- Email: [email protected]
O2-03-03: Depletion of GGA3 regulates BACE in vivo
T136
Oral O2-03: Disease Mechanisms: Others
fied homolog of NEP, is sensitive to thiorphan and phosphoramidon, and has been shown to degrade A. Therefore, NEP2 may be an important A degrading enzyme. Methods: Knockout mice deficient for NEP2 and NEP were analyzed in this study. Specific ELISA for A was used for quantification in vitro and in vivo. Immunoblot and flow cytometry utilizing specific antibodies were used for molecular characterization of NEP2. Results: We have investigated the role of NEP2 in A degradation in vivo. Aged mice deficient for the NEP2 gene showed a 3 fold increase in A42 levels compared to age and strain matched controls. Furthermore, knockout mice lacking NEP, showed a gene dose dependent increase in A42 levels inversely correlated with NEP2 gene copy number. Only mild effects were seen on A40 levels. We have also characterized the human homolog of NEP2. hNEP2 was shown to mediate the degradation of cell produced A40 and A42, however, not all splice forms were active. The active variant of NEP2 was shown to be a type-II integral membrane protein similar to NEP. It was also found to be sensitive to thiorphan and phosphoramidon. Finally, NEP2 expression was detected in human brain tissue by RT-PCR. Conclusions: These findings suggest NEP2 is required for maintenance of normal A levels in the brain. O2-03-03
DEPLETION OF GGA3 REGULATES BACE IN VIVO
Giuseppina Tesco1, Andrew N. Cameron1, Eugene L. Kang1, Rudolph E. Tanzi1, Michael J. Whalen2, 1Genetics and Aging Research Unit, Massachusetts General Hospital, Charlestown, MA, USA; 2 Department of Pediatric Critical Care Medicine, Massachusetts General Hospital, Charlestown, MA, USA. Contact e-mail: [email protected] Background: BACE levels are elevated in Alzheimer’s disease (AD) brains, and increasing evidence reveals BACE to be a stress-related protease that is upregulated following cerebral ischemia and traumatic brain injury (TBI), risk factors for AD. However, the molecular mechanism responsible for BACE upregulation in AD, ischemia and TBI has not been identified. We recently reported that BACE and -secretase activity increase following cerebral ischemia in vivo and caspase activation in vitro due to post-translational stabilization of BACE protein. We also found that the impaired degradation of BACE is due to caspase-mediated depletion of GGA3, an adaptor protein involved in BACE trafficking. Moreover, we have found that GGA3 levels are decreased while BACE levels are increased in the brain of AD subjects (Tesco et al. Neuron 2007). Objective(s): We set out to determine whether 1) caspase-mediated depletion of GGA3 and increased levels of BACE are observed following TBI in a mouse model; 2) GGA3 genetic ablation regulates levels of BACE in mouse brain. Methods: GGA3 deficient mice were created from microinjection of E14Tg2a.4 from 129P2/OlaHsd embryonic stem (ES) cells generated by BayGenomics. Head trauma was induced by controlled cortical impact model (CCI). Results: We have found that GGA3 is depleted following TBI while BACE protein levels increase with a pattern similar to the one observed following cerebral ischemia. Furthermore, inter-crosses of GGA3⫹/mice produced GGA3-/- mice in the normal Mendelian frequency of ⬃25%. GGA3-/- mice are healthy, fertile and appear normal at 6 months of age. However, levels of BACE are increased by ⬃70 and ⬃20 in the brain of GGA3-/- and GGA3-/⫹ mice, respectively. Conclusions: These new findings indicate that GGA3 depletion, mediated by caspase cleavage, and consequent BACE upregulation may be the common underlying mechanism of increased A production following cerebral ischemia and TBI. This mechanism may also explain how TBI leads to increased risk of developing AD over time. Furthermore, the lack or decreased levels of GGA3 results in increase cerebral levels of BACE suggesting that subjects with lower levels of GGA3 could be at greater risk of developing AD.
