- Email: [email protected]
O3-03-01
S56
Oral O3-03: Disease Mechanisms (Presenilins/␥-Secretase) 2
of the Akt pathway in AD, and also examined IGF-1 (IGF-1R) and insulin receptors (IR), major Akt-activating receptors in brain. Results discovered a disease-stage related loss of Akt containing neurons selective to areas of the brain that degenerate in AD. This was detected together with a significantly increased Akt activity in AD, and hyperphosphorylation of many Akt downstream targets, including GSK3, tau, mTOR and p27kip1. Hyperphosphorylation of tau occurs at Ser214, an optimal consensus sequence for Akt, important for PHF formation, linking Akt activation to tau pathology. Mechanistically, increased Akt activation may be due to a significant loss of PTEN, and its altered subcellular localization, which was detected in neurons that degenerate in AD. We also discovered key defects in IGF-1R and IR, upstream to Akt signalling, indicating impaired normal growth factor activation of Akt in AD. This included a significant loss of IRS1/2, key adaptor proteins for IR and IGF-1R-activation of PI3-kinase, and loss of p85␣ and p100␣ subunits of PI3-kinase. Importantly, in other systems downregulation of IRS1/2 is a feedback response to heightened Akt activity, with resultant inhibition of normal receptor responses through PI3kinase-Akt. Further work detected more pronounced defects in IGF-1R status in AD compared to IR, including increased IGF-1R levels in glia, and within and surrounding A plaques. Together results indicate that Akt is hyper-activated in AD neurons, and that loss of normal regulated Akt target phosphorylation in AD may link to loss of Akt neurons, and impaired normal growth factor and insulin responses through this pathway in AD. O3-02-08
THE PHOSPHORYLATION STATE OF TYR687 ON APP CONTRIBUTES TO PROTEIN-PROTEIN INTERACTIONS AND ABETA PRODUCTION
Sandra Rebelo1, Sara S. Domingues1, Sandra I. Vieira1, Hermann Esselman2, Jens Wiltfang2, Edgar F. Cruz e Silva3, Odete A. da Cruz e Silva1, 1Laborato´rio de Neurocieˆncias, Centro de Biologia Celular, Universidade de Aveiro, Portugal; 2Erlangen University, Groningen, Germany; 3Laborato´rio de Transduc¸a˜o de Sinais, Centro de Biologia Celular, Universidade de Aveiro, Portugal. Background: The Alzheimer’s amyloid precursor protein (APP) is a transmembrane protein whose cytoplasmic domain contains several phosphorylatable residues. Objective: We have addressed the role of Tyr687 phosphorylation on APP metabolism, and the production of different cleavage products including Abeta. Methods: Constitutive phosphorylation or dephosphorylation of APP was mimicked by mutating Tyr687 to Glu (Y687E) or Phe (Y687F), respectively. In order to aid visualization GFP-fusion constructs were prepared. Results: Our results show that the phosphorylation state of Tyr687 is crucial in determining the fate of APP. Y687E-APP-GFP was delayed in the TGN, co-localized more to the ER and was distributed throughout the cell, apparently in a non-vesicular manner. In marked contrast, Y687FAPP-GFP was readily incorporated into vesicles. Expression of Y687EAPP-GFP resulted in a dramatic decrease in Abeta production. Multimeric structures are important in determining the fate of specific proteins and protein phosphorylation can play an important role in their formation. FE65, a protein which binds APP, was closely studied in terms of binding APP in a phosphorylation regulated manner. This interaction was also affected by the phosphorylation state of APP. Conclusions: Our results provide novel insights into the role of direct APP phosphorylation on processing and Abeta production, pointing towards potential intervention strategies for the treatment of Alzheimer’s disease. Acknowledgments: Supported by the EU V (Project DIADEM) and VI Framework Program (Project APOPIS), Fundac¸a˜o para a Cieˆncia e Tecnologia of the Portuguese Ministry of Science and Technology, and Centro de Biologia Celular, of the Universidade de Aveiro.
