- Email: [email protected]
P3-392
Poster P3:: Tuesday Posters mutations on those are currently under investigation. The progress on these studies will be presented. P3-392
P24 FAMILY PROTEINS HAVE DIFFERENT FUNCTION IN APP PROCESSING
Hiroshi Hasegawa1, Fusheng Chen2, Gerold Schmitt-Ulms2, Toshitaka Kawarai2, Christopher Bohm2, Taiichi Katayama2, Yong-Jun Gu2, Paul E. Fraser2, Peter St. George-Hyslop2,3, 1MNRC, Shiga University of Medical Science, Otsu, Japan; 2CRND, University of Toronto, Toronto, ON, Canada; 3University Health Network/ Toronto Western Hospital Research Institute, Toronto, ON, Canada. Contact e-mail: [email protected] Background: Presenilin complexes with gamma-secretase activities consist of at least four proteins, presenilin, nicastrin, Aph-1 and PEN-2. Transfection of these four proteins reconstitute gamma-secretase activities, but presenilin complexes cleave lots of type I membrane proteins as a substrate and are responsible to at least two independent cleavages in the transmembrane domain of APP. Thus, it might be possible that there is a regulatory mechanism in substrate specificity and cleavagesite control. Recently, we identified a novel associating protein that modulates APP processing. Objective(s): Identified protein is one of the members of p24 family. p24 family proteins work as hetero-oligomer in the vesicular trafficking machinery in the early secretary pathway. We investigated whether the modulation of gamma-secretase activities is a common function in p24 family in order to clarify the mechanism, although any other p24 family was not detected in the initial protein interaction screening. Methods: Another p24 family protein whose homologous protein, sel-9, was reported to modify mutant Notch signal pathway in C. elegans. was analyzed in HEK293 cells. The interaction with the components of presenilin complexes, was studied by immunoprecipitation. The effect of overexpression or knockdown on gamma- and epsilon-secretase activities was assessed in whole cell and in vitro assay. In addition, Notch S3 cleavage was analyzed by metabolic labeling. Results & Conclusions: Endogenous p24 family protein corresponding to sel-9 did not pull-down any components in presenilin complexes, different from another p24 protein identified as a modulator of APP processing, while apparent interaction between p24 family proteins was detected as reported previously. Neither overexpression nor knock-down of sel-9 homologous protein has any effect on Abeta or AICD production at least in endogenous and Swedish mutant APP. In addition, no significant difference in wild-type Notch S3 cleavage was observed. One of the explanations of this discrepancy between human and C. elegans is that Sel-9 in C. elegans might affect just mutant Notch through quality control mechanism. Besides, these results also revealed that p24 protein identified as a modulator of APP processing has a clearly different function from the other members in APP processing. P3-393
FAMILIAL ALZHEIMER’S DISEASE PRESENILIN MUTANTS REDUCE CALCIUM LEVELS OF INTRACELLULAR STORES. A CRITICAL REEVALUATION OF THE “CALCIUM OVERLOAD” HYPOTHESIS
Paola Pizzo1, Giancarlo Zatti1, Andrea Burgo1, Marta Giacomello1, Cristina Florean1, Giulietta Sinigaglia1, Laura Barbiero2, Roberta Ghidoni2, Silvia Bagnoli3, Benedetta Nacmias3, Sandro Sorbi3, Giuliano Binetti2, Cristina Fasolato1, 1University of Padua, Padova, Italy; 2IRCCS “Centro San Giovanni di Dio-FBF”-AFaR, Brescia, Italy; 3 University of Florence, Firenze, Italy. Contact e-mail: [email protected] Background: Presenilin-1 and -2 (PS1 and PS2) mutations, the major cause of Familial Alzheimer’s Disease (FAD), have been causally implicated in the pathogenesis of neuronal cell death through a perturbation of cellular Ca2⫹ homeostasis. We have recently shown that, at variance with
S491
