The GTPase Rab21 is required for neuronal development and migration in the cerebral cortex.

Development of the mammalian neocortex requires proper inside-out migration of developing cortical neurons from the germinal ventricular zone toward the cortical plate. The mechanics of this migration requires precise coordination of different cellular phenomena including cytoskeleton dynamics, membrane trafficking, and cell adhesion. The small GTPases play a central role in all these events. The small GTPase Rab21 regulates migration and neurite growth in developing neurons. Moreover, regulators and effectors of Rab21 have been implicated in brain pathologies with cortical malformations, suggesting a key function for the Rab21 signaling pathway in cortical development. Mechanistically, it has been posited that Rab21 influences cell migration by controlling the trafficking of endocytic vesicles containing adhesion molecules. However, direct evidence of the participation of Rab21 or its mechanism of action in the regulation of cortical migration is still incomplete. In this study, we demonstrate that Rab21 plays a critical role in the differentiation and migration of pyramidal neurons by regulating the levels of the amyloid precursor protein on the neuronal cell surface. Rab21 loss of function increased the levels of membrane-exposed APP, resulting in impaired cortical neuronal differentiation and migration. These findings further our understanding of the processes governing the development of the cerebral cortex and shed light onto the molecular mechanisms behind cortical development disorders derived from the malfunctioning of Rab21 signaling effectors.

Estrategias para mitigar hipoglucemias y variabilidad glucémica / Strategies to mitigate hypoglycemia and glycemic variability

La última década se ha destacado por los importantes avances en el desarrollo de nuevas tecnologías para pacientes que viven con diabetes mellitus (DM). Las innovaciones han estado orientadas principalmente a: mejorar la calidad de vida, reducir el impacto que genera la ocurrencia de hipoglucemias y reducir la carga de la enfermedad colaborando en la toma diaria de decisiones1. El monitoreo continuo de glucosa (MCG) es una herramienta que ha experimentado un importante avance al aportar información dinámica del estado metabólico en los pacientes y permitir la toma de decisiones, demostrado por un control metabólico estable, menores excursiones glucémicas, y una reducción significativa en la aparición y gravedad de las hipoglucemias2-5. Las presentes recomendaciones tienen como objetivo brindar herramientas rápidas para la interpretación de datos metabólicos y la consiguiente toma de decisiones terapéuticas. A tal fin se realizó una exhaustiva revisión de las principales guías y recomendaciones vigentes; posteriormente, el Grupo de Trabajo adaptó esa información según una serie de preguntas con criterio clínico práctico. El avance de los MCG es innegable, no solo en el desarrollo tecnológico, sino que se han convertido en una herramienta educativa para las personas con DM, su entorno y el equipo de salud al posibilitar un ajuste dinámico del tratamiento, prevenir complicaciones agudas y mejorar la calidad de vida. En esta ecuación enfatizamos la importancia de la educación diabetológica continua de la persona con DM y su entorno, participando activamente en la toma de decisiones para, de esta manera, cumplir con los objetivos propuestos: mejorar la calidad de vida, reducir la carga de la enfermedad y disminuir las excursiones glucémicas agudas.

The last decade has been highlighted by important advances in the development of new technologies for patients living with diabetes. The innovations have been oriented above all to improve the quality of life, reduce the impact generated by the occurrence of hypoglycemia and reduce the burden of the disease by collaborating in daily decision-making1. Continuous glucose monitoring (CGM) is a tool that has undergone significant progress, providing dynamic information on the metabolic status of patients, allowing decision making, demonstrated by stable metabolic control, lower glycemic excursions and a significant reduction in the occurrence and severity of hypoglycemia2-5. The purpose of these recommendations, developed by members of the Innovation Committee of the Argentine Society of Diabetes, is to provide rapid tools for the interpretation of metabolic data and the subsequent therapeutic decisionmaking. To this end, an exhaustive review of the main current guidelines and recommendations has been carried out, later the working group adapted this information according to a series of questions with practical clinical criteria. The progress of CGMs is undeniable, not only in technological development, but it has become an educational tool for people with diabetes, their environment, and the health team, offering the possibility of a dynamic adjustment of treatment, prevention of acute complications and improving quality of life. In this equation, we emphasize the importance of continuous diabetes education for the person with diabetes and their environment, actively participating in decision-making, and in this way, meeting the proposed objectives: improving quality of life, reducing the burden of disease, and decreasing acute glycemic excursions.

