PTEN and Akt signalling in Alzheimer´s disease

Sammanfattning: Akt / protein kinase B is an important cell survival kinase that modulates the activity of a range of pro-apoptotic proteins. A major downstream target of Akt is glycogen synthase kinase-3beta (GSK3beta) that phosphorylates the microtubule-associated protein tau. Previous studies have shown that GSK3beta activity is increased in Alzheimer s disease (AD) brain in parallel to the accumulation of neurofibrillary changes. This study aimed to determine whether signal transduction pathways upstream of GSK3beta are also deregulated in AD brain. The study focussed on Akt and PTEN. The latter is a dual lipid and protein phosphatase the functions of which include inhibition of Akt. Akt and PTEN activity states and levels were determined in AD brain as compared to material from other diseases and controls. Also studied was the basic biology of PTEN and the role of this enzyme as a modulator of rat primary cortical neuron cell death. Paper I studied the levels and activity of Akt in post-mortem brain material from sporadic AD, controls and other neurodegenerative disorders. Using enzyme immunoprecipitations and phosphorylation of exogenous substrate, as well as immunoblotting with phospho-specific antibodies, we showed that Akt enzyme activities were significantly increased in soluble fraction of mid-temporal cortex from AD cases as compared to both control groups. This increase correlated with Braak staging for neurofibrillary changes. Immunohistochemistry revealed that this increase also occurred partly due to gliosis. Paper II determined whether altered levels or distribution of PTEN are also found in AD brain. No significant changes of PTEN protein levels were seen in the nuclear, membrane or homogenates of medial AD temporal cortex as compared to control cases. A small but significant decrease in the levels of Ser380PTEN was found in AD medial temporal cortex as shown by dot-blot analysis. Immunohistochemistry of layer III temporal cortex showed the majority of pyramidal neurons in this region were PTEN immunopositive (93-100%). The total numbers of pyramidal neurons and PTEN immunopositive neurons were significantly lower in AD as compared to control cases. Paper III is a methodological study designed to optimise conditions for studying toxic effects of Abeta, HNE and TNFalpha on rat primary cortical neurons using propidium iodide/annexin V staining and flow cytometry analyses. These stressors were chosen as being implicated in AD and as having reported effects on Akt signalling. We showed time and dose-responses for all stressors and chose optimal conditions (24h 10muM Abeta, 24h 1muM or 5muM HNE, 24h 500 ng/ml TNFalpha) that demonstrated effects on numbers of viable, apoptotic and late apoptotic cells. Flow cytometry generated data were confirmed by immunocytochemistry of treated cells. Our results also provided important information on the culture conditions of primary neuron cultures. Paper IV assessed the regulation of Akt and PTEN during neuronal death induced by the stress factors of TNFalpha and HNE. We used optimal conditions from study III to determine TNFalpha and HNE effects on the regulation of PTEN, Akt and GSK3beta levels and activity. The inhibitors bpV(Pic) and LY294002 were used to determine PTEN and PI3K involvement, respectively, in TNFalpha and HNE modulation of neuronal cell death. Immunoblotting with phospho-epitope specific antibodies was used to determine effects on PTEN/PI3K/Akt/GSK3beta signalling. In parallel, flow cytometry was used to determine effects on populations of viable and early apoptotic cells. Immunoblotting showed that neither PTEN nor PI3K inhibition alone had an effect on Ser473Akt, or Ser9GSK3beta phosphorylation. Instead, effects of these inhibitors were apparent when used together with TNFalpha and to a lesser extent HNE. TNFalpha together with PTEN inhibition increased phosphorylation on Ser473Akt and Ser9GSK3beta that was paralleled by an increased number of viable and decreased number of early apoptotic neurons. These effects were all reversed by PI3K inhibition. Thus, inhibition of PTEN results in a mild neuroprotection against TNFalpha induced toxicity. HNE did not show this protective effect. These studies provide evidence for a deregulated Akt and PTEN signalling in AD brain.