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.
