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

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.

Cytotoxicity and Mitochondrial Dysregulation Caused by α-Synuclein in Dictyostelium discoideum.

Alpha synuclein has been linked to both sporadic and familial forms of Parkinson's disease (PD) and is the most abundant protein in Lewy bodies a hallmark of Parkinson's disease. The function of this protein and the molecular mechanisms underlying its toxicity are still unclear, but many studies have suggested that the mechanism of α-synuclein toxicity involves alterations to mitochondrial function. Here we expressed human α-synuclein and two PD-causing α-synuclein mutant proteins (with a point mutation, A53T, and a C-terminal 20 amino acid truncation) in the eukaryotic model Dictyostelium discoideum. Mitochondrial disease has been well studied in D. discoideum and, unlike in mammals, mitochondrial dysfunction results in a clear set of defective phenotypes. These defective phenotypes are caused by the chronic hyperactivation of the cellular energy sensor, AMP-activated protein kinase (AMPK). Expression of α-synuclein wild type and mutant forms was toxic to the cells and mitochondrial function was dysregulated. Some but not all of the defective phenotypes could be rescued by down regulation of AMPK revealing both AMPK-dependent and -independent mechanisms. Importantly, we also show that the C-terminus of α-synuclein is required and sufficient for the localisation of the protein to the cell cortex in D. discoideum.

Misfolded α-synuclein causes hyperactive respiration without functional deficit in live neuroblastoma cells.

The misfolding and aggregation of the largely disordered protein, α-synuclein, is a central pathogenic event that occurs in the synucleinopathies, a group of neurodegenerative disorders that includes Parkinson's disease. While there is a clear link between protein misfolding and neuronal vulnerability, the precise pathogenic mechanisms employed by disease-associated α-synuclein are unresolved. Here, we studied the pathogenicity of misfolded α-synuclein produced using the protein misfolding cyclic amplification (PMCA) assay. To do this, previous published methods were adapted to allow PMCA-induced protein fibrillization to occur under non-toxic conditions. Insight into potential intracellular targets of misfolded α-synuclein was obtained using an unbiased lipid screen of 15 biologically relevant lipids that identified cardiolipin (CA) as a potential binding partner for PMCA-generated misfolded α-synuclein. To investigate whether such an interaction can impact the properties of α-synuclein misfolding, protein fibrillization was carried out in the presence of the lipid. We show that CA both accelerates the rate of α-synuclein fibrillization and produces species that harbour enhanced resistance to proteolysis. Because CA is virtually exclusively expressed in the inner mitochondrial membrane, we then assessed the ability of these misfolded species to alter mitochondrial respiration in live non-transgenic SH-SY5Y neuroblastoma cells. Extensive analysis revealed that misfolded α-synuclein causes hyperactive mitochondrial respiration without causing any functional deficit. These data give strong support for the mitochondrion as a target for misfolded α-synuclein and reveal persistent, hyperactive respiration as a potential upstream pathogenic event associated with the synucleinopathies.This article has an associated First Person interview with the first author of the paper.