- Email: [email protected]
P3-246
Poster P3:: Tuesday Posters the b-amyloid precursor protein (bAPP) and the cellular prion protein (PrPc), the two culprit membrane proteins in AD and prion-associated pathologies respectively, are similarly processed in the middle of their toxic sequence. Indeed, the so-called a-secretase cleavage precludes the integrity of the amyloid-b peptide (Ab) and the 106-126 sequence of PrPc. The recent identification of two members of the disintegrin family of proteases, ADAM10 and ADAM17, as constitutive and PKC-regulated a-secretase activities cleaving both bAPP and PrPc further reinforced the link between Alzheimer’s and prion diseases. We showed very recently that a third ADAM protease, namely ADAM9, also participates in the processing of bAPP and PrPc. However, ADAM9 does not directly cleave bAPP and PrPc but rather acts through the modulation of ADAM10 likely by contributing to its shedding. A well-documented aspect of the regulation of the a-secretase pathway concerns its activation by protein kinase C agonists. Interestingly, it has been suggested that some but not all PKC isozymes contribute the cleavage of bAPP at the a-site. We established that the conventional a and b as well as the novel e isoforms are involved in the regulated cleavage of both bAPP and PrPc while a fourth member, the novel PKCd, seems to only modulate PrPc processing. This tends to prove that the molecular mechanisms governing the PKC-mediated cleavage of both proteins, although very similar, display subtle differences. Finally, in the search for the receptors and ligands acting upstream of PKC activation which ultimately leads to the up-regulation of PrPc processing, we demonstrated that the M1/M3 subclass of muscarinic receptors which are coupled to PLC/PKC, but not the M2/M4 (linked to adenylate cyclase/ PKA) was a key component of the PKC-mediated PrPc processing. Altogether, these data suggest that any medication aiming at increasing the a-secretase cleavage could ameliorate two apparently unrelated groups of diseases. P3-244
THE IMPACT OF LRP ON APP METABOLISM ALONG THE SECRETORY PATHWAY
Elaine A. Waldron, Catherine Heilig, Andrea Schweitzer, Claus U. Pietrzik, University of Mainz, Mainz, Germany. Contact e-mail: [email protected] Background: Despite the recent advance in the understanding of amyloid precursor protein (APP) metabolism, much remains to be learned. In order to enhance our present level of knowledge, other Alzheimer’s disease (AD) influential proteins should be accounted for, in the APP metabolic setting. Increasing evidence has implicated the low density lipoprotein receptorrelated protein (LRP) in AD pathogenesis. Through a complex formation of APP, FE65, and LRP, LRP may mediate APP processing. APP internalization is mediated by LRP, while in the absence of LRP the internalization rate of APP is reduced by approximately 40%, thereby increasing cell surface APP and decreasing A secretion. Furthermore, the APP-CTF levels are augmented in the presence of LRP, which is likely due to an LRP stabilizing effect. Objective(s): Since LRP may traffic with APP to the cell surface, we asked the question has LRP the ability to retain APP in compartments along the secretory pathway and if so, what are its effects on APP metabolism. Methods: Truncated LRP-ER and LRP-Golgi retention constructs were expressed in LRP -/- cells and LRP -/- cells stably expressing APP695. APP retention was analyzed by confocal microscopy and cell surface biotinylation. A secretion was measured by immunoprecipitation and Western blotting. APP intracellular domain (AICD) generation was measured using a well defined in vitro assay followed by Western blotting. Results: LRP can hold APP back in these compartments. A secretion from cells where the LRP is retained in the ER is dramatically less than that secreted in the presence of normally transported LRP. A secretion resulting from retention of APP through LRP in the Golgi is less than that for normally transported LRP but more than that for APP retention in the ER. Since, we believe the AICD and A generating steps to be a sequential process, we hypothesized to see a similar situation for AICD as A, which surprisingly was not the case. AICD production was reduced upon ER retention but was not comparable to A secretion. Conclusions:
S447
