T. Vandebroek

Characterization of α-synuclein aggregation and synergistic toxicity with protein tau in yeast

A yeast model was generated to study the mechanisms and phenotypical repercussions of expression of α‐synuclein as well as the coexpression of protein tau. The data show that aggregation of α‐synuclein is a nucleation–elongation process initiated at the plasma membrane. Aggregation is consistently enhanced by dimethyl sulfoxide, which is known to increase the level of phospholipids and membranes in yeast cells. Aggregation of α‐synuclein was also triggered by treatment of the yeast cells with ferrous ions, which are known to increase oxidative stress. In addition, data are presented in support of the hypothesis that degradation of α‐synuclein occurs via autophagy and proteasomes and that aggregation of α‐synuclein disturbs endocytosis. Reminiscent of observations in double‐transgenic mice, coexpression of α‐synuclein and protein tau in yeast cells is synergistically toxic, as exemplified by inhibition of proliferation. Taken together, the data show that these yeast models recapitulate major aspects of α‐synuclein aggregation and cytotoxicity, and offer great potential for defining the underlying mechanisms of toxicity and synergistic actions of α‐synuclein and protein tau.

Microtubule Binding and Clustering of Human Tau-4R and Tau-P301L Proteins Isolated from Yeast Deficient in Orthologues of Glycogen Synthase Kinase-3β or cdk5

Phosphorylation of Tau protein and binding to microtubules is complex in neurons and was therefore studied in the less complicated model of humanized yeast. Human Tau was readily phosphorylated at pathological epitopes, but in opposite directions regulated by kinases Mds1 and Pho85, orthologues of glycogen synthase kinase-3β and cdk5, respectively (1). We isolated recombinant Tau-4R and mutant Tau-P301L from wild type, Δmds1 and Δpho85 yeast strains and measured binding to Taxol-stabilized mammalian microtubules in relation to their phosphorylation patterns. Tau-4R isolated from yeast lacking mds1 was less phosphorylated and bound more to microtubules than Tau-4R isolated from wild type yeast. Paradoxically, phosphorylation of Tau-4R isolated from kinase Pho85-deficient yeast was dramatically increased resulting in very poor binding to microtubules. Dephosphorylation promoted binding to microtubules to uniform high levels, excluding other modifications. Isolated hyperphosphorylated, conformationally altered Tau-4R completely failed to bind microtubules. In parallel to Tau-4R, we expressed, isolated, and analyzed mutant Tau-P301L. Total dephosphorylated Tau-4R and Tau-P301L bound to microtubules very similarly. Surprisingly, Tau-P301L isolated from all yeast strains bound to microtubules more extensively than Tau-4R. Atomic force microscopy demonstrated, however, that the high apparent binding of Tau-P301L was due to aggregation on the microtubules, causing their deformation and bundling. Our data explain the pathological presence of granular Tau aggregates in neuronal processes in tauopathies.

Serine‐409 phosphorylation and oxidative damage define aggregation of human protein tau in yeast

Unraveling the biochemical and genetic alterations that control the aggregation of protein tau is crucial to understand the etiology of tau-related neurodegenerative disorders. We expressed wild type and six clinical frontotemporal dementia with parkinsonism (FTDP) mutants of human protein tau in wild-type yeast cells and cells lacking Mds1 or Pho85, the respective orthologues of the tau kinases GSK3β and cdk5. We compared tau phosphorylation with the levels of sarkosyl-insoluble tau (SinT), as a measure for tau aggregation. The deficiency of Pho85 enhanced significantly the phosphorylation of serine-409 (S409) in all tau mutants, which coincided with marked increases in SinT levels. FTDP mutants tau-P301L and tau-R406W were least phosphorylated at S409 and produced the lowest levels of SinT, indicating that S409 phosphorylation is a direct determinant for tau aggregation. This finding was substantiated by the synthetic tau-S409A mutant that failed to produce significant amounts of SinT, while its pseudophosphorylated counterpart tau-S409E yielded SinT levels higher than or comparable to wild-type tau. Furthermore, S409 phosphorylation reduced the binding of protein tau to preformed microtubules. The highest SinT levels were found in yeast cells subjected to oxidative stress and with mitochondrial dysfunction. Under these conditions, the aggregation of tau was enhanced although the protein is less phosphorylated, suggesting that additional mechanisms are involved. Our results validate yeast as a prime model to identify the genetic and biochemical factors that contribute to the pathophysiology of human tau.

P2-033: Molecular determinants of the phosphorylation and aggregation of human protein tau in a yeast model

aim of this study was to elucidate functional properties of IMPAS proteins. Methods: We applied mammalian cell culture proteolytic assays and soil nematode C.elegans model. Results and Conclusions: Three IMPAS genes in C.elegans (Ce-imp-1, Ce-imp-2, Ce-imp-3) and five paralogous human genes genes (IMP1-IMP5) were cloned and studied. Based on original alignment comparisons we predicted that human hIMP1/SPP and Ce-imp-2 are structural and functional orthologs. Inhibition of Ce-imp-2 gene by dsRNAi or in knockout mutant strains resulted in slow growth, uncoordinated movement, reduced brood size and larval death due to the incomplete shedding of cuticle (ICS) in the animals. The progeny of heterozygous deletion mutant Ce-imp-2 was analyzed by single worm PCR. The data demonstrated that homozygous mutants showed development pathology of late embryo death and ISC defects that is unrelated to Notch defects described for presenilin (sel-12, hop-1) mutants. This specific phenotype mimics features observed in worms with low function of megalin gene or on cholesterol diet. Inhibition of other IMPAS homologues in C.elegans (Ce-imp-1, Ce-imp-3) did not result in any obvious phenotype. We revealed that in co-expressing experiments with PS1 both human IMP1 and Ce-imp-2 were capable to cleave C-terminal part of PS1 in transmembrane domain. The data indicate that Ce-imp-2 is likely orthologous gene to hIMP1/SPP with similar proteolytic functions. The IMP1/Ce-imp-2induced and PS1-induced proteolytic cleavage assays were used to delineate essential domains and amino-acid signatures important for enzymatic or conformation properties critical for proteolytic functions of two types of intramembrane proteases (IMPAS and presenilin families).