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P4-175 The proteasome regulates presenilin 1 levels during apoptosis
Poster Session P4: Molecular Mechanisms of Neurodegeneration - Enzyme Activities mutations are used for studying modulators of the APP processing pathway (e.g. HEK293-APP swe/l°n cells studied herein). Conclusions: NT2N cells however provide a cell culture system that more closely mimics native human neurons in that they express wild type forms APP and BACE proteins at modest levels. We developed a 96 well format assay to assess the potency of peptidic and non-peptidic BACE inhihitors to suppress the production of secreted AIM0 from NT2N cells and compared this to our HEK293 APP swe/l°n stably transfected cell line.
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NEUROPEPTIDES REGULATE BRAIN AMYLOID BETA LEVELS THROUGH A MODULATION OF NEPRILYSIN ACTIVITY
Takashi Saito*, Nobuhisa Iwata, Yoshie Takaki, Makoto Higuchi, Emi Hosoki, Misaki Sekiguchi, Yukio Matsuba, Takaomi C. Saido. RIKEN Brain Science Institute, Wako-shi, Saitama, Japan. Contact e-mail: takasai @brain.riken.jp The deposition of amyloid beta peptide (Abeta) initiates the pathological cascade leading to Alzheimer's disease (AD). While in familial AD cases, the deposition is known to be caused by increased Abeta production, in most of the other cases, decreased clearance may be involved, which also would lead to increased steady-state levels of Abeta in the brain. We previously reported that neprilysin, a neutral endopeptidase family member, is a major Abeta -degrading enzyme in vivo. Although the enzymology of neprilysin is well known, the regulatory mechanism associated with its actions, particularly in the brain, are not well understood. We established in vitro assay system to evaluate neprilysin activity, and found that some neuropeptides regulates its activity. In particular, somatostatin was found to induce an increase in neprilysin activity. In addition, roles of neuropeptides in regulation of neprilysin activity are also analyzed by using reverse genetic systems. An understanding of precisely how neprilysin activity is regulated could lead to the development of prevention and therapeutic strategies for decreasing Abeta levels. Supported by: RIKEN BSI and Grant-in-Aid for Young Scientists (B) from MEXT.
~ T H E PROTEASOME REGULATES PRESENILIN 1 LEVELS DURING APOPTOSIS Maria Ankarcrona*, Angel Cedazo-Minguez, Bengt Winblad, Bogdan O. Popescu. Karolinska Institutet, Huddinge, Sweden. Contact e-mail: maria.ankarcrona @neurotec.ki.se Background: Mutations of genes encoding presenilins are found in a majority of familial Alzheimer's disease cases. Mutant presenilins increase the production of longer [3-amyloid species and sensitize cells to death by triggering various pro-apoptotic mechanisms. Presenilins are subject to complex proteolytic processing (eg. endopmteolysis, caspases, calpains, proteasome activity) and alternative presenilin fragments seem to play a role in regulation of cell death. Objectives: To study the processing of presenilin 1 (PS 1) and the fate of PS 1 resultant species during staurosporine-induced apoptosis. Methods: For these studies we used SH-SY5Y neuroblastoma cells non-transfected or transfected with either wild type or exon 9 deleted PSI. Cells were treated with staurosporin in the presence or absence of proteasome or caspase inhibitors. Immunoblotting was used to detect the different PS1 fragments generated during apoptosis. Results: We report that PS1 is processed both by caspases and the proteasome during apoptosis. Proteasome inhibitors were able to block the degradation of fulllength PS 1 and caspase cleaved N-terminal and C-terminal PS 1 fragments. Interestingly, staurosporine treatment resulted in complete proteasome mediated degradation of full length PS 1 in wild-type cells, while the fulllength exon 9 deleted variant of PS 1 was resistant to proteasome degradation. Conclusions: We conclude that the levels of full-length wild-type PS 1 and alternative PS 1 fragments generated during apoptosis can be modulated by proteasome activation or inhibition.
