- Email: [email protected]
IMMUNOGLOBULIN-GENERAL AMYLOID INTERACTION MOTIF (IG-GAIM) MOLECULES TARGET BETA AMYLOID AND NEUROFIBRILLARY TANGLES IN VITRO AND IN VIVO
P494
Poster Presentations: P2
antioxidants (GSH and catalase activities) were assessed. Furthermore, the Kaplan-Meier test was used to calculate the average life expectancy. Results: 3xTgAD mice showed lower values of macrophage functions, lymphocyte chemotaxis, NK cells proportion and activity, and GSH from the onset of illness, whereas lymphoproliferative response was exacerbated. Moreover, GSSG/GSH ratio and xanthine oxidase activity are higher in these animals during the establishment of the disease. The 3xTgAD mice showed a shorter lifespan (p<0,05) than NTg. Conclusions: The impaired innate immune functions as well as the higher levels of OS in leucocytes seem to show a premature immunosenescence in 3xTgAD at the age when the amyloid plaques begin to appear (4 months). In contrast, the specific lymphocyte functions showed an over-activation. Although, the differences with NTg lessen as the age increases, the premature immune impairment of 3xTgAD supports the higher mortality shown in this model. P2-067
IMMUNOGLOBULIN-GENERAL AMYLOID INTERACTION MOTIF (IG-GAIM) MOLECULES TARGET BETA AMYLOID AND NEUROFIBRILLARY TANGLES IN VITRO AND IN VIVO
Haim Tsubery1, Rajaraman Krishnan1, Ming Proschitsky1, Eva Asp1, Michal Lulu1, Sharon Gilead1, Myra Gartner1, Kimberley S. Gannon2, Jonathan M. Levenson2, Sally Schroeter1, Valerie Cullen1, Jenna Carroll1, Jason Wright1, Beka Solomon3, David Myszka4, Eliezer Masliah5, Edward Rockenstein6, Jonathon Waltho7, Peter Davis7, Elliott Jay Mufson8, Muhammad Nadeem9, Richard Alan Fisher2, 1Neurophage Pharmaceuticals, Cambridge, Massachusetts, United States; 2NeuroPhage Pharmaceuticals, Cambridge, Massachusetts, United States; 3Tel Aviv University, Tel Aviv, Israel; 4Biosensor Tools, Salt Lake City, Utah, United States; 5University of California San Diego, La Jolla, California, United States; 6Univ. of California San Diego, La Jolla, California, United States; 7 University of Manchester, Sheffield, United Kingdom; 8Rush University, Chicago, Illinois, United States; 9Rush University Medical Center, Chicago, Illinois, United States. Contact e-mail: [email protected] Background: Protein misfolding that produces assemblies of toxic and transmissible aggregates is a central feature of the pathobiology of neurodegenerative diseases. A serendipitous discovery that direct exposure to filamentous bacteriophage M13 mediates reductions of both Ab and tau deposits in brains of transgenic mouse models of Alzheimer’s disease led to a search for the mechanism. We have isolated and characterized a fragment of the phage capsid protein responsible for the amyloid targeting activities of M13, and we show that an immunoglobulin fusion of this motif (Ig-GAIM) recapitulates the efficacy of M13 both in vitro and in transgenic Alzheimer’s disease model mice following either intrahippocampal or chronic systemic administration. Methods: SPR, ELISA and dot blot based assays were used for binding analysis of fibers and oligomers. Fibers remodeling was shown by filter retention assay. Fiber assembly inhibition was monitored using ThT binding assays and transmission electron microscopy. Nuclear magnetic resonance spectroscopy was used to map binding of GAIM to Ab fibers using H/D exchange technique. Blocking of oligomer-Induced cytotoxicity using Ig-GAIM is shown as well. Efficacy in an aged Tg2576 AD mouse model was measured by Ab ELISA and immunohistochemical Ab plaque load in the hippocampus 7 days after a single bilateral intracranial injection of Ig-GAIM. Results: Assays for amyloid fiber binding and remodeling, fiber assembly inhibition, and neuroprotection from cytotoxic oligomers together suggest this protein motif mediates these activities by potently preventing edge-to-edge beta strand aggregation. We also show that the GAIM activity mechanism is highly related to the natural role of bacteriophage infection mechanism. This targeting hypothesis is further substantiated by NMR H/D exchange spectroscopy of complexes of fibrilar Ab and the capsid protein and by binding specificity studies on an array of misfolded and natively folded proteins. Conclusions: Collectively, these results indicate on a novel general mechanism of amyloid specific recognition, disruption and a potentially broad therapeutic modality. Systemic administration of
GAIM Ig fusion reverses behavioral, biochemical and neuropathologic endpoints in both Ab and Tau mouse models. The Ig-GAIM fusions represent a novel, potent, and specific therapeutic approach for reducing pathologic misfolded protein assemblies that are central players in neurodegenerative diseases.
