Astrocyte-synthesized oleic acid behaves as a neurotrophic factor for neurons.

Unlike in the adult brain, the newborn brain specifically takes up serum albumin during the postnatal period, coinciding with the stage of maximal brain development. Here we shall summarize our knowledge about the role played by albumin in brain development. The role of this protein in brain development is intimately related to its ability to carry fatty acids. Thus, albumin stimulates oleic acid synthesis by astrocytes from the main metabolic substrates available during brain development. Astrocytes internalize albumin in vesicle-like structures by receptor-mediated endocytosis, which is followed by transcytosis, including passage through the endoplasmic reticulum (ER). The presence of albumin in the ER activates the sterol regulatory element-binding protein-1 (SREBP-1) and increases stearoyl-CoA 9-desaturase (SCD) mRNA, the key enzyme in oleic acid synthesis. Oleic acid released by astrocytes is used by neurons for the synthesis of phospholipids and is specifically incorporated into growth cones. In addition, oleic acid promotes axonal growth, neuronal clustering, and the expression of the axonal growth associated protein, GAP-43. All of these observations indicate neuronal differentiation. The effect of oleic acid on GAP-43 synthesis is brought about by the activation of protein kinase C. The expression of GAP-43 is significantly increased by the presence of albumin in neurons co-cultured with astrocytes, indicating that neuronal differentiation takes place by the presence of oleic acid synthesized and released by astrocytes in situ. In conclusion, during brain development the presence of albumin could play an important role by triggering the synthesis and release of oleic acid by astrocytes, thereby inducing neuronal differentiation.     

Albumin promotes neuronal survival by increasing the synthesis and release of glutamate

It is well known that the presence of albumin within the brain and the CSF is developmentally regulated. However, the physiological relevance of this phenomenon is not well established. We have previously shown that albumin specifically increases the flux of glucose and lactate through the pyruvate dehydrogenase reaction in astrocytes. Here we show that, in neurones, albumin also increases the oxidation of glucose and lactate through the pyruvate dehydrogenase-catalysed reaction, the final purpose of this being the synthesis of glutamate. Thus, in neurones, the presence of albumin strongly increased the synthesis and release of glutamate to the extracellular medium. Our results also suggest that glutamate release caused by albumin is designed to promote neuronal survival. Thus, under culture conditions in which neurones die by apoptosis, the presence of albumin promoted neuronal survival and maintained the differentiation programme of these cells, as judged by the expression of the axonal protein, GAP-43. The effect of albumin on neuronal survival was counteracted by the presence of DNQX, an antagonist of non-NMDA-glutamate receptors, suggesting that the glutamate synthesized and released due to the presence of albumin is responsible for neuronal survival. In addition, the effect of albumin seemed to depend on the activity of the NGF receptor, TrkA, suggesting that the glutamate synthesized and released due to the presence of albumin promotes neuronal survival through the activity of TrkA.     

Albumin endocytosis via megalin in astrocytes is caveola- and Dab-1 dependent and is required for the synthesis of the neurotrophic factor oleic acid

The synthesis and release of the neurotrophic factor oleic acid requires internalization of albumin into the astrocyte, which is mediated by megalin. In this study, we show that the binding and internalization of albumin involve its interaction with megalin, caveolin-1, caveolin-2 and cavin, but not with clathrin in astrocytes from primary culture. Electron microscopy analyses revealed albumin-gold complexes localized in caveolae, but not in clathrin-coated vesicles. Neither chlorpromazine nor silencing clathrin expression modified albumin uptake. Silencing caveolin-1 strongly reduced the binding and internalization of albumin and the distribution of megalin in the plasma membrane. However, silencing caveolin-2 only decreased albumin internalization, suggesting that caveolin-1 is responsible for megalin recruitment to the caveolae and that caveolin-2 participates in caveolae internalization. In most tissues, the cytosolic adaptor protein disabled (Dab)-2 connects megalin to clathrin, astrocytes lack Dab-2; instead, they express Dab-1, which interacts with caveolin-1 and megalin and is required for albumin internalization. The transcytosis of albumin in astrocytes, including the passage through the endoplasmic reticulum, which is a compulsory step for oleic acid synthesis, was confirmed by electron microscopy analyses. Thus, whereas silencing clathrin did not modify the synthesis and release of oleic acid, the knock-down of caveolin-1, caveolin-2 and Dab-1 strongly reduced the synthesis and release of this neurotrophic factor. In conclusion, caveola-mediated endocytosis of albumin requires megalin and the adaptor protein Dab-1 in cultured astrocytes. Albumin endocytosis may be a key step in brain development because it stimulates the synthesis of oleic acid, which in turn promotes neuronal differentiation.     

