- Email: [email protected]
Amyloid beta induces changes in NMDA and AMPA receptor trafficking in primary neuronal cultures and brain slices
P304
Poster Presentations P2
Acknowledgment: Alz.Association Grant IIRG-06-27105 and Italian Institute for Technology grant. P2-145
THE MOLECULAR CHAPERONE HUMAN Ab CRYSTALLIN MODULATES AMYLOID-b NEUROTOXICITY
Joy G. Ghosh1, Denise Fabian1, Ashley Mallat1, Steve Ramirez1, Mark Burton1, Juliet Moncaster1, Noel Casey1, Anca Mocofanescu1, Dean Hartley2, Patric Stanton3, Lee E. Goldstein1, 1Molecular Aging & Development Laboratory, Boston University School of Medicine, College of Engineering, & Photonics Center, Boston, MA, USA; 2Rush University Medical Center, Chicago, IL, USA; 3New York Medical College, New York, NY, USA. Contact e-mail: [email protected] Background: The low-molecular-weight (LMW) chaperone aB crystallin, co-localizes with amyloid b (Ab) in Alzheimer’s disease (AD) neuritic plaques, co-aggegregates in the cytoplasm of AD lens fiber cells, and is upregulated in an AD transgenic Caenorhabditis elegans model. Previously, these molecular chaperones were conceptualized as relatively non-specific components of the unfolding protein response. Recent studies suggest that the interaction of LMW chaperones with amyloidogenic proteins may in fact be pathogenic. Here we investigate the interaction of the archetypic LMW molecular chaperone aB crystallin with Ab. Methods: Immunogold electron microscopy, SDS-PAGE and immunoblotting, spectral reflectance imaging biosensor, and cell culture. Results: Double-immunogold electron microscopy showed that aB crystallin arrests amyloid-b fibrillogenesis and facilitates sequestration of amyloid-b peptides into soluble hetero-oligomeric chaperone complexes. Subsequently, we used synthetic amyloid-b and recombinant human aB crystallin in cell culture experiments to ascertain whether aB crystallin modulates amyloid-b toxicity in SH-SY5Y neuroblastoma cells. We utilized a novel label-free spectral reflectance imaging biosensor (SRIB) to determine that the apparent affinity constant for the aB crystallin/amyloid-b interaction was in the low nanomolar range making this interaction exceedingly stable and resistant to degradation. Conclusions: Our data are consistent with a specific and important role for the LMW chaperone aB crystallin in Alzheimer’s disease pathogenesis. P2-146
EPIGENETIC CHANGES RELATED TO BETAAMYLOID-IMPLICATIONS FOR ALZHEIMER’S DISEASE
Christina Unger Lithner1, Caterina M. Hernandez2, Agneta Nordberg1, J. David Sweatt3, 1Karolinska Institutet, Stockholm, Sweden; 2University of Texas Medical Branch, Galveston, TX, USA; 3University of Alabama at Birmingham, Birmingham, AL, USA. Contact e-mail: Christina.Unger@ki. se Background: Transcriptional dysfunction has been implicated in the pathology of Alzheimer’s disease (AD). Long-term memory formation involves biochemical signaling cascades that lead to a change in neuronal gene expression. Regulation of histones, and thus chromatin structure, plays an important role in gene transcription and facilitate long-term changes in neuronal physiology and cell survival. Methods: In this study we used Tg2576 mice and two transfected cell lines (SH-SY5Y cells and HEK293 cells) carrying the human APPswe mutation to examine the consequences of the excess of Ab on the regulation of histones. Results: In the Tg2576 mice we observed an increase in histone H3 acetylation and phosphorylation in prefrontal cortex (PRF) and cortex. Methylation of histone H3 was increased in PRF but decreased in the striatum. We also found an increase in histone H4 acetylation in the CA1 of the hippocampus. Treatment with sodium butyrate (NaB), a histone deacetylase (HDAC) inhibitor increased the histone H3 acetylation in SH-SY5Ycells, but not in HEK293 cells. Treatment with the g-secretase inhibitor DAPT caused a decrease in histone H3 acetylation in SH-SY5Y cells. Conclusions: The results strongly suggest that Ab triggers changes in chromatin structure in the central nervous system (CNS). Understanding these functions is of importance to further investigate the roles and mechanisms of persistent Ab exposure in the aging-related CNS dysfunction. This might hopefully provide new insights into this disease and into new
