- Email: [email protected]
P1-174 The role of the APP intracellular domain (AICD) in gene expression: implications for Alzheimer's disease
Poster Session PI : Molecular Mechanisms of Neurodegeneration - ~-Amyloidosis
•J]
PROTEOMIC ANALYSIS OF THE HIPPOCAMPUS IN TRANSGENIC RATS CARRYING HUMAN APP
Kate E. Wilson* 1, John Prime 2, Paul Pashby 2, Paul Orange 2, Edward Hawkins 2, Rita Marouga 3 , Helene B ergling 3, Chris M. Morris 1.
1Institute for Ageing and Health, Newcastle upon Tyne, United Kingdom; 2Amersham Biosciences, Little Chalfont, United Kingdom; 3Amersham Biosciences, UppsaIa, Sweden. Contact e-mail: [email protected] Background: Transgenic animal models of Alzheimer's disease (AD) are useful tools in the analysis of the molecular basis of this disease. Transgenic rats carrying the human Swedish amyloid precursor protein mutation have been well characterised using genomic studies. However, since post translational modifications and alternative splice variants can alter the final protein, proteomic studies allow a global approach to the analysis of the 'business end' of the cell. Objectlve(s): In the current study proteomic techniques have been used in order to identify changes in the APP Swedish mutation model compared to wild type animals. Methods: 2D electrophnresis is a widely used technique capable of separating thousands of proteins from complex mixtures. Recently 2D-difference gel electrophoresis (DIGE) has been developed allowing accurate quantitation of changes in protein abundance between control and test samples, a key aspect in proteomic studies. Labelling different samples with spectrally resolvable dyes allows the incorporation of the same internal standard into all gels within an experiment. When this standard is multiplexed with the analytical sample on a single gel, gel-to-gel variation is reduced, improving both reproducibility and accuracy of results. Laser capture microdissection has been used to obtain a sample enriched for the CA1 region of the hippocampus and analysed using CyDye DIGE Fluors from Amersham's Scarce Sample Labelling Kit for the comparison of tissue obtained from transgenic and wild type animals by LCM. Conclusions: Of the 128 differentially expressed proteins those identified are involved in both normal cellular function and pathological events, including energy metabolism, synaptic function, protein degradation pathways and oxidative stress. These results provide an insight into possible mechanisms and interactions which may contribute to the pathogenesis of AD.
~ T H E
ROLE OF THE APP INTRACELLULAR DOMAIN (AICD) IN GENE EXPRESSION: IMPLICATIONS FOR ALZHEIMER'S DISEASE
Rupert Egensperger* 1, Thorsten Mueller 1, Donat K6gel 2, Jochen H. Prehn 3. 1University Hospital Muenster, Muenster, Germany; 2Johann
Wolfgang Goethe University Frankfurt, Frankfurt, Germany; 3Royal College of Surgeons in Ireland, Department of Physiology, Dublin, Ireland. Contact e-mail: [email protected] Background: The beta-Amyloid Precursor Protein (APP), a type-1 transmembrane protein, plays a fundamental role in the pathophysiology of Alzheimer's disease (AD). Thereby APP is cleaved by beta-secretase at the N-terminus of the Abeta domain. The generated cleavage fragment C99 can be further processed by the activity of gamma-secretase complex to yield Abeta, the major constituent of Alzheimer Plaques, and AICD, the APP intracellular domain. AICD has been shown to bind to several interaction partners, among them Fe65 and Tip60. Recent findings indicate that in analogy to the Notch intracellular domain, AICD (in complex with other proteins) may serve as a membrane-to-nucleus messenger, thus linking APP to transcriptional processes. As another function of AICD, its capability to induce apoptosis via Tip60 has been reported. Objective(s): Our study first aimed to elucidate putative transcriptional target genes of AICD using GeneChips. Furthermore, we analyzed AICD dependent Caspase-3 activity. Methods: We cloned two AICD variants (AICD50 and AICD59) into Doxycycline dependent inducible expression vectors and generated human neuroblastoma cell lines (SHEP-SF) inducible expressing one of the two AICD variants (Tet-Off system). Affymetrix U133A GeneChips were employed to compare the expression profile of SHEP-SF cells under AICD induced versus non-induced conditions. Caspase-3 activity in constitutively stable expressing AICD versus mock cells was measured using fluorometric activity assay following 1 ttM Thapsigargin stimulation. Results: Analyses
S 145
of microarray data with Expressionist software (GeneData) revealed a more than 1,5-fold increase or decrease in expression of 12 genes, when using stringent statistical criteria for differential expression. These genes were involved in regulation of transcription, DNA repair, and others. Caspase-3 activity measurement revealed a significant role of AICD in cell death induced by ER Ca 2÷ depletion. Conclusions: We show that AICD overexpression significantly enhances Caspase-3 activation after Ca 2÷ depletion from the ER stores. As we could not find AICD dependent expression of genes involved in apoptosis, our data suggest a transcriptional independent mechanism for AICD induced cell death.