TUESDAY, JULY 18, 2006 ORAL O3-03 DISEASE MECHANISMS (PRESENILINS/␥-SECRETASE) 2 O3-03-01
PRESENILIN-DEPENDENT TRANSCRIPTIONAL CONTROL OF THE A-DEGRADING ENZYME NEPRILYSIN BY AICD
Raphaelle Pardossi-Piquard1, Agnes Petit2, Julie Dunys2, Toshitaka Kawarai1, Claire Sunyach2, Cristine Alves da Costa2, Bruno Vincent2, Sabine Ring3, Luciano D’Adamio4, David Westaway1, Jie Shen5, Ulrike Mu¨ller3, Peter St. George-Hyslop1, Frederic Checler2, 1 Centre for research in Neurodegenerative Diseases, Toronto, ON, Canada; 2CNRS IPMC, Valbonne, France; 3Max-Planck-Institute, Frankfurt, Germany; 4Albert Einstein College of Medicine, New York, NY, USA; 5Center for Neurologic Diseases, Harvard Medical School, Boston, MA, USA. Contact e-mail: [email protected] Amyloid -peptide (A), which plays a central role in Alzheimer’s disease, is generated by presenilin-dependent ␥-secretase cleavage of -amyloid precursor protein (APP). It is now believed that presenilin-dependent ␥-secretase activity resides in a multimeric membrane-protein complex including presenilin (PS1 or PS2), nicastrin (Nct), aph-1 and pen-2. We have demonstrated that this presenilin-dependent ␥-secretase activity also regulates A degradation. Indeed, presenilin-deficient cells fail to degrade A and have drastic reductions in the transcription, expression, and activity of neprilysin, a key A-degrading enzyme. Neprilysin activity and expression are also drastically reduced in nicastrin-deficient cells, and lowered by ␥-secretase inhibitors and by PS1/PS2 deficiency in mouse brain. In contrast, expressions of two other putative A-degrading enzymes, endothelin-converting enzyme (ECE) and Insulin-degrading enzyme (IDE) are unchanged. We have reported that neprilysin activity and expression is restored in presenilin-deficient cells by transient expression of PS1 or PS2. Nicastrin also restores neprilysin activity and expression in nicastrin-deficient but not in presenilin-deficient fibroblasts, indicating that the control of neprilysin requires the completeness of the ␥-secretase complex harboring its four reported components. Furthermore, we have clearly established that deficiency of APP, APLP1 or APLP2 drastically reduces neprilysin expression and activity in both fibroblasts and in brain tissues. We have also shown that neprilysin activity is restored by expression of the amyloid intracellular domain (AICD), which is cogenerated with A, during ␥-secretase cleavage of APP. Several lines of evidence indicate that the APP-binding proteins Fe65 and Tip60 stabilize AICD and favor its translocation to the nucleus. Our experiments have shown also that AICD translocates to the nucleus in the presence of Fe65 and Tip60 and the AICD-induced up-regulation of neprilysin is strongly potentiated by co-expression of AICD with Fe65 or Tip60. Finally we have demonstrated that neprilysin gene promoters are transactivated by AICDs from APP-like proteins (APP, APLP1, and APLP2), but not by A or by the ␥-secretase cleavage products of Notch, N- or E- cadherins. These results provide evidence that presenilin-dependent ␥-secretase activity regulates neprilysin activity. In addition they support the hypothesis that AICD provides a physiological role within the nucleus to activate neprilysin gene transcription. O3-03-02
PS1/␥-SECRETASE REGULATE THE EPHBINDUCED SRC SIGNALING
Anastasios Georgakopoulos, Claudia Litterst, Enrico Ghersi, Lia Baki, Chijie Xu, Geo Serban, Nikolaos K. Robakis, Mount Sinai School of Medicine, New York, NY, USA. Contact e-mail: [email protected]
Oral O3-03: Disease Mechanisms (Presenilins/␥-Secretase) 2
