathfinding, the mechanism by which growing axons find an appropriate route through the developing embryo to their target cells. In the growth cone, many intracellular signaling pathways that are activated by guidance cues converge on the growth cone cytoskeleton and regulate its dynamics. Most of the research effort in this area has focussed on the actin, microfilament cytoskeleton of the growth cone, principally because it underlies growth cone motility, the extension and retraction of filopodia and lamellipodia, and these structures are the first to encounter guidance cues during growth cone advance. However, more recently, it has become apparent that the microtubule cytoskeleton also has a role in growth cone pathfinding and is also regulated by guidance cues operating through intracellular signaling pathways via engagement with cell membrane receptors. Furthermore, recent work has revealed an interaction between these two components of the growth cone cytoskeleton that is probably essential for growth cone turning, a fundamental growth cone behavior during pathfinding. In this short review I discuss recent experiments that uncover the function of microtubules in growth cones, how their behavior is regulated, and how they interact with the actin filaments.     

The promotion of neurite formation in Neuro2A cells by mouse Mob2 protein

The molecular mechanism of neuritogenesis has been extensively studied but remains unclear. In this study, we identified Mob2 protein which plays a significant role in promoting neurite formation in Neuro2A (N2A) cells. Our results showed that Mob2 was expressed in developing N2A cells. To study whether Mob2 was involved in neurite formation, we downregulated Mob2 expression using RNA interference and found that neurite formation decreased in low serum induced N2A cells. In addition, we found that overexpression of Mob2 promoted neurite formation in N2A cells. Furthermore, downregulation of Mob2 expression altered the rearrangement of the actin cytoskeleton and decreased the expression of phosphorylated Moesin. Together, these results provide information on the role of Mob2 in mediating neurite formation.     

Neurite formation by neurons derived from adult rat hippocampal progenitor cells is susceptible to myelin inhibition

Myelin-associated inhibitors expressed following injury to the adult central nervous system (CNS) induce growth cone collapse and retraction of the axonal cytoskeleton. Myelin-associated glycoprotein (MAG) is a bi-functional molecule that promotes neuritogenesis in some immature neurons during development then becomes inhibitory to neurite outgrowth as neurons mature. Progress is being made towards the elucidation of the downstream events that regulate myelin inhibition of regeneration in neuronal populations. However it is not known how adult-derived neural stem cells or progenitors respond to myelin during neuronal differentiation and neuritogenesis. Here we examine the effect of MAG on neurons derived from an adult rat hippocampal progenitor cell line (AHPCs). We show that, unlike their developmental counterparts, AHPC-derived neurons are susceptible to MAG inhibition of neuritogenesis during differentiation and display a 57% reduction in neurite outgrowth when compared with controls. We demonstrate that this effect can be overcome (by up to 69%) by activation of the neurotrophin, cyclic AMP and protein kinase A pathways or by Rho-kinase suppression. We also demonstrate that combination of these factors enhanced neurite outgrowth from differentiating neurons in the presence of MAG. This work provides important information for the successful generation of new neurons from adult neural stem cell populations within compromised adult circuitry and is thus directly relevant to endogenous repair and regeneration of the adult CNS.     

Shared and unique roles of CAP23 and GAP43 in actin regulation, neurite outgrowth, and anatomical plasticity

CAP23 is a major cortical cytoskeleton-associated and calmodulin binding protein that is widely and abundantly expressed during development, maintained in selected brain structures in the adult, and reinduced during nerve regeneration. Overexpression of CAP23 in adult neurons of transgenic mice promotes nerve sprouting, but the role of this protein in process outgrowth was not clear. Here, we show that CAP23 is functionally related to GAP43, and plays a critical role to regulate nerve sprouting and the actin cytoskeleton. Knockout mice lacking CAP23 exhibited a pronounced and complex phenotype, including a defect to produce stimulus-induced nerve sprouting at the adult neuromuscular junction. This sprouting deficit was rescued by transgenic overexpression of either CAP23 or GAP43 in adult motoneurons. Knockin mice expressing GAP43 instead of CAP23 were essentially normal, indicating that, although these proteins do not share homologous sequences, GAP43 can functionally substitute for CAP23 in vivo. Cultured sensory neurons lacking CAP23 exhibited striking alterations in neurite outgrowth that were phenocopied by low doses of cytochalasin D. A detailed analysis of such cultures revealed common and unique functions of CAP23 and GAP43 on the actin cytoskeleton and neurite outgrowth. The results provide compelling experimental evidence for the notion that CAP23 and GAP43 are functionally related intrinsic determinants of anatomical plasticity, and suggest that these proteins function by locally promoting subplasmalemmal actin cytoskeleton accumulation.     

