- Email: [email protected]
The role of autophagy in Alzheimer's disease
Poster Presentations: P1 a "hierarchical pathogenic map" developing as a consequence of APP loss will inform the identification of novel therapeutic targets.
P1-104
THE BINDING OF BETA-AMYLOID 42 TO LIPID RAFTS CONTAINING DETERGENT-RESISTANT MEMBRANES OF RBC GHOST IS ENHANCED BY DIETARY DOCOSAHEXAENOIC ACID IN RATS: AN IMPLICATION OF ERYTHROCYTE FUNCTIONS TO ALZHEIMER’S DISEASE
Michio Hashimoto1, Hossain Shahdat1, Masahiro Katakura1, Yoko Tanabe1, Hiroyuki Arai2, Osamu Shido1, 1Shimane University, Izumo, Japan; 2Tohoku University School of Medicine, Sendai, Japan. Contact e-mail: [email protected] Background: The interactions of RBC and amyloid beta peptide (Ab) are implicated in Alzheimer’s disease (AD), however, the mechanisms are far from clarity. In AD, circulating Abs increase in the plasma, interact with RBCs, and affect their functions/morphology. We investigated whether Ab 1-42 interacts with caveolin1-containing lipid-rafts of detergent resistance membranes (DRMs) and whether such interactions could be modulated by oral administration of docosahexaenoic acid (DHA, C22:6, n3), an essential brain fatty acid, whose plasma levels is lower in AD patients. Methods: RBC hemolysis, RBC ghost and DRMs were prepared. DRMS-bound A b 1-42, caveolin1, and cathepsin were measured by immunomicrofluorescence assay or ELISA. Results: A b 1-42 -induced hemolysis was reduced in the RBCs of DHA-preadministered rats. When fluorescent-labeled A b 1-42 (TAMRA-A b 1-42) was incubated with DRMs, TAMRA-A b 1-42 occupancy significantly increased in the DRMs of the DHA-preadministered rats. The levels of caveolin-1, DHA, cathepsin and membrane fluidity were high er, while cholesterol levels were truncated in the DRMs of DHA-preadministered rats, when compared with those of control rats. Additionally, when TAMRA-A b 1-42 was infused directly into the carotid veins of the rats, the TAMRA- A b 1-42 was found to a greater level in the DRMs of the DHA- preadministered rats, thus indicating that circulating A b s interact with the caveolin-1 containing lipid-rafts/caveola of the DRMs. The plasma levels of TAMRA-A b 1-42 were reduced, while that of the TAMRA-A b 1-42 in hepatic-DRMs and -cytosolic protease such as cathepsin activity also significantly increased in the post-infused DHA-preadministered rats. This demonstrates, at least partially, the contribution of hepatic DRMs-bound proteins in clearance of plasma A b 1-42 levels of DHA rats. The resistance to A b 1-42 -induced hemolysis was associated with decreases in levels of lipid peroxide, reactive oxygen species and increases in GSH levels in the RBC of DHA rats. Conclusions: [i] The detection of A b s, at least in part, on the surface of the RBCs of AD patients might constitute the basis of biomarkers of AD, and [ii] the enrichment of RBCs with DHA helps in plasma clearance of the amyloids, probably, by increased lipid-rafts-dependent degradative pathways and also by membrane-bound increased protease like catehpsin acivity.