•
DEVELOPMENT OF CONFORMATION-SPECIFIC ANTIBODIES FOR NEUTRALIZATION OF 13-AMYLOID OLIGOMERS
Young Soo Kim*, Jason A. Moss, Kim D. Janda. The Scripps Research Institute, La Jolla, CA, USA. Contact e-mail: [email protected]
Background: Several immunotherapeutic strategies have been proposed to delay the onset of AD or reverse its degenerative effects. While insoluble beta-Amyloid (A[~) fibrils were originally believed to be the primary pathogenic species in AD, recent data suggest a pathogenic role for soluble A[~ oligomers. In this session, we present our synthetic results in the preparation of these macromolecular haptens. Objective(s): We have synthesized conformationally-defined A[~ haptens for the selection of antibodies that can selectively sequester these folding intermediates. Methods & Results: Herein, we report high-yielding synthesis and purification of native A[~(142) by optimized solid phase peptide synthesis protocols. Our purified native A~(1-42) peptide shows identical HPLC and mass spectrometry characteristics to commercially derived material. Critical in our synthesis and purification of A[3(1-42) is several modifications inspired by the known biophysical properties of A[~. Conclusions: With reliable synthetic protocols in hand, we are now preparing telechelic A[3(1-42) analogues required for the selection of conformation-specific anti-A[3(1-42) human scFv antibodies. We envision that these antibodies may prove useful for therapeutic and/or diagnostic purposes.
•
AGE-RELATED CHANGES OF ALZHEIMER'S DISEASE-ASSOCIATED PROTEINS IN CYNOMOLGUS MONKEY BRAINS
Nobuyu!d Kimura* i Kentaro Tanemura 2, Shin-Ichiro Nakamura 3, Akihiko Takashima 2, Katsuhiko Yanagisawa 4 , Keiji Terao 5, Fumiko Ono 5, Yoshiyuld Ishii 1, Shigeru Kyuwa 1, Yasuhiro Yoshikawa 1.
1Graduate School of Agricultural and Life Sciences, The University of Tokyo, Tokyo, Japan; 2Laboratory for Alzheimer's Disease, Brain Science Institute, RIKEN, Saitama, Japan; 3Department of Pathology, Nippon Veterinary and Animal Science University, Tokyo, Japan; 4Department of Dementia Research, National Institute for Longevity Sciences, Aichi, Japan; 5The Tsukuba Primate Center, National Institute of lnfectious Diseases, Ibaraki, Japan. Contact e-mail: [email protected] We characterized senile plaques (SPs) immunohistochemically in cynomolgus monkey brains, and also examined age-related biochemical changes of SP-associated proteins in these brains from monkeys of various ages. In the neocortex of aged monkeys (>20 years old), antibodies against [%amyloid precursor protein (APP) or apolipoprotein E (ApoE) stained SPs; however, the pattern of immunostaining was different for the two antigens. APP was present only in swollen neurites, but ApoE was present throughout all parts of SPs. Western blot analysis revealed that the pattern of APP expression changed with age. Although full-length APP695 protein was mainly expressed in brains from young monkeys (4 years old), the expression of full-length APP751 protein was increased in brains from older monkeys (> 20 years old). We also biochemically characterized intracellular A~, and it revealed that intracellular A[3 generation changed with age. In the microsomai fraction, AIM3 was mainly generated in brains from young monkey (4 years of age). The amount of A~42 significantly increased in brains from older monkeys (>30 years of age), and the amount of AI343
•J]
PROTEOMIC ANALYSIS OF THE HIPPOCAMPUS IN TRANSGENIC RATS CARRYING HUMAN APP
Kate E. Wilson* 1, John Prime 2, Paul Pashby 2, Paul Orange 2, Edward Hawkins 2, Rita Marouga 3 , Helene B ergling 3, Chris M. Morris 1.
