- Email: [email protected]
EFFECT OF INCREASING APOLIPOPROTEIN E ON INFLAMMATION IN THE BRAIN
Poster Presentations: Wednesday, July 19, 2017 P4-436
PROMINENT MICROGLIAL ACTIVATION IN CORTICAL WHITE MATTER IS SELECTIVELY ASSOCIATED WITH CORTICAL ATROPHY IN PRIMARY PROGRESSIVE APHASIA
Daniel T. Ohm, Garam Kim, Tamar Gefen, Alfred Rademaker, Sandra Weintraub, Eileen H. Bigio, Marsel Mesulam, Emily J. Rogalski, Changiz Geula, Northwestern University, Chicago, IL, USA. Contact e-mail: danielohm2012@u. northwestern.edu Background: The neuropathologic correlates of cortical atrophy in neurodegenerative disorders remain poorly understood. Primary progressive aphasia (PPA) is a clinical syndrome characterized by selective language impairments that reflect focal and asymmetric cortical atrophy in the language dominant (LD) hemisphere. Brains of individuals with PPA can be pathologically diagnosed with Alzheimer’s disease (AD), FTLD-tau, or FTLD-TDP, and they also display significant accumulation of activated microglia in the gray and white matter. Activated microglia can initiate an inflammatory cascade that may contribute to neurodegeneration, but their quantitative distribution in cortical white matter and their relationship with cortical atrophy are unknown. The present study investigated 10 PPA cases with AD or FTLD-TDP pathology to determine the density of white matter activated microglia and its association with gray matter atrophy. Methods: In each PPA case, two language regions with peak cortical atrophy and one non-atrophied region were selected within the LD hemisphere using photographs of intact postmortem brains, clinical MRI, or quantitative metrics using FreeSurfer with MRI scans acquired close to death. Non-atrophied contralateral homologues of LD regions were also examined for analyses of hemispheric asymmetry. Activated microglia were visualized immunohistochemically using a specific antibody to HLA-DR, and quantified with optical density measures of immunoreactivity. Results: Qualitatively, greater densities of activated microglia were observed in cortical white matter when compared to gray matter. Quantitative analyses showed that the white matter of atrophied language regions displayed significantly greater densities of activated microglia than the non-atrophied regions in the LD hemisphere (p<0.01). LD atrophied regions also showed a trend towards more activated microglia compared to non-language dominant regions (p¼0.0568). Conclusions: Activated microglia in PPA showed a greater propensity for white matter compared to gray matter, had higher densities in atrophied language regions than non-atrophied regions in the LD hemisphere, and often showed asymmetry favoring the LD atrophied regions consistent with the PPA clinical profile. Thus, activated microglia in cortical white matter are a proximal marker for gray matter atrophy. Given that activated microglia are thought to promote neuroinflammation, which may potentially exacerbate the disease progression, they may also be detrimental to the integrity of white matter. P4-437
sponses to amyloid beta (Ab) and aggregated a-synuclein. Activation of these receptors can amplify inflammation, but also promote phagocytosis of Ab. Activation of TLR-9 resulted in significant reduction of Ab in animal AD models. Activation of TLR-3 could have therapeutic potential due to its induction of beta-interferon responses. The premise of this study was observation of increased numbers of TLR-3 immunoreactive cells in AD affected brains. Methods: This study had two components; firstly staged series of human brain samples from low plaque (LP) and high plaque (HP) nondemented (ND) cases and AD cases were used to identify TLR-3 cellular localization using immunohistochemistry, and to quantify changes of expression of TLR-3 mRNA with disease by quantitative polymerase chain reaction (PCR). The second component involved in vitro studies of TLR-3 expression and activation using human postmortem brain-derived microglia and vascular endothelial cells. Results: Quantification of TLR-3 immunoreactivity in middle temporal gyrus using Image J software to measure area occupied showed increase of 101.8% in AD cases compared to LPND (p¼0.0036), and 16.5% increase between LPND and HPND (NS). TLR-3 immunoreactivity showed significant correlation with scores for brain plaque loads (Spearman r¼0.464, p<0.01) and tangle loads (r¼0.561, p<0.001). TLR-3 immunoreactivity was primarily in microglia, but also visible in subsets of astrocytes and vascular endothelial cells. Increased TLR-3 immunoreactivity was noticeable in microglia associated with Ab plaques. Quantitative PCR showed significant increase in TLR-3 mRNA between AD and ND cases (p¼0.043), interferon-b (p¼0.036) and interferon regulatory factor-3 (p¼0.013). Significant increase in TLR-9 mRNA expression in AD cases was also detected in these samples (p¼0.0019). In vitro experiments showed that treatment with TLR-3 ligand poly I:C did not induce significant increased expression of TLR-3 mRNA in microglia, but a tenfold increase in brain-derived endothelial cells. Conclusions: Different ways of manipulating microglia to promote removal of aggregated proteins has been explored as therapeutic strategies for AD. Increasing TLR-3 signaling could be one method for achieving this with the accumulation of TLR-3 expressing microglia associated with Ab plaques.
