Journal: Chemistry & biology

Article Title: Discovery of oxysterol-derived pharmacological chaperones for NPC1: implication for the existence of second sterol-binding site.

doi: 10.1016/j.chembiol.2013.02.009

Figure Lengend Snippet: Figure 2. Identification of Sterol Derivatives with Greater Potency by Means of Chemical Optimization (A) Structures of representative sterol derivatives used in this article. (B) NPC1I1061T colocalization assay, showing dose-response curves for selected oxysterols and sterol derivatives. The extent of colocalization of the NPC1 mutant and LAMP1 was quantified as described in Experimental Procedures, and the data points represent the averages (n = 10) with SE depicted by error bars. (C) Representative images of the experiments in (B). Calibration bar represents 20 mm. See also Figure S1A.

Article Snippet: For immunoblotting of endogenous NPC1 protein, rabbit anti-NPC1 polyclonal antibody (Novus Biologicals) combined with HRP-conjugated anti-rabbit antibody (R&D Systems) was used.

Techniques: Mutagenesis

Journal: Chemistry & biology

Article Title: Discovery of oxysterol-derived pharmacological chaperones for NPC1: implication for the existence of second sterol-binding site.

doi: 10.1016/j.chembiol.2013.02.009

Figure Lengend Snippet: Figure 3. Effect of Oxysterol Derivatives on Steady-State Expression Level and Maturation Status of NPC1I1061T Mutant (A) The steady-state expression level of FLAG-NPC1-GFP (WT or I1061T) was quantified by measuring GFP fluorescence in the lysate with or without oxysterol derivatives. The GFP fluorescence was normalized with respect to total protein concentration. Data points represent the averages (n = 3) with SD depicted by error bars. (B) Acquisition of EndoH resistance upon treatment with 25HC and its derivative. Cells stably expressing either WT or I1061T version of FLAG-NPC1-GFP were treated as indicated for 24 hr and lysed. The lysates were digested with EndoH and immunoprecipitated with anti-FLAG beads. The immunoprecipitated proteins were subjected to western blot analysis (immunoblotted with anti-FLAG antibody).

Article Snippet: For immunoblotting of endogenous NPC1 protein, rabbit anti-NPC1 polyclonal antibody (Novus Biologicals) combined with HRP-conjugated anti-rabbit antibody (R&D Systems) was used.

Techniques: Expressing, Mutagenesis, Protein Concentration, Stable Transfection, Immunoprecipitation, Western Blot

Journal: Chemistry & biology

Article Title: Discovery of oxysterol-derived pharmacological chaperones for NPC1: implication for the existence of second sterol-binding site.

doi: 10.1016/j.chembiol.2013.02.009

Figure Lengend Snippet: Figure 5. Functional Rescue of Patient- Derived Fibroblasts (A) Comparison of the expression levels and band patterns of endogenous WT (HEK293) and I1061T NPC1 proteins (NPC fibroblast). The filled arrow- head indicates the mature form, and the open arrowhead indicates the immature form. (B) Effects of 25HC and mo56HC on expression level and band pattern of endogenous NPC1I1061T. NPC fibroblasts were treated with the indicated compound for 48 hr and processed for western blot analysis using anti-NPC1 antibody. (C) Effect of other sterol derivatives on expression level and band pattern of I1061T mutant. To facil- itate comparison between the compounds, the concentrations normalized with their EC50s are also shown. (D) Alleviation of intracellular cholesterol accumu- lation by oxysterol derivative. NPC fibroblasts were cultured in the presence of the indicated compound for 48 hr, and processed for filipin staining. Calibration bar represents 100 mm. The intracellular cholesterol accumulation was quanti- fied as described in Experimental Procedures. Error bar represents SD (n = 12). See also Figure S2.

Article Snippet: For immunoblotting of endogenous NPC1 protein, rabbit anti-NPC1 polyclonal antibody (Novus Biologicals) combined with HRP-conjugated anti-rabbit antibody (R&D Systems) was used.

Techniques: Functional Assay, Derivative Assay, Comparison, Expressing, Western Blot, Mutagenesis, Cell Culture, Staining

Journal: Chemistry & biology

Article Title: Discovery of oxysterol-derived pharmacological chaperones for NPC1: implication for the existence of second sterol-binding site.

doi: 10.1016/j.chembiol.2013.02.009

Figure Lengend Snippet: Figure 6. Dispensability of NTD for Oxysterol Derivative-Mediated Rescue of Mutant NPC1 Protein (A) Schematic representation of the NTD-deleted NPC1-GFP (DNTD). (B) Subcellular localization of the DNTD-WT and DNTD-I1061T. Cells stably expressing the DNTD-NPC1-GFP construct were treated as indicated for 24 hr and colocalization of the NPC1 with LAMP1 was examined. Calibration bar represents 20 mm. (C) Dose-dependent rescue of DNTD-I1061T localization by representative oxysterol derivatives. The graph shows the dose-response curves of representative compounds and the table shows calculated EC50 values. For 25HC, the extrapolated value is shown. Error bar, SE (n = 12).

