Research reportOleic acid induces GAP-43 expression through a protein kinase C-mediated mechanism that is independent of NGF but synergistic with NT-3 and NT-4/5
Introduction
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 [21], [30]. This protein has been found in neurons and glial cells during development [14] but not in the adult brain. The presence of albumin in the brain may be due to the existence of a mechanism through which albumin is transferred from the blood to the brain and CSF that is active only in immature rats [6], [17]. In addition, the in situ synthesis of albumin can also contribute to the increased albumin levels found in the newborn brain [4]. We have recently shown that the presence of albumin in astrocytes could be important in triggering the synthesis and release of oleic acid, which behaves as a neurotrophic factor for neurons [27], [29]. Thus, albumin is internalized by receptor-mediated endocytosis [29] and once inside the astrocytes it increases the flux of glucose and lactate through the pyruvate dehydrogenase-catalyzed reaction [28]. Albumin internalization is followed by transcytosis, including passage through the endoplasmic reticulum (ER). The presence of albumin in the ER activates sterol regulatory element-binding protein-1 (SREBP-1) and increases stearoyl-CoA 9-desaturase (SCD) mRNA [29]. SREBP-1 is a basic helix-loop-helix-leucine zipper transcription factor responsible for the induction of the enzymes involved in oleic acid synthesis, including the rate-limiting enzyme stearoyl-CoA 9-desaturase (SCD), which introduces the double bond in oleic acid synthesis [1], [10], [18], [23].
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 [27]. All these observations point to oleic acid as a key factor in neuronal differentiation.
Growth-associated protein, GAP-43, is a marker of neuronal differentiation. This protein is located in growing axons, where it is bound to the membrane, possibly by two acylated cysteine residues at positions 3 and 4 of the N-terminus [24]. The carboxyl terminal part of the protein contains an F motif that binds to F-actin, thus building the internal structure of axons (for a review, see [16]). GAP-43 expression is restricted to the nervous system during development and regeneration (for a review, see [2]) and can be regulated by several growth factors, including neurotrophins [8], [12], [19], [22], NDF [31], IL-6 [32], EGF [32], IGF [25] and FGF [13]. Since GAP-43 expression is upregulated by oleic acid [27], in this work we attempted to elucidate whether the mechanism by which oleic acid exerts its neurotrophic effect includes the participation of other neurotrophic factors.
Section snippets
Reagents
Dulbecco’s modified Eagle’s medium (DMEM), DMEM+Ham’s F12, insulin, transferrin, pyruvate, penicillin, streptomycin, poly-l-lysine, oleic acid, palmitic acid, NGF, NT-3, NT-4/5, H-89, H-7, anti-mouse Ig FITC, protease inhibitors, monoclonal antibodies against GAP-43 and α-tubulin, anti-mouse Ig biotin, avidin–peroxidase conjugate, AEC substrate staining kit and bovine serum albumin (BSA, fatty acid free) were purchased from Sigma–Aldrich (Madrid, Spain). BSA was dialyzed three times against
PKC mediates the synthesis of GAP-43 promoted by oleic acid
Oleic acid directly activates PKC [3], [7], [9], [15] and this indeed appears to be the mechanism by which oleic acid increases GAP-43 mRNA [27]. In this work we investigated whether the increase in protein synthesis was also mediated by PKC activation. Fig. 1 shows that the inhibition of PKC by H-7 prevented GAP-43 expression both in the absence or presence of oleic acid. However, the inhibition of protein kinase A (PKA) by H-89 had no effect on the induction of GAP-43 synthesis caused by
Discussion
We have recently shown that astrocytes synthesize and release oleic acid [29], which behaves as a neurotrophic factor for neurons [27]. Thus, oleic acid promotes axonal growth and neuronal differentiation, characterized by the increase in the synthesis of the growth-associated protein, GAP-43. In this work, we show that oleic acid increases GAP-43 synthesis by a PKC-dependent mechanism since the effect of oleic acid was not observed when PKC was inhibited (Fig. 1, see also: [27]). Since oleic
Acknowledgements
This work was supported by FIS, Fundación Ramón Areces (A.T.) and the Junta de Castilla y León, Spain. We thank Professor D. Martin-Zanca for TrkA cDNA and for helping with the discussion. We are grateful for the technical assistance of T. del Rey and thank N. Skinner for help in writing the manuscript.
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