treatment options by allowing us to rescue cells that otherwise would be destined to die. P2-147
AMYLOID BETA INDUCES CHANGES IN NMDA AND AMPA RECEPTOR TRAFFICKING IN PRIMARY NEURONAL CULTURES AND BRAIN SLICES
Warren D. Hirst, Katie E. Kubek, Jeannette Golembieski, Roland G. W. Staal, Menelas N. Pangalos, Peter H. Reinhart, Stephen P. Braithwaite, Wyeth Discovery Research, Princeton, NJ, USA. Contact e-mail: [email protected] Background: Alzheimer’s disease is a chronic neurodegenerative disorder that is characterized by elevated levels, and deposition of, amyloid beta. Numerous studies have implicated the 42 amino acid amyloid beta peptide (Ab1-42) as key to underlying the disease. However transgenic mouse models expressing high levels, or mutant forms of amyloid precursor protein, leading to elevated Ab1-42, exhibit cognitive deficits prior to deposition of amyloid plaques and without neurodegenerative pathology. These deficits in cognition may therefore be the result of synaptic dysfunction rather than neuronal death. Objective: To determine whether Ab1-42 modulates key mechanisms in synaptic function by assessing changes in receptor trafficking at synaptic membranes in primary neuronal cultures and in acutely dissected hippocampal slices from adult rats. Methods: Following exposure to 1 uM Ab1-42 or scrambled peptide surface proteins were labeled in either rat primary cortical neurons (10-14 days in vitro) or acutely dissected rat hippocampal slices by incubation with a non-cell permeable form of biotin. NeutrAvidin agarose beads were used to pull down the biotinylated proteins and these were quantified by immunoblot (with antibodies to NMDA and AMPA receptor subunits and GABAA and EGF receptors), together with the total lysates in order to determine a ratio of surface to total proteins. Results: In primary cortical neuronal cultures biotinylation experiments have demonstrated that Ab1-42 selectively mediates endocytosis of both NR2A and NR2B containing NMDA receptors, but not AMPA, GABAA or EGF receptors, from the neuronal surface. Treatment with 1 mM Ab1-42 for one hour reduces surface expressed NMDA by 25-40%. In the acute hippocampal slices, Ab1-42 selectively mediates endocytosis of both NR2A and NR2B containing NMDA receptors as well as GluR2 subunits of AMPA receptors, but not EGF receptors, from the neuronal surface. Treatment with 1 mM Ab142 for one hour reduces surface expressed NMDA and AMPA receptors by approximately 50%. Conclusions: These studies demonstrate that the synapse is a dynamically regulated system that is influenced by multiple external influences such as Ab1-42. However, not all synaptic proteins are affected by Ab1-42. The receptor changes induced by Ab1-42 may underlie some of the symptoms of Alzheimer’s disease. P2-148
THE ANXIOGENIC EFFECTS OF ABETA (1- 40) AND ABETA (1-42) IN THE DORSAL RAPHE NUCLEUS IN MALE RATS
Shervin Gholizadeh, Tooka Aavani, Fereshteh Motamedi, Neuroscience Research Center, Shaheed Beheshti University of Medical Sciences, Tehran, Iran, Islamic Republic of. Contact e-mail: [email protected] Background: The pathological accumulation of Abeta in the brain leads to oxidative stress which plays both a key role in Alzheimer’s disease (AD) and anxiety. Furthermore, numerous studies have documented the role of serotonergic neurons, which mostly originate from the dorsal raphe nucleus (DRN), in the modulation of anxious states. As 5-Hydroxytryptamine (5-HT) sites are significantly decreased in the DRN in AD, the present study was designed to examine the effects of A 40 and A 42 microinjection into the DRN. Methods: Abeta 40 and Abeta 42 (2 mg/ml) were injected into the DRN in male wistar rats and 1 week, 15 days and 1 month later, anxiety levels were evaluated using the elevated plus maze (EPM) and the light/dark paradigm. Sham-exposed and naive rats receiving saline were used as controls and diazepam (2 mg/kg, i.p.) was used as a positive control drug. Results: Abeta 40 and Abeta 42 both decreased open arm exploratory behavior in the EPM and decreased the time spent in the lighted box in the light/dark
