- Email: [email protected]
P2-130 A nuclear transcription factor involved in the downregulation of the amyloid Aβ peptide levels
Poster Session P2: Animal and Cellular Models - Cellular Models
$262 ~
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NUCLEAR TRANSCRIPTION FACTOR INVOLVED IN T H E D O W N R E G U L A T I O N O F T H E A M Y L O I D AI3 PEPTIDE LEVELS
Ira Espuny Camacho . t , Kurt Spittaels 2, Pascal Merchiers 2, Lutgarde Serneels 1, Diana Dominguez 1, Bart De Strooper 1.1KU Leuven, Leuven, Belgium; 2Galapagos Genomics NV, Mechelen, Belgium. Contact e-mail: [email protected] Background: The brains of Alzheimer's disease patients are characterized by two major features: Neurofibrillary tangles and Amyloid Beta plaques. The Amyloid Beta plaques consist mainly of Amyloid Beta peptide fibrils. Increasing evidence suggest that the aggregation and subsequent deposition of the A[~ peptide is a key factor for the generation of Alzheimer's Disease. Therefore, several strategies were developed in recent years aiming to lower the A~ steady-state levels by: 1) decreasing the production of the A[3 peptide, by the specific inhibition of either the [3-secretase or the y-secretase cleavage, or 2) increasing the degradation of the Af3 peptide. Objective(s): The present work has as an objective, to look for modulators of the A[3 steady-state levels, as possible therapeutic targets for Alzheimer's Disease. Methods: Infection of cells in culture with recombinant adenovirus and analyse the processing of APP by Western Blot and ELISA detection assays. Results: Our experiments specifically show that over expression, or activation at the endogenous levels of a nuclear transcription factor in different cell lines, provokes a dramatic reduction in the steady state levels of secreted and cellular A[3 peptide. This was not accompanied by any other modification in the processing of APP and/or changes in gamma- or betasecretase activity, or Notch processing. Furthermore, we confirm through a specific stability assay, that Amyloid Beta peptide is being rapidly degraded from conditioned medium of cells when expression of this transcription factor is being induced. Conclusions: The obtained results implicate the transcription factor, or the pathway regulated by this transcription factor, as a possible therapeutic target to decrease the AI3 load in the brains of Alzheimer's disease patients.
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E S T R O G E N R E S T O R E S A G I N G RAT N E U R O N MITOCHONDRIAL FUNCTION
Gregory J. Brewer*, M.S. Parihar, Torrie T. Jones, Jason Nash. Southern Illinois University Sch Med, Springfield, IL, USA. Contact e-mail: gbrewer @siumed, edu Background: The mitochondrial oxyradical theory of aging is gaining increased support from studies of brain, heart and muscle tissue, and may explain the aging requirement for Alzheimer's disease. For neuronspecific function, we have established culture techniques that allow us to compare neurons isolated from embryonic, middle-age (12 month) and old (24 month) rat hippocampus (Brewer, 1997). Previously, we found that susceptibility to glutamate and A-beta toxicity increased with age of the animal from which the neurons were isolated, even though the old neurons regenerated just as well as the middle-age neurons in the uniform culture conditions (Brewer, 1998). Objective(s): To link aging mitochondria in neurons to increased susceptibility to glutamate and A-beta toxicity and to determine the effects of estrogen treatment on this susceptibility. Methods: For use with these cultured neurons, we monitored cytochrome oxidase activity (COX), cardiolipin specific staining of mitochondria with nonylacrydine orange (NAt), as well as labeling with mitotracker-red and immunostain fox cytochrome C, fluorescent NADH and glutathione levels and respiration. Results: Cytochrome C immunostaln and mitotracker red stained equal numbers of mitochondria per cell (350) for middle-age and old neurons, compared to 750 per cell for embryonic neurons. In contrast to mitochondrial numbers, COX activity/cell declined sharply with age of the neurons: levels of old neurons were reduced by 30% of those of middle-age neurons. Similarly, N A t stain for mitochondria was 40% lower for old neurons compared to middle-age neurons. By addition of FGF2 and low density passage, old neurons divide in culture (Brewer, 1999). COX activity in these dividing old neurons was equivalent to that of middle-age neurons. Age-related declines in glutathione levels were exacerbated by exposure to glutamate. Although NADH levels and respiration were normal
in old neurons, in response to glutamate, old neurons showed dramatic deficits. Treatment of old neurons with 17-beta-estradiol restored NAO staining levels/cell to those of middle-age neurons, restored control of calcium responses to glutamate and provided full neuroprotection from glutamate and Abeta toxicity. Conclusions: These results demonstrate that mitochondrial function is impaired in old neurons, especially with stress, but function can be restored by treatment with FGF2 or estrogen.
