High-content siRNA screening of the kinome identifies kinases involved in Alzheimer's disease-related tau hyperphosphorylation

Abstract

Background: Neurofibrillary tangles (NFT), a cardinal neuropathological feature of Alzheimer's disease (AD) that is highly correlated with synaptic loss and dementia severity, appear to be partly attributable to increased phosphorylation of the microtubule stabilizing protein tau at certain AD-related residues. Identifying the kinases involved in the pathologic phosphorylation of tau may provide targets at which to aim new AD-modifying treatments.

Results: We report results from a screen of 572 kinases in the human genome for effects on tau hyperphosphorylation using a loss of function, high-throughput RNAi approach. We confirm effects of three kinases from this screen, the eukaryotic translation initiation factor 2 alpha kinase 2 (EIF2AK2), the dual-specificity tyrosine-(Y)-phosphorylation regulated kinase 1A (DYRK1A), and the A-kinase anchor protein 13 (AKAP13) on tau phosphorylation at the 12E8 epitope (serine 262/serine 356). We provide evidence that EIF2AK2 effects may result from effects on tau protein expression, whereas DYRK1A and AKAP13 are likely more specifically involved in tau phosphorylation pathways.

Conclusions: These findings identify novel kinases that phosphorylate tau protein and provide a valuable reference data set describing the kinases involved in phosphorylating tau at an AD-relevant epitope.

Figure 1

Quantitative immunofluorescence detection of reductions in 12E8 tau and total tau protein levels using MAPT siRNA. Shown in A is reduction of both pS262 tau and total tau levels using an siRNA directed at the tau transcript (all splice variants). Using this siRNA, pS262 tau and total tau levels are reduced to 45% and 54% of control values, respectively, following 4 days of transfection with siRNA. pS262 tau is visualized in red. Total tau is in green. Blue staining represents DAPI stained nuclei. Note that significant total tau expression remains following siRNA treatment, likely resulting from the extremely high constitutive levels of tau expression in this cell line. Shown in B is secondary quantitation of the MAPT siRNA effects on 12E8 and total tau using western analyses. Results support the immunofluorescence quantitation shown in A. Representative images are shown. Percent control numbers (± SD) in A represent the average of three independent assays. Westerns in B were performed in triplicate. NS refers to the non-silencing siRNA control.

Figure 2

Quantitative reduction of 12E8 tau with siRNA against MARK2. Shown in A is detection of pS262 tau and total tau levels following transfection with MARK2 siRNA. Our quantitative assay detects significant reduction of pS262 tau using siRNA directed at the microtubule affinity regulating kinase 2 (MARK2) gene (red panels; p = 0.029 for three replicates). MARK2 siRNA has no effect on the overall levels of total tau protein (green panels), demonstrating that this assay can specifically detect reductions in pS262 tau. Representative images are shown. Percent control numbers represent the average of three independent assays. NS refers to the non-silencing siRNA control. In B, qRT-PCR results confirmed >95% knockdown of MARK2 mRNA relative to non-silencing siRNA control (NS).

Figure 3

DYRK1A is required for efficient phosphorylation of 12E8 tau. Shown in A are results of siRNA screening for DYRK1A siRNA. 12E8 tau is shown in red and total tau is shown in green (see Experimental Methods). For B, H4 cells overexpressing four repeat tau (4R0N) were transfected with siRNA targeting the DYRK1A transcript. Silencing of DYRK1A was confirmed with anti-DYRK1A antibody (top panel). Percent control values represent the average of three independent siRNA transfections and westerns. In A and B, NS refers to the nonsilencing control.

Figure 4

AKAP13 is required for efficient phosphorylation of 12E8 tau. Shown in A are results of siRNA screening for AKAP13 siRNA. 12E8 tau is shown in red and total tau is shown in green (see Experimental Methods). For B, H4 cells overexpressing four repeat tau (4R0N) were transfected with siRNA targeting the AKAP13 transcript. Silencing of AKAP13 was confirmed with anti-AKAP13 antibody (top panel). Percent control values represent the average of three independent siRNA transfections and westerns shown. In A and B, NS refers to the non-silencing control.

Figure 5

EIF2AK2 is required for maintenance of normal 12E8 tau levels and total tau expression. Shown in A is the kinome library screening result for EIF2AK2 siRNA. Effects on 12E8 tau (red) and total tau (green) are shown to the right of each image. In B, triplicate western analyses confirm that EIF2AK2 is required for maintenance of tau and 12E8 tau levels. The EIF2AK2 siRNA reduced expression of EIF2AK2 by 95%, resulting in an 85% reduction of 12E8 tau and a greater than 50% reduction of total tau levels. NS is the non-silencing control siRNA.