- Email: [email protected]
SOLUBLE AMYLOID-BETA AGGREGATES FROM HUMAN ALZHEIMER’S DISEASE BRAINS
Poster Presentations: Monday, July 17, 2017
during life. We studied the prevalence and topographical distribution of microbleeds and microinfarcts on ex vivo MRI in CAA cases. Moreover, we determined number of microbleeds and microinfarcts on microscopy of standard samples to compare to the numbers detected by MRI. We specifically sought to compare MRI’s methodologic advantage of providing full brain coverage to microscopy’s advantage of greater sensitivity for detection of these two lesion types. Methods: We included four patients with a diagnosis of CAA during life who donated their brains to CAA research (mean age at death 73.466.7 years). One control case was included from our neuropathology database, a 90-year old individual without cognitive impairment. At autopsy, the brains were removed and formalin-fixed. The hemisphere without macrohemorrhage was packed in a bag with periodate-lysine-paraformaldehyde and scanned overnight at 3T MRI. The protocol included a flash (resolution 500x500x500mm3) and a T2-weighted sequence (resolution 500x500x500mm3). On the acquired scans, cortical microbleeds and microinfarcts were assessed. In parallel, the hemisphere was cut in 10mm-thick coronal slabs. Subsequently, four pre-defined standard areas were sampled, blinded to MRI (one area from the frontal, parietal, temporal, and occipital cortex), embedded in paraffin, and cut in 6mmthick sections. One section was stained with H&E, and screened microscopically for microbleeds and microinfarcts. The adjacent section underwent Ab immunohistochemistry to determine CAA severity. Results: On ex vivo MRI 426 microbleeds [range 39-261] and 313 microinfarcts [range 21-144] were identified in the four CAA cases. Microbleeds and microinfarcts did not spatially overlap on MRI, but showed distinct topographical patterns. Microscopy revealed 3 microbleeds [range 0-2 per case] and 25 microinfarcts [range 0-15 per case] in the 16 examined sections. All cases showed CAA, ranging from mild-to-severe. The control case had 3 microinfarcts and 0 microbleeds on MRI, and microscopy revealed no lesions of either type and absence of CAA. Conclusions: In CAA, superior brain coverage provided by MRI resulted in more sensitive microbleed detection. In contrast, MRI appears to substantially undercount microinfarcts, which are often better detectable by the more sensitive method of histopathology. P2-068
SOLUBLE AMYLOID-BETA AGGREGATES FROM HUMAN ALZHEIMER’S DISEASE BRAINS
Thomas J. Esparza1, Norelle C. Wildburger1, Hao Jiang1, Mihika Gangolli1, Randall J. Bateman1,2,3, David L. Brody1,2, 1 Washington University School of Medicine, St. Louis, MO, USA; 2 Hope Center for Neurological Disorders, St. Louis, MO, USA; 3 Knight Alzheimer’s Disease Research Center, St. Louis, MO, USA. Contact e-mail: [email protected] Background: Soluble amyloid-beta (Ab) aggregates likely contribute substantially to the dementia that characterizes Alzheimer’s disease. However, despite intensive study of in vitro preparations and animal models, little is known about the characteristics of soluble Ab aggregates in the human Alzheimer’s disease brain. Here we present a new method for extracting soluble Ab aggregates from human brains, separating them from insoluble aggregates and Ab monomers using differential ultracentrifugation, and purifying them >6000 fold by dual antibody immunoprecipitation. Methods: Our approach to isolating soluble Ab aggregates from human brain involved using 2-3 grams of frontal or parietal cortex from pathologically confirmed CDR3 (severe) AD cases. Tissue was homogenized in 1xPBS containing protease inhibitors and subjected to differential ultracentrifugation, and dual antibody immunoprecipitation. We examined the effect of multiple extractions, the use of detergents during extraction,
P631
and chromatographic methods to improve enrichment of the Ab aggregates prior to immunopurification. The stability of the aggregates was characterized during purification using size-exclusion chromatography, sucrose density gradient centrifugation, immunoelectron microscopy, and quantitative tracking of the Ab aggregates at each step. Results: We developed a method that results in <40% loss of starting material and purifying them >6000 fold with no detectible ex vivo aggregation of monomeric Ab, and no apparent ex vivo alterations in soluble aggregate sizes. Loss of Ab was greatly reduced by universal blocking of materials with albumin. By immunoelectron microscopy, soluble Ab aggregates typically appear as clusters of 10–20 nanometer diameter ovoid structures with 2-3 amino-terminal Ab antibody binding sites, distinct from previously characterized structures. Conclusions: A major challenge has been that the specific forms of soluble Ab aggregates most relevant to human disease have not been determined. Ab can potentially aggregate into a vast number of forms, consisting of different numbers of Ab peptides, various size forms of Ab, multiple Ab post-translational modifications and alternative structural configurations of Ab. Here we present a method to purify soluble Ab aggregates directly from frozen human AD brain tissue, reasoning this would be the most relevant approach to facilitate investigation into the characteristics of native soluble Ab aggregates, and deepen our understanding of Alzheimer’s dementia. P2-069