INCREASED EXPRESSION OF TOLL-LIKE RECEPTOR (TLR)-3 IN ALZHEIMER’S DISEASE BRAINS: A PROTECTIVE OR PATHOGENIC RESPONSE?
Douglas Gordon Walker1, Tiffany M. Tang1, Geidy E. Serrano2, Lucia I. Sue2, Thomas G. Beach2, Lih-Fen Lue1, 1Arizona State University, Tempe, AZ, USA; 2Banner Sun Health Research Institute, Sun City, AZ, USA. Contact e-mail: [email protected] Background: Toll-like receptors (TLR) are pattern recognition re-
ceptors involved in immune responses to microbial organisms. Studies have also shown their involvement in inflammatory re-
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P4-438
EFFECT OF INCREASING APOLIPOPROTEIN E ON INFLAMMATION IN THE BRAIN
Gayathri Ramaswamy, Pfizer Internal Medicine Research Unit, Cambridge, MA, USA. Contact e-mail: Gayathri.Ramaswamy@ pfizer.com
P1500
Poster Presentations: Wednesday, July 19, 2017
Background: Apolipoprotein E (apoE) is a key lipid transport protein in brain and plasma. Humans have three apoE isoforms; namely, apoE2, apoE3 and apoE4. Of the three isoforms, apoE4 is strongly associated with late-onset and sporadic forms of Alzheimer’s disease (AD). In addition to its role in neuronal repair, synaptogenesis, and clearance of toxic Aß fragments from brain, apoE has been suggested to play a critical role in inflammation in the brain. Using pan LXR agonists as a tool compound for increasing brain and astrocytic apoE levels, we sought to investigate the effect of increasing astrocytic apoE on brain inflammation induced by lipopolysaccharide (LPS) both in vitro and in vivo. Methods: Using primary mouse microglia and astrocytes and C57/bl6 wild type mice as our model system, we evaluated the effect of increasing apoE protein levels using T0901317 (pan LXR agonist) on LPS induced inflammation. We used RT-qPCR to evaluate the mRNA levels of apoE and apoE lipidating genes and MSD assays to evaluate cytokine protein levels in cell media and brain homogenates. Results: TO901317 treatment led to an increase in brain apoE levels in vivo in wild type mice and in vitro, in both primary murine astrocytes and microglia. This increase in apoE was accompanied by an increase in apoE lipidating genes (Abca1 and Abcg1). Treatment with LPS led to a significant inflammatory response as seen by an increase in pro-inflammatory cytokines, TNFa and IL6. However, TO901317 treatment led to a significant reduction in the levels of the pro-inflammatory cytokines both in vitro (astrocytes and microglia) and in vivo in the brain. Conclusions: Our results indicate that increasing brain and gial apoE using a pan LXR agonist attenuates LPS induced inflammation. Our data suggests that apoE could play an important role in promoting anti- inflammatory effects in the brain. Given the strong association of apoE with AD, increasing lipidated apoE in the brain could have multi-prong benefits including anti-inflammatory effects.
