Protein phosphatases and kinases implicated in Alzheimer's disease abnormal tau phosporylation

Abstract

This thesis describes the distribution, levels and activities of a number of protein kinases and protein phosphatases (PPs) implicated in the abnormal phosphorylation of the tau protein that forms the paired helical filaments (PHFs) of Alzheimer's disease (AD) neurofibrillary tangles (NFTs). Paper I describes the immunocytochemical localization of PP-1, PP-2A, PP-2B and PTP-1B, as well as the protein kinase MAP kinase and p34cdc2 in the hippocampal formation of AD and control brains. All the protein phosphatases and protein kinases studied were localized to both granular and pyramidal neurons. In the pyramidal neurons, enzyme staining was observed in neuronal soma and neurites. PTP-1B, PP-1 and PP-2A were also highly expressed in microglial-like cells. The topographical distributions of all the enzymes studied were similar and expression of all enzymes was observed in the tangle-bearing neurons. The PP-2B staining of tangle-bearing neurons was weaker than unaffected neurons in AD cases. Paper II shows that the proline-directed protein kinase, glycogen synthase kinase-3 (GSK-3) is prominently present in neuronal cell bodies and their processes and colocalizes with neurofibrillary changes in AD brain. Furthermore, the levels of GSK-3 as determined by indirect ELISA were approximately 50% increased in the postsynaptosomal supernatant from AD brains as compared to controls, although no increase in GSK-3 enzyme activity was detected. Paper III describes levels and activities of PP-1, PP-2A, and PP-2B in superior temporal cortical grey matter of AD and control brains. The protein levels of PP-2A and PP-2B were significantly increased in postsynaptosomal supernatant 2 (S2) of the AD group, and this alteration showed a significant linear correlation with levels of hyperphosphorylated tau. In contrast, PP-1 and PTP-1B levels were not significantly changed in any of the AD fractions. The activity levels of none of the phosphatases investigated were significantly different between the AD and control groups, whereas the PP-2B specific activity (activity/protein) showed a significant linear inverse correlation with hyperphosphorylated tau. Paper IV describes a follow up to study III where it is shown using immunocytochernistry that astroglia positive for PP-2A and PP-2B immunoreactivities Were greater in numbers in AD medial temporal cortex, compared to controls. Tissue fractionation and ELISA studies confirmed that PP-2A and PP-2B levels correlated with those for GFAP, suggesting that astrogliosis probably accounts for the increased levels of PP-2A and PP-2B in AD brain. In paper V the immunocytochernical localisation of cyclin-dependent kinase 5 (cdk5) was compared with that obtained using antibodies to AD abnormally hyperphosphorylated tau (AD p-tau) and ubiquitin as markers of early and late stage NFTs, respectively in hippocampal, entorhinal, transentorhinal, temporal and frontal cortices and cerebellum of AD and control brains. Immunoreactivities of cdk5 and AD p-tau were found in neuronal perikarya and processes and an apparent increase of cdk5 immunoreactivity was seen in pretangle neurons and in neurons bearing early stage NFTs, suggesting that this kinase might be involved in the formation of NFTs at a relatively early stage in neocortex. Paper VI is a follow up to paper II where it is shown that immunostaining for the active, but not inactive form of GSK-3 shows a similar regional distribution to the spread of neurofibrillary changes described according to the Braak and Braak staging protocol. This, together with evidence that active GSK-3 accumulates in the cytoplasm of pre-tangle neurons, is consistent with an early involvement of GSK-3 in AD p-tau formation. Taken together, this thesis provides substantial support for the hypothesis that aberrant tau hyperphosphorylation in AD brain results from a protein kinase / phosphatase imbalance that most likely involves increased GSK-3 and cdk-5 and decreased PP-2B levels and activities.