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Identification and validation of human proteins that control tau seeding in cell-based and in vivo models

Periodic Reporting for period 1 - Tau Seeding (Identification and validation of human proteins that control tau seeding in cell-based and in vivo models)

Reporting period: 2017-11-01 to 2019-10-31

A broad range of human diseases is known to be associated with aberrations in the process of proteins reaching or maintaining their functional three-dimensional structure or conformation. There are ~50 currently documented disorders with a multitude of symptoms that are associated with the misfolding (i.e. the loss of their functional conformation) of normally soluble, functional peptides that subsequently convert into intractable aggregates leading to disease onset. A group of neurodegenerative diseases known as tauopathies, including its most prevalent form Alzheimer’s disease (AD), are characterized by the misfolding and aggregation of the microtubule-associated protein tau. Although tau is natively unfolded and therefore has no fixed three-dimensional structure, under pathological conditions it forms well-ordered, periodic and β-sheet-rich aggregates, which are common to most neurological illnesses and are held responsible to drive disease progression through templated protein misfolding or ‘seeding’. Understanding the processes by which this protein transitions from a soluble, functional protein to pathogenic aggregates as well as how these initial aggregates are able to template and thereby propagate misfolding, are key to identifying the very fundamental disease mechanisms of this variety of dementias.

While increasing evidence is emerging that tau pathology progression is based on seeding and spreading mechanisms reminiscent of prion protein pathology, an in-depth understanding of the cellular pathways and cofactors that drive disease progression is, however, still lacking. In this project, we set out to identify tau seeding modulators by undertaking systematic investigations in mammalian cells deleting every human gene via CRISPR genome editing, and observing which ones affect the aggregation of tau. We found the cellular machinery for degrading unnecessary or dysfunctional components, called autophagy, to be highly relevant. To complete the project, we are currently using human cells and an in vivo fruit fly model to mechanistically unravel the interplay between tau seeing and processes of the autophagy machinery. Genes identified to robustly influence the formation or degradation of tau aggregates could be targeted for AD therapy.
In order to identify tau seeding modulators, a previously described HEK293T biosensor cell line was successfully applied, which co-expresses cyan- and yellow fluorescent protein (CFP- and YFP)-tagged tau repeat domain (TauRD, aa243-375) fusion proteins with the disease-associated mutation P301S. While TauRD fusion proteins are soluble in untreated cells, upon addition of preformed, fibrillar tau seeds, their co-aggregation is induced, which can be quantified by Förster resonance energy transfer (FRET). Previously, this FRET-based reporter assay was applied to quantify the abundance of seeding-competent tau aggregates in brain homogenates of P301S transgenic mice, suggesting that this assay can be utilized as a screening tool to identify proteins that modulate tau seeding. In this project, the established FRET-based biosensor assay was optimized to identify human proteins that influence tau seeded TauRD-CFP/YFP co-aggregation in HEK293T cells using a fluorescence-activated cell sorting (FACS)-based genome-wide loss-of-function CRISPR screen. After screening a library of sgRNAs targeting all genes of the human genome, a vast number of genes that enhanced and reduced tau seeding were identified. Among the top hits that influenced tau-seeded aggregation of the FRET reporter were many autophagy-related genes (ATGs). The genes identified suggest that perturbation of crucial early events in the autophagy pathway, namely the formation of autophagosomes, influences seeded TauRD-CFP/YFP co-aggregation. Next, building on the insights revealed by our CRISPR screen, we wanted to understand in mechanistic detail how the loss of function of the ATG genes impacts tau seeding. In order to address this question, we generated different isogenic knockout HEK293T cell lines expressing the TauRD-CFP/YFP reporter proteins using specific sgRNAs. The initial results suggest that the loss of these ATG genes is fundamental in prohibiting the fusion of relevant connector proteins with the autophagosome membrane, a step crucial for cellular debris, such as aggregates, to be taken up into autophagosomes and being degraded (see Summary Figure). The exact details of this process are still under investigation first in mammalian cells followed by transferring the findings to in vivo Drosophila models. Once the project is complete and the interplay between tau seeding and processes of the autophagy pathway has been unraveled in mechanistic detail, the results will be disseminated via publication. Exploitation will be pursued by sharing relevant target genes with industry partners as these modulators have high potential to be relevant for disease-modifying therapeutics, whose identification is an ambitious, long sought-after aim shared by researchers across the protein misfolding field.
In an ageing society, dementias are a major public health challenge. AD has been labelled the 21st century plague with worldwide 44 million affected, a number that is set to double by 2030. With global costs estimated to be on the order of US $600 billion, public health strategies to combat neurodegenerative diseases are a priority on the European political agenda and worldwide. Research strategies addressing tau in general, and tau seeding more specifically, are highly innovative and have enormous potential to provide disease-modifying first- in-class therapeutics for AD.

While it has been shown that tau aggregates are able to function as seeds which can induce aggregation of endogenous protein, the molecular pathways and co-factors that may promote or may be able to hinder this process are unknown, despite the enormous opportunity to capitalize on their potential to interfere with disease progression. The novel output of the action is the identification of target genes and molecular pathways, which will delineate causal relationships between modifiers and tau seeding. In our CRISPR screen we have uncovered autophagy to be the top modifying pathway of tau seeding and are currently in the process of understanding the full mechanistic details of how specific processes of the autophagy pathway are able to impact on tau seeding. In the longer term we are aiming to suggest new targets to industry partners, based on which new drug screens can be established. In the medium term, this study may lay the essential foundation to identify chemical compounds that slow or inhibit tau seeding.
Suggested pathogenic mechanism of compromised tau aggregate degradation in autophagy-impaired cells