Signaling mechanisms of daidzein-induced axonal outgrowth in hippocampal neurons

We aim to study the mechanisms underlying the neurotrophic effect of daidzein (Dz) in hippocampal neurons. Dz-enhanced axonal outgrowths manifested growth cone formation and increased immunostaining intensity of growth-associated protein 43 (GAP-43) in growth cones. Consistent with this, Dz increased GAP-43 phosphorylation and its membrane translocation without affecting total GAP-43 levels. In the presence of Dz, significant increase in the immunoreactivity for estrogen receptor (ER) β, but not ERα, was observed on the membrane of cell bodies and growing axons. Dz also induced the activation of protein kinase C α (PKCα), which was inhibited by the ICI182,780 pretreatment. Similarly, Dz-promoted axonal elongation was blocked by ICI182,780 and Gö6976. Moreover, Dz-stimulated activation of GAP-43 was specifically abolished by Gö6976, suggesting PKCα being the upstream effector of GAP-43. Taken together, our data suggest that Dz triggers an ERβ/PKCα/GAP-43 signaling cascade to promote axonal outgrowths in cultured hippocampal neurons.     

Molecular analysis of the function of the neuronal growth-associated protein GAP-43 by genetic intervention

GAP-43 is a presynaptic membrane phosphoprotein that has been implicated in both the development and the modulation of neural connections. The availability of cDNA clones for GAP-43 makes it possible to examine with greater precision its role in neuronal outgrowth and physiology. We used Northern blots and in situ hybridization with GAP-43 antisense RNA probes to show that GAP-43 is expressed selectively in associative regions of the adult brain. Immunocytochemical analyses showed alterations in the pattern of GAP-43 expression in the hippocampus during reactive synaptogenesis following lesions of the perforant pathway. Genetic intervention methodology was used to analyze the molecular nature of GAP-43 involvement in synaptic plasticity. GAP-43-transfected PC12 cells displayed an enhanced response to nerve growth factor, suggesting that GAP-43 may be directly involved in neurite extension and in the modulation of the neuronal response to extrinsic trophic factors. Studies of PC12 cell transfectants, in which the synthesis of GAP-43 was blocked by expression of GAP-43 antisense RNA, showed that evoked dopamine release was significantly attenuated in these cells. The use of gene transfer into neurons with the HSV-1 vector is presented as a method of analyzing the interaction of GAP-43 with signal transduction systems during neurotransmitter release.     

GAP-43, a protein associated with axon growth, is phosphorylated at three sites in cultured neurons and rat brain.

GAP-43 is a neuronal calmodulin-binding phosphoprotein that is concentrated in growth cones and presynaptic terminals. By sequencing tryptic and endoproteinase Asp-N phosphopeptides and directly determining the release of radioactive phosphate, we have identified three sites (serines 41 and 96 and threonine 172) that are phosphorylated, both in cultured neurons and in neonatal rat brain. These three sites account for most of the 32PO4 that was incorporated into GAP-43 in cultured neurons; 8-15% of each site was occupied with phosphate in GAP-43 isolated from neonatal rat brain. Phosphorylation of serine 41 in cultured neurons was stimulated by phorbol ester, indicating that it is the only site phosphorylated by protein kinase C. The resemblance of the sequence surrounding the other two sites suggests that they may be substrates for the same protein kinase. None of the sites phosphorylated by casein kinase II in vitro was phosphorylated in living cells or in neonatal rat brain. These results show that GAP-43 is a substrate for at least one protein kinase in addition to protein kinase C in living cells and brain.     

