Abstract
Alzheimer's disease (AD) afflicts millions of people worldwide and is caused by accumulated amyloid beta and tau pathology. Progression of tau pathology in AD may utilize prion mechanisms of propagation in which pathological tau aggregates released from one cell are taken up by neighboring or connected cells and act as templates for their own replication, a process termed 'seeding'. We have used HEK293T cells to model various aspects of pathological tau propagation, including uptake of tau aggregates, induced seeding by exogenous aggregates, seeding caused by Lipofectamine-mediated delivery to the cell interior, and stable maintenance of aggregates in dividing cells. The factors that regulate these processes are not well understood, and we hypothesized that AD risk modifier genes might play a role. We identified 22 genes strongly linked to AD via meta-analysis of genome-wide association study (GWAS). We used CRISPR/Cas9 to individually knock out each gene in HEK293T cells and verified disruption using genomic sequencing. We then tested the effect of gene knockout in tau aggregate uptake, naked and Lipofectamine-mediated seeding, and aggregate maintenance in these cultured cell lines. GWAS gene knockouts had no effect in these models of tau pathology. With obvious caveats due to the model systems used, these results imply that the 22 AD risk modifier genes are unlikely to directly modulate tau uptake, seeding, or aggregate maintenance in a cell-autonomous fashion.
Highlights
Tauopathies are neurodegenerative diseases characterized accumulation of tau protein in ordered assemblies
We first identified the candidate genes based on the reported genome-wide association studies (GWAS) of Alzheimer’s disease (AD) [14]
AD GWAS genes were individually targeted in HEK293T RD(P301S)-C/R biosensor cells using CRISPR/Cas9 to create polyclonal knockout cell lines, which were cultured for 2 weeks in the presence of puromycin
Summary
Tauopathies are neurodegenerative diseases characterized accumulation of tau protein in ordered assemblies. Concurrent work from the Tolnay group demonstrated that inoculation of mouse brain with tau aggregates induced local pathology in a transgenic mouse model[6] This led us initially to propose that tau had properties similar to the prion protein, PrP[7]. We propagated distinct tau strains in cultured cells that we used to create transmissible tauopathy in mouse models, with faithful, inter-animal propagation of defined pathology[7]. This was the first evidence that an infectious form of tau created in vitro, would faithfully transmit unique conformations between animals, and we referred to tau as a prion. We tested the impact of AD GWAS genes through systematic genetic knockout via CRISPR/Cas-9
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