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Item INPP5D expression is associated with risk for Alzheimer’s disease and induced by plaque-associated microglia(Elsevier, 2021-06) Tsai, Andy P.; Bor-Chian, Lin Peter; Dong, Chuanpeng; Moutinho, Miguel; Casali, Brad T.; Liu, Yunlong; Lamb, Bruce T.; Landreth, Gary E.; Oblak, Adrian L.; Nho, Kwangsik; Medical and Molecular Genetics, School of MedicineAlzheimer's disease (AD) is a progressive neurodegenerative disorder characterized by cognitive decline, robust microgliosis, neuroinflammation, and neuronal loss. Genome-wide association studies recently highlighted a prominent role for microglia in late-onset AD (LOAD). Specifically, inositol polyphosphate-5-phosphatase (INPP5D), also known as SHIP1, is selectively expressed in brain microglia and has been reported to be associated with LOAD. Although INPP5D is likely a crucial player in AD pathophysiology, its role in disease onset and progression remains unclear. We performed differential gene expression analysis to investigate INPP5D expression in AD and its association with plaque density and microglial markers using transcriptomic (RNA-Seq) data from the Accelerating Medicines Partnership for Alzheimer's Disease (AMP-AD) cohort. We also performed quantitative real-time PCR, immunoblotting, and immunofluorescence assays to assess INPP5D expression in the 5xFAD amyloid mouse model. Differential gene expression analysis found that INPP5D expression was upregulated in LOAD and positively correlated with amyloid plaque density. In addition, in 5xFAD mice, Inpp5d expression increased as the disease progressed, and selectively in plaque-associated microglia. Increased Inpp5d expression levels in 5xFAD mice were abolished entirely by depleting microglia with the colony-stimulating factor receptor-1 antagonist PLX5622. Our findings show that INPP5D expression increases as AD progresses, predominantly in plaque-associated microglia. Importantly, we provide the first evidence that increased INPP5D expression might be a risk factor in AD, highlighting INPP5D as a potential therapeutic target. Moreover, we have shown that the 5xFAD mouse model is appropriate for studying INPP5D in AD.Item SHIP1 therapeutic target enablement: Identification and evaluation of inhibitors for the treatment of late‐onset Alzheimer's disease(Wiley, 2023) Jesudason, Cynthia D.; Mason, Emily R.; Chu, Shaoyou; Oblak, Adrian L.; Javens-Wolfe, June; Moussaif, Mustapha; Durst, Greg; Hipskind, Philip; Beck, Daniel E.; Dong, Jiajun; Amarasinghe, Ovini; Zhang, Zhong-Yin; Hamdani, Adam K.; Singhal, Kratika; Mesecar, Andrew D.; Souza, Sarah; Jacobson, Marlene; Di Salvo, Jerry; Soni, Disha M.; Kandasamy, Murugesh; Masters, Andrea R.; Quinney, Sara K.; Doolen, Suzanne; Huhe, Hasi; Sukoff Rizzo, Stacey J.; Lamb, Bruce T.; Palkowitz, Alan D.; Richardson, Timothy I.; Medicine, School of MedicineIntroduction: The risk of developing Alzheimer's disease is associated with genes involved in microglial function. Inositol polyphosphate-5-phosphatase (INPP5D), which encodes Src homology 2 (SH2) domain-containing inositol polyphosphate 5-phosphatase 1 (SHIP1), is a risk gene expressed in microglia. Because SHIP1 binds receptor immunoreceptor tyrosine-based inhibitory motifs (ITIMs), competes with kinases, and converts PI(3,4,5)P3 to PI(3,4)P2, it is a negative regulator of microglia function. Validated inhibitors are needed to evaluate SHIP1 as a potential therapeutic target. Methods: We identified inhibitors and screened the enzymatic domain of SHIP1. A protein construct containing two domains was used to evaluate enzyme inhibitor potency and selectivity versus SHIP2. Inhibitors were tested against a construct containing all ordered domains of the human and mouse proteins. A cellular thermal shift assay (CETSA) provided evidence of target engagement in cells. Phospho-AKT levels provided further evidence of on-target pharmacology. A high-content imaging assay was used to study the pharmacology of SHIP1 inhibition while monitoring cell health. Physicochemical and absorption, distribution, metabolism, and excretion (ADME) properties were evaluated to select a compound suitable for in vivo studies. Results: SHIP1 inhibitors displayed a remarkable array of activities and cellular pharmacology. Inhibitory potency was dependent on the protein construct used to assess enzymatic activity. Some inhibitors failed to engage the target in