- Email: [email protected]
P3-359
S480
Poster P3:: Tuesday Posters
2 Monash University, Melbourne, Australia; 3The University of Queensland, Brisbane, Australia; 4Howard Florey Institute of Medical Research, Parkville, Australia; 5Mental Health Research Institute of Victoria, Parkville, Australia. Contact e-mail: [email protected]
Background: The Abeta peptide is toxic but the mechanisms of toxicity are still subject of much debate. A number of different hypotheses have been proposed, but most recent interest has been directed at the formation of soluble oligomers of Abeta and their interactions with lipid membranes. In addition to membrane composition other factors that might affect Abeta toxicity include the presence of metal ions that induce peptide aggregation and the generation of reactive oxygen species. Objective(s): To elucidate factors affecting Abeta toxicity. Methods: We have investigated the interactions of Abeta with a range of co-factors and examined the effects on Abeta toxicity. In addition we have prepared a number of Abeta peptides where key residues have been altered and examined changes in the chemistry of the peptide, which have then been correlated to the neurotoxic activity of Abeta. Results: Our results indicate that specific metallated forms of Abeta are more toxic than non-metalled forms. The consequences of Abeta metal interactions include the formation of soluble oligomeric species as a result of oxidative modifications such dityrosine linkages. For Abeta to be toxic it must associate with the cell surface membrane, oligomeric metallated Abeta has a higher affinity for lipid membranes than non-metalled Abeta. Once bound to the lipid there is no correlation between toxicity and the peptide’s ability to perturb the bulk fluidity of the lipid membrane, but there is a correlation with the ability of the peptide to induce lipid peroxidation via the formation of peptide centred radicals. Conclusions: Metal-induced oxidative modifications to Abeta and lipid peroxidation can be prevented by inhibiting Abeta/metal interactions. We have developed a class of metal-protein attenuating compounds (MPAC) that are capable of inhibiting Abeta/metal interactions, these compounds have shown efficacy in cell and animal models of AD as well as some therapeutic potential. P3-358
NOVEL FUNCTIONS OF THE ALPHAKETOGLUTARATE DEHYDROGENASE COMPLEX MAY MEDIATE OXIDANT-INDUCED DIVERSE CHANGES IN MITOCHONDRIAL ENZYMES IN ALZHEIMER’S DISEASE
Gary E. Gibson1, Hui Xu2, Wayne Kleinman2, Qingli Shi2, 1Weill Medical College of Cornell University, Burke Medical Research Institute, NY, USA; 2Weill Medical College of Cornell University, Burke Medical Research Institute, White Plains, NY, USA. Contact e-mail: [email protected] Background: Specific activities of some mitochondrial enzymes of energy metabolism decline with AD whereas others increase. ␣-Ketoglutarate dehydrogenase complex (KGDHC) activity declines in brains of patients with AD, and the reduction is highly correlated to the clinical dementia rating score before death. KGDHC catalyzes an irreversible dehydrogenase and decarboxylating step that is an important control point for the tricarboxylic acid (TCA) cycle. The specific activity of malate dehydrogenase (MDH) that catalyzes a reversible dehydrogenase step of the TCA cycle, increases in AD. The experiments test the hypothesis that the opposite response of the two enzymes is due to cross-talk within the TCA cycle that is stimulated by oxidative stress and mediated by E2k, one of the catalytic subunits of the KGDHC complex. Objective(s): To understand the relations of diminished KGDHC, elevated MDH and oxidative stress. Methods: HEK 293 lines expressing E2k antisense RNA in which E2k protein contents were reduced to 46% (E2k-46) and 23% (E2k-23) of a sense control line (E2k-100) were developed. These cells were used to test the consequences of different levels of E2k on KGDHC and MDH activities in response to oxidative stress induced by oxidant (H2O2) and/or by depletion of glutathione with buthionine sulfoximine (BSO). Results: Glutathione levels declined similarly regardless of the level of E2k protein in response
