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

High-content siRNA screen of the kinome

To identify the kinases that are important in AD relevant pathologic phosphorylation of tau protein, we developed a cell-based, high-throughput immunofluorescence assay for the rapid detection and quantitation of both total tau and 12E8 tau (pS262 and pS356) protein expression. The assay uses an H4 neuroglioma cell line engineered to overexpress four repeat tau (4R0N). We confirmed that the assay was sensitive to changes in both total tau protein levels and phosphorylated tau protein levels using siRNA directed at the tau (MAPT) mRNA (Figure 1A). Importantly, we are able to achieve >95% siRNA transfection efficiency with the H4-tau cell line as assessed by transfection with a lethal siRNA (data not shown). The accuracy of the immunofluorescence quantitation was confirmed by measuring the effects of the MAPT siRNA on total and phosphorylated tau protein levels using western analysis (Figure 1B). We next tested if reduced 12E8 tau levels could be detected using siRNA directed against microtubule affinity regulating kinase 2 (MARK2), which has previously been shown to phosphorylate tau protein on serine 262[19]. The MARK2 siRNA, which reduced MARK2 expression >95% at the mRNA level, led to a 26% reduction in 12E8 phosphorylated tau (p = 0.029 for three replicates) and to an insignificant change in total tau protein expression (Figure 2). This result demonstrates that this immunofluorescence assay can detect changes to phosphorylated tau protein independently of significant changes in total tau protein levels.

siRNA screening results

Following assay validation, we screened the validated kinome siRNA library (Qiagen) for kinases that affect tau phosphorylation status. Cataloging of kinases from the human genome has identified at least 518 kinases[23, 24]. This library contains siRNAs to 572 known and predicted kinases. Two siRNAs per target kinase were screened in triplicate for a total of 3,432 target siRNAs screened. Standard paired, two-tailed T-tests were used to determine significant effects relative to non-silencing siRNA controls, which were present in triplicate on each plate. All siRNA screening data are presented as Additional File 1.

Notably, the microtubule affinity regulating kinase 2 (MARK2) protein, which has been shown to phosphorylate serine 262 of tau protein[19], showed the largest reductions in the ratio of 12E8 tau/total tau when knocked down (69% of control; p = 0.012). This is comparable to the effects seen in our validation assays with MARK2 siRNA, suggesting that the screening assay is performing as expected. Because this kinase is already well known to be a serine 262 tau kinase, we did not confirm this hit via secondary measures. Rather, we chose to validate two additional candidates. These were A-kinase anchor protein 13 (AKAP13) and dual specificity tyrosine phosphorylation regulated kinase 1A (DYRK1A), which were selected both for the significance of the effect (based on p values for the ratio of 12E8/total tau) and for the known biology of these candidates (see Discussion). The gene encoding DYRK1A is located within the Down Syndrome critical region (DSCR) on chromosome 21. Screening results showed 12E8 tau levels to be reduced to 77 ± 5% of control (p = 0.017), total tau to be 85 ± 10% of control (p = 0.127), which lead to a significant reduction in the ratio of 12E8 tau/total tau to 86 ± 4% of control (p = 0.023; Table 1 and Figure 3A). We confirmed via western analyses that DYRK1A is required for efficient phosphorylation of 12E8 tau. We retransfected our cell line with siRNA targeting DYRK1A and quantitated effects on 12E8 tau and total tau levels via western (Figure 3B). Results confirmed that DYRK1A is required for maintaining normal levels of 12E8 tau. The DYRK1A siRNA reduced DYRK1A protein expression by over 60%, causing a nearly 40% reduction of 12E8 tau compared to the nonsilencing siRNA control. DYRK1A siRNA had no significant effect on total tau levels in this assay. Interestingly, the effects on tau phosphorylation are strikingly larger than observed in the fluorescence screening assay. Reasons for this are unclear, however it suggests that the screening assay may be underestimating the magnitude of effects on tau phosphorylation. Since DYRK1A is a proline- directed serine/threonine kinase and serine 262 is a non-proline directed site on tau, these effects of DYRK1A may result from indirect effects through additional kinases (see Discussion).

Tests of AKAP13 showed that this protein kinase A (PKA) associated protein is also involved in 12E8 tau phosphorylation. Screening results showed 12E8 tau levels to be reduced to 74 ± 7% (p = 0.023) of control non-silencing siRNA samples, total tau levels to be 93 ± 7% (p = 0.223) of control, leading to a reduction in the ratio of 12E8 tau/total tau to 79 ± 9% (p = 0.054) of control (Table 1 and Figure 4A). Because this phosphorylation profile resembled that of MARK2, a confirmed S262 tau kinase, we proceeded to confirm these effects via secondary measures. Western results demonstrated a role for AKAP13 in maintaining 12E8 tau phosphorylation levels. The AKAP13 siRNA reduced AKAP13 protein expression to undetectable levels (Figure 4B), causing a nearly 50% reduction of 12E8 tau. AKAP13 siRNA had no significant effect on total tau levels in this assay.