P4-439
COMMON PATHWAYS BETWEEN AGING AND ALZHEIMER’S DISEASE REVEALED BY SYSTEMS-BIOLOGICAL APPROACH
Hae Young Chung1,2, Hyoung Oh Jeong1,2, Jaewon Lee1,2, 1 Pusan National University, Busan, Republic of South Korea; 2 Molecular Inflammation Research Center for Aging Intervention, Busan, Republic of South Korea. Contact e-mail: hyjung@pusan. ac.kr Background: Alzheimer’s disease (AD) is one of the neurodegenerative diseases occurring over 65 years old. Aging is a risk factor of AD. Systems-biological analysis is potential tool for integration of biological information, for example on the pathogenesis of diseases. In particular, gene expression and protein-protein interaction (PPI) analysis have been very powerful in finding target molecules for the treatment of diseases. To identify therapeutic target molecules and biological pathways of AD and brain aging process, we have done PPI network analysis of proteins using microarray big data. Methods: Bioinformatics approaches including microarray analysis, transcription factor binding site prediction, and protein– protein interaction (PPI) network analysis were used in this study. Results: We identified about 300 genes that were significantly differently expressed in the brain tissues of patients with AD and normal aging process. To identify hub molecules, we tried PPI analysis.
The PPI network was found to include 27 molecules, which were found to be related to ‘hematopoietic cell lineage’, ‘cytokine-cytokine receptor interaction’, and ‘Jak-STAT signaling pathway’. These pathways might be important in the progression of AD and brain aging. In particular, the Jak-STAT signaling pathway, which is known to play a critical role in AD development, was up-regulated in both AD and aging. SOCS3, IL4R, and CSF3R were upregulated, which was found to be involved in the Jak-STAT signaling pathway. Furthermore, the current study on RNA-Seq analysis of aged hippocampus revealed that pro-inflammatory genes were upregulated in brain aging process. Conclusions: Cytokine-cytokine interaction and Jak-STAT signaling pathway play an important role in the common mechanisms underlying both AD and brain aging. These systems-biological approach is powerful tool for discovery of new key molecules involved in AD.
P4-440
CHARACTERIZATION OF BIOFLUID STREM2 USING A NOVEL STREM2 ASSAY
Hong Wang1, Nicholas Proctor1, Robert Spencer2, Caroline Hersley1, Francis David Tingley, III,1, Sarah E. Raines1, John Ryder1, Stuart Allen Kuhstoss1, Audrey McConnell3, Mansuo L. Hayashi1, Yaming Wang1, 1Eli Lilly and Company, Indianapolis, IN, USA; 2Eli Lilly BioA Sourcing, Indianapolis, IN, USA; 3Eli Lilly and Company, San Diego, CA, USA. Contact e-mail: [email protected] Background: Genetic variants of triggering receptor expressed on
myeloid cells 2 (TREM2) have been linked to Alzheimer’s disease (AD) and other neurodegenerative diseases. TREM2 is expressed as a transmembrane protein on the surface of myeloid cells, including microglia from the brain. sTREM2 (soluble TREM2) lacking the transmembrane domain can be detected in the extracellular space as a product of either enzymatic cleavage or alternative splicing. Recently, sTREM2 in cerebral spinal fluid (CSF) has been shown to be elevated in early AD, suggesting sTREM2 is a candidate biofluid biomarker for AD. Here we aimed to better understand the biology of sTREM2 release from monocytic cells as well as characterize sTREM2 levels in biofluids and brain tissues. Methods: To analyze sTREM2 levels in conditioned media, biofluids and brain tissue, we developed and validated a robust novel sTREM2 assay using the MSD platform. This assay specifically detects sTREM2 with a LLOD of w6 pg/mL sensitivity. Using this assay, endogenous sTREM2 was analyzed in cell media, mouse plasma, mouse CSF, mouse brain lysate, human plasma, and human CSF. In addition, immunoprecipitation and Western Blotting (IP/WB) were also employed to characterize sTREM2 in biofluid and brain lysate. Results: We demonstrate that sTREM2 is constitutively released from monocytic cell lines in vitro and is readily detectable in rodent brain and in both rodent and human plasma and CSF. IP/WB reveals sTrem2 runs as smeared bands around 25-50 kDa on SDS PAGE. Levels of sTREM2 measured by MSD and IP/WB align well. In mouse, sTREM2 levels increase in biofluids with age, but there is no correlation observed between CSF and plasma. sTREM2 levels in biofluids and brain tissues from AD animal models were examined and their relationship with pathology was analyzed. Conclusions: sTREM2 is constitutively released from monocytic cells and readily detectable in biofluids. Levels of CSF sTREM2 increase in AD animal models.