We are currently investigating if AICD production and A production destined for secretion may be segregated depending on the location of LRP. P3-245
METHYLAMINE OXIDATION BY HUMAN VAP-1/ SSAO INDUCES CYTOTOXICITY ON VASCULAR CELLS: ITS CONTRIBUTION TO CAA-AD
Montserrat Sole´1, Mar Herna`ndez1, Merce` Boada2, Mercedes Unzeta1, 1 Universitat Auto`noma de Barcelona, Bellaterra (Barcelona), Spain; 2 Servei de Neurologia, Hospital G. U. Vall d’Hebron. Fundacio´ ACE. Institut Catala` de Neurocie`ncies Aplicades, Barcelona, Spain. Contact e-mail: [email protected] Background: Semicarbazide sensitive amine oxidase (SSAO), is a multifunctional enzyme (O’Sullivan J. et al., 2004) that shows different biological roles depending on the tissue where it’s expressed. SSAO is present in almost all mammalian tissues (Andres N. et al., 2001), specially those highly vascularized. It metabolizes primary amines (Lyles GA., 1996) generating hydogen peroxide (H2O2), ammonia (NH3) and the corresponding aldehyde as final toxic products able to contribute to cellular oxidative stress. SSAO shows also an insulinomimetic effect in adipocytes (EnriqueTaranco´n G. et al., 1998), and it behaves as a vascular adhesion protein (VAP-1) in endothelial cells. However, its physiological function is still far from clear. SSAO/VAP-1 is expressed in the cells in a membrane-bound form and is also present as a soluble enzyme in plasma; in this concern, it has been postulated (Abella A, et al., 2004) that the soluble form comes from the membrane-bound one by a shedding process metalloproteasedependent. We have described that SSAO/VAP-1 is overexpressed in human cerebrovascular tissue from cerebral amyloid angiopathy linked to Alzheimer’s disease (CAA-AD) (Ferrer I. et al., 2002). We have also reported that the plasma SSAO activity increases significantly with the severity of Alzheimer’s disease (del Mar Hernandez M. et al., 2005). These results showed for the first time, a role of the enzyme in this neurodegenerative disorder. Objective(s): The aim of this work has been to study the possible contribution of this enzyme to the vascular damage underlying the CAA-AD. Methods: Because the SSAO/VAP-1 expression phenotype is lost in cultured cells, A7r5 (smooth muscle cells from rat aorta) and HUVEC (human umbilical vein endothelial cells) cells, were stably transfected with vector pcDNA 3.1 containing hVAP-1/SSAO. Cells were treated with different concentrations of methylamine (1-3mM), the physiological SSAO substrate. Results: A time-dependent and dose-response cytotoxic effect was observed. The activation of caspase-3 corroborates that apoptosis is involved in this cell death. Conclusions: These results allow us to suggest that in pathological conditions such as CAA-AD, in which SSAO is overexpressed (Ferrer I. et al., 2002), this enzyme through its catalytic action could contribute to the vascular tissue damage observed in CAA-AD. P3-246
EFFECTS OF NITRATION ON PS/␥-SECRETASE
Kohji Mori, Masayasu Okochi, Akio Fukumori, Jingwei Jiang, Akiko Ikuta, Kanta Yanagida, Taisuke Nakayama, Yoshiko Ishizuka-Katsura, Hong Tao, Takashi Morihara, Kojin Kamino, Toshihisa Tanaka, Takashi Kudo, Shinji Tagami, Masatoshi Takeda, Osaka University Graduate School of Medicine, Osaka, Japan. Contact e-mail: [email protected] Background: Accumulating evidence suggests that oxidative stress plays roles in Alzheimer’s disease (AD) pathology. A elicits the production of reactive nitrogen and oxygen intermediates from microglia and other glial cells in accordance with proinflamatory cytokines. Inducible NO synthase (iNOS) and nitrated proteins were detected in the AD brain. Genetic ablation of iNOS reduced amyloid plaque formation in double transgenic mice expressing familial AD-associated mutant forms of both APP and PS1. Objective(s): Protein modulation by NO and its derivative peroxynitrite modify the function of some targeted protein. Nitration of ADassociated proteins such as members of PS/␥-secretase complex were analyzed. Methods: Cultured cells were directly exposed to peroxynitrite
S448
Poster P3:: Tuesday Posters
