GSK-3beta regulates phosphorylation of CRMP-2 and neuronal polarity

Neurons are highly polarized and comprised of two structurally and functionally distinct parts, an axon and dendrites. We previously showed that collapsin response mediator protein-2 (CRMP-2) is critical for specifying axon/dendrite fate, possibly by promoting neurite elongation via microtubule assembly. Here, we showed that glycogen synthase kinase-3beta (GSK-3beta) phosphorylated CRMP-2 at Thr-514 and inactivated it. The expression of the nonphosphorylated form of CRMP-2 or inhibition of GSK-3beta induced the formation of multiple axon-like neurites in hippocampal neurons. The expression of constitutively active GSK-3beta impaired neuronal polarization, whereas the nonphosphorylated form of CRMP-2 counteracted the inhibitory effects of GSK-3beta, indicating that GSK-3beta regulates neuronal polarity through the phosphorylation of CRMP-2. Treatment of hippocampal neurons with neurotrophin-3 (NT-3) induced inactivation of GSK-3beta and dephosphorylation of CRMP-2. Knockdown of CRMP-2 inhibited NT-3-induced axon outgrowth. These results suggest that NT-3 decreases phosphorylated CRMP-2 and increases nonphosphorylated active CRMP-2, thereby promoting axon outgrowth.     

Ras regulates neuronal polarity via the PI3-kinase/Akt/GSK-3beta/CRMP-2 pathway

The establishment of a polarized morphology is an essential event in the differentiation of neurons into a single axon and dendrites. We previously showed that glycogen synthase kinase-3beta (GSK-3beta) is critical for specifying axon/dendrite fate by the regulation of the phosphorylation of collapsin response mediator protein-2 (CRMP-2). Here, we found that the overexpression of the small GTPase Ras induced the formation of multiple axons in cultured hippocampal neurons, whereas the ectopic expression of the dominant negative form of Ras inhibited the formation of axons. Inhibition of phosphatidylinositol-3-kinase (PI3-kinase) or extracellular signal-related kinase (ERK) kinase (MEK) suppressed the Ras-induced formation of multiple axons. The expression of the constitutively active form of PI3-kinase or Akt (also called protein kinase B) induced the formation of multiple axons. The overexpression of Ras prevented the phosphorylation of CRMP-2 by GSK-3beta. Taken together, these results suggest that Ras plays critical roles in establishing neuronal polarity upstream of the PI3-kinase/Akt/GSK-3beta/CRMP-2 pathway and mitogen-activated protein kinase cascade.     

Role of the integrin-linked kinase (ILK) in determining neuronal polarity

The establishment of axon-dendrite polarity in mammalian neurons has recently been shown to involve the kinases Akt and GSK-3beta. Here we report the function of the integrin-linked kinase (ILK) in neuronal polarization. ILK distribution is differential: with more of it present in the axonal tips than that in the dendritic tips of a polarized neuron. Inactivation of ILK by chemical inhibitors, a kinase-inactive mutant or siRNAs inhibited axon formation, whereas a kinase hyperactive ILK mutant induced the formation of multiple axons. Biochemical studies indicate that ILK is upstream of Akt and GSK-3beta. Manipulations of multiple intracellular components indicate that ILK is functionally upstream of Akt and GSK-3beta but downstream of PI3K in neuronal polarity. These results reveal a key role of ILK in the formation of neuronal polarity and suggest a signaling pathway important for neuronal polarity.     

Regulation of neuronal polarity

The distinctive polarized morphology of neuronal cells is essential for the proper wiring of the nervous system. The rodent hippocampal neuron culture established about three decades ago has provided an amenable in vitro system to uncover the molecular mechanisms underlying neuronal polarization, a process relying on highly regulated cytoskeletal dynamics, membrane traffic and localized protein degradation. More recent research in vivo has highlighted the importance of the extracellular environment and cell–cell interactions in neuronal polarity. Here, I will review some key signaling pathways regulating neuronal polarization and provide some insights on the complexity of this process gained from in vivo studies.     

Maintenance of neuronal polarity

Neurons are highly polarized cells with distinct domains responsible for receiving, transmitting, and propagating electrical signals. Central to these functions is the axon initial segment (AIS), a short region of the axon adjacent to the cell body that is enriched in voltage-gated ion channels, cell adhesion molecules, and cytoskeletal scaffolding proteins. Traditionally, the function of the AIS has been limited to its role in action potential initiation and modulation. However, recent experiments indicate that it also plays essential roles in neuronal polarity. Here, we review how initial segments are assembled, and discuss proposed mechanisms for how the AIS contributes to maintenance of neuronal polarity.     

