Alzheimer's Disease tissue culture models
JSW provides different cell culture models for Alzheimer's Disease including induced neuronal cell death, analyses of human APP cleavage and Tau phosphorylation.
1) In vitro lesion models
It is meanwhile well described, that neuronal loss in case of AD is due to various types of stress conditions. These comprise oxidative stress, excitotoxicity, Ca++-overload and others beyond amyloid toxicity.
To induce suchlike conditions embryonic neuronal cultures and neuron/glia co-cultures are treated with agents that induce lesions mimicking important aspects of Alzheimer's Disease:
- Aß1-42
- H2O2
- NMDA
- Glutamate
- Ionomycin
- Kainate
- Okadaic acid
Cultures are analysed for cell survival, apoptotic and necrotic phenotypes and expression of different synaptic markers (see Analyses of culture models).
2) Screening of secretase inhibitors
Generation of Aß1-42 species is a hallmark of Alzheimer's Disease. This assay allows to identify agents that interfere with the cleavage of human APP. We provide two different cell culture models to analyse the effect of pharmacological agents on generation of different Aß and sAPP peptides. One model is based on the cultivation of primary cultured chicken neurons. Chicken and human amyloid share the same amino acid sequence. Using geniune neurons the system was found to be extremely useful to study secretase modulation and inhibition. Additonally it was seen to be highly predictive regarding the conditions in AD brains.
In the second system transgenic neuroglioma cells provide secretase activity and hAPP as a substrate. These cells proved to be the second valid system to study secretase inihibiton and kinetics.
3) Analyses of Tau phosphorylation
Increased Tau phosphorylation and the formation of neurofibrillary tangles are critical appearances of Alzheimer's Disease. We use embryonic hippocampal neurons at day in vitro 14 to induce Tau phopshorylation using okadaic acid, a phosphatase inhibitor. Different phospho-Tau epitopes can be analysed including:
- Ser396/404
- Ser202/Thr205
- Thr231
- Thr181
- and others
Phospo-Tau epitopes can be examined by immunocytochemistry and Western Blotting (see Analyses of culture models).
