scholarly journals TREM2 knockout, but not the R47H Alzheimer’s variant, reduces neural phagocytosis and survival of human iPSC-derived macrophages

2020 ◽  
Author(s):  
Hazel Hall-Roberts ◽  
Juliane Obst ◽  
Thomas B. Smith ◽  
Elena Di Daniel ◽  
Emma Mead ◽  
...  
Keyword(s):  
Inflammatory Cytokine ◽  
Induced Pluripotent Stem Cell ◽  
Surface Expression ◽  
Cell Surface Receptor ◽  
Calcium Flux ◽  
Loss Of Function ◽  
Human Microglia ◽  
Surface Receptor ◽  
Induced Pluripotent ◽  
Human Ipsc

Abstract BACKGROUND: TREM2 is a microglial cell surface receptor, with risk mutations linked to Alzheimer’s disease (AD), including R47H. Binding of ligands to TREM2 triggers Syk-dependent signalling through the DAP12 co-receptor, leading to phagocytosis, survival, and changes to microglial activation state. In biochemical assays, R47H impairs TREM2 binding to phosphatidylserine, a lipid “eat-me” signal exposed by apoptotic neurons. The effect of R47H TREM2 upon phagocytosis of apoptotic neurons by human microglia has not yet been reported. METHODS: We generated human microglia-like iPSC-macrophages (pMac) from isogenic induced pluripotent stem cell (iPSC) lines with homozygous R47H mutation or TREM2 knockout (KO). To assess microglial phenotypic function in the mutants, we measured: (1) pro-inflammatory cytokine responses by ELISA; (2) survival after growth factor-withdrawal; (3) phagocytosis by novel high-content imaging assays, using two neuron-derived cargoes that expose phosphatidylserine (fixed SH-SY5Ys and synaptosomes). Downstream signalling resulting from TREM2 activation was additionally assessed by assaying Syk phosphorylation and calcium flux. RESULTS: We demonstrated that TREM2 KO strongly diminished both pMac survival and neural phagocytosis, while having little impact on inflammatory cytokine response. R47H TREM2 modified surface expression and shedding of TREM2, but did not impair TREM2-mediated signalling, survival, or phagocytosis. CONCLUSIONS: Under healthy conditions in culture, the R47H mutation is not sufficient to cause defects in phagocytosis or survival of human pMac, unlike more severe T66M or W50C TREM2 loss-of-function mutations. We hypothesise that R47H TREM2 affects other microglia phenotypes yet to be explored, and/or that pathogenic manifestation requires other stressors relating to neurodegenerative disease.

scholarly journals TREM2 Alzheimer’s variant R47H causes similar transcriptional dysregulation to knockout, yet only subtle functional phenotypes in human iPSC-derived macrophages

2020 ◽  
Vol 12 (1) ◽  
Author(s):  
Hazel Hall-Roberts ◽  
Devika Agarwal ◽  
Juliane Obst ◽  
Thomas B. Smith ◽  
Jimena Monzón-Sandoval ◽  
...  
Keyword(s):  
Gene Expression ◽  
Microglial Activation ◽  
Induced Pluripotent Stem Cell ◽  
Surface Expression ◽  
Culture Conditions ◽  
Cell Surface Receptor ◽  
Loss Of Function ◽  
Human Microglia ◽  
Surface Receptor ◽  
Induced Pluripotent