Neurofibromin interacts with CRMP-2 and CRMP-4 in rat brain

Neurofibromin, encoded by the neurofibromatosis type 1 (NF1) gene, regulates the Ras and cAMP pathways and plays a role in proliferation and neuronal morphogenesis. The details of the molecular mechanism of neurofibromin action in these processes are still unclear. In this study, immunoprecipitation and proteomics were used to identify novel proteins from rat brain that interact with neurofibromin. Mass spectrometry analysis showed that two proteins, the collapsin response mediator protein-2 (CRMP-2) and propionyl-CoA carboxylase alpha chain (PCCA), associated with neurofibromin. Immunoprecipitation-immunoblotting analysis confirmed the interactions between neurofibromin and CRMP-2 and CRMP-4, but not CRMP-1, in rat brain. CDK5, a kinase that regulates CRMP-2 in axonal outgrowth, was required for the interaction between neurofibromin and CRMP-2. Since both neurofibromin and CRMP proteins are involved in proliferation and axonal morphogenesis, these results suggest that the interaction with CRMPs contributes to the function of neurofibromin in tumorigenesis and neuronal morphogenesis.     

Key regulators in neuronal polarity

Neurons are highly polarized cells, most of which develop a single axon and several dendrites. These two compartments acquire specific characteristics that enable neurons to transmit intercellular signals from several dendrites to an axon. A wealth of recent studies has shown that PI 3-kinase, Rho family GTPases, the Par complex, and cytoskeleton-related proteins participate in the initial events of neuronal polarization. Here, we review the role of polarity-regulating molecules and the potential mechanisms underlying the specification of an axon and dendrites.     

p80 ROKalpha binding protein is a novel splice variant of CRMP-1 which associates with CRMP-2 and modulates RhoA-induced neuronal morphology

Using antibody against the Rho binding domain of ROKalpha, two neuronal phosphoproteins of 62 and 80 kDa were co-immunoprecipitated from brain extracts. Peptide analysis revealed their identity as collapsin response mediator proteins (CRMPs); p62 was CRMP-2 whereas p80 was a novel splice form of CRMP-1 with an extended N-terminus. p80 CRMP-1 was able to complex with CRMP-2, suggesting that p80 CRMP-1 and CRMP-2 form oligomers. CRMP-2 was the major substrate of ROK. p80 CRMP-1 interacted with the kinase domain of ROKalpha, resulting in inhibition of the catalytic activity towards other substrates. Over-expression of p80 CRMP-1 and CRMP-2 together counteracted the effects of RhoA on neurite retraction, an effect enhanced by mutation of the ROK phosphorylation site in CRMP-2. p80 CRMP-1 and CRMP-2 may be modulators of RhoA-dependent signaling, through interaction with and regulation of ROKalpha.     

Role of the PAR-3-KIF3 complex in the establishment of neuronal polarity

Neurons polarize to form elaborate multiple dendrites and one long axon. The establishment and maintenance of axon/dendrite polarity are fundamentally important for neurons. Recent studies have demonstrated that the polarity complex PAR-3-PAR-6-atypical protein kinase C (aPKC) is involved in polarity determination in many tissues and cells. The function of the PAR-3-PAR-6-aPKC protein complex depends on its subcellular localization in polarized cells. PAR-3 accumulates at the tip of growing axons in cultured rat hippocampal neurons, but the molecular mechanism of this localization remains unknown. Here we identify a direct interaction between PAR-3 and KIF3A, a plus-end-directed microtubule motor protein, and show that aPKC can associate with KIF3A through its interaction with PAR-3. The expression of dominant-negative PAR-3 and KIF3A fragments that disrupt PAR-3-KIF3A binding inhibited the accumulation of PAR-3 and aPKC at the tip of the neurites and abolished neuronal polarity. These results suggest that PAR-3 is transported to the distal tip of the axon by KIF3A and that the proper localization of PAR-3 is required to establish neuronal polarity.     

Par proteins and neuronal polarity

A hallmark of neurons is their ability to polarize with dendrite and axon specification to allow the proper flow of information through the nervous system. Over the past decade, extensive research has been performed in an attempt to understand the molecular and cellular machinery mediating this neuronal polarization process. It has become evident that many of the critical regulators involved in establishing neuronal polarity are evolutionarily conserved proteins that had previously been implicated in controlling the polarization of other cell types. At the forefront of this research are the partition defective (Par) proteins. In this review,we will provide a commentary on the progress of work regarding the central importance of Parproteins in the establishment of neuronal polarity.     

