Journal: Journal of Cell Science

Article Title: Isoform-dependent lysosomal degradation and internalization of apolipoprotein E requires autophagy proteins

doi: 10.1242/jcs.258687

Figure Lengend Snippet: APOE4 alters autophagic flux in in HEK293 cells stably expressing fluorescently tagged APOE. (A,C) HEK293 cells expressing APOE3–mCh or APOE4–mCh or mCh vector were analyzed by western blotting. (B) HEK293 cells stably expressing APOE3–mCh, APOE4–mCh, or mCh vector were treated with Baf and analyzed as for A and C (50 nM 4 h), Rap (10 nM 4 h) or both. Quantitative results are mean±s.e.m. Revert, protein stain; NT, no treatment. * P <0.05, ** P <0.01, **** P <0.0001 (one-way ANOVA with multiple comparisons correction).

Article Snippet: APOE3–Myc–Flag was purchased from Origene (catalog number RC200395).

Techniques: Stable Transfection, Expressing, Plasmid Preparation, Western Blot, Staining

Journal: Journal of Cell Science

Article Title: Isoform-dependent lysosomal degradation and internalization of apolipoprotein E requires autophagy proteins

doi: 10.1242/jcs.258687

Figure Lengend Snippet: APOE is turned over by autophagy in HEK293 cells with stable APOE expression. Representative images and fluorescence intensity of APOE3–mCh, APOE4–mCh, or mCh cells treated with Baf (50 nM 4 h), Epox (100 nM), or MG132 (50 µM). Quantitative results are mean±s.e.m. Bars over graphs indicate time points at which P <0.05 on two-way ANOVA with post-hoc Dunnett test.

Article Snippet: APOE3–Myc–Flag was purchased from Origene (catalog number RC200395).

Techniques: Expressing, Fluorescence

Journal: Journal of Cell Science

Article Title: Isoform-dependent lysosomal degradation and internalization of apolipoprotein E requires autophagy proteins

doi: 10.1242/jcs.258687

Figure Lengend Snippet: APOE4 colocalizes with enlarged lysosomes. (A) HEK293 cells stably expressing APOE3– or APOE4–mCh (red) and transfected with CellLight Lamp1–GFP (green), and stained with DAPI (blue). (B) HEK293 cells stably expressing APOE3– or APOE4–mCh–SepH. SepH shown in green. (C) HEK293 cells expressing APOE2, APOE3, APOE4 or vector were treated with oleic acid (OA, overnight) and Baf (4 h). NT, no treatment. Quantitative results are mean±s.e.m. In A–C, three wells were imaged per APOE isoform (three images per well) by confocal microscopy. Images were analyzed using Imaris software. Quantitative results are mean±s.e.m. * P <0.05, ** P <0.01, *** P <0.001, **** P <0.0001 [Student's two-tailed unpaired t -test (A,B) or one-way ANOVA with a post-hoc Tukey–Kramer test (C)].

Article Snippet: APOE3–Myc–Flag was purchased from Origene (catalog number RC200395).

Techniques: Stable Transfection, Expressing, Transfection, Staining, Plasmid Preparation, Confocal Microscopy, Software, Two Tailed Test

Journal: Journal of Cell Science

Article Title: Isoform-dependent lysosomal degradation and internalization of apolipoprotein E requires autophagy proteins

doi: 10.1242/jcs.258687

Figure Lengend Snippet: APOE transiently overexpressed in ST14A cells is degraded by LAMP2A-dependent autophagy. (A) Schematic of dual-tag fluorescent APOE with quenching of green SepHluorin in lysosomes. (B) APOE3–mCh–SepH and mCh–SepH tag fluorescence intensity in ST14A cells with Baf (50 nM, 4 h). (C) APOE3 mRNA in ST14A cells expressing APOE3–Myc–flag with Baf treatment. (D) APOE3–Myc–Flag abundance in ST14A cells following Baf treatment (4 h 50 nM). (E) APOE3–mCh–SepH fluorescence intensity following BFA (5 μg/ml) treatment. (F) ST14A cells expressing APOE3-myc-flag and treated with BFA (5 μg/ml, 4 h) were analyzed by western blotting. Quantitative results are mean±s.e.m. Revert, protein stain; NT, no treatment. * P <0.05, *** P <0.001 [Student's unpaired two-tailed t -test was used (D); bars above graphs indicate time points at which FDR<0.05 by two-way ANOVA with post-hoc Tukey–Kramer test (B,E)].

