ABSTRACT
GRN mutations cause frontotemporal dementia (GRN-FTD) due to deficiency in progranulin (PGRN), a lysosomal and secreted protein with unclear function. Here, we found that Grn-/- mice exhibit a global deficiency in bis(monoacylglycero)phosphate (BMP), an endolysosomal phospholipid we identified as a pH-dependent PGRN interactor as well as a redox-sensitive enhancer of lysosomal proteolysis and lipolysis. Grn-/- brains also showed an age-dependent, secondary storage of glucocerebrosidase substrate glucosylsphingosine. We investigated a protein replacement strategy by engineering protein transport vehicle (PTV):PGRN-a recombinant protein linking PGRN to a modified Fc domain that binds human transferrin receptor for enhanced CNS biodistribution. PTV:PGRN rescued various Grn-/- phenotypes in primary murine macrophages and human iPSC-derived microglia, including oxidative stress, lysosomal dysfunction, and endomembrane damage. Peripherally delivered PTV:PGRN corrected levels of BMP, glucosylsphingosine, and disease pathology in Grn-/- CNS, including microgliosis, lipofuscinosis, and neuronal damage. PTV:PGRN thus represents a potential biotherapeutic for GRN-FTD.
Subject(s)
Biological Products/therapeutic use , Brain/metabolism , Lysosomal Storage Diseases/therapy , Progranulins/therapeutic use , Animals , Bone Morphogenetic Proteins/metabolism , Endosomes/metabolism , Female , Frontotemporal Dementia/blood , Frontotemporal Dementia/cerebrospinal fluid , Gliosis/complications , Gliosis/pathology , Humans , Induced Pluripotent Stem Cells/metabolism , Inflammation/pathology , Lipid Metabolism , Lipofuscin/metabolism , Lysosomes/metabolism , Macrophages/metabolism , Male , Membrane Glycoproteins/metabolism , Mice , Mice, Inbred C57BL , Mice, Transgenic , Microglia/metabolism , Nerve Degeneration/pathology , Phenotype , Progranulins/deficiency , Progranulins/metabolism , Receptors, Immunologic/metabolism , Receptors, Transferrin/metabolism , Tissue DistributionABSTRACT
Haploinsufficiency of the progranulin (PGRN)-encoding gene (GRN) causes frontotemporal lobar degeneration (GRN-FTLD) and results in microglial hyperactivation, TREM2 activation, lysosomal dysfunction, and TDP-43 deposition. To understand the contribution of microglial hyperactivation to pathology, we used genetic and pharmacological approaches to suppress TREM2-dependent transition of microglia from a homeostatic to a disease-associated state. Trem2 deficiency in Grn KO mice reduced microglia hyperactivation. To explore antibody-mediated pharmacological modulation of TREM2-dependent microglial states, we identified antagonistic TREM2 antibodies. Treatment of macrophages from GRN-FTLD patients with these antibodies led to reduced TREM2 signaling due to its enhanced shedding. Furthermore, TREM2 antibody-treated PGRN-deficient microglia derived from human-induced pluripotent stem cells showed reduced microglial hyperactivation, TREM2 signaling, and phagocytic activity, but lysosomal dysfunction was not rescued. Similarly, lysosomal dysfunction, lipid dysregulation, and glucose hypometabolism of Grn KO mice were not rescued by TREM2 ablation. Synaptic loss and neurofilament light-chain (NfL) levels, a biomarker for neurodegeneration, were further elevated in the Grn/Trem2 KO cerebrospinal fluid (CSF). These findings suggest that TREM2-dependent microglia hyperactivation in models of GRN deficiency does not promote neurotoxicity, but rather neuroprotection.