Estrategias para mitigar hipoglucemias y variabilidad glucémica / Strategies to mitigate hypoglycemia and glycemic variability

Resumen La última década se ha destacado por los importantes avances en el desarrollo de nuevas tecnologías para pacientes que viven con diabetes mellitus (DM). Las innovaciones han estado orientadas principalmente a: mejorar la calidad de vida, reducir el impacto que genera la ocurrencia de hipoglucemias y reducir la carga de la enfermedad colaborando en la toma diaria de decisiones1. El monitoreo continuo de glucosa (MCG) es una herramienta que ha experimentado un importante avance al aportar información dinámica del estado metabólico en los pacientes y permitir la toma de decisiones, demostrado por un control metabólico estable, menores excursiones glucémicas, y una reducción significativa en la aparición y gravedad de las hipoglucemias2-5. Las presentes recomendaciones tienen como objetivo brindar herramientas rápidas para la interpretación de datos metabólicos y la consiguiente toma de decisiones terapéuticas. A tal fin se realizó una exhaustiva revisión de las principales guías y recomendaciones vigentes; posteriormente, el Grupo de Trabajo adaptó esa información según una serie de preguntas con criterio clínico práctico. El avance de los MCG es innegable, no solo en el desarrollo tecnológico, sino que se han convertido en una herramienta educativa para las personas con DM, su entorno y el equipo de salud al posibilitar un ajuste dinámico del tratamiento, prevenir complicaciones agudas y mejorar la calidad de vida. En esta ecuación enfatizamos la importancia de la educación diabetológica continua de la persona con DM y su entorno, participando activamente en la toma de decisiones para, de esta manera, cumplir con los objetivos propuestos: mejorar la calidad de vida, reducir la carga de la enfermedad y disminuir las excursiones glucémicas agudas.

Abstract The last decade has been highlighted by important advances in the development of new technologies for patients living with diabetes. The innovations have been oriented above all to improve the quality of life, reduce the impact generated by the occurrence of hypoglycemia and reduce the burden of the disease by collaborating in daily decision-making1. Continuous glucose monitoring (CGM) is a tool that has undergone significant progress, providing dynamic information on the metabolic status of patients, allowing decision making, demonstrated by stable metabolic control, lower glycemic excursions and a significant reduction in the occurrence and severity of hypoglycemia2-5. The purpose of these recommendations, developed by members of the Innovation Committee of the Argentine Society of Diabetes, is to provide rapid tools for the interpretation of metabolic data and the subsequent therapeutic decisionmaking. To this end, an exhaustive review of the main current guidelines and recommendations has been carried out, later the working group adapted this information according to a series of questions with practical clinical criteria. The progress of CGMs is undeniable, not only in technological development, but it has become an educational tool for people with diabetes, their environment, and the health team, offering the possibility of a dynamic adjustment of treatment, prevention of acute complications and improving quality of life. In this equation, we emphasize the importance of continuous diabetes education for the person with diabetes and their environment, actively participating in decision-making, and in this way, meeting the proposed objectives: improving quality of life, reducing the burden of disease, and decreasing acute glycemic excursions.

BARS Influences Neuronal Development by Regulation of Post-Golgi Trafficking.

Neurons are highly polarized cells requiring precise regulation of trafficking and targeting of membrane proteins to generate and maintain different and specialized compartments, such as axons and dendrites. Disruption of the Golgi apparatus (GA) secretory pathway in developing neurons alters axon/dendritic formation. Therefore, detailed knowledge of the mechanisms underlying vesicles exiting from the GA is crucial for understanding neuronal polarity. In this study, we analyzed the role of Brefeldin A-Ribosylated Substrate (CtBP1-S/BARS), a member of the C-terminal-binding protein family, in the regulation of neuronal morphological polarization and the exit of membrane proteins from the Trans Golgi Network. Here, we show that BARS is expressed during neuronal development in vitro and that RNAi suppression of BARS inhibits axonal and dendritic elongation in hippocampal neuronal cultures as well as largely perturbed neuronal migration and multipolar-to-bipolar transition during cortical development in situ. In addition, using plasma membrane (PM) proteins fused to GFP and engineered with reversible aggregation domains, we observed that expression of fission dominant-negative BARS delays the exit of dendritic and axonal membrane protein-containing carriers from the GA. Taken together, these data provide the first set of evidence suggesting a role for BARS in neuronal development by regulating post-Golgi membrane trafficking.