O2-03-04
APH1B GAMMA-SECRETASE GENERATES LONG ABETA PEPTIDES AND GENETIC ABLATION IMPROVES ALZHEIMER’S DISEASE PHENOTYPES WITHOUT AFFECTING NOTCH SIGNALING IN THE MOUSE
Lutgarde Serneels1, Je´roˆme Van Biervliet1, Katleen Craessaerts1, Tim Dejaegere1, Katrien Horre´1, Tine Van Houtvin1, Hermann Esselmann2, Sabine Paul3, Ben Sprangers4, Raf Sciot5, Lieve Moons6, Mathias Jucker7, Jens Wiltfang2, Rudi D’Hooge8, Bart De Strooper1, 1Department for Molecular and Developmental Genetics, VIB, and Center for Human Genetics, K.U. Leuven, Leuven, Belgium; 2Department of Psychiatry and Psychotherapy, Rhine State Hospital, Essen, University of Duisburg-Essen, Essen, Germany; 3 Department of Psychiatry and Psychotherapy, University of ErlangenNuremberg, Erlangen, Germany; 4Laboratory of Experimental Transplantation, K.U. Leuven, Leuven, Belgium; 5Laboratory of Morphology and Molecular Pathology, K.U. Leuven, Leuven, Belgium; 6 Department for Transgene Technology and Gene Therapy, VIB, and Center for Transgene Technology and Gene Therapy (CTG), K.U. Leuven, Leuven, Belgium; 7Department of Cellular Neurology, HertieInstitute for Clinical Brain Research, University of Tu¨bingen, Tu¨bingen, Germany; 8Laboratory of Biological Psychology, Department of Psychology, K.U. Leuven, Leuven, Belgium. Contact e-mail: [email protected] Background: Gamma-secretase cleavage releases the Abeta peptides of Alzheimer’s disease (AD), but is also involved in regulated intramembrane proteolysis of Notch and other important signaling pathways. Epsiloncleavage releases intracellular domains and is physiologically essential, while gamma-cleavages process the remnant peptide until it is soluble and is released. Abeta is heterogeneous as a consequence of gamma-activity and the ratio of long versus short Abeta is central to AD pathogenesis. Gamma-secretase activity is mediated by a multiprotein complex, minimally consisting of Presenilin 1 or 2 (Psen), Aph1A or B/C, Pen2 and Nicastrin. Differential association of Psen or Aph1 isoforms results in structural heterogeneity in the gamma-secretase complex in vivo. This structural diversity raises questions with regard to specificity and biological activity of the different complexes. Methods: In the current work, we analyze the activity in reconstituted knock-out cell lines, in purified complexes from mouse and human brain and test the in vivo consequences of this functional heterogeneity in a murine AD model. Results: In contrast with recent in vitro overexpression studies, we find significant differences in the profiles of the Abeta peptides generated by complexes with different Aph1 components. Furthermore, when we specifically inactivate the Aph1B/C gamma-secretase complex in a mouse AD model, we find remarkable improvements of disease-relevant phenotypes without serious Notch side effects. Conclusions: We show that gamma-secretase exists as structurally heterogeneous complexes and that the Aph1 subunit affects gamma-cleavage through a shift in the longer versus shorter Abeta species, but does not affect the release of intracellular domains. This allows dissociation of toxicity and therapeutic effects because specific inactivation of Aph1B(C) in an AD model results in decreased amyloid plaque, Abeta and Abeta oligomer accumulation, and rescue of AD-related phenotypes without significantly altering Notch signalling. Our work suggests that the different Aph1 gamma-secretases contribute differentially to biological and pathological functions and opens a new avenue for more specific and less toxic AD therapy. O2-03-05
MICROGLIA AND ALZHEIMER’S DISEASE: IMPLICATIONS OF A IMMUNOTHERAPY
Delphine Boche, Elina Zotova, Clive Holmes, James A. R. Nicoll, University of Southampton, Southampton, United Kingdom. Contact email: [email protected] Background: Over the past decade many studies have focused on neuroinflammation as a harmful feature of AD, with the implication that
