CRISPRi screening reveals regulators of tau pathology shared between exosomal and vesicle-free tau

The aggregation of the microtubule-associated protein tau is a defining feature of Alzheimer’s disease and other tauopathies. Tau pathology is believed to be driven by free tau aggregates and tau carried within exosome-like extracellular vesicles, both of which propagate trans-synaptically and induce tau pathology in recipient neurons by a corrupting process of seeding.

Researchers at the University of Queensland performed a genome-wide CRISPRi screen in tau biosensor cells and identified cellular regulators shared by both mechanisms of tau seeding. The researchers identified ANKLE2, BANF1, NUSAP1, EIF1AD, and VPS18 as the top validated regulators that restrict tau aggregation initiated by both exosomal and vesicle-free tau seeds. None of their validated hits affected the uptake of either form of tau seeds, supporting the notion that they operate through a cell-autonomous mechanism downstream of the seed uptake. Lastly, validation studies with human brain tissue also revealed that several of the identified protein hits are down-regulated in the brains of Alzheimer’s patients, suggesting that their decreased activity may be required for the emergence or progression of tau pathology in the human brain.

Functional validation of bioinformatic hits using individual CRISPRi knockdowns followed by incubation with exosome-like EVs and vesicle-free tau seeds

(A) Schematic representation of the workflow for functional validation. Individual sgRNAs were used to silence the corresponding genes in combination with KRAB-dCas9 in tau biosensor cells (BSKRAB-KD), then treated with either sarkosyl-insoluble tau (vesicle-free tau seeds) or exosome-like EVs for 72 h, followed by detection and quantification of tau aggregation using FRET flow cytometry. A fraction of the same BSKRAB-KD cells was also grown for 72 h to corroborate the knockdown of protein expression using Western blots. (B, C, D, E, F, G) Integrated FRET intensities represent levels of tau aggregation upon knocking down the different targets. The control black column (NT) is the average obtained with three independent non-targeting sgRNAs (n = 3) assessed in triplicate. Control cells were compared with knockdown cells targeted individually (1, 2, and 3). Error bars represent the SEM for n = 3, *P < 0.05; **P < 0.01; ***P < 0.001; and ****P < 0.0001. Each single targeting sgRNA increased tau aggregation with both exosomal and vesicle-free tau seeds. (C, D) Interestingly, ANKLE2 and BANF1 (C, D) appear to induce a stronger effect on tau aggregation induced by exosome-like EVs. (H) Quantitative Western blot analysis of BSKRAB-KD knockdown cells. Each sgRNA generated a protein knockdown of the targeted gene. Note that the ANKLE2-specific antibody reacted with several isoforms, including the canonical variant sized 104–117 kD (red box outline); however, all isoforms were down-regulated when the ANKLE2 locus was silenced. Similarly, the EIF1AD antibody recognized the canonical isoform of 19 kD and one additional variant of lower molecular weight, both being silenced with the individual sgRNA against EIF1AD. (I) Quantification of the extent of protein knockdown for the different targeted genes. Error bars represent the SEM for n = 3, *P < 0.05; **P < 0.01; and ****P < 0.0001.