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$525
DETERMINING THE MINIMAL SUBSTRATE SEQUENCE REQUIREMENT FOR BACE
Hsiu-Chinng Yang*, Raymond Patch, Marian Mosior, Wu-Kuang Yeh, Don McClure, Sheila Little, Patrick May, Patricia Gonzalez-DeWhitt, Yuan Tu. Eli Lilly and Company, Indianapolis, IN, USA. Contact e-mail: Ginnie_Yang @Lilly. corn Background: Beta-secretase performs the first proteolytic cleavage of the amyloid precursor protein (APP) to generate sAPP~ and C99. Subsequently, gamma-secretase hydrolyzes C99 and produces amyloid-beta (AI3) peptides. The accumulation of A~ peptides in brain parenchyma is believed to be essential to progression of Alzheimer Disease. Therefore, the inhibition of BACE limits the availability of substrate (C99) for gamma-secretase and, thereby, reduces the Abeta production. The BACE-gene knockout mouse studies demonstrated that BACE is the sole beta-secretase in neurons. Objectives: The minimal substrate sequence requirement is an important information for the design of protease inhibitors. This information is also critical for the design of a sensitive and selective protease assay. Methods: Peptides derived from sequential amino- and carboxyl-terminal truncations of a decahexapeptide, derived from the APP sequence, were prepared by parallel peptide synthesis. All peptides were tested for their ability to compete with the labeled substrate in the BACE FRET (Fluorescent Resonance Energy Transfer) assay. Peptides that showed inhibitory effect in the original assay, were resynthesized as FRET peptides for further confirmation. Results and Conclusions: Using this approach, we have quickly identified the shortest peptide sequence for the BACE substrate. This information provided input for the design of a sensitive BACE assay.
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HIERARCHICAL PHOSPHORYLATION OF CHOLINE ACETYLTRANSFERASE BY PROTEIN KINASE-C AND REGULATION OF ITS ENZYMATIC ACTIVITY
Tomas Dobransky* 1, Amanda Doherty-Kirby 2, Gilles Lajoie 2, Rebecca Jane Rylett 2,I . JRobarts Research Institute, London, ON, Canada; 2University of Western Ontario, London, ON, Canada. Contact e-mail: tomas.dobransky @find. uwo. ca Background: The expression and activity of choline acetyltransferase (CHAT), the enzyme that catalyzes the synthesis of acetylcholine (ACh), decreases during the course of Alzheimer disease leading to dysfunction of cholinergic neurotransmission. ChAT undergoes multi-site phosphorylation involving several protein kinases, and thus alterations in either the activity or subcellular dislribution of protein kinases brought about by neuronal perturbations or pathology could result in changes in ChAT function and ACh production. Objectives: To better understand the regulation of ChAT by protein kinase-C (PK-C), we undertook to identify all of the sites in purified recombinant human 69-kDa ChAT that are phosphorylated by PK-C in vitro. Methods and Results: Using a combination of MALDI-TOF and ESI-MS/MS analysis and site-directed mutagenesis, we determined that Ser-346, Ser-347 and Ser-476 and Thr-255 are phosphorytated by various PK-C isoenzymes; we identified Ser-440 as a PK-C phosphorylation site previously and that this played a role in regulating ChAT activity and subcellular distribution. Phosphorylation of ChAT by PK-C isoenzymes was found to have a hierarchical relationship in that phosphorylation at Ser-476 was required prior to phosphorylation of any of the other serine residues, but not the threonine residue. Several loss-of-function spontaneous single nucleotide polymorphisms (SNPs) have been reported recently for CHAT, including an Arg-442 to histidine mutation; this results in a catalyticallyinactive form of the enzyme. We predicted that this arginine residue forms an important part of the consensus sequence for recognition of Ser-440 by PK-C. To test this, we mutated Arg-442 to both histidine and alanine residues and found that PK-C is unable to phosphorylate Set-440 and that the enzyme is essentially inactive. Conclusions: These studies provide a new line of investigation into cellular events that may control ChAT activity and contribute to dysregulation of communication by cholinergic neurons. (This work is supported by a grant from Canadian Institute for Health Research to RJR)