M-calpain-mediated cleavage of GAP-43 near Ser41 is negatively regulated by protein kinase C, calmodulin and calpain-inhibiting fragment GAP-43-3

Neuronal protein GAP-43 performs multiple functions in axon guidance, synaptic plasticity and regulation of neuronal death and survival. However, the molecular mechanisms of its action in these processes are poorly understood. We have shown that in axon terminals GAP-43 is a substrate for calcium-activated cysteine protease m-calpain, which participates in repulsion of axonal growth cones and induction of neuronal death. In pre-synaptic terminals in vivo, in synaptosomes, and in vitro, m-calpain cleaved GAP-43 in a small region near Ser41, on either side of this residue. In contrast, micro-calpain cleaved GAP-43 in vitro at several other sites, besides Ser41. Phosphorylation of Ser41 by protein kinase C or GAP-43 binding to calmodulin strongly suppressed GAP-43 proteolysis by m-calpain. A GAP-43 fragment, lacking about forty N-terminal residues (named GAP-43-3), was produced by m-calpain-mediated cleavage of GAP-43 and inhibited m-calpain, but not micro-calpain. This fragment prevented complete cleavage of intact GAP-43 by m-calpain as a negative feedback. GAP-43-3 also blocked m-calpain activity against casein, a model calpain substrate. This implies that GAP-43-3, which is present in axon terminals in high amount, can play important role in regulation of m-calpain activity in neurons. We suggest that GAP-43-3 and another (N-terminal) GAP-43 fragment produced by m-calpain participate in modulation of neuronal response to repulsive and apoptotic signals.     

Specific Proteolysis of Neuronal Protein GAP-43 by Calpain: Characterization,Regulation, and Physiological Role

The mechanism of specific proteolysis of the neuronal protein GAP-43 in axonal terminals has been investigated. In synaptic terminals in vivo and in synaptosomes in vitro GAP-43 is cleaved only at the single peptide bond formed by Ser41; this is within the main effector domain of GAP-43. Proteolysis at this site involves the cysteine calcium-dependent neutral protease calpain. The following experimental evidences support this conclusion: 1) calcium-dependent proteolysis of GAP-43 in synaptosomes is insensitive to selective inhibitor of micro-calpain (PD151746), but it is completely blocked by micro- and m-calpain inhibitor PD150606; 2) GAP-43 proteolysis in the calcium ionophore A23187-treated synaptosomes is activated by millimolar concentration of calcium ions; 3) the pattern of fragmentation of purified GAP-43 by m-calpain (but not by micro-calpain) is identical to that observed in synaptic terminals in vivo. GAP-43 phosphorylated at Ser41 by protein kinase C (PKC) is resistant to the cleavage by calpain. In addition, calmodulin binding to GAP-43 decreases the rate of calpain-mediated GAP-43 proteolysis. Our results indicate that m-calpain-mediated GAP-43 proteolysis regulated by PKC and calmodulin is of physiological relevance, particularly in axonal growth cone guidance. We suggest that the function of the N-terminal fragment of GAP-43 (residues 1-40) formed during cleavage by m-calpain consists in activation of neuronal heterotrimeric GTP-binding protein G(o); this results in growth cone turning in response to repulsive signals.     

GAP-43 is key to mitotic spindle control and centrosome-based polarization in neurons

In neurons, the position of the centrosome during final mitosis marks the point of emergence of the future axon. However, the molecular underpinnings linking centrosome position to axon emergence are unknown. GAP-43 is a calmodulin-binding IQ motif protein that regulates neuronal cytoskeletal architecture by interacting with F-actin in a phosphorylation dependent manner. Here we show that GAP-43 is associated with the centrosome and plays a critical role in mitosis and acquisition of neuronal polarity in cerebellar granule neurons. In the absence of GAP-43, the centrosome position is delinked from process outgrowth and is only capable of mediating morphological polarization, however molecular specification of the axonal compartment does not take place. These results show that GAP-43 is required to link centrosome position to process outgrowth in order to generate neuronal polarity in cerebellar granule cells.     