THE RELATIONSHIP OF PRION PROTEIN AND NEUROGENESIS IN APP/PS1 DOUBLE TRANSGENIC MOUSE MODEL OF ALZHEIMER’S DISEASE
Yingxin Yu, Navy General Hospital, Beijing, China. Contact e-mail: [email protected] Background: Alzheimer’s disease (AD)is the most common adult onset dementia. Cellular prion protein is a high affinity receptor for Ab oligomersis, one of main protein related to cognition function. Prion expression and neurogenesis are all increased in AD patient’s brain hippocampus. Are prion protein and neurogenesis related? Actually, prion participates to embryo and adult neurogenesis. Prion protein is important for neural precursors differentiation to neuron. Is prion protein important to neurogenesis or regulate neurogenesis in AD? Methods: Based on this idea, we design this experiment using APP/PS1 double transgenic mice through in vivo and in vitro experiments. The study will focus on prion protein and neurogenesis in AD through neural stem cell culture, cell differentiation, behavior test, immunohistochemistry. Results: We found that short term anti-PrP treatment have a recovery in cognitive learning and spatial memory acquisition of the mice in APP/PS1 mice compared with wild mice. Amyloid pathology in the hippocampus of APP/PS1 Tg mice was increased compared with wild mice. Number of Ab plaques was decreased in anti-PrP treated APP/PS1 compared with non-treated Tg mice. Anti-Prion antibody treatment suppressed the prion protein levels in mice brian of 9month- old APP/PS1 mice compared with wild mice. Conclusions: We got a conclusion that anti-PrP treated APP/PS1 mice could increased neurogenesis and recovery cognitive function.
P2-070
HYPERTENSION INDUCES ALZHEIMER’S DISEASE-RELATED PATHOLOGIES IN MICE AND PIGS
Yu-Min Kuo1, Yao-Hsiang Shih1,2, 1National Cheng Kung University, Tainan, Taiwan; 2Academia Sinica, Taipei, Taiwan. Contact e-mail: [email protected]
during life. We studied the prevalence and topographical distribution of microbleeds and microinfarcts on ex vivo MRI in CAA cases. Moreover, we determined number of microbleeds and microinfarcts on microscopy of standard samples to compare to the numbers detected by MRI. We specifically sought to compare MRI’s methodologic advantage of providing full brain coverage to microscopy’s advantage of greater sensitivity for detection of these two lesion types. Methods: We included four patients with a diagnosis of CAA during life who donated their brains to CAA research (mean age at death 73.466.7 years). One control case was included from our neuropathology database, a 90-year old individual without cognitive impairment. At autopsy, the brains were removed and formalin-fixed. The hemisphere without macrohemorrhage was packed in a bag with periodate-lysine-paraformaldehyde and scanned overnight at 3T MRI. The protocol included a flash (resolution 500x500x500mm3) and a T2-weighted sequence (resolution 500x500x500mm3). On the acquired scans, cortical microbleeds and microinfarcts were assessed. In parallel, the hemisphere was cut in 10mm-thick coronal slabs. Subsequently, four pre-defined standard areas were sampled, blinded to MRI (one area from the frontal, parietal, temporal, and occipital cortex), embedded in paraffin, and cut in 6mmthick sections. One section was stained with H&E, and screened microscopically for microbleeds and microinfarcts. The adjacent section underwent Ab immunohistochemistry to determine CAA severity. Results: On ex vivo MRI 426 microbleeds [range 39-261] and 313 microinfarcts [range 21-144] were identified in the four CAA cases. Microbleeds and microinfarcts did not spatially overlap on MRI, but showed distinct topographical patterns. Microscopy revealed 3 microbleeds [range 0-2 per case] and 25 microinfarcts [range 0-15 per case] in the 16 examined sections. All cases showed CAA, ranging from mild-to-severe. The control case had 3 microinfarcts and 0 microbleeds on MRI, and microscopy revealed no lesions of either type and absence of CAA. Conclusions: In CAA, superior brain coverage provided by MRI resulted in more sensitive microbleed detection. In contrast, MRI appears to substantially undercount microinfarcts, which are often better detectable by the more sensitive method of histopathology. P2-068