and their membrane fractions were collected. We also collected the membrane fractions of the cells cultured under non-peroxynitrite stimulation condition and then exposed them to peroxynitrite. Results: Following nitration treatment, the membrane fractions were served for cell-free ␥-secretase assay. de novo A and AICD generation was analyzed by immunoprecipitation-immunoblotting and combined immunoprecipitationMS. Conclusions: We will report effects of nitration on AD-associated molecules such as members of ␥-secretase complex. P3-247
FUNCTIONAL CONSEQUENCES OF LRP EXPRESSION ON FAMILIAR ALZHEIMER DISEASE APP MUTATIONS
Sebastian Jaeger1, Barbara S. Rogge1, Sascha Weggen1, Edward H. Koo2, Claus U. Pietrzik1, 1Johannes-Gutenberg-University Mainz, Mainz, Germany; 2University of California, San Diego, CA, USA. Contact e-mail: [email protected] Background: Increasing evidence has implicated the low density lipoprotein receptor related protein (LRP) and FE65 in Alzheimer’s disease (AD) pathogenesis. We have been able to show that LRP mediates wildtype (wt) APP processing by influencing A-, APPs-secretion and APP-CTF generation. This influence of LRP on APP processing is dependent on a tripartite complex formation linking LRP to APP by Fe65. Objective(s): Here we set out to identify whether A-generation is generally dependent on LRP expression or whether different APP mutations alter the LRP dependent processing effect on APP. Methods: As A production, APPs secretion, and the APP C-terminal fragments (CTFs) are frequently correlated with each other, LRP⫹/- or LRP-/- stable expressing APP695swe were assayed for these metabolic products and compared to APPwt stable expressing cells. Results: Interestingly the levels of the ␣-secretase CTF (C83) and -secretase cleaved CTF (C99) were unchanged in LRP-/- cells in contrast to APPwt expressing cells. Since the internalization rate in APPwt expressing LRP-/- cells was reduced by approximately 40%, we measured the internalization rate of APPswe expressing LRP-/- cells. Surprisingly we observed similar APP endocytosis rates. Although the internalization of APPswe was reduced in LRP-/- cells we did not see any reduction of A-secretion compared to LRP expressing control cells, arguing that Ageneration from APPswe expressing cells occurs on the way to the cell surface rather than after internalization. To further prove this observation, we generated C99- and C99⌬NPXY-deletion constructs and expressed them in LRP expressing and deficient cells to verify whether the internalization of BACE1 cleaved substrate is necessary for A-generation. Conclusions: Our results suggest that the effect seen by LRP on APP processing might affect wildtype rather than swedish APP, indicating that processing of familiar APP mutations occurs mostly on the way to or at the cell surface. P3-248
ACTIVATION OF CALPAIN I IN ALZHEIMER DISEASE BRAIN NEGATIVELY REGULATES CREB
Fei Liu1,2, Inge Grundke-Iqbal1, Khalid Iqbal1, Cheng-Xin Gong1, 1New York State Institute for Basic Research in Developmental Disabilities, Staten Island, NY, USA; 2Nantong University, Nantong, China. Contact e-mail: [email protected] Alzheimer disease (AD) is the most common cause of dementia in adults, resulting in impairment of cognition and memory. The molecular pathogenesis of AD is not well understood. Dysregulation of intracellular calcium signaling has been implicated in the pathogenesis of AD. Many putative etiologic factors of AD, including excitotoxicity, -amyloid neurotoxicity, and free-radical injury, have in common the potential for disrupting intracellular calcium homeostasis. Here, we studied two major Ca2⫹-regulated proteins in the central neuron system, calpain I and calcineurin, and found that calpain I was activated by seven-fold in AD brain as compared with control brain, which in turn cleaved and activated calcineurin. Mass spectrometric analysis indicated that the cleavage of
calcineurin by calpain I was at lysine 501, a position C-terminal of the autoinhibitory domain, which produced a 57-kDa truncated form. The 57-kDa calcineurin maintained its Ca2⫹/camodulin-dependence of the phosphatase activity, but the activity was increased by three-fold upon truncation. In addition to calcineurin A, calpain I also cleaved cAMP responsive element binding protein (CREB), a switcher molecule for learning and memory, and in turn decreased the CREB level in AD brain. Inhibition of calpain I restored the decreased level of CREB induced by calcium influx. Since calcineurin negatively regulates CREB phosphorylation and activity by dephosphorylating DARPP32 and activating PP1, these findings suggest that the over-activation of calpain I and calcineurin due to calcium homeostatic disturbance may inactivate CREB by direct degradation and dephosphorylation and may play an important role in learning and memory impairment in AD patients. P3-249