previous suggestions obtained in cells expressing other FAD-linked PS mutations, PS2-M239I and PS2-T122R cause a reduction and not an increase in cytosolic Ca2⫹ rises induced by Ca2⫹ release from stores (1,2). Objective(s): In this study we aim at investigating whether other FAD-linked PS mutations induce a similar dysregulation of Ca2⫹ homeostasis. Methods: Different cell models have been used: human fibroblasts from controls and FAD patients, cell lines (SH-SY5Y, HeLa, HEK293, MEFs) and rat primary neurons expressing a number of PS mutations, e.g. P117L, M146L, L286V, and A246E in PS1 and M239I, T122R, and N141I in PS2. The effects of FAD-linked PS mutations on cytosolic Ca2⫹ changes have been monitored either by using fura-2 or recombinant cytosolic aequorin as the probe. Independently of the cell model or the employed probe, the cytosolic Ca2⫹ increases, caused by agonist stimulation or full store depletion by drug treatment, were reduced or unchanged in cells expressing the PS mutations. Using aequorins, targeted to the endoplasmic reticulum or the Golgi apparatus, we here show that FAD-linked PS mutants lower the Ca2⫹ content of intracellular stores. The phenomenon was most prominent in cells expressing PS2 mutants, and was observed also in cells expressing the non-pathogenic, “loss-of-function” PS2-D366A mutation. Conclusions: Taken as a whole, our findings, while confirming the capability of presenilins to modify Ca2⫹ homeostasis, suggest a re-evaluation of the “Ca2⫹ hypothesis” in AD and a new working hypothesis is presented. 1. Zatti G, Ghidoni R, Barbiero L, Binetti G, Pozzan T, Fasolato C, Pizzo P. (2004). Neurobiology of disease, 15, 269-278. 2. Giacomello M, Barbiero L, Zatti G, Squitti R, Binetti G, Pozzan T, Fasolato C, Ghidoni R, Pizzo P. (2005). Neurobiology of disease, 18, 638-648. P3-394
PROTEOLYTIC CLEAVAGE OF LAR RECEPTOR TYROSINE PHOSPHATASE BY GAMMASECRETASE REGULATES ASSOCIATION WITH BETA-CATENIN
Annakaisa Haapasalo1, Doo Y. Kim2, Bryce W. Carey2, Warren H. Pettingell2, Dora M. Kovacs2, 1University of Kuopio, Kuopio, Finland; 2Massachusetts General Hospital/Harvard Medical School, Charlestown, MA, USA. Contact e-mail: [email protected] Background: Presenilin (PS)/gamma-secretase proteolytically cleaves many type-I membrane proteins in addition to the Alzheimer’s diseaseassociated beta-amyloid precursor protein (APP). Leukocyte-common antigen related (LAR) receptor tyrosine phosphatase is a type-I transmembrane protein regulating cell adhesion, neurite outgrowth, and synaptic function. LAR consists of noncovalently linked extracellular E- and membrane-bound P-subunits, and undergoes inducible ectodomain shedding resulting in the release of the E-subunit. However, the proteases cleaving LAR have thus far remained unidentified. Objective: We asked whether LAR is proteolytically cleaved by PS/gamma-secretase and how this cleavage might affect LAR physiological function. Methods: We used BLASTbased sequence homology search, cell culture, Western blotting, in vitro gamma-secretase assay and coimmunoprecipitation. Results and Conclusions: We discovered that LAR shares sequence homology with APP gamma-secretase epsilon-cleavage site, suggesting that LAR may be a new PS/gamma-secretase substrate. Indeed, LAR C-terminal fragments (LARCTFs) accumulated upon inhibited gamma-secretase activity in stable LAR-overexpressing Chinese hamster ovary (LAR-CHO) cells and mouse cortical neurons endogenously expressing LAR. Inhibition of alfa-secretase activity blocked LAR-CTF accumulation, demonstrating that LAR cleavage by gamma-secretase required prior ectodomain shedding. Furthermore, LAR-CTFs accumulated in cells expressing dominant negative mutant of PS1 or lacking both PS1 and PS2, indicating that gamma-secretase-mediated cleavage of LAR was PS-dependent. LAR intracellular domain (LICD) fragment was generated in an in vitro gamma-secretase assay and in vivo in CHO cells overexpressing LAR-CTF in a PS/gamma-secretasedependent manner. Finally, we examined if PS/gamma-secretase cleavage
