- Email: [email protected]
The anxiogenic effects of Abeta (1- 40) and Abeta (1-42) in the dorsal raphe nucleus in male rats
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 test, 1 week, 15 days, and 1 month after microinjection. Conclusions: The present results illuminate the anxiogenic role of beta amyloid accumulation in the DRN. Our data support the notion that the oxidative stress and the neuronal loss resulting from Abeta accumulation in the DRN are involved in inducing anxiety-like behavior. P2-149
MOLECULAR SIGNATURE OF PRODROMAL ALZHEIMER’S DISEASE: A MICROARRAY STUDY IN POST-MORTEM CORTICAL TISSUE
Concepcion Conejero-Goldberg1, Thomas M. Hyde2, Shufen Chen1, Ute Dreses-Werringloer1, Mary Herman2, Joel E. Kleinman2, Peter Davies1, Terry Goldberg1, 1Litwin-Zucker Alzheimer’s Disease Research Center, FIMR, Manhasset, NY, USA; 2Clinical Brain Disorder Branch, NIMH, Bethesda, MD, USA. Contact e-mail: [email protected] Background: A number of studies have suggested that there are differences among cortical regions in vulnerability to Alzheimer’s disease (AD). Primary sensory cortices, including BA 1/2/3 remain relatively spared, while medial temporal and association cortices (including BA 21) are susceptible to plaque and tangle formation, even early in the disease. Though hypotheses have been put forth, the reasons for this difference remain obscure. In this study we used microarray technology to address this question by determining differences in inter-regional transcript expression from human post-mortem brain tissue from individuals at high risk for AD (positive for the APOE4 genotype) and individuals at low risk (negative for the APOE4 genotype). Subjects were chosen in whom AD was not present, as we did not want to measure ‘‘downstream’’ changes consequent to frank pathology. Methods: Fresh-frozen human brain tissue from normal control subjects (N ¼ 41) at autopsy was obtained from the Clinical Brain Disorders Branch, NIMH. Demographic characteristics of this cohort include: Age ¼ 41.9 6 10.3; Sex ¼ 35 M/6 F; pH ¼ 6.6 6 0.29; PMI ¼ 31.9 6 16.4; RIN ¼ BA 21 (8.0 6 0.5) and BA 1/2/3 (8.3 6 0.5); Genotype: APOE3¼ 28 & APOE4¼13. WG6 BeadChip arrays (Illumina) were processed according to the manufacturer’s guidelines. For statistical analyses, the data was processed using a ‘‘double subtraction’’ method. We determined expression differences between the two regions (BA 1/2/3 and BA 21) and contrasted these difference scores between APOE4 positive and negative groups, thus isolating intrinsic vulnerability or protective factors. Results: We identified 70 transcripts corresponding to genes from RefSeq and UniGene databases that differed significantly (at p< .001) between the APOE3 and APOE4 using ‘‘double subtraction’’ analyses. Fifty one of these transcripts survived FDR analyses. Conclusions: This study indicates that E4 promotes changes in calcium regulation, mitochondrial dysfunction, cell cycle activity, apoptosis, and Wnt signaling that are associated with disease pathogenesis. By contrasting expression profiles for brain regions relatively invulnerable and highly vulnerable to AD pathology, we identified novel genes and/or signaling pathways that might account for differential vulnerability. These findings may have implications for treatment targets. P2-150
LECITHIN: CHOLESTEROL ACYLTRANSFERASE (LCAT) IS REQUIRED FOR NORMAL BRAIN APOE METABOLISM IN MICE
Veronica Hirsch-Reinshagen1, James Donkin1, Sophie Stukas1, Jianjia Fan1, John S. Parks2, Jan Albert Kuivenhoven3, Dieter Lu¨tjohann4, Haydn Pritchard1, Cheryl L. Wellington1, 1The University of British Columbia, Vancouver, BC, Canada; 2Wake Forest University Health Sciences, Winston-Salem, NC, USA; 3Department of Vascular Medicine, Academic Medical Center, Amsterdam, Netherlands; 4University of Bonn, Bonn, Germany. Contact e-mail: [email protected] Background: The lipidation status of apoE has been shown critical for the development of amyloid plaques in animal models of Alzheimer Disease (AD). Poor lipidation of apoE, brought about by the absence of the cholesterol transporter ABCA1, significantly worsens amyloid pathology. Conversely, overexpression of ABCA1 and increased apoE lipidation virtually eliminates the deposition of amyloid in the brain. These data sug-
P305