P2-068
REGULATION OF AMYLOID PRECURSOR PROTEIN EXPRESSION BY TETRATRICOPEPTIDE REPEAT PROTEIN 2 (TPR2)
Hideo Hara1, Takeshi Tabira2, 1Saga University Faculty of Medicine, Saga City, Japan; 2Graduate School of Juntendo University, Tokyo, Japan. Contact e-mail: [email protected] Background: An inflammatory response, with pro-inflammatory cytokines has been found throughout the brain of individuals with Alzheimer’s disease (AD). There were reports that inflammatory cytokines could alter the metabolism of b APP. Furthermore, adult mouse astrocytes were found to bind and degrade A b 1-42 in vitro. We hypothesized that the pro-inflammatory cytokine IFN- r could promote A b degradation by astrocytes, and attempted to identify the gene induced in astrocytes responsible for Ab degradation. Methods: We produced a subtracted cDNA library from a mouse astrocyte cell line treated with IFN- r and screened for the gene responsible for APP or Ab metabolism. After screening the cDNA library (approximately 1 x 10 6 clones), 100 positive clones were selected and sequenced. These selected 100 clones were transfected individually to the SHAPP cell line, which stably expressed APP and A b by transfection of APP695wt-expressing vector, then A b levels in the supernatant was measured by ELISA. Results: We found that co-chaperone tetratriopeptide repeat protein 2 (Tpr2) lowered the production of Ab and APP. Co-chaperone Tpr2 binds to APP through its TPR domain. Overexpression of Tpr2 dramatically reduced APP and A b levels. To determine the involvement of chaperones in the down-regulation of APP, we analyzed the influence of proteasome inhibition
Figure 1. All SHSY5Y cell transfections experiment used 160ng APP plasmids, while doses of Tpr2 plasmids varied. The control was APP transfection alone. Western blot shows the dose-related effect of Tpr2 on sAPPa and Ab production. The level of sAPPa in growth medium was lowered with increasing amounts of Tpr2 plasmid DNA transfected. The total level of cytosolic Ab was also significantly reduced to w50% of control in the highest Tpr2 condition.
Poster Presentations: P2
antioxidants (GSH and catalase activities) were assessed. Furthermore, the Kaplan-Meier test was used to calculate the average life expectancy. Results: 3xTgAD mice showed lower values of macrophage functions, lymphocyte chemotaxis, NK cells proportion and activity, and GSH from the onset of illness, whereas lymphoproliferative response was exacerbated. Moreover, GSSG/GSH ratio and xanthine oxidase activity are higher in these animals during the establishment of the disease. The 3xTgAD mice showed a shorter lifespan (p<0,05) than NTg. Conclusions: The impaired innate immune functions as well as the higher levels of OS in leucocytes seem to show a premature immunosenescence in 3xTgAD at the age when the amyloid plaques begin to appear (4 months). In contrast, the specific lymphocyte functions showed an over-activation. Although, the differences with NTg lessen as the age increases, the premature immune impairment of 3xTgAD supports the higher mortality shown in this model. P2-067
IMMUNOGLOBULIN-GENERAL AMYLOID INTERACTION MOTIF (IG-GAIM) MOLECULES TARGET BETA AMYLOID AND NEUROFIBRILLARY TANGLES IN VITRO AND IN VIVO
Haim Tsubery1, Rajaraman Krishnan1, Ming Proschitsky1, Eva Asp1, Michal Lulu1, Sharon Gilead1, Myra Gartner1, Kimberley S. Gannon2, Jonathan M. Levenson2, Sally Schroeter1, Valerie Cullen1, Jenna Carroll1, Jason Wright1, Beka Solomon3, David Myszka4, Eliezer Masliah5, Edward Rockenstein6, Jonathon Waltho7, Peter Davis7, Elliott Jay Mufson8, Muhammad Nadeem9, Richard Alan Fisher2, 1Neurophage Pharmaceuticals, Cambridge, Massachusetts, United States; 2NeuroPhage Pharmaceuticals, Cambridge, Massachusetts, United States; 3Tel Aviv University, Tel Aviv, Israel; 4Biosensor Tools, Salt Lake City, Utah, United States; 5University of California San Diego, La Jolla, California, United States; 6Univ. of California San Diego, La Jolla, California, United States; 7 University of Manchester, Sheffield, United Kingdom; 8Rush University, Chicago, Illinois, United States; 9Rush University Medical Center, Chicago, Illinois, United States. Contact e-mail: [email protected] Background: Protein misfolding that produces assemblies of toxic and transmissible aggregates is a central feature of the pathobiology of neurodegenerative diseases. A serendipitous