Article Snippet: For immunoblotting of endogenous NPC1 protein, rabbit anti-NPC1 polyclonal antibody (Novus Biologicals) combined with HRP-conjugated anti-rabbit antibody (R&D Systems) was used.

Techniques: Mutagenesis, Stable Transfection, Expressing, Construct

Journal: Chemistry & biology

Article Title: Discovery of oxysterol-derived pharmacological chaperones for NPC1: implication for the existence of second sterol-binding site.

doi: 10.1016/j.chembiol.2013.02.009

Figure Lengend Snippet: Figure 7. Existence of Non-NTD Sterol-Binding Site (A) Sterol-mediated stabilization of DNTD-I1061T. The steady-state expression level of DNTD-I1061T was quantified as in Figure 3A. Error bar, SD (n = 3). (B) Schematic representation of the NTD-tail-GFP construct. See also Figure S3. (C) Photoaffinity labeling experiments of NTD-deleted NPC1 and NTD-tail NPC1. Membranes from cells stably expressing either FLAG-tagged DNTD-I1061T or NTD-tail-GFP were labeled with mo56AZK as in Figure 4. Right panel shows the labeling of DNTD-WT. Because of the low expression level of the stable cell line, the longer exposure time was used for DNTD-WT. ns, nonspecific labeling/staining. See also Figure S3C. (D) The quantified results of (C).

Article Snippet: For immunoblotting of endogenous NPC1 protein, rabbit anti-NPC1 polyclonal antibody (Novus Biologicals) combined with HRP-conjugated anti-rabbit antibody (R&D Systems) was used.

Techniques: Binding Assay, Expressing, Construct, Labeling, Stable Transfection, Staining

Journal: PLoS ONE

Article Title: Pulmonary Abnormalities in Animal Models Due to Niemann-Pick Type C1 (NPC1) or C2 (NPC2) Disease

doi: 10.1371/journal.pone.0067084

Figure Lengend Snippet: Western blot of wild type (W) littermates, NPC1 (Mut) or NPC2 (Mut) mutant mouse lungs using anti-NPC1 or -NPC2 antibody. β-actin used as a loading control. 30 µg protein/lane.

Article Snippet: Primary antibody against rabbit anti-NPC1 (diluted 1∶200 Novus Biologicals, Littleton, CO), NPC2 (diluted 1∶750, Sigma, St. Louis, MO, Cat# HPA000835), secondary antibody goat anti-rabbit IgG horseradish peroxidase-conjugated (diluted 1∶3,000, Upstate Millipore, Billerica, MA), and ECL Plus Western Blotting Detection Reagent (GE Healthcare, Amersham, Piscataway Township, NJ) were used.

Techniques: Western Blot, Mutagenesis, Control

Journal: PLoS ONE

Article Title: Pulmonary Abnormalities in Animal Models Due to Niemann-Pick Type C1 (NPC1) or C2 (NPC2) Disease

doi: 10.1371/journal.pone.0067084

Figure Lengend Snippet: Lungs from BALB/c wild type (95 days), NPC1 mutant (70 days) and NPC2 mutant mice (88 days). NPC1 and NPC2 mutant mice show “nests” of macrophages and alveolar macrophages with large inclusions.

Article Snippet: Primary antibody against rabbit anti-NPC1 (diluted 1∶200 Novus Biologicals, Littleton, CO), NPC2 (diluted 1∶750, Sigma, St. Louis, MO, Cat# HPA000835), secondary antibody goat anti-rabbit IgG horseradish peroxidase-conjugated (diluted 1∶3,000, Upstate Millipore, Billerica, MA), and ECL Plus Western Blotting Detection Reagent (GE Healthcare, Amersham, Piscataway Township, NJ) were used.