gest that genes involved in the lipidation of apoE may play a role in the pathogenesis of AD. LCAT catalyses the esterification of free cholesterol on circulating plasma lipoproteins, a key step in their maturation and function in lipid transport. LCAT is also synthesized within the brain, but its role in brain apoE lipoprotein metabolism and in brain disorders has not been evaluated. Here we describe the first characterization of brain LCAT expression and function. Methods: Wild-type and LCAT-deficient animal models were used to investigate the impact of LCAT on brain apoE metabolism. Primary glial and neuronal cultures were used to determine the cell-type specificity of LCAT expression in the brain. Glial-conditioned media (GCM) was used to characterize LCAT enzymatic activity. The ability of LCAT to esterify cholesterol on glia-derived lipoproteins and the effects of LCAT deficiency on apoE levels in GCM, brain and CSF were measured by Western blot. Results: In vivo, LCAT deficiency results in a 75% increase in brain apoE levels. In CSF, absence of LCAT leads to a 200% increase in 13-14 nm CSF apoE particles and virtually no apoA-I. In vitro, we show that astrocytes are the primary producers of LCAT and that apoE is the main activator of LCAT in GCM. Astrocyte-derived LCAT esterifies free cholesterol on nascent, glia-derived apoE lipopoproteins, and both apoE and ABCA1 are required for this reaction. Finally, LCAT deficiency leads to the appearance of abnormal, small w8 nm particles in GCM. Conclusions: These data show that brain LCAT esterifies cholesterol on glial-derived apoE-lipoproteins, and influences CSF apoE and apoA-I levels. Because a modulatory effect of LCAT on apoE levels and lipidation has now been demonstrated, further studies will evaluate the impact of LCAT deficiency in AD mouse models. P2-151
APOE AND AMYLOID BETA DEFLATE THE CHOLINERGIC NEUROTRANSMISSION BY BOOSTING THE ACTIVITY AND STABILITY OF CHOLINESTERASES IN THE BRAIN
Taher Darreh-Shori, Negar Modiri, Agneta Nordberg, Karolinska Institutet, Stockholm, Sweden. Contact e-mail: [email protected] Background: In a recent study, we found that high CSF ApoE protein was strongly associated with increased levels of functional butyrylcholinesterase (F-BuChE) molecules to the total CSF BuChE protein. In addition, a high BuChE level in cortex is associated with a steeper annual cognitive decline in patients with dementia, and inhibition of BuChE rather than acetylcholinesterase (AChE) seems to correlate best with cognitive test result. The central cholinergic neurotransmission is one of the first neuronal systems affected by AD. Intriguingly, the underlying mechanisms are highly obscure. We hence investigated whether high levels of ApoE, which is particularly observed in CSF of AD patients with ApoE e4, plays a role in this regard. Methods: Purified human BuChE and ApoE proteins or recombinant human AChE, ApoE e2, e3 and e4 isoproteins, and synthetic Ab40 and Ab42 peptides were used. The enzymes activity was measured by Ellman’s assay. Results: The most unexpected and striking findings were that both ApoE protein and Ab peptides in a concentration-dependent manner strongly augmented the intrinsic activity of the AChE (6 folds) and BuChE (22-40 folds) at physiologically relevant concentrations. In the presence of high Ab concentration, the increased intrinsic AChE activity was however temporarily, so that within a few hours of incubation completely diminished. In contrast, the augmented intrinsic activity of BuChE in the presence of Ab, ApoE or both remained highly stable for at least 70 hours of incubation. Intriguingly, the functional stability of BuChE (in addition to its dependency on ApoE concentration), was highly ApoE e-isoprotein dependent, where the e4-isoprotein had the highest and the e2-isoprotein the least effect. Conclusions: These results, together with findings in the AD CSF and brain, provide a highly plausible mechanistic hypothesis that links the AD-induced cholinergic deficiency, with the ApoE e4 allele through high level of ApoE protein expression in the e4-carriers. This together with Ab strongly boost the ACh-hydrolyzing capacities of cholinesterases in the brain, reduce duration of action of ACh at synapses of the cholinergic neurons and/or the cholinergic non-neuronal cells such as microglia and oligodendrocytes responsible for the maintenance of myelin sheet, causing the inflammatory and neuronal disconnectivity features of AD brain.