THE A AND ECTODOMAIN COPPER BINDING SITES ON THE AMYLOID PRECURSOR PROTEIN MARKEDLY AFFECT PROCESSING
Susan-Marie E. Harding1,2, James Camakaris1,2, Roberto Cappai3,4, Andrew F. Hill5,3, Ashley I. Bush2,3, 1The University of Melbourne, Department of Genetics, Victoria, Australia; 2The Mental Health Research Institute, Victoria, Australia; 3The University of Melbourne, Department of Pathology, Victoria, Australia; 4The University of Melbourne, Centre for Neuroscience, Victoria, Australia; 5Bio21, Department of Biochemistry and Molecular Biology, Australia. Contact e-mail: [email protected] Background: Over-expression of the amyloid precursor protein (APP) results in significantly reduced copper and iron levels in transgenic mice brains. Conversely, APP and APLP2 genetic ablation both cause neuronal copper accumulation. Cellular copper up-regulates the expression of the APP gene. APP has two histidine-mediated ectodomain Cu2⫹ binding sites, one in the amino terminal region, and another in the A domain. Both sites reduce Cu2⫹ to Cu1⫹. BACE1 interacts with the copper chaperone of SOD1, which plays an important role in copper homeostasis. We hypothesized that the APP Cu2⫹ binding sites may be key in sensing Cu2⫹ and then regulating the processing of APP for its possible participation in copper homeostasis. Objective(s): To investigate the differential processing of APP in response to the ablation of the copper binding sites and/or the manipulation of cellular copper levels. Methods: Chinese hamster ovary (CHO) cells were transfected with human APP695 (wtAPP) or mutant APP including; Swedish mutant, London mutant, a mutant of the amino-terminal Cu binding site (APP⌬Cu1), or APP with the A Cu binding site mutated (APP⌬Cu2). Cells were cultivated in basal medium, medium containing excess copper, or copper chelators. Extracellular, soluble (sAPP) and membrane (full length, flAPP) fractions were analyzed for APP levels (using Western blot) and copper content (using ICP mass spectrometry). Results: The processing of APP⌬Cu1and APP⌬Cu2 was markedly different from that of wtAPP. The most significant differences were; 1. The APP⌬Cu1 mutant exhibited a marked increase in membranous flAPP compared to wtAPP. 2. The APP⌬Cu2 mutant was processed such that the ration of flAPP was higher than that of sAPP compared to wtAPP. Further analysis of APP fragments and the effects of modulating copper levels will be reported. Conclusions: These results indicate that the integrity of the APP copper binding sites are important in APP processing. P3-250
UNNATURAL AMINO ACID-SUBSTITUTED (HYDROXYETHYL)UREA PEPTIDOMIMETICS INHIBIT GAMMA-SECRETASE WITH ENHANCED STABILITY
Yung-Feng Liao1, Bo-Jeng Wang1, Chia-Yin Liao2, Shin-Ying Wu1, Ming-Kuan Hu2, 1Academia Sinica, Taipei, Taiwan, Province of China; 2 National Defense Medical Center, Taipei, Taiwan, Province of China. Contact e-mail: [email protected]
THE IMPACT OF LRP ON APP METABOLISM ALONG THE SECRETORY PATHWAY
Elaine A. Waldron, Catherine Heilig, Andrea Schweitzer, Claus U. Pietrzik, University of Mainz, Mainz, Germany. Contact e-mail: [email protected] Background: Despite the recent advance in the understanding of amyloid precursor protein (APP) metabolism, much remains to be learned. In order to enhance our present level of knowledge, other Alzheimer’s disease (AD) influential proteins should be accounted for, in the APP metabolic setting. Increasing evidence has implicated the low density lipoprotein receptorrelated protein (LRP) in AD pathogenesis. Through a complex formation of APP, FE65, and LRP, LRP may mediate APP processing. APP internalization is mediated by LRP, while in the absence of LRP the internalization rate of APP is reduced by approximately 40%, thereby increasing cell surface APP and decreasing A secretion. Furthermore, the APP-CTF levels are augmented in the presence of LRP, which is likely due to an LRP stabilizing effect. Objective(s): Since LRP may traffic with APP to the cell surface, we asked the question has LRP the ability to retain APP in