P1-105
THE ROLE OF AUTOPHAGY IN ALZHEIMER’S DISEASE
Miranda Orr1, Salvatore Oddo2, 1University of Texas Health Science Center at San Antonio, San Antonio, Texas, United States; 2University of Texas Health Science Center at San Antonio, San Antonio, Texas, United States. Contact e-mail: [email protected] Background: The accumulation of extracellular Ab plaques and intracellular neurofibrillary tangles comprised of hyperphosphorylated tau protein coincide with synaptic dysfunction, cognitive decline, and neuron loss in patients afflicted with Alzheimer’s disease (AD). A prominent hypothesis suggests that elevated Ab initiates the pathogenic cascade; however, mechanisms linking Ab to downstream events remain unclear. Due to the accumulation of proteinaceous aggregates in patient brains, pathways involved with protein degradation and clearance likely contribute to disease. One
P189
such system, the autophagic-lysosomal system, is crucial for catabolizing excess proteins, including amyloid precursor protein (APP), Ab, and tau. While specific autophagic pathways regulating Ab and tau accumulation remain largely unknown, deficits in chaperone-mediated autophagy (CMA) generate aggregate-prone tau fragments. Methods: Here we used complementary in vivo and in vitro approaches to elucidate the role of macroautophagy and CMA on protein accumulation in AD. Results: CMA activity directly correlates to the levels of the lysosome-associated membrane protein type 2a (Lamp2a); when we compared lysosomal Lamp2a levels between 6-month-old 3xTg-AD mice and age-matched controls, we saw a significant decrease in 3xTg-AD mice suggesting CMA deficits. We also found that modulating macroautophagy, the prominent and less-selective autophagic-lysosomal pathway, can directly affect Ab and tau levels. Since macroautophagy inhibition can lead to increased CMA, we are exploring crosstalk between these pathways. Conclusions: Understanding the specific autophagic pathways involved in AD pathogenesis may lead to the identification of new therapeutic targets. Since decreased Lamp2a levels are indicative of decreased CMA activity, our results suggest that CMA dysfunction occurs in 6-month-old 3xTg-AD mice. While 3xTgAD mice display elevated Ab at this age, insoluble tau aggregates have not yet formed. Therefore this result associates elevated Ab to CMA dysregulation, and suggests that both may be upstream of tau misprocessing.
P1-106
USING PRIMARY NEURONS FROM TRANSGENIC MICE TO IDENTIFY GENES ON HUMAN CHROMOSOME 21 INFLUENCING ALZHEIMER’S NEUROPATHOLOGY
Xun Yu Choong1, Alex Raha2, Victor Tybulewicz3, Frances Wiseman1, Elizabeth Fisher1, 1Institute of Neurology, University College London, London, United Kingdom; 2MRC Prion Unit, London, United Kingdom; 3 MRC National Institute for Medical Research, London, United Kingdom. Contact e-mail: [email protected] Background: Virtually all individuals with Down Syndrome (DS) develop features of Alzheimer neuropathology before their 40s, and are at a greatly increased risk of Alzheimer Disease (AD). DS is caused by trisomy of chromosome 21 (Hsa21), resulting in the presence of an extra copy of its genes. This may result in an increased expression of these genes, including the gene for amyloid precursor protein (APP), which plays a key role in the development of AD. While the duplication of the APP locus alone is sufficient to cause early-onset AD, we have shown using transgenic mouse models that another gene or genes on Hsa21 may contribute to the increased risk of AD associated with DS. Methods: Tc1 mice contain a freely segregating copy of Hsa21 without functionally trisomic APP, while J20 mice express a human APP transgene with three AD-associated mutations (APP-SwInd). By investigating a novel Tc1 x J20 mouse cross, we have recently shown that trisomy of chromosome 21 leads to a doubling of the area covered by APP/A b plaques at 6 and 16 months of age, compared to the J20 model alone. In order to determine which Hsa21 genes influence APP/A b pathology, we seek to modulate the expression of candidate genes in primary cortical cultures grown from our mouse models, using lentivirus-mediated RNA interference (RNAi). These candidate genes will be selected if they are trisomic in our cross and are known to have an increased expression when trisomic. Results: We have characterized primary cortical neurons from Tc1 x J20 mice for the first time, including a comparison of APP protein and RNA expression levels between Tc1, J20, Tc1 x J20 and wildtype mice. Subsequently we are developing RNAi methods to reduce the expression of candidate genes from trisomic to disomic levels of expression. Conclusions: By assessing the effects of reduced candidate gene expression on levels of APP and other measures of pathology, this method will offer a more efficient approach to identify novel contributors to AD risk. It will also allow the study of potential combinatorial gene effects, which will otherwise be impractical in mouse models.