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 test, 1 week, 15 days, and 1 month after microinjection. Conclusions: The present results illuminate the anxiogenic role of beta amyloid accumulation in the DRN. Our data support the notion that the oxidative stress and the neuronal loss resulting from Abeta accumulation in the DRN are involved in inducing anxiety-like behavior. P2-149
MOLECULAR SIGNATURE OF PRODROMAL ALZHEIMER’S DISEASE: A MICROARRAY STUDY IN POST-MORTEM CORTICAL TISSUE
Concepcion Conejero-Goldberg1, Thomas M. Hyde2, Shufen Chen1, Ute Dreses-Werringloer1, Mary Herman2, Joel E. Kleinman2, Peter Davies1, Terry Goldberg1, 1Litwin-Zucker Alzheimer’s Disease Research Center, FIMR, Manhasset, NY, USA; 2Clinical Brain Disorder Branch, NIMH, Bethesda, MD, USA. Contact e-mail: [email protected] Background: A number of studies have suggested that there are differences among cortical regions in vulnerability to Alzheimer’s disease (AD). Primary sensory cortices, including BA 1/2/3 remain relatively spared, while medial temporal and association cortices (including BA 21) are susceptible to plaque and tangle formation, even early in the disease. Though hypotheses have been put forth, the reasons for this difference remain obscure. In this study we used microarray technology to address this question by determining differences in inter-regional transcript expression from human post-mortem brain tissue from individuals at high risk for AD (positive for the APOE4 genotype) and individuals at low risk (negative for the APOE4 genotype). Subjects were chosen in whom AD was not present, as we did not want to measure ‘‘downstream’’ changes consequent to frank pathology. Methods: Fresh-frozen human brain tissue from normal control subjects (N ¼ 41) at autopsy was obtained from the Clinical Brain Disorders Branch, NIMH. Demographic characteristics of this cohort include: Age ¼ 41.9 6 10.3; Sex ¼ 35 M/6 F; pH ¼ 6.6 6 0.29; PMI ¼ 31.9 6 16.4; RIN ¼ BA 21 (8.0 6 0.5) and BA 1/2/3 (8.3 6 0.5); Genotype: APOE3¼ 28 & APOE4¼13. WG6 BeadChip arrays (Illumina) were processed according to the manufacturer’s guidelines. For statistical analyses, the data was processed using a ‘‘double subtraction’’ method. We determined expression differences between the two regions (BA 1/2/3 and BA 21) and contrasted these difference scores between APOE4 positive and negative groups, thus isolating intrinsic vulnerability or protective factors. Results: We identified 70 transcripts corresponding to genes from RefSeq and UniGene databases that differed significantly (at p< .001) between the APOE3 and APOE4 using ‘‘double subtraction’’ analyses. Fifty one of these transcripts survived FDR analyses. Conclusions: This study indicates that E4 promotes changes in calcium regulation, mitochondrial dysfunction, cell cycle activity, apoptosis, and Wnt signaling that are associated with disease pathogenesis. By contrasting expression profiles for brain regions relatively invulnerable and highly vulnerable to AD pathology, we identified novel genes and/or signaling pathways that might account for differential vulnerability. These findings may have implications for treatment targets. P2-150
LECITHIN: CHOLESTEROL ACYLTRANSFERASE (LCAT) IS REQUIRED FOR NORMAL BRAIN APOE METABOLISM IN MICE
Veronica Hirsch-Reinshagen1, James Donkin1, Sophie Stukas1, Jianjia Fan1, John S. Parks2, Jan Albert Kuivenhoven3, Dieter Lu¨tjohann4, Haydn Pritchard1, Cheryl L. Wellington1, 1The University of British Columbia, Vancouver, BC, Canada; 2Wake Forest University Health Sciences, Winston-Salem, NC, USA; 3Department of Vascular Medicine, Academic Medical Center, Amsterdam, Netherlands; 4University of Bonn, Bonn, Germany. Contact e-mail: [email protected] Background: The lipidation status of apoE has been shown critical for the development of amyloid plaques in animal models of Alzheimer Disease (AD). Poor lipidation of apoE, brought about by the absence of the cholesterol transporter ABCA1, significantly worsens amyloid pathology. Conversely, overexpression of ABCA1 and increased apoE lipidation virtually eliminates the deposition of amyloid in the brain. These data sug-
P305
