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Here, will review current evidence regarding the signaling pathways and mechanisms underlying membrane addition at sites of active growth during axon formation. © 2017 Wiley Periodicals, Inc. Develop Neurobiol 78: 170-180, 2018., (© 2017 Wiley Periodicals, Inc.)

Axonal growth cone motility requires precise regulation of adhesion to navigate the complex environment of the nervous system and reach its target. Myristoylated alanine-rich C kinase substrate (MARCKS) protein is enriched in the developing brain and plays an important, phosphorylation-dependent role in the modulation of axonal growth cone adhesion. The ratio of phospho-MARCKS (MARCKS-P) to total MARCKS controls adhesion modulation and spreading of the axonal growth cone. Pin1, a peptidyl-prolyl cis/trans isomerase (PPIase) that recognizes and binds to phosphorylated serine/threonine residues preceded by a proline (pSer/Thr-Pro) is also expressed in the developing brain. Here, we show that Pin1 is present in the growth cone, interacts with MARCKS-P, and regulates its dephosphorylation. We also described morphological alterations in the corpus callosum and cerebral cortex fibers of the Pin1 knockout mouse brain that may be caused by the misregulation of MARCKS-P and alterations of neuronal adhesion. We have shown that MARCKS, a critical protein in the movement of neuronal growth cones, is in turn regulated by both phosphorylation and cis-trans peptidyl isomerization mediated by Pin1. In the absence of Pin1, MARCKS is hyperphosphorylated, leading to loss of adhesions, and collapse of the growth cone. The Pin1 KO mice exhibited disturbed neuronal projections from the cerebral cortex and reduced white matter tracks such as the corpus callosum. This study highlights a novel function of Pin1 in neurodevelopment., (© 2016 International Society for Neurochemistry.)

Axonal regeneration is an essential condition to re-establish functional neuronal connections in the injured adult central nervous system (CNS), but efficient regrowth of severed axons has proven to be very difficult to achieve. Although significant progress has been made in identifying the intrinsic and extrinsic mechanisms involved, many aspects remain unresolved. Axonal development in embryonic CNS (hippocampus) requires the obligate activation of the insulin-like growth factor 1 receptor (IGF-1R). Based on known similarities between axonal growth in fetal compared to mature CNS, we decided to examine the expression of the IGF-1R, using an antibody to the βgc subunit or a polyclonal anti-peptide antibody directed to the IGF-R (C20), in an in vitro model of adult CNS axonal regeneration, namely retinal ganglion cells (RGC) derived from adult rat retinas. Expression of both βgc and the β subunit recognized by C20 antibody were low in freshly isolated adult RGC, but increased significantly after 4 days in vitro. As in embryonic axons, βgc was localised to distal regions and leading growth cones in RGC. IGF-1R-βgc co-localised with activated p85 involved in the phosphatidylinositol-3 kinase (PI3K) signaling pathway, upon stimulation with IGF-1. Blocking experiments using either an antibody which neutralises IGF-1R activation, shRNA designed against the IGF-1R sequence, or the PI3K pathway inhibitor LY294002, all significantly reduced axon regeneration from adult RGC in vitro (∼40% RGC possessed axons in controls vs 2-8% in the different blocking studies). Finally, co-transfection of RGC with shRNA to silence IGF-1R together with a vector containing a constitutively active form of downstream PI3K (p110), fully restored axonal outgrowth in vitro. Hence these data demonstrate that axonal regeneration in adult CNS neurons requires re-expression and activation of IGF-1R, and targeting this system may offer new therapeutic approaches to enhancing axonal regeneration following trauma.

Axonal elongation is one of the hallmarks of neuronal polarization. This phenomenon requires axonal membrane growth by exocytosis of plasmalemmal precursor vesicles (PPVs) at the nerve growth cone, a process regulated by IGF-1 activation of the PI3K (phosphatidylinositol-3 kinase) pathway. Few details are known, however, about the targeting mechanisms for PPVs. Here, we show, in cultured hippocampal pyramidal neurons and growth cones isolated from fetal rat brain, that IGF-1 activates the GTP-binding protein TC10, which triggers translocation to the plasma membrane of the exocyst component exo70 in the distal axon and growth cone. We also show that TC10 and exo70 function are necessary for addition of new membrane and, thus, axon elongation stimulated by IGF-1. Moreover, expression silencing of either TC10 or exo70 inhibit the establishment of neuronal polarity by hindering the insertion of IGF-1 receptor in one of the undifferentiated neurites. We conclude that, in hippocampal pyramidal neurons in culture, (1) membrane expansion at the axonal growth cone is regulated by IGF-1 via a cascade involving TC10 and the exocyst complex, (2) TC10 and exo70 are essential for the polarized externalization of IGF-1 receptor, and (3) this process is necessary for axon specification.

