Biology subjects

Maxfield, F. R.

Publications and source records attributed to Maxfield, F. R..

11 recordsLinked to original sources

bioRxiv2026

Aggregated Low-Density Lipoprotein (agLDL) Triggers Inflammatory Responses in Macrophages through Toll-Like Receptor 4 (TLR4)

Atherosclerosis is a chronic inflammatory disease primarily initiated by the sub-endothelial retention and modification of low-density lipoproteins (LDL), such as oxidation and aggregation. While oxidized LDL (oxLDL) exists in atherosclerotic lesions, aggregated lipoproteins are also prominent. Aggregated LDL (agLDL), for instance, is viewed as a passive substrate for macrophages to uptake and form foam cells. Recent studies show that agLDL, through toll-like receptor 4 (TLR4), induces formation of lysosomal synapses, which drive agLDL degradation by macrophages, a process described as "digestive exophagy". As TLR4 is one of the pattern-recognition receptors for inflammatory responses in macrophages, we postulated that agLDL could initiate macrophage inflammation along with lipoprotein degradation/internalization, thereby contributing to a low degree of chronic inflammation in atherosclerosis. Here, we demonstrate that agLDL directly activates the canonical NF-{kappa}B signaling pathway and triggers a pro-inflammatory transcriptional program through TLR4, which closely resembles classical lipopolysaccharide (LPS)-mediated TLR4 activation. In addition, agLDL also activates TLR4 to generate inflammatory macrophages that recruit immune cells, a key process in the development of atherosclerotic lesions. Our findings therefore support a novel dual functionality of agLDL: a lipid donor and an inflammatory agonist. This establishes the agLDL-TLR4-NF-{kappa}B axis as a direct link between LDL aggregation in the intima and the initiation of sterile vascular inflammation. It also strengthens the understanding of the role of digestive exophagy with new mechanistic details.

cell biology↗

bioRxiv2026

Intravital pH Imaging using the sensor ApHID Reveals Incomplete Lysosomal Acidification in Resident Meningeal Phagocytes.

Tracking lysosomes and quantifying their pH in the living, intact brain has been challenging owing to the lack of suitable in vivo tools. Here we report the use of dextran polymers labeled with the pH sensor ApHID to visualize and quantify lysosomal pH in mouse brain resident phagocytes. ApHID-dextrans delivered systemically are rapidly endocytosed by phagocytes in the meninges -mainly dural and subdural macrophages- and accumulate in lysosomal compartments. Intravital pH imaging revealed a striking heterogeneity in lysosomal acidification, with only ~5% of meningeal phagocytes reaching pH values below 5.0, in marked contrast with measurements in macrophage cell cultures. Photothrombotic ischemic lesions induced progressive lysosomal acidification in local phagocytes, while acute chloroquine treatment led to rapid alkalinization. Our methodology enables direct, quantitative pH measurements without genetic manipulations, and unveils a previously unappreciated heterogeneity in lysosomal acidification among brain-resident myeloid cells.

neuroscience↗

bioRxiv2025

The Na V1.5 auxiliary subunit FGF13 modulates channels by regulating membrane cholesterol independent of channel binding

Fibroblast growth factor homologous factors (FHFs) bind to the cytoplasmic carboxy terminus of voltage-gated sodium channels (VGSCs) and modulate channel function. Variants in FHFs or VGSCs perturbing that bimolecular interaction are associated with arrhythmias. Like some channel auxiliary subunits, FHFs exert additional cellular regulatory roles, but whether these alternative roles affect VGSC regulation is unknown. Using a separation-of-function strategy, we show that a structurally guided, binding incompetent mutant FGF13 (the major FHF in mouse heart), confers complete regulation of VGSC steady-state inactivation (SSI), the canonical effect of FHFs. In cardiomyocytes isolated from Fgf13 knockout mice, expression of the mutant FGF13 completely restores wild-type regulation of SSI. FGF13 regulation of SSI derives from effects on local accessible membrane cholesterol, which is unexpectedly polarized and concentrated in cardiomyocytes at the intercalated disc (ID) where most VGSCs localize. Fgf13 knockout eliminates the polarized cholesterol distribution and causes loss of VGSCs from the ID. Moreover, we show that the previously described FGF13-dependent stabilization of VGSC currents at elevated temperatures depends on the cholesterol mechanism. These results provide new insights into how FHFs affect VGSCs and alter the canonical model by which channel auxiliary exert influence.

