- Email: [email protected]
P1-205 Amyloid beta peptide assembly: effect of antifibrillogenic peptides
$154
Poster Session PI: Molecular Mechanisms of Neurodegeneration - ~-Amyloidosis
Activation of NMDA and tuACh receptors lead to significant enhancement of PARP activity in hippocampus. Moreover, stimulation of mACh. receptor transduces the signal in to PARP also in the other parts of the brain. These data indicated that AB and NAC peptides through free radicals dependent processes significantly influence the basic and receptors mediated activity of PARP. Alteration of PARP may affect the function of several transcription factors, gene expression and signal transduction.
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INHIBITION OF THE TGF-BETA SIGNALING PATHWAY RESULTS IN INCREASED N E U R O D E G E N E R A T I O N AND A M Y L O I D D E P O S I T I O N IN H U M A N A P P TRANSGENIC M I C E
Ina C. Tesseur* 1, Kun Zou 1, Eliezer Masliah 2, Jacob Corn 3, Thomas Brionne I , Frederic Bard 4, Tony Wyss-Coray 1,5. 1Stanford
University, Stanford, CA, USA; 2University of California San Diego, La Jolla, CA, USA; 3Gladstone Institute of Neurological Disease, University of California San Francisco, San Francisco, CA, USA; 4Elan Pharmaceuticals, Inc., South San Francisco, CA, USA; SGRECC, VA Palo Alto Health Care System, Palo Alto, CA, USA. Contact e-mail: itesseur@ stanford.edu Background: Recent evidence suggests that TGF-bl, a multifunctional cytokine involved in development and tissue homeostasis, could also play a role in neurodegeneration and the pathogenesis of Alzheimer's Disease. Overexpression of TGF-bl in astrocytes of human APP transgenic mice reduced parenchymal amyloid deposition, but also increased vascular amyloid. Both in vitro and recent in vivo studies in TGF-bl knock-out and overexpressing mice showed that TGF-bl is neuroprotective. Objective: To test the hypothesis that in neurons stressed by amyloid, decreased TGF-bl signaling leads to increased neurodegeneration and amyloid deposition. Methods: We crossed human APP mice 020 line) with heterozygous TGFbl knock-out mice, which express half the level of TGF-bl from wildtype littermates. To inhibit TGF-b signaling in neurons and glial cells we also crossed human APP mice with mice expressing a truncated, dominant negative, TGF-b type II receptor. Amyloid deposition was measured using thioflavin-S and 3D6 immunohistochemistry. Ab-levels was measured via ELISA and neurodegeneration was analyzed using synaptophysin and NeuN immunohistochemistry. Results: In 12-15 month old mice, reduced levels of TGF-bl in hAPP mice resulted in fewer synaptophysin positive presynaptic terminals as well as fewer numbers of NeuN positive cells. The levels of Ab42/totaiAb were increased but no significant changes in thioflavin-S positive amyloid, 3D6 immunoreactive Ab deposits, or APP protein levels were found. As homozygous TGF-bl knock-out mice die around weaning, we inhibited TGFb signaling in hAPP mice by crossing them with mice expressing a dominant negative TGF-b type II receptor. Twenty-month-old hAPP mice with deficient TGF-b signaling had twofold higher levels of 3D6- and Ab42-immunoreactive deposits than hAPP littermate controls and a 60% increase in the number of thioflavin S-positive plaques. Conclusions: These data support the concept that TGF-bl acts as a neuroprotective factor in vivo and that a relative increase in Ab42/totalAb rather than total Ab is associated with more neurodegeneration and AD-like pathology. Interfering or reducing growth factor signaling in the brain may therefore promote age-related neurodegeneration and Alzheimer's disease. Support Contributed By: NIA, N1NDS, John Douglas French Alzheimer's Foundation
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EFFECTS OF UBIQUILIN-1 RNA I N T E R F E R E N C E (RNAI) ON APP PROCESSING
Mikko Hiltunen*, Lars Bertram, Alice Lu, Zhongcong Xie, Rudolph E. Tanzi. Genetics and Aging Research Unit, Massachusetts General
Hospital and Harvard Medical School, Charlestown, MA, USA. Contact e-mail: mhiltunenl @parmers.org Background: The ubiquilin-1 (UBQLN1) gene located on chromosome 9@2 has recently been suggested to be a novel Alzheimer's disease (AD) susceptibility gene according to our genetic studies of late-onset
