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Modulation of Amyloid Peptide Oligomerization and Toxicity by Extracellular Hsp70
444a
Tuesday, February 14, 2017
volumes, strengthen cognition capabilities and facilitate creative inspiration productions. Information storages in the sensory cortices confer them to signify the sources of the retrieved memory signals. This study is supported by National Basic Research Program (2013CB531304 and 2016YFC1307100) and Natural Science Foundation China (81671071 and 81471123) to JHW. 2181-Pos Board B501 Bilobalide Protects against Focal Cerebral Ischemia Reperfusion Injury by Inhibiting of Cell Death Pathways and Promoting of Angiogenesis Yongqiu Zheng1, Mingjiang Yao1, Frank Yi2, Bin Yang1, Xiaodi Fan1, Jianxun Liu1, Matthew Orange3, Hua Zhu2. 1 Xiyuan Hospital, Beijing, China, 2The Ohio State University, Columbus, OH, USA, 3Department of Physical Education and Human Performance, Central Connecticut State University, New Britain, CT, USA. Ischemic stroke is a leading cause of long-term disability. Although previous studies have identified some promising neuroprotective agents, such as cinnamophilin, EGCG, and oxyresveratrol, their protective effects are still very limited. Thus, identification of novel neuroprotective agents is highly desired for treatment of this disease. Previous studies have shown that Ginkgo biloba extract has protective effects against neurodegenerative disorders such as cerebral insufficiency, Alzheimer’s disease, and dementia. In this present study, we sought to test the potential protective role of an active component of Ginkgo biloba extract, Bilobalide, in a rat model of middle cerebral artery occlusion (MCAO). We found that intraperitoneal (IP) delivery of various Bilobalide doses during ischemia can protect against brain injury, as evidenced by reduced infarct size and improved neurological scores after surgery. Histochemical analysis revealed that treatment with Bilobalide can significantly reduce apoptosis, autophagy, and promote angiogeneis following ischemia/reperfusion injury to the brain. In order to determine the molecular mechanisms underlying Bilobalide mediated neuroprotective effects, biochemical assays were performed, and the results suggested that Bilobalide can activate Akt prosurvival and eNOS pathways to promote cell survival and angiogenesis, respectively. To confirm these biochemical observations, we cotreated animals with a combination of Bilobalide and inhibitors of Akt and/or eNOS pathways. We found that treatment with inhibitors can partially abolish the beneficial effects of Bilobalide, indicating that Bilobalide indeed provides protection from neuronal injury through activation of these signaling pathways. Taken together, our results suggested that Bilobalide benefits stroke symptoms by reducing cell death pathways (apoptosis and autophagy) and promoting angiogenesis. As such, Bilobalide may be a potential agent for improving self-repair after ischemic stroke. 2182-Pos Board B502 Using Light-Sheet Microscopy to Understand Evoked Motor Sequence Generation Amicia D. Elliott1, Feici Diao2, Sarav Shah2, Daniel Yasoshima2, Yicong Wu3, Hari Shroff3, Benjamin White2. 1 NIGMS, National Institutes of Health, Bethesda, MD, USA, 2NIMH, National Institutes of Health, Bethesda, MD, USA, 3NIBIB, National Institutes of Health, Bethesda, MD, USA. Motor sequences are critical elements of everyday behavior, but how they are produced by central neural circuits is poorly understood. The complexity and scale of the circuitry involved makes motor sequence generation very difficult to study at the cellular level in large, mammalian brains. However, the neural circuits of the fruitfly also drive complex motor sequences, and are small enough to investigate at a brain-wide scale via emerging methods in microscopy. A critical behavioral sequence for the fruitfly, called ecdysis, is required for molting at each developmental stage and consists of three serially executed, stereotyped behavioral programs at the pupal stage. The neural circuit controlling the pupal ecdysis sequence includes approximately 300 peptidergic neurons that express the Ecdysis Triggering Hormone receptor (ETHR) and are activated by peripheral release of Ecdysis Triggering Hormone (ETH). The activation of these neurons leads directly to the three phases of associated motor neuron activity that mediate the ecdysis sequence, and existing data indicate that specific subpopulations are required for each behavioral phase of ecdysis. However, the identities of the individual neurons that control each behavioral phase remain largely unknown, as do the mechanisms by which they regulate motor output. To achieve a detailed cellular-level understanding of the ecdysis circuit, we have built a light-sheet microscope that is capable of imaging the Drosophila pupal CNS rapidly at high resolution. We are currently using calcium biosensors to monitor the neural activity in ETHR-expressing neurons and motor neurons of excised brains in response to ETH. Single-cell activity imaging on the light-sheet microscope confirms that individual neurons respond to ETH with varying onset times and distinct activity profiles that suggest the generation of fictive ecdysis behavior. Analysis of these data, and data collected from other populations of neurons, are being used to generate a pre-