biophysics↗

bioRxiv2024

A high-content microscopy drug screening platform for regulators of the extracellular digestion of lipoprotein aggregates by macrophages

The recruitment of macrophages to the intima of arteries is a critical event in atherosclerotic progression. These macrophages accumulate excessive lipid droplets and become "foam cells", a hallmark of atherosclerosis. Most studies focus on lipid accumulation through macrophage interaction with modified monomeric low-density lipoprotein (LDL). However, in the intima, macrophages predominantly encounter aggregated LDL (agLDL), an interaction that has been studied far less. Macrophages digest agLDL and generate free cholesterol in an extracellular, acidic, hydrolytic compartment. They form a tight seal around agLDL through actin polymerization and deliver lysosomal contents into this space in a process termed digestive exophagy. There is some evidence that inhibiting digestive exophagy to slow cholesterol accumulation in macrophages protects them from becoming foam cells and slows the progression of atherosclerotic lesions. Thus, understanding the mechanisms of digestive exophagy is critical. Here, we describe a high-content microscopy screen on a library of repurposed drugs for compounds that inhibit lysosome exocytosis during digestive exophagy. We identified many hit compounds and further characterized the effects that five of these compounds have on various aspects of digestive exophagy. In addition, three of the five compounds do not inhibit oxidized LDL-induced foam cell formation, indicating the two pathways to foam cell formation can be targeted independently. We demonstrate that this high-content screening platform has great potential as a drug discovery tool with the ability to effectively and efficiently screen for modulators of digestive exophagy.

cell biology↗

bioRxiv2024

Ratiometric pH Imaging of Macrophage Lysosomes Using the Novel pH-sensitive Dye ApHID.

Lysosomes actively regulate their lumenal pH, which is necessary for optimal enzymatic activity. Endocytic processes are involved in many diseases, including Alzheimers disease, in which sub-optimal lysosomal function has been reported. To measure acidification, pH-sensitive probes can be delivered to endosomes and lysosomes using labeled dextran polymers or proteins. However, many commercially available probes have limited sensitivity in the acidic range of lysosomes, and their fluorescence is subject to enzymatic degradation and photobleaching. Herein, we describe the preparation, characterization, and use of a novel pH-sensitive probe, ApHID, a green-emitting fluorescent dye with optimal dynamic range within the acidity of endosomes and lysosomes. ApHID has a pKa near 5, increasing brightness with acidity, and it is robustly resistant to oxidation and photobleaching. We used ApHID ratiometric imaging to measure lysosomal pH in macrophages, yielding virtually identical results when compared with fluorescein and Oregon Green. Overall, ApHID circumvents limitations presented by most commercially available pH-sensitive probes and can be useful in demanding imaging applications such as intravital imaging of tissues.

biochemistry↗

bioRxiv2022

Screening of small molecule chemical library identifies leads that reduce cholesterol accumulation in human NPC1 fibroblasts

Although considerable progress has been made in finding a therapy for Niemann-Pick type C1 (NPC1) disease, there still is no effective approved treatment. Previously, we reported results from a small molecule screen that was conducted on Chinese Hamster Ovary (CHO) cells (CT60) that lacked NPC1 expression. The hit compounds that were effective in reducing cholesterol accumulation in CT60 cells were not effective on human cells bearing disease-causing mutations in NPC1. Hence, we conducted a new small molecule screen on patient-derived NPC1 skin fibroblasts using a combinatorial library of ~48,000 compounds. We have identified several compounds that corrected the phenotype in human heterozygous mutant NPC1 skin fibroblasts (GM03123) bearing the inactivating I1061T mutation in one allele and another set of inactivating mutations in the other. Among the hit compounds were several histone deacetylase inhibitors, which we have explored further and reported previously. With the determination of the structure of NPC1 protein and the development of computational docking algorithms, it seems worthwhile to show the structures of 128 compounds that reproducibly reduced cholesterol storage in mutant NPC1 fibroblasts but with no known mechanism of action. These compounds were not toxic and specifically effective on mutant cells but not on NPC1-/- cells, indicating on-target effect directly or indirectly.

