- Email: [email protected]
BLOCKADE OF ALPHA4 INTEGRINS AMELIORATES COGNITIVE DYSFUNCTION AND NEUROPATHOLOGICAL CHANGES IN TRANSGENIC ANIMALS WITH ALZHEIMER'S-LIKE DISEASE
P660 P2-126
Poster Presentations: Monday, July 25, 2016 BLOCKADE OF ALPHA4 INTEGRINS AMELIORATES COGNITIVE DYSFUNCTION AND NEUROPATHOLOGICAL CHANGES IN TRANSGENIC ANIMALS WITH ALZHEIMER’SLIKE DISEASE
Gennj Piacentino, Vittorina Della Bianca, Elena Zenaro, Enrica Caterina Pietronigro, Tommaso Carlucci, Silvia Dusi, Gabriela Constantin, University of Verona, Verona, Italy. Contact e-mail: [email protected] Background: Blood-derived leukocyte subpopulations, including
lymphocytes, monocytes and neutrophils, have been identified in the brains of patients with Alzheimer’s disease (AD) and in corresponding animal models, but their role in disease pathogenesis is unclear. We have recently demonstrated that neutrophils infiltrate the AD brain and that neutrophil depletion has therapeutic effect in AD transgenic mouse models. However, leukocyte trafficking mechanisms in the central nervous system (CNS) during AD are largely unknown and our goal was to determine the role of alpha4 integrins in disease pathogenesis. Methods: Intravital microscopy studies were performed to visualize and analyze leukocyte-vascular interactions in 3xTg-AD mice, which present both amyloid and tau pathology. Contextual fear conditioning and Y maze tests were performed to analyze the cognitive deficit. Neuropathological studies were performed to evaluate amyloid beta deposition, tau hyperphosphorylation, microglial activation and expression of synaptic proteins. Results: Intravital microscopy studies performed in the CNS microcirculation of 3xTg-AD mice showed that blockade of alpha4 integrins prevents activated T cell rolling and firm adhesion on brain endothelium. Treating 3xTg-AD mice with an anti-alpha4 integrin antibody starting during early disease phases in mice already presenting cognitive deficits clearly improved memory function compared to mice treated with a control antibody in Y maze and contextual fear conditioning tests. These findings were supported by neuropathological data showing a reduction in Abeta deposition and lower density and activation state of microglia in the cortex and hippocampus of 3xTg-AD mice treated with anti-alpha4 antibody compared to animals treated with an isotype control antibody. Moreover, anti-apha4 treatment reduced tau hyperphosphorylation and restored synaptic protein expression compared to control animals. Notably, restoration of cognitive function in mice with temporary anti-alpha4 treatment during early disease was maintained also at later time points in aged animals, suggesting that therapeutic blockade of leukocyte adhesion during the early stages of disease provides a long-term beneficial effect on cognition in older mice. Conclusions: Antibodies anti-alpha4 integrin are currently used for the treatment of patients with autoimmune diseases and our data suggest that blockade of alpha4 integrins may represent a novel therapeutic approach in AD that has the potential for rapid translation into the clinic.
We found that neuronal death is not mediated by pro-inflammatory mechanisms that rapidly kill neurons in CVN-AD mice. Instead, immune-mediated nutrient deprivation and metabolic disruption caused by prolonged immunosuppression are involved in neuodegeneration. Methods: We determined gene expression levels in isolated subtypes of microglia from brains of CVN-AD mice using flow cytometry, followed by RNA amplification and an Agilent Whole Mouse Genome Oligo Microarray. Data were analyzed using Agilent Feature Extraction Software and Partek Genomics Suite, and an analysis of variance with the four experimental groups to identify significantly differentially expressed genes. Amino acids and metabolites were extracted from cryo-pulverized 20-35 weekold mouse brain tissue and utilized for MS-based quantitation. In some cases brain lysates were used for the AbsoluteIDQp180 Kit metabolomics assay, with LC/MS based-analysis. Results: RNA expression analysis of flow-sorted CD11c+ microglia and control groups demonstrated significantly increased expression levels for genes associated with arginase 1, phagocytic vesicle processing and blockade of effector T cells. Microglial expression of arginase-1 mRNA and protein was seen within the microglia (and some astrocytes) in the hippocampus and other regions heavily populated with CD11c+microglia and where neuronal death was also found. CVN-AD brains also exhibited decreased arginine bioavailability, implicating immune-mediated arginine catabolism in neuronal death in this model. Interrelated brain metabolic pathways that depend on arginine and its breakdown product ornithine, were also changed, including polyamine, proline and creatine levels. Blockade of two key enzymes in the arginine utilization pathway, arginase and ornithine decarboxylase, with difluoromethylornithine (DFMO) prevented the development of the key features of AD, including beta amyloid plaques and memory deficits, and reversed metabolomic changes. Conclusions: We hypothesize that neuronal loss in AD is caused by prolonged immune responses within the brain microenvironment that lead to amino acid catabolism, altered local metabolites and chronic neuronal starvation. Further characterization of these changes will allow us to define a specific metabolic signature for AD, allowing for the identification of future diagnostic and therapeutic targets in AD.