1Institute for Ageing and Health, Newcastle upon Tyne, United Kingdom; 2Amersham Biosciences, Little Chalfont, United Kingdom; 3Amersham Biosciences, UppsaIa, Sweden. Contact e-mail: [email protected] Background: Transgenic animal models of Alzheimer's disease (AD) are useful tools in the analysis of the molecular basis of this disease. Transgenic rats carrying the human Swedish amyloid precursor protein mutation have been well characterised using genomic studies. However, since post translational modifications and alternative splice variants can alter the final protein, proteomic studies allow a global approach to the analysis of the 'business end' of the cell. Objectlve(s): In the current study proteomic techniques have been used in order to identify changes in the APP Swedish mutation model compared to wild type animals. Methods: 2D electrophnresis is a widely used technique capable of separating thousands of proteins from complex mixtures. Recently 2D-difference gel electrophoresis (DIGE) has been developed allowing accurate quantitation of changes in protein abundance between control and test samples, a key aspect in proteomic studies. Labelling different samples with spectrally resolvable dyes allows the incorporation of the same internal standard into all gels within an experiment. When this standard is multiplexed with the analytical sample on a single gel, gel-to-gel variation is reduced, improving both reproducibility and accuracy of results. Laser capture microdissection has been used to obtain a sample enriched for the CA1 region of the hippocampus and analysed using CyDye DIGE Fluors from Amersham's Scarce Sample Labelling Kit for the comparison of tissue obtained from transgenic and wild type animals by LCM. Conclusions: Of the 128 differentially expressed proteins those identified are involved in both normal cellular function and pathological events, including energy metabolism, synaptic function, protein degradation pathways and oxidative stress. These results provide an insight into possible mechanisms and interactions which may contribute to the pathogenesis of AD.
~ T H E
ROLE OF THE APP INTRACELLULAR DOMAIN (AICD) IN GENE EXPRESSION: IMPLICATIONS FOR ALZHEIMER'S DISEASE
Rupert Egensperger* 1, Thorsten Mueller 1, Donat K6gel 2, Jochen H. Prehn 3. 1University Hospital Muenster, Muenster, Germany; 2Johann
Wolfgang Goethe University Frankfurt, Frankfurt, Germany; 3Royal College of Surgeons in Ireland, Department of Physiology, Dublin, Ireland. Contact e-mail: [email protected] Background: The beta-Amyloid Precursor Protein (APP), a type-1 transmembrane protein, plays a fundamental role in the pathophysiology of Alzheimer's disease (AD). Thereby APP is cleaved by beta-secretase at the N-terminus of the Abeta domain. The generated cleavage fragment C99 can be further processed by the activity of gamma-secretase complex to yield Abeta, the major constituent of Alzheimer Plaques, and AICD, the APP intracellular domain. AICD has been shown to bind to several interaction partners, among them Fe65 and Tip60. Recent findings indicate that in analogy to the Notch intracellular domain, AICD (in complex with other proteins) may serve as a membrane-to-nucleus messenger, thus linking APP to transcriptional processes. As another function of AICD, its capability to induce apoptosis via Tip60 has been reported. Objective(s): Our study first aimed to elucidate putative transcriptional target genes of AICD using GeneChips. Furthermore, we analyzed AICD dependent Caspase-3 activity. Methods: We cloned two AICD variants (AICD50 and AICD59) into Doxycycline dependent inducible expression vectors and generated human neuroblastoma cell lines (SHEP-SF) inducible expressing one of the two AICD variants (Tet-Off system). Affymetrix U133A GeneChips were employed to compare the expression profile of SHEP-SF cells under AICD induced versus non-induced conditions. Caspase-3 activity in constitutively stable expressing AICD versus mock cells was measured using fluorometric activity assay following 1 ttM Thapsigargin stimulation. Results: Analyses
S 145
of microarray data with Expressionist software (GeneData) revealed a more than 1,5-fold increase or decrease in expression of 12 genes, when using stringent statistical criteria for differential expression. These genes were involved in regulation of transcription, DNA repair, and others. Caspase-3 activity measurement revealed a significant role of AICD in cell death induced by ER Ca 2÷ depletion. Conclusions: We show that AICD overexpression significantly enhances Caspase-3 activation after Ca 2÷ depletion from the ER stores. As we could not find AICD dependent expression of genes involved in apoptosis, our data suggest a transcriptional independent mechanism for AICD induced cell death.