gest that genes involved in the lipidation of apoE may play a role in the pathogenesis of AD. LCAT catalyses the esterification of free cholesterol on circulating plasma lipoproteins, a key step in their maturation and function in lipid transport. LCAT is also synthesized within the brain, but its role in brain apoE lipoprotein metabolism and in brain disorders has not been evaluated. Here we describe the first characterization of brain LCAT expression and function. Methods: Wild-type and LCAT-deficient animal models were used to investigate the impact of LCAT on brain apoE metabolism. Primary glial and neuronal cultures were used to determine the cell-type specificity of LCAT expression in the brain. Glial-conditioned media (GCM) was used to characterize LCAT enzymatic activity. The ability of LCAT to esterify cholesterol on glia-derived lipoproteins and the effects of LCAT deficiency on apoE levels in GCM, brain and CSF were measured by Western blot. Results: In vivo, LCAT deficiency results in a 75% increase in brain apoE levels. In CSF, absence of LCAT leads to a 200% increase in 13-14 nm CSF apoE particles and virtually no apoA-I. In vitro, we show that astrocytes are the primary producers of LCAT and that apoE is the main activator of LCAT in GCM. Astrocyte-derived LCAT esterifies free cholesterol on nascent, glia-derived apoE lipopoproteins, and both apoE and ABCA1 are required for this reaction. Finally, LCAT deficiency leads to the appearance of abnormal, small w8 nm particles in GCM. Conclusions: These data show that brain LCAT esterifies cholesterol on glial-derived apoE-lipoproteins, and influences CSF apoE and apoA-I levels. Because a modulatory effect of LCAT on apoE levels and lipidation has now been demonstrated, further studies will evaluate the impact of LCAT deficiency in AD mouse models. P2-151
APOE AND AMYLOID BETA DEFLATE THE CHOLINERGIC NEUROTRANSMISSION BY BOOSTING THE ACTIVITY AND STABILITY OF CHOLINESTERASES IN THE BRAIN
Taher Darreh-Shori, Negar Modiri, Agneta Nordberg, Karolinska Institutet, Stockholm, Sweden. Contact e-mail: [email protected] Background: In a recent study, we found that high CSF ApoE protein was strongly associated with increased levels of functional butyrylcholinesterase (F-BuChE) molecules to the total CSF BuChE protein. In addition, a high BuChE level in cortex is associated with a steeper annual cognitive decline in patients with dementia, and inhibition of BuChE rather than acetylcholinesterase (AChE) seems to correlate best with cognitive test result. The central cholinergic neurotransmission is one of the first neuronal systems affected by AD. Intriguingly, the underlying mechanisms are highly obscure. We hence investigated whether high levels of ApoE, which is particularly observed in CSF of AD patients with ApoE e4, plays a role in this regard. Methods: Purified human BuChE and ApoE proteins or recombinant human AChE, ApoE e2, e3 and e4 isoproteins, and synthetic Ab40 and Ab42 peptides were used. The enzymes activity was measured by Ellman’s assay. Results: The most unexpected and striking findings were that both ApoE protein and Ab peptides in a concentration-dependent manner strongly augmented the intrinsic activity of the AChE (6 folds) and BuChE (22-40 folds) at physiologically relevant concentrations. In the presence of high Ab concentration, the increased intrinsic AChE activity was however temporarily, so that within a few hours of incubation completely diminished. In contrast, the augmented intrinsic activity of BuChE in the presence of Ab, ApoE or both remained highly stable for at least 70 hours of incubation. Intriguingly, the functional stability of BuChE (in addition to its dependency on ApoE concentration), was highly ApoE e-isoprotein dependent, where the e4-isoprotein had the highest and the e2-isoprotein the least effect. Conclusions: These results, together with findings in the AD CSF and brain, provide a highly plausible mechanistic hypothesis that links the AD-induced cholinergic deficiency, with the ApoE e4 allele through high level of ApoE protein expression in the e4-carriers. This together with Ab strongly boost the ACh-hydrolyzing capacities of cholinesterases in the brain, reduce duration of action of ACh at synapses of the cholinergic neurons and/or the cholinergic non-neuronal cells such as microglia and oligodendrocytes responsible for the maintenance of myelin sheet, causing the inflammatory and neuronal disconnectivity features of AD brain.