dictive model of the circuit underlying the ecdysis sequence with the general goal of understanding how nervous systems orchestrate complex motor sequences in response to input stimuli. 2183-Pos Board B503 Structural Polymorphism of Amyloid Fibrils in Alzheimer’s Disease Liu Jiliang1, John Badger2, Biel Roig Solvas3, Lee Makowski3. 1 Brookhaven National Lab, Upton, NY, USA, 2Delta G Technologies, San Diego, CA, USA, 3Northeastern University, Boston, MA, USA. X-ray microdiffraction of histological sections of human tissue has been used to map variations in the wide-angle scattering from amyloid fibrils in human brain in Alzheimer’s Disease (AD). These studies indicate that tissue derived from subjects with different clinical histories may contain different ensembles of fibrillar structures; plaques harboring distinct amyloid structures can coexist within a single tissue section; and within individual plaques there may be a gradient of fibrillar structure from core to margins. The significance of these polymorphisms is uncertain. One possibility is that amyloid fibrils with different structures may contribute differentially to disease. However, testing this hypothesis will require significantly more information about the structure and structural variation of amyloid. Here we report initial results of modeling amyloid fibril structures to explain the observed variations in x-ray scattering. By characterizing the polymorphisms in fibril structure and their distribution throughout the brain, we seek to better understand the nucleation and maturation of plaques, the relationship between fibril structure and packing within the plaques and the contribution of these processes to the inception and inexorable progression of neurodegeneration and disease. 2184-Pos Board B504 Modulation of Amyloid Peptide Oligomerization and Toxicity by Extracellular Hsp70 Antonio De Maio1, Isabel Rivera2, David M. Cauvi3, Nelson Arispe4. 1 Department of Surgery, Department of Neurosciences, School of Medicine, University of California, San Diego, La Jolla, CA, USA, 2Initiative for Maximizing Student Development (IMSD), University of California, San Diego, La Jolla, CA, USA, 3Department of Surgery, University of California, San Diego, La Jolla, CA, USA, 4Department of Anatomy, Pysiology and Genetics, Uniformed Services University, Bethesda, MD, USA. Alzheimer’s disease (AD) is a progressive neurodegenerative disorder leading to dementia due to advanced neuronal dysfunction and death. Treatment for AD is a major burden for the health care system in the U.S. and the disease has a large emotional, social, and economic impact on the family members. Currently, the predominant theory for AD is the ‘‘amyloid hypothesis,’’ which states that abnormally increased levels of amyloid b (Ab) peptides results in the production of a variety of Ab aggregates that are neurotoxic. The specific mechanism(s) for Ab-induced cytotoxicity has not been completely elucidated yet. However, since the majority of Ab is released in the extracellular milieu, it is reasonable to assume that toxicity begins outside the cells and makes its way inside where it disrupts the basic cellular process that results in cell stress and death. Cells exposed to stressful conditions respond by the expression of heat shock proteins (hsps) that are involved in processes to preserve cell viability. Hsps are molecular chaperones that play a major role in protein folding and the solubilization of misfolded aggregated polypeptides. Recently, it has been demonstrated that hsps, in particular Hsp70, are exported into the extracellular milieu by an active export mechanism independent of cell death. Therefore, both Ab peptides and Hsp70 may coexist in a common extracellular pool during pathological conditions. We observed that incubation of extracellular Hsp70 affected the Ab assembling process preventing oligomer formation in vitro. Moreover, the combination of Hsp70 and Ab peptides reduced the cytotoxicity of the peptide on cultured neuron (N2A) cells. These results suggest a potential mechanism to reduce the detrimental effects of Ab peptides in AD. 2185-Pos Board B505 Calcium Calmodulin Regulates Zinc Mediated Changes in the Structure, Self-Association, and Activity of CaMKII Laurel Hoffman1, Lin Li2, Emil Alexov2, M. Neal Waxham1, Hugo Sanabria2. 1 The University of Texas Health Science Center, Houston, TX, USA, 2 Clemson University, Clemson, SC, USA. Ca2þ-Calmodulin-dependent protein kinase II (CaMKII) is an interpreter of Ca2þ signaling and has been shown to be a critical component of learning and memory. This dodecameric oligomer also functions in part as a scaffolding molecule, serving to promote the formation of higher order signaling complexes within synapses. We set to study the effect that divalent cations, known to be critically important in neuronal signaling, have on the structure and function CaMKII. First, we computed the electrostatic surface potential of the dodecameric complex as a means to predict possible binding sites for divalent