SOLUBLE AMYLOID-BETA AGGREGATES FROM HUMAN ALZHEIMER’S DISEASE BRAINS
Thomas J. Esparza1, Norelle C. Wildburger1, Hao Jiang1, Mihika Gangolli1, Randall J. Bateman1,2,3, David L. Brody1,2, 1 Washington University School of Medicine, St. Louis, MO, USA; 2 Hope Center for Neurological Disorders, St. Louis, MO, USA; 3 Knight Alzheimer’s Disease Research Center, St. Louis, MO, USA. Contact e-mail: [email protected] Background: Soluble amyloid-beta (Ab) aggregates likely contribute substantially to the dementia that characterizes Alzheimer’s disease. However, despite intensive study of in vitro preparations and animal models, little is known about the characteristics of soluble Ab aggregates in the human Alzheimer’s disease brain. Here we present a new method for extracting soluble Ab aggregates from human brains, separating them from insoluble aggregates and Ab monomers using differential ultracentrifugation, and purifying them >6000 fold by dual antibody immunoprecipitation. Methods: Our approach to isolating soluble Ab aggregates from human brain involved using 2-3 grams of frontal or parietal cortex from pathologically confirmed CDR3 (severe) AD cases. Tissue was homogenized in 1xPBS containing protease inhibitors and subjected to differential ultracentrifugation, and dual antibody immunoprecipitation. We examined the effect of multiple extractions, the use of detergents during extraction,
P631
and chromatographic methods to improve enrichment of the Ab aggregates prior to immunopurification. The stability of the aggregates was characterized during purification using size-exclusion chromatography, sucrose density gradient centrifugation, immunoelectron microscopy, and quantitative tracking of the Ab aggregates at each step. Results: We developed a method that results in <40% loss of starting material and purifying them >6000 fold with no detectible ex vivo aggregation of monomeric Ab, and no apparent ex vivo alterations in soluble aggregate sizes. Loss of Ab was greatly reduced by universal blocking of materials with albumin. By immunoelectron microscopy, soluble Ab aggregates typically appear as clusters of 10–20 nanometer diameter ovoid structures with 2-3 amino-terminal Ab antibody binding sites, distinct from previously characterized structures. Conclusions: A major challenge has been that the specific forms of soluble Ab aggregates most relevant to human disease have not been determined. Ab can potentially aggregate into a vast number of forms, consisting of different numbers of Ab peptides, various size forms of Ab, multiple Ab post-translational modifications and alternative structural configurations of Ab. Here we present a method to purify soluble Ab aggregates directly from frozen human AD brain tissue, reasoning this would be the most relevant approach to facilitate investigation into the characteristics of native soluble Ab aggregates, and deepen our understanding of Alzheimer’s dementia. P2-069
THE RELATIONSHIP OF PRION PROTEIN AND NEUROGENESIS IN APP/PS1 DOUBLE TRANSGENIC MOUSE MODEL OF ALZHEIMER’S DISEASE
Yingxin Yu, Navy General Hospital, Beijing, China. Contact e-mail: [email protected] Background: Alzheimer’s disease (AD)is the most common adult onset dementia. Cellular prion protein is a high affinity receptor for Ab oligomersis, one of main protein related to cognition function. Prion expression and neurogenesis are all increased in AD patient’s brain hippocampus. Are prion protein and neurogenesis related? Actually, prion participates to embryo and adult neurogenesis. Prion protein is important for neural precursors differentiation to neuron. Is prion protein important to neurogenesis or regulate neurogenesis in AD? Methods: Based on this idea, we design this experiment using APP/PS1 double transgenic mice through in vivo and in vitro experiments. The study will focus on prion protein and neurogenesis in AD through neural stem cell culture, cell differentiation, behavior test, immunohistochemistry. Results: We found that short term anti-PrP treatment have a recovery in cognitive learning and spatial memory acquisition of the mice in APP/PS1 mice compared with wild mice. Amyloid pathology in the hippocampus of APP/PS1 Tg mice was increased compared with wild mice. Number of Ab plaques was decreased in anti-PrP treated APP/PS1 compared with non-treated Tg mice. Anti-Prion antibody treatment suppressed the prion protein levels in mice brian of 9month- old APP/PS1 mice compared with wild mice. Conclusions: We got a conclusion that anti-PrP treated APP/PS1 mice could increased neurogenesis and recovery cognitive function.
P2-070
HYPERTENSION INDUCES ALZHEIMER’S DISEASE-RELATED PATHOLOGIES IN MICE AND PIGS
Yu-Min Kuo1, Yao-Hsiang Shih1,2, 1National Cheng Kung University, Tainan, Taiwan; 2Academia Sinica, Taipei, Taiwan. Contact e-mail: [email protected]