Subject(s)
Frontotemporal Lobar Degeneration/pathology , Membrane Glycoproteins/metabolism , Microglia/physiology , Monocytes/metabolism , Progranulins/deficiency , Receptors, Immunologic/metabolism , Animals , Antibodies/immunology , Antibodies/pharmacology , Brain/diagnostic imaging , Brain/physiopathology , Disease Models, Animal , Female , Frontotemporal Lobar Degeneration/metabolism , Humans , Lysosomes/metabolism , Lysosomes/pathology , Male , Membrane Glycoproteins/genetics , Membrane Glycoproteins/immunology , Mice, Inbred C57BL , Mice, Knockout , Microglia/drug effects , Monocytes/drug effects , Receptors, Immunologic/genetics , Receptors, Immunologic/immunology , Syk Kinase/metabolismABSTRACT
Heteromeric interactions between the catalytically impaired human epidermal growth factor receptor (HER3/ERBB3) and its catalytically active homologs EGFR and HER2 are essential for their signaling. Different ligands can activate these receptor pairs but lead to divergent signaling outcomes through mechanisms that remain largely unknown. We used stochastic optical reconstruction microscopy (STORM) with pair-correlation analysis to show that EGF and neuregulin (NRG) can induce different extents of HER3 clustering that are dependent on the nature of the coexpressed HER receptor. We found that the presence of these clusters correlated with distinct patterns and mechanisms of receptor phosphorylation. NRG induction of HER3 phosphorylation depended on the formation of the asymmetric kinase dimer with EGFR in the absence of detectable higher-order oligomers. Upon EGF stimulation, HER3 paralleled previously observed EGFR behavior and formed large clusters within which HER3 was phosphorylated via a noncanonical mechanism. HER3 phosphorylation by HER2 in the presence of NRG proceeded through still another mechanism and involved the formation of clusters within which receptor phosphorylation depended on asymmetric kinase dimerization. Our results demonstrate that the higher-order organization of HER receptors is an essential feature of their ligand-induced behavior and plays an essential role in lateral cross-activation of the receptors. We also show that HER receptor ligands exert unique effects on signaling by modulating this behavior.
Subject(s)
Epidermal Growth Factor/metabolism , ErbB Receptors/metabolism , Neuregulin-1/metabolism , Receptor, ErbB-3/metabolism , Animals , Cell Line , Epidermal Growth Factor/pharmacology , ErbB Receptors/genetics , HEK293 Cells , Humans , Mice , Microscopy/methods , Molecular Imaging/methods , Neuregulin-1/pharmacology , Phosphorylation/drug effects , Protein Multimerization , Receptor, ErbB-2/genetics , Receptor, ErbB-2/metabolism , Receptor, ErbB-3/geneticsABSTRACT
Based on their structural similarity to previously described compound AMG 009, indole-phenyl acetic acids were proposed to be potent dual inhibitors of chemoattractant receptor-homologous molecule expressed on Th2 cells (CRTH2 or DP2) and prostanoid D receptor (DP or DP1). This series was equipotent to AMG 009 in binding assays against both receptors but exhibited decreased serum shift. We discovered early in the optimization of these indole-phenylacetic acid compounds that they demonstrated CYP3A4 time-dependent inhibition (TDI). Hypothesizing that the source of TDI was the indole core we modified the 1,2,3-substitution to eventually afford a highly potent modulator of CRTH2 and DP which did not exhibit TDI.
Subject(s)
Cytochrome P-450 CYP3A/metabolism , Enzyme Inhibitors/pharmacology , Indoles/pharmacology , Phenylacetates/pharmacology , Receptors, Immunologic/antagonists & inhibitors , Receptors, Prostaglandin/antagonists & inhibitors , Dose-Response Relationship, Drug , Enzyme Inhibitors/chemical synthesis , Enzyme Inhibitors/chemistry , Humans , Indoles/chemistry , Molecular Structure , Phenylacetates/chemistry , Structure-Activity Relationship , Time FactorsABSTRACT
Membrane fusion consists of a complex rearrangement of lipids and proteins that results in the merger of two lipid bilayers. We have developed a model system that employs synthetic DNA-lipid conjugates as a surrogate for the membrane proteins involved in the biological fusion reaction. We previously showed that complementary DNA-lipids, inserted into small unilamellar vesicles, can mediate membrane fusion in bulk. Here, we use a model membrane architecture developed in our lab to directly observe single-vesicle fusion events using fluorescence microscopy. In this system, a planar tethered membrane patch serves as the target membrane for incoming vesicles. This allows us to quantify the kinetics and characteristics of individual fusion events from the perspective of the lipids or the DNA-lipids involved in the process. We find that the fusion pathways are heterogeneous, with an arrested hemi-fusion state predominating, and we quantitate the outcome and rate of fusion events to construct a mechanistic model of DNA-mediated vesicle fusion. The waiting times between docking and fusion are distributed exponentially, suggesting that fusion occurs in a single step. Our analysis indicates that when two lipid bilayers are brought into close proximity, fusion occurs spontaneously, with little or no dependence on the number of DNA hybrids formed.