P24 FAMILY PROTEINS HAVE DIFFERENT FUNCTION IN APP PROCESSING
Hiroshi Hasegawa1, Fusheng Chen2, Gerold Schmitt-Ulms2, Toshitaka Kawarai2, Christopher Bohm2, Taiichi Katayama2, Yong-Jun Gu2, Paul E. Fraser2, Peter St. George-Hyslop2,3, 1MNRC, Shiga University of Medical Science, Otsu, Japan; 2CRND, University of Toronto, Toronto, ON, Canada; 3University Health Network/ Toronto Western Hospital Research Institute, Toronto, ON, Canada. Contact e-mail: [email protected] Background: Presenilin complexes with gamma-secretase activities consist of at least four proteins, presenilin, nicastrin, Aph-1 and PEN-2. Transfection of these four proteins reconstitute gamma-secretase activities, but presenilin complexes cleave lots of type I membrane proteins as a substrate and are responsible to at least two independent cleavages in the transmembrane domain of APP. Thus, it might be possible that there is a regulatory mechanism in substrate specificity and cleavagesite control. Recently, we identified a novel associating protein that modulates APP processing. Objective(s): Identified protein is one of the members of p24 family. p24 family proteins work as hetero-oligomer in the vesicular trafficking machinery in the early secretary pathway. We investigated whether the modulation of gamma-secretase activities is a common function in p24 family in order to clarify the mechanism, although any other p24 family was not detected in the initial protein interaction screening. Methods: Another p24 family protein whose homologous protein, sel-9, was reported to modify mutant Notch signal pathway in C. elegans. was analyzed in HEK293 cells. The interaction with the components of presenilin complexes, was studied by immunoprecipitation. The effect of overexpression or knockdown on gamma- and epsilon-secretase activities was assessed in whole cell and in vitro assay. In addition, Notch S3 cleavage was analyzed by metabolic labeling. Results & Conclusions: Endogenous p24 family protein corresponding to sel-9 did not pull-down any components in presenilin complexes, different from another p24 protein identified as a modulator of APP processing, while apparent interaction between p24 family proteins was detected as reported previously. Neither overexpression nor knock-down of sel-9 homologous protein has any effect on Abeta or AICD production at least in endogenous and Swedish mutant APP. In addition, no significant difference in wild-type Notch S3 cleavage was observed. One of the explanations of this discrepancy between human and C. elegans is that Sel-9 in C. elegans might affect just mutant Notch through quality control mechanism. Besides, these results also revealed that p24 protein identified as a modulator of APP processing has a clearly different function from the other members in APP processing. P3-393
FAMILIAL ALZHEIMER’S DISEASE PRESENILIN MUTANTS REDUCE CALCIUM LEVELS OF INTRACELLULAR STORES. A CRITICAL REEVALUATION OF THE “CALCIUM OVERLOAD” HYPOTHESIS
Paola Pizzo1, Giancarlo Zatti1, Andrea Burgo1, Marta Giacomello1, Cristina Florean1, Giulietta Sinigaglia1, Laura Barbiero2, Roberta Ghidoni2, Silvia Bagnoli3, Benedetta Nacmias3, Sandro Sorbi3, Giuliano Binetti2, Cristina Fasolato1, 1University of Padua, Padova, Italy; 2IRCCS “Centro San Giovanni di Dio-FBF”-AFaR, Brescia, Italy; 3 University of Florence, Firenze, Italy. Contact e-mail: [email protected] Background: Presenilin-1 and -2 (PS1 and PS2) mutations, the major cause of Familial Alzheimer’s Disease (FAD), have been causally implicated in the pathogenesis of neuronal cell death through a perturbation of cellular Ca2⫹ homeostasis. We have recently shown that, at variance with
S491
