P3-326: Expression of human truncated tau protein in long term culture of rat hippocampal neurons derived from Axon transgenic AD rat model

Abstract

Truncated and hyperphosphorylated tau protein is one of the most important molecular and histopathological hallmarks found in Alzheimer‘s disease. Mutual occurrence of both events – truncation and hyperphosphorylation – is essential for disease progression and therefore transgenic models expressing non–mutated truncated tau could be successfully employed for detailed analysis of molecular pathways leading to neurodegeneration. To investigate the role of human truncated tau in neurofibrillary degeneration in long term rat primary hippocampal neuronal cultures. All experiments were performed using long–term hippocampal neuronal cultures prepared from transgenic rats expressing truncated human AlzTau. Neurons were cultured in vitro for 6–84 days (DIV). Distribution of AlzTau within the cell body and neurites was analyzed by staining with anti–human tau mAb – HT7. Tau protein phosphorylation at epitope 199/202,205 was detected with AT8 mAb. Pictures of neurons were taken at constant exposure times using confocal microscope (Olympus–Fluoview). Expression of transgenic human AlzTau in hippocampal neurons was observed from 6DIV. Intraneuronal AlzTau was not evenly distributed within the cell body and neurites. In the time period of 6th – 37th DIV AlzTau was present exclusively in bigger (wider) processes. However, on 52nd DIV it emerged also in peripheral neurites of neurons. Alzheimer‘s tau expression (gene dose dependent) pattern was enhanced in homozygous animals. Neurons derived from these animals displayed AlzTau in all neurites already on 25th DIV. Embryonic tau protein phosphorylation was detected up to 6th DIV in both, wild type and transgenic cultures. Strikingly AT–8 phosphorylation in transgenic neurons persisted up to 84 DIV. Detailed analysis of aged AT8 positive neurons (84DIV) showed the presence of granular and filamentous tau protein structures, localized within the cell bodies, suggesting early stage of NFT maturation. We have shown the expression pattern and intraneuronal distribution of human truncated tau protein in transgenic hippocampal neurons. Transgenic neuronal primary cultures (6 – 84 DIV) strongly suggest that truncated tau induced phosphorylation and formation of altered tau that leads to assembly into filamentous and granular structures.