Papel de Akt y sus isoformas en cultivos neuronales

Tesis doctoral inédita leída en la Universidad Autónoma de Madrid, Facultad de Ciencias, Departamento de Biología Molecular. Fecha de lectura: 26-11-2010Class I PI3K>Akt is one of the most studied cell signaling pathways, and it has been involved in many cell processes, such as survival, growth and polarity; most of the works published in this field use the pan-PI3K inhibitor LY294002. In mammals, there are three isoforms of Akt coded by three different genes: Akt1, Akt2 and Akt3. We decided to study the function of each of them in neuron cultures. For that purpose, we firstly characterized the evolution of Akt and its substrates GSK3 and the mTORC1 pathway on cortical and hippocampal neurons in the first ten days of culture. Our data indicated that the three isoforms are present in both neuron types and they undergo changes along the development. Secondly, we used a lentiviral-ShRNA approach to study the functions of each of them in the cell signalling of cortical neurons. Although apparently there were no changes on the GSK3 or MAPK pathways, our data suggested that Akt2 is involved in the insulin response and that both Akt2 and 3 regulate the S6 kinase activity in a mTORC1-independent manner. Akt2 protein levels are downregulated by rapamycin and by the Akt3 ShRNAs. Considering the little changes observed by knocking down each isoform, we used a complementary double pharmacological or ShRNA experimental approach to analyze the functions of Akt on cell survival and axon growth. Treatment of cortical neurons with specific Akt inhibitors (AktV and AktVIII) lead to a decrease on cell viability associated with apoptotic (caspase 3) but not autophagic (LC3) markers and with a fall on GSK3α/β S21/9 phosphorylation. Lentiviral-ShRNA approach showed a slight but significative effect of Akt2 and 3. However, our preliminary data suggested that GSK3 inhibition does not rescue the Akt-inactivation induced cell death. Hippocampal neurons showed a reduction on axon length when treated with AktVIII that was linked to a S6 kinase activity impairment. Akt2 and Akt3 ShRNA nucleofection also reduced axon length. Neither global inhibition nor isoform-specific knock down showed great changes on hippocampal neuron polarity. As Akt has been proposed to play an important role in this process, we decided to check the assumptions that lead to this model. First, direct inhibition of Akt does not change growth cone localized GSK3 phosphorylation. Second, LY294002, the PI3K inhibitor used in previous experiments, inhibited mTORC1 substrates in an Akt-independent manner. Third, a constitutively active Akt mutant induced no change on neuron polarity. Then, we decided to analyze the functions of class I PI3K on axon growth. The specific inhibitor GDC-0941 did not alter hippocampal neuron polarity. In summary, our data suggests that Akt is involved in neuron survival and axon growth but not in the establishment of neuronal polarity and that its isoforms show functional redundancy in many cell processes