Some drosophila models have shown that increased tau expression can lead to neurodegeneration, albeit in the absence of NFT pathology[25]. Thus the potential for a tau therapeutic that affects both phosphorylated and total tau levels cannot at this point be diminished. We therefore performed a second analysis of the data to identify those kinases that significantly affected both 12E8 and total tau levels simultaneously. To identify those candidates, we pulled out kinases that had statistically significant effects on both total tau levels and 12E8 tau levels relative to controls, irrespective of the resulting effects on the ratio of 12E8 tau/total tau. These are presented in Table 2. Interestingly, two siRNAs to the eukaryotic translation initiation factor 2 α kinase 2 (EIF2AK2) gene significantly reduced 12E8 and total tau levels (Figure 5 and Table 2). These siRNAs reduced 12E8 tau levels to 61 ± 8% (p = 0.012) and 72 ± 7% (p = 0.018) of non-silencing siRNA controls and reduced total tau levels to 65 ± 10% (p = 0.026) and 71 ± 8% (p = 0.025) of control values, respectively. The comparable eductions of both phosphorylated and total tau levels led to no change in the ratio of 12E8 tau/total tau.

		12EB Tau/Control			Total Tau			12E8 Tau/Total Tau
Gene Name	Cell Count	Average	St Dev	p value	Average	St Dev	p value	Average	St Dev	p value
EIF2AK2	217	0.606	0.075	0.012	0.647	0.102	0.026	0.962	0.237	0.806
EIF2AK2	318	0.719	0.069	0.018	0.706	0.082	0.025	1.038	0.257	0.822
CDKL1	398	0.852	0.032	0.015	0.761	0.085	0.040	1.132	0.157	0.281
DCK	363	1.068	0.015	0.017	0.701	0.051	0.010	1.530	0.107	0.013
DGKQ	406	1.119	0.042	0.039	0.805	0.059	0.029	1.394	0.111	0.026
PFKFB3	458	1.204	0.046	0.016	0.813	0.029	0.008	1.483	0.088	0.011
ERK8	466	1.234	0.063	0.024	0.807	0.009	0.001	1.528	0.084	0.008
STK19	347	1.334	0.103	0.030	0.654	0.115	0.035	2.065	0.231	0.015
PRKG2	413	1.384	0.152	0.048	0.721	0.107	0.046	1.959	0.412	0.056
MAP2K1IP1	436	1.393	0.052	0.006	0.831	0.048	0.026	1.679	0.123	0.011

Kinases that affect bioth 12E8 tau and total tau levels. Shown are those kinases that affect total tau levels and 12E8 tau levels, irrespective of significant effects to the ratio of 12E8 tau/total tau. Columns are labeled as in Table 1.

To confirm these screening results for EIF2AK2, we retransfected our cell line with EIF2AK2 siRNA. Western blotting results confirmed that EIF2AK2 is required for maintaining normal levels of 12E8 tau and total tau protein. EIF2AK2 siRNA treatment reduced EIF2AK2 levels to 5% of control non-silencing siRNA, leading to a reduction of 12E8 tau levels to 14% of control non-silencing siRNA (Figure 5B). Total tau levels were reduced to 47% of control values. Again, this effect is strikingly larger than revealed in the immunofluorescence screening assay, providing additional confirmation that the assay is likely to be underestimating the true magnitude of the effects of some gene candidates.

High-throughput, high-content siRNA screening in neurodegenerative diseases

We report the combination of a high-throughput survey of the entire kinome with a new assay approach to understand a critical component of tau pathology, and provide new targets for the discovery of tau-modifying AD treatments. We have developed a sensitive and specific assay for the detection of hyperphosphorylated tau protein on an AD-relevant site. The assay is not only amenable to additional tau phosphorylation sites, but also to those identifiable cell-based endpoints related to other neurodegenerative diseases. Imaging technology has only recently become available to enable these high-content analyses. However, with a persistent need for the identification of novel drug targets to treat increasingly common neurodegenerative diseases, RNA interference screening technology now provides a promising approach.

EIF2AK2 and DYRK1A as important mediators of AD-related tau hyperphosphorylation

The cell line used in these studies overexpresses 4R0N tau protein. This is technically necessary to achieve levels of phosphorylated tau protein sufficient enough for detection of modulation of phosphorylation status. Because of this overexpression there is the possibility of creating a significant cytoplasmic pool of tau protein that is not bound to microtubules. This raises the possibility that some phospho-epitopes that are normally protected (via microtubule binding or interactions with other proteins at physiologic concentrations) may be exposed and phosphorylation pathways that do not normally regulate tau would have an effect in this assay. However, the identification of MARK2 as a significant pS262 tau phosphorylator (Table 1) suggests that tau regulatory pathways are functioning on the 12E8 epitope in this cell line. Nevertheless, the true applicability of the kinases that we identify here will emerge only after subsequent follow up studies in neuronal cell lines and in vivo in animal models of AD and of tauopathies.