to either BSO or H2O2. Reduction in E2k, however, altered the response of KGDHC activities to H2O2. H2O2 (1hr) increased total MDH specific activity (15%) only in the E2k-23 line. However, H2O2 (24 hr) reduced total MDH specific activity dramatically (-72%) in the E2k-23 line. BSO had no effects on either KGDHC or MDH activities. H2O2 significantly increased MDH mRNA by 15-20% (cMDH) and 25% (mMDH) only in the E2k-23 line. Conclusions: The responses of KGDHC and MDH to oxidant varied with the level of E2k protein. The results suggest that oxidants induce diverse responses in TCA cycle enzymes and that the E2k component of KGDHC may have roles independent of the complex activity that mediate the oxidant induced changes. P3-359
REDUCTION OF MITOCHONDRIAL SUPEROXIDE DISMUTASE MODULATES ALZHEIMER’S DISEASE-LIKE PATHOLOGY IN HUMAN AMYLOID PRECURSOR PROTEIN TRANSGENIC MICE
Luke Esposito1, Fengrong Yan1, Gui-Qiu Yu1, Xin Wang1, Jukka Puolivali1, Kimberly Scearce-Levie1, Eliezer Masliah2, Lennart Mucke1, 1Glaldstone Institute of Neurological Disease, University of California San Francisco, San Francisco, CA, USA; 2 University of California at San Diego, LaJolla, CA, USA. Contact e-mail: [email protected] Background: Alzheimer’s disease (AD) results in the accumulation of amyloid- (A) peptides in the brain, oxidative damage, degeneration of neuronal processes, gliosis, dementia, and death. Whether oxidative damage and mitochondrial decline contribute directly to the characteristic pathology or are a secondary effect of the disease process has not been fully established in vivo. Objective: We tested the hypothesis that the mitochondrial antioxidant enzyme superoxide dismutase-2 (Sod2) plays a critical role in defending against A-induced AD-like alterations. Methods: To test this hypothesis, we inactivated one Sod2 allele (Sod2⫹/-) in human amyloid precursor protein (hAPP) transgenic mice. Results: Levels of Sod2 mRNA, protein and activity in Sod2⫹/- mice were roughly 50% of those in Sod2 wild-type (Sod2⫹/⫹) mice. In the presence of normal Sod2 levels, hAPP/A expression resulted in deposition of A predominantly in the brain parenchyma, decreases in levels of the dendritic marker microtubule-associated protein-2 (MAP-2), neuritic dystrophy and behavioral alterations, including deficits in prepulse inhibition of acoustic startle. Sod2 reduction in hAPP mice caused a striking redistribution of amyloid deposits to the cerebrovasculature by middle age and a decrease in parenchymal amyloid plaques by old age, as well as an increased microgliosis and the presence of macrophages with abnormal accumulation of lipid vacuoles near some vessels. Compared with hAPP/Sod2⫹/⫹ mice, hAPP/Sod2⫹/mice also had an earlier age of onset of behavioral deficits, a greater loss of MAP-2, and a more prominent and widespread neuritic dystrophy, much of which was plaque-independent. Hippocampi of hAPP/Sod2⫹/- mice had increased DNA binding activity of the injury-responsive transcription factor nuclear factor--B (NFB). Conclusions: These results suggest that Sod2 protects the aging brain against A-induced impairments. While reductions in Sod2 would be expected to trigger or exacerbate neuronal and vascular pathology in AD, increasing Sod2 activity might be of therapeutic benefit. Supported by NIH grants AG022074 and NS43945. P3-360
VAV IS REQUIRED FOR -AMYLOIDSTIMULATED NADPH OXIDASE ACTIVATION AND PHAGOSOME FORMATION IN PRIMARY MICROGLIA
Brandy L. Wilkinson1, Jessica Koenigsknecht-Talboo2, Gary E. Landreth2, 1Case Western Reserve University, Cleveland, OH, USA; 2Case Western Reserve University, Cleveland, USA. Contact e-mail: [email protected]