of the Akt pathway in AD, and also examined IGF-1 (IGF-1R) and insulin receptors (IR), major Akt-activating receptors in brain. Results discovered a disease-stage related loss of Akt containing neurons selective to areas of the brain that degenerate in AD. This was detected together with a significantly increased Akt activity in AD, and hyperphosphorylation of many Akt downstream targets, including GSK3, tau, mTOR and p27kip1. Hyperphosphorylation of tau occurs at Ser214, an optimal consensus sequence for Akt, important for PHF formation, linking Akt activation to tau pathology. Mechanistically, increased Akt activation may be due to a significant loss of PTEN, and its altered subcellular localization, which was detected in neurons that degenerate in AD. We also discovered key defects in IGF-1R and IR, upstream to Akt signalling, indicating impaired normal growth factor activation of Akt in AD. This included a significant loss of IRS1/2, key adaptor proteins for IR and IGF-1R-activation of PI3-kinase, and loss of p85␣ and p100␣ subunits of PI3-kinase. Importantly, in other systems downregulation of IRS1/2 is a feedback response to heightened Akt activity, with resultant inhibition of normal receptor responses through PI3kinase-Akt. Further work detected more pronounced defects in IGF-1R status in AD compared to IR, including increased IGF-1R levels in glia, and within and surrounding A plaques. Together results indicate that Akt is hyper-activated in AD neurons, and that loss of normal regulated Akt target phosphorylation in AD may link to loss of Akt neurons, and impaired normal growth factor and insulin responses through this pathway in AD. O3-02-08
THE PHOSPHORYLATION STATE OF TYR687 ON APP CONTRIBUTES TO PROTEIN-PROTEIN INTERACTIONS AND ABETA PRODUCTION
Sandra Rebelo1, Sara S. Domingues1, Sandra I. Vieira1, Hermann Esselman2, Jens Wiltfang2, Edgar F. Cruz e Silva3, Odete A. da Cruz e Silva1, 1Laborato´rio de Neurocieˆncias, Centro de Biologia Celular, Universidade de Aveiro, Portugal; 2Erlangen University, Groningen, Germany; 3Laborato´rio de Transduc¸a˜o de Sinais, Centro de Biologia Celular, Universidade de Aveiro, Portugal. Background: The Alzheimer’s amyloid precursor protein (APP) is a transmembrane protein whose cytoplasmic domain contains several phosphorylatable residues. Objective: We have addressed the role of Tyr687 phosphorylation on APP metabolism, and the production of different cleavage products including Abeta. Methods: Constitutive phosphorylation or dephosphorylation of APP was mimicked by mutating Tyr687 to Glu (Y687E) or Phe (Y687F), respectively. In order to aid visualization GFP-fusion constructs were prepared. Results: Our results show that the phosphorylation state of Tyr687 is crucial in determining the fate of APP. Y687E-APP-GFP was delayed in the TGN, co-localized more to the ER and was distributed throughout the cell, apparently in a non-vesicular manner. In marked contrast, Y687FAPP-GFP was readily incorporated into vesicles. Expression of Y687EAPP-GFP resulted in a dramatic decrease in Abeta production. Multimeric structures are important in determining the fate of specific proteins and protein phosphorylation can play an important role in their formation. FE65, a protein which binds APP, was closely studied in terms of binding APP in a phosphorylation regulated manner. This interaction was also affected by the phosphorylation state of APP. Conclusions: Our results provide novel insights into the role of direct APP phosphorylation on processing and Abeta production, pointing towards potential intervention strategies for the treatment of Alzheimer’s disease. Acknowledgments: Supported by the EU V (Project DIADEM) and VI Framework Program (Project APOPIS), Fundac¸a˜o para a Cieˆncia e Tecnologia of the Portuguese Ministry of Science and Technology, and Centro de Biologia Celular, of the Universidade de Aveiro.