Subject(s)
DNA/metabolism , Membrane Fusion , Unilamellar Liposomes/metabolism , Cholesterol/chemistry , DNA/chemistry , Lipid Bilayers/chemistry , Lipid Bilayers/metabolism , Phosphatidylcholines/chemistry , Phosphatidylethanolamines/chemistry , Unilamellar Liposomes/chemistryABSTRACT
Loss-of-function variants of TREM2 are associated with increased risk of Alzheimer's disease (AD), suggesting that activation of this innate immune receptor may be a useful therapeutic strategy. Here we describe a high-affinity human TREM2-activating antibody engineered with a monovalent transferrin receptor (TfR) binding site, termed antibody transport vehicle (ATV), to facilitate blood-brain barrier transcytosis. Upon peripheral delivery in mice, ATV:TREM2 showed improved brain biodistribution and enhanced signaling compared to a standard anti-TREM2 antibody. In human induced pluripotent stem cell (iPSC)-derived microglia, ATV:TREM2 induced proliferation and improved mitochondrial metabolism. Single-cell RNA sequencing and morphometry revealed that ATV:TREM2 shifted microglia to metabolically responsive states, which were distinct from those induced by amyloid pathology. In an AD mouse model, ATV:TREM2 boosted brain microglial activity and glucose metabolism. Thus, ATV:TREM2 represents a promising approach to improve microglial function and treat brain hypometabolism found in patients with AD.
Subject(s)
Alzheimer Disease , Induced Pluripotent Stem Cells , Humans , Animals , Mice , Microglia , Blood-Brain Barrier , Tissue Distribution , Antibodies , Brain , Disease Models, Animal , Membrane Glycoproteins , Receptors, Immunologic/geneticsABSTRACT
Our first generation CRTH2 and DP dual antagonists, represented by AMG 009, are more potent toward the CRTH2 receptor than to the DP receptor. Here we report our efforts in the discovery of CRTH2 and DP dual antagonists with more balanced potencies to both receptors, such as compound 15.
Subject(s)
Drug Design , Phenylacetates/chemical synthesis , Receptors, Immunologic/antagonists & inhibitors , Receptors, Prostaglandin/antagonists & inhibitors , HEK293 Cells , Humans , Inhibitory Concentration 50 , Molecular Structure , Phenylacetates/chemistry , Phenylacetates/pharmacology , Protein Binding/drug effectsABSTRACT
Synthetic lipid-oligonucleotide conjugates inserted into lipid vesicles mediate fusion when one population of vesicles displays the 5'-coupled conjugate and the other the 3'-coupled conjugate, so that anti-parallel hybridization allows the membrane surfaces to come into close proximity. Improved assays show that lipid mixing proceeds more quickly and to a much greater extent than content mixing, suggesting the latter is rate limiting. To test the effect of membrane-membrane spacing on fusion, a series of conjugates was constructed by adding 2-24 noncomplementary bases at the membrane-proximal ends of two complementary sequences. Increasing linker lengths generally resulted in progressively reduced rates and extents of lipid and content mixing, in contrast to higher vesicle docking rates. The relatively flexible, single-stranded DNA linker facilitates docking but allows greater spacing between the vesicles after docking, thus making the transition into fusion less probable, but not preventing it altogether. These experiments demonstrate the utility of DNA as a model system for fusion proteins, where sequence can easily be modified to systematically probe the effect of distance between bilayers in the fusion reaction.