Poster P3:: Tuesday Posters
2 Monash University, Melbourne, Australia; 3The University of Queensland, Brisbane, Australia; 4Howard Florey Institute of Medical Research, Parkville, Australia; 5Mental Health Research Institute of Victoria, Parkville, Australia. Contact e-mail: [email protected]
Background: The Abeta peptide is toxic but the mechanisms of toxicity are still subject of much debate. A number of different hypotheses have been proposed, but most recent interest has been directed at the formation of soluble oligomers of Abeta and their interactions with lipid membranes. In addition to membrane composition other factors that might affect Abeta toxicity include the presence of metal ions that induce peptide aggregation and the generation of reactive oxygen species. Objective(s): To elucidate factors affecting Abeta toxicity. Methods: We have investigated the interactions of Abeta with a range of co-factors and examined the effects on Abeta toxicity. In addition we have prepared a number of Abeta peptides where key residues have been altered and examined changes in the chemistry of the peptide, which have then been correlated to the neurotoxic activity of Abeta. Results: Our results indicate that specific metallated forms of Abeta are more toxic than non-metalled forms. The consequences of Abeta metal interactions include the formation of soluble oligomeric species as a result of oxidative modifications such dityrosine linkages. For Abeta to be toxic it must associate with the cell surface membrane, oligomeric metallated Abeta has a higher affinity for lipid membranes than non-metalled Abeta. Once bound to the lipid there is no correlation between toxicity and the peptide’s ability to perturb the bulk fluidity of the lipid membrane, but there is a correlation with the ability of the peptide to induce lipid peroxidation via the formation of peptide centred radicals. Conclusions: Metal-induced oxidative modifications to Abeta and lipid peroxidation can be prevented by inhibiting Abeta/metal interactions. We have developed a class of metal-protein attenuating compounds (MPAC) that are capable of inhibiting Abeta/metal interactions, these compounds have shown efficacy in cell and animal models of AD as well as some therapeutic potential. P3-358
NOVEL FUNCTIONS OF THE ALPHAKETOGLUTARATE DEHYDROGENASE COMPLEX MAY MEDIATE OXIDANT-INDUCED DIVERSE CHANGES IN MITOCHONDRIAL ENZYMES IN ALZHEIMER’S DISEASE
Gary E. Gibson1, Hui Xu2, Wayne Kleinman2, Qingli Shi2, 1Weill Medical College of Cornell University, Burke Medical Research Institute, NY, USA; 2Weill Medical College of Cornell University, Burke Medical Research Institute, White Plains, NY, USA. Contact e-mail: [email protected] Background: Specific activities of some mitochondrial enzymes of energy metabolism decline with AD whereas others increase. ␣-Ketoglutarate dehydrogenase complex (KGDHC) activity declines in brains of patients with AD, and the reduction is highly correlated to the clinical dementia rating score before death. KGDHC catalyzes an irreversible dehydrogenase and decarboxylating step that is an important control point for the tricarboxylic acid (TCA) cycle. The specific activity of malate dehydrogenase (MDH) that catalyzes a reversible dehydrogenase step of the TCA cycle, increases in AD. The experiments test the hypothesis that the opposite response of the two enzymes is due to cross-talk within the TCA cycle that is stimulated by oxidative stress and mediated by E2k, one of the catalytic subunits of the KGDHC complex. Objective(s): To understand the relations of diminished KGDHC, elevated MDH and oxidative stress. Methods: HEK 293 lines expressing E2k antisense RNA in which E2k protein contents were reduced to 46% (E2k-46) and 23% (E2k-23) of a sense control line (E2k-100) were developed. These cells were used to test the consequences of different levels of E2k on KGDHC and MDH activities in response to oxidative stress induced by oxidant (H2O2) and/or by depletion of glutathione with buthionine sulfoximine (BSO). Results: Glutathione levels declined similarly regardless of the level of E2k protein in response