Abstract Background TREM2 is a microglial cell surface receptor, with risk mutations linked to Alzheimer’s disease (AD), including R47H. TREM2 signalling via SYK aids phagocytosis, chemotaxis, survival, and changes to microglial activation state. In AD mouse models, knockout (KO) of TREM2 impairs microglial clustering around amyloid and prevents microglial activation. The R47H mutation is proposed to reduce TREM2 ligand binding. We investigated cell phenotypes of the R47H mutant and TREM2 KO in a model of human microglia, and compared their transcriptional signatures, to determine the mechanism by which R47H TREM2 disrupts function. Methods We generated human microglia-like iPSC-macrophages (pMac) from isogenic induced pluripotent stem cell (iPSC) lines, with homozygous R47H mutation or TREM2 knockout (KO). We firstly validated the effect of the R47H mutant on TREM2 surface and subcellular localization in pMac. To assess microglial phenotypic function, we measured phagocytosis of dead neurons, cell morphology, directed migration, survival, and LPS-induced inflammation. We performed bulk RNA-seq, comparing significant differentially expressed genes (DEGs; p < 0.05) between the R47H and KO versus WT, and bioinformatically predicted potential upstream regulators of TREM2-mediated gene expression. Results R47H modified surface expression and shedding of TREM2, but did not impair TREM2-mediated signalling, or gross phenotypes that were dysregulated in the TREM2 KO (phagocytosis, motility, survival). However, altered gene expression in the R47H TREM2 pMac overlapped by 90% with the TREM2 KO and was characterised by dysregulation of genes involved with immunity, proliferation, activation, chemotaxis, and adhesion. Downregulated mediators of ECM adhesion included the vitronectin receptor αVβ3, and consequently, R47H TREM2 pMac adhered weakly to vitronectin compared with WT pMac. To counteract these transcriptional defects, we investigated TGFβ1, as a candidate upstream regulator. TGFβ1 failed to rescue vitronectin adhesion of pMac, although it improved αVβ3 expression. Conclusions The R47H mutation is not sufficient to cause gross phenotypic defects of human pMac under standard culture conditions. However, overlapping transcriptional defects with TREM2 KO supports the hypothesised partial loss-of-function effects of the R47H mutation. Furthermore, transcriptomics can guide us to more subtle phenotypic defects in the R47H cells, such as reduced cell adhesion, and can be used to predict targets for therapeutic intervention.

scholarly journals Robust Expression of Functional NMDA Receptors in Human Induced Pluripotent Stem Cell-Derived Neuronal Cultures Using an Accelerated Protocol

2021 ◽  
Vol 14 ◽  
Author(s):  
Jacob B. Ruden ◽  
Mrinalini Dixit ◽  
José C. Zepeda ◽  
Brad A. Grueter ◽  
Laura L. Dugan
Keyword(s):  
Nmda Receptors ◽  
Neural Progenitor Cells ◽  
Induced Pluripotent Stem Cell ◽  
Calcium Flux ◽  
Neuronal Cultures ◽  
Mature Neurons ◽  
Health And Disease ◽  
Nervous System Function ◽  
Induced Pluripotent ◽  
Human Ipsc

N-methyl-D-aspartate (NMDA) receptors are critical for higher-order nervous system function, but in previously published protocols to convert human induced pluripotent stem cells (iPSCs) to mature neurons, functional NMDA receptors (NMDARs) are often either not reported or take an extended time to develop. Here, we describe a protocol to convert human iPSC-derived neural progenitor cells (NPCs) to mature neurons in only 37 days. We demonstrate that the mature neurons express functional NMDARs exhibiting ligand-activated calcium flux, and we document the presence of NMDAR-mediated electrically evoked postsynaptic current. In addition to being more rapid than previous procedures, our protocol is straightforward, does not produce organoids which are difficult to image, and does not involve co-culture with rodent astrocytes. This could enhance our ability to study primate/human-specific aspects of NMDAR function and signaling in health and disease.

scholarly journals MEK1/2 activity modulates TREM2 cell surface recruitment

2020 ◽  
pp. jbc.RA120.014352
Author(s):  
Jason Schapansky ◽  
Yelena Y Grinberg ◽  
David M Osiecki ◽  
Emily A Freeman ◽  
Stephen G Walker ◽  
...  
Keyword(s):  
Cell Surface ◽  
Myeloid Cells ◽  
Surface Expression ◽  
Mek Inhibitor ◽  
Therapeutic Benefit ◽  
Cell Surface Receptor ◽  
Cell Surface Expression ◽  
Loss Of Function ◽  
Downstream Effector ◽  
Surface Receptor