A simple electrochemical immunosensor for sensitive detection of transgenic soybean protein CP4-EPSPS in seeds.

Soybean is the most produced crop in Argentina, and 99 % corresponds to genetically modified soybean. One of the main produced varieties is Roundup Ready® soybean (RR), which was modified to express the enzyme CP4 5-enolpyruvylshikimate 3-phosphate synthase (CP4 EPSPS), which confers resistance to glyphosate, the main herbicide worldwide used. The possible impact of genetically modified organisms (GMO) has generated public concerns, thus increasing interest in the development of GMOs detection devices. In this work, an electrochemical immunosensor for CP4 EPSPS detection in soybean seeds was obtained, by using a gold electrode modified with an anti-CP4 EPSPS polyclonal antibody produced in our laboratory. The presented immunosensor resulted in a simple, low-cost, fast, and reproducible device. Also, labeling and/or signal amplification system was not necessary, since the sensor showed high sensibility with a low detection limit (lower at 0,038 % RR soybean, 38 ng mL-1 CP4 EPSPS).

Sec3 exocyst component knockdown inhibits axonal formation and cortical neuronal migration during brain cortex development.

Neurons are the largest known cells, with complex and highly polarized morphologies and consist of a cell body (soma), several dendrites, and a single axon. The establishment of polarity necessitates initial axonal outgrowth in concomitance with the addition of new membrane to the axon's plasmalemma. Axolemmal expansion occurs by exocytosis of plasmalemmal precursor vesicles primarily at the neuronal growth cone membrane. The multiprotein exocyst complex drives spatial location and specificity of vesicle fusion at plasma membrane. However, the specific participation of its different proteins on neuronal differentiation has not been fully established. In the present work we analyzed the role of Sec3, a prominent exocyst complex protein on neuronal differentiation. Using mice hippocampal primary cultures, we determined that Sec3 is expressed in neurons at early stages prior to neuronal polarization. Furthermore, we determined that silencing of Sec3 in mice hippocampal neurons in culture precluded polarization. Moreover, using in utero electroporation experiments, we determined that Sec3 knockdown affected cortical neurons migration and morphology during neocortex formation. Our results demonstrate that the exocyst complex protein Sec3 plays an important role in axon formation in neuronal differentiation and the migration of neuronal progenitors during cortex development.

Kinesin-1-mediated axonal transport of CB1 receptors is required for cannabinoid-dependent axonal growth and guidance.

Endocannabinoids (eCB) modulate growth cone dynamics and axonal pathfinding through the stimulation of cannabinoid type-1 receptors (CB1R), the function of which depends on their delivery and precise presentation at the growth cone surface. However, the mechanism involved in the axonal transport of CB1R and its transport role in eCB signaling remains elusive. As mutations in the kinesin-1 molecular motor have been identified in patients with abnormal cortical development and impaired white matter integrity, we studied the defects in axonal pathfinding and fasciculation in mice lacking the kinesin light chain 1 (Klc1-/-) subunit of kinesin-1. Reduced levels of CB1R were found in corticofugal projections and axonal growth cones in Klc1-/- mice. By live-cell imaging of CB1R-eGFP we characterized the axonal transport of CB1R vesicles and described the defects in transport that arise after KLC1 deletion. Cofilin activation, which is necessary for actin dynamics during growth cone remodeling, is impaired in the Klc1-/- cerebral cortex. In addition, Klc1-/- neurons showed expanded growth cones that were unresponsive to CB1R-induced axonal elongation. Together, our data reveal the relevance of kinesin-1 in CB1R axonal transport and in eCB signaling during brain wiring.

