P2-155: Phosphorylation of SC35 by Dyrk1A inhibits its promotion of tau exon 10 inclusion

T416

Poster Presentations P2:

is critical for the phosphorylation state of Ser 404. This kind of modification is not phosphorylation. Prephosphorylation of Ser 214, Ser262 and Ser409 can up-regulate phosphorylation level of Thr231. P2-155

PHOSPHORYLATION OF SC35 BY DYRK1A INHIBITS ITS PROMOTION OF TAU EXON 10 INCLUSION

Hongwei Liang1, Jianhua Shi1, Khalid Iqbal2, Inge Grundke-Iqbal2, Cheng-Xin Gong2, Fei Liu2, 1Neuroregeneration Key Laboratory of Jiangsu, Nantong, China; 2New York State Institute for Basic Research in Developmental Disability, New York, NY, USA. Contact e-mail: [email protected] Background: SC35 is a member of serine/arginine rich protein of splicing factors. It plays very important role in alternative splicing of pre-mRNAs. Tau is the major neuronal microtubule-associated protein. Its abnormally hyperphosphorylation and aggregation into neurofibrillary tangles is a characteristic of tauopathies, including Alzheimer’s disease. In adult human brain, six tau isoforms are expressed from a single gene by alternative splicing of exons 2, 3, and 10. Alternative splicing of exon 10 generates tau isoforms with either three or four microtubule-binding repeats (3R- or 4R-tau). Normal human expresses equal levels of 3R-tau and 4R-tau. Dysregulation of E10 10 causes tauopathies, such as frototemporal dementia with Parkinsonism linked with chromosome 17, progressive supranuclear palsy, Pick’s disease, and corticobasal degeneration, suggesting that the balance of 3R-tau and 4R-tau is critical for normal function of brain. Methods: We cotransfected mini-tau gene, consisting of exons 9, 10, 11, a part of intron 9 and full length of intron 10 with same amount of pCEP4/SC35 plasmids into HEK-293T cells and detected E10 splicing by using RT-PCR. To test whether SC35 is phosphorylated by PKA, we phosphorylated GST-SC35 in vitro by PKA. To map the phosphorylation sites, we constructed deletion mutations of SC35 and then phosphorylated them with PKA. To learn whether PKA affects the function of SC35 in tau E10 splicing, Results: we found SC35 significantly promoted tau E10 inclusion, resulting in an increase in 4R-tau production. Dyrk1A (dualspecificity tyrosine phosphorylation-regulated kinase 1A), coded by a gene located at the Down syndrome critical region, interacted with SC35 in vitro and in vivo and phosphorylated it efficiently. Overexpression of Dyrk1A in HEK-293T cells significant inhibited tau E10 inclusion promoted by SC35. Conclusions: In Down syndrome brain, Dyrk1A is overexpressed due to trisomy 21 and 3R/4R tau ratio is increased. Dyrk1A inhibits SC35promoted tau E10 inclusion by phosphorylating SC35. Imbalance of 3R-tau and 4R-tau induced by Dyrk1A overexpression might contribute to neurofibrillary degeneration in Down syndrome. P2-156

TAU OVEREXPRESSION INHIBITS APOPTOSIS INDUCED BY STAUROSPORINE AND THE UNDERLYING MECHANISMS 1

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Hai-Hong Wang , Jian-Zhi Wang , Southern Medical University, Guangzhou, China; 2Tongji Medical College, Wuhan, China. Contact e-mail: [email protected] Background: Neurofibrillary tangles (NFTs) in the brain neurons is one of the pathological hallmarks of Alzheimer’s disease (AD) and related tauopathies. It is little known why the NFTs-bearing neurons do not die of apoptosis but rather degeneration even though they are constantly exposed to viarious apoptotic stimuli in the brains of patients with AD and related tauopathies. To reveal the effect of tau protein on the anti-apoptosis of NFTs-bearing neurons and the mechanisms. Methods: Neuroblastoma 2a (N2a) cells were transiently transfected with tau or its vector as a control, and treated with staurosporine, an apoptosis inducer, after transfection for 48 hours. Apoptosis was assayed using an Annexin V- Propidium Iodide (PI) staining kit by following the manufacture’s procedure and apoptotic rate was automatically quantified by flow cytometry with the standardized program of the instrument. Double- or triple-labeling immunofluorescence showed the staining pattern of overexpressed EGFP-tau or hyperphosphor-