P2-128
MITOCHONDRIA AND MITOCHONDRIAL DNA INDUCE NEUROINFLAMMATION AND ALTER APP HOMEOSTASIS
Heather M. Wilkins1, Scott Koppel2, Nairita Roy2, Lauren Ryan3, John A. Stanford2, Russell H. Swerdlow1,2, 1University of Kansas Alzheimer’s Disease Center, Fairway, KS, USA; 2University of Kansas Medical Center, Kansas City, KS, USA; 3University of Kansas Medical Center Alzheimer’s Disease Center, Kansas City, KS, USA. Contact e-mail: [email protected] Background: Mitochondrial components induce inflammatory re-
P2-127
IMMUNE-MEDIATED NUTRIENT DEPRIVATION AND METABOLIC DISRUPTION IN AN ALZHEIMER’S DISEASE MOUSE MODEL
Carol A. Colton, Ram S. Puranam, Michael P. Vitek, Matthew J. Kan, Duke University Medical Center, Durham, NC, USA. Contact e-mail: Carol. [email protected] Background: We have established a unique mouse model (APPSwDI/mNos2-/-; CVN-AD mice) that recapitulates major features of AD, (beta amyloid plaques, spontaneously hyper-phosphorylated tau, memory loss, and hippocampal neuronal death).
sponses in pathological conditions such as arthritis, cardiovascular disease, and trauma. For reasons such as this, a number of mitochondrial components, including mitochondrial DNA (mtDNA), have been labeled damage-associated molecular pattern (DAMP) molecules. We have previously shown that mitochondrial lysates prepared from cells containing mtDNA, but not from those lacking detectable mtDNA, induce inflammation and increase the expression of APP in vitro. Methods: To extend our in vitro findings, C57BL/6 male mice underwent stereotactic surgery in which mitochondrial lysates, mtDNA, or vehicle (saline) were injected into the hippocampus. Seven days post-surgery tissue was analyzed for
Poster Presentations: Monday, July 25, 2016 BLOCKADE OF ALPHA4 INTEGRINS AMELIORATES COGNITIVE DYSFUNCTION AND NEUROPATHOLOGICAL CHANGES IN TRANSGENIC ANIMALS WITH ALZHEIMER’SLIKE DISEASE
Gennj Piacentino, Vittorina Della Bianca, Elena Zenaro, Enrica Caterina Pietronigro, Tommaso Carlucci, Silvia Dusi, Gabriela Constantin, University of Verona, Verona, Italy. Contact e-mail: [email protected] Background: Blood-derived leukocyte subpopulations, including
lymphocytes, monocytes and neutrophils, have been identified in the brains of patients with Alzheimer’s disease (AD) and in corresponding animal models, but their role in disease pathogenesis is unclear. We have recently demonstrated that neutrophils infiltrate the AD brain and that neutrophil depletion has therapeutic effect in AD transgenic mouse models. However, leukocyte trafficking mechanisms in the central nervous system (CNS) during AD are largely unknown and our goal was to determine the role of alpha4 integrins in disease pathogenesis. Methods: Intravital microscopy studies were performed to visualize and analyze leukocyte-vascular interactions in 3xTg-AD mice, which present both amyloid and tau pathology. Contextual fear conditioning and Y maze tests were performed to analyze the cognitive deficit. Neuropathological studies were performed to evaluate amyloid beta deposition, tau hyperphosphorylation, microglial activation and expression of synaptic proteins. Results: Intravital microscopy studies performed in the CNS microcirculation of 3xTg-AD mice showed that blockade of alpha4 integrins prevents activated T cell rolling and firm adhesion on brain endothelium. Treating 3xTg-AD mice with an anti-alpha4 integrin antibody starting during early disease phases in mice already presenting cognitive deficits clearly improved memory function compared to mice treated with a control antibody in Y maze and contextual fear conditioning tests. These findings were supported by neuropathological data showing a reduction in Abeta deposition and lower density and activation state of microglia in the cortex and hippocampus of 3xTg-AD mice treated with anti-alpha4 antibody compared to animals treated with an isotype control antibody. Moreover, anti-apha4 treatment reduced tau hyperphosphorylation and restored synaptic protein expression compared to control animals. Notably, restoration of cognitive function in mice with temporary anti-alpha4 treatment during early disease was maintained also at later time points in aged animals, suggesting that therapeutic blockade of leukocyte adhesion during the early stages of disease provides a long-term beneficial effect on cognition in older mice. Conclusions: Antibodies anti-alpha4 integrin are currently used for the treatment of patients with autoimmune diseases and our data suggest that blockade of alpha4 integrins may represent a novel therapeutic approach in AD that has the potential for rapid translation into the clinic.