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E F F E C T O F AI3 S P E C I E S ON RAT P R I M A R Y C O R T I C A L N E U R O N SURVIVAL A N D C Y T O K I N E SECRETION FROM MICROGLIAL CELLS
Peter E. Soden*, Karen L. Philpott, Colin Dingwall, Tsu Tshen Chuang. Neurology and GI Centre of Excellence for Drug Discovery, GlaxoSmithKline R&D Limited, Harlow, United Kingdom. Contact e-mail: Peter.E.Soden@ gsk.com Background: Recent studies have switched the focus from fibrillar amyloid to monomers and soluble oligomers of AI~ as the active species of peptide that ultimately bring about the synaptic loss and associated dementia in Alzheimer's disease (AD). In addition, inflammatory processes involving A~ activation of microglial cells are believed to play a significant role in AD progression. Objective(s): Establish which form of AI3 (monomeric, oligomeric and pre-fibrillised) can cause neuronal death or microglial activation. Methods: Three in-vitro models were employed in this study - a) rat primary cortical neurons b) rat primary microglial cells c) NTW8 mouse microglial cell line. AI3 1-42 peptides, purchased from California Peptide Research Inc., were added to the cultures, either as freshly solnhilised, monomeric peptides, low molecular weight oligomers or pre-fibrillised solutions at concentrations up to 30 I~M. Induction of apoptosis was measured using a caspase 3/7 activity assay, and the cellular viability of neuronal cultures was measured by LDH release at either 24 or 48 hrs post administration. Microglial activation was measured via the production of cytokines, including TNFct, IL-6, IL-I[5, MIPlct and MCP-1 and an MTT assay used to establish cell viability at 24 or 48 hrs. The presence of different species of amyloid were confirmed using western blot analysis. Results: Preparations containing AI3 1-42 in the oligomeric and monomeric states induced neuronal death at 48 hrs at 30 p,M concentration, whereas fibrillar AI3 showed no effect on cell viability. By contrast all forms of A~ showed an ability to induce cytoldne secretion from microglial cells. Conclusions: Neuronal death could be induced by oligomeric and monomeric forms of A~3 1-42. Cytokine production from microglia could be stimulated by all three A~ preparations.
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A S S E M B L Y O F F I L A M E N T O U S TAU A G G R E G A T E S IN C O N D I T I O N A L T R A N S F E C T A N T S O F H U M A N NEURONAL LINEAGE THAT OVERPRODUCE W I L D - T Y P E H U M A N B R A I N TAU IN T R I P L E ISOFORMS
Li-wen Ko* 1, Toni Rush 1, Naruhiko Sahara 2, Jay S. Kersh 1, Michael DeTure 1, Wen-Lang Lin I , Colin Easson 1, Yamicia D. Connor 1, Shu-I-Iui C. Yen 1. I May o Clinic Jacksonville, Jacksonville, FL, USA; 2Riken, Saitama, Japan. Contact e-mail: [email protected] Background: Formation of filamentous tan aggregates constitutes a pathological hallmark of neurofibrillary degeneration that is characteristic of Alzheimer's disease (AD) and related disorders known collectively as taupathies. Our current understanding of the natural history of how neurofibrillary pathology evolves remains limited. However, this can be improved by the development of animal or cellular models with which cardinal features of such neurodegeneration are recapitulated. Objective(s): Our goal is to develop cellular models for the intraneuronal assembly of filamentous tan aggregates. Methods: We undertook sequential transfections of human neuroblastoma M17D cells in conjunction with construction of a tan minigene and succeeded in generating conditional transfectants of human neuronal lineage overproducing the wild-type human brain tan in isoforms 4RON, 3R1N and 4R1N - the principal constituents of neurofibril-
$262 ~
A
NUCLEAR TRANSCRIPTION FACTOR INVOLVED IN T H E D O W N R E G U L A T I O N O F T H E A M Y L O I D AI3 PEPTIDE LEVELS