discovery that direct exposure to filamentous bacteriophage M13 mediates reductions of both Ab and tau deposits in brains of transgenic mouse models of Alzheimer’s disease led to a search for the mechanism. We have isolated and characterized a fragment of the phage capsid protein responsible for the amyloid targeting activities of M13, and we show that an immunoglobulin fusion of this motif (Ig-GAIM) recapitulates the efficacy of M13 both in vitro and in transgenic Alzheimer’s disease model mice following either intrahippocampal or chronic systemic administration. Methods: SPR, ELISA and dot blot based assays were used for binding analysis of fibers and oligomers. Fibers remodeling was shown by filter retention assay. Fiber assembly inhibition was monitored using ThT binding assays and transmission electron microscopy. Nuclear magnetic resonance spectroscopy was used to map binding of GAIM to Ab fibers using H/D exchange technique. Blocking of oligomer-Induced cytotoxicity using Ig-GAIM is shown as well. Efficacy in an aged Tg2576 AD mouse model was measured by Ab ELISA and immunohistochemical Ab plaque load in the hippocampus 7 days after a single bilateral intracranial injection of Ig-GAIM. Results: Assays for amyloid fiber binding and remodeling, fiber assembly inhibition, and neuroprotection from cytotoxic oligomers together suggest this protein motif mediates these activities by potently preventing edge-to-edge beta strand aggregation. We also show that the GAIM activity mechanism is highly related to the natural role of bacteriophage infection mechanism. This targeting hypothesis is further substantiated by NMR H/D exchange spectroscopy of complexes of fibrilar Ab and the capsid protein and by binding specificity studies on an array of misfolded and natively folded proteins. Conclusions: Collectively, these results indicate on a novel general mechanism of amyloid specific recognition, disruption and a potentially broad therapeutic modality. Systemic administration of
GAIM Ig fusion reverses behavioral, biochemical and neuropathologic endpoints in both Ab and Tau mouse models. The Ig-GAIM fusions represent a novel, potent, and specific therapeutic approach for reducing pathologic misfolded protein assemblies that are central players in neurodegenerative diseases.
P2-068
REGULATION OF AMYLOID PRECURSOR PROTEIN EXPRESSION BY TETRATRICOPEPTIDE REPEAT PROTEIN 2 (TPR2)
Hideo Hara1, Takeshi Tabira2, 1Saga University Faculty of Medicine, Saga City, Japan; 2Graduate School of Juntendo University, Tokyo, Japan. Contact e-mail: [email protected] Background: An inflammatory response, with pro-inflammatory cytokines has been found throughout the brain of individuals with Alzheimer’s disease (AD). There were reports that inflammatory cytokines could alter the metabolism of b APP. Furthermore, adult mouse astrocytes were found to bind and degrade A b 1-42 in vitro. We hypothesized that the pro-inflammatory cytokine IFN- r could promote A b degradation by astrocytes, and attempted to identify the gene induced in astrocytes responsible for Ab degradation. Methods: We produced a subtracted cDNA library from a mouse astrocyte cell line treated with IFN- r and screened for the gene responsible for APP or Ab metabolism. After screening the cDNA library (approximately 1 x 10 6 clones), 100 positive clones were selected and sequenced. These selected 100 clones were transfected individually to the SHAPP cell line, which stably expressed APP and A b by transfection of APP695wt-expressing vector, then A b levels in the supernatant was measured by ELISA. Results: We found that co-chaperone tetratriopeptide repeat protein 2 (Tpr2) lowered the production of Ab and APP. Co-chaperone Tpr2 binds to APP through its TPR domain. Overexpression of Tpr2 dramatically reduced APP and A b levels. To determine the involvement of chaperones in the down-regulation of APP, we analyzed the influence of proteasome inhibition
Figure 1. All SHSY5Y cell transfections experiment used 160ng APP plasmids, while doses of Tpr2 plasmids varied. The control was APP transfection alone. Western blot shows the dose-related effect of Tpr2 on sAPPa and Ab production. The level of sAPPa in growth medium was lowered with increasing amounts of Tpr2 plasmid DNA transfected. The total level of cytosolic Ab was also significantly reduced to w50% of control in the highest Tpr2 condition.