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Alzheimer's amyloidosis induces global changes in protein homeostasis in the brains of transgenic mice
S562
Poster Presentations P3
Alzheimer gene regulation. These gene changes appear to correlate with known pathologic changes observed in AD and further support the contention that infection with C. pneumoniae plays a role in AD pathogenesis. P3-144
ALZHEIMER’S AMYLOIDOSIS INDUCES GLOBAL CHANGES IN PROTEIN HOMEOSTASIS IN THE BRAINS OF TRANSGENIC MICE 1
2
1
of JIP1b with KLC in respective proteins. These regions are also required for an effective anterograde APP transport by kinesin-1. Aberrant regulation of complex formation composed of APP, JIP1b and kinesin-1 can increase the generation of A beta and may cause the pathogenic state of neurons in sporadic Alzheimer’s disease. P3-146
1
Guilian Xu , Stanley Stevens , Firas Kobeissy , Mark Gold , David Borchelt1, 1University of Florida, Gainesville, Florida, United States; 2University of South Florida, Tampa, Florida, United States. Background: The deposition of amyloid beta peptide (Ab) is viewed as a key trigger in the early pathogenesis of Alzheimer’s Disease (AD) that precedes or is coincident with the appearance of intracellular neurofibrillary tangles (NFTs). The latter pathology could be viewed as evidence for alterations protein homeostasis. Transgenic mice that model Alzheimer-type amyloidosis do not develop NFTs, providing a means to specifically assess whether amyloid deposition impacts protein homeostasis. Methods: Using a heat-shock model of cultured cells we determined that loss of protein solubility and an accumulation of polyubiquitin chains linked via lysine 48 residues, indicating an accumulation of excess proteasome substrates, are key biomarkers of altered protein homeostasis. We then use mass spectrometry approaches to determine whether these biomarkers were present in the brains of mice that model Alzheimer amyloidosis. Results: We identified numerous cytosolic proteins that show specific losses insolubility in the brains of mice with high amyloid burden. Importantly we also detected an accumulation of lysine 48 linked polyubiquitin in these animals. Conclusions: We conclude that amyloid deposition is sufficient to trigger changes in intracellular protein homeostasis and as such could be a critical component of the amyloid cascade.
P3-145
MOLECULAR MECHANISM OF JIP1B-MEDIATED APP ASSOCIATION TO KINESIN-1 IN APP TRANSPORT AND METABOLIC REGULATION
Kyoko Chiba1, Keisuke Nozawa1, Masahiko Araseki1, Masataka Kinjo2, Hidenori Taru3, Toshiharu Suzuki1, 1Laboratory of Neuroscience, Graduate School of Pharmaceutical Sciences, Hokkaido University, Sapporo, Japan; 2 Laboratory of Molecular Cell Dynamics, Faculty of Advance of Life Science, Hokkaido University, Sapporo, Japan; 3Creative Research Institute Sousei, Hokkaido University and Laboratory of Neuronal Cell Biology, Graduate School of Pharmaceutical Sciences, Hokkaido University, Sapporo, Japan. Background: Amyloid beta-protein precursor (APP) is transported anterogradely by kinesin-1 and thought to work as a cargo receptor. JNK-interacting protein1b (JIP1b) associates APP with kinesin light chain (KLC) of kinesin-1 (J. Biol. Chem. [2002] 277, 20070; EMBO J. [2007] 26, 1475). However, the molecular mechanisms how JIP1b mediate the association of APP with KLC1 and what function does JIP1b play in the role of APP transport regulation remain unclear. Dysfunction and/or aberration of APP transport system by kinesin-1 facilitate amyloidogenic processing of APP and cause the increased generation of A beta. Therefore, it is important to understand the molecular regulation of interactions among APP, JIP1b and KLC. Methods: To determine regions which required for interaction between JIP1b and KLC, regional-deleted mutant proteins for JIP1b and KLC1 are expressed in cells and their ability for interaction was examined by co-immunoprecipitation assay. Along with binding ability of JIP1b to KLC, APP transport ability of JIP1 mutant proteins was tested in differentiating CAD cells expressing APP-GFP under the observation with TIRF microscopy. Results: Binding regions of JIP1b to KLC1 were analyzed in detail and a novel region for JIP1b-binding of KLC1 was found. Knocked-down of endogenous JIP1b and expression of mutant JIP1b proteins, which lack regions required for KLC-binding, resulted in the slower anterograde transport of APP when compared to physiological velocity of APP transport. Conclusions: We identified regions required for interaction