Effects of anti-ß2GPI antibodies on VWF release from human umbilical vein endothelial cells and ADAMTS13 activity.

BACKGROUND: Antiphospholipid syndrome (APS) is characterized by recurrent thromboembolic events in the setting of pathologic autoantibodies, some of which are directed to ß2-Glycoprotein 1 (ß2GPI). The mechanisms of thrombosis in APS appear to be multifactorial and likely include a component of endothelial activation. Among other things, activated endothelium secretes von Willebrand factor, a hemostatic protein that in excess can increase the risk of thrombosis. OBJECTIVE: We hypothesized that anti-ß2GPI antibodies could regulate the release and modulation of VWF from endothelial cells. PATIENTS/METHODS: Isolated anti-ß2GPI antibodies from patients with APS were assayed for their ability to induced VWF release from HUVECs and modulate the effects of ADAMTS13 in a shear-dependent assay. RESULTS: We observed that anti-ß2GPI antibodies from some patients with APS induced VWF release from human endothelial cells but did not induce formation of cell-anchored VWF-platelet strings. Finally, we also determined that one of the Anti-ß2GPI antibodies tested can inhibit the function of ADAMTS13, the main modulator of extracellular VWF. CONCLUSIONS: These results suggest that VWF and ADAMTS13 may play a role in the prothrombotic phenotype of APS.

IGF-1 receptor regulates dynamic changes in neuronal polarity during cerebral cortical migration.

During cortical development, neurons undergo polarization, oriented migration and layer-type differentiation. The biological and biochemical mechanisms underlying these processes are not completely understood. In neurons in culture we showed that IGF-1 receptor activation is important for growth cone assembly and axonal formation. However, the possible roles of the insulin like growth factor-1 receptor (IGF-1R) on neuronal differentiation and polarization in vivo in mammals have not yet been studied. Using in utero electroporation, we show here that the IGF-1R is essential for neocortical development. Neurons electroporated with a shRNA targeting IGF-1 receptor failed to migrate to the upper cortical layers and accumulated at the ventricular/subventricular zones. Co-electroporation with a constitutively active form of PI3K rescued migration. The change of the morphology from multipolar to bipolar cells was also attenuated. Cells lacking the IGF-1 receptor remain arrested as multipolar forming a highly disorganized tissue. The typical orientation of the migrating neurons with the Golgi complex oriented toward the cortical upper layers was also affected by electroporation with shRNA targeting IGF-1 receptor. Finally, cells electroporated with the shRNA targeting IGF-1 receptor were unable to form an axon and, therefore, neuron polarity was absent.

The physiological role of the amyloid precursor protein as an adhesion molecule in the developing nervous system.

The amyloid precursor protein (APP) is a type I transmembrane glycoprotein better known for its participation in the physiopathology of Alzheimer disease as the source of the beta amyloid fragment. However, the physiological functions of the full length protein and its proteolytic fragments have remained elusive. APP was first described as a cell-surface receptor; nevertheless, increasing evidence highlighted APP as a cell adhesion molecule. In this review, we will focus on the current knowledge of the physiological role of APP as a cell adhesion molecule and its involvement in key events of neuronal development, such as migration, neurite outgrowth, growth cone pathfinding, and synaptogenesis. Finally, since APP is over-expressed in Down syndrome individuals because of the extra copy of chromosome 21, in the last section of the review, we discuss the potential contribution of APP to the neuronal and synaptic defects described in this genetic condition. Read the Editorial Highlight for this article on page 9. Cover Image for this issue: doi. 10.1111/jnc.13817.

The Motor KIF5C Links the Requirements of Stable Microtubules and IGF-1 Receptor Membrane Insertion for Neuronal Polarization.