to either BSO or H2O2. Reduction in E2k, however, altered the response of KGDHC activities to H2O2. H2O2 (1hr) increased total MDH specific activity (15%) only in the E2k-23 line. However, H2O2 (24 hr) reduced total MDH specific activity dramatically (-72%) in the E2k-23 line. BSO had no effects on either KGDHC or MDH activities. H2O2 significantly increased MDH mRNA by 15-20% (cMDH) and 25% (mMDH) only in the E2k-23 line. Conclusions: The responses of KGDHC and MDH to oxidant varied with the level of E2k protein. The results suggest that oxidants induce diverse responses in TCA cycle enzymes and that the E2k component of KGDHC may have roles independent of the complex activity that mediate the oxidant induced changes. P3-359
REDUCTION OF MITOCHONDRIAL SUPEROXIDE DISMUTASE MODULATES ALZHEIMER’S DISEASE-LIKE PATHOLOGY IN HUMAN AMYLOID PRECURSOR PROTEIN TRANSGENIC MICE
Luke Esposito1, Fengrong Yan1, Gui-Qiu Yu1, Xin Wang1, Jukka Puolivali1, Kimberly Scearce-Levie1, Eliezer Masliah2, Lennart Mucke1, 1Glaldstone Institute of Neurological Disease, University of California San Francisco, San Francisco, CA, USA; 2 University of California at San Diego, LaJolla, CA, USA. Contact e-mail: [email protected] Background: Alzheimer’s disease (AD) results in the accumulation of amyloid- (A) peptides in the brain, oxidative damage, degeneration of neuronal processes, gliosis, dementia, and death. Whether oxidative damage and mitochondrial decline contribute directly to the characteristic pathology or are a secondary effect of the disease process has not been fully established in vivo. Objective: We tested the hypothesis that the mitochondrial antioxidant enzyme superoxide dismutase-2 (Sod2) plays a critical role in defending against A-induced AD-like alterations. Methods: To test this hypothesis, we inactivated one Sod2 allele (Sod2⫹/-) in human amyloid precursor protein (hAPP) transgenic mice. Results: Levels of Sod2 mRNA, protein and activity in Sod2⫹/- mice were roughly 50% of those in Sod2 wild-type (Sod2⫹/⫹) mice. In the presence of normal Sod2 levels, hAPP/A expression resulted in deposition of A predominantly in the brain parenchyma, decreases in levels of the dendritic marker microtubule-associated protein-2 (MAP-2), neuritic dystrophy and behavioral alterations, including deficits in prepulse inhibition of acoustic startle. Sod2 reduction in hAPP mice caused a striking redistribution of amyloid deposits to the cerebrovasculature by middle age and a decrease in parenchymal amyloid plaques by old age, as well as an increased microgliosis and the presence of macrophages with abnormal accumulation of lipid vacuoles near some vessels. Compared with hAPP/Sod2⫹/⫹ mice, hAPP/Sod2⫹/mice also had an earlier age of onset of behavioral deficits, a greater loss of MAP-2, and a more prominent and widespread neuritic dystrophy, much of which was plaque-independent. Hippocampi of hAPP/Sod2⫹/- mice had increased DNA binding activity of the injury-responsive transcription factor nuclear factor--B (NFB). Conclusions: These results suggest that Sod2 protects the aging brain against A-induced impairments. While reductions in Sod2 would be expected to trigger or exacerbate neuronal and vascular pathology in AD, increasing Sod2 activity might be of therapeutic benefit. Supported by NIH grants AG022074 and NS43945. P3-360
VAV IS REQUIRED FOR -AMYLOIDSTIMULATED NADPH OXIDASE ACTIVATION AND PHAGOSOME FORMATION IN PRIMARY MICROGLIA
Brandy L. Wilkinson1, Jessica Koenigsknecht-Talboo2, Gary E. Landreth2, 1Case Western Reserve University, Cleveland, OH, USA; 2Case Western Reserve University, Cleveland, USA. Contact e-mail: [email protected]