Interactions and Cytotoxicity of Human Neurodegeneration- Associated Proteins Tau and α-Synuclein in the Simple Model Dictyostelium discoideum

Abstract

The abnormal accumulation of the tau protein into aggregates is a hallmark in neurodegenerative diseases collectively known as tauopathies. In normal conditions, tau binds off and on microtubules aiding in their assembly and stability dependent on the phosphorylation state of the protein. In disease-affected neurons, hyperphosphorylation leads to the accumulation of the tau protein into aggregates, mainly neurofibrillary tangles (NFT) which have been seen to colocalise with other protein aggregates in neurodegeneration. One such protein is α-synuclein, the main constituent of Lewy bodies (LB), a hallmark of Parkinson’s disease (PD). In many neurodegenerative diseases, including PD, the colocalisation of tau and α-synuclein has been observed, suggesting possible interactions between the two proteins. To explore the cytotoxicity and interactions between these two proteins, we expressed full length human tau and α-synuclein in Dictyostelium discoideum alone, and in combination. We show that tau is phosphorylated in D. discoideum and colocalises closely (within 40 nm) with tubulin throughout the cytoplasm of the cell as well as with α-synuclein at the cortex. Expressing wild type α-synuclein alone caused inhibited growth on bacterial lawns, phagocytosis and intracellular Legionella proliferation rates, but activated mitochondrial respiration and non-mitochondrial oxygen consumption. The expression of tau alone impaired multicellular morphogenesis, axenic growth and phototaxis, while enhancing intracellular Legionella proliferation. Direct respirometric assays showed that tau impairs mitochondrial ATP synthesis and increased the “proton leak,” while having no impact on respiratory complex I or II function. In most cases depending on the phenotype, the coexpression of tau and α-synuclein exacerbated (phototaxis, fruiting body morphology), or reversed (phagocytosis, growth on plates, mitochondrial respiratory function, Legionella proliferation) the defects caused by either tau or α-synuclein expressed individually. Proteomics data revealed distinct patterns of dysregulation in strains ectopically expressing tau or α-synuclein or both, but down regulation of expression of cytoskeletal proteins was apparent in all three groups and most evident in the strain expressing both proteins. These results indicate that tau and α-synuclein exhibit different but overlapping patterns of intracellular localisation, that they individually exert distinct but overlapping patterns of cytotoxic effects and that they interact, probably physically in the cell cortex as well as directly or indirectly in affecting some phenotypes. The results show the efficacy of using D. discoideum as a model to study the interaction of proteins involved in neurodegeneration.

Dictyostelium discoideum: A Model System for Neurological Disorders

“…Strains expressing both α-synuclein and Tau showed that these two human neurodegeneration-associated proteins colocalised at the cortex where α-synuclein is most prominent. The close proximity (within 10 nm) suggests that Tau and α-synuclein have a physical interaction, not just a colocalization [14].…”

“…The longest isoform of Tau was used to create a D. discoideum model. It was found that Tau was present throughout the cytosol where it was also seen colocalized in close association with tubulin [14]. In human cells, Tau is phosphorylated on many sites and in the phosphorylated state dissociates from microtubules [75].…”

“…The aggregation and accumulation of α-synuclein is thought to involve impaired interactions with other proteins and studying these interactions in model systems can assist in characterising the cytopathological disease pathways [13,14]. D. discoideum has been used to investigate the interaction of α-synuclein with the human microtubule-associated Tau protein [14]. D. discoideum lacks endogenous Tau and α-synuclein which allowed investigation of the effects of human Tau alone and in combination with human α-synuclein.…”

“…The strains expressing Tau or α-synuclein were examined phenotypically and shown to cause different patterns of phenotypic abnormalities [14]. Whilst expression of WT αsynuclein resulted in growth defects, impaired phagocytosis and increased mitochondrial respiratory activity, expression of Tau resulted in impaired phototaxis, thermotaxis, growth in liquid and an isolated respiratory complex V defect in oxidative phosphorylation.…”

“…The genetic tractability of the organism allows genes related to such disorders to be easily manipulated and phenotypically analysed allowing insight into their conserved cellular functions. In addition to altering endogenous genes, several human genes have been expressed in D. discoideum and studied in the absence of endogenous copies, for example, human αsynuclein and Tau protein have been expressed singly and in combination in D. discoideum to elucidate the mechanisms of cellular toxicity in synucleinopathies and tauopathies [13,14]. Furthermore, many cellular processes are highly conserved in D. discoideum allowing for the investigation of underlying cytopathological mechanisms.…”