AD. In addition, yeast two-hybrid experiments have previously shown that UBQLN1 interacts with both presenilin-1 and 2 (PS) proteins and that overexpression of UBQLN1 in non-neuronal cell lines promotes PS protein accumulation. According to immunohistochemical analyses, anti-ubiquilin antibodies stain the neurofibrillary tangles in AD brain as well as the Lewy bodies in Parkinson's disease. Objective(s): Because previous genetic and biochemical findings indicate UBQLN1 as a plausible determinant in AD, we studied the effects of UBQLN1 protein down regulation on amyloid precursor protein (APP) processing using RNA interference (RNAi). Methods: In vitro transcription was used to produce multiple short interfering RNA (siRNA) hairpins specifically targeted against UBQLN1 mRNA. siRNA hairpins were nucleofected to H4 neuroglioma cell line overexpressing APP751 and after 48 hours of nucleofection, C83, C99 and full length levels of APP were quantified using immunoblotting. Results: Depending on the siRNA target site in UBQLN1 rnRNA, 40 to 90% reduction in UBQLNt expression was observed both in rnRNA and protein levels. Decrease in UBQLN1 expression was followed by approximately 80% and 40% increases in C83 and C99 levels, respectively. More precise analyses for determining the effects of UBQLN1 down regulation on ysecretase function, half life of C-terminal and full length fragments of APP, as well as sAPPc~,I3 and A[3 levels are in preparation. Conclusions: Down regulation of UBQLN1 expression likely affects APP processing by increasing levels of C83 and C99 levels, providing further support for the pivotal role of UBQLN1 in AD pathogenesis.
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AMYLOID BETA PEPTIDE ASSEMBLY: EFFECT OF ANTIFIBRILLOGENIC PEPTIDES
Utpal Das*, Pallavi Manral, Brajesh Agrawal, Rao G. Hariprasad, Kolandaivelu Saravanan, Baskar Singh, Savita Yadav, Tej P. Singh, Alagiri Srinivasan. All India Institute of Medical Sciences, New Delhi,
India. Contact e-mail: [email protected] Amyloid beta peptides (Abeta) are generated by the proteolytic processing of the amyloid precursor protein. Abeta is thought to be the causal agent for the amyloid plaque formation and subsequent cognitive decline in Alzheimer's Disease (AD). Assembly of monomeric amyloid beta peptide into oligomeric structure is an important pathogenic feature of this disease. Detailed knowledge of structure of Abeta and the dynamics of its aggregation is thus important to understand the pathogenic process. Since the emphasis of clinical interference in AD is slightly towards antiaggregation process, this knowledge is critical for the development of properly targeted AD therapeutics. The assembly of Abeta follows a distinct path rather than a random oligomerization. We have studied the oligomerization of synthetic Abeta1-42 in presence of salts and Abeta interacting peptide. Size exclusion chromatography, dynamic light scattering, circular dichroism spectroscopy and electron microscopy have been combined to elucidate the fundamental features of this Abeta assembly. Our studies have shown that the oligomerization property of Abetal-42 in presence of these salts and peptides can be modulated. The effect of different pH values, temperature and concentration of Abeta1-42 and time have been studied. In the presence of salts and peptide, aggregation is slowed down. Our results also show that these peptides can disaggregate the preformed aggregates. The results of these experiments will be presented.
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CAPACITATIVE AND NON-CAPACITATIVE CA 2+ ENTRY MODULATE Aft L E V E L S
Yama Akbari* 1, Nabil Dagher 1, M. Paul Murphy 2, J. Ashot Kozak 1, Brian Hitt I , Salvatore Oddo 1, Bert Tseng 1, Todd E. Golde 2, Michael Cahalan 1, Malcolm A. Leissring 3, Frank M. LaFerla 1. I UC
Irvine, Irvine, CA, USA, 2Mayo Clinic, Jacksonville, FL, USA; 3Harvard Medical School, Boston, MA, USA. Contact e-mail: [email protected] Background: Mutations in presenilin, responsible for the majority of earlyonset Alzheimer's disease (AD), lead to higher endoplasmic reticulum (ER) Ca 2+ stores and lower capacitative Ca 2+ entry (CCE). CCE is a mechanism by which cells replenish intracellular Ca 2+ stores after depletion due to
Poster Session PI: Molecular Mechanisms of Neurodegeneration - ~-Amyloidosis
Activation of NMDA and tuACh receptors lead to significant enhancement of PARP activity in hippocampus. Moreover, stimulation of mACh. receptor transduces the signal in to PARP also in the other parts of the brain. These data indicated that AB and NAC peptides through free radicals dependent processes significantly influence the basic and receptors mediated activity of PARP. Alteration of PARP may affect the function of several transcription factors, gene expression and signal transduction.