Megalin is a receptor for albumin in astrocytes and is required for the synthesis of the neurotrophic factor oleic acid

We have previously shown that the uptake and transcytosis of albumin in astrocytes promote the synthesis of the neurotrophic factor oleic acid. Although the mechanism by which albumin induces oleic acid synthesis is well known, the mechanism of albumin uptake in astrocytes remains unknown. In this work, we found that astrocytes express megalin, an endocytic receptor for multiple ligands including albumin. In addition, when the activity of megalin is blocked by specific antibodies or by silencing megalin with specific siRNA, albumin binding and internalization is strongly reduced indicating that megalin is required for albumin binding and internalization in the astrocyte. Since the uptake of albumin in astrocytes aims at synthesizing the neurotrophic factor oleic acid, we tested the ability of megalin-silenced astrocytes to synthesize and release oleic acid in the presence of albumin. Our results showed that the amount of oleic acid found in the extracellular medium of megalin-silenced astrocytes was strongly reduced as compared with their controls. Together, the results of this work indicate that megalin is a receptor for albumin in astrocytes and is required for the synthesis of the neurotrophic factor oleic acid. Consequently, the possible involvement of albumin in the holoprosencephalic syndrome observed in megalin-deficient mice is suggested.     

ERK-mediated production of neurotrophic factors by astrocytes promotes neuronal stem cell differentiation by erythropoietin

Erythropoietin (EPO), a hematopoietic factor, is also required for normal brain development, and its receptor is localized in brain. Our previous study showed that EPO promotes differentiation of neuronal stem cells into astrocytes. Since astrocytes have influence on the neuronal function, we investigated whether EPO-activated astrocytes could stimulate differentiation of neuronal stem cells into neurons. EPO did not promote neuronal differentiation of neuronal stem cells isolated from 17 day embryos, however, neuronal differentiation was promoted when the neuronal stem cells were co-cultured with astrocyte isolated from post neonatal (Day 1) rat brain. Moreover, neuronal differentiation was further promoted when the neuronal stem cells were cultured with astrocyte culture medium treated by EPO (10U/ml) showing increase of morphological differentiation, and expression of neuronal differentiation marker proteins, neurofilament, and tyrosine hydroxylase. The promoting effect of EPO-treated astrocyte medium was also found in the differentiation of PC12 cells. EPO-promoted morphological differentiation of neuronal stem cells as well as astrocytes was dose dependently reduced by treatment with anti-EPO receptor antibodies in culture with astrocyte culture medium. To clarify whether EPO itself or via production of well-known neurotropic factor could promote neuronal cell differentiation, we determined the level of neurotropic factors in the EPO-treated astrocytes. Compared to untreated astrocytes, EPO-treated astrocytes increased about 2-fold in beta-NGF and 3-4-fold in BMP2, but did not increase BNDF and NT-3 levels. Since the previous study showed that extracellular signal-regulated kinase (ERK) is involved in activation of astrocytes by EPO, we determined whether generation of neurotrophic factor may also be involved with the ERK pathway. In the presence of ERK inhibitor, PD98059, the generation of beta-NGF was diminished in a dose dependent manner consistent with the inhibiting effect on neuronal differentiation. These data demonstrate that EPO promotes neuronal cell differentiation through increased release of beta-NGF and BMP2 from astrocytes, and this effect may be associated with ERK pathway signals.     

Model system for live imaging of neuronal responses to injury and repair

Although it has been well established that induction of growth-associated protein-43 (GAP-43) during development coincides with axonal outgrowth and early synapse formation, the existence of neuronal plasticity and neurite outgrowth in the adult central nervous system after injuries is more controversial. To visualize the processes of neuronal injury and repair in living animals, we generated reporter mice for bioluminescence and fluorescence imaging bearing the luc (luciferase) and gfp (green fluorescent protein) reporter genes under the control of the murine GAP-43 promoter. Reporter functionality was first observed during the development of transgenic embryos. Using in vivo bioluminescence and fluorescence imaging, we visualized induction of the GAP-43 signals from live embryos starting at E10.5, as well as neuronal responses to brain and peripheral nerve injuries (the signals peaked at 14 days postinjury). Moreover, three-dimensional analysis of the GAP-43 bioluminescent signal confirmed that it originated from brain structures affected by ischemic injury. The analysis of fluorescence signal at cellular level revealed colocalization between endogenous protein and the GAP-43-driven gfp transgene. Taken together, our results suggest that the GAP-43-luc/gfp reporter mouse represents a valid model system for real-time analysis of neurite outgrowth and the capacity of the adult nervous system to regenerate after injuries.