Techniques: Mutagenesis

Journal: PLoS ONE

Article Title: Pulmonary Abnormalities in Animal Models Due to Niemann-Pick Type C1 (NPC1) or C2 (NPC2) Disease

doi: 10.1371/journal.pone.0067084

Figure Lengend Snippet: Wild type lung (A–C). A. Overview of section of lung with alveolar type II cell (AT2) and endothelial cell (Endo). B. Capillary (Ca). C. Respiratory membrane with type I cell (T1) and endothelial cell (Endo). AS, alveolar space. NPC1 mutant lung (D–H). D. Overview of lung with leukocytes in the capillary and excess surfactant in alveolar space. AL, alveolar lipidosis. d. Enlargement of area in D showing vacuolar leukocyte (L). Similar leukocytes were seen in NPC2 mutant lung. E. Alveolar type II cell (AT2) with a foamy alveolar macrophage (AM) in close proximity in the alveolar space. e. Enlargement of area in E showing type II cell-macrophage contact. *Indicates vacuolar inclusions in endothelial cell. F. Alveolar macrophage with lipid-like material and vacuolar inclusions. G. Type II cells with excess surfactant (white arrowhead). H. Endothelial cell with vesicular inclusions (*). NPC2 mutant lung (I–P). I. Overview of lung with surfactant completely filling the alveolar space characteristic of alveolar lipidosis. J. AT2 with an alveolar macrophage containing multivesicular whirls and a foamy circulating macrophage (CM). K. Respiratory membrane of endothelial cell, basement membrane (BM) and type I cell and demonstrating large amounts of surfactant as tubular myelin (TM) and aggregate (Ag) structures. L. Endothelial cell with vesicular structures. M. Alveolar space with black arrowhead indicating proteinaceous material. Similar material was seen in NPC1 mutant lung. N. Large aggregate structure with tightly packed phospholipid-type whirls (gray arrowheads) or string-like structures (white arrowhead) in alveolar space. O. Surfactant vesicles (white arrowheads) filling the alveolar space. P. Type II cell with inset (p) showing autophagosome-like structures (ap) in enlargement.

Article Snippet: Primary antibody against rabbit anti-NPC1 (diluted 1∶200 Novus Biologicals, Littleton, CO), NPC2 (diluted 1∶750, Sigma, St. Louis, MO, Cat# HPA000835), secondary antibody goat anti-rabbit IgG horseradish peroxidase-conjugated (diluted 1∶3,000, Upstate Millipore, Billerica, MA), and ECL Plus Western Blotting Detection Reagent (GE Healthcare, Amersham, Piscataway Township, NJ) were used.

Techniques: Membrane, Mutagenesis

Journal: PLoS ONE

Article Title: Pulmonary Abnormalities in Animal Models Due to Niemann-Pick Type C1 (NPC1) or C2 (NPC2) Disease

doi: 10.1371/journal.pone.0067084

Figure Lengend Snippet: Using electron microscopic photographs of type II cells from wild type, NPC1 and NPC2 mutant mice, the size of the lamellar bodies was analyzed using ImageJ. Top. Electron micrographs of typical type II cells from wild type (WT, left), NPC1 (middle) and NPC2 (right) mutant mice. LB, lamellar body. Bottom. Histogram of the lamellar bodies from wild type (white triangles), NPC1 (gray circles) and NPC2 (black circles) type II cells. Frequency of each lamellar body size in micron 2 is expressed as a % of the total numbers of lamellar bodies. Inset: size of lamellar bodies in grouped bins. Wild type, 60 type II cells, 451 LBs, 3 mice; NPC1, 53 type II cells, 435 LBs, 3 mice; NPC2, 37 type II cells, 459 LBs, 3 mice. *Statistically significant difference, P<0.05. E. Lamellar body sizes in grouped bins of ranges of areas.

Article Snippet: Primary antibody against rabbit anti-NPC1 (diluted 1∶200 Novus Biologicals, Littleton, CO), NPC2 (diluted 1∶750, Sigma, St. Louis, MO, Cat# HPA000835), secondary antibody goat anti-rabbit IgG horseradish peroxidase-conjugated (diluted 1∶3,000, Upstate Millipore, Billerica, MA), and ECL Plus Western Blotting Detection Reagent (GE Healthcare, Amersham, Piscataway Township, NJ) were used.

Techniques: Mutagenesis

Journal: PLoS ONE

Article Title: Pulmonary Abnormalities in Animal Models Due to Niemann-Pick Type C1 (NPC1) or C2 (NPC2) Disease

doi: 10.1371/journal.pone.0067084

Figure Lengend Snippet: A–C Cholesterol and phospholipid content of mice lungs. A. Lipid content of the lavaged lung as µg lipid/mg lung protein. Data are mean±SE, n = 4 separate mice. B. Broncho-alveolar lavage (BAL) of mice lungs. Data as µg lipid/gm weight of lung and are mean±SE, n = 4 separate mice. C. Lipid content of lamellar bodies isolated from the lungs of the wild type and mutant mice. Four (NPC1 or wild type littermates) or three (NPC2 or wild type littermates) lamellar body preparation isolated from the lungs of 2–6 mice. *Statistically significant difference versus wild type littermates, # statistically significant difference between wild types. δ Statistically significant difference between mutants, P <0.05. D. Degradation of 3 H-labeled DPPC as percentage of total label uptake. 3 H-DPPC liposome degradation by the isolated, perfused lungs of three wild type or NPC1 mutant mice after intratracheal instillation. Values are means±SE. *Statistically significant difference from wild type. P<0.001, n = 3. Total degradation is the sum of lysophosphatidyl choline (lysoPC), unsaturated PC (unsatPC), and the aqueous fractions.

