Flow cytometry-based method to analyze the change in Tau phosphorylation in a hGSK-3beta and hTau over-expressing EcR-293 cell line.

Neurofibrillary tangles are composed of insoluble aggregates of microtubule-associated protein Tau. In the pathology of Alzheimer's disease (AD), accumulation of hyperphosphorylated Tau results in formation of paired helical filaments. One of the main candidate to hyperphosphorylate Tau in AD is glycogen synthase kinase 3beta (GSK-3beta). Here we introduce a non-neuronal cell line, stably co-expressing human Tau and GSK-3beta proteins, where the effect of potential kinase inhibitors on Tau phosphorylation can be monitored. The aim of our study was to establish a new flow-cytometry-based method to quantitatively analyze the changing of Tau phosphorylation, which is a suitable alternative to the well-accepted but non-quantitative Western blot technique. Our results demonstrate that the flow cytometry-based method is a convenient tool to analyze the effect of GSK-3beta inhibitors on Tau phosphorylation. This new approach provides appropriate throughput for screening purposes in preclinical research for characterization of GSK-3beta inhibitors, as potential drug candidate to cure Alzheimer's disease.

Progress toward identification of protease activity involved in proteolysis of apolipoprotein e in human brain.

Apolipoprotein E (apoE) genotype is the single most important genetic risk factor for the most common (sporadic) form of Alzheimer's disease (AD). Increasing evidence supports the hypothesis that the presence of the E4 isoform of this cholesterol-binding protein contributes directly to disease risk, age of onset, and severity of the neuropathology. For example, studies in transgenic mice demonstrate that apoE is necessary for the formation of plaques with neuritic pathology. The precise mechanism by which apoE contributes to the disease remains unknown. However, several lines of investigation from a number of laboratories now point to a role for proteolytic fragments of apoE in the formation of both plaques and tangles, the two pathological hallmarks of the disease. In particular, the C-terminal portion of apoE has been implicated in binding to amyloid and is localized to plaques. The N-terminal domain, on the other hand, is neurotoxic in culture and has been localized to, and implicated in the formation of, neurofibrillary tangles. These results suggest that inhibition of apoE proteolysis is a potential therapeutic strategy for AD. Using human brain homogenates, we have determined that proteolysis of apoE is greatest at acidic pH and can be inhibited by compounds targeting aspartic proteases. The feasibility of screening candidate inhibitors is supported by both ELISA and immunoblotting methods. Future studies will use a combination of in vitro and in vivo assays to test the efficacy of the most effective compounds for their ability to inhibit apoE proteolysis in human brain and apoE transgenic mouse brain tissue.

Evidence against increased glycoxidation in patients with Alzheimer's disease.

Neuropathological findings of Alzheimer's disease (AD) are intracellular (neurofibrillary tangles) and extracellular (senile plaques) filamentous protein aggregates. Non-enzymatic glycation has been proposed as a primary factor in this pathogenesis, leading to increased insolubility of tau protein and beta-amyloid. The aim of our study was to test the hypothesis that increased glycoxidation, i.e. increased levels of oxidized products from non-enzymatic glycation could be found in brains of patients with AD and of aged Down syndrome (DS) subjects with abundant AD-like neuropathological lesions. Frontal cortex specimens were assayed for pentosidine (Pent) and N-epsilon-carboxymethyl-lysine (CML) by reversed phase high performance liquid chromatographical methods. Pent and CML levels in AD (n = 10; Pent, 35.5 +/- 4.84 mumol/g wet-weight tissue; CML, 135.2 +/- 5.0 mumol/g wet-weight tissue) were comparable to DS (n = 9; Pent, 36.4 +/- 3.21; CML, 133.5 +/- 4.7) and controls (n = 10; Pent, 35.2 +/- 3.55; CML, 136.9 +/- 3.3). We conclude that the results are not compatible with the concept of increased glycoxidation in AD compared to normal aging.

Neurofibrillary tangles have no obligatory predilection for acetylcholinesterase-rich neurons.

Parts of the brain that are prone to NFT formation normally contain many neurons that are intensely acetylcholinesterase (AChE)-positive. In this study, we used thioflavin-S immunofluorescence, AChE histochemistry, and AChE immunocytochemistry to investigate the possibility that intense AChE positivity may act as a perikaryal marker for the vulnerability to NFT formation. Our observations in entorhinal and motor cortices and in the subthalamic nucleus demonstrate major mismatches between the distribution of AChE-rich neurons in normal brains and the distribution of NFT in AD. There is therefore no obligatory relationship between intense AChE positivity in the premorbid period and subsequent vulnerability to tangle formation.