Article Snippet: APOE3–Myc–Flag was purchased from Origene (catalog number RC200395).

Techniques: Fluorescence, Expressing, Western Blot, Staining, Two Tailed Test

Journal: Journal of Cell Science

Article Title: Isoform-dependent lysosomal degradation and internalization of apolipoprotein E requires autophagy proteins

doi: 10.1242/jcs.258687

Figure Lengend Snippet: LAMP2A is required for autophagy of APOE3 in ST14A cells. (A,B) ST14A cells were co-transfected with shRNA targeting LAMP2A and (A) APOE3–Myc–Flag or (B) APOE3–mCh, and APOE3 levels were assessed by western blot or fluorescence intensity. shCtrl, control shRNA. Bars above graph in B indicates time points at which FDR<0.05 by two-way ANOVA. (C) APOE levels in mouse brain tissue from 2-year-old wild-type and LAMP2 knockout mice. Quantitative results are mean±s.e.m. * P <0.05, ** P <0.01, **** P <0.0001 [Student's two-tailed unpaired t -test (A,C)].

Article Snippet: APOE3–Myc–Flag was purchased from Origene (catalog number RC200395).

Techniques: Transfection, shRNA, Western Blot, Fluorescence, Knock-Out, Two Tailed Test

Journal: Journal of Cell Science

Article Title: Isoform-dependent lysosomal degradation and internalization of apolipoprotein E requires autophagy proteins

doi: 10.1242/jcs.258687

Figure Lengend Snippet: Fluorescently tagged APOE is endocytosed in an isoform-dependent manner and alters endosomal morphology. (A,B) APOE–mCh-conditioned medium was collected from HEK293T cells and applied to (A) HepG2 and (B) ST14A cells. Cells were imaged and red fluorescence/phase area calculated. Bars represent times when FDR<0.05 between APOE isoforms by two-way ANOVA. (C) ST14A cells were treated for 24 h with conditioned medium with APOE3–mCh or APOE4–mCh (red), and immunocytochemistry for EEA1 or Rab7 (green) was performed, and cells stained with DAPI (blue). Cells were imaged by confocal microscopy and analyzed using Imaris image. Magnified view indicates enlarged images from white boxes in merge panel. (D) HepG2 cells were treated for 24 h with APOE3–mCh or APOE4–mCh conditioned medium, lysosomes were immunoprecipitated and proteomic contents analyzed by mass spectrometry. Proteins reduced in APOE4 lysosomes that were not reduced in mCh-treated cells included mitochondrial proteins such as prohibitin. Western blot analysis for prohibitin was performed on lyso-depleted flow-through. Revert, protein stain. HepG2 cells treated with APOE-mCh conditioned medium were also analyzed by qPCR. Quantitative results are mean±s.e.m. * P <0.05, ** P <0.01, *** P <0.001; ns, not significant (one-way ANOVA with Tukey–Kramer test).

Article Snippet: APOE3–Myc–Flag was purchased from Origene (catalog number RC200395).

Techniques: Fluorescence, Immunocytochemistry, Staining, Confocal Microscopy, Immunoprecipitation, Mass Spectrometry, Western Blot

Journal: Journal of Cell Science

Article Title: Isoform-dependent lysosomal degradation and internalization of apolipoprotein E requires autophagy proteins

doi: 10.1242/jcs.258687

Figure Lengend Snippet: Knockdown of Rubicon or ATG7 reduces APOE internalization. (A,B) ATG7 and Rubicon were knocked down using siRNA in HepG2 cells, and (A) conditioned medium with APOE3–mCh or (B) medium containing LDL-pHrodo was applied. siCtrl, control siRNA. (C,D) HepG2 cells with siRNA knockdown of (C) ATG7 or (D) Rubicon were analyzed by qPCR. (E) HepG2 cells were treated with inhibitors and APOE3–mCh conditioned medium. Concentrations of inhibitors used were: 50 nM Bafilomycin A1, 20 mM ammonium chloride. NT, no treatment. Cells were imaged and fluorescence quantified by incucyte. Imaging and fluorescence quantification by incucyte. Quantitative results are mean±s.e.m. **** P <0.0001 [Student's two-tailed unpaired t -test (C,D); bars represent times when FDR<0.05 between APOE isoforms by two-way ANOVA with a post-hoc Dunnett's test (A,B,E)]

Article Snippet: APOE3–Myc–Flag was purchased from Origene (catalog number RC200395).