compartments along the secretory pathway and if so, what are its effects on APP metabolism. Methods: Truncated LRP-ER and LRP-Golgi retention constructs were expressed in LRP -/- cells and LRP -/- cells stably expressing APP695. APP retention was analyzed by confocal microscopy and cell surface biotinylation. A secretion was measured by immunoprecipitation and Western blotting. APP intracellular domain (AICD) generation was measured using a well defined in vitro assay followed by Western blotting. Results: LRP can hold APP back in these compartments. A secretion from cells where the LRP is retained in the ER is dramatically less than that secreted in the presence of normally transported LRP. A secretion resulting from retention of APP through LRP in the Golgi is less than that for normally transported LRP but more than that for APP retention in the ER. Since, we believe the AICD and A generating steps to be a sequential process, we hypothesized to see a similar situation for AICD as A, which surprisingly was not the case. AICD production was reduced upon ER retention but was not comparable to A secretion. Conclusions:
S447
We are currently investigating if AICD production and A production destined for secretion may be segregated depending on the location of LRP. P3-245
METHYLAMINE OXIDATION BY HUMAN VAP-1/ SSAO INDUCES CYTOTOXICITY ON VASCULAR CELLS: ITS CONTRIBUTION TO CAA-AD
Montserrat Sole´1, Mar Herna`ndez1, Merce` Boada2, Mercedes Unzeta1, 1 Universitat Auto`noma de Barcelona, Bellaterra (Barcelona), Spain; 2 Servei de Neurologia, Hospital G. U. Vall d’Hebron. Fundacio´ ACE. Institut Catala` de Neurocie`ncies Aplicades, Barcelona, Spain. Contact e-mail: [email protected] Background: Semicarbazide sensitive amine oxidase (SSAO), is a multifunctional enzyme (O’Sullivan J. et al., 2004) that shows different biological roles depending on the tissue where it’s expressed. SSAO is present in almost all mammalian tissues (Andres N. et al., 2001), specially those highly vascularized. It metabolizes primary amines (Lyles GA., 1996) generating hydogen peroxide (H2O2), ammonia (NH3) and the corresponding aldehyde as final toxic products able to contribute to cellular oxidative stress. SSAO shows also an insulinomimetic effect in adipocytes (EnriqueTaranco´n G. et al., 1998), and it behaves as a vascular adhesion protein (VAP-1) in endothelial cells. However, its physiological function is still far from clear. SSAO/VAP-1 is expressed in the cells in a membrane-bound form and is also present as a soluble enzyme in plasma; in this concern, it has been postulated (Abella A, et al., 2004) that the soluble form comes from the membrane-bound one by a shedding process metalloproteasedependent. We have described that SSAO/VAP-1 is overexpressed in human cerebrovascular tissue from cerebral amyloid angiopathy linked to Alzheimer’s disease (CAA-AD) (Ferrer I. et al., 2002). We have also reported that the plasma SSAO activity increases significantly with the severity of Alzheimer’s disease (del Mar Hernandez M. et al., 2005). These results showed for the first time, a role of the enzyme in this neurodegenerative disorder. Objective(s): The aim of this work has been to study the possible contribution of this enzyme to the vascular damage underlying the CAA-AD. Methods: Because the SSAO/VAP-1 expression phenotype is lost in cultured cells, A7r5 (smooth muscle cells from rat aorta) and HUVEC (human umbilical vein endothelial cells) cells, were stably transfected with vector pcDNA 3.1 containing hVAP-1/SSAO. Cells were treated with different concentrations of methylamine (1-3mM), the physiological SSAO substrate. Results: A time-dependent and dose-response cytotoxic effect was observed. The activation of caspase-3 corroborates that apoptosis is involved in this cell death. Conclusions: These results allow us to suggest that in pathological conditions such as CAA-AD, in which SSAO is overexpressed (Ferrer I. et al., 2002), this enzyme through its catalytic action could contribute to the vascular tissue damage observed in CAA-AD. P3-246
EFFECTS OF NITRATION ON PS/␥-SECRETASE