Subject(s)
DNA/metabolism , Lipids/chemistry , Membrane Fusion , Oligonucleotides/metabolism , Base Sequence , DNA/genetics , Lipid Bilayers/metabolism , Models, Biological , Molecular Sequence Data , Oligonucleotides/genetics , Organophosphorus Compounds , SNARE Proteins/metabolismABSTRACT
Synaptic transmission is achieved by exocytosis of small, synaptic vesicles containing neurotransmitters across the plasma membrane. Here, we use a DNA-tethered freestanding bilayer as a target architecture that allows observation of content transfer of individual vesicles across the tethered planar bilayer. Tethering and fusion are mediated by hybridization of complementary DNA-lipid conjugates inserted into the two membranes, and content transfer is monitored by the dequenching of an aqueous content dye. By analyzing the diffusion profile of the aqueous dye after vesicle fusion, we are able to distinguish content transfer across the tethered bilayer patch from vesicle leakage above the patch.
Subject(s)
Lipid Bilayers/metabolism , Membrane Fusion , Unilamellar Liposomes/metabolism , Biological Transport , Coloring Agents/metabolism , Spectrometry, Fluorescence , Water/metabolismABSTRACT
Specific membrane interactions such as lipid vesicle docking and fusion can be mediated by synthetic DNA-lipid conjugates as a model for the protein-driven processes that are ubiquitous in biological systems. Here we present a method of tethering vesicles to a supported lipid bilayer that allows the simultaneous deposition of cognate vesicle partners displaying complementary DNA, resulting in well-mixed populations of tethered vesicles that are laterally mobile. Vesicles are covalently attached to a supporting lipid bilayer using a DNA-templated click reaction; then DNA-mediated interactions between tethered vesicles are triggered by spiking the salt concentration. These interactions, such as docking and fusion, can then be observed for individual vesicles as they collide on the surface. The architecture of this new system also permits control over the number of lipid anchors that tether the vesicle to the supporting bilayer. The diffusion coefficient of tethered vesicles anchored by two lipids is approximately 1.6-fold slower than that of vesicles anchored only with a single lipid, consistent with a simple physical model.
Subject(s)
DNA/chemistry , Lipid Bilayers , Chromatography, High Pressure Liquid , Spectrometry, Mass, Matrix-Assisted Laser Desorption-IonizationABSTRACT
Human genetic data indicate that microglial dysfunction contributes to the pathology of Alzheimer's disease (AD), exemplified by the identification of coding variants in triggering receptor expressed on myeloid cells 2 (TREM2) and, more recently, in PLCG2, a phospholipase-encoding gene expressed in microglia. Although studies in mouse models have implicated specific Trem2-dependent microglial functions in AD, the underlying molecular mechanisms and translatability to human disease remain poorly defined. In this study, we used genetically engineered human induced pluripotent stem cell-derived microglia-like cells to show that TREM2 signals through PLCγ2 to mediate cell survival, phagocytosis, processing of neuronal debris, and lipid metabolism. Loss of TREM2 or PLCγ2 signaling leads to a shared signature of transcriptional dysregulation that underlies these phenotypes. Independent of TREM2, PLCγ2 also signals downstream of Toll-like receptors to mediate inflammatory responses. Therefore, PLCγ2 activity regulates divergent microglial functions via distinct TREM2-dependent and -independent signaling and might be involved in the transition to a microglial state associated with neurodegenerative disease.
