Utilizing Drosophila Primary Neurons To Study Human Tau Propagation

Aggregates of the microtubule stabilizing protein, tau, are found in the neurofibrillary tangles (NFTs) of Alzheimer's disease (AD) patients. When phosphorylated, the protein is altered from an endogenous form to a pathogenic form. These aggregations, or tauopothies, are known to disrupt cell transport and destabilize the microtubule in its diseased state. Although these tauopothies have been accepted by the scientific community as a potential cause of AD, the mechanisms behind which this aggregated tau protein can spread and further the progression of the disease are unknown. New evidence suggests that these pathogenic forms of tau can infect neighboring neurons in a prion-like manner, meaning they have the potential to induce a conformational change in a normal tau protein, altering it to a diseased state. This trans-synaptic propagation is a hypothesized method of propagation in AD neurons. The purpose of this research project is to investigate the cellular mechanisms of the release of tau in a cellular model of Alzheimer's disease. Our preliminary results have shown that a Drosophila primary cell model can be used to express an aggregation prone pathogenic version of human tau protein (2N4R) in cholinergic neurons in vitro. Expression of hTau was confirmed by western blot of highly specific immunoprecipitated adult fly brain protein and in primary culture neurons by immunofluorescence using an anti hTau antibody. Tau protein was released extracellularly by inducing membrane depolarization in primary cultured neurons after incubation with 50 mM KCl in conditioned media and in Locke's Buffer. A fluorescence intensity assay measuring tau protein level after KCl treatment suggested that these neurons had a lower level of intracellular hTau when compared with untreated, 2N4R expressing neurons. Addition of this conditioned media to control neurons (Cha-GFP) demonstrated cellular uptake of hTau protein into the soma. Western blot analysis of the immunoprecipitated conditioned media (using hTau antibody) and then probed with anti - PHF tau (phosphotau) demonstrated that released tau was phosphorylated. These results suggest that our model may be useful for studying the release and uptake of tau protein occurring in AD pathogenesis.