Ira Espuny Camacho . t , Kurt Spittaels 2, Pascal Merchiers 2, Lutgarde Serneels 1, Diana Dominguez 1, Bart De Strooper 1.1KU Leuven, Leuven, Belgium; 2Galapagos Genomics NV, Mechelen, Belgium. Contact e-mail: [email protected] Background: The brains of Alzheimer's disease patients are characterized by two major features: Neurofibrillary tangles and Amyloid Beta plaques. The Amyloid Beta plaques consist mainly of Amyloid Beta peptide fibrils. Increasing evidence suggest that the aggregation and subsequent deposition of the A[~ peptide is a key factor for the generation of Alzheimer's Disease. Therefore, several strategies were developed in recent years aiming to lower the A~ steady-state levels by: 1) decreasing the production of the A[3 peptide, by the specific inhibition of either the [3-secretase or the y-secretase cleavage, or 2) increasing the degradation of the Af3 peptide. Objective(s): The present work has as an objective, to look for modulators of the A[3 steady-state levels, as possible therapeutic targets for Alzheimer's Disease. Methods: Infection of cells in culture with recombinant adenovirus and analyse the processing of APP by Western Blot and ELISA detection assays. Results: Our experiments specifically show that over expression, or activation at the endogenous levels of a nuclear transcription factor in different cell lines, provokes a dramatic reduction in the steady state levels of secreted and cellular A[3 peptide. This was not accompanied by any other modification in the processing of APP and/or changes in gamma- or betasecretase activity, or Notch processing. Furthermore, we confirm through a specific stability assay, that Amyloid Beta peptide is being rapidly degraded from conditioned medium of cells when expression of this transcription factor is being induced. Conclusions: The obtained results implicate the transcription factor, or the pathway regulated by this transcription factor, as a possible therapeutic target to decrease the AI3 load in the brains of Alzheimer's disease patients.
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•
E S T R O G E N R E S T O R E S A G I N G RAT N E U R O N MITOCHONDRIAL FUNCTION
Gregory J. Brewer*, M.S. Parihar, Torrie T. Jones, Jason Nash. Southern Illinois University Sch Med, Springfield, IL, USA. Contact e-mail: gbrewer @siumed, edu Background: The mitochondrial oxyradical theory of aging is gaining increased support from studies of brain, heart and muscle tissue, and may explain the aging requirement for Alzheimer's disease. For neuronspecific function, we have established culture techniques that allow us to compare neurons isolated from embryonic, middle-age (12 month) and old (24 month) rat hippocampus (Brewer, 1997). Previously, we found that susceptibility to glutamate and A-beta toxicity increased with age of the animal from which the neurons were isolated, even though the old neurons regenerated just as well as the middle-age neurons in the uniform culture conditions (Brewer, 1998). Objective(s): To link aging mitochondria in neurons to increased susceptibility to glutamate and A-beta toxicity and to determine the effects of estrogen treatment on this susceptibility. Methods: For use with these cultured neurons, we monitored cytochrome oxidase activity (COX), cardiolipin specific staining of mitochondria with nonylacrydine orange (NAt), as well as labeling with mitotracker-red and immunostain fox cytochrome C, fluorescent NADH and glutathione levels and respiration. Results: Cytochrome C immunostaln and mitotracker red stained equal numbers of mitochondria per cell (350) for middle-age and old neurons, compared to 750 per cell for embryonic neurons. In contrast to mitochondrial numbers, COX activity/cell declined sharply with age of the neurons: levels of old neurons were reduced by 30% of those of middle-age neurons. Similarly, N A t stain for mitochondria was 40% lower for old neurons compared to middle-age neurons. By addition of FGF2 and low density passage, old neurons divide in culture (Brewer, 1999). COX activity in these dividing old neurons was equivalent to that of middle-age neurons. Age-related declines in glutathione levels were exacerbated by exposure to glutamate. Although NADH levels and respiration were normal
in old neurons, in response to glutamate, old neurons showed dramatic deficits. Treatment of old neurons with 17-beta-estradiol restored NAO staining levels/cell to those of middle-age neurons, restored control of calcium responses to glutamate and provided full neuroprotection from glutamate and Abeta toxicity. Conclusions: These results demonstrate that mitochondrial function is impaired in old neurons, especially with stress, but function can be restored by treatment with FGF2 or estrogen.