Subject(s)
Inflammation/metabolism , Membrane Glycoproteins/metabolism , Microglia/metabolism , Phospholipase C gamma/metabolism , Receptors, Immunologic/metabolism , Signal Transduction/physiology , Animals , Cell Survival/physiology , Humans , Induced Pluripotent Stem Cells/metabolism , Membrane Glycoproteins/genetics , Mice , Mice, Knockout , Neurons/metabolism , Phagocytosis/physiology , Phospholipase C gamma/genetics , Receptors, Immunologic/geneticsABSTRACT
Loss-of-function (LOF) variants of TREM2, an immune receptor expressed in microglia, increase Alzheimer's disease risk. TREM2 senses lipids and mediates myelin phagocytosis, but its role in microglial lipid metabolism is unknown. Combining chronic demyelination paradigms and cell sorting with RNA sequencing and lipidomics, we find that wild-type microglia acquire a disease-associated transcriptional state, while TREM2-deficient microglia remain largely homeostatic, leading to neuronal damage. TREM2-deficient microglia phagocytose myelin debris but fail to clear myelin cholesterol, resulting in cholesteryl ester (CE) accumulation. CE increase is also observed in APOE-deficient glial cells, reflecting impaired brain cholesterol transport. This finding replicates in myelin-treated TREM2-deficient murine macrophages and human iPSC-derived microglia, where it is rescued by an ACAT1 inhibitor and LXR agonist. Our studies identify TREM2 as a key transcriptional regulator of cholesterol transport and metabolism under conditions of chronic myelin phagocytic activity, as TREM2 LOF causes pathogenic lipid accumulation in microglia.
Subject(s)
Brain/metabolism , Cholesterol/metabolism , Macrophages/metabolism , Membrane Glycoproteins/genetics , Microglia/metabolism , Myelin Sheath/metabolism , Phagocytosis/genetics , Receptors, Immunologic/genetics , Acetyl-CoA C-Acetyltransferase/antagonists & inhibitors , Alzheimer Disease/genetics , Alzheimer Disease/metabolism , Animals , Cholesterol Esters/metabolism , Disease Models, Animal , Flow Cytometry , Humans , Induced Pluripotent Stem Cells , Lipid Metabolism/genetics , Lipidomics , Liver X Receptors/agonists , Mice , Mice, Knockout , Mice, Knockout, ApoE , RNA-SeqABSTRACT
(R)-N-{1-[3-(4-Ethoxy-phenyl)-4-oxo-3,4-dihydro-pyrido[2,3-d]-pyrimidin-2-yl]-ethyl}-N-pyridin-3-yl-methyl-2-(4-trifluoromethoxyphenyl)-acetamide (AMG 487) is a potent and selective orally bioavailable chemokine (C-X-C motif) receptor 3 (CXCR3) antagonist that displays dose- and time-dependent pharmacokinetics in human subjects after multiple oral dosing. Although AMG 487 exhibited linear pharmacokinetics on both days 1 and 7 at the 25-mg dose, dose- and time-dependent kinetics were evident at the two higher doses. Nonlinear kinetics were more pronounced after multiple dosing. Area under the plasma concentration-time curve from 0 to 24 h [AUC((0-24 h))] increased 96-fold with a 10-fold increase in dose on day 7 compared with a 28-fold increase in AUC((0-24 h)) on day 1. These changes were correlated with time- and dose-dependent decreases in the metabolite to parent plasma concentrations, suggesting that these changes result from a decrease in the oral clearance (CL) of AMG 487 (e.g., intestinal/hepatic first-pass metabolism and systemic CL). The biotransformation of AMG 487 is dependent on CYP3A and results in the formation of two primary metabolites, a pyridyl N-oxide AMG 487 (M1) and an O-deethylated AMG 487 (M2). One of these metabolites, M2, undergoes further metabolism by CYP3A. M2 has also been demonstrated to inhibit CYP3A in a competitive (K(i)=0.75 microM) manner as well as via mechanism-based inhibition (unbound K(I)=1.4 microM, k(inact)=0.041 min(-1)). Data from this study implicate M2-mediated CYP3A mechanism-based inhibition as the proximal cause for the time-dependent pharmacokinetics of AMG 487. However, the sequential metabolism of M2, nonlinear AMG 487 pharmacokinetics, and the inability to accurately determine the role of intestinal AMG 487 metabolism complicates the correlation between M2 plasma concentrations and the time-dependent AMG 487 pharmacokinetic changes.
