ABSTRACT
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.
Competing Interest Statement
Malm and Giniatullin are inventors of PCT patent application related to this topic.
LIST OF ABBREVIATIONS
- 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