Tuesday, February 14, 2017
volumes, strengthen cognition capabilities and facilitate creative inspiration productions. Information storages in the sensory cortices confer them to signify the sources of the retrieved memory signals. This study is supported by National Basic Research Program (2013CB531304 and 2016YFC1307100) and Natural Science Foundation China (81671071 and 81471123) to JHW. 2181-Pos Board B501 Bilobalide Protects against Focal Cerebral Ischemia Reperfusion Injury by Inhibiting of Cell Death Pathways and Promoting of Angiogenesis Yongqiu Zheng1, Mingjiang Yao1, Frank Yi2, Bin Yang1, Xiaodi Fan1, Jianxun Liu1, Matthew Orange3, Hua Zhu2. 1 Xiyuan Hospital, Beijing, China, 2The Ohio State University, Columbus, OH, USA, 3Department of Physical Education and Human Performance, Central Connecticut State University, New Britain, CT, USA. Ischemic stroke is a leading cause of long-term disability. Although previous studies have identified some promising neuroprotective agents, such as cinnamophilin, EGCG, and oxyresveratrol, their protective effects are still very limited. Thus, identification of novel neuroprotective agents is highly desired for treatment of this disease. Previous studies have shown that Ginkgo biloba extract has protective effects against neurodegenerative disorders such as cerebral insufficiency, Alzheimer’s disease, and dementia. In this present study, we sought to test the potential protective role of an active component of Ginkgo biloba extract, Bilobalide, in a rat model of middle cerebral artery occlusion (MCAO). We found that intraperitoneal (IP) delivery of various Bilobalide doses during ischemia can protect against brain injury, as evidenced by reduced infarct size and improved neurological scores after surgery. Histochemical analysis revealed that treatment with Bilobalide can significantly reduce apoptosis, autophagy, and promote angiogeneis following ischemia/reperfusion injury to the brain. In order to determine the molecular mechanisms underlying Bilobalide mediated neuroprotective effects, biochemical assays were performed, and the results suggested that Bilobalide can activate Akt prosurvival and eNOS pathways to promote cell survival and angiogenesis, respectively. To confirm these biochemical observations, we cotreated animals with a combination of Bilobalide and inhibitors of Akt and/or eNOS pathways. We found that treatment with inhibitors can partially abolish the beneficial effects of Bilobalide, indicating that Bilobalide indeed provides protection from neuronal injury through activation of these signaling pathways. Taken together, our results suggested that Bilobalide benefits stroke symptoms by reducing cell death pathways (apoptosis and autophagy) and promoting angiogenesis. As such, Bilobalide may be a potential agent for improving self-repair after ischemic stroke. 2182-Pos Board B502 Using Light-Sheet Microscopy to Understand Evoked Motor Sequence Generation Amicia D. Elliott1, Feici Diao2, Sarav Shah2, Daniel Yasoshima2, Yicong Wu3, Hari Shroff3, Benjamin White2. 1 NIGMS, National Institutes of Health, Bethesda, MD, USA, 2NIMH, National Institutes of Health, Bethesda, MD, USA, 3NIBIB, National Institutes of Health, Bethesda, MD, USA. Motor sequences are critical elements of everyday behavior, but how they are produced by central neural circuits is poorly understood. The complexity and scale of the circuitry involved makes motor sequence generation very difficult to study at the cellular level in large, mammalian brains. However, the neural circuits of the fruitfly also drive complex motor sequences, and are small enough to investigate at a brain-wide scale via emerging methods in microscopy. A critical behavioral sequence for the fruitfly, called ecdysis, is required for molting at each developmental stage and consists of three serially executed, stereotyped behavioral programs at the pupal stage. The neural circuit controlling the pupal ecdysis sequence includes approximately 300 peptidergic neurons that express the Ecdysis Triggering Hormone receptor (ETHR) and are activated by peripheral release of Ecdysis Triggering Hormone (ETH). The activation of these neurons leads directly to the three phases of associated motor neuron activity that mediate the ecdysis sequence, and existing data indicate that specific subpopulations are required for each behavioral phase of ecdysis. However, the identities of the individual neurons that control each behavioral phase remain largely unknown, as do the mechanisms by which they regulate motor output. To achieve a detailed cellular-level understanding of the ecdysis circuit, we have built a light-sheet microscope that is capable of imaging the Drosophila pupal CNS rapidly at high resolution. We are currently using calcium biosensors to monitor the neural activity in ETHR-expressing neurons and motor neurons of excised brains in response to ETH. Single-cell activity imaging on the light-sheet microscope confirms that individual neurons respond to ETH with varying onset times and distinct activity profiles that suggest the generation of fictive ecdysis behavior. Analysis of these data, and data collected from other populations of neurons, are being used to generate a pre-