TUESDAY, JULY 18, 2006 ORAL O3-03 DISEASE MECHANISMS (PRESENILINS/␥-SECRETASE) 2 O3-03-01
PRESENILIN-DEPENDENT TRANSCRIPTIONAL CONTROL OF THE A-DEGRADING ENZYME NEPRILYSIN BY AICD
Raphaelle Pardossi-Piquard1, Agnes Petit2, Julie Dunys2, Toshitaka Kawarai1, Claire Sunyach2, Cristine Alves da Costa2, Bruno Vincent2, Sabine Ring3, Luciano D’Adamio4, David Westaway1, Jie Shen5, Ulrike Mu¨ller3, Peter St. George-Hyslop1, Frederic Checler2, 1 Centre for research in Neurodegenerative Diseases, Toronto, ON, Canada; 2CNRS IPMC, Valbonne, France; 3Max-Planck-Institute, Frankfurt, Germany; 4Albert Einstein College of Medicine, New York, NY, USA; 5Center for Neurologic Diseases, Harvard Medical School, Boston, MA, USA. Contact e-mail: [email protected] Amyloid -peptide (A), which plays a central role in Alzheimer’s disease, is generated by presenilin-dependent ␥-secretase cleavage of -amyloid precursor protein (APP). It is now believed that presenilin-dependent ␥-secretase activity resides in a multimeric membrane-protein complex including presenilin (PS1 or PS2), nicastrin (Nct), aph-1 and pen-2. We have demonstrated that this presenilin-dependent ␥-secretase activity also regulates A degradation. Indeed, presenilin-deficient cells fail to degrade A and have drastic reductions in the transcription, expression, and activity of neprilysin, a key A-degrading enzyme. Neprilysin activity and expression are also drastically reduced in nicastrin-deficient cells, and lowered by ␥-secretase inhibitors and by PS1/PS2 deficiency in mouse brain. In contrast, expressions of two other putative A-degrading enzymes, endothelin-converting enzyme (ECE) and Insulin-degrading enzyme (IDE) are unchanged. We have reported that neprilysin activity and expression is restored in presenilin-deficient cells by transient expression of PS1 or PS2. Nicastrin also restores neprilysin activity and expression in nicastrin-deficient but not in presenilin-deficient fibroblasts, indicating that the control of neprilysin requires the completeness of the ␥-secretase complex harboring its four reported components. Furthermore, we have clearly established that deficiency of APP, APLP1 or APLP2 drastically reduces neprilysin expression and activity in both fibroblasts and in brain tissues. We have also shown that neprilysin activity is restored by expression of the amyloid intracellular domain (AICD), which is cogenerated with A, during ␥-secretase cleavage of APP. Several lines of evidence indicate that the APP-binding proteins Fe65 and Tip60 stabilize AICD and favor its translocation to the nucleus. Our experiments have shown also that AICD translocates to the nucleus in the presence of Fe65 and Tip60 and the AICD-induced up-regulation of neprilysin is strongly potentiated by co-expression of AICD with Fe65 or Tip60. Finally we have demonstrated that neprilysin gene promoters are transactivated by AICDs from APP-like proteins (APP, APLP1, and APLP2), but not by A or by the ␥-secretase cleavage products of Notch, N- or E- cadherins. These results provide evidence that presenilin-dependent ␥-secretase activity regulates neprilysin activity. In addition they support the hypothesis that AICD provides a physiological role within the nucleus to activate neprilysin gene transcription. O3-03-02
PS1/␥-SECRETASE REGULATE THE EPHBINDUCED SRC SIGNALING
Anastasios Georgakopoulos, Claudia Litterst, Enrico Ghersi, Lia Baki, Chijie Xu, Geo Serban, Nikolaos K. Robakis, Mount Sinai School of Medicine, New York, NY, USA. Contact e-mail: [email protected]