DECLINE IN MITOCHONDRIAL BIOENERGETICS UNDERLIES CENTRAL INSULIN RESISTANCE IN STREPTOZOCIN-INDUCED MODEL OF SPORADIC ALZHEIMER’S DISEASE
Sonia Correia1, George Perry2, Xiongwei Zhu3, Mark Smith3, Paula Moreira4, 1Center for Neuroscience and Cell Biology (CNC), Coimbra, Portugal; 27UTSA Neurosciences Institute and Department of Biology, University of Texas at San Antonio, San Antonio, Texas, United States; 3Case Western Reserve University, Cleveland, Ohio, United States; 4 Center for Neuroscience and Cell Biology, Coimbra, Portugal. Background: Early abnormalities in cerebral glucose/energy metabolism are consistent antecedents to sporadic Alzheimer’s disease (sAD) development, suggesting that impaired insulin signaling is present at the earliest symptomatic stages of the disease. Additionally, cerebral metabolism decline also points for a potential causal role of mitochondrial bioenergetics in sAD pathogenesis. Methods: In this study we investigated the impact of central insulin resistance on mitochondrial bioenergetics by using rats intracerebroventricularly (icv) treated with streptozotocin, an animal model of sAD. Three-month-old male Wistar rats were investigated 5 weeks after a single bilateral icv injection of STZ (3mg/Kg) or vehicle. Results: Both cortical and hippocampal mitochondria from icvSTZ rats exhibit impaired oxidative phosphorylation system characterized by decreased mitochondrial transmembrane potential and ADP depolarization and increased lag phase of repolarization, when compared with mitochondria isolated from vehicle-treated rats. Consistently, icvSTZ rats also present decreased mitochondrial respiratory state 3, respiratory control ratio, ADP/O index and DNPstimulated respiration, in the presence of complexes I (glutamate/malate) or II (succinate) substrates, whereas state 4 remained unaltered. A decrease in pyruvate dehydrogenase, a-ketoglutarate dehydrogenase and cytochrome c oxidade activities was also observed, which reinforces the existence of mitochondrial bioenergetic deficits in this animal model of sAD. icvSTZ rats also display an increased susceptibility to calcium-induced mitochondrial permeability transition, a phenomenon that could be related with the decrease in hexokinase activity. Conclusions: Altogether, these results strongly suggest that mitochondrial bioenergetics decline is intimately associated with the "insulin-resistant brain state" in this animal model of sAD, highlighting the central importance of mitochondrial (dys)function in the pathology of the disease. P3-147
AXONAL TRANSPORT OF THE UBIQUITINPROTEASOME SYSTEM AND IT RELEVANCE FOR PROTEIN DEGRADATION
Maria Gabriela Otero, Lucas Cromberg, Tomas Falzone, IBCN-UBA-CONICET, Buenos Aires, Argentina. Background: Aberrant accumulation of misfolded protein in AD suggests that protein degradation defects by the Ubiquitin Proteasome System (UPS) are implicated in disease pathogenesis. Amyloid dependent impaired UPS degradation was observed in brain areas suggesting that UPS localization is crucial for proper protein removal. Consequently, it is important to understand how UPS is moved along axons and how defects in UPS delivery can lead to dysfunction in protein degradation as is observed in AD. Methods: To understand the axonal transport properties of proteasome subunits we generated an expression vector driving PSMA-7 proteasome subunit fused to YFP. To unravel if neuronal proteasomes can be transported associated with membrane compartment we performed flotation experiments in continuous sucrose density gradients. To understand if proteasome function can regulate its own axonal transport we perform PSMA-YFP movies in the presence of proteasome inhibition.