neuroscience↗

bioRxiv2021

HSP90 inhibitors reduce cholesterol storage in Niemann-Pick type C1 mutant fibroblasts

Niemann Pick type C1 (NPC1) disease is a lysosomal lipid storage disorder caused by mutations of the NPC1 gene. More than 300 disease-associated mutations are reported in patients, resulting in abnormal accumulation of unesterified cholesterol, glycosphingolipids and other lipids in late endosomes and lysosomes (LE/Ly) of many cell types. Previously, we showed that treatment of many different NPC1 mutant fibroblasts with histone deacetylase inhibitors resulted in reduction of cholesterol storage, and we found that this was associated with enhanced exit of the NPC1 protein from the endoplasmic reticulum and delivery to LE/Ly. This suggested that histone deacetylase inhibitors may work through changes in protein chaperones to enhance the folding of NPC1 mutants, allowing them to be delivered to LE/Ly. In this study we evaluated the effect of several HSP90 inhibitors on NPC1I1061T skin fibroblasts. We found that HSP90 inhibition resulted in clearance of cholesterol from LE/Ly, and this was associated with enhanced delivery of the mutant NPC1I1061T protein to LE/Ly. We also observed that inhibition of HSP90 increased the expression of HSP70, and overexpression of HSP70 also reduced cholesterol storage in NPC1I1061T fibroblasts. However, we did not see correction of cholesterol storage by arimoclomol, a drug that is reported to increase HSP70 expression, at doses up to 0.5 mM. These results indicate that manipulation of molecular chaperones may lead to effective treatments for NPC1 disease, but further investigation of mechanisms will be required.

cell biology↗

bioRxiv2019

Cholesterol and matrisome pathways dysregulated in human APOE ε4 glia

Apolipoprotein E (APOE) {varepsilon}4 is the strongest genetic risk factor for Alzheimers disease (AD). Although its association with AD is well-established, the impact of APOE {varepsilon}4 on human brain cell function remains unclear. Here we investigated the effects of APOE {varepsilon}4 on several brain cell types derived from human induced pluripotent stem cells and human APOE targeted replacement mice. Gene set enrichment and pathway analyses of whole transcriptome profiles showed that APOE {varepsilon}4 is associated with dysregulation of cholesterol homeostasis in human but not mouse astrocytes and microglia. Elevated matrisome signaling associated with chemotaxis, glial activation and lipid biosynthesis in APOE {varepsilon}4 mixed neuron/astrocyte cultures parallels altered pathways uncovered in cell-type deconvoluted transcriptomic data from APOE {varepsilon}4 glia and AD post-mortem brains. Experimental validation of the transcriptomic findings showed that isogenic APOE {varepsilon}4 is associated with increased lysosomal cholesterol levels and decreased cholesterol efflux, demonstrating decoupled lipid metabolism. APOE {varepsilon}4 glia also secrete higher levels of proinflammatory chemokines, cytokines and growth factors, indicative of glial activation. Thus, APOE {varepsilon}4 induces human glia-specific dysregulation that may initiate AD risk.

neuroscience↗

bioRxiv2019

Lysosomal enzyme tripeptidyl peptidase 1 plays a role in degradation of beta amyloid fibrils

Alzheimers disease (AD) is characterized by the accumulation of amyloid plaques surrounded by microglia. In cell culture, microglia internalize fibrillar {beta}-amyloid but do not degrade it efficiently. Unactivated microglia have a relatively high lysosomal pH, which impairs the activity of lysosomal proteases. Previous studies showed that activation of microglia with macrophage colony stimulating factor decreases lysosomal pH and enhances fibrillar {beta}-amyloid degradation. We investigated the role of the lysosomal protease tripeptidyl peptidase 1 (TPP1) in cell culture and in a mouse model of Alzheimers disease. Increased levels of TPP1 in unactivated microglia enhanced fibrillar {beta}-amyloid degradation. Conversely, reduction of TPP1 led to decreased fibrillar {beta}-amyloid degradation in activated microglia, macrophages, and other cells that degrade fibrillar {beta}-amyloid efficiently. Reduction of TPP1 in an AD model mouse using a gene-targeted hypomorphic Tpp1 allele increased plaque burden. These results suggest that decreased TPP1 potentiates AD pathogenesis and that strategies to increase TPP1 activity may have therapeutic value. Highlights*In microglia, TPP1 is important for the degradation of fibrillar {beta}-amyloid. *Increased TPP1 in microglia results in enhanced fibrillar {beta}-amyloid degradation. *In an AD mouse model, reduction of TPP1 led to increased amyloid plaque deposition.