P1-104
THE BINDING OF BETA-AMYLOID 42 TO LIPID RAFTS CONTAINING DETERGENT-RESISTANT MEMBRANES OF RBC GHOST IS ENHANCED BY DIETARY DOCOSAHEXAENOIC ACID IN RATS: AN IMPLICATION OF ERYTHROCYTE FUNCTIONS TO ALZHEIMER’S DISEASE
Michio Hashimoto1, Hossain Shahdat1, Masahiro Katakura1, Yoko Tanabe1, Hiroyuki Arai2, Osamu Shido1, 1Shimane University, Izumo, Japan; 2Tohoku University School of Medicine, Sendai, Japan. Contact e-mail: [email protected] Background: The interactions of RBC and amyloid beta peptide (Ab) are implicated in Alzheimer’s disease (AD), however, the mechanisms are far from clarity. In AD, circulating Abs increase in the plasma, interact with RBCs, and affect their functions/morphology. We investigated whether Ab 1-42 interacts with caveolin1-containing lipid-rafts of detergent resistance membranes (DRMs) and whether such interactions could be modulated by oral administration of docosahexaenoic acid (DHA, C22:6, n3), an essential brain fatty acid, whose plasma levels is lower in AD patients. Methods: RBC hemolysis, RBC ghost and DRMs were prepared. DRMS-bound A b 1-42, caveolin1, and cathepsin were measured by immunomicrofluorescence assay or ELISA. Results: A b 1-42 -induced hemolysis was reduced in the RBCs of DHA-preadministered rats. When fluorescent-labeled A b 1-42 (TAMRA-A b 1-42) was incubated with DRMs, TAMRA-A b 1-42 occupancy significantly increased in the DRMs of the DHA-preadministered rats. The levels of caveolin-1, DHA, cathepsin and membrane fluidity were high er, while cholesterol levels were truncated in the DRMs of DHA-preadministered rats, when compared with those of control rats. Additionally, when TAMRA-A b 1-42 was infused directly into the carotid veins of the rats, the TAMRA- A b 1-42 was found to a greater level in the DRMs of the DHA- preadministered rats, thus indicating that circulating A b s interact with the caveolin-1 containing lipid-rafts/caveola of the DRMs. The plasma levels of TAMRA-A b 1-42 were reduced, while that of the TAMRA-A b 1-42 in hepatic-DRMs and -cytosolic protease such as cathepsin activity also significantly increased in the post-infused DHA-preadministered rats. This demonstrates, at least partially, the contribution of hepatic DRMs-bound proteins in clearance of plasma A b 1-42 levels of DHA rats. The resistance to A b 1-42 -induced hemolysis was associated with decreases in levels of lipid peroxide, reactive oxygen species and increases in GSH levels in the RBC of DHA rats. Conclusions: [i] The detection of A b s, at least in part, on the surface of the RBCs of AD patients might constitute the basis of biomarkers of AD, and [ii] the enrichment of RBCs with DHA helps in plasma clearance of the amyloids, probably, by increased lipid-rafts-dependent degradative pathways and also by membrane-bound increased protease like catehpsin acivity.