Three early signals of asymmetry have been described to occur in a single neurite of neurons at stage 2 of differentiation (before polarization) and shown to be essential for neuronal polarization: (i) accumulation of stable microtubules, (ii) enrichment of the plasma membrane with activatable IGF-1r, and (iii) polarized transport of the microtubular motor KIF5C. Here, we studied the possible relationship between these three phenomena. Our results show that the activatable (membrane-inserted) IGF-1r and stable microtubules accumulate in the same neurite of cells at stage 2. The polarized insertion of IGF-1r depends on microtubule dynamics as shown using drugs which modify microtubule stability. Silencing of KIF5C expression prevents the polarized insertion of IGF-1r into the neuronal plasmalemma and neuronal polarization. Syntaxin 6 and VAMP4, necessary for the polarized insertion of the IGF-1r, are associated to vesicles carried by the microtubular motor KIF5C and is transported preferentially to the neurite where KIF5C accumulates. We conclude that the enrichment of stable microtubules in the future axon enhances KIF5C-mediated vesicular transport of syntaxin 6 and VAMP4, which in turn mediates the polarized insertion of IGF-1r in the plasmalemma, a key step for neuronal polarization. We herewith establish a mechanistic link between three early polarity events necessary for the establishment of neuronal polarity.

Protein interacting with NIMA (never in mitosis A)-1 regulates axonal growth cone adhesion and spreading through myristoylated alanine-rich C kinase substrate isomerization.

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.

Selected SNARE proteins are essential for the polarized membrane insertion of igf-1 receptor and the regulation of initial axonal outgrowth in neurons.

The establishment of polarity necessitates initial axonal outgrowth and, therefore, the addition of new membrane to the axon's plasmalemma. Axolemmal expansion occurs by exocytosis of plasmalemmal precursor vesicles (PPVs) primarily at the neuronal growth cone. Little is known about the SNAREs family proteins involved in the regulation of PPV fusion with the neuronal plasmalemma at early stages of differentiation. We show here that five SNARE proteins (VAMP2, VAMP4, VAMP7, Syntaxin6 and SNAP23) were expressed by hippocampal pyramidal neurons before polarization. Expression silencing of three of these proteins (VAMP4, Syntaxin6 and SNAP23) repressed axonal outgrowth and the establishment of neuronal polarity, by inhibiting IGF-1 receptor exocytotic polarized insertion, necessary for neuronal polarization. In addition, stimulation with IGF-1 triggered the association of VAMP4, Syntaxin6 and SNAP23 to vesicular structures carrying the IGF-1 receptor and overexpression of a negative dominant form of Syntaxin6 significantly inhibited exocytosis of IGF-1 receptor containing vesicles at the neuronal growth cone. Taken together, our results indicated that VAMP4, Syntaxin6 and SNAP23 functions are essential for regulation of PPV exocytosis and the polarized insertion of IGF-1 receptor and, therefore, required for initial axonal elongation and the establishment of neuronal polarity.

Dosage of amyloid precursor protein affects axonal contact guidance in Down syndrome.

Amyloid precursor protein (APP), encoded on Hsa21, functions as a cell adhesion molecule (CAM) in axonal growth cones (GCs) of the developing brain. We show here that axonal GCs of human fetal Down syndrome (DS) neurons (and of a DS mouse model) overexpress APP protein relative to euploid controls. We investigated whether DS neurons generate an abnormal, APP-dependent GC phenotype in vitro. On laminin, which binds APP and ß1 integrins (Itgb1), DS neurons formed enlarged and faster-advancing GCs compared to controls. On peptide matrices that bind APP only, but not on those binding exclusively Itgb1 or L1CAM, DS GCs were significantly enlarged (2.0-fold), formed increased close adhesions (1.8-fold), and advanced faster (1.4-fold). In assays involving alternating stripes of monospecific matrices, human control GCs exhibited no preference for any of the substrates, whereas DS GCs preferred the APP-binding matrix (cross-over decreased significantly from 48.2 to 27.2%). Reducing APP expression in DS GCs with siRNA normalized most measures of the phenotype, including substrate choice. These experiments show that human DS neurons exhibit an APP-dependent, abnormal GC phenotype characterized by increased adhesion and altered contact guidance. The results suggest that APP overexpression may perturb axonal pathfinding and circuit formation in developing DS brain.

Amyloid precursor protein is an autonomous growth cone adhesion molecule engaged in contact guidance.

