Periodic Reporting for period 1 - REVERSING TAUOPATHY (Identifying and deciphering the mechanism of the cellular machinery responsible for disaggregation of intracellular prion-like Tau aggregates) Periodo di rendicontazione: 2017-06-01 al 2019-05-31 Sintesi del contesto e degli obiettivi generali del progetto Protein aggregation is a phenomenon which results due to inadequacy of the cellular machinery to deal with misfolded or non-native proteins. It has been found to be associated with a number of human pathological disorders including neurodegenerative diseases such as Alzheimer’s, Parkinson’s, Huntington’s, and Prion diseases. Aggregation of the neuronal protein Tau results in the formation of fibrillar, rod-like structures (known as amyloid fibrils) in the brain, a phenomenon that has been implicated in a number of neurodegenerative diseases (tauopathies), including Alzheimer’s disease, frontotemporal dementia, progressive supranuclear palsy, Pick disease, primary age related tauopathy, and corticobasal degeneration. According to the World Health Organization, more than 47 million people worldwide are affected by dementia, with Alzheimer’s disease being the most common and widespread among them (60-70%). In this study, we were able to identify the conditions wherein intracellular Tau aggregates were found to be disassembled or disaggregated. Moreover, we were able to pinpoint the cellular machinery responsible for the disaggregation process. This study provides an opportunity to design therapeutic strategies for neurodegeneration affecting the disaggregation machinery for improved aggregate clearance.Inside cells, protein misfolding is an unfavorable event that can result in the loss of biological activity or the formation of soluble and insoluble aggregates, the latter being associated with toxic effects underlying a number of human pathological disorders. Cells have mechanisms in place to prevent these aggregates from forming, either by preventing protein misfolding with molecular chaperones or by degrading misfolded proteins via proteolytic machineries. However, cellular machinery very frequently cannot take care of misfolded proteins, leading to their aggregation and accumulation in cells and tissues. Such protein aggregation has been implicated in a number of human pathological disorders including neurodegenerative diseases. Aggregation of the protein Tau has been implicated in Alzheimer’s disease, frontotemporal dementia and other pathologies referred to as tauopathies. Despite major research efforts world-wide, the mechanism of Tau aggregation and its pathogenesis giving rise to disease is not yet understood. Therapeutics to cure these tauopathies, including Alzheimer’s disease, are urgently needed. In our study, we were able to identify the conditions wherein intracellular Tau aggregates were found to be disassembled or disaggregated. Moreover, we were able to pinpoint the cellular machinery responsible for the disaggregation process. Lavoro eseguito dall’inizio del progetto fino alla fine del periodo coperto dalla relazione e principali risultati finora ottenuti Initially, conditions were identified for disaggregation of Tau aggregates in mammalian cell model expressing a fragment of Tau. There were two main conditions identified wherein the aggregates were disassembled:1. Starvation of the cellular model using EBSS (Earle's Balanced Salt Solution) medium which contains only the essential salts and glucose and is devoid of any essential or non-essential amino acids (Figure 1A). The aggregates appeared to disassemble after 24 hours of the starvation period.2. Turning off the expression of the protein Tau in the cellular model also resulted in the disappearance of the aggregates (Figure 1B). However, it must be mentioned that the aggregates are difficult to disassemble and therefore it was surprising to observe the disaggregation of the aggregates even after switching off the expression of the monomeric protein.Identification of the conditions where we could achieve the disassembly of intracellular aggregates provided us with the opportunity to study the system in detail and identify the cellular machinery responsible for the disaggregation process. Therefore, our first approach was to carry out the interactome analysis of intracellular Tau aggregates. For this purpose, we utilized the approach of immune precipitation followed by SILAC (stable isotope labeling with amino acids in cell culture) based mass spectrometery. A few chaperones as well as a hexameric AAA+ ATPase involved in the unfolding of substrates known as p97 or VCP (valosin containing protein) were identified as aggregate interactors in our studies. Our results demonstrated that VCP is the main component involved in the initial disaggregation of large aggregates of protein Tau in the cellular model.This study which was carried out in the Hartl laboratory was presented to a wider scientific and non-scientific public in the Open House Day of the Max Planck Institutes in Martinsried on November 17th, 2018. Approximately 4500 visitors were welcomed to our institute with lectures and guided tours through the laboratories. Apart from this broad level presentation, the results of the project “Reversing Tauopathy” were also presented as a poster at an international scientific meeting, the EMBO workshop on “Protein quality control: From mechanisms to disease” on April/May 2019. A manuscript describing these results is in preparation for publication in a widely read journal with open access. Progressi oltre lo stato dell’arte e potenziale impatto previsto (incluso l’impatto socioeconomico e le implicazioni sociali più ampie del progetto fino ad ora) The current project provided a detailed understanding of the cellular components which can disaggregate preformed Tau aggregates. As mentioned above, aggregation of Tau in neuronal cells in the brain is associated with several neurodegenerative diseases, including Alzheimer’s disease and other tauopathies. According to the World Health Organization, more than 47 million people worldwide are affected by dementia, with Alzheimer’s disease being the most common and widespread among them (60-70%). Despite major research efforts, the mechanism of Tau aggregation and its pathogenic consequences are not yet understood. Therapeutics to cure these tauopathies, including Alzheimer’s disease, are urgently needed. In our study, we were able to identify the conditions and cellular components involved in the disassembly of Tau aggregates. Therefore, the focus of the current project on neurodegenerative diseases, such as Alzheimer’s disease, and specifically the role of the Tau protein and the cellular components involved in its disaggregation, will contribute towards finding a cure for these diseases.