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. 2019 Sep;370(3):823-833.
doi: 10.1124/jpet.119.257345. Epub 2019 May 17.

δ-Tocopherol Effect on Endocytosis and Its Combination with Enzyme Replacement Therapy for Lysosomal Disorders: A New Type of Drug Interaction?

Rachel L Manthe  1 , Jeffrey A Rappaport  1 , Yan Long  1 , Melani Solomon  1 , Vinay Veluvolu  1 , Michael Hildreth  1 , Dencho Gugutkov  1 , Juan Marugan  1 , Wei Zheng  1 , Silvia Muro  2
Affiliations

δ-Tocopherol Effect on Endocytosis and Its Combination with Enzyme Replacement Therapy for Lysosomal Disorders: A New Type of Drug Interaction?

Rachel L Manthe et al. J Pharmacol Exp Ther. 2019 Sep.

Abstract

Induction of lysosomal exocytosis alleviates lysosomal storage of undigested metabolites in cell models of lysosomal disorders (LDs). However, whether this strategy affects other vesicular compartments, e.g., those involved in endocytosis, is unknown. This is important both to predict side effects and to use this strategy in combination with therapies that require endocytosis for intracellular delivery, such as lysosomal enzyme replacement therapy (ERT). We investigated this using δ-tocopherol as a model previously shown to induce lysosomal exocytosis and cell models of type A Niemann-Pick disease, a LD characterized by acid sphingomyelinase (ASM) deficiency and sphingomyelin storage. δ-Tocopherol and derivative CF3-T reduced net accumulation of fluid phase, ligands, and polymer particles via phagocytic, caveolae-, clathrin-, and cell adhesion molecule (CAM)-mediated pathways, yet the latter route was less affected due to receptor overexpression. In agreement, δ-tocopherol lowered uptake of recombinant ASM by deficient cells (known to occur via the clathrin pathway) and via _targeting intercellular adhesion molecule-1 (associated to the CAM pathway). However, the net enzyme activity delivered and lysosomal storage attenuation were greater via the latter route. Data suggest stimulation of exocytosis by tocopherols is not specific of lysosomes and affects endocytic cargo. However, this effect was transient and became unnoticeable several hours after tocopherol removal. Therefore, induction of exocytosis in combination with therapies requiring endocytic uptake, such as ERT, may represent a new type of drug interaction, yet this strategy could be valuable if properly timed for minimal interference.