ylated tau and activated caspase-3 or fragmented nuclei. Western blotting was used to observed the related proteins level. Results: Here we report that cells overexpressing tau protein are more resistant to apoptosis induced by staurosporine. Inverse staining pattern of overexpressed EGFP-tau or hyperphosphorylated tau and activated caspase-3 or fragmented nuclei was observed in cells overexpressing tau, suggesting that tau’s anti-apoptotic effect involves its hyperphosphorylation. Furthermore, tau overexpression decreases the phosphorylation of p53 at Ser33 site and p53 level, arrests release of cytochrome C from mitochondrial and inhibits activity of caspases-9/-3. In addition, tau overexpression decreases phosphorylated ␤-catenin, increases ␤-catenin and nuclear translocation of ␤-catenin. Knock down of ␤-catenin antagonized the antiapoptotic effect of tau. Conclusions: These findings together reveal an anti-apoptotic function of tau which involves hyperphosphorylation of tau, p53-mediated pathway and stabilization of ␤-catenin. P2-157

SERINE422 PHOSPHORYLATION OF TAU PROTEIN BY OXIDATIVE STRESS VIA C-JUN N-TERMINAL KINASE (JNK) ACTIVATION

Nobuo Sanjo, Katsuyoshi Takahashi, Jin Haifeng, Mutsufusa Watanabe, Hidehiro Mizusawa, Tokyo Medical and Dental University, Tokyo, Japan. Contact e-mail: [email protected] Background: Mechanisms of tau hyperphosphorylation have not yet been well understood. In order to clarify mechanisms of tau protein abnormal phosphorylation, we have investigated quantitative changes of phosphorylated tau proteins and various kinases, such as JNK and glycogen synthase kinase-3beta (GSK-3beta), in culture cells under oxidative stress condition. Methods: Nickel chloride was added to culture media of 4 repeat tau protein overexpressing HEK293 cells to produce chemical oxidative stress condition, and then those cells were harvested at regular time intervals. Those samples were analyzed by western blotting, and were checked the quantitative changes of phosphorylated tau proteins and activated kineses. Results: Twenty-four hours after addition of nickel chloride, there was an increased activation of JNK and related kinases in the cell lysate, and at the same time, we detected an increased amount of phosphorylated serine422 in tau proteins. Conclusions: It is known that tau proteins are hyperphosphorylated by kinases which are activated under various stress conditions or an accumulation of amyloid proteins. Our results indicate JNK is one of important kinases for tau hyperphosphorylation under oxidative stress condition. P2-158

COENZYME Q10 ATTENUATES HYPERPHOSPHORYLATION OF TAU WITH UPREGULATION OF AKT SIGNALING IN THE AGED TRANSGENIC MICE WITH ALZHEIMER PRESENILIN 1 MUTATION

Xifei Yang1,2, Ying Yang2, Jing Gu1, Raymond Chuen-Chung Chang3, Geng Li1, Jianzhi Wang2, Edward S. Yang1, 1Jockey Club MRI Centre, The University of Hong Kong, Hong Kong, China; 2Department of Pathophysiology, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, China; 3Laboratory of Neurodegenerative Diseases, Department of Anatomy, The University of Hong Kong, Hong Kong, China. Contact e-mail: [email protected] Background: Alzheimer’s disease (AD) is characterized by ␤-amyloid (A␤) overproduction and tau hyperphosphorylation. Familial Alzheimer disease mutations of presenilin 1 (PS-1) enhance the generation of A␤42 and intracellular accumulation. However, the effect of PS-1 mutations on tau phosphorylation and the underlying mechanisms were poorly understood, and there is no treatment to arrest tau pathology. Methods: Western blot, Immunohistochemistry and SOD activity assay were performed in this study. Results: Tau phosphorylation at Ser396, Ser404 and Tau-1 (Ser198/ 199/202) epitopes was increased in hippocampus of the aged transgenic mice overexpressing Alzheimer presenilin 1-L235P(leucine-to-proline mu-