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NEUROPEPTIDES REGULATE BRAIN AMYLOID BETA LEVELS THROUGH A MODULATION OF NEPRILYSIN ACTIVITY
Takashi Saito*, Nobuhisa Iwata, Yoshie Takaki, Makoto Higuchi, Emi Hosoki, Misaki Sekiguchi, Yukio Matsuba, Takaomi C. Saido. RIKEN Brain Science Institute, Wako-shi, Saitama, Japan. Contact e-mail: takasai @brain.riken.jp The deposition of amyloid beta peptide (Abeta) initiates the pathological cascade leading to Alzheimer's disease (AD). While in familial AD cases, the deposition is known to be caused by increased Abeta production, in most of the other cases, decreased clearance may be involved, which also would lead to increased steady-state levels of Abeta in the brain. We previously reported that neprilysin, a neutral endopeptidase family member, is a major Abeta -degrading enzyme in vivo. Although the enzymology of neprilysin is well known, the regulatory mechanism associated with its actions, particularly in the brain, are not well understood. We established in vitro assay system to evaluate neprilysin activity, and found that some neuropeptides regulates its activity. In particular, somatostatin was found to induce an increase in neprilysin activity. In addition, roles of neuropeptides in regulation of neprilysin activity are also analyzed by using reverse genetic systems. An understanding of precisely how neprilysin activity is regulated could lead to the development of prevention and therapeutic strategies for decreasing Abeta levels. Supported by: RIKEN BSI and Grant-in-Aid for Young Scientists (B) from MEXT.
~ T H E PROTEASOME REGULATES PRESENILIN 1 LEVELS DURING APOPTOSIS Maria Ankarcrona*, Angel Cedazo-Minguez, Bengt Winblad, Bogdan O. Popescu. Karolinska Institutet, Huddinge, Sweden. Contact e-mail: maria.ankarcrona @neurotec.ki.se Background: Mutations of genes encoding presenilins are found in a majority of familial Alzheimer's disease cases. Mutant presenilins increase the production of longer [3-amyloid species and sensitize cells to death by triggering various pro-apoptotic mechanisms. Presenilins are subject to complex proteolytic processing (eg. endopmteolysis, caspases, calpains, proteasome activity) and alternative presenilin fragments seem to play a role in regulation of cell death. Objectives: To study the processing of presenilin 1 (PS 1) and the fate of PS 1 resultant species during staurosporine-induced apoptosis. Methods: For these studies we used SH-SY5Y neuroblastoma cells non-transfected or transfected with either wild type or exon 9 deleted PSI. Cells were treated with staurosporin in the presence or absence of proteasome or caspase inhibitors. Immunoblotting was used to detect the different PS1 fragments generated during apoptosis. Results: We report that PS1 is processed both by caspases and the proteasome during apoptosis. Proteasome inhibitors were able to block the degradation of fulllength PS 1 and caspase cleaved N-terminal and C-terminal PS 1 fragments. Interestingly, staurosporine treatment resulted in complete proteasome mediated degradation of full length PS 1 in wild-type cells, while the fulllength exon 9 deleted variant of PS 1 was resistant to proteasome degradation. Conclusions: We conclude that the levels of full-length wild-type PS 1 and alternative PS 1 fragments generated during apoptosis can be modulated by proteasome activation or inhibition.