previous suggestions obtained in cells expressing other FAD-linked PS mutations, PS2-M239I and PS2-T122R cause a reduction and not an increase in cytosolic Ca2⫹ rises induced by Ca2⫹ release from stores (1,2). Objective(s): In this study we aim at investigating whether other FAD-linked PS mutations induce a similar dysregulation of Ca2⫹ homeostasis. Methods: Different cell models have been used: human fibroblasts from controls and FAD patients, cell lines (SH-SY5Y, HeLa, HEK293, MEFs) and rat primary neurons expressing a number of PS mutations, e.g. P117L, M146L, L286V, and A246E in PS1 and M239I, T122R, and N141I in PS2. The effects of FAD-linked PS mutations on cytosolic Ca2⫹ changes have been monitored either by using fura-2 or recombinant cytosolic aequorin as the probe. Independently of the cell model or the employed probe, the cytosolic Ca2⫹ increases, caused by agonist stimulation or full store depletion by drug treatment, were reduced or unchanged in cells expressing the PS mutations. Using aequorins, targeted to the endoplasmic reticulum or the Golgi apparatus, we here show that FAD-linked PS mutants lower the Ca2⫹ content of intracellular stores. The phenomenon was most prominent in cells expressing PS2 mutants, and was observed also in cells expressing the non-pathogenic, “loss-of-function” PS2-D366A mutation. Conclusions: Taken as a whole, our findings, while confirming the capability of presenilins to modify Ca2⫹ homeostasis, suggest a re-evaluation of the “Ca2⫹ hypothesis” in AD and a new working hypothesis is presented. 1. Zatti G, Ghidoni R, Barbiero L, Binetti G, Pozzan T, Fasolato C, Pizzo P. (2004). Neurobiology of disease, 15, 269-278. 2. Giacomello M, Barbiero L, Zatti G, Squitti R, Binetti G, Pozzan T, Fasolato C, Ghidoni R, Pizzo P. (2005). Neurobiology of disease, 18, 638-648. P3-394
PROTEOLYTIC CLEAVAGE OF LAR RECEPTOR TYROSINE PHOSPHATASE BY GAMMASECRETASE REGULATES ASSOCIATION WITH BETA-CATENIN
Annakaisa Haapasalo1, Doo Y. Kim2, Bryce W. Carey2, Warren H. Pettingell2, Dora M. Kovacs2, 1University of Kuopio, Kuopio, Finland; 2Massachusetts General Hospital/Harvard Medical School, Charlestown, MA, USA. Contact e-mail: [email protected] Background: Presenilin (PS)/gamma-secretase proteolytically cleaves many type-I membrane proteins in addition to the Alzheimer’s diseaseassociated beta-amyloid precursor protein (APP). Leukocyte-common antigen related (LAR) receptor tyrosine phosphatase is a type-I transmembrane protein regulating cell adhesion, neurite outgrowth, and synaptic function. LAR consists of noncovalently linked extracellular E- and membrane-bound P-subunits, and undergoes inducible ectodomain shedding resulting in the release of the E-subunit. However, the proteases cleaving LAR have thus far remained unidentified. Objective: We asked whether LAR is proteolytically cleaved by PS/gamma-secretase and how this cleavage might affect LAR physiological function. Methods: We used BLASTbased sequence homology search, cell culture, Western blotting, in vitro gamma-secretase assay and coimmunoprecipitation. Results and Conclusions: We discovered that LAR shares sequence homology with APP gamma-secretase epsilon-cleavage site, suggesting that LAR may be a new PS/gamma-secretase substrate. Indeed, LAR C-terminal fragments (LARCTFs) accumulated upon inhibited gamma-secretase activity in stable LAR-overexpressing Chinese hamster ovary (LAR-CHO) cells and mouse cortical neurons endogenously expressing LAR. Inhibition of alfa-secretase activity blocked LAR-CTF accumulation, demonstrating that LAR cleavage by gamma-secretase required prior ectodomain shedding. Furthermore, LAR-CTFs accumulated in cells expressing dominant negative mutant of PS1 or lacking both PS1 and PS2, indicating that gamma-secretase-mediated cleavage of LAR was PS-dependent. LAR intracellular domain (LICD) fragment was generated in an in vitro gamma-secretase assay and in vivo in CHO cells overexpressing LAR-CTF in a PS/gamma-secretasedependent manner. Finally, we examined if PS/gamma-secretase cleavage