P1-366: Glycogen synthase kinase activity in platelets of patients with Alzheimer's disease, mild cognitive impairment and controls

Abstract

Glycogen synthase kinase-3β (GSK3) is a highly expressed kinase in the central nervous system, and its activity is regulated by phosphorylation and desphosphorylation of serine-threonine epitopes. GSK3 activity is increased in the AD brain, being implicated in the hyperphosphorylation of TAU and abnormal APP processing. This abnormality has been recently detected in human leukocytes, and for this reason GSK3 has been suggested as a candidate biomarker of AD, which can be measured in peripheral tissues and may help indicate the risk of conversion from MCI to AD. Recent, unpublished determinations from our group showed that GSK3 is highly expressed in platelets, bearing both phosphorylated and non-phosphorylated forms. The objective of the present study is to validate a semi-quantitative method to determine GSK3 activity in human platelets, with the aid of Western Blot. Platelet samples from patients with AD, MCI and age-matched controls were obtained from peripheral blood. Aliquots holding 25μg of total protein in sample buffer were submitted to electrophoresis in 10% poliacrylamide gels and transferred to nitrocellulose membranes. Membranes were incubated with primary antibodies raised against total- and phospho-GSK, following incubations with species-specific biotinilated secondary antibodies, and a streptavidin horseradish peroxidase conjugate. Development was performed with enhanced chemiluminescence (ECL-Plus), prior to densitometry of the correspondent bands. GSK3 activity was estimated by ratio between its inactive (phosphorylated) form and the total GSK amount in platelet samples (phospho-GSK3/ total-GSK3). In this preliminary analysis (n=27), the method proved adequate for the semi-quantitative determination of GSK3 expression and activity in human platelets. There was a clear tendency toward an increased GSK3 activity in patients with AD and MCI as compared to controls, although not within significant limits: AD (n=9), 0.64±0.09; MCI (n=9), 0.75±0.10; Controls (n=9), 0.85±0.08. Recruitment in under way (n=51 at present), and a larger sample of patients and controls will presented at the ICAD-Chicago. If confirmed in a larger sample, the current findings will support the notion that GSK3 expression and activity can be determined in human platelets, with findings largely similar to the ones presented in leukocytes.