Karl H. Pfenninger

Plasma membrane expansion: a neuron's Herculean task.

The formation of axons and dendrites and maintenance of the neurons vastly expanded surface require the continuous addition of new membrane. This is achieved by membrane synthesis through the secretory pathway followed by regulated vesicle fusion with the plasma membrane, typically in the distal neurite. However, it is far from simple: multiple distinct membrane carriers are used to target specific membrane domains, dendrites seem to operate semi-autonomously from the rest of the neuron, and exocytosis for membrane expansion is different from that for release of synaptic vesicles. Current knowledge of this process and its implications for neuronal development, function and repair are reviewed.

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.

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.

NPY and its involvement in axon guidance, neurogenesis, and feeding

OBJECTIVES The role of neuropeptides in nervous system function is still in many cases undefined. In the present study we examined a possible role of the 36-amino acid neuropeptide Y (NPY) with regard to three functions: axon guidance and attraction/repulsion, adult neurogenesis, and control of food intake. METHODS Growth cones from embryonic dorsal root ganglion neurons were studied in culture during asymmetrical gradient application of NPY. Growth cones were monitored over a 60-min period, and final turning angle and growth rate were recorded. In the second part the NPY Y(1) and Y(2) receptors were studied in the subventricular zone, the rostral migratory stream, and the olfactory bulb in normal mice and mice with genetically deleted NPY Y(1) or Y(2) receptors. In the third part an anorectic mouse was analyzed with immunohistochemistry. RESULTS 1) NPY elicited an attractive turning response and an increase in growth rate, effects exerted via the NPY Y(1) receptor. 2) The NPY Y(1) receptor was expressed in neuroblasts in the anterior rostral migratory stream. Mice deficient in the Y(1) or Y(2) receptor had fewer proliferating precursor cells and neuroblasts in the subventricular zone and rostral migratory stream and fewer neurons in the olfactory bulb expressing calbindin, calretinin or tyrosine hydroxylase. 3) In the anorectic mouse markers for microglia were strongly upregulated in the arcuate nucleus and in projection areas of the NPY/agouti gene-related protein arcuate system. CONCLUSION NPY participates in several mechanisms involved in the development of the nervous system and is of importance in the control of food intake.

Arachidonic Acid Turnover and Phospholipase A2 Activity in Neuronal Growth Cones

Abstract: We analyzed de novo synthesis and local turnover of phospholipids in the growing neuron and the isolated nerve growth cone. The metabolism of phosphatidylinositol (PI) was studied with regard to the incorporation of saturated and unsaturated fatty acids and inositol. A comparison of de novo phospholipid synthesis in the intact neuron (whole brain, cell cultures) versus local turnover in isolated growth cone particles (GCPs) from fetal rat brain revealed different incorporation patterns and, in particular, high arachidonic acid (AA) turnover in PI of GCPs. These observations, together with elevated levels of free AA (2.5% of total AA content) in GCPs, demonstrate the predominance of acylation/deacylation in the sn‐2 position of PI. GCP phospholipase A2 (PLA2) activity was demonstrated using [3H]‐or [14C]AA‐phosphatidylcholine (PC) or ‐PI as the substrate in vitro and GCPs or a cytosolic GCP extract as the source of enzyme. In contrast to PC, which is hydrolyzed very slowly, PI is a very good GCP PLA2 substrate. PLA2 activity is much higher in GCPs than that of phospholipase C, as demonstrated by the comparison of AA and inositol turnover, by the low levels of 1,2‐diacylglycerol generated by GCPs, and by the resistance of AA release to treatment of GCPs with RHC‐80267, a specific inhibitor of diacylglycerol lipase. The predominance of PLA2 activity in GCPs raises questions regarding its regulation and the functional roles of PI metabolites, especially lysocompounds, in growth cones.

Dynamic adhesions and MARCKS in melanoma cells

Cell motility necessitates the rapid formation and disassembly of cell adhesions. We have studied adhesions in a highly motile melanoma cell line using various biochemical approaches and microscopic techniques to image close adhesions. We report that WM-1617 melanoma cells contain at least two types of close adhesion: classic focal adhesions and more extensive, irregularly shaped adhesions that tend to occur along lamellipodial edges. In contrast to focal adhesions, these latter adhesions are highly dynamic and can be disassembled rapidly via protein kinase C (PKC) activation (e.g. by eicosanoid) and MARCKS phosphorylation. MARCKS overexpression, however, greatly increases the area of close adhesions and renders them largely refractory to PKC stimulation. This indicates that nonphosphorylated MARCKS is an adhesion stabilizer. Unlike focal adhesions, the dynamic adhesions contain α3 integrin and MARCKS, but they do not contain the focal adhesion marker vinculin. Overall, these results begin to define the molecular and functional properties of dynamic close adhesions involved in cell motility.

Growth cone responses to growth and chemotropic factors

During nervous system development axons reach their target areas under the influence of numerous guidance cues that affect rate and direction of growth. This report addresses the unsettled question of whether and to what extent growth velocity and turning responses (attraction, repulsion) are interdependent. We exposed individual growth cones of fetal rat dorsal root ganglion neurons in culture asymmetrically to gradients of seven different factors and recorded their growth rates and turning angles. Growth cones exhibited divergent patterns of turning and growth responses. For example, hepatocyte growth factor (HGF), insulin‐like growth factor‐1 (IGF‐1) and thrombin all promoted growth, but HGF was a powerful attractant, thrombin a potent repellent and IGF‐1 did not elicit turning. Galanin and neuropeptide Y also affected growth and/or turning differentially. Finally, nerve growth factor in the culture medium not only inhibited the turning responses to HGF, but also converted growth promotion of HGF and IGF‐1 into inhibition. Overall, our studies indicate that: (i) turning and advance are regulated independently, except that strong attractive or repulsive responses generally are accompanied by growth promotion; (ii) asymmetric growth factor application per se does not elicit attraction; (iii) regulation of the two parameters may occur through a single receptor; and (iv) the effects of combined growth factors may not be additive and can be inhibitory.