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Development of a valid cell model of Alzheimer's type neurodegeneration


O.G. Tatarnikova – Post-graduate Student, Junior Research Scientist, Institute of Biophysics of a Cage of RAS (Pushchino, Moscow Region)
N.V. Bobkova – Ph.D. (Biol.), Head of Laboratory, Institute of Biophysics of a Cage of RAS (Pushchino, Moscow Region)

For screening of drugs potentially useable for AD treatment, in vivo animal models of the disease were predominantly used [1–3]. The efficacy of the compounds was evaluated through their effects on behavioral, biochemical, and morphological hallmarks of AD [4, 5]. However, in vivo studies are very expensive, as they need a huge amount of both transgenic animals and long-lasting experiments. Alternatively, a co-cultivation of primary hippocampal cultures and cells secreting the toxic phosphorylated form of Tau-protein (transfected cells 3T3-4R-Tau) or beta-amyloid (primary culture of brain cells of transgenic mice line 5ХFAD) is able to reproduce in vitro those conditions which are observed in the brain during progression of AD-type neurodegeneration [6]. For this, it is necessarily to prove, firstly, the viability of cultures obtained from the brains of 5XFAD animals, and, secondly, both transgenic specificity of cultured cells and their ability to produce beta-amyloid. The same has to be proven for the fibroblasts line 3T3-4R-Tau, transfected with plasmid DNA encoding, in turn, Tau-protein gene with 4 sites of phosphorylation, which is characteristic for AD patients. The reaching of satisfactory viability and effective functioning of these cultures is the main aim of the present study.
Beta-amyloid producing cells were formed in the neuronal culture obtained from hippocampal tissue of newborn 5XFAD mice, which were previously genotyped by PCR analysis followed by direct testing of cultivated cells transgenity. The transgenic cassette was detected using specific primers, yielding a 377 bp product.
We selected good conditions for cell cultivation and right medium composition to form primary hippocampal neuronal culture from 5XFAD mice, with more than 21-day viability.
On the 8th day of the 5XFAD transgenic culture surviving, the immunocytochemical assay with polyclonal antibodies to beta-amyloid revealed both beta-amyloid (Fig. 3b) and neuronal networks in the culture that were associated with the immu-nopositivity to III-β-tubulin.
Tau-protein expression by 3T3-4R-Tau cell was confirmed by immunofluorescence assay with use of monoclonal anti-bodies (clone HT7) recognizing the whole set of human Tau-protein isoforms.
Co-cultivation of 3T3-4R-Tau cells with native primary hippocampal culture of the newborn rats increased a death rate of hippocampal cells up to 42.7 %, while it was about 29.3% when the hippocampal cells were co-cultivated with control, un-transfected, 3T3 fibroblasts (Fig. 5). This evidently indicates the toxic effect 3T3-4R-Tau cells on the viability of neuronal cultures from native animals. Monoclonal antibodies HT7 to Tau-protein markedly reduced the cellular death (17.7%) in the native culture, which was co-cultured with 3T3-4R-Tau fibroblasts, meaning relatively high functional activity of the 3T3-4R-Tau cells and their ability to enrich the cultural medium by Tau-proteins toxicity. In this study, developing an in vitro model of a genetic form of Alzheimer's disease (AD), optimal conditions supporting vital activity of primary culture of hippocampal cells of newborn 5XFAD mice were revealed. With use of antibodies to beta-amyloid protein (the main AD hallmark) and immunocytochemical approach, the ability of this neuronal culture to produce the beta-amyloid was demonstrated. Furthermore, with use of antibodies against Tau-protein, steady expression and toxicity of human Tau-protein transfected by 3T3-4R-Tau cells was shown as well.
This work was supported by the Program for Basic Research of the Presidium of RAS «FRMT2016» and by grant of RScF № 14-14-00879.

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June 24, 2020
May 29, 2020

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