Santiago Quiroga

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 neurons 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.

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.

The Cdk5‐p35 kinase associates with the Golgi apparatus and regulates membrane traffic

We show here that an active Cdk5‐p35 kinase is present in Golgi membranes, where it associates with a detergent‐insoluble fraction containing actin. In addition, Cdk5‐p35‐dependent phosphorylation of α‐PAK immunoreactive protein species was detected in Golgi membranes, as well as an interaction with the small GTPase, Cdc42. Moreover, antisense oligonucleotide suppression of Cdk5 or p35 in young cultured neurons, as well as inhibition of Cdk5 activity with olomoucine, blocks the formation of membrane vesicles from the Golgi apparatus. Taken together, these results show a novel subcellular localization of this kinase and suggest a role for Cdk5‐p35 in membrane traffic during neuronal process outgrowth.

Regulation of membrane expansion at the nerve growth cone

Exocytotic incorporation of plasmalemmal precursor vesicles (PPVs) into the cell surface is necessary for neurite extension and is known to occur mainly at the growth cone. This report examines whether this is a regulated event controlled by growth factors. The Golgi complex and nascent PPVs of hippocampal neurons in culture were pulse-labeled with fluorescent ceramide. We studied the dynamics of labeled PPVs upon arrival at the axonal growth cone. In controls and cultures stimulated with brain-derived neurotrophic factor (BDNF), PPV clusters persisted in growth cones with a half-life (t1/2) of >14 minutes. Upon challenge with IGF-1, however, fluorescent elements cleared from the growth cones with a t1/2 of only 6 minutes. Plasmalemmal expansion was measured directly as externalization of membrane glycoconjugates in resealed growth cone particles (GCPs) isolated from fetal forebrain. These assays demonstrated that membrane expansion could be stimulated by IGF-1 in a dose-dependent manner but not by BDNF, even though intact, functional BDNF receptor was present on GCPs. Because both BDNF and IGF-1 are known to enhance neurite growth, but BDNF did not stimulate membrane expansion at the growth cone, we studied the effect of BDNF on the IGF-1 receptor. BDNF was found to cause the translocation of the growth-cone-specific IGF-1 receptor subunitβ gc to the distal axon, in a KIF2-dependent manner. We conclude that IGF-1 stimulates axonal assembly at the growth cone, and that this occurs via regulated exocytosis of PPVs. This mechanism is affected by BDNF only indirectly, by regulation of the βgc level at the growth cone.

Growth-regulated proteins and neuronal plasticity

Growth-regulated proteins (GRPs) of the neuron are synthesized during outgrowth and regeneration at an increased rate and enriched in nerve growth cones. Therefore, they can be used to some degree as markers of neurite growth. However, these proteins are not unique to the growing neuron, and their properties are not known sufficiently to assign them a functional and/or causal role in the mechanisms of outgrowth. During synaptogenesis, GRPs decrease in abundance, and growth cone functions of motility and organelle assembly are being replaced by junctional contact and transmitter release. However, there is a stage during which growth cone and synaptic properties overlap to some degree. We propose that it is this overlap and its continuation that allow for synaptic plasticity in developing and adult nervous systems. We also propose a hypothesis involving (a) trophic factor(s) that might explain the regulation of synaptic sizes and collateral sprouting. Some GRPs, especially GAP43/B50/pp46/F1, are more prominent in adult brain regions of high plasticity, and they undergo change, such as phosphorylation, during long-term potentiation (LTP). Without precise functional knowledge of GRPs, it is impossible to use changes in such proteins to explain the plasticity mechanism. However, changes in these “growth markers” are likely to be an indication of sprouting activity, which would explain well the various phenomena associated with plasticity and learning in the adult. Thus, plasticity and memory may be viewed as a continuation of the developmental process into adulthood.

Protein Kinase D Regulates Trafficking of Dendritic Membrane Proteins in Developing Neurons

In non-neuronal cells, inactivation of protein kinase D (PKD) blocks fission of trans-Golgi network (TGN) transport carriers, inducing the appearance of long tubules filled with cargo. We now report on the function of PKD1 in neuronal protein trafficking. In cultured hippocampal pyramidal cells, the transferrin receptor (TfR) and the low-density receptor-related protein (LRP) are predominantly transported to dendrites and excluded from axons. Expression of kinase-inactive PKD1 or its depletion by RNA interference treatment dramatically and selectively alter the intracellular trafficking and membrane delivery of TfR- and LRP-containing vesicles, without inhibiting exit from the TGN or inducing Golgi tubulation. After PKD1 suppression, dendritic membrane proteins are mispackaged into carriers that transport VAMP2; these vesicles are distributed to both axons and dendrites, but are rapidly endocytosed from dendrites and preferentially delivered to the axonal membrane. A kinase-defective mutant of PKD1 lacking the ability to bind diacylglycerol and hence its Golgi localization does not cause missorting of TfR or LRP. These results suggest that in neurons PKD1 regulates TGN-derived sorting of dendritic proteins and hence has a role in neuronal polarity.

Axonal origin and purity of growth cones isolated from fetal rat brain

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 gradients 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.

Tau Protein Function in Axonal Formation

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.

The insulin-like growth factor 1 receptor is essential for axonal regeneration in adult central nervous system neurons.

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.