cells. Inhibitors that were active in the CETSA consistently destabilized the protein and reduced pAKT levels. Many SHIP1 inhibitors were cytotoxic either at high concentration due to cell stress or they potently induced cell death depending on the compound and cell type. One compound activated microglia, inducing phagocytosis at concentrations that did not result in significant cell death. A pharmacokinetic study demonstrated brain exposures in mice upon oral administration. Discussion: 3-((2,4-Dichlorobenzyl)oxy)-5-(1-(piperidin-4-yl)-1H-pyrazol-4-yl) pyridine activated primary mouse microglia and demonstrated exposures in mouse brain upon oral dosing. Although this compound is our recommended chemical probe for investigating the pharmacology of SHIP1 inhibition at this time, further optimization is required for clinical studies. Highlights: Cellular thermal shift assay (CETSA) and signaling (pAKT) assays were developed to provide evidence of src homology 2 (SH2) domain-containing inositol phosphatase 1 (SHIP1) target engagement and on-target activity in cellular assays. A phenotypic high-content imaging assay with simultaneous measures of phagocytosis, cell number, and nuclear intensity was developed to explore cellular pharmacology and monitor cell health. SHIP1 inhibitors demonstrate a wide range of activity and cellular pharmacology, and many reported inhibitors are cytotoxic. The chemical probe 3-((2,4-dichlorobenzyl)oxy)-5-(1-(piperidin-4-yl)-1H-pyrazol-4-yl) pyridine is recommended to explore SHIP1 pharmacology.Item The Role of INPP5D in Microglial Function and Amyloid Pathogenesis(2023-07) Lin, Peter Bor-Chian; Block, Michelle L.; Oblak, Adrian L.; Landreth, Gary E.; Kim, Jungsu; Territo, Paul R.Alzheimer’s disease (AD) is a neurodegenerative disorder and the most common cause of dementia. Genetic studies implicate the involvement of microglia-mediated immune responses during disease progression. Importantly, inositol polyphosphate-5-phosphatase D (INPP5D) serves as a regulator of microglial functions and its variants have been identified as risk of late-onset AD. The primary object of this thesis was to study the role of INPP5D in AD pathogenesis. First, increased levels of INPP5D were detected in brain regions of LOAD patients, and a positive association was noted between INPP5D expression and amyloid plaque density. Importantly, increased INPP5D expression was also observed in the amyloidogenic 5xFAD mouse model, with a similar pattern of elevated expression predominately in plaque-associated microglia. These results demonstrated that INPP5D plays an important role in AD. Second, we determined the effect of Inpp5d haplodeficiency on amyloid pathology and microglial functions in 5xFAD mice. The results revealed that Inpp5d haploinsufficiency reduced amyloid plaque burdens and reversed behavioral deficits in 5xFAD mice. Inpp5d haploinsufficiency enhanced microglial engagement to plaques while increasing amyloid plaque compaction in the brains. Furthermore, Inpp5d haploinsufficiency activates TREM2 signaling and suppresses proinflammatory cytokines release in cortical tissues. Spatial transcriptomic analysis highlights that Inpp5d haploinsufficiency modulated the functional pathways including immune cell activation, cytokines production, protein degradation, memory, and synaptic plasticity. Our study suggests that reducing INPP5D expression alters microglial responses and mitigates amyloid pathology during AD progression. Finally, we prepared primary microglial cultures from the wild-type and Inpp5d-haplodeficient mice. The microglial cultures were treated with fibrillar beta-amyloid (fAβ) to investigate the effect of INPP5D inhibition on microglial signaling. Our results demonstrate an increased fAβ uptake and decreased fAβ cytotoxicity in the Inpp5d-deficient microglia. Inpp5d haplodeficiency alters microglial functional pathways, including phagocytosis, apoptosis, cytokines production, and the complement system. Importantly, Inpp5d haplodeficiency elevates the expression of homeostatic microglia signatures. Furthermore, treatment of microglia with INPP5D antagonist (TAD32, 1 μM) showed similar effect as the Inpp5d deficiency in microglia. Collectively, our study validates the hypothesis that INPP5D inhibition may help protect against AD pathology. Treatments utilizing INPP5D antagonists to target microglia-mediated immune responses may be beneficial as an AD therapy.