Poster Presentations P2
Acknowledgment: Alz.Association Grant IIRG-06-27105 and Italian Institute for Technology grant. P2-145
THE MOLECULAR CHAPERONE HUMAN Ab CRYSTALLIN MODULATES AMYLOID-b NEUROTOXICITY
Joy G. Ghosh1, Denise Fabian1, Ashley Mallat1, Steve Ramirez1, Mark Burton1, Juliet Moncaster1, Noel Casey1, Anca Mocofanescu1, Dean Hartley2, Patric Stanton3, Lee E. Goldstein1, 1Molecular Aging & Development Laboratory, Boston University School of Medicine, College of Engineering, & Photonics Center, Boston, MA, USA; 2Rush University Medical Center, Chicago, IL, USA; 3New York Medical College, New York, NY, USA. Contact e-mail: [email protected] Background: The low-molecular-weight (LMW) chaperone aB crystallin, co-localizes with amyloid b (Ab) in Alzheimer’s disease (AD) neuritic plaques, co-aggegregates in the cytoplasm of AD lens fiber cells, and is upregulated in an AD transgenic Caenorhabditis elegans model. Previously, these molecular chaperones were conceptualized as relatively non-specific components of the unfolding protein response. Recent studies suggest that the interaction of LMW chaperones with amyloidogenic proteins may in fact be pathogenic. Here we investigate the interaction of the archetypic LMW molecular chaperone aB crystallin with Ab. Methods: Immunogold electron microscopy, SDS-PAGE and immunoblotting, spectral reflectance imaging biosensor, and cell culture. Results: Double-immunogold electron microscopy showed that aB crystallin arrests amyloid-b fibrillogenesis and facilitates sequestration of amyloid-b peptides into soluble hetero-oligomeric chaperone complexes. Subsequently, we used synthetic amyloid-b and recombinant human aB crystallin in cell culture experiments to ascertain whether aB crystallin modulates amyloid-b toxicity in SH-SY5Y neuroblastoma cells. We utilized a novel label-free spectral reflectance imaging biosensor (SRIB) to determine that the apparent affinity constant for the aB crystallin/amyloid-b interaction was in the low nanomolar range making this interaction exceedingly stable and resistant to degradation. Conclusions: Our data are consistent with a specific and important role for the LMW chaperone aB crystallin in Alzheimer’s disease pathogenesis. P2-146
EPIGENETIC CHANGES RELATED TO BETAAMYLOID-IMPLICATIONS FOR ALZHEIMER’S DISEASE
Christina Unger Lithner1, Caterina M. Hernandez2, Agneta Nordberg1, J. David Sweatt3, 1Karolinska Institutet, Stockholm, Sweden; 2University of Texas Medical Branch, Galveston, TX, USA; 3University of Alabama at Birmingham, Birmingham, AL, USA. Contact e-mail: Christina.Unger@ki. se Background: Transcriptional dysfunction has been implicated in the pathology of Alzheimer’s disease (AD). Long-term memory formation involves biochemical signaling cascades that lead to a change in neuronal gene expression. Regulation of histones, and thus chromatin structure, plays an important role in gene transcription and facilitate long-term changes in neuronal physiology and cell survival. Methods: In this study we used Tg2576 mice and two transfected cell lines (SH-SY5Y cells and HEK293 cells) carrying the human APPswe mutation to examine the consequences of the excess of Ab on the regulation of histones. Results: In the Tg2576 mice we observed an increase in histone H3 acetylation and phosphorylation in prefrontal cortex (PRF) and cortex. Methylation of histone H3 was increased in PRF but decreased in the striatum. We also found an increase in histone H4 acetylation in the CA1 of the hippocampus. Treatment with sodium butyrate (NaB), a histone deacetylase (HDAC) inhibitor increased the histone H3 acetylation in SH-SY5Ycells, but not in HEK293 cells. Treatment with the g-secretase inhibitor DAPT caused a decrease in histone H3 acetylation in SH-SY5Y cells. Conclusions: The results strongly suggest that Ab triggers changes in chromatin structure in the central nervous system (CNS). Understanding these functions is of importance to further investigate the roles and mechanisms of persistent Ab exposure in the aging-related CNS dysfunction. This might hopefully provide new insights into this disease and into new
