Azorsa et al. BMC Genomics 2010, 11:25 http://www.biomedcentral.com/1471-2164/11/25 Page 6 of 10 Table 2 Candidate kinases affecting total tau and 12E8 tau expression Gene Name EIF2AK2 EIF2AK2 CDKL1 DCK DGKQ PFKFB3 ERK8 STK19 PRKG2 MAP2K1IP1 Cell Count 217 318 398 363 406 458 466 347 413 436 12EB Tau/Control Average St Dev p value 0.606 0.075 0.012 0.719 0.069 0.018 0.852 0.032 0.015 1.068 0.015 0.017 1.119 0.042 0.039 1.204 0.046 0.016 1.234 0.063 0.024 1.334 0.103 0.030 1.384 0.152 0.048 1.393 0.052 0.006 Average 0.647 0.706 0.761 0.701 0.805 0.813 0.807 0.654 0.721 0.831 Total Tau St Dev 0.102 0.082 0.085 0.051 0.059 0.029 0.009 0.115 0.107 0.048 p value 0.026 0.025 0.040 0.010 0.029 0.008 0.001 0.035 0.046 0.026 12E8 Tau/Total Tau Average St Dev p value 0.962 0.237 0.806 1.038 0.257 0.822 1.132 0.157 0.281 1.530 0.107 0.013 1.394 0.111 0.026 1.483 0.088 0.011 1.528 0.084 0.008 2.065 0.231 0.015 1.959 0.412 0.056 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 Table1. 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 immunofluores- cence screening assay, providing additional confirmation that the assay is likely to be underestimating the true magnitude of the effects of some gene candidates. Discussion 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 under- stand a critical component of tau pathology, and provide new targets for the discovery of tau-modifying AD treat- ments. 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 neuro- degenerative diseases. Imaging technology has only recently become available to enable these high-content analyses. However, with a persistent need for the identifi- cation 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 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.