Poster Presentations P3
Alzheimer gene regulation. These gene changes appear to correlate with known pathologic changes observed in AD and further support the contention that infection with C. pneumoniae plays a role in AD pathogenesis. P3-144
ALZHEIMER’S AMYLOIDOSIS INDUCES GLOBAL CHANGES IN PROTEIN HOMEOSTASIS IN THE BRAINS OF TRANSGENIC MICE 1
2
1
of JIP1b with KLC in respective proteins. These regions are also required for an effective anterograde APP transport by kinesin-1. Aberrant regulation of complex formation composed of APP, JIP1b and kinesin-1 can increase the generation of A beta and may cause the pathogenic state of neurons in sporadic Alzheimer’s disease. P3-146
1
Guilian Xu , Stanley Stevens , Firas Kobeissy , Mark Gold , David Borchelt1, 1University of Florida, Gainesville, Florida, United States; 2University of South Florida, Tampa, Florida, United States. Background: The deposition of amyloid beta peptide (Ab) is viewed as a key trigger in the early pathogenesis of Alzheimer’s Disease (AD) that precedes or is coincident with the appearance of intracellular neurofibrillary tangles (NFTs). The latter pathology could be viewed as evidence for alterations protein homeostasis. Transgenic mice that model Alzheimer-type amyloidosis do not develop NFTs, providing a means to specifically assess whether amyloid deposition impacts protein homeostasis. Methods: Using a heat-shock model of cultured cells we determined that loss of protein solubility and an accumulation of polyubiquitin chains linked via lysine 48 residues, indicating an accumulation of excess proteasome substrates, are key biomarkers of altered protein homeostasis. We then use mass spectrometry approaches to determine whether these biomarkers were present in the brains of mice that model Alzheimer amyloidosis. Results: We identified numerous cytosolic proteins that show specific losses insolubility in the brains of mice with high amyloid burden. Importantly we also detected an accumulation of lysine 48 linked polyubiquitin in these animals. Conclusions: We conclude that amyloid deposition is sufficient to trigger changes in intracellular protein homeostasis and as such could be a critical component of the amyloid cascade.
P3-145
MOLECULAR MECHANISM OF JIP1B-MEDIATED APP ASSOCIATION TO KINESIN-1 IN APP TRANSPORT AND METABOLIC REGULATION
Kyoko Chiba1, Keisuke Nozawa1, Masahiko Araseki1, Masataka Kinjo2, Hidenori Taru3, Toshiharu Suzuki1, 1Laboratory of Neuroscience, Graduate School of Pharmaceutical Sciences, Hokkaido University, Sapporo, Japan; 2 Laboratory of Molecular Cell Dynamics, Faculty of Advance of Life Science, Hokkaido University, Sapporo, Japan; 3Creative Research Institute Sousei, Hokkaido University and Laboratory of Neuronal Cell Biology, Graduate School of Pharmaceutical Sciences, Hokkaido University, Sapporo, Japan. Background: Amyloid beta-protein precursor (APP) is transported anterogradely by kinesin-1 and thought to work as a cargo receptor. JNK-interacting protein1b (JIP1b) associates APP with kinesin light chain (KLC) of kinesin-1 (J. Biol. Chem. [2002] 277, 20070; EMBO J. [2007] 26, 1475). However, the molecular mechanisms how JIP1b mediate the association of APP with KLC1 and what function does JIP1b play in the role of APP transport regulation remain unclear. Dysfunction and/or aberration of APP transport system by kinesin-1 facilitate amyloidogenic processing of APP and cause the increased generation of A beta. Therefore, it is important to understand the molecular regulation of interactions among APP, JIP1b and KLC. Methods: To determine regions which required for interaction between JIP1b and KLC, regional-deleted mutant proteins for JIP1b and KLC1 are expressed in cells and their ability for interaction was examined by co-immunoprecipitation assay. Along with binding ability of JIP1b to KLC, APP transport ability of JIP1 mutant proteins was tested in differentiating CAD cells expressing APP-GFP under the observation with TIRF microscopy. Results: Binding regions of JIP1b to KLC1 were analyzed in detail and a novel region for JIP1b-binding of KLC1 was found. Knocked-down of endogenous JIP1b and expression of mutant JIP1b proteins, which lack regions required for KLC-binding, resulted in the slower anterograde transport of APP when compared to physiological velocity of APP transport. Conclusions: We identified regions required for interaction