treatment options by allowing us to rescue cells that otherwise would be destined to die. P2-147
AMYLOID BETA INDUCES CHANGES IN NMDA AND AMPA RECEPTOR TRAFFICKING IN PRIMARY NEURONAL CULTURES AND BRAIN SLICES
Warren D. Hirst, Katie E. Kubek, Jeannette Golembieski, Roland G. W. Staal, Menelas N. Pangalos, Peter H. Reinhart, Stephen P. Braithwaite, Wyeth Discovery Research, Princeton, NJ, USA. Contact e-mail: [email protected] Background: Alzheimer’s disease is a chronic neurodegenerative disorder that is characterized by elevated levels, and deposition of, amyloid beta. Numerous studies have implicated the 42 amino acid amyloid beta peptide (Ab1-42) as key to underlying the disease. However transgenic mouse models expressing high levels, or mutant forms of amyloid precursor protein, leading to elevated Ab1-42, exhibit cognitive deficits prior to deposition of amyloid plaques and without neurodegenerative pathology. These deficits in cognition may therefore be the result of synaptic dysfunction rather than neuronal death. Objective: To determine whether Ab1-42 modulates key mechanisms in synaptic function by assessing changes in receptor trafficking at synaptic membranes in primary neuronal cultures and in acutely dissected hippocampal slices from adult rats. Methods: Following exposure to 1 uM Ab1-42 or scrambled peptide surface proteins were labeled in either rat primary cortical neurons (10-14 days in vitro) or acutely dissected rat hippocampal slices by incubation with a non-cell permeable form of biotin. NeutrAvidin agarose beads were used to pull down the biotinylated proteins and these were quantified by immunoblot (with antibodies to NMDA and AMPA receptor subunits and GABAA and EGF receptors), together with the total lysates in order to determine a ratio of surface to total proteins. Results: In primary cortical neuronal cultures biotinylation experiments have demonstrated that Ab1-42 selectively mediates endocytosis of both NR2A and NR2B containing NMDA receptors, but not AMPA, GABAA or EGF receptors, from the neuronal surface. Treatment with 1 mM Ab1-42 for one hour reduces surface expressed NMDA by 25-40%. In the acute hippocampal slices, Ab1-42 selectively mediates endocytosis of both NR2A and NR2B containing NMDA receptors as well as GluR2 subunits of AMPA receptors, but not EGF receptors, from the neuronal surface. Treatment with 1 mM Ab142 for one hour reduces surface expressed NMDA and AMPA receptors by approximately 50%. Conclusions: These studies demonstrate that the synapse is a dynamically regulated system that is influenced by multiple external influences such as Ab1-42. However, not all synaptic proteins are affected by Ab1-42. The receptor changes induced by Ab1-42 may underlie some of the symptoms of Alzheimer’s disease. P2-148
THE ANXIOGENIC EFFECTS OF ABETA (1- 40) AND ABETA (1-42) IN THE DORSAL RAPHE NUCLEUS IN MALE RATS
Shervin Gholizadeh, Tooka Aavani, Fereshteh Motamedi, Neuroscience Research Center, Shaheed Beheshti University of Medical Sciences, Tehran, Iran, Islamic Republic of. Contact e-mail: [email protected] Background: The pathological accumulation of Abeta in the brain leads to oxidative stress which plays both a key role in Alzheimer’s disease (AD) and anxiety. Furthermore, numerous studies have documented the role of serotonergic neurons, which mostly originate from the dorsal raphe nucleus (DRN), in the modulation of anxious states. As 5-Hydroxytryptamine (5-HT) sites are significantly decreased in the DRN in AD, the present study was designed to examine the effects of A 40 and A 42 microinjection into the DRN. Methods: Abeta 40 and Abeta 42 (2 mg/ml) were injected into the DRN in male wistar rats and 1 week, 15 days and 1 month later, anxiety levels were evaluated using the elevated plus maze (EPM) and the light/dark paradigm. Sham-exposed and naive rats receiving saline were used as controls and diazepam (2 mg/kg, i.p.) was used as a positive control drug. Results: Abeta 40 and Abeta 42 both decreased open arm exploratory behavior in the EPM and decreased the time spent in the lighted box in the light/dark