Subject(s)
Acetamides/pharmacokinetics , Pyrimidinones/pharmacokinetics , Receptors, CXCR3/antagonists & inhibitors , Acetamides/administration & dosage , Adult , Area Under Curve , Chromatography, Liquid , Cohort Studies , Cytochrome P-450 Enzyme Inhibitors , Drug Administration Schedule , Humans , Male , Pyrimidinones/administration & dosage , Tandem Mass SpectrometryABSTRACT
Growth factor binding to EGFR drives conformational changes that promote homodimerization and transphosphorylation, followed by adaptor recruitment, oligomerization, and signaling through Ras. Whether specific receptor conformations and oligomerization states are necessary for efficient activation of Ras is unclear. We therefore evaluated the sufficiency of a phosphorylated EGFR dimer to activate Ras without growth factor by developing a chemical-genetic strategy to crosslink and "trap" full-length EGFR homodimers on cells. Trapped dimers become phosphorylated and recruit adaptor proteins at stoichiometry equivalent to that of EGF-stimulated receptors. Surprisingly, these phosphorylated dimers do not activate Ras, Erk, or Akt. In the absence of EGF, phosphorylated dimers do not further oligomerize or reorganize on cell membranes. These results suggest that a phosphorylated EGFR dimer loaded with core signaling adapters is not sufficient to activate Ras and that EGFR ligands contribute to conformational changes or receptor dynamics necessary for oligomerization and efficient signal propagation through the SOS-Ras-MAPK pathway.
Subject(s)
ErbB Receptors/metabolism , Protein Multimerization , ras Proteins/metabolism , Clathrin-Coated Vesicles/drug effects , Clathrin-Coated Vesicles/metabolism , Cross-Linking Reagents/chemistry , Cross-Linking Reagents/metabolism , Epidermal Growth Factor/pharmacology , ErbB Receptors/chemistry , HEK293 Cells , Humans , Ligands , Phosphorylation/drug effects , Phosphotyrosine/metabolism , Protein Conformation , Signal Transduction/drug effectsABSTRACT
The synthesis of @-tide beta-strand peptidomimetics has been improved such that oligomers now can be obtained from solution- and solid-phase synthesis protocols approaching the efficiency and flexibility of peptide chemistry. These methods enable the synthesis of @-tide oligomers with a variety of amino acids and with lengths up to 13 units. [reaction: see text]
Subject(s)
Combinatorial Chemistry Techniques , Oligopeptides/chemical synthesis , Molecular MimicryABSTRACT
Prostaglandin D2 (PGD2) plays a key role in mediating allergic reactions seen in asthma, allergic rhinitis, and atopic dermatitis. PGD2 exerts its activity through two G protein-coupled receptors (GPCRs), prostanoid D receptor (DP or DP1), and chemoattractant receptor-homologous molecule expressed on Th2 cells (CRTH2 or DP2). We report the optimization of a series of phenylacetic acid derivatives in an effort to improve the dual activity of AMG 009 against DP and CRTH2. These efforts led to the discovery of AMG 853 (2-(4-(4-(tert-butylcarbamoyl)-2-(2-chloro-4-cyclopropylphenyl sulfonamido)phenoxy)-5-chloro-2-fluorophenyl)acetic acid), which is being evaluated in human clinical trials for asthma.
ABSTRACT
A general method for synthesizing 5(')- and 3(')-coupled DNA-lipid conjugates has been developed and employed in DNA-mediated vesicle fusion. Vesicles presenting complementary DNA fuse, resulting in both outer and inner leaflet mixing as well as content mixing. Fusion is maximized using 5(')- and 3(')-coupled DNA on opposite vesicle partners, rather than only 5(')-coupled DNA, showing the importance of DNA orientation to the process. Lipid and content mixing assays show a dependence of fusion kinetics on the sequence and average number of DNA per vesicle. Vesicles without DNA or presenting noncomplementary sequences also appear to undergo some degree of lipid mixing or exchange, but no content mixing. Total lipid mixing appears to occur more efficiently than inner leaflet mixing and content mixing, and this may be explained by the observed nonspecific lipid mixing and/or the rise of a hemifused intermediate. The ability to control DNA sequence and the relative experimental simplicity of this system make it highly attractive to probe fundamental questions of membrane fusion using both ensemble and single vesicle assays.