•f•
INHIBITION OF THE TGF-BETA SIGNALING PATHWAY RESULTS IN INCREASED N E U R O D E G E N E R A T I O N AND A M Y L O I D D E P O S I T I O N IN H U M A N A P P TRANSGENIC M I C E
Ina C. Tesseur* 1, Kun Zou 1, Eliezer Masliah 2, Jacob Corn 3, Thomas Brionne I , Frederic Bard 4, Tony Wyss-Coray 1,5. 1Stanford
University, Stanford, CA, USA; 2University of California San Diego, La Jolla, CA, USA; 3Gladstone Institute of Neurological Disease, University of California San Francisco, San Francisco, CA, USA; 4Elan Pharmaceuticals, Inc., South San Francisco, CA, USA; SGRECC, VA Palo Alto Health Care System, Palo Alto, CA, USA. Contact e-mail: itesseur@ stanford.edu Background: Recent evidence suggests that TGF-bl, a multifunctional cytokine involved in development and tissue homeostasis, could also play a role in neurodegeneration and the pathogenesis of Alzheimer's Disease. Overexpression of TGF-bl in astrocytes of human APP transgenic mice reduced parenchymal amyloid deposition, but also increased vascular amyloid. Both in vitro and recent in vivo studies in TGF-bl knock-out and overexpressing mice showed that TGF-bl is neuroprotective. Objective: To test the hypothesis that in neurons stressed by amyloid, decreased TGF-bl signaling leads to increased neurodegeneration and amyloid deposition. Methods: We crossed human APP mice 020 line) with heterozygous TGFbl knock-out mice, which express half the level of TGF-bl from wildtype littermates. To inhibit TGF-b signaling in neurons and glial cells we also crossed human APP mice with mice expressing a truncated, dominant negative, TGF-b type II receptor. Amyloid deposition was measured using thioflavin-S and 3D6 immunohistochemistry. Ab-levels was measured via ELISA and neurodegeneration was analyzed using synaptophysin and NeuN immunohistochemistry. Results: In 12-15 month old mice, reduced levels of TGF-bl in hAPP mice resulted in fewer synaptophysin positive presynaptic terminals as well as fewer numbers of NeuN positive cells. The levels of Ab42/totaiAb were increased but no significant changes in thioflavin-S positive amyloid, 3D6 immunoreactive Ab deposits, or APP protein levels were found. As homozygous TGF-bl knock-out mice die around weaning, we inhibited TGFb signaling in hAPP mice by crossing them with mice expressing a dominant negative TGF-b type II receptor. Twenty-month-old hAPP mice with deficient TGF-b signaling had twofold higher levels of 3D6- and Ab42-immunoreactive deposits than hAPP littermate controls and a 60% increase in the number of thioflavin S-positive plaques. Conclusions: These data support the concept that TGF-bl acts as a neuroprotective factor in vivo and that a relative increase in Ab42/totalAb rather than total Ab is associated with more neurodegeneration and AD-like pathology. Interfering or reducing growth factor signaling in the brain may therefore promote age-related neurodegeneration and Alzheimer's disease. Support Contributed By: NIA, N1NDS, John Douglas French Alzheimer's Foundation
~
EFFECTS OF UBIQUILIN-1 RNA I N T E R F E R E N C E (RNAI) ON APP PROCESSING
Mikko Hiltunen*, Lars Bertram, Alice Lu, Zhongcong Xie, Rudolph E. Tanzi. Genetics and Aging Research Unit, Massachusetts General
Hospital and Harvard Medical School, Charlestown, MA, USA. Contact e-mail: mhiltunenl @parmers.org Background: The ubiquilin-1 (UBQLN1) gene located on chromosome 9@2 has recently been suggested to be a novel Alzheimer's disease (AD) susceptibility gene according to our genetic studies of late-onset
