Phosphorylation of p66Shc and forkhead proteins mediates Aß toxicity

Wanli W. Smith¹, Darrell D. Norton¹, Myriam Gorospe¹, Haibing Jiang², Shino Nemoto³, Nikki J. Holbrook⁵, Toren Finkel³, , and John W. Kusiak⁴

¹ Molecular Neurobiology Unit, Laboratory of Cellular and Molecular Biology, Intramural Research Program, National Institute on Aging, National Institutes of Health, Baltimore, MD 21224
² Johns Hopkins University School of Medicine, Baltimore MD 21205
³ National Heart, Lung and Blood Institute
⁴ Molecular and Cellular Neurobiology Program, National Institute of Dental and Craniofacial Research, National Institutes of Health, Bethesda, MD 20892
⁵ Yale University School of Medicine, New Haven, CT 06520

Correspondence to Wanli W. Smith: wsmith60{at}jhmi.edu

Abstract: Excessive accumulation of amyloid ß-peptide (Aß) plays an early and critical role in synapse and neuronal loss in Alzheimer's Disease (AD). Increased oxidative stress is one of the mechanisms whereby Aß induces neuronal death. Given the lessened susceptibility to oxidative stress exhibited by mice lacking p66Shc, we investigated the role of p66Shc in Aß toxicity. Treatment of cells and primary neuronal cultures with Aß caused apoptotic death and induced p66Shc phosphorylation at Ser36. Ectopic expression of a dominant-negative SEK1 mutant or chemical JNK inhibition reduced Aß-induced JNK activation and p66Shc phosphorylation (Ser36), suggesting that JNK phosphorylates p66Shc. Aß induced the phosphorylation and hence inactivation of forkhead transcription factors in a p66Shc-dependent manner. Ectopic expression of p66ShcS36A or antioxidant treatment protected cells against Aß-induced death and reduced forkhead phosphorylation, suggesting that p66Shc phosphorylation critically influences the redox regulation of forkhead proteins and underlies Aß toxicity. These findings underscore the potential usefulness of JNK, p66Shc, and forkhead proteins as therapeutic targets for AD.

W.W. Smith's present address is Dept. of Psychiatry, Division of Neurobiology, Johns Hopkins University School of Medicine, Baltimore, MD 21205.

Abbreviations used in this paper: Aß, amyloid ß-peptide; AD, Alzheimer's Disease; DCF, 2',7'-dichlorofluorescein; DCFDA, DCF diacetate; DHR, dihydrorhodamine; DIV, day in vitro; FKH, forkhead transcription factor; HE, hydroethidine; MnSOD, manganese superoxide dismutase; NAC, N-acetylcysteine; PP2A1, serine/threonine protein phosphatase 2A1; ROS, reactive oxygen species.

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