Background: Neurite outgrowth is an important aspect of neuronal plasticity and regeneration after neuronal injury. Alpha-lipoic acid (LA) has been used as a therapeutic approach for a variety of neural disorders. We recently reported that LA prevents local anesthetics-induced neurite loss. In this study, we hypothesized that LA administration promotes neurite outgrowth. Methods: To test our hypothesis, we treated mouse neuroblastoma N2a cells and primary neurons with LA. Neurite outgrowth was evaluated by examination of morphological changes and by immunocytochemistry for α-tubulin-3. ROS production was examined, as were the phosphorylation levels of ERK and Akt. In separate experiments, we determined the effects of the inhibition of ERK or PI3K/Akt as well as ROS production on LA-induced neurite outgrowth. Results: LA promoted significantly neurite outgrowth in a time- and concentration-dependent manner. LA stimulation significantly increased the phosphorylation levels of both Akt and ERK and transiently induced ROS production. PI3K/Akt inhibition did not affect LA-induced neurite outgrowth. However, the inhibition of ERK activation completely abolished LA-induced neurite outgrowth. Importantly, the prevention of ROS production by antioxidants attenuated LA-stimulated ERK activation and completely abolished LA-promoted neurite outgrowth. Conclusion: Our data suggest that LA stimulates neurite outgrowth through the activation of ERK signaling, an effect mediated through a ROS-dependent mechanism. This article is part of a Special Issue entitled: 11th European Symposium on Calcium.

In vielen humanen Neoplasien findet sich eine erhöhte Aktivität des Raf-Mek-Erk-Signaltransduktionsweges. Zunächst wurde davon ausgegangen, dass diese erhöhte Aktivität hauptsächlich durch die Ras-Onkogene hervorgerufen wird. Doch mittlerweile konnten auch Mutationen der Raf Gene in humanen Neoplasien nachgewiesen werden. Gegen Raf und Mek konnten eine Anzahl von Enzyminhibitoren entwickelt werden. Der Nachteil vieler dieser Inhibitoren ist, dass sie nicht zwischen den einzelnen Kinaseisoformen unterscheiden können. In dieser Arbeit ist es nun das erste Mal gelungen, jede Komponente des Raf-Mek-Erk-Signaltransduktionsweges einzeln mittels RNA Interferenz effizient zu inhibieren. Dabei konnte die Rolle der verschiedenen Isoformen in der Proliferation, Morphologie und Genex-pression von transformierten Zellen definiert werden. In den NIH3T3-pEJ Zellen konnte A-Raf erstmals eine antiapoptotische Rolle zugewiesen werden. Diese Hemmung der Apoptose läuft möglicherweise über einen Mek2-abhängigen Weg und ist an die Mitochondrien gekoppelt. Für die beiden Mek Kinasen konnten unter-schiedliche Funktionen in der Signalweiterleitung gezeigt werden. Mek2 spielt die Hauptrolle in der Aktivierung der beiden Substratkinasen Erk1 und Erk2. Der Verlust der Mek1 Isoform wird dagegen möglicherweise durch eine erhöhte Expression von Mek2 kompensiert und wirkt sich nicht so stark auf die Phosphorylierung von Erk1/2 aus. Durch die Verwendung von Erk1 und Erk2 spezifischen siRNAs konnte eine Trennung zwischen der Proliferationsre-gulation und der Kontrolle der morphologischen Transformation herausgearbeitet werden. Durch die Verwendung von Mikroarrays ist es gelungen, beiden Phänotypen ein Genexpres-sionsprofil zuzuordnen. Neben Unterschieden zwischen den verschiedenen Kinaseisoformen konnten neue, potentielle Feedbacks beschrieben werden. / In many human neoplasia an increased activity of the RAF/MEK/ERK- signaling pathway is found. First it was assumed that this raised activity is caused primarily by the RAS onco-genes. However, meanwhile mutations in the RAF genes could be also proved in human neo-plasia. A number of enzyme inhibitors have been developed against the RAF and MEK pro-teins. The disadvantage of many of these inhibitors is that they cannot distinguish between the different kinase isoforms. In this work it has succeeded the first time to inhibit every compo-nent of the RAF/MEK/ERK- signaling pathway individually by means of interference RNA. Beside this, the role of the different isoforms in the proliferation, morphology and genetic profile of transformed cells could be defined. For the first time A-Raf could be assigned an anti-apoptotic role in NIH3T3-pEJ cells. This inhibition of the apoptosis possibly runs through a Mek2-dependent way and is coupled to the mitochondria. For both Mek kinases different functions could be shown in the downstream signaling. Mek2 plays the leading role in the activation of both downstream kinases Erk1 and Erk2. The loss of the Mek1 isoform expression is possibly compensated through an increased expression of Mek2 and does not affect the phosphorylation of Erk1 / 2 so strongly. A discri-mination between the regulation of proliferation and the control of the morphological trans-formation could be worked out by the use of Erk1 and Erk2 specific siRNAs. By the use of micorarray an expression profile of both phenotypes has assigned. Beside differences between the different kinases new, potential feedback pathways could be described.