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  of neurodegeneration. This has revealed common activation states including disease associated microglia, antigen presentation microglia, and a type 1 interferon-responsive microglia (IRM). To truly make use of this data, we now must move towards understanding the functional consequence of these microglial activation states and uncovering the mechanisms that drive changes in cell states during disease.
Materials and Methods: To uncover the molecular pathways that regulate the IRM state, I conducted a CRISPR-interference (CRISPRi) screen on human induced pluripotent stem cell-derived microglia. We tested ~2300 genes from a library of kinases and phosphatases (druggable genome) and assessed induction of the IRM state via IFIT1 expression. Top genes promoting or inhibiting IRM were validated via pharmacological inhibition and functional analyses.
Results: As expected, knockdown of known regulators of interferon signaling including IFNAR1/2, JAK1, and TYK2 were identified as negative regulators of IRM. More interestingly, we uncovered microglial- specific regulators of IRM including several Alzheimer’s disease (AD) risk loci. This data informs ongoing studies of mechanisms driving IRM as well as functional characterization of the IRM state via neuron co- cultures, cytokine secretion profiles, and migration to chemotactic cues. Discussion: The discovery that AD risk loci modulate IRM induction suggests a novel role for IRM in the progression of neurodegenerative disease. Furthermore, the results of my CRISPRi screen highlight clinically relevant modifiers of interferon-responses that are specific to microglia and may be used to modulate IRM in disease contexts in vivo. Conclusions: Understanding mechanisms that promote/inhibit transition to the disease-relevant IRM state deepens our understanding of how this activation state may influence neurodegeneration. Uncovering AD risk loci as modifiers of IRM suggests this state may represent a novel therapeutic strategy.
Results: We observe distinct lipidomic signatures between neurons, astrocytes and microglia, suggesting that the effect of disease- associated mutations may be cell-type specific. In microglia, we observe differential responses of cells carrying the APOE4 AD risk variant compared to the APOE2 protective variant, in response to lipid accumulation.
Conclusion: These preliminary results suggest an altered microglial response to lipid dyshomeostasis, which appears to be APOE genotype- dependent. Uncovering the molecular mechanisms driving the differential responses in microglia may contribute to our understanding of early AD pathology. Collectively, the Neurolipid Atlas pipeline will inform us on the effect of early disease mechanisms on the cell-type specific lipidome and represent a unique platform to identify novel candidate therapeutic interventions.
Glial and vascular contributions to neurodegenerative diseases
Severe reactive astrocytes precipitate pathological hallmarks of Alzheimer’s disease via H2O2−production
Jiwoon Lim1,7,8, Heejung Chun1,2,3, Hyeonjoo Im3, You Jung Kang4, Yunha Kim3, Jin Hee Shin5, Woojin Won1,6, Yeonha Ju1,7,8, Yongmin Mason Park1,7,8, Sunpil Kim1,6, Seung Eun Lee9, Jaekwang Lee2, Junsung Woo2, Yujin Hwang3, Hyesun Cho3,10, Seonmi Jo2,11, Jong-Hyun Park12, Daesoo Kim11, Doo Yeon Kim13, Jeong-Sun Seo10,14, Byoung Joo Gwag5, Young Soo Kim15, Ki Duk Park8,12,16, Bong-Kiun Kaang17, Hansang Cho4,18,19, Hoon Ryu3,20, C. Justin Lee1,2,6,7
1Center for Cognition and Sociality, Institute For Basic Science, Daejeon, Republic of Korea, 2Center for Glia-Neuron Interaction, Brain Science Institute, Korea Institute of Science and Technology, Seoul, Republic of Korea, 3Center for Neuroscience, Brain Science Institute, Korea Institute of Science and Technology, Seoul, Republic of Korea, 4Department of Mechanical Engineering and Engineering Science, Center for Biomedical Engineering and Science, Department of Biological Sciences, University of North Carolina at Charlotte, Charlotte, USA, 5GNT Pharma Co. Ltd., Yongin, Republic of Korea, 6Korea Uivresity-Korea Institute of Science and Technology, Graduate School of Convergence Technology, Korea University, Seoul, Republic of Korea, 7IBS School, University of Science and Technology, Daejeon, Republic of Korea, 8Neuroscience Program, University of Science and Technology, Daejeon, Republic