The c-Fes tyrosine kinase cooperates with the breakpoint cluster region protein (Bcr) to induce neurite extension in a Rac- and Cdc42-dependent manner

The c-fes locus encodes a cytoplasmic protein-tyrosine kinase (Fes) previously shown to accelerate nerve growth factor (NGF)-induced neurite outgrowth in rat PC12 cells. Here, we investigated the role of the Rho family small GTPases Rac1 and Cdc42 in Fes-mediated neuritogenesis, which have been implicated in neuronal differentiation in other systems. Fes-induced acceleration of neurite outgrowth in response to NGF treatment was completely blocked by the expression of dominant-negative Rac1 or Cdc42. Expression of a kinase-active mutant of Fes induced constitutive relocalization of endogenous Rac1 to the cell periphery in the absence of NGF, and led to dramatic actin reorganization and spontaneous neurite extension. We also investigated the breakpoint cluster region protein (Bcr), which possesses the Dbl and PH domains characteristic of guanine nucleotide exchange factors for Rho family GTPases, as a possible link between Fes, Rac/Cdc42 activation, and neuritogenesis. Coexpression of a GFP-Bcr fusion protein containing the Fes binding and tyrosine phosphorylation sites (amino acids 162-413) completely suppressed neurite outgrowth triggered by Fes. Conversely, coexpression of full-length Bcr with wild-type Fes in PC12 cells induced NGF-independent neurite formation. Taken together, these data suggest that Fes and Bcr cooperate to activate Rho family GTPases as part of a novel pathway regulating neurite extension in PC12 cells, and provide more evidence for an emerging role for Fes in neuronal differentiation.     

Microtubule-Associated Type II Protein Kinase A Is Important for Neurite Elongation

Neuritogenesis is a process through which neurons generate their widespread axon and dendrites. The microtubule cytoskeleton plays crucial roles throughout neuritogenesis. Our previous study indicated that the amount of type II protein kinase A (PKA) on microtubules significantly increased upon neuronal differentiation and neuritogenesis. While the overall pool of PKA has been shown to participate in various neuronal processes, the function of microtubule-associated PKA during neuritogenesis remains largely unknown. First, we showed that PKA localized to microtubule-based region in different neurons. Since PKA is essential for various cellular functions, globally inhibiting PKA activity will causes a wide variety of phenotypes in neurons. To examine the function of microtubule-associated PKA without changing the total PKA level, we utilized the neuron-specific PKA anchoring protein MAP2. Overexpressing the dominant negative MAP2 construct that binds to type II PKA but cannot bind to the microtubule cytoskeleton in dissociated hippocampal neurons removed PKA from microtubules and resulted in compromised neurite elongation. In addition, we demonstrated that the association of PKA with microtubules can also enhance cell protrusion using the non-neuronal P19 cells. Overexpressing a MAP2 deletion construct which does not target PKA to the microtubule cytoskeleton caused non-neuronal cells to generate shorter cell protrusions than control cells overexpressing wild-type MAP2 that anchors PKA to microtubules. Finally, we demonstrated that the ability of microtubule-associated PKA to promote protrusion elongation was independent of MAP2 phosphorylation. This suggests other proteins in close proximity to the microtubule cytoskeleton are involved in this process.     

Transient neuritogenesis in NB2a/d1 neuroblastoma cells induced by glial-derived protease inhibitors

The initial outgrowth of neuritogenesis in mouse NB2a/d1 neuroblastoma cells may be regulated by thrombin or a thrombin-like protease, present either in serum or adsorbed to the plasma membrane, since neuritogenesis is induced by serum deprivation and treatment with the specific thrombin inhibitor, hirudin (Shea et al., 1991, J. Neurochem., 56:842). Cultured astroglial cells secrete factors that promote neuritogenesis, including protease inhibitors active against thrombin, leading to suggestions that the inhibition of specific neuronal surface proteases by the surrounding glial environment may represent an initial step in axonal outgrowth in situ. To examine the relative importance of glial-derived protease inhibitory activities on neurine outgrowth, we tested the neurite promoting effect of glial-conditioned medium (GCM) on NB2a/d1 cells. Like serum deprivation and hirudin treatment, GCM induced neurite outgrowth within 4 hr. Exogenous thrombin inhibited the effect of GCM, and cell-free enzyme assays confirmed the presence of thrombin-inhibitory activity in GCM, suggesting that GCM induces neuritogenesis by inhibition of a thrombin-like protease. Unlike neurites induced by serum removal or hirudin addition, which are rapidly resorbed following serum replenishment or hirudin depletion, however, GCM-induced neurites continued to elongate after GCM removal. Furthermore, cultures treated simultaneously with GCM and thrombin exhibited delayed outgrowth of neurites following GCM removal which were insensitive to further thrombin treatment. These findings indicate that the initial elaboration of neurites can be mediated by glial-derived protease inhibitor(s) active against a thrombin-like protease, but indicate the requirement of additional glial-derived factors for the maintenance and continued elaboration of these neurites.     

Role of phosphatidylcholine during neuronal differentiation

Neuronal differentiation is characterized by neuritogenesis and neurite outgrowth, processes, which are critically dependent on membrane biosynthesis, and therefore, on the expression and regulation of enzymes involved in phospholipid biosynthesis. During the last decade a great effort was made to clarify where membrane lipids are synthesized, how the newly synthesized membrane components reach the membrane and are inserted during neuritogenesis and to elucidate the mechanism by which the supply of new membrane components is coordinated with the demand for growth. Phosphatidylcholine is the principal and essential component for mammalian membranes. This review updates the mechanism by which phosphatidylcholine biosynthesis takes place and how it is coordinately regulated during neuronal differentiation.