We found that neuronal death is not mediated by pro-inflammatory mechanisms that rapidly kill neurons in CVN-AD mice. Instead, immune-mediated nutrient deprivation and metabolic disruption caused by prolonged immunosuppression are involved in neuodegeneration. Methods: We determined gene expression levels in isolated subtypes of microglia from brains of CVN-AD mice using flow cytometry, followed by RNA amplification and an Agilent Whole Mouse Genome Oligo Microarray. Data were analyzed using Agilent Feature Extraction Software and Partek Genomics Suite, and an analysis of variance with the four experimental groups to identify significantly differentially expressed genes. Amino acids and metabolites were extracted from cryo-pulverized 20-35 weekold mouse brain tissue and utilized for MS-based quantitation. In some cases brain lysates were used for the AbsoluteIDQp180 Kit metabolomics assay, with LC/MS based-analysis. Results: RNA expression analysis of flow-sorted CD11c+ microglia and control groups demonstrated significantly increased expression levels for genes associated with arginase 1, phagocytic vesicle processing and blockade of effector T cells. Microglial expression of arginase-1 mRNA and protein was seen within the microglia (and some astrocytes) in the hippocampus and other regions heavily populated with CD11c+microglia and where neuronal death was also found. CVN-AD brains also exhibited decreased arginine bioavailability, implicating immune-mediated arginine catabolism in neuronal death in this model. Interrelated brain metabolic pathways that depend on arginine and its breakdown product ornithine, were also changed, including polyamine, proline and creatine levels. Blockade of two key enzymes in the arginine utilization pathway, arginase and ornithine decarboxylase, with difluoromethylornithine (DFMO) prevented the development of the key features of AD, including beta amyloid plaques and memory deficits, and reversed metabolomic changes. Conclusions: We hypothesize that neuronal loss in AD is caused by prolonged immune responses within the brain microenvironment that lead to amino acid catabolism, altered local metabolites and chronic neuronal starvation. Further characterization of these changes will allow us to define a specific metabolic signature for AD, allowing for the identification of future diagnostic and therapeutic targets in AD.
P2-128
MITOCHONDRIA AND MITOCHONDRIAL DNA INDUCE NEUROINFLAMMATION AND ALTER APP HOMEOSTASIS
Heather M. Wilkins1, Scott Koppel2, Nairita Roy2, Lauren Ryan3, John A. Stanford2, Russell H. Swerdlow1,2, 1University of Kansas Alzheimer’s Disease Center, Fairway, KS, USA; 2University of Kansas Medical Center, Kansas City, KS, USA; 3University of Kansas Medical Center Alzheimer’s Disease Center, Kansas City, KS, USA. Contact e-mail: [email protected] Background: Mitochondrial components induce inflammatory re-
P2-127
IMMUNE-MEDIATED NUTRIENT DEPRIVATION AND METABOLIC DISRUPTION IN AN ALZHEIMER’S DISEASE MOUSE MODEL
Carol A. Colton, Ram S. Puranam, Michael P. Vitek, Matthew J. Kan, Duke University Medical Center, Durham, NC, USA. Contact e-mail: Carol. [email protected] Background: We have established a unique mouse model (APPSwDI/mNos2-/-; CVN-AD mice) that recapitulates major features of AD, (beta amyloid plaques, spontaneously hyper-phosphorylated tau, memory loss, and hippocampal neuronal death).
sponses in pathological conditions such as arthritis, cardiovascular disease, and trauma. For reasons such as this, a number of mitochondrial components, including mitochondrial DNA (mtDNA), have been labeled damage-associated molecular pattern (DAMP) molecules. We have previously shown that mitochondrial lysates prepared from cells containing mtDNA, but not from those lacking detectable mtDNA, induce inflammation and increase the expression of APP in vitro. Methods: To extend our in vitro findings, C57BL/6 male mice underwent stereotactic surgery in which mitochondrial lysates, mtDNA, or vehicle (saline) were injected into the hippocampus. Seven days post-surgery tissue was analyzed for