Kohji Mori, Masayasu Okochi, Akio Fukumori, Jingwei Jiang, Akiko Ikuta, Kanta Yanagida, Taisuke Nakayama, Yoshiko Ishizuka-Katsura, Hong Tao, Takashi Morihara, Kojin Kamino, Toshihisa Tanaka, Takashi Kudo, Shinji Tagami, Masatoshi Takeda, Osaka University Graduate School of Medicine, Osaka, Japan. Contact e-mail: [email protected] Background: Accumulating evidence suggests that oxidative stress plays roles in Alzheimer’s disease (AD) pathology. A elicits the production of reactive nitrogen and oxygen intermediates from microglia and other glial cells in accordance with proinflamatory cytokines. Inducible NO synthase (iNOS) and nitrated proteins were detected in the AD brain. Genetic ablation of iNOS reduced amyloid plaque formation in double transgenic mice expressing familial AD-associated mutant forms of both APP and PS1. Objective(s): Protein modulation by NO and its derivative peroxynitrite modify the function of some targeted protein. Nitration of ADassociated proteins such as members of PS/␥-secretase complex were analyzed. Methods: Cultured cells were directly exposed to peroxynitrite
S448
Poster P3:: Tuesday Posters
and their membrane fractions were collected. We also collected the membrane fractions of the cells cultured under non-peroxynitrite stimulation condition and then exposed them to peroxynitrite. Results: Following nitration treatment, the membrane fractions were served for cell-free ␥-secretase assay. de novo A and AICD generation was analyzed by immunoprecipitation-immunoblotting and combined immunoprecipitationMS. Conclusions: We will report effects of nitration on AD-associated molecules such as members of ␥-secretase complex. P3-247
FUNCTIONAL CONSEQUENCES OF LRP EXPRESSION ON FAMILIAR ALZHEIMER DISEASE APP MUTATIONS
Sebastian Jaeger1, Barbara S. Rogge1, Sascha Weggen1, Edward H. Koo2, Claus U. Pietrzik1, 1Johannes-Gutenberg-University Mainz, Mainz, Germany; 2University of California, San Diego, CA, USA. Contact e-mail: [email protected] Background: Increasing evidence has implicated the low density lipoprotein receptor related protein (LRP) and FE65 in Alzheimer’s disease (AD) pathogenesis. We have been able to show that LRP mediates wildtype (wt) APP processing by influencing A-, APPs-secretion and APP-CTF generation. This influence of LRP on APP processing is dependent on a tripartite complex formation linking LRP to APP by Fe65. Objective(s): Here we set out to identify whether A-generation is generally dependent on LRP expression or whether different APP mutations alter the LRP dependent processing effect on APP. Methods: As A production, APPs secretion, and the APP C-terminal fragments (CTFs) are frequently correlated with each other, LRP⫹/- or LRP-/- stable expressing APP695swe were assayed for these metabolic products and compared to APPwt stable expressing cells. Results: Interestingly the levels of the ␣-secretase CTF (C83) and -secretase cleaved CTF (C99) were unchanged in LRP-/- cells in contrast to APPwt expressing cells. Since the internalization rate in APPwt expressing LRP-/- cells was reduced by approximately 40%, we measured the internalization rate of APPswe expressing LRP-/- cells. Surprisingly we observed similar APP endocytosis rates. Although the internalization of APPswe was reduced in LRP-/- cells we did not see any reduction of A-secretion compared to LRP expressing control cells, arguing that Ageneration from APPswe expressing cells occurs on the way to the cell surface rather than after internalization. To further prove this observation, we generated C99- and C99⌬NPXY-deletion constructs and expressed them in LRP expressing and deficient cells to verify whether the internalization of BACE1 cleaved substrate is necessary for A-generation. Conclusions: Our results suggest that the effect seen by LRP on APP processing might affect wildtype rather than swedish APP, indicating that processing of familiar APP mutations occurs mostly on the way to or at the cell surface. P3-248
ACTIVATION OF CALPAIN I IN ALZHEIMER DISEASE BRAIN NEGATIVELY REGULATES CREB
Fei Liu1,2, Inge Grundke-Iqbal1, Khalid Iqbal1, Cheng-Xin Gong1, 1New York State Institute for Basic Research in Developmental Disabilities, Staten Island, NY, USA; 2Nantong University, Nantong, China. Contact e-mail: [email protected] Alzheimer disease (AD) is the most common cause of dementia in adults, resulting in impairment of cognition and memory. The molecular pathogenesis of AD is not well understood. Dysregulation of intracellular calcium signaling has been implicated in the pathogenesis of AD. Many putative etiologic factors of AD, including excitotoxicity, -amyloid neurotoxicity, and free-radical injury, have in common the potential for disrupting intracellular calcium homeostasis. Here, we studied two major Ca2⫹-regulated proteins in the central neuron system, calpain I and calcineurin, and found that calpain I was activated by seven-fold in AD brain as compared with control brain, which in turn cleaved and activated calcineurin. Mass spectrometric analysis indicated that the cleavage of
