Hyperphosphorylation and aggregation of protein tau are typical for neurodegenerative tauopathies, including Alzheimer's disease (AD). We demonstrate here that human tau expressed in yeast acquired pathological phosphoepitopes, assumed a pathological conformation, and formed aggregates. These processes were modulated by yeast kinases Mds1 and Pho85, orthologues of GSK-3beta and cdk5, respectively. Surprisingly, inactivation of Pho85 increased phosphorylation of tau-4R, concomitant with increased conformational change defined by antibody MC1 and a 40-fold increase in aggregation. Soluble protein tau, purified from yeast lacking PHO85, spontaneously and rapidly formed tau filaments in vitro. Further fractionation of tau by anion-exchange chromatography yielded a hyperphosphorylated monomeric subfraction, termed hP-tau/MC1, with slow electrophoretic mobility and enriched with all major epitopes, including MC1. Isolated hP-tau/MC1 vastly accelerated in vitro aggregation of wild-type tau-4R, demonstrating its functional capacity to initiate aggregation, as well as its structural stability. Combined, this novel yeast model recapitulates hyperphosphorylation, conformation, and aggregation of protein tau, provides insight in molecular changes crucial in tauopathies, offers a source for isolation of modified protein tau, and has potential for identification of modulating compounds and genes.

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.

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.

Phosphorylation of Tau protein and binding to microtubules is complex in neurons and was therefore studied in the less complicatedmodelofhumanizedyeast.HumanTauwasreadilyphosphorylated at pathological epitopes, but in opposite directions regulated by kinases Mds1 and Pho85, orthologues of glycogen synthase kinase-3␤ and cdk5, respectively (1). We isolated recombinant Tau-4R and mutant Tau-P301L from wild type, ⌬mds1 and ⌬pho85 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.

Minimally invasive surgery is now a well established field in surgery but continuous efforts are made to reduce invasiveness even further. This paper proposes a novel concept of small-diameter multi-arm instrument for Single-Port Access Surgery. The concept introduces a novel combination of backbone and actuation principles in a macro-micro fashion to achieve an excellent decoupling of the triangulation platform (macro) and of the end-effectors (micro). Concentric tube robots are used for the triangulation platform, while compliant fluidic-actuated bending segments are used as end-effectors. The fluidic actuation is advantageous as it minimally interferes with the triangulation platform. The triangulation platform on the other hand provides a stable base for the end-effectors such that large distal actuation bandwidth can be achieved. A specific embodiment for Spina Bifida repair is developed and proposed. The surgical and technical requirements as well as the mechanical design are presented in details. A first prototype is built and characterization experiments are conducted to evaluate its performance.