Total and phosphorylated Akt/PKB in the parietal cortex of patients with Alzheimer’s disease

Sundhya Raman, Robert J Williams and Paul T Francis, Wolfson Centre for Age-Related Diseases, King’s College London, Guy’s Campus, St Thomas Street, London SE1 1UL, UK.

Alzheimers disease (AD) is the most common form of dementia, however the pathogenic processes leading to neuronal cell loss remain poorly understood. The serine/threonine kinase Akt (PKB) is thought to play a central role in maintaining cell survival in part by inhibiting apoptosis. Glutamate and oxidative stress acutely increase Akt phosphorylation although prolonged exposure produces deficits in Akt signalling which have been associated with neuronal damage (Crossthwaite et al., 2002) The aim of this study was to investigate whether the levels and/or activity of this potentially protective protein were altered in AD.

Western Blot analysis was performed using samples from the parietal cortex (Brodmann area 39) of a group of AD patients and controls. Homogenates were prepared from cortical tissue as described (Minger et al, 2000) and 40µg of protein per sample was subjected to SDS-PAGE and transferred to nitrocellulose membranes. Proteins were detected using polyclonal antibodies against Ser473 Phospho Akt or Total Akt, both at a dilution of 1:1000 (Cell Signaling Technologies). Subsequently, membranes were incubated in horseradish peroxidase conjugated secondary antibodies before the immunoreactivity of the bands was detected using Enhanced ChemiLuminescence reagent. Resulting duplicate blots for phospho Akt and total Akt were scanned and quantified using the BioImage Intelligent Quantifier densitometry programme and integrals of band density relative to rat cortex obtained. In addition where both measurements were available in the same sample the ratio phospho Akt/total Akt was calculated. Group means were compared using the Student’s t-test.

Table 1. Total and phospho akt levels in parietal cortex.

Values are mean ± SEM (n) and are in arbitrary units.

Although a trend towards an increase in phospho Akt levels in AD patients was seen (Table 1), consistent with the findings of Griffin et al, (2005) and Rickle et al, (2004), a Student’s T-test revealed no significant difference in the mean levels of phospho Akt (t=0.2; p=0.7), total Akt (t=0.6; p=0.5) or the ratio of phospho/total Akt (t=3.3; p=0.08). This result may in part be due to the low n number for Phospho/Total Akt. A positive linear correlation was found between total Akt and Synaptophysin levels in AD (β=0.51; p=0.018) as well as with VGLUT1 (β=0.46; p=0.036), however no correlation was found between final Mini Mental State Examination score and Akt levels.

Crossthwaite AJ et al (2002) J Neurochem 80: 24-35.
Griffin , RJ et al. (2005) J Neurochem. 93:105-117.
Minger SL et al. (2000) Neurology. 55:1460-1467.
Rick le A et al. (2004) Neuroreport. 15:955-959.