AD. In addition, yeast two-hybrid experiments have previously shown that UBQLN1 interacts with both presenilin-1 and 2 (PS) proteins and that overexpression of UBQLN1 in non-neuronal cell lines promotes PS protein accumulation. According to immunohistochemical analyses, anti-ubiquilin antibodies stain the neurofibrillary tangles in AD brain as well as the Lewy bodies in Parkinson's disease. Objective(s): Because previous genetic and biochemical findings indicate UBQLN1 as a plausible determinant in AD, we studied the effects of UBQLN1 protein down regulation on amyloid precursor protein (APP) processing using RNA interference (RNAi). Methods: In vitro transcription was used to produce multiple short interfering RNA (siRNA) hairpins specifically targeted against UBQLN1 mRNA. siRNA hairpins were nucleofected to H4 neuroglioma cell line overexpressing APP751 and after 48 hours of nucleofection, C83, C99 and full length levels of APP were quantified using immunoblotting. Results: Depending on the siRNA target site in UBQLN1 rnRNA, 40 to 90% reduction in UBQLNt expression was observed both in rnRNA and protein levels. Decrease in UBQLN1 expression was followed by approximately 80% and 40% increases in C83 and C99 levels, respectively. More precise analyses for determining the effects of UBQLN1 down regulation on ysecretase function, half life of C-terminal and full length fragments of APP, as well as sAPPc~,I3 and A[3 levels are in preparation. Conclusions: Down regulation of UBQLN1 expression likely affects APP processing by increasing levels of C83 and C99 levels, providing further support for the pivotal role of UBQLN1 in AD pathogenesis.
•
AMYLOID BETA PEPTIDE ASSEMBLY: EFFECT OF ANTIFIBRILLOGENIC PEPTIDES
Utpal Das*, Pallavi Manral, Brajesh Agrawal, Rao G. Hariprasad, Kolandaivelu Saravanan, Baskar Singh, Savita Yadav, Tej P. Singh, Alagiri Srinivasan. All India Institute of Medical Sciences, New Delhi,
India. Contact e-mail: [email protected] Amyloid beta peptides (Abeta) are generated by the proteolytic processing of the amyloid precursor protein. Abeta is thought to be the causal agent for the amyloid plaque formation and subsequent cognitive decline in Alzheimer's Disease (AD). Assembly of monomeric amyloid beta peptide into oligomeric structure is an important pathogenic feature of this disease. Detailed knowledge of structure of Abeta and the dynamics of its aggregation is thus important to understand the pathogenic process. Since the emphasis of clinical interference in AD is slightly towards antiaggregation process, this knowledge is critical for the development of properly targeted AD therapeutics. The assembly of Abeta follows a distinct path rather than a random oligomerization. We have studied the oligomerization of synthetic Abeta1-42 in presence of salts and Abeta interacting peptide. Size exclusion chromatography, dynamic light scattering, circular dichroism spectroscopy and electron microscopy have been combined to elucidate the fundamental features of this Abeta assembly. Our studies have shown that the oligomerization property of Abetal-42 in presence of these salts and peptides can be modulated. The effect of different pH values, temperature and concentration of Abeta1-42 and time have been studied. In the presence of salts and peptide, aggregation is slowed down. Our results also show that these peptides can disaggregate the preformed aggregates. The results of these experiments will be presented.
•
CAPACITATIVE AND NON-CAPACITATIVE CA 2+ ENTRY MODULATE Aft L E V E L S
Yama Akbari* 1, Nabil Dagher 1, M. Paul Murphy 2, J. Ashot Kozak 1, Brian Hitt I , Salvatore Oddo 1, Bert Tseng 1, Todd E. Golde 2, Michael Cahalan 1, Malcolm A. Leissring 3, Frank M. LaFerla 1. I UC
Irvine, Irvine, CA, USA, 2Mayo Clinic, Jacksonville, FL, USA; 3Harvard Medical School, Boston, MA, USA. Contact e-mail: [email protected] Background: Mutations in presenilin, responsible for the majority of earlyonset Alzheimer's disease (AD), lead to higher endoplasmic reticulum (ER) Ca 2+ stores and lower capacitative Ca 2+ entry (CCE). CCE is a mechanism by which cells replenish intracellular Ca 2+ stores after depletion due to