PROMINENT MICROGLIAL ACTIVATION IN CORTICAL WHITE MATTER IS SELECTIVELY ASSOCIATED WITH CORTICAL ATROPHY IN PRIMARY PROGRESSIVE APHASIA
Daniel T. Ohm, Garam Kim, Tamar Gefen, Alfred Rademaker, Sandra Weintraub, Eileen H. Bigio, Marsel Mesulam, Emily J. Rogalski, Changiz Geula, Northwestern University, Chicago, IL, USA. Contact e-mail: danielohm2012@u. northwestern.edu Background: The neuropathologic correlates of cortical atrophy in neurodegenerative disorders remain poorly understood. Primary progressive aphasia (PPA) is a clinical syndrome characterized by selective language impairments that reflect focal and asymmetric cortical atrophy in the language dominant (LD) hemisphere. Brains of individuals with PPA can be pathologically diagnosed with Alzheimer’s disease (AD), FTLD-tau, or FTLD-TDP, and they also display significant accumulation of activated microglia in the gray and white matter. Activated microglia can initiate an inflammatory cascade that may contribute to neurodegeneration, but their quantitative distribution in cortical white matter and their relationship with cortical atrophy are unknown. The present study investigated 10 PPA cases with AD or FTLD-TDP pathology to determine the density of white matter activated microglia and its association with gray matter atrophy. Methods: In each PPA case, two language regions with peak cortical atrophy and one non-atrophied region were selected within the LD hemisphere using photographs of intact postmortem brains, clinical MRI, or quantitative metrics using FreeSurfer with MRI scans acquired close to death. Non-atrophied contralateral homologues of LD regions were also examined for analyses of hemispheric asymmetry. Activated microglia were visualized immunohistochemically using a specific antibody to HLA-DR, and quantified with optical density measures of immunoreactivity. Results: Qualitatively, greater densities of activated microglia were observed in cortical white matter when compared to gray matter. Quantitative analyses showed that the white matter of atrophied language regions displayed significantly greater densities of activated microglia than the non-atrophied regions in the LD hemisphere (p<0.01). LD atrophied regions also showed a trend towards more activated microglia compared to non-language dominant regions (p¼0.0568). Conclusions: Activated microglia in PPA showed a greater propensity for white matter compared to gray matter, had higher densities in atrophied language regions than non-atrophied regions in the LD hemisphere, and often showed asymmetry favoring the LD atrophied regions consistent with the PPA clinical profile. Thus, activated microglia in cortical white matter are a proximal marker for gray matter atrophy. Given that activated microglia are thought to promote neuroinflammation, which may potentially exacerbate the disease progression, they may also be detrimental to the integrity of white matter. P4-437
sponses to amyloid beta (Ab) and aggregated a-synuclein. Activation of these receptors can amplify inflammation, but also promote phagocytosis of Ab. Activation of TLR-9 resulted in significant reduction of Ab in animal AD models. Activation of TLR-3 could have therapeutic potential due to its induction of beta-interferon responses. The premise of this study was observation of increased numbers of TLR-3 immunoreactive cells in AD affected brains. Methods: This study had two components; firstly staged series of human brain samples from low plaque (LP) and high plaque (HP) nondemented (ND) cases and AD cases were used to identify TLR-3 cellular localization using immunohistochemistry, and to quantify changes of expression of TLR-3 mRNA with disease by quantitative polymerase chain reaction (PCR). The second component involved in vitro studies of TLR-3 expression and activation using human postmortem brain-derived microglia and vascular endothelial cells. Results: Quantification of TLR-3 immunoreactivity in middle temporal gyrus using Image J software to measure area occupied showed increase of 101.8% in AD cases compared to LPND (p¼0.0036), and 16.5% increase between LPND and HPND (NS). TLR-3 immunoreactivity showed significant correlation with scores for brain plaque loads (Spearman r¼0.464, p<0.01) and tangle loads (r¼0.561, p<0.001). TLR-3 immunoreactivity was primarily in microglia, but also visible in subsets of astrocytes and vascular endothelial cells. Increased TLR-3 immunoreactivity was noticeable in microglia associated with Ab plaques. Quantitative PCR showed significant increase in TLR-3 mRNA between AD and ND cases (p¼0.043), interferon-b (p¼0.036) and interferon regulatory factor-3 (p¼0.013). Significant increase in TLR-9 mRNA expression in AD cases was also detected in these samples (p¼0.0019). In vitro experiments showed that treatment with TLR-3 ligand poly I:C did not induce significant increased expression of TLR-3 mRNA in microglia, but a tenfold increase in brain-derived endothelial cells. Conclusions: Different ways of manipulating microglia to promote removal of aggregated proteins has been explored as therapeutic strategies for AD. Increasing TLR-3 signaling could be one method for achieving this with the accumulation of TLR-3 expressing microglia associated with Ab plaques.