•P-2•
E F F E C T O F AI3 S P E C I E S ON RAT P R I M A R Y C O R T I C A L N E U R O N SURVIVAL A N D C Y T O K I N E SECRETION FROM MICROGLIAL CELLS
Peter E. Soden*, Karen L. Philpott, Colin Dingwall, Tsu Tshen Chuang. Neurology and GI Centre of Excellence for Drug Discovery, GlaxoSmithKline R&D Limited, Harlow, United Kingdom. Contact e-mail: Peter.E.Soden@ gsk.com Background: Recent studies have switched the focus from fibrillar amyloid to monomers and soluble oligomers of AI~ as the active species of peptide that ultimately bring about the synaptic loss and associated dementia in Alzheimer's disease (AD). In addition, inflammatory processes involving A~ activation of microglial cells are believed to play a significant role in AD progression. Objective(s): Establish which form of AI3 (monomeric, oligomeric and pre-fibrillised) can cause neuronal death or microglial activation. Methods: Three in-vitro models were employed in this study - a) rat primary cortical neurons b) rat primary microglial cells c) NTW8 mouse microglial cell line. AI3 1-42 peptides, purchased from California Peptide Research Inc., were added to the cultures, either as freshly solnhilised, monomeric peptides, low molecular weight oligomers or pre-fibrillised solutions at concentrations up to 30 I~M. Induction of apoptosis was measured using a caspase 3/7 activity assay, and the cellular viability of neuronal cultures was measured by LDH release at either 24 or 48 hrs post administration. Microglial activation was measured via the production of cytokines, including TNFct, IL-6, IL-I[5, MIPlct and MCP-1 and an MTT assay used to establish cell viability at 24 or 48 hrs. The presence of different species of amyloid were confirmed using western blot analysis. Results: Preparations containing AI3 1-42 in the oligomeric and monomeric states induced neuronal death at 48 hrs at 30 p,M concentration, whereas fibrillar AI3 showed no effect on cell viability. By contrast all forms of A~ showed an ability to induce cytoldne secretion from microglial cells. Conclusions: Neuronal death could be induced by oligomeric and monomeric forms of A~3 1-42. Cytokine production from microglia could be stimulated by all three A~ preparations.
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A S S E M B L Y O F F I L A M E N T O U S TAU A G G R E G A T E S IN C O N D I T I O N A L T R A N S F E C T A N T S O F H U M A N NEURONAL LINEAGE THAT OVERPRODUCE W I L D - T Y P E H U M A N B R A I N TAU IN T R I P L E ISOFORMS
Li-wen Ko* 1, Toni Rush 1, Naruhiko Sahara 2, Jay S. Kersh 1, Michael DeTure 1, Wen-Lang Lin I , Colin Easson 1, Yamicia D. Connor 1, Shu-I-Iui C. Yen 1. I May o Clinic Jacksonville, Jacksonville, FL, USA; 2Riken, Saitama, Japan. Contact e-mail: [email protected] Background: Formation of filamentous tan aggregates constitutes a pathological hallmark of neurofibrillary degeneration that is characteristic of Alzheimer's disease (AD) and related disorders known collectively as taupathies. Our current understanding of the natural history of how neurofibrillary pathology evolves remains limited. However, this can be improved by the development of animal or cellular models with which cardinal features of such neurodegeneration are recapitulated. Objective(s): Our goal is to develop cellular models for the intraneuronal assembly of filamentous tan aggregates. Methods: We undertook sequential transfections of human neuroblastoma M17D cells in conjunction with construction of a tan minigene and succeeded in generating conditional transfectants of human neuronal lineage overproducing the wild-type human brain tan in isoforms 4RON, 3R1N and 4R1N - the principal constituents of neurofibril-