Techniques: Fluorescence, Imaging, Two Tailed Test

Journal: bioRxiv

Article Title: APOE traffics to astrocyte lipid droplets and modulates triglyceride saturation and droplet size

doi: 10.1101/2023.04.28.538740

Figure Lengend Snippet: ( A ) Cartoon schematic of the FPP assay to test the topology of fluorescently-tagged proteins in cells. Cells are treated with 30 µM digitonin for 1 minute, which selectively permeabilizes the plasma membrane but not the ER. After permeabilization, cells are treated with 50 µg/mL proteinase K, which enters the permeabilized plasma membrane and degrades all cytoplasmic-facing fluorophores (green). Because the ER membrane is not permeabilized, proteinase K does not enter into the ER lumen and ER lumen-facing fluorophores are retained (red). (B) Representative confocal slices of FPP performed on primary cortical rat astrocytes transiently transfected with APOE3-mEm, the ER marker TagBFP2-KDEL, and labelled for LDs with BODIPY 665/676. After digitonin permeabilization and proteinase K treatment, APOE signal on the surface of LDs is lost, but the luminal ER marker fluorescence is retained. A Gaussian filter with a radius of 1 pixel was applied to all images to improve visibility for print. Scale bars: 10 µm (left), 5 µm for zoom (right). (C) For APOE3 as well as two representative LD proteins, PLIN2 and LiveDrop, the fluorescence intensity ratio was calculated by dividing the mean fluorescence intensity after both permeabilization and proteinase K treatment by the mean fluorescence intensity after permeabilization. The ER ratio was calculated by measuring the whole cell intensity of the ER fluorescence at the two timepoints. The LD ratio was calculated by measuring the fluorescence on the surface of BODIPY-labelled LDs at the two timepoints. Ratios close to 1 indicate minimal loss of signal after proteinase K treatment, as observed with the ER marker TagBFP2-KDEL. Lower ratios indicate loss of fluorescence upon proteinase K treatment. N = 8-18 cells per condition, collected from three independent experiment. *p<0.05, **** p<0.0001. Dig., 30 µM digitonin. PK, +50 µg/mL Proteinase K. P-values were calculated via the Wilcoxon rank sum test and Bonferonni-corrected for multiple comparisons.

Article Snippet: A plasmid containing human APOE3-TurboGFP was purchased from Origene (Cat# RG200395).

Techniques: Membrane, Transfection, Marker, Fluorescence

Journal: bioRxiv

Article Title: APOE traffics to astrocyte lipid droplets and modulates triglyceride saturation and droplet size

doi: 10.1101/2023.04.28.538740

Figure Lengend Snippet: ( A ) Representative frames from fast Airyscan movies showing the localization of LD-associated APOE relative to the ER after 4 hours of treatment with 400 µM OA in TRAE3-H cells. Cells were transfected with APOE3-mEm and the ER marker TagBFP2-KDEL and labelled for LDs with BODIPY 665/676. In the merged images, the ER is in magenta and APOE is in green. The yellow lines across the merged images indicate the line of pixels used to create the linescan graphs to the right of the images. In the linescan graphs, the relative fluorescence intensity of BODIPY-labelled LDs is in cyan, APOE3-mEm is green, and the ER is magenta. Two different localization patterns were observed: “half rings”, in which APOE partially covers the LD surface and colocalizes with the ER and “full rings”, where APOE fully encloses the surface of the LD and only partially colocalizes with the ER. Scale bars, 500 nm. (B) Immunogold electron micrographs of endogenous APOE localization at membrane contact sites between the ER and LDs in TRAE3-H cells treated with 400 µM OA for 5 hours. The blue arrow points to a direct membrane contact between the ER and an LD. Yellow arrows mark APOE localized to the cytoplasmic face of the ER membrane. The red arrow marks APOE localized to the cytoplasmic surface of the LD. Scale bars, 200 nm. (C) Representative frames from confocal FRAP movies of APOE3-Em on the surface of BODIPY 665/676 labelled LDs in primary rat cortical astrocytes during an OA pulse (200 µM OA for 4 hours) or an OA pulse-chase (200 µM OA for 4 hours followed by 2 hours chase in complete media – OA). APOE fluorescence was bleached at the 0 s timepoint. Scale bar, 1 µm. (D) Normalized intensity of APOE signal within the bleach ROI over time, with t = 0 sec denoting the time at which APOE was bleached. The bold center line is the mean normalized intensity, and the upper and lower bounds of the ribbon represent ± standard deviation (SD). N = 28 cells per condition, collected from three independent experiments. (E) Comparison of the rate constant of recovery k between OA pulse and pulse-chase conditions. The rate constant was derived by fitting each recovery curve to the equation y = C (1 – e - k t ). N = 28 cells per condition, collected from three independent experiments. * p<0.05 (F) Comparison of the mobile fraction between OA pulse and pulse-chase conditions. The mobile fraction was derived by fitting each recovery curve to the equation y = C (1 – e - k t ), where C is equal to the asymptote of the curve i.e. the mobile fraction. N = 28 cells per condition, collected from three independent experiments. **** p<0.0001. P-values were calculated via the Wilcoxon rank-sum test. (G) Schematic illustrating interpretation of the results of the FRAP experiment. When APOE on the LD is bleached during the OA pulse, it recovers very rapidly with a high mobile fraction. This indicates that bleached APOE on the LD is rapidly exchanged for unbleached APOE. After a short washout, LD-associated APOE recovers slowly or not at all, indicating that unbleached APOE molecules are unable to replace bleached ones on the LD. We hypothesize LD-APOE exchanges with APOE on the cytoplasmic face of the ER via membrane bridges during OA loading. These bridges are reduced or lost after OA washout, preventing exchange of APOE between LDs and the ER.