INCREASED EXPRESSION OF TOLL-LIKE RECEPTOR (TLR)-3 IN ALZHEIMER’S DISEASE BRAINS: A PROTECTIVE OR PATHOGENIC RESPONSE?
Douglas Gordon Walker1, Tiffany M. Tang1, Geidy E. Serrano2, Lucia I. Sue2, Thomas G. Beach2, Lih-Fen Lue1, 1Arizona State University, Tempe, AZ, USA; 2Banner Sun Health Research Institute, Sun City, AZ, USA. Contact e-mail: [email protected] Background: Toll-like receptors (TLR) are pattern recognition re-
ceptors involved in immune responses to microbial organisms. Studies have also shown their involvement in inflammatory re-
P1499
P4-438
EFFECT OF INCREASING APOLIPOPROTEIN E ON INFLAMMATION IN THE BRAIN
Gayathri Ramaswamy, Pfizer Internal Medicine Research Unit, Cambridge, MA, USA. Contact e-mail: Gayathri.Ramaswamy@ pfizer.com
P1500
Poster Presentations: Wednesday, July 19, 2017
Background: Apolipoprotein E (apoE) is a key lipid transport protein in brain and plasma. Humans have three apoE isoforms; namely, apoE2, apoE3 and apoE4. Of the three isoforms, apoE4 is strongly associated with late-onset and sporadic forms of Alzheimer’s disease (AD). In addition to its role in neuronal repair, synaptogenesis, and clearance of toxic Aß fragments from brain, apoE has been suggested to play a critical role in inflammation in the brain. Using pan LXR agonists as a tool compound for increasing brain and astrocytic apoE levels, we sought to investigate the effect of increasing astrocytic apoE on brain inflammation induced by lipopolysaccharide (LPS) both in vitro and in vivo. Methods: Using primary mouse microglia and astrocytes and C57/bl6 wild type mice as our model system, we evaluated the effect of increasing apoE protein levels using T0901317 (pan LXR agonist) on LPS induced inflammation. We used RT-qPCR to evaluate the mRNA levels of apoE and apoE lipidating genes and MSD assays to evaluate cytokine protein levels in cell media and brain homogenates. Results: TO901317 treatment led to an increase in brain apoE levels in vivo in wild type mice and in vitro, in both primary murine astrocytes and microglia. This increase in apoE was accompanied by an increase in apoE lipidating genes (Abca1 and Abcg1). Treatment with LPS led to a significant inflammatory response as seen by an increase in pro-inflammatory cytokines, TNFa and IL6. However, TO901317 treatment led to a significant reduction in the levels of the pro-inflammatory cytokines both in vitro (astrocytes and microglia) and in vivo in the brain. Conclusions: Our results indicate that increasing brain and gial apoE using a pan LXR agonist attenuates LPS induced inflammation. Our data suggests that apoE could play an important role in promoting anti- inflammatory effects in the brain. Given the strong association of apoE with AD, increasing lipidated apoE in the brain could have multi-prong benefits including anti-inflammatory effects.