Cadherins as regulators of neuronal polarity

A compelling amount of data is accumulating about the polyphonic role of neuronal cadherins during brain development throughout all developmental stages, starting from the involvement of cadherins in the organization of neurulation up to synapse development and plasticity. Recent work has confirmed that specifically N-cadherins play an important role in asymmetrical cellular processes in developing neurons that are at the basis of polarity. In this review we will summarize recent data, which demonstrate how N-cadherin orchestrates distinct processes of polarity establishment in neurons.     

Cadherins as regulators of neuronal polarity

A compelling amount of data is accumulating about the polyphonic role of neuronal cadherins during brain development throughout all developmental stages, starting from the involvement of cadherins in the organization of neurulation up to synapse development and plasticity. Recent work has confirmed that specifically N-cadherins play an important role in asymmetrical cellular processes in developing neurons that are at the basis of polarity. In this review we will summarize recent data, which demonstrate how N-cadherin orchestrates distinct processes of polarity establishment in neurons.     

N-cadherin: A new player in neuronal polarity

Comment on: Gärtner A, et al. EMBO J 2012; <span class="b">31</span>:1893-903     

N-cadherin: A new player in neuronal polarity

Comment on: Gärtner A, et al. EMBO J 2012; 31:1893-903     

CRMP-2 regulates polarized Numb-mediated endocytosis for axon growth

Axon growth during neural development is highly dependent on both cytoskeletal re-organization and polarized membrane trafficking. Previously, we demonstrated that collapsin response mediator protein-2 (CRMP-2) is critical for specifying axon/dendrite fate and axon growth in cultured hippocampal neurons, possibly by interacting with tubulin heterodimers and promoting microtubule assembly. Here, we identify Numb as a CRMP-2-interacting protein. Numb has been shown to interact with alpha-adaptin and to be involved in endocytosis. We found that Numb was associated with L1, a neuronal cell adhesion molecule that is endocytosed and recycled at the growth cone, where CRMP-2 and Numb were colocalized. Furthermore, expression of dominant-negative CRMP-2 mutants or knockdown of CRMP-2 message with small-interfering (si) RNA inhibited endocytosis of L1 at axonal growth cones and suppressed axon growth. These results suggest that in addition to regulating microtubule assembly, CRMP-2 is involved in polarized Numb-mediated endocytosis of proteins such as L1.     

Systems biology of symmetry breaking during neuronal polarity formation

Polarization, in which a single axon and multiple dendrites are formed, is crucial for neuronal functions, and symmetry breaking is the initial step of this process. Accumulating studies have revealed a number of molecules that act asymmetrically in neurons, and thereby regulate neuronal polarity. Thus, one of the major goals of current research is to understand how asymmetric signals are generated during the symmetry-breaking step. Current models of neuronal symmetry breaking generally involve "local activation" for induction of axon outgrowth and "global inhibition" to suppress formation of multiple axons and can be categorized into "one-takes-all" and "activator-inhibitor" models. Both types of model incorporate a positive feedback loop to execute local activation, but differ in the manner of global inhibition. Quantitative experimentation combined with computational modeling is a powerful strategy in systems biology, and analyses in this direction have begun to yield a more profound understanding of how neurons break their symmetry during polarity formation.     

In vitro control of neuronal polarity by glycosaminoglycans.

We have studied the effects of proteoglycans (PGs) and glycosaminoglycans (GAGs) on the growth and morphology of neurons in culture. PGs from glial cells or Engelbreth-Holm-Swarm tumor cells (EHS), pure bovine kidney heparan sulfate (HS), shark cartilage type C chondro?tin sulfate (CSc) and bovine mucosa dermatan sulfate (DS) added to embryonic rat neurons strongly enhanced total neurite growth after 48 h in vitro. No trophic effects were seen when PGs treated with a mixture of glycanases were used. PGs, CSc and HS not only enhanced neurite growth but induced the appearance of a majority of neurons with a single long axon whereas, in contrast, DS increased dendrite growth. GAGs bound to the cell surface and were rapidly internalized, a feature that correlated well with the absence of neurotrophicity of GAGs previously immobilized on the culture substratum. Although the mechanisms involved in GAGs neurotrophic effects and in the separate regulation of neuronal polarity by HS and DS were not elucidated, we found that, as opposed to HS, DS was able to enhance neuronal adhesion and spreading and to maintain a high level of expression of microtubule-associated protein 2 (MAP2), a specific dendritic marker. This finding confirms and extends our previous observations on the role of adhesion in the regulation of dendrite growth.