Both the eukaryotic translation initiation factor 2 α kinase 2 gene (EIF2AK2) and the dual specificity tyrosine phosphorylation regulated kinase 1A gene (DYRK1A) have been implicated previously in neurodegeneration in AD. Polymorphisms within the eukaryotic translation initiation factor 2 α kinase 2 (EIF2AK2) gene have been genetically associated with AD[26]. In addition, EIF2AK2 has been shown to be activated in AD brain[27, 28] and has been implicated in neuronal apoptosis resulting from toxic β-amyloid peptides[28]. EIF2AK2 has also been implicated in the extrastriatal neurodegeneration of Parkinson's disease and Huntington's disease[29]. In our study, two siRNAs that reduced EIF2AK2 protein expression by 95% caused a significant reduction of pS262 tau levels and a lesser but significant reduction in total tau levels (Table 2). These findings suggest an important role for EIF2AK2 in the expression of total and 12E8 phosphorylated tau protein. Moreover, the comparable effects seen for total tau and 12E8 tau suggest the possibility that EIF2AK2 may be required for the maintenance of multiple forms of phosphorylated tau, in addition to 12E8 tau. Deciphering the mechanisms through which this regulation occurs will require additional experimentation.

Polymorphisms within the DYRK1A locus also have been associated with AD in some AD patient populations[30]. Moreover, DYRK1A has been implicated in phosphorylation of tau protein on threonine 212, serine 202, and serine 404[31, 32]. In this work we demonstrate a role for DYRK1A in the phosphorylation of tau protein on the 12E8 epitope, independent of significant effects on total tau protein expression. Interestingly, DYRK1A is a proline- directed serine/threonine kinase. Serine 262 is not a proline-directed phosphorylation site on tau. One possible scenario is that DYRK1A effects on S262 could occur through activities of additional kinases downstream of DYRK1A function. Presumably, silencing of those kinases would also affect 12E8 tau levels in our assay and strong candidates for downstream kinases through which DYRK1A may be signaling are those additional kinases in Table 1. Ongoing work is directed at identifying the additional signaling components involved in mediating the effects of DYRK1A on 12E8 tau phosphorylation and at determining if DYRK1A is capable of directly phosphorylating tau protein on serine 262/serine 356.

A possible role for AKAP13 in the regulation of tau phosphorylation

Although it is clear that AKAP13 has significant effects on 12E8 tau levels in our cell line, the mechanisms through which this protein may affect tau are unclear. The AKAP13 protein is an ~320 kD protein that functions as an anchor protein for the regulatory subunit of protein kinase A, effectively localizing the PKA holoenzyme to discrete locations within the cell. Protein kinase A has been reported to increase tau phosphorylation in vitro on the 12E8 epitope under some conditions, such as in response to the binding of α-synuclein to tau protein[33]. Additionally, AKAP13, and other members of the AKAP family, have a Dbl homology (DH) domain that functions as a guanine nucleotide exchange activation domain for the Rho/Rac family of GTP binding proteins. Thus, AKAP13 coordinates a Rho signaling pathway that ultimately leads to cytoskeletal reorganization. In addition, other AKAP family members have been implicated in regulating synaptic plasticity and long-term memory formation[34, 35]. These combined observations suggest interesting possibilities wherein AKAP13 could anchor a signaling complex at the cell membrane that ultimately regulates tau phosphorylation and microtubule dynamics in response to extracellular stimuli. However, direct connections to tau phosphorylation based on the currently known functions of AKAP13 are unclear but certainly worthy of further study.

Generation of 4R0N tau overexpressing H4 cell line

The Human H4 neuroglioma cell line (ATCC) was transfected with a 4R0N tau construct in pcDNA3.1. Following positive selection using geneticin (Invitrogen), individual clones were isolated and screened for tau expression using immunocytochemistry with a rabbit anti-tau antibody (Dako). A single stable tau overexpressing cell line was selected for subsequent siRNA studies. Cells were maintained in Dulbecco's Modified Eagle Medium (Invitrogen) supplemented with 10% fetal bovine serum (Invitrogen), 1% penicillin-streptomycin, geneticin (0.25 mg/ml), and 2 mM L-Glutamine (Invitrogen). Cells were split 1:10 at 90% confluency, twice a week.

High Throughput siRNA Kinome Screen