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1.  Global Analysis of Phosphorylation of Tau by the Checkpoint Kinases Chk1 and Chk2 in vitro 
Journal of proteome research  2013;12(6):2654-2665.
Hyperphosphorylation of microtubule-associated protein tau is thought to contribute to Alzheimer’s disease (AD) pathogenesis. We previously showed that DNA damage-activated cell cycle checkpoint kinases Chk1 and Chk2 phosphorylate tau at an AD-related site and enhance tau toxicity, suggesting potential roles of these kinases in AD. The purpose of this study is to systematically identify which sites in tau are directly phosphorylated by Chk1 and Chk2. Using recombinant human tau phosphorylated by Chk1 and Chk2 in vitro, we firstly analyzed tau phosphorylation at the AD-related sites by Western blot with phospho-tau-specific antibodies. Secondly, to globally identify phosphorylated sites in tau, liquid chromatography-tandem mass spectrometry (LC-MS/MS) was employed. These systematic analyses identified a total of 27 Ser/Thr residues as Chk1- or Chk2- target sites. None of them were proline-directed kinase targets. Many of these sites are located within the microtubule-binding domain and C-terminal domain, whose phosphorylation has been shown to reduce tau binding to microtubules and/or has been implicated in tau toxicity. Among these 27 sites, 13 sites have been identified to be phosphorylated in AD brains. Since DNA damage is accumulated in diseased brains, Chk1 and Chk2 may be involved in tau phosphorylation and toxicity in AD pathogenesis.
doi:10.1021/pr400008f
PMCID: PMC3757556  PMID: 23550703
Alzheimer’s disease; checkpoint kinase 1; checkpoint kinases 2; liquid chromatography; mass spectrometry; microtubule-associated protein tau; phosphorylation
2.  Loss of Axonal Mitochondria Promotes Tau-Mediated Neurodegeneration and Alzheimer's Disease–Related Tau Phosphorylation Via PAR-1 
PLoS Genetics  2012;8(8):e1002918.
Abnormal phosphorylation and toxicity of a microtubule-associated protein tau are involved in the pathogenesis of Alzheimer's disease (AD); however, what pathological conditions trigger tau abnormality in AD is not fully understood. A reduction in the number of mitochondria in the axon has been implicated in AD. In this study, we investigated whether and how loss of axonal mitochondria promotes tau phosphorylation and toxicity in vivo. Using transgenic Drosophila expressing human tau, we found that RNAi–mediated knockdown of milton or Miro, an adaptor protein essential for axonal transport of mitochondria, enhanced human tau-induced neurodegeneration. Tau phosphorylation at an AD–related site Ser262 increased with knockdown of milton or Miro; and partitioning defective-1 (PAR-1), the Drosophila homolog of mammalian microtubule affinity-regulating kinase, mediated this increase of tau phosphorylation. Tau phosphorylation at Ser262 has been reported to promote tau detachment from microtubules, and we found that the levels of microtubule-unbound free tau increased by milton knockdown. Blocking tau phosphorylation at Ser262 site by PAR-1 knockdown or by mutating the Ser262 site to unphosphorylatable alanine suppressed the enhancement of tau-induced neurodegeneration caused by milton knockdown. Furthermore, knockdown of milton or Miro increased the levels of active PAR-1. These results suggest that an increase in tau phosphorylation at Ser262 through PAR-1 contributes to tau-mediated neurodegeneration under a pathological condition in which axonal mitochondria is depleted. Intriguingly, we found that knockdown of milton or Miro alone caused late-onset neurodegeneration in the fly brain, and this neurodegeneration could be suppressed by knockdown of Drosophila tau or PAR-1. Our results suggest that loss of axonal mitochondria may play an important role in tau phosphorylation and toxicity in the pathogenesis of AD.
Author Summary
Abnormal phosphorylation and toxicity of a microtubule-associated protein tau are involved in the pathogenesis of Alzheimer's disease (AD). Tau is phosphorylated at multiple sites, and phosphorylation of tau regulates its microtubule binding and physiological functions such as regulation of microtubule stability. Abnormal phosphorylation of tau occurs in the AD brains and is thought to cause tau toxicity; however, what pathological conditions trigger abnormal phosphorylation and toxicity of tau in AD is not fully understood. Since a reduction in the number of mitochondria in the axon has been observed in the AD brains, we investigated whether and how loss of axonal mitochondria promotes tau phosphorylation and toxicity. Using transgenic flies expressing human tau, we found that knockdown of milton or Miro, an adaptor protein essential for axonal transport of mitochondria, enhanced human tau-induced neurodegeneration. This study demonstrates that loss of axonal mitochondria caused by milton knockdown increases tau phosphorylation at an AD–related site through partitioning defective-1 (PAR-1), promotes detachment of tau from microtubules, and enhances tau-mediated neurodegeneration. Our results suggest that loss of axonal mitochondria may play an important role in tau phosphorylation and toxicity in the pathogenesis of AD.
doi:10.1371/journal.pgen.1002918
PMCID: PMC3431335  PMID: 22952452

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