dictive model of the circuit underlying the ecdysis sequence with the general goal of understanding how nervous systems orchestrate complex motor sequences in response to input stimuli. 2183-Pos Board B503 Structural Polymorphism of Amyloid Fibrils in Alzheimer’s Disease Liu Jiliang1, John Badger2, Biel Roig Solvas3, Lee Makowski3. 1 Brookhaven National Lab, Upton, NY, USA, 2Delta G Technologies, San Diego, CA, USA, 3Northeastern University, Boston, MA, USA. X-ray microdiffraction of histological sections of human tissue has been used to map variations in the wide-angle scattering from amyloid fibrils in human brain in Alzheimer’s Disease (AD). These studies indicate that tissue derived from subjects with different clinical histories may contain different ensembles of fibrillar structures; plaques harboring distinct amyloid structures can coexist within a single tissue section; and within individual plaques there may be a gradient of fibrillar structure from core to margins. The significance of these polymorphisms is uncertain. One possibility is that amyloid fibrils with different structures may contribute differentially to disease. However, testing this hypothesis will require significantly more information about the structure and structural variation of amyloid. Here we report initial results of modeling amyloid fibril structures to explain the observed variations in x-ray scattering. By characterizing the polymorphisms in fibril structure and their distribution throughout the brain, we seek to better understand the nucleation and maturation of plaques, the relationship between fibril structure and packing within the plaques and the contribution of these processes to the inception and inexorable progression of neurodegeneration and disease. 2184-Pos Board B504 Modulation of Amyloid Peptide Oligomerization and Toxicity by Extracellular Hsp70 Antonio De Maio1, Isabel Rivera2, David M. Cauvi3, Nelson Arispe4. 1 Department of Surgery, Department of Neurosciences, School of Medicine, University of California, San Diego, La Jolla, CA, USA, 2Initiative for Maximizing Student Development (IMSD), University of California, San Diego, La Jolla, CA, USA, 3Department of Surgery, University of California, San Diego, La Jolla, CA, USA, 4Department of Anatomy, Pysiology and Genetics, Uniformed Services University, Bethesda, MD, USA. Alzheimer’s disease (AD) is a progressive neurodegenerative disorder leading to dementia due to advanced neuronal dysfunction and death. Treatment for AD is a major burden for the health care system in the U.S. and the disease has a large emotional, social, and economic impact on the family members. Currently, the predominant theory for AD is the ‘‘amyloid hypothesis,’’ which states that abnormally increased levels of amyloid b (Ab) peptides results in the production of a variety of Ab aggregates that are neurotoxic. The specific mechanism(s) for Ab-induced cytotoxicity has not been completely elucidated yet. However, since the majority of Ab is released in the extracellular milieu, it is reasonable to assume that toxicity begins outside the cells and makes its way inside where it disrupts the basic cellular process that results in cell stress and death. Cells exposed to stressful conditions respond by the expression of heat shock proteins (hsps) that are involved in processes to preserve cell viability. Hsps are molecular chaperones that play a major role in protein folding and the solubilization of misfolded aggregated polypeptides. Recently, it has been demonstrated that hsps, in particular Hsp70, are exported into the extracellular milieu by an active export mechanism independent of cell death. Therefore, both Ab peptides and Hsp70 may coexist in a common extracellular pool during pathological conditions. We observed that incubation of extracellular Hsp70 affected the Ab assembling process preventing oligomer formation in vitro. Moreover, the combination of Hsp70 and Ab peptides reduced the cytotoxicity of the peptide on cultured neuron (N2A) cells. These results suggest a potential mechanism to reduce the detrimental effects of Ab peptides in AD. 2185-Pos Board B505 Calcium Calmodulin Regulates Zinc Mediated Changes in the Structure, Self-Association, and Activity of CaMKII Laurel Hoffman1, Lin Li2, Emil Alexov2, M. Neal Waxham1, Hugo Sanabria2. 1 The University of Texas Health Science Center, Houston, TX, USA, 2 Clemson University, Clemson, SC, USA. Ca2þ-Calmodulin-dependent protein kinase II (CaMKII) is an interpreter of Ca2þ signaling and has been shown to be a critical component of learning and memory. This dodecameric oligomer also functions in part as a scaffolding molecule, serving to promote the formation of higher order signaling complexes within synapses. We set to study the effect that divalent cations, known to be critically important in neuronal signaling, have on the structure and function CaMKII. First, we computed the electrostatic surface potential of the dodecameric complex as a means to predict possible binding sites for divalent