P4-439
COMMON PATHWAYS BETWEEN AGING AND ALZHEIMER’S DISEASE REVEALED BY SYSTEMS-BIOLOGICAL APPROACH
Hae Young Chung1,2, Hyoung Oh Jeong1,2, Jaewon Lee1,2, 1 Pusan National University, Busan, Republic of South Korea; 2 Molecular Inflammation Research Center for Aging Intervention, Busan, Republic of South Korea. Contact e-mail: hyjung@pusan. ac.kr Background: Alzheimer’s disease (AD) is one of the neurodegenerative diseases occurring over 65 years old. Aging is a risk factor of AD. Systems-biological analysis is potential tool for integration of biological information, for example on the pathogenesis of diseases. In particular, gene expression and protein-protein interaction (PPI) analysis have been very powerful in finding target molecules for the treatment of diseases. To identify therapeutic target molecules and biological pathways of AD and brain aging process, we have done PPI network analysis of proteins using microarray big data. Methods: Bioinformatics approaches including microarray analysis, transcription factor binding site prediction, and protein– protein interaction (PPI) network analysis were used in this study. Results: We identified about 300 genes that were significantly differently expressed in the brain tissues of patients with AD and normal aging process. To identify hub molecules, we tried PPI analysis.
The PPI network was found to include 27 molecules, which were found to be related to ‘hematopoietic cell lineage’, ‘cytokine-cytokine receptor interaction’, and ‘Jak-STAT signaling pathway’. These pathways might be important in the progression of AD and brain aging. In particular, the Jak-STAT signaling pathway, which is known to play a critical role in AD development, was up-regulated in both AD and aging. SOCS3, IL4R, and CSF3R were upregulated, which was found to be involved in the Jak-STAT signaling pathway. Furthermore, the current study on RNA-Seq analysis of aged hippocampus revealed that pro-inflammatory genes were upregulated in brain aging process. Conclusions: Cytokine-cytokine interaction and Jak-STAT signaling pathway play an important role in the common mechanisms underlying both AD and brain aging. These systems-biological approach is powerful tool for discovery of new key molecules involved in AD.
P4-440
CHARACTERIZATION OF BIOFLUID STREM2 USING A NOVEL STREM2 ASSAY
Hong Wang1, Nicholas Proctor1, Robert Spencer2, Caroline Hersley1, Francis David Tingley, III,1, Sarah E. Raines1, John Ryder1, Stuart Allen Kuhstoss1, Audrey McConnell3, Mansuo L. Hayashi1, Yaming Wang1, 1Eli Lilly and Company, Indianapolis, IN, USA; 2Eli Lilly BioA Sourcing, Indianapolis, IN, USA; 3Eli Lilly and Company, San Diego, CA, USA. Contact e-mail: [email protected] Background: Genetic variants of triggering receptor expressed on
myeloid cells 2 (TREM2) have been linked to Alzheimer’s disease (AD) and other neurodegenerative diseases. TREM2 is expressed as a transmembrane protein on the surface of myeloid cells, including microglia from the brain. sTREM2 (soluble TREM2) lacking the transmembrane domain can be detected in the extracellular space as a product of either enzymatic cleavage or alternative splicing. Recently, sTREM2 in cerebral spinal fluid (CSF) has been shown to be elevated in early AD, suggesting sTREM2 is a candidate biofluid biomarker for AD. Here we aimed to better understand the biology of sTREM2 release from monocytic cells as well as characterize sTREM2 levels in biofluids and brain tissues. Methods: To analyze sTREM2 levels in conditioned media, biofluids and brain tissue, we developed and validated a robust novel sTREM2 assay using the MSD platform. This assay specifically detects sTREM2 with a LLOD of w6 pg/mL sensitivity. Using this assay, endogenous sTREM2 was analyzed in cell media, mouse plasma, mouse CSF, mouse brain lysate, human plasma, and human CSF. In addition, immunoprecipitation and Western Blotting (IP/WB) were also employed to characterize sTREM2 in biofluid and brain lysate. Results: We demonstrate that sTREM2 is constitutively released from monocytic cell lines in vitro and is readily detectable in rodent brain and in both rodent and human plasma and CSF. IP/WB reveals sTrem2 runs as smeared bands around 25-50 kDa on SDS PAGE. Levels of sTREM2 measured by MSD and IP/WB align well. In mouse, sTREM2 levels increase in biofluids with age, but there is no correlation observed between CSF and plasma. sTREM2 levels in biofluids and brain tissues from AD animal models were examined and their relationship with pathology was analyzed. Conclusions: sTREM2 is constitutively released from monocytic cells and readily detectable in biofluids. Levels of CSF sTREM2 increase in AD animal models.