•
DEVELOPMENT OF CONFORMATION-SPECIFIC ANTIBODIES FOR NEUTRALIZATION OF 13-AMYLOID OLIGOMERS
Young Soo Kim*, Jason A. Moss, Kim D. Janda. The Scripps Research Institute, La Jolla, CA, USA. Contact e-mail: [email protected]
Background: Several immunotherapeutic strategies have been proposed to delay the onset of AD or reverse its degenerative effects. While insoluble beta-Amyloid (A[~) fibrils were originally believed to be the primary pathogenic species in AD, recent data suggest a pathogenic role for soluble A[~ oligomers. In this session, we present our synthetic results in the preparation of these macromolecular haptens. Objective(s): We have synthesized conformationally-defined A[~ haptens for the selection of antibodies that can selectively sequester these folding intermediates. Methods & Results: Herein, we report high-yielding synthesis and purification of native A[~(142) by optimized solid phase peptide synthesis protocols. Our purified native A~(1-42) peptide shows identical HPLC and mass spectrometry characteristics to commercially derived material. Critical in our synthesis and purification of A[3(1-42) is several modifications inspired by the known biophysical properties of A[~. Conclusions: With reliable synthetic protocols in hand, we are now preparing telechelic A[3(1-42) analogues required for the selection of conformation-specific anti-A[3(1-42) human scFv antibodies. We envision that these antibodies may prove useful for therapeutic and/or diagnostic purposes.
•
AGE-RELATED CHANGES OF ALZHEIMER'S DISEASE-ASSOCIATED PROTEINS IN CYNOMOLGUS MONKEY BRAINS
Nobuyu!d Kimura* i Kentaro Tanemura 2, Shin-Ichiro Nakamura 3, Akihiko Takashima 2, Katsuhiko Yanagisawa 4 , Keiji Terao 5, Fumiko Ono 5, Yoshiyuld Ishii 1, Shigeru Kyuwa 1, Yasuhiro Yoshikawa 1.
1Graduate School of Agricultural and Life Sciences, The University of Tokyo, Tokyo, Japan; 2Laboratory for Alzheimer's Disease, Brain Science Institute, RIKEN, Saitama, Japan; 3Department of Pathology, Nippon Veterinary and Animal Science University, Tokyo, Japan; 4Department of Dementia Research, National Institute for Longevity Sciences, Aichi, Japan; 5The Tsukuba Primate Center, National Institute of lnfectious Diseases, Ibaraki, Japan. Contact e-mail: [email protected] We characterized senile plaques (SPs) immunohistochemically in cynomolgus monkey brains, and also examined age-related biochemical changes of SP-associated proteins in these brains from monkeys of various ages. In the neocortex of aged monkeys (>20 years old), antibodies against [%amyloid precursor protein (APP) or apolipoprotein E (ApoE) stained SPs; however, the pattern of immunostaining was different for the two antigens. APP was present only in swollen neurites, but ApoE was present throughout all parts of SPs. Western blot analysis revealed that the pattern of APP expression changed with age. Although full-length APP695 protein was mainly expressed in brains from young monkeys (4 years old), the expression of full-length APP751 protein was increased in brains from older monkeys (> 20 years old). We also biochemically characterized intracellular A~, and it revealed that intracellular A[3 generation changed with age. In the microsomai fraction, AIM3 was mainly generated in brains from young monkey (4 years of age). The amount of A~42 significantly increased in brains from older monkeys (>30 years of age), and the amount of AI343