DECLINE IN MITOCHONDRIAL BIOENERGETICS UNDERLIES CENTRAL INSULIN RESISTANCE IN STREPTOZOCIN-INDUCED MODEL OF SPORADIC ALZHEIMER’S DISEASE
Sonia Correia1, George Perry2, Xiongwei Zhu3, Mark Smith3, Paula Moreira4, 1Center for Neuroscience and Cell Biology (CNC), Coimbra, Portugal; 27UTSA Neurosciences Institute and Department of Biology, University of Texas at San Antonio, San Antonio, Texas, United States; 3Case Western Reserve University, Cleveland, Ohio, United States; 4 Center for Neuroscience and Cell Biology, Coimbra, Portugal. Background: Early abnormalities in cerebral glucose/energy metabolism are consistent antecedents to sporadic Alzheimer’s disease (sAD) development, suggesting that impaired insulin signaling is present at the earliest symptomatic stages of the disease. Additionally, cerebral metabolism decline also points for a potential causal role of mitochondrial bioenergetics in sAD pathogenesis. Methods: In this study we investigated the impact of central insulin resistance on mitochondrial bioenergetics by using rats intracerebroventricularly (icv) treated with streptozotocin, an animal model of sAD. Three-month-old male Wistar rats were investigated 5 weeks after a single bilateral icv injection of STZ (3mg/Kg) or vehicle. Results: Both cortical and hippocampal mitochondria from icvSTZ rats exhibit impaired oxidative phosphorylation system characterized by decreased mitochondrial transmembrane potential and ADP depolarization and increased lag phase of repolarization, when compared with mitochondria isolated from vehicle-treated rats. Consistently, icvSTZ rats also present decreased mitochondrial respiratory state 3, respiratory control ratio, ADP/O index and DNPstimulated respiration, in the presence of complexes I (glutamate/malate) or II (succinate) substrates, whereas state 4 remained unaltered. A decrease in pyruvate dehydrogenase, a-ketoglutarate dehydrogenase and cytochrome c oxidade activities was also observed, which reinforces the existence of mitochondrial bioenergetic deficits in this animal model of sAD. icvSTZ rats also display an increased susceptibility to calcium-induced mitochondrial permeability transition, a phenomenon that could be related with the decrease in hexokinase activity. Conclusions: Altogether, these results strongly suggest that mitochondrial bioenergetics decline is intimately associated with the "insulin-resistant brain state" in this animal model of sAD, highlighting the central importance of mitochondrial (dys)function in the pathology of the disease. P3-147
AXONAL TRANSPORT OF THE UBIQUITINPROTEASOME SYSTEM AND IT RELEVANCE FOR PROTEIN DEGRADATION
Maria Gabriela Otero, Lucas Cromberg, Tomas Falzone, IBCN-UBA-CONICET, Buenos Aires, Argentina. Background: Aberrant accumulation of misfolded protein in AD suggests that protein degradation defects by the Ubiquitin Proteasome System (UPS) are implicated in disease pathogenesis. Amyloid dependent impaired UPS degradation was observed in brain areas suggesting that UPS localization is crucial for proper protein removal. Consequently, it is important to understand how UPS is moved along axons and how defects in UPS delivery can lead to dysfunction in protein degradation as is observed in AD. Methods: To understand the axonal transport properties of proteasome subunits we generated an expression vector driving PSMA-7 proteasome subunit fused to YFP. To unravel if neuronal proteasomes can be transported associated with membrane compartment we performed flotation experiments in continuous sucrose density gradients. To understand if proteasome function can regulate its own axonal transport we perform PSMA-YFP movies in the presence of proteasome inhibition.