Rare sequence variants in the microglial cell surface receptor TREM2 have been shown to increase the risk for Alzheimer’s disease (AD). Disease-linked TREM2 mutations seem to confer a partial loss of function, and increasing TREM2 cell surface expression and thereby its function(s) might have therapeutic benefit in AD. However, druggable targets that could modulate microglial TREM2 surface expression are not known. To identify such targets, we conducted a screen of small molecule compounds with known pharmacology using human myeloid cells, searching for those that enhance TREM2 protein at the cell surface Inhibitors of the kinases MEK1/2 displayed the strongest and most consistent increases in cell surface TREM2 protein, identifying a previously unreported pathway for TREM2 regulation. Unexpectedly, inhibitors of the downstream effector ERK kinases did not have the same effect, suggesting that non-canonical MEK signaling regulates TREM2 trafficking. In addition, siRNA knockdown experiments confirmed that decreased MEK1 and MEK2 was required for this recruitment. In iPSC-derived microglia, MEK inhibition increased cell surface TREM2 only modestly, so various cytokines were used to alter iPSC microglia phenotype, making cells more sensitive to MEK inhibitor-induced TREM2 recruitment. Of those tested, only IFN-gamma priming prior to MEK inhibitor treatment resulted in greater TREM2 recruitment. These data identify the first known mechanisms for increasing surface TREM2 protein and TREM2-regulated function in human myeloid cells, and are the first to show a role for MEK1/MEK2 signaling in TREM2 activity.

scholarly journals TREM2 regulates purinergic receptor-mediated calcium signaling and motility in human iPSC-derived microglia

2021 ◽  
Author(s):  
Amit Jairaman ◽  
Amanda McQuade ◽  
You Jung Kang ◽  
Alberto Granzotto ◽  
Jean Paul Chadarevian ◽  
...  
Keyword(s):  
Stem Cell ◽  
Pluripotent Stem Cell ◽  
Purinergic Receptor ◽  
Induced Pluripotent Stem Cell ◽  
Loss Of Function ◽  
Increased Risk ◽  
P2y12 Receptor Antagonist ◽  
Genetic Deletion ◽  
Induced Pluripotent ◽  
Human Ipsc

The membrane protein TREM2 (Triggering Receptor Expressed on Myeloid cells 2) regulates key microglial functions including phagocytosis and chemotaxis. Loss?of?function variants of TREM2 are associated with increased risk of Alzheimer's disease (AD). Because abnormalities in Ca2+ signaling have been observed in several AD models, we investigated TREM2 regulation of Ca2+ signaling via genetic deletion in human induced pluripotent stem cell-derived microglia (iPSC microglia). We found that iPSC-microglia lacking TREM2 (TREM2 KO) show exaggerated Ca2+ signals in response to purinergic agonists such as ADP that shape microglial injury responses. This ADP hypersensitivity, driven by increased expression of P2Y12 and P2Y13 receptors, results in sustained Ca2+ influx and alters cell motility and process extension in TREM2 KO microglia. In a 'Ca2+ clamp' assay using iPSC-microglia expressing the genetically encoded Ca2+ probe, Salsa6f, we found that cytosolic Ca2+ tunes motility to a greater extent in TREM2 KO microglia. Despite showing greater overall displacement, TREM2 KO microglia exhibit reduced directional chemotaxis along ADP gradients. Accordingly, the chemotactic defect in TREM2 KO microglia was rescued by reducing cytosolic Ca2+ using a P2Y12 receptor antagonist. Our results show that loss of TREM2 confers a defect in microglial Ca2+ response to purinergic signals, suggesting a window of Ca2+ signaling for optimal microglial motility.

scholarly journals A high-throughput screen for TMPRSS2 expression identifies FDA-approved compounds that can limit SARS-CoV-2 entry

