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  Materials and Methods: Here, we investigated the mechanisms by which a homozygous stop-gain TREM2 variant (Q33X) contributes to NHD. Induced pluripotent stem cell (iPSC)-derived microglia (iMGLs) [6] were generated from two siblings homozygous for the TREM2 p.Q33X mutation, one related non-carrier, and one unrelated non-carrier. Results: Transcriptomic analysis and biochemical assays revealed that iMGLs from NHD patients carrying the TREM2 p.Q33X mutation exhibited lysosomal dysfunction, downregulation of cholesterol metabolism genes, and reduced lipid droplets compared to related and unrelated controls. Also, NHD iMGLs displayed defective activation, HLA antigen presentation, and NF-kB signaling. Alteration in lysosomal gene expression, such as decreased expression of genes implicated in lysosomal acidification (ATP6AP2) and chaperone mediated autophagy (LAMP2), and reduction in lipid droplets were also observed in post- mortem brain tissues from NHD patients, thus closely recapitulating in vivo the phenotype observed in iMGLs in vitro.
Discussion: Thus far, murine models are not able to recapitulate the key phenotypic features observed in patients affected by NHD. This has become an increasingly pressing challenge as rare variants in TREM2 have been identified as risk factors in AD.
Conclusions: Our study provides the first cellular and molecular evidence that lack of TREM2 in microglia leads to a defect in lysosomal function. A better understanding of how microglial lipid metabolism and lysosomal machinery are altered in NHD may provide new insights into mechanisms underlying NHD and AD pathogenesis.
Background: BIN1, the second most important risk locus for Late-Onset Alzheimer’s Disease (LOAD) after ApoE, is expressed as more than 10 isoforms in the brain. In LOAD, BIN1 isoform 1 expression decreases concomitantly with neuronal loss, whereas the ubiquitous isoform 9 (BIN1iso9) levels increase. Previous research by our group has shown that ubiquitous BIN1iso9 is highly expressed in mature oligodendrocytes and the white matter in rodents and humans. However, how BIN1iso9 is involved in AD progression and contributes to neurodegeneration remains enigmatic. This study aims to investigate the role of BIN1iso9 in neuronal dysfunction and AD neuropathology using an inducible transgenic mouse model that overexpresses human BIN1iso9 in mature oligodendrocytes in 5XFAD genetic background (5XFAD:PLP:BIN1iso9). Methods: We generated PLP:BIN1iso9 transgenic mice to achieve cell-type specific PLP-CreERT-dependent expression of BIN1iso9 in mature oligodendrocytes. To assess whether BIN1iso9 overexpression affected myelination or AD pathophysiology, we analyzed myelin marker expression in 7-month-old 5XFAD:PLP:BIN1iso9 and control. To characterize the influence of BIN1iso9 overexpression on amyloid pathology, we measured the insoluble amyloid-beta levels in forebrain homogenates. The cognitive functions of the animals were assessed between 6-7 months using different behavioral tests.
Results: We confirmed the cell-type-specific expression of human BIN1iso9 with no change in endogenous mouse BIN1. qPCR analyses of myelin-associated genes showed a decrease in expression levels of myelin basic protein (Mbp) and 2',3'-Cyclic-nucleotide 3'-phosphodiesterase (Cnp) compared to 5XFAD:PLP controls. These results were accompanied by a significant decline in the protein expression of MBP in 5XFAD:PLP:BIN1iso9. On the other hand, the elevation of BIN1iso9 expression in mature oligodendrocytes altered neither insoluble amyloid-beta levels nor the behavioral pathology. Conclusion: Based on the above results, we conclude that the elevated expression of BIN1iso9 in mature oligodendrocytes might contribute to AD pathophysiology through myelin integrity rather than amyloid pathology.
Funded by NIH grants AG054223 and AG056061.
Glial and vascular contributions to neurodegenerative diseases
The effects of elevated BIN1 isoform 9 expression in mature oligodendrocytes on myelination and AD pathophysiology in the 5XFAD mouse model
Melike Yuksel1, Mitch Hansen1, Robert J. Andrew2, Moorthi Ponnusamy, Gopal Thinakaran1
1Department of Molecular Medicine and USF Health Byrd Alzheimer’s Center and Research Institute, University of South Florida, Tampa, USA, 2Department of Neurobiology, The University of Chicago, Chicago, USA
Identification of novel regulators of the TREM2 pathway using arrayed CRISPR screens with iPSC derived human microglia-like cells Fangfang Bai1, Jean Paul Chadarevian2, Aaron Wolman1, Alex Tamburino1, Carlo Ramil1, Sidney Hsieh1, Choya Yoon1, Tobias Ehrenberger1, Aleksandra Olow1, Federica Piccioni1, An Chi1, Matthew Kennedy1, Hayk Davtyan2, Matthew
Blurton-Jones2, Vanessa Peterson1, Rebecca Mathew1
1MSD, Cambridge, USA, 2UCI Department of Neurobiology and Behavior, Irvine, USA
 Designing reverse genetic experiments to ascertain the function of microglial Alzheimer's Disease (AD)-associated genes are hindered by both the inability to obtain and manipulate postmortem tissue from AD patients and the absence of functionally relevant human microglia cell lines. Human-induced pluripotent stem cell (hiPSC)-derived microglia cells represent a novel strategy to examine the relationship between genetic risk factors and late-onset AD. Functional analysis of induced microglial-like cells (iMGLs) reveals that they secrete cytokines in response to inflammatory stimuli and CNS disease- associated substrates, including amyloid beta fibrils. The ability to precisely introduce disease-associated genetic mutations in iMGLs using CRISPR/Cas9 technology will help define the contribution and function of genes associated with late-onset AD. We have established an arrayed CRISPR screening platform that can be used to evaluate the functional consequences of genetic perturbations in iMGLs. A CRISPR screen was performed using a library of 150 genes that were identified by IP-MS with myeloid cells after stimulation with an anti- TREM2 agonistic antibody. Putative hits from the arrayed CRISPR screen include genes that are involved in: (I) regulation of microglial functions including morphological change, migration, and phagocytosis; (II) a component of a dysfunctional TREM2-dependent signaling node; (III) direct targets of a microRNA circuit that regulates chronic peripheral neuropathic pain. Hits from the arrayed CRISPR screen were evaluated by a secondary assay to determine impact on cytokine secretion; both positive and negative regulators of cytokine production were identified from this assay. This functional genomics screen demonstrates the utility of iMGLs as a platform to identify novel targets and regulators of microglial activation states.

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