Oral O2-03: Disease Mechanisms: Others
fied homolog of NEP, is sensitive to thiorphan and phosphoramidon, and has been shown to degrade A. Therefore, NEP2 may be an important A degrading enzyme. Methods: Knockout mice deficient for NEP2 and NEP were analyzed in this study. Specific ELISA for A was used for quantification in vitro and in vivo. Immunoblot and flow cytometry utilizing specific antibodies were used for molecular characterization of NEP2. Results: We have investigated the role of NEP2 in A degradation in vivo. Aged mice deficient for the NEP2 gene showed a 3 fold increase in A42 levels compared to age and strain matched controls. Furthermore, knockout mice lacking NEP, showed a gene dose dependent increase in A42 levels inversely correlated with NEP2 gene copy number. Only mild effects were seen on A40 levels. We have also characterized the human homolog of NEP2. hNEP2 was shown to mediate the degradation of cell produced A40 and A42, however, not all splice forms were active. The active variant of NEP2 was shown to be a type-II integral membrane protein similar to NEP. It was also found to be sensitive to thiorphan and phosphoramidon. Finally, NEP2 expression was detected in human brain tissue by RT-PCR. Conclusions: These findings suggest NEP2 is required for maintenance of normal A levels in the brain. O2-03-03
DEPLETION OF GGA3 REGULATES BACE IN VIVO
Giuseppina Tesco1, Andrew N. Cameron1, Eugene L. Kang1, Rudolph E. Tanzi1, Michael J. Whalen2, 1Genetics and Aging Research Unit, Massachusetts General Hospital, Charlestown, MA, USA; 2 Department of Pediatric Critical Care Medicine, Massachusetts General Hospital, Charlestown, MA, USA. Contact e-mail: [email protected] Background: BACE levels are elevated in Alzheimer’s disease (AD) brains, and increasing evidence reveals BACE to be a stress-related protease that is upregulated following cerebral ischemia and traumatic brain injury (TBI), risk factors for AD. However, the molecular mechanism responsible for BACE upregulation in AD, ischemia and TBI has not been identified. We recently reported that BACE and -secretase activity increase following cerebral ischemia in vivo and caspase activation in vitro due to post-translational stabilization of BACE protein. We also found that the impaired degradation of BACE is due to caspase-mediated depletion of GGA3, an adaptor protein involved in BACE trafficking. Moreover, we have found that GGA3 levels are decreased while BACE levels are increased in the brain of AD subjects (Tesco et al. Neuron 2007). Objective(s): We set out to determine whether 1) caspase-mediated depletion of GGA3 and increased levels of BACE are observed following TBI in a mouse model; 2) GGA3 genetic ablation regulates levels of BACE in mouse brain. Methods: GGA3 deficient mice were created from microinjection of E14Tg2a.4 from 129P2/OlaHsd embryonic stem (ES) cells generated by BayGenomics. Head trauma was induced by controlled cortical impact model (CCI). Results: We have found that GGA3 is depleted following TBI while BACE protein levels increase with a pattern similar to the one observed following cerebral ischemia. Furthermore, inter-crosses of GGA3⫹/mice produced GGA3-/- mice in the normal Mendelian frequency of ⬃25%. GGA3-/- mice are healthy, fertile and appear normal at 6 months of age. However, levels of BACE are increased by ⬃70 and ⬃20 in the brain of GGA3-/- and GGA3-/⫹ mice, respectively. Conclusions: These new findings indicate that GGA3 depletion, mediated by caspase cleavage, and consequent BACE upregulation may be the common underlying mechanism of increased A production following cerebral ischemia and TBI. This mechanism may also explain how TBI leads to increased risk of developing AD over time. Furthermore, the lack or decreased levels of GGA3 results in increase cerebral levels of BACE suggesting that subjects with lower levels of GGA3 could be at greater risk of developing AD.