calcineurin by calpain I was at lysine 501, a position C-terminal of the autoinhibitory domain, which produced a 57-kDa truncated form. The 57-kDa calcineurin maintained its Ca2⫹/camodulin-dependence of the phosphatase activity, but the activity was increased by three-fold upon truncation. In addition to calcineurin A, calpain I also cleaved cAMP responsive element binding protein (CREB), a switcher molecule for learning and memory, and in turn decreased the CREB level in AD brain. Inhibition of calpain I restored the decreased level of CREB induced by calcium influx. Since calcineurin negatively regulates CREB phosphorylation and activity by dephosphorylating DARPP32 and activating PP1, these findings suggest that the over-activation of calpain I and calcineurin due to calcium homeostatic disturbance may inactivate CREB by direct degradation and dephosphorylation and may play an important role in learning and memory impairment in AD patients. P3-249
THE A AND ECTODOMAIN COPPER BINDING SITES ON THE AMYLOID PRECURSOR PROTEIN MARKEDLY AFFECT PROCESSING
Susan-Marie E. Harding1,2, James Camakaris1,2, Roberto Cappai3,4, Andrew F. Hill5,3, Ashley I. Bush2,3, 1The University of Melbourne, Department of Genetics, Victoria, Australia; 2The Mental Health Research Institute, Victoria, Australia; 3The University of Melbourne, Department of Pathology, Victoria, Australia; 4The University of Melbourne, Centre for Neuroscience, Victoria, Australia; 5Bio21, Department of Biochemistry and Molecular Biology, Australia. Contact e-mail: [email protected] Background: Over-expression of the amyloid precursor protein (APP) results in significantly reduced copper and iron levels in transgenic mice brains. Conversely, APP and APLP2 genetic ablation both cause neuronal copper accumulation. Cellular copper up-regulates the expression of the APP gene. APP has two histidine-mediated ectodomain Cu2⫹ binding sites, one in the amino terminal region, and another in the A domain. Both sites reduce Cu2⫹ to Cu1⫹. BACE1 interacts with the copper chaperone of SOD1, which plays an important role in copper homeostasis. We hypothesized that the APP Cu2⫹ binding sites may be key in sensing Cu2⫹ and then regulating the processing of APP for its possible participation in copper homeostasis. Objective(s): To investigate the differential processing of APP in response to the ablation of the copper binding sites and/or the manipulation of cellular copper levels. Methods: Chinese hamster ovary (CHO) cells were transfected with human APP695 (wtAPP) or mutant APP including; Swedish mutant, London mutant, a mutant of the amino-terminal Cu binding site (APP⌬Cu1), or APP with the A Cu binding site mutated (APP⌬Cu2). Cells were cultivated in basal medium, medium containing excess copper, or copper chelators. Extracellular, soluble (sAPP) and membrane (full length, flAPP) fractions were analyzed for APP levels (using Western blot) and copper content (using ICP mass spectrometry). Results: The processing of APP⌬Cu1and APP⌬Cu2 was markedly different from that of wtAPP. The most significant differences were; 1. The APP⌬Cu1 mutant exhibited a marked increase in membranous flAPP compared to wtAPP. 2. The APP⌬Cu2 mutant was processed such that the ration of flAPP was higher than that of sAPP compared to wtAPP. Further analysis of APP fragments and the effects of modulating copper levels will be reported. Conclusions: These results indicate that the integrity of the APP copper binding sites are important in APP processing. P3-250
UNNATURAL AMINO ACID-SUBSTITUTED (HYDROXYETHYL)UREA PEPTIDOMIMETICS INHIBIT GAMMA-SECRETASE WITH ENHANCED STABILITY
Yung-Feng Liao1, Bo-Jeng Wang1, Chia-Yin Liao2, Shin-Ying Wu1, Ming-Kuan Hu2, 1Academia Sinica, Taipei, Taiwan, Province of China; 2 National Defense Medical Center, Taipei, Taiwan, Province of China. Contact e-mail: [email protected]