In cultured neurons, axon formation is preceded by the appearance in one of the multiple neurites of a large growth cone containing a labile actin network and abundant dynamic microtubules. The invasion-inducing T-lymphoma and metastasis 1 (Tiam1) protein that functions as a guanosine nucleotide exchange factor for Rac1 localizes to this neurite and its growth cone, where it associates with microtubules. Neurons overexpressing Tiam1 extend several axon-like neurites, whereas suppression of Tiam1 prevents axon formation, with most of the cells failing to undergo changes in growth cone size and in cytoskeletal organization typical of prospective axons. Cytochalasin D reverts this effect leading to multiple axon formation and penetration of microtubules within neuritic tips devoid of actin filaments. Taken together, these results suggest that by regulating growth cone actin organization and allowing microtubule invasion within selected growth cones, Tiam1 promotes axon formation and hence participates in neuronal polarization.

Tau protein is a predominantly neuronal microtubule-associated protein that is enriched in axons and is capable of promoting microtubule assembly and stabilization. In the present article we review some of the key experiments directed to obtain insights about tau protein function in developing neurons. Aspects related to whether or not tau has essential, unique, or complementary functions during axonal formation are discussed.

Cultures of cerebellar macroneurons were used to study the pattern of expression, subcellular localization, and function of the neuronal cdk5 activator p35 during laminin-enhanced axonal growth. The results obtained indicate that laminin, an extracellular matrix molecule capable of selectively stimulating axonal extension and promoting MAP1B phosphorylation at a proline-directed protein kinase epitope, selectively stimulates p35 expression, increases its association with the subcortical cytoskeleton, and accelerates its redistribution to the axonal growth cones. Besides, suppression of p35, but not of a highly related isoform designated as p39, by antisense oligonucleotide treatment selectively reduces cdk5 activity, laminin-enhanced axonal elongation, and MAP1b phosphorylation. Taken collectively, the present results suggest that cdk5/p35 may serve as an important regulatory linker between environmental signals (e.g., laminin) and constituents of the intracellular machinery (e.g., MAP1B) involved in axonal elongation.

The investigation of the molecular properties of nerve growth cones depends to a significant degree on their isolation from fetal brain in the form of 'growth cone particles' (GCPs). The availability of markers for developing axons and dendrites, as well as glial cells, has made it possible to characterize the GCP fraction in much greater detail than before and to optimize its yield. Marker analyses show that a member of the N-CAM family (5B4-CAM), synaptophysin, and especially GAP-43 and non-phosphorylated tau, are enriched in the GCP fraction. In contrast, MAP2 and, particularly, glial fibrillary acidic protein and vimentin are fractionated away from GCPs. Furthermore, GCP yield can be doubled relative to the original procedure, without compromising purity, by raising the sucrose concentration of the fractionation gradient's uppermost layer. The results indicate that GCPs are highly purified growth cone fragments with very little glial contamination, and that they are primarily of axonal origin.

The question of whether growth cones generated by different neurons contain distinctive membrane glycoproteins was examined. Growth cone particles (GCPs) were isolated from specific regions of fetal or early postnatal brain, and their membrane proteins were analyzed by 2D gel electrophoresis and Western blotting, using WGA as a probe. These blots were compared to those generated by synaptosomes from adult brain. The patterns reveal a number of WGA-binding glycoproteins that are uniformly present in these subcellular fractions and others that are found in GCPs from selected brain regions only. The results indicate, therefore, substantial pattern diversity for the different, restricted growth cone populations. Some of the WGA-binding glycoproteins seen in GCPs disappear with increasing age and are absent from synaptosomes, while others seem to become more prominent. One of the glycoprotein complexes present in all GCP and synaptosome fractions analyzed is gp93. It has an apparent molecular weight of 90-97 kDa and exhibits unusually high heterogeneity in GCPs from whole fetal brain. The gp93 complex covers a pI range from about 4.9 to about 6.4 and consists of at least 12 different species, probably isoelectric variants. In GCPs from different brain regions, the sets of gp93 species observed are different and characteristic. Neuraminidase digestion shifts the gp93 pattern to a more neutral pI but simplifies it only partially, indicating that variable sialic acid content explains the molecular diversity to some extent. Thus, gp93 is a glycoprotein complex whose members are expressed and/or posttranslationally processed differentially in different growth cone populations. Such a glycoprotein family may be involved in selective cell-cell recognition.

Abstract: Neurons are highly polarized cells, composed of one axon and several branching dendrites. One important issue in neurobiology is to understand the molecular factors that determine the neuron to develop polarized structures. A particularly early event, in neurons still lacking a discernible axon, is the segregation of IGF-1 (Insulin like Growth Factor-1) receptors in one neurite. This receptor can be activated by insulin in bulk, but, it is not known if changes of insulin organization as a monomolecular film may affect neuron polarization. To this end, in this work we developed solid-supported Langmuir–Blodgett films of insulin with different surface packing density. Hyppocampal pyramidal neurons, in early stage of differentiation, were cultured onto those substrates and polarization was studied after 24h by confocal microscopy. Also we used surface reflection interference contrast microscopy and confocal microscopy to study attachment patterns and morphology of growth cones. We observed that insulin films packed at 14mN/m induced polarization in a similar manner to high insulin concentration in bulk, but insulin packed at 44mN/m did not induce polarization. Our results provide novel evidence that the neuron polarization through IGF-1 receptor activation can be selectively modulated by the lateral packing of insulin organized as a monomolecular surface for cell growth. [Copyright &y& Elsevier]