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Yanwen Chen ◽  
Travis B. Lear ◽  
John W. Evankovich ◽  
Mads B. Larsen ◽  
Bo Lin ◽  
...  
Keyword(s):  
Rational Design ◽  
Surface Expression ◽  
In Vitro Models ◽  
Lung Epithelial Cells ◽  
Cell Surface Receptor ◽  
Pharmacokinetic Data ◽  
Drug Induced ◽  
Global Pandemic ◽  
Surface Receptor

AbstractSARS-CoV-2 (2019-nCoV) is the pathogenic coronavirus responsible for the global pandemic of COVID-19 disease. The Spike (S) protein of SARS-CoV-2 attaches to host lung epithelial cells through the cell surface receptor ACE2, a process dependent on host proteases including TMPRSS2. Here, we identify small molecules that reduce surface expression of TMPRSS2 using a library of 2,560 FDA-approved or current clinical trial compounds. We identify homoharringtonine and halofuginone as the most attractive agents, reducing endogenous TMPRSS2 expression at sub-micromolar concentrations. These effects appear to be mediated by a drug-induced alteration in TMPRSS2 protein stability. We further demonstrate that halofuginone modulates TMPRSS2 levels through proteasomal-mediated degradation that involves the E3 ubiquitin ligase component DDB1- and CUL4-associated factor 1 (DCAF1). Finally, cells exposed to homoharringtonine and halofuginone, at concentrations of drug known to be achievable in human plasma, demonstrate marked resistance to SARS-CoV-2 infection in both live and pseudoviral in vitro models. Given the safety and pharmacokinetic data already available for the compounds identified in our screen, these results should help expedite the rational design of human clinical trials designed to combat active COVID-19 infection.

scholarly journals Contribution of two-pore K+ channels to cardiac ventricular action potential revealed using human iPSC-derived cardiomyocytes

2017 ◽  
Vol 312 (6) ◽  
pp. H1144-H1153 ◽  
Author(s):  
Sam Chai ◽  
Xiaoping Wan ◽  
Drew M. Nassal ◽  
Haiyan Liu ◽  
Christine S. Moravec ◽  
...  
Keyword(s):  
Action Potential ◽  
Expression Profile ◽  
Cardiac Electrophysiology ◽  
Human Heart ◽  
Induced Pluripotent Stem Cell ◽  
Heart Tissue ◽  
Induced Pluripotent ◽  
Interfering Rna ◽  
Human Ipsc ◽  
Physiological Systems

Two-pore K+ (K2p) channels have been described in modulating background conductance as leak channels in different physiological systems. In the heart, the expression of K2p channels is heterogeneous with equivocation regarding their functional role. Our objective was to determine the K2p expression profile and their physiological and pathophysiological contribution to cardiac electrophysiology. Induced pluripotent stem cells (iPSCs) generated from humans were differentiated into cardiomyocytes (iPSC-CMs). mRNA was isolated from these cells, commercial iPSC-CM (iCells), control human heart ventricular tissue (cHVT), and ischemic (iHF) and nonischemic heart failure tissues (niHF). We detected 10 K2p channels in the heart. Comparing quantitative PCR expression of K2p channels between human heart tissue and iPSC-CMs revealed K2p1.1, K2p2.1, K2p5.1, and K2p17.1 to be higher expressed in cHVT, whereas K2p3.1 and K2p13.1 were higher in iPSC-CMs. Notably, K2p17.1 was significantly lower in niHF tissues compared with cHVT. Action potential recordings in iCells after K2p small interfering RNA knockdown revealed prolongations in action potential depolarization at 90% repolarization for K2p2.1, K2p3.1, K2p6.1, and K2p17.1. Here, we report the expression level of 10 human K2p channels in iPSC-CMs and how they compared with cHVT. Importantly, our functional electrophysiological data in human iPSC-CMs revealed a prominent role in cardiac ventricular repolarization for four of these channels. Finally, we also identified K2p17.1 as significantly reduced in niHF tissues and K2p4.1 as reduced in niHF compared with iHF. Thus, we advance the notion that K2p channels are emerging as novel players in cardiac ventricular electrophysiology that could also be remodeled in cardiac pathology and therefore contribute to arrhythmias. NEW & NOTEWORTHY Two-pore K+ (K2p) channels are traditionally regarded as merely background leak channels in myriad physiological systems. Here, we describe the expression profile of K2p channels in human-induced pluripotent stem cell-derived cardiomyocytes and outline a salient role in cardiac repolarization and pathology for multiple K2p channels.