P1-105
THE ROLE OF AUTOPHAGY IN ALZHEIMER’S DISEASE
Miranda Orr1, Salvatore Oddo2, 1University of Texas Health Science Center at San Antonio, San Antonio, Texas, United States; 2University of Texas Health Science Center at San Antonio, San Antonio, Texas, United States. Contact e-mail: [email protected] Background: The accumulation of extracellular Ab plaques and intracellular neurofibrillary tangles comprised of hyperphosphorylated tau protein coincide with synaptic dysfunction, cognitive decline, and neuron loss in patients afflicted with Alzheimer’s disease (AD). A prominent hypothesis suggests that elevated Ab initiates the pathogenic cascade; however, mechanisms linking Ab to downstream events remain unclear. Due to the accumulation of proteinaceous aggregates in patient brains, pathways involved with protein degradation and clearance likely contribute to disease. One
P189
such system, the autophagic-lysosomal system, is crucial for catabolizing excess proteins, including amyloid precursor protein (APP), Ab, and tau. While specific autophagic pathways regulating Ab and tau accumulation remain largely unknown, deficits in chaperone-mediated autophagy (CMA) generate aggregate-prone tau fragments. Methods: Here we used complementary in vivo and in vitro approaches to elucidate the role of macroautophagy and CMA on protein accumulation in AD. Results: CMA activity directly correlates to the levels of the lysosome-associated membrane protein type 2a (Lamp2a); when we compared lysosomal Lamp2a levels between 6-month-old 3xTg-AD mice and age-matched controls, we saw a significant decrease in 3xTg-AD mice suggesting CMA deficits. We also found that modulating macroautophagy, the prominent and less-selective autophagic-lysosomal pathway, can directly affect Ab and tau levels. Since macroautophagy inhibition can lead to increased CMA, we are exploring crosstalk between these pathways. Conclusions: Understanding the specific autophagic pathways involved in AD pathogenesis may lead to the identification of new therapeutic targets. Since decreased Lamp2a levels are indicative of decreased CMA activity, our results suggest that CMA dysfunction occurs in 6-month-old 3xTg-AD mice. While 3xTgAD mice display elevated Ab at this age, insoluble tau aggregates have not yet formed. Therefore this result associates elevated Ab to CMA dysregulation, and suggests that both may be upstream of tau misprocessing.
P1-106
USING PRIMARY NEURONS FROM TRANSGENIC MICE TO IDENTIFY GENES ON HUMAN CHROMOSOME 21 INFLUENCING ALZHEIMER’S NEUROPATHOLOGY
Xun Yu Choong1, Alex Raha2, Victor Tybulewicz3, Frances Wiseman1, Elizabeth Fisher1, 1Institute of Neurology, University College London, London, United Kingdom; 2MRC Prion Unit, London, United Kingdom; 3 MRC National Institute for Medical Research, London, United Kingdom. Contact e-mail: [email protected] Background: Virtually all individuals with Down Syndrome (DS) develop features of Alzheimer neuropathology before their 40s, and are at a greatly increased risk of Alzheimer Disease (AD). DS is caused by trisomy of chromosome 21 (Hsa21), resulting in the presence of an extra copy of its genes. This may result in an increased expression of these genes, including the gene for amyloid precursor protein (APP), which plays a key role in the development of AD. While the duplication of the APP locus alone is sufficient to cause early-onset AD, we have shown using transgenic mouse models that another gene or genes on Hsa21 may contribute to the increased risk of AD associated with DS. Methods: Tc1 mice contain a freely segregating copy of Hsa21 without functionally trisomic APP, while J20 mice express a human APP transgene with three AD-associated mutations (APP-SwInd). By investigating a novel Tc1 x J20 mouse cross, we have recently shown that trisomy of chromosome 21 leads to a doubling of the area covered by APP/A b plaques at 6 and 16 months of age, compared to the J20 model alone. In order to determine which Hsa21 genes influence APP/A b pathology, we seek to modulate the expression of candidate genes in primary cortical cultures grown from our mouse models, using lentivirus-mediated RNA interference (RNAi). These candidate genes will be selected if they are trisomic in our cross and are known to have an increased expression when trisomic. Results: We have characterized primary cortical neurons from Tc1 x J20 mice for the first time, including a comparison of APP protein and RNA expression levels between Tc1, J20, Tc1 x J20 and wildtype mice. Subsequently we are developing RNAi methods to reduce the expression of candidate genes from trisomic to disomic levels of expression. Conclusions: By assessing the effects of reduced candidate gene expression on levels of APP and other measures of pathology, this method will offer a more efficient approach to identify novel contributors to AD risk. It will also allow the study of potential combinatorial gene effects, which will otherwise be impractical in mouse models.