Microglial amyloid beta clearance is driven by PIEZO1 channels

Background Microglia are the endogenous immune cells of the brain and act as sensors of pathology to maintain brain homeostasis and eliminate potential threats. In Alzheimer’s disease (AD), toxic amyloid beta (Aβ) accumulates in the brain and forms stiff plaques. In late-onset AD accounting for 95% of all cases, this is thought to be due to reduced clearance of Aβ. Human genome-wide association studies and animal models suggest that reduced clearance results from aberrant function of microglia. While the impact of neurochemical pathways on microglia have been broadly studied, mechanical receptors regulating microglial functions remain largely unexplored. Methods Here we showed that a mechanotransduction ion channel, PIEZO1, is expressed and functional in human and mouse microglia. We used a small molecule agonist, Yoda1, to study how activation of PIEZO1 affects AD-related functions in human induced pluripotent stem cell (iPSC) -derived microglia-like cells (iMGL) under controlled laboratory experiments. Cell survival, metabolism, phagocytosis and lysosomal activity were assessed using real-time functional assays. To evaluate the effect of activation of PIEZO1 in vivo , 5-month-old 5xFAD male mice were infused daily with Yoda1 for two weeks through intracranial cannulas. Microglial Iba1 expression and Aβ pathology were quantified with immunohistochemistry and confocal microscopy. Published human and mouse AD datasets were used for in-depth analysis of PIEZO1 gene expression and related pathways in microglial subpopulations. Results We show that PIEZO1 orchestrates Aβ clearance by enhancing microglial survival, phagocytosis, and lysosomal activity. Aβ inhibited PIEZO1-mediated calcium transients, whereas activation of PIEZO1 with a selective agonist, Yoda1, improved microglial phagocytosis resulting in Aβ clearance both in human and mouse models of AD. Moreover, PIEZO1 expression was associated with a unique microglial transcriptional phenotype in AD as indicated by assessment of cellular metabolism, and human and mouse single cell datasets. Conclusion These results indicate that the compromised function of microglia in AD could be improved by controlled activation of PIEZO1 channels resulting in alleviated Aβ burden. Pharmacological regulation of these mechanoreceptors in microglia could represent a novel therapeutic paradigm for AD. ![Figure][1] ### Competing Interest Statement Malm and Giniatullin are inventors of PCT patent application related to this topic. * AD : Alzheimer’s disease ADP : Adenosine diphosphate ANOVA : Analysis of variance APOC1 : Apolipoprotein C1 APOE : Apolipoprotein E APP : Amyloid-beta precursor protein ARRIVE : Animal Research: Reporting of In Vivo Experiments ATP : Adenosine triphosphate AUC : Area under curve Aβ : Amyloid beta BF : Bright field Cas9 : CRISPR-associated protein 9 CCD : Charge-coupled device CD14 : Cluster of differentiation 14 CD14M : CD14+ monocyte cDNA : Complementary DNA Cox6a1 : Cytochrome c oxidase subunit 6A1 CRISPR : Clustered Regularly Interspaced Short Palindromic Repeats Ctsb : Cathepsin B Ctsd : Cathepsin D Ctsl : Cathepsin L Ctsz : Cathepsin z CX3CR1 : CX3C chemokine receptor 1 DAM : Disease associated microglia DAPI : 4′,6-diamidino-2-phenylindole DC : Dendritic cell DEG : Differentially expressed gene DMEM : Dulbecco’s Modified Eagle Medium DMSO : Dimethyl sulfoxide DNA : Deoxyribonucleic acid DNAse : Deoxyribonuclease dNTP : Deoxynucleoside triphosphate DRC : Dose-response curves EBiSC : European bank for Induced Pluripotent Stem cells ECAR : Extracellular acidification EDTA : Ethylenediaminetetraacetic acid EOAD : Early-onset Alzheimer’s disease Expr. : Expression level F12 : Nutrient Mixture F-12 FBS : Fetal bovine serum FCCP : Carbonyl cyanide-p-trifluoromethoxyphenylhydrazon FIMM : Institute for Molecular Medicine Finland FPKM : Fragments Per Kilobase of transcript per Million mapped reads Freq. : frequency FRMD4A : FERM domain containing 4A Fth1 : Ferritin heavy chain 1 GCU : Green calibrated Unit GFAP : Glial fibrillary acidic protein Glul : Glutamate-ammonia ligase GSVA : Gene set variation analysis GWAS : Genome-wide association study HEK293 : Human embryonic kidney 293 cells HiLiFE : Helsinki Institute of Life Science HOS : Hypo-osmotic solution IBA1 : Ionized calcium binding adaptor molecule 1 IFNγ : Interferon Gamma IHC : Immunocytochemistry iHPC : Induced hematopoietic progenitor cells IL-13 : Interleukin 13 IL-4 : Interleukin 4 iMGL : iPSC-derived microglia-like cells iPSC : Induced pluripotent stem cell ISSCR : The International Society for Stem Cell Research LDH : Lactate dehydrogenase LINGO1 : Leucine rich repeat and Ig domain containing 1 LOAD : Late-onset Alzheimer’s disease LPS : Lipopolysaccharide Malat1 : Metastasis associated lung adenocarcinoma transcript 1 Marcks : Myristoylated alanine rich protein kinase C substrate MG : Microglia miRNA : MicroRNA MPP+ : 1-Methyl-4-phenylpyridinium iodide MT-ATP6 : Mitochondrially encoded ATP synthase membrane subunit 6 MT-CO2 : Mitochondrially encoded cytochrome c oxidase II MT- CO3 : Mitochondrially encoded cytochrome c oxidase III MT-CYB : Mitochondrially encoded cytochrome b MT-ND2 : Mitochondrially encoded NADH:ubiquinone oxidoreductase core subunit 2 MT-ND4 : Mitochondrially encoded NADH:ubiquinone oxidoreductase core subunit 4 NGS : Normal goat serum Npm1 : Nucleophosmin 1 OCR : Oxygen consumption rate OXPHOS : Oxidative phosphorylation P/S : Penicillin Streptomycin solution P2RY12 : Purinergic Receptor P2Y12 PB : Phosphate buffer PBS : Phosphate-buffered saline PC : Positive control PCA : Principal component analysis PCR : Polymerase chain reaction PCT : Patent Cooperation Treaty PDL : Poly-d-lysine PFA : Paraformaldehyde PIEZO1 : Piezo Type Mechanosensitive Ion Channel Component 1 PLL : Poly-L-lysine PSEN1 : Presenilin 1 RIN : RNA integrity number RNA : Ribonucleic acid ROCK : Rho-associated protein kinase inhibitor RPKM : Reads Per Kilobase of transcript per Million mapped reads Rpl22l1 : Ribosomal protein L22 like 1 Rpl35a : Ribosomal protein L35a Rpl5 : Ribosomal protein L5 Rplp2 : Ribosomal protein lateral stalk subunit P2 Rps11 : Ribosomal protein s11 Rpsa : Ribosomal protein SA RT-qPCR : Rreal-time quantitative polymerase chain reaction scRNA-seq : Single-cell RNA sequencing SEM : Standard error of mean Serinc3 : Serine incorporator 3 SNP : Single nucleotide polymorphism snRNA-seq : Single nucleus RNA sequencing SNX6 : Sorting nexin 6 TMEM119 : Transmembrane Protein 119 Tmem173 : Stimulator of interferon response cGAMP interactor 1 TREM2 : Triggering Receptor Expressed On Myeloid Cells 2 UEF : University of Eastern Finland UMAP : Uniform Manifold Approximation and Projection UMI : Unique Molecular Identifier VEH : Vehicle WMA : World Medical Association WT : Wild type [1]: pending:yes