scholarly journals Establishment of a Human Induced Pluripotent Stem Cell-Derived Neuromuscular Co-Culture Under Optogenetic Control

2020 ◽  
Author(s):  
Elliot W. Swartz ◽  
Greg Shintani ◽  
Jijun Wan ◽  
Joseph S. Maffei ◽  
Sarah H. Wang ◽  
...  
Keyword(s):  
Motor Neurons ◽  
Induced Pluripotent Stem Cell ◽  
Cell Types ◽  
Calcium Flux ◽  
Electrode Arrays ◽  
Spinal Motor Neurons ◽  
Culture Model ◽  
Skeletal Myotubes ◽  
Induced Pluripotent

SummaryThe failure of the neuromuscular junction (NMJ) is a key component of degenerative neuromuscular disease, yet how NMJs degenerate in disease is unclear. Human induced pluripotent stem cells (hiPSCs) offer the ability to model disease via differentiation toward affected cell types, however, the re-creation of an in vitro neuromuscular system has proven challenging. Here we present a scalable, all-hiPSC-derived co-culture system composed of independently derived spinal motor neurons (MNs) and skeletal myotubes (sKM). In a model of C9orf72-associated disease, co-cultures form functional NMJs that can be manipulated through optical stimulation, eliciting muscle contraction and measurable calcium flux in innervated sKM. Furthermore, co-cultures grown on multi-electrode arrays (MEAs) permit the pharmacological interrogation of neuromuscular physiology. Utilization of this co-culture model as a tunable, patient-derived system may offer significant insights into NMJ formation, maturation, repair, or pathogenic mechanisms that underlie NMJ dysfunction in disease.

scholarly journals Lithium inhibits tryptophan catabolism via the inflammation-induced kynurenine pathway in human microglia

2020 ◽  
Author(s):  
Ria Göttert ◽  
Pawel Fidzinski ◽  
Larissa Kraus ◽  
Ulf Christoph Schneider ◽  
Martin Holtkamp ◽  
...  
Keyword(s):  
Chromatin Immunoprecipitation ◽  
Pluripotent Stem Cell ◽  
Induced Pluripotent Stem Cell ◽  
Kynurenine Pathway ◽  
Mrna Transcription ◽  
Human Microglia ◽  
Tryptophan Catabolism ◽  
Induced Pluripotent ◽  
Sb 216763 ◽  
Gsk3 Inhibitor

SummaryActivation of the kynurenine pathway may lead to depletion of the serotonin precursor tryptophan, which has been implicated in the neurobiology of depression. This study describes a mechanism whereby lithium inhibits inflammatory tryptophan breakdown. Upon activation, immortalized human microglia showed a robust increase in indoleamine-2,3-dioxygenase (IDO1) mRNA transcription, IDO1 protein expression, and activity. Further, chromatin immunoprecipitation verified enriched binding of both STAT1 and STAT3 to the IDO1 promoter. Lithium counteracted these effects, increasing inhibitory GSK3βS9 phosphorylation and reducing STAT1S727 and STAT3Y705 phosphorylation levels in activated cells. Experiments in primary human microglia and human induced pluripotent stem cell (hiPSC)-derived microglia corroborated lithium’s effects. Moreover, IDO activity was reduced by GSK3 inhibitor SB-216763 and STAT inhibitor nifuroxazide via downregulation of P-STAT1S727 and P-STAT3Y705. Our study demonstrates that lithium inhibits the inflammatory kynurenine pathway in the microglia compartment of the human brain.

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