Genome-wide CRISPR screen identifies protein pathways modulating tau protein levels in neurons

Abstract

Aggregates of hyperphosphorylated tau protein are a pathological hallmark of more than 20 distinct neurodegenerative diseases, including Alzheimer's disease, progressive supranuclear palsy, and frontotemporal dementia. While the exact mechanism of tau aggregation is unknown, the accumulation of aggregates correlates with disease progression. Here we report a genome-wide CRISPR screen to identify modulators of endogenous tau protein for the first time. Primary screens performed in SH-SY5Y cells, identified positive and negative regulators of tau protein levels. Hit validation of the top 43 candidate genes was performed using Ngn2-induced human cortical excitatory neurons. Using this approach, genes and pathways involved in modulation of endogenous tau levels were identified, including chromatin modifying enzymes, neddylation and ubiquitin pathway members, and components of the mTOR pathway. TSC1, a critical component of the mTOR pathway, was further validated in vivo, demonstrating the relevance of this screening strategy. These findings may have implications for treating neurodegenerative diseases in the future.

Fig. 1. Characterization of SH-SHY5Y cell lines expressing Cas9.

a Schematic representation of primary genome-wide screen performed in SH-SY5Y cells expressing Cas9. b Immunocytochemistry of endogenous tau (SP70), Cas9 and FLAG proteins in parental and stable cell lines expressing Cas9. Pooled cells (PC1) and single clones (SC2) are depicted. Scale bar represents 50 µm. c Tau (SP70) and Cas9 western blot analysis on SH-SH5Y/Cas9 (PC1) cells infected with control or MAPT gRNAs for 14, 21, or 30 days of editing. d Immunocytochemistry of endogenous tau in SH-SH5Y/Cas9 (PC1) cells infected with control or MAPT gRNAs for 7 days of editing. Scale bars represent 200 µm. Data is represented as mean fluorescence intensity ± SEM. Control is a non-target gRNA.

Fig. 2. Gene knockouts upregulating and downregulating tau protein in SH-SY5Y cells after CRISPR editing.

a, b Scatter plots with RSA scores (Y-axis) vs. z-score quartiles (X-axis), for 30 day edited group from primary genome-wide screen. Plots shows genes downregulating (a) and upregulating (b) tau protein levels. Highly significant candidate genes are labeled. c Heat maps representing candidate genes with an RSA score ≤−3.0 in primary genome-wide screens (21 and 30 day time points). d, e STRING enrichment pathways for highly validated genes (primary and mini array screens). Links between nodes are highly confident connections. d Pathways identified for genes downregulating tau protein levels. e Pathways identified for genes upregulating tau protein levels. KO knockout, Q quartile, Dn down, RSA redundant siRNA activity score.

Fig. 3. Candidate genes validated in a human neurons.

a Immunocytochemistry of endogenous tau (SP70) and Cas9 in iNgn2 neuronal cultures 14 days of post-differentiation. Scale bar represents 1000 µm. b Schematic representation of secondary well-based screen in human neurons. c Heat maps showing top 44 genes candidate genes with an RSA score ≤−5.0. Twenty-one downregulating genes (left) and 23 upregulating genes (right). d Quantitative analysis of tau protein levels in neurons following treatment with individual gRNAs. Gene knockouts downregulating tau (blue) and gene knockouts upregulating tau (green). Data is represented as fold change of tau protein levels compared to non-targeting control gRNAs ± SEM for seven biological replicates. e Z-score analysis on fold change for individually tested gRNAs, for gene knockouts downregulating tau (blue) or gene knockouts upregulating tau (green). Validated genes 1 standard deviation above or below the mean are labeled. KO knockout, DIV days in vitro, hESC human embryonic stem cells, Control is a non-target gRNA.

Fig. 4. TSC1 knockout in mice increased tau protein levels.

a Phosphorylation of S6 (Serine 240/244) levels at postnatal day 30 (p30, squares) and postnatal day 38 (p38, triangles) compared to aged matched Cre- control mice (circles). Western blot data is represented as mean ± SEM, as the percentage of control animals. Each data point represents an individual animal. b Total tau levels at p30 (squares) and p38 (triangles) compared to aged matched Cre- control mice (circles). Data is represented as ng of tau protein/mg total protein ± SEM. One-way ANOVA was performed; ****p < 0.0001 as compared to control. Each data point represents an individual animal.