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  microglial processes – lysosomal activity and inflammatory response – are aberrantly activated in AD. How these microglial processes, critical for brain development and function, become dysfunctional in the context of aging or disease remains largely unexamined. I propose to study lysosomal and immune responses of microglia in vivo using zebrafish in light of numerous observations showing that microglia cultured in vitro rapidlylosetheiridentityanddisplayaberrantexpressionofdisease- associated genes. I have previously defined a lysosomal network centered around the key lysosomal transcription factors Tfeb and Tfe3. Using RNA-Sequencing of macrophages overexpressing Tfeb and Tfe3, I have uncovered the lysosomal targets of these transcription factors in the macrophage lineage. I find that many lysosomal targets of Tfeb and Tfe3 are dysregulated in AD and my ongoing experiments will reveal mechanisms through which aberrant Tfeb and Tfe3 activity contributes to the pathology in AD. In parallel, I am leveraging the experimental amenability of zebrafish for performing large-scale CRISPR screens to study genes implicated in AD. I have identified zebrafish homologs of genes mutated in AD, prioritized them based on microglial expression or lysosomal function, and performed a CRISPR mutagenesis screen. My preliminary data confirm that indeed many of these AD risk-associated genes function in microglia. Collectively, my research renders microglial biology accessible to live imaging and functional characterization in vivo, and will bridge the gap between genomic resources available for AD and molecular mechanisms underlying the pathology of this devastating disease.
Discussion: Through unbiased RNA and proteomic profiling and a series of pharmacological manipulations, we demonstrate that reduction of INPP5D activity induces changes in immune signaling through the activation of the inflammasome. Further, we show functional consequences of inflammasome activation in microglia on neuronal gene expression profiles.
Conclusions: This work implicates INPP5D as a regulator of inflammasome signaling in human microglia.
Glial and vascular contributions to neurodegenerative diseases
Canonical amyloid-β (Aβ) and tau pathologies regulate disease stage-specific microglia subtypes in AD
Dong Won Kim2, Kevin Tu1, Alice Wei1, Ashley Lau1, Anabel Gonzalez-Gil3, Tianyu Cao1, Kerstin Braunstein1, Jonathan Ling1, Juan Troncoso1, Philip Wong1,2, Seth Blackshaw2, Ronald Schnaar3, Tong Li1
1Department of Pathology, Johns Hopkins University School of Medicine, Baltimore, USA 2Solomon H. Snyder Department of Neuroscience, Johns Hopkins University School of Medicine, Baltimore, USA
3Department of Pharmacology and Molecular Sciences, Johns Hopkins University School of Medicine, Baltimore, USA
Regulation of inflammasome activation in human microglia by Inositol Polyphosphate-5- Phosphatase D (INPP5D/SHIP1)
Vicky Chou1, RIchard Pearse1, Aimee Aylward1, Seeley Fancher1, Hyo Lee1, Matti Lam2, Nicholas Seyfried3, David Bennett4, Phil De Jager2, Vilas Menon2, Tracy L. Young- Pearse1
1Brigham and Women's Hospital and Harvard Medical School, Boston, USA 2Columbia University Medical Center, New York, USA
3Emory University, Atlanta, USA
4Rush University Medical Center, Chicago, USA
 Background: Microglia and neuroinflammation are implicated in the development and progression of Alzheimer’s disease (AD). To better understand microglia-mediated processes in AD, we studied the function of Inositol Polyphosphate-5-Phosphatase D (INPP5D/SHIP1), a gene linked to AD through GWAS.
Materials and Methods: Immunostaining, single nucleus RNA sequencing (RNAseq), and Western blotting were used to interrogate INPP5D expression in human postmortem brain and iPSC-derived microglia (iMGs). CRISPR Cas9 targeting and pharmacological inhibitors of INPP5D were employed to generate an experimental model of lower of INPP5D activity using human iMGs. To interrogate the function of INPP5D in human microglia, proteomic profiling, ELISA, and a series of pharmacological treatments were employed. Single cell RNAseq was utilized in both human co-culture systems and a conditional knock out mouse model to investigate the consequences of reduced INPP5D activity on neurons and astrocytes.
Results: INPP5D expression in the human brain is largely restricted to microglia, and examination of prefrontal cortex across a large cohort revealed reduced full length INPP5D protein levels in AD patient brains compared to cognitively normal controls. INPP5D inhibition or copy number reduction in iMGs induced the secretion of IL-1β and IL-18. These elevated levels were rescued with caspase-1 and NLRP3 inhibitors, suggesting activation of the inflammasome with reduced INPP5D activity. Inflammasome activation with INPP5D reduction was further confirmed through ASC immunostaining and observation of increased cleaved caspase-1.
Background: Amongst risk alleles associated with late-onset Alzheimer’s disease (AD), those that converged on the regulation of microglia activity have emerged as central to disease progression. Yet, how canonical amyloid-β (Aβ) and tau pathologies regulate microglia subtypes during the progression of AD remains poorly understood. Methods: We use single-cell RNA-sequencing to profile novel microglia subtypes that are induced in response to both Aβ and tau pathologies in a disease stage-specific manner. To validate the observation in AD mouse models, we also generated snRNA-Seq dataset from the human superior frontal gyrus (SFG) and entorhinal cortex (ERC) at different Braak stages.
Results: We show that during early-stage disease, interferon signalling induces a subtype of microglia termed Early-stage AD-Associated Microglia (EADAM) in response to both Aβ and tau pathologies. During late-stage disease, a second microglia subtype termed Late-stage AD- Associated Microglia (LADAM) is detected. Importantly, the pattern of EADAM- and LADAM-associated gene expression is observed in microglia from AD brains, during the early (Braak II)- or late (Braak VI/V)- stage of the disease, respectively. Furthermore, we show that several Siglec genes are selectively expressed in either EADAM or LADAM. For example, Siglecg is expressed in white-matter-associated LADAM at late stage, while Siglec-10, the human orthologue of Siglecg, is progressively elevated in an AD-stage-dependent manner but not shown in non- AD tauopathy, suggesting that Siglec family members could serve as specific markers for the stage-dependent microglia subtypes in AD. Conclusions: Using scRNA-Seq in mouse models bearing amyloid-β and/or tau pathologies, we identify novel microglia subtypes induced by the combination of Aβ and tau pathologies in a disease stage-specific manner. Our findings suggest that both Aβ and tau pathologies are required for the disease stage-specific induction of EADAM and LADAM. In addition, we revealed Siglecs as biomarkers of AD progression, and potential therapeutic targets.

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