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Figures

Fig. 1.
Fig. 1.
Effect of tocopherols on sphingomyelin storage and lysosomal exocytosis in diseased endothelial cells. (A) Micrograph quantification of the perinuclear lysosomal staining of sphingomyelin using fluorescent lysenin, in control (Ctrl) vs. imipramine-diseased endothelial cells (Dis), prior or after 48-hour incubation with 40 µM δ-tocopherol (δ-Toc) or 20 µM CF3-T. Data are normalized to untreated control cells (horizontal dashed line). (B) Fluorimetric quantification of the enzymatic activity of lysosomal β-hexosaminidase in the cell medium (exocytosed) or the cell fraction (lysosomal) upon treatment of imipramine-diseased endothelial cells with δ-Toc, as in (A). Control endothelial cells treated or not with 10 µM ionomycin to cause lysosomal secretion are shown for a comparison (A and B). Data are mean ± S.E.M. (n ≥ 4 independent wells). *Comparison with untreated control cells; †comparison with untreated diseased cells (P < 0.05 by Student’s t test).
Fig. 2.
Fig. 2.
Effect of tocopherols on bulk fluid-phase uptake in diseased endothelial cells. (A) Fluorescence microscopy of control (Ctrl) and imipramine-diseased endothelial cells (Dis) incubated for 1-hour pulse with Texas Red dextran, 1 hour after treating cells for 48 hours with 40 µM δ-tocopherol or 20 µM CF3-T. Dotted lines mark the cell borders, observed by phase-contrast microscopy. Scale bar, 10 µm. (B) Dextran uptake was quantified per cell and normalized to untreated diseased cells (horizontal solid line). Data are mean ± S.E.M. (n ≥ 4 independent wells). *Comparison with untreated control cells; †comparison with untreated diseased cells (P < 0.05 by Student’s t test).
Fig. 3.
Fig. 3.
Effect of δ-tocopherol on receptor-mediated uptake in diseased endothelial cells. (A) Microscopy quantification of the uptake (2-hour) of fluorescent ligands of individual endocytic pathways in imipramine-diseased endothelial cells (Dis) vs. control (Ctrl) cells. (B) Uptake of fluorescent ligands (3-hour) in imiprimine-diseased cells treated for 48 hours with 40 µM δ-tocopherol under noninflammatory or (C) inflammatory-like conditions (overnight incubation with TNFα). Ligands were CTB (caveolae-mediated endocytosis), Tf (clathrin-mediated endocytosis), 200 nm polymer nanocarriers _targeted to ICAM-1 (anti-ICAM NCs; CAM-mediated endocytosis), and 1 µm IgG-coated microparticles (phagocytosis; Phag.). (A–C) After ligand incubation, cells were washed and fixed, and cell surface–bound ligands were immunostained with antibodies fluorescently labeled in a different color to distinguish internalized vs. surface-bound localization (see Materials and Methods). Data are mean ± S.E.M. (n ≥ 4 independent wells), normalized to conditions shown in the horizontal solid lines. *Comparison with untreated diseased cells; †comparison with untreated diseased cells; #comparison with the CAM pathway; $comparison with the clathrin pathway (P < 0.05 by Student’s t test).
Fig. 4.
Fig. 4.
δ-Tocopherol modulation of the TNFα effect on endocytosis in diseased endothelial cells. Imipramine-diseased endothelial cells (Dis) treated for 48 hours with 40 µM δ-tocopherol were left quiescent or activated overnight with TNFα to mimic inflammation. Cells were then incubated for 3 hours with (A) fluorescent anti-ICAM NCs or (B) fluorescent ligands of all individual endocytic pathways described in Fig. 3. Cells were washed and fixed, and cell surface ligands were fluorescently immunostained in a different color to distinguish internalized vs. surface-bound counterparts. (A) Binding was quantified as the total number of cell-associated fluorescent NCs, of which also the percentage of NCs internalized was measured. Data were normalized to untreated diseased cells (horizontal solid line). (B) Uptake of fluorescent ligands in TNFα-activated cells was normalized to nonactivated cells (horizontal solid line). (A and B) Data are mean ± S.E.M. (n ≥ 4 independent wells). *Comparison with untreated diseased cells; †comparison with nonactivated cells (P < 0.05 by Student’s t test).
Fig. 5.
Fig. 5.
δ-Tocopherol reduction of the number of EEA-1–positive compartments in diseased cells. Imipramine-diseased endothelial cells and NPD-A patient fibroblasts were left untreated (Ctr) or were treated with 40 µM δ-tocopherol for 15 minutes, 1 hour, or 3 hours. Cells were washed, fixed, and permeabilized, and early endosomes were labeled using anti–EEA-1, followed by secondary antibody conjugated to green Alexa Fluor 488. (A) Fluorescence microscopy images of endothelial cells, shown as an example. Scale bar, 10 µm. (B) Number of EEA-1–positive compartments per cell compared with absence of δ-tocopherol (Ctr), expressed as a percentage. Data are mean ± S.E.M. (n ≥ 4 independent wells). *Comparison with Ctrl cells (P < 0.05 by Student’s t test).
Fig. 6.
Fig. 6.
Effect of δ-tocopherol on the activity provided by recombinant ASM to diseased cells. (A) ASM activity in wild-type (Ctrl) or NPD-A patient fibroblasts (Dis) incubated for 4 hours in the absence vs. presence of 2.3 µg/ml recombinant ASM. (B) ASM activity in healthy (Ctrl) vs. imipramine-diseased endothelial cells or NPD-A fibroblasts (Dis) after incubation for 4 hours with 2.3 µg/ml recombinant ASM, which was added after treatment with 40 µM δ-tocopherol. Data are normalized to (A) wild-type fibroblasts without ASM addition and to (B) untreated cells (respective horizontal dashed lines). (C) NPD-A fibroblasts treated for 48 hours with 40 µM δ-tocopherol and labeled with fluorescent sphingomyelin were washed and incubated with 5 µg/ml recombinant ASM for 4 hours, after which the sphingomyelin levels were measured in a plate reader. Data show sphingomyelin levels as a percentage of that found in cells not incubated with ASM. All data are mean ± S.E.M. (n ≥ 4 independent wells). (A and B) *Comparison with wild-type fibroblasts without ASM addition; †comparison with untreated cells; (C) *comparison with untreated cells (all P < 0.05 by Student’s t test).
Fig. 7.
Fig. 7.
Effect of δ-tocopherol on uptake of recombinant ASM via the clathrin vs. CAM pathways. Imipramine-diseased endothelial cells (Dis), activated overnight with TNFα and treated for 48 hours with 40 µM δ-tocopherol, were incubated for 2 hours with 2.1 µg/ml naked 125I-ASM or 125I-ASM coupled to anti-ICAM NCs (anti-ICAM/ASM NCs). After washing cells, an acid glycine solution was used to elute noninternalized ASM from the cell surface. ASM delivered into cells was then measured in the cell lysates. (A) Internalized ASM in cell lysates. (B) ASM uptake after treatment with δ-tocopherol as a percentage of untreated diseased cells (horizontal solid line). Data are mean ± S.E.M. (n ≥ 4 independent wells). *Comparison with untreated diseased cells; †comparison with naked ASM (P < 0.05 by Student’s t test).
Fig. 8.
Fig. 8.
Kinetics of tocopherol effect on bulk fluid-phase uptake and sphingomyelin levels. (A) Control (Ctrl) and imipramine-diseased endothelial cells (Dis) were incubated with Texas Red dextran (1 hour pulse) 1 to 8 hours after a 48-hour treatment with 40 µM δ-tocopherol or 20 µM CF3-T. Dextran uptake was normalized to untreated diseased cells (horizontal solid line). Dextran uptake by untreated control cells is shown by the horizontal dashed line. (B) Sphingomyelin was stained with fluorescent lysenin 1 to 24 hours after a 48-hour treatment with δ-tocopherol or CF3-T, quantified as described in Fig. 1, and normalized to untreated control cells (horizontal dashed line). Sphingomyelin levels in untreated diseased cells are shown by the horizontal solid line. Data are mean ± S.E.M. (n ≥ 4 independent wells). *Comparison with untreated control cells; †comparison with untreated diseased cells (P < 0.05 by Student’s t test).
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