Article Snippet: A plasmid containing human APOE3-TurboGFP was purchased from Origene (Cat# RG200395).

Techniques: Transfection, Marker, Fluorescence, Membrane, Pulse Chase, Standard Deviation, Comparison, Derivative Assay

Journal: bioRxiv

Article Title: APOE traffics to astrocyte lipid droplets and modulates triglyceride saturation and droplet size

doi: 10.1101/2023.04.28.538740

Figure Lengend Snippet: ( A ) Cartoon illustrating the conditions used in the APOE rescue experiment. Endogenous APOE3 protein is present in cells transfected with a non-targeting siRNA. APOE protein is depleted upon APOE knockdown. The siRNA used to knock down APOE targets the mRNA sequence encoding the N-terminal signal peptide. The RNAi-resistant full-length APOE (RR FL) rescue construct consists of APOE3 with synonymous mutations in the signal peptide that impart resistance to the APOE siRNA. The LD-only APOE construct has the signal sequence removed, making it insensitive to the APOE siRNA , and replaced with the hairpin domain of the LD protein GPAT4. This version of APOE only targets LDs and never enters the ER lumen. (B) Western blot of lysates of TRAE3-H cells transfected with the indicated siRNA and transduced with the indicated lentivirus. The same samples were run on two separate SDS-PAGE gels, with 20 µg of total protein loaded into each well. Gels were transferred onto nitrocellulose membranes at the same time and then blotted with anti-HA or anti-APOE antibody together with an anti-tubulin antibody. Both the RR FL and LD-only APOE constructs are expressed in an endogenous APOE knockdown background. Moreover, the HA tag obstructs the epitope of the APOE antibody, allowing endogenous APOE and exogenous, HA-tagged APOE to be distinguished. (C) Representative confocal slices of cells transfected with non-targeting siRNA or APOE siRNA and transduced with an empty vector control, RR FL APOE, or LD-only APOE. Cells were subjected to an OA pulse-chase as described in 5A, fixed, and stained for LDs with BODIPY 493/507. Scale bar, 10 µm. (D-F) Quantification of LD parameters the conditions described in (A) after an OA pulse-chase assay. (D) Total LD area was measured as the area of the entire LD mask per cell in µm 2 . (E) Average LD size was calculated as the mean LD area per cell in µm 2 . (F) Number of LDs per cell. Each data point represents one cell, and each color represents data collected from a separate, independent experiment. N = 60 cells per condition and independent experiment. ns p>0.05, **** p<0.0001. P-values were calculated using a clustered Wilcox rank sum test via the Rosner-Glynn-Lee method and Bonferonni-corrected for multiple comparisons.

Article Snippet: A plasmid containing human APOE3-TurboGFP was purchased from Origene (Cat# RG200395).

Techniques: Transfection, Knockdown, Sequencing, Construct, Western Blot, Transduction, SDS Page, Plasmid Preparation, Control, Pulse Chase, Staining