Amyloid precursor protein (APP), a transmembrane glycoprotein, is well known for its involvement in the pathogenesis of Alzheimer disease of the aging brain, but its normal function is unclear. APP is a prominent component of the adult as well as the developing brain. It is enriched in axonal growth cones (GCs) and has been implicated in cell adhesion and motility. We tested the hypothesis that APP is an extracellular matrix adhesion molecule in experiments that isolated the function of APP from that of well-established adhesion molecules. To this end we plated wild-type, APP-, or ß1-integrin (Itgb1)- misexpressing mouse hippocampal neurons on matrices of either laminin, recombinant L1, or synthetic peptides binding specifically to Itgb1 s or APP. We measured GC adhesion, initial axonal outgrowth, and substrate preference on alternating matrix stripes and made the following observations: Substrates of APP-binding peptide alone sustain neurite outgrowth; APP dosage controls GC adhesion to laminin and APP-binding peptide as well as axonal outgrowth in Itgb1- independent manner; and APP directs GCs in contact guidance assays. It follows that APP is an independently operating cell adhesion molecule that affects the GC's phenotype on APP-binding matrices including laminin, and that it is likely to affect axon pathfinding in vivo.

Functional complexity of the axonal growth cone: a proteomic analysis.

The growth cone, the tip of the emerging neurite, plays a crucial role in establishing the wiring of the developing nervous system. We performed an extensive proteomic analysis of axonal growth cones isolated from the brains of fetal Sprague-Dawley rats. Approximately 2000 proteins were identified at ≥ 99% confidence level. Using informatics, including functional annotation cluster and KEGG pathway analysis, we found great diversity of proteins involved in axonal pathfinding, cytoskeletal remodeling, vesicular traffic and carbohydrate metabolism, as expected. We also found a large and complex array of proteins involved in translation, protein folding, posttranslational processing, and proteasome/ubiquitination-dependent degradation. Immunofluorescence studies performed on hippocampal neurons in culture confirmed the presence in the axonal growth cone of proteins representative of these processes. These analyses also provide evidence for rough endoplasmic reticulum and reveal a reticular structure equipped with Golgi-like functions in the axonal growth cone. Furthermore, Western blot revealed the growth cone enrichment, relative to fetal brain homogenate, of some of the proteins involved in protein synthesis, folding and catabolism. Our study provides a resource for further research and amplifies the relatively recently developed concept that the axonal growth cone is equipped with proteins capable of performing a highly diverse range of functions.

The TC10-Exo70 complex is essential for membrane expansion and axonal specification in developing neurons.

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.

IGF-1 receptor is essential for the establishment of hippocampal neuronal polarity.

How a neuron becomes polarized remains largely unknown. Results obtained with a function-blocking antibody and an siRNA targeting the insulin-like growth factor-1 (IGF-1) receptor suggest that an essential step in the establishment of hippocampal neuronal polarity and the initiation of axonal outgrowth is the activation of the phosphatidylinositol 3-kinase (PI3k)-Cdc42 pathway by the IGF-1 receptor, but not by the TrkA or TrkB receptors.

PI3K activation by IGF-1 is essential for the regulation of membrane expansion at the nerve growth cone.

Exocytotic incorporation of plasmalemmal precursor vesicles (PPVs) into the cell surface is necessary for axonal outgrowth and is known to occur mainly at the nerve growth cone. We have demonstrated recently that plasmalemmal expansion is regulated at the growth cone by IGF-1, but not by BDNF, in a manner that is quasi independent of the neuron's perikaryon. To begin elucidating the signaling pathway by which exocytosis of the plasmalemmal precursor is regulated, we studied activation of the IRS/PI3K/Akt pathway in isolated growth cones and hippocampal neurons in culture stimulated with IGF-1 or BDNF. Our results show that IGF-1, but not BDNF, significantly and rapidly stimulates IRS/PI3K/Akt and membrane expansion. Inhibition of PI3K with Wortmannin or LY294002 blocked IGF-1-stimulated plasmalemmal expansion at the growth cones of cultured neurons. Finally, our results show that, upon stimulation with IGF-1, most active PI3K becomes associated with distal microtubules in the proximal or central domain of the growth cone. Taken together, our results suggest a critical role for IGF-1 and the IRS/PI3K/Akt pathway in the process of membrane assembly at the axonal growth cone.