of Korea, 9Virus Facility, Research Animal Resource Center, Korea Institute of Science and Technology, Seoul, Republic of Korea, 10Precision Medicine Center, Seoul National University Bundang Hospital, Seongnam, Republic of Korea, 11Department of Biological Sciences, Korea Advanced Institute of Science and Technology, Daejeon, Republic of Korea, 12Convergence Research Center for Diagnosis, Treatment and Care System of Dementia, Korea Institute of Science and Technology, Seoul, Republic of Korea, 13Genetics and Aging Research Unit, Mass General Institute for Neurodegenerative Disease, Massachusetts General Hospital, Harvard Medical School, Charlestown, USA, 14Genomic Institute, Macrogen Inc, Seoul, Republic of Korea, 15Integrated Science and Engineering Division, Department of Pharmacy, and Yonsei Institute of Pharmaceutical Sciences, Yonsei University, Incheon, Republic of Korea, 16Division of Bio-Med Science & Technology, KIST School, Korea University of Science and Technology, Seoul, Republic of Korea, 17School of Biological Sciences, Seoul National University, Seoul, Republic of Korea, 18The Nanoscale Science Program, University of North Carolina at Charlotte, Charlotte, USA, 19Department of Biophysics, Institute of Quantum Biophysics, Sungkyunkwan
University, Suwon, Republic of Korea, 20Boston University Alzheimer’s Disease Center and Department of Neurology, Boston University School of Medicine, Boston, USA
Investigating the effect of lipid dyshomeostasis in Alzheimer’s disease microglia
Aiko Robert1,2, Amanda McQuade3, Martin Giera4, Lena Erlebach5,6, Deborah Kronenberg-Versteeg5,6, Martin Kampmann3,7, Rik van der Kant1,2
1Department of Functional Genomics, Center for Neurogenomics and Cognitive Research (CNCR), VU University Amsterdam, Amsterdam, Netherlands, 2Alzheimer Center Amsterdam, Department of Neurology, Amsterdam Neuroscience, Amsterdam University Medical Center, Amsterdam, Netherlands, 3Institute for Neurodegenerative Diseases, University of California, San Francisco, San Francisco, USA, 4Center for Proteomics and Metabolomics, Leiden, Netherlands, 5Hertie Institute for Clinical Brain Research, University of Tübingen, Tübingen, Germany, 6German Center for Neurodegenerative Diseases, Tübingen, Germany, 7Department of Biochemistry and Biophysics, University of California, San Francisco, San Francisco, USA
 Background: Genome-wide association studies have identified a number of Alzheimer’s disease (AD) associated genetic risk variants highly expressed in glial cells and heavily implicated in lipid metabolism. Notably, the APOE4 variant is the largest risk factor for late-onset AD, the protein product of which mediates lipid transport between neuronal and glial cells. Several lines of research have supported an intimate link between altered lipid metabolism and AD neuropathology, and recent data from human and mouse microglia indicate a strong coupling between aberrant lipid droplet formation and a pro-inflammatory transcriptional and phenotypic signature.
Materials and methods: We have developed protocols to induce and study the functional effects of microglial lipid accumulation in a reproducible manner. This work forms part of the Neurolipid Atlas, a collaborative effort to combine gene-editing of human iPSC-derived neurons, astrocytes and microglia and advanced lipidomic analysis to understand the role of lipids in neurodegenerative diseases, which will be available as an open-access webtool. In addition, we will perform a high throughput CRISPR/Cas9 genetic screen to correlate functional changes in human iPSC-derived microglia with aberrant lipid accumulation.
Background: Although the pathological contributions of reactive astrocytes have been implicated in Alzheimer’s disease (AD), their in vivo functions remain elusive due to the lack of appropriate experimental models and precise molecular mechanisms.
Materials and methods: We newly developed animal model of reactive astrocytes, GiD, where the reactivity of astrocytes can be manipulated as mild (GiDm) or severe (GiDs). Also, we utilized three-dimensional culture AD model, virus-infected APP/PS1 mice and the brains of patients with AD. We conducted immunohistochemistry, western blot analysis, RT- PCR, in vivo hydrogen peroxide (H2O2) assay, nitric oxide measurement electrophysiology and behavior tests.
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