cell biology↗

bioRxiv2019

TLR4-dependent signaling drives extracellular catabolism of low-density lipoprotein aggregates

ObjectiveAggregation and modification of low-density lipoproteins (LDL) promotes their retention and accumulation in the arteries. This is a critical initiating factor during atherosclerosis. Macrophage catabolism of aggregated LDL (agLDL) occurs using a specialized extracellular, hydrolytic compartment, the lysosomal synapse (LS). Compartment formation by local actin polymerization and delivery of lysosomal contents by exocytosis promotes acidification of the compartment and degradation of agLDL. Internalization of metabolites such as cholesterol promotes foam cell formation, a process that drives atherogenesis. Further, there is accumulating evidence for the involvement of TLR4 and its adaptor protein MyD88 in atherosclerosis. Here, we investigated the role of TLR4 in catabolism of agLDL using the LS and foam cell formation.\n\nApproach and ResultsUsing bone marrow-derived macrophages (BMMs) from knockout mice, we find that TLR4 and MyD88 regulate compartment formation, lysosome exocytosis, acidification of the compartment and foam cell formation. Using siRNA, pharmacological inhibition and knockout BMMs, we implicate SYK, PI3 kinase and Akt in agLDL catabolism using the LS. Using bone marrow transplantation of LDL receptor knockout mice with TLR4KO bone marrow, we show that deficiency of TLR4 protects macrophages from lipid accumulation during atherosclerosis. Finally, we demonstrate that macrophages in vivo form an extracellular compartment and exocytose lysosome contents similar to that observed in vitro for degradation of agLDL.\n\nConclusionsWe present a mechanism in which interaction of macrophages with agLDL initiates a TLR4 signaling pathway, resulting in formation of the LS, catabolism of agLDL and lipid accumulation in vitro and in vivo.

cell biology↗

bioRxiv2017

A Carbon Nanotube Optical Reporter Maps Endolysosomal Lipid Flux

Lipid accumulation within the lumen of endolysosomal vesicles is observed in various pathologies including atherosclerosis, liver disease, neurological disorders, lysosomal storage disorders, and cancer. Current methods cannot measure lipid flux specifically within the lysosomal lumen of live cells. We developed an optical reporter, composed of a photoluminescent carbon nanotube of a single chirality, which responds to lipid accumulation via modulation of the nanotubes optical bandgap. The engineered nanomaterial, composed of short-single stranded DNA and a single nanotube chirality, localizes exclusively to the lumen of endolysosomal organelles without adversely affecting cell viability or proliferation, or organelle morphology, integrity, or function. The emission wavelength of the reporter can be spatially resolved from within the endolysosomal lumen to generate quantitative maps of lipid content in live cells. Endolysosomal lipid accumulation in cell lines, an example of drug-induced phospholipidosis (DIPL), was observed for multiple drugs in macrophages, and measurements of patient-derived Niemann-Pick type C fibroblasts identified lipid accumulation and phenotypic reversal of this lysosomal storage disease. Single-cell measurements using the reporter discerned sub-cellular differences in equilibrium lipid content, illuminating significant intracellular heterogeneity among endolysosomal organelles of differentiating bone marrow-derived monocytes. Single-cell kinetics of lipoprotein-derived cholesterol accumulation within macrophages revealed rates that differed among cells by an order of magnitude. This carbon nanotube optical reporter of endolysosomal lipid content in live cells confers new capabilities for drug development processes and the investigation of lipid-linked diseases.

bioengineering↗