O2-03-04
APH1B GAMMA-SECRETASE GENERATES LONG ABETA PEPTIDES AND GENETIC ABLATION IMPROVES ALZHEIMER’S DISEASE PHENOTYPES WITHOUT AFFECTING NOTCH SIGNALING IN THE MOUSE
Lutgarde Serneels1, Je´roˆme Van Biervliet1, Katleen Craessaerts1, Tim Dejaegere1, Katrien Horre´1, Tine Van Houtvin1, Hermann Esselmann2, Sabine Paul3, Ben Sprangers4, Raf Sciot5, Lieve Moons6, Mathias Jucker7, Jens Wiltfang2, Rudi D’Hooge8, Bart De Strooper1, 1Department for Molecular and Developmental Genetics, VIB, and Center for Human Genetics, K.U. Leuven, Leuven, Belgium; 2Department of Psychiatry and Psychotherapy, Rhine State Hospital, Essen, University of Duisburg-Essen, Essen, Germany; 3 Department of Psychiatry and Psychotherapy, University of ErlangenNuremberg, Erlangen, Germany; 4Laboratory of Experimental Transplantation, K.U. Leuven, Leuven, Belgium; 5Laboratory of Morphology and Molecular Pathology, K.U. Leuven, Leuven, Belgium; 6 Department for Transgene Technology and Gene Therapy, VIB, and Center for Transgene Technology and Gene Therapy (CTG), K.U. Leuven, Leuven, Belgium; 7Department of Cellular Neurology, HertieInstitute for Clinical Brain Research, University of Tu¨bingen, Tu¨bingen, Germany; 8Laboratory of Biological Psychology, Department of Psychology, K.U. Leuven, Leuven, Belgium. Contact e-mail: [email protected] Background: Gamma-secretase cleavage releases the Abeta peptides of Alzheimer’s disease (AD), but is also involved in regulated intramembrane proteolysis of Notch and other important signaling pathways. Epsiloncleavage releases intracellular domains and is physiologically essential, while gamma-cleavages process the remnant peptide until it is soluble and is released. Abeta is heterogeneous as a consequence of gamma-activity and the ratio of long versus short Abeta is central to AD pathogenesis. Gamma-secretase activity is mediated by a multiprotein complex, minimally consisting of Presenilin 1 or 2 (Psen), Aph1A or B/C, Pen2 and Nicastrin. Differential association of Psen or Aph1 isoforms results in structural heterogeneity in the gamma-secretase complex in vivo. This structural diversity raises questions with regard to specificity and biological activity of the different complexes. Methods: In the current work, we analyze the activity in reconstituted knock-out cell lines, in purified complexes from mouse and human brain and test the in vivo consequences of this functional heterogeneity in a murine AD model. Results: In contrast with recent in vitro overexpression studies, we find significant differences in the profiles of the Abeta peptides generated by complexes with different Aph1 components. Furthermore, when we specifically inactivate the Aph1B/C gamma-secretase complex in a mouse AD model, we find remarkable improvements of disease-relevant phenotypes without serious Notch side effects. Conclusions: We show that gamma-secretase exists as structurally heterogeneous complexes and that the Aph1 subunit affects gamma-cleavage through a shift in the longer versus shorter Abeta species, but does not affect the release of intracellular domains. This allows dissociation of toxicity and therapeutic effects because specific inactivation of Aph1B(C) in an AD model results in decreased amyloid plaque, Abeta and Abeta oligomer accumulation, and rescue of AD-related phenotypes without significantly altering Notch signalling. Our work suggests that the different Aph1 gamma-secretases contribute differentially to biological and pathological functions and opens a new avenue for more specific and less toxic AD therapy. O2-03-05
MICROGLIA AND ALZHEIMER’S DISEASE: IMPLICATIONS OF A IMMUNOTHERAPY
Delphine Boche, Elina Zotova, Clive Holmes, James A. R. Nicoll, University of Southampton, Southampton, United Kingdom. Contact email: [email protected] Background: Over the past decade many studies have focused on neuroinflammation as a harmful feature of AD, with the implication that