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$525
DETERMINING THE MINIMAL SUBSTRATE SEQUENCE REQUIREMENT FOR BACE
Hsiu-Chinng Yang*, Raymond Patch, Marian Mosior, Wu-Kuang Yeh, Don McClure, Sheila Little, Patrick May, Patricia Gonzalez-DeWhitt, Yuan Tu. Eli Lilly and Company, Indianapolis, IN, USA. Contact e-mail: Ginnie_Yang @Lilly. corn Background: Beta-secretase performs the first proteolytic cleavage of the amyloid precursor protein (APP) to generate sAPP~ and C99. Subsequently, gamma-secretase hydrolyzes C99 and produces amyloid-beta (AI3) peptides. The accumulation of A~ peptides in brain parenchyma is believed to be essential to progression of Alzheimer Disease. Therefore, the inhibition of BACE limits the availability of substrate (C99) for gamma-secretase and, thereby, reduces the Abeta production. The BACE-gene knockout mouse studies demonstrated that BACE is the sole beta-secretase in neurons. Objectives: The minimal substrate sequence requirement is an important information for the design of protease inhibitors. This information is also critical for the design of a sensitive and selective protease assay. Methods: Peptides derived from sequential amino- and carboxyl-terminal truncations of a decahexapeptide, derived from the APP sequence, were prepared by parallel peptide synthesis. All peptides were tested for their ability to compete with the labeled substrate in the BACE FRET (Fluorescent Resonance Energy Transfer) assay. Peptides that showed inhibitory effect in the original assay, were resynthesized as FRET peptides for further confirmation. Results and Conclusions: Using this approach, we have quickly identified the shortest peptide sequence for the BACE substrate. This information provided input for the design of a sensitive BACE assay.
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HIERARCHICAL PHOSPHORYLATION OF CHOLINE ACETYLTRANSFERASE BY PROTEIN KINASE-C AND REGULATION OF ITS ENZYMATIC ACTIVITY
Tomas Dobransky* 1, Amanda Doherty-Kirby 2, Gilles Lajoie 2, Rebecca Jane Rylett 2,I . JRobarts Research Institute, London, ON, Canada; 2University of Western Ontario, London, ON, Canada. Contact e-mail: tomas.dobransky @find. uwo. ca Background: The expression and activity of choline acetyltransferase (CHAT), the enzyme that catalyzes the synthesis of acetylcholine (ACh), decreases during the course of Alzheimer disease leading to dysfunction of cholinergic neurotransmission. ChAT undergoes multi-site phosphorylation involving several protein kinases, and thus alterations in either the activity or subcellular dislribution of protein kinases brought about by neuronal perturbations or pathology could result in changes in ChAT function and ACh production. Objectives: To better understand the regulation of ChAT by protein kinase-C (PK-C), we undertook to identify all of the sites in purified recombinant human 69-kDa ChAT that are phosphorylated by PK-C in vitro. Methods and Results: Using a combination of MALDI-TOF and ESI-MS/MS analysis and site-directed mutagenesis, we determined that Ser-346, Ser-347 and Ser-476 and Thr-255 are phosphorytated by various PK-C isoenzymes; we identified Ser-440 as a PK-C phosphorylation site previously and that this played a role in regulating ChAT activity and subcellular distribution. Phosphorylation of ChAT by PK-C isoenzymes was found to have a hierarchical relationship in that phosphorylation at Ser-476 was required prior to phosphorylation of any of the other serine residues, but not the threonine residue. Several loss-of-function spontaneous single nucleotide polymorphisms (SNPs) have been reported recently for CHAT, including an Arg-442 to histidine mutation; this results in a catalyticallyinactive form of the enzyme. We predicted that this arginine residue forms an important part of the consensus sequence for recognition of Ser-440 by PK-C. To test this, we mutated Arg-442 to both histidine and alanine residues and found that PK-C is unable to phosphorylate Set-440 and that the enzyme is essentially inactive. Conclusions: These studies provide a new line of investigation into cellular events that may control ChAT activity and contribute to dysregulation of communication by cholinergic neurons. (This work is supported by a grant from Canadian Institute for Health Research to RJR)