Article Snippet: Primary antibody against rabbit anti-NPC1 (diluted 1∶200 Novus Biologicals, Littleton, CO), NPC2 (diluted 1∶750, Sigma, St. Louis, MO, Cat# HPA000835), secondary antibody goat anti-rabbit IgG horseradish peroxidase-conjugated (diluted 1∶3,000, Upstate Millipore, Billerica, MA), and ECL Plus Western Blotting Detection Reagent (GE Healthcare, Amersham, Piscataway Township, NJ) were used.

Techniques: Isolation, Mutagenesis, Labeling

Journal: PLoS ONE

Article Title: Pulmonary Abnormalities in Animal Models Due to Niemann-Pick Type C1 (NPC1) or C2 (NPC2) Disease

doi: 10.1371/journal.pone.0067084

Figure Lengend Snippet: Physical parameters of NPC mice and wild type littermates.

Article Snippet: Primary antibody against rabbit anti-NPC1 (diluted 1∶200 Novus Biologicals, Littleton, CO), NPC2 (diluted 1∶750, Sigma, St. Louis, MO, Cat# HPA000835), secondary antibody goat anti-rabbit IgG horseradish peroxidase-conjugated (diluted 1∶3,000, Upstate Millipore, Billerica, MA), and ECL Plus Western Blotting Detection Reagent (GE Healthcare, Amersham, Piscataway Township, NJ) were used.

Techniques:

Journal: PLoS ONE

Article Title: Pulmonary Abnormalities in Animal Models Due to Niemann-Pick Type C1 (NPC1) or C2 (NPC2) Disease

doi: 10.1371/journal.pone.0067084

Figure Lengend Snippet: Left, Western blots of SP-A or actin. Right, quantitation of Western blots. A. Arbitrary units (AU) of SP-A relative to actin from lungs of NPC1 or NPC2 mutant mice (C1 or C2) or age-matched wild type controls (W1 or W2) (n = 6–8) or B. Arbitrary units of SP-A in surfactant (n = 4–9). All samples were loaded at equal protein values. *Significant difference from wild type, ( P <0.05). The two SP-A bands are due to differences in glycosylation.

Article Snippet: Primary antibody against rabbit anti-NPC1 (diluted 1∶200 Novus Biologicals, Littleton, CO), NPC2 (diluted 1∶750, Sigma, St. Louis, MO, Cat# HPA000835), secondary antibody goat anti-rabbit IgG horseradish peroxidase-conjugated (diluted 1∶3,000, Upstate Millipore, Billerica, MA), and ECL Plus Western Blotting Detection Reagent (GE Healthcare, Amersham, Piscataway Township, NJ) were used.

Techniques: Western Blot, Quantitation Assay, Mutagenesis, Glycoproteomics

Journal: PLoS ONE

Article Title: Pulmonary Abnormalities in Animal Models Due to Niemann-Pick Type C1 (NPC1) or C2 (NPC2) Disease

doi: 10.1371/journal.pone.0067084

Figure Lengend Snippet: A. Type II cells were isolated from NPC1 (Top) or NPC2 (Bottom) mutant mice or their corresponding wild type littermates and placed in culture for 24 hrs. The cells were fixed and stained with anti-ABCA3 antibody (green) to mark the lamellar body limiting membrane or filipin (Fil, gray or blue) to mark cholesterol. The merged pictures with anti-ABCA3 in green and filipin in blue are enlarged. Scale bar = 5 µm. B . Alveolar macrophages from NPC mutant mice contain cholesterol. Alveolar macrophages were isolated from the lung lavage from NPC1 (Top) or NPC2 (Bottom) mutant mice or their corresponding wild type littermates and placed in culture for 2 hrs. The cells were fixed and stained with filipin which labels free unesterified cholesterol. Lt, Phase micrograph; Fil, Filipin stain in gray. Merge of phase and filipin (blue) are enlarged. Scale bar = 10 µm.

Article Snippet: Primary antibody against rabbit anti-NPC1 (diluted 1∶200 Novus Biologicals, Littleton, CO), NPC2 (diluted 1∶750, Sigma, St. Louis, MO, Cat# HPA000835), secondary antibody goat anti-rabbit IgG horseradish peroxidase-conjugated (diluted 1∶3,000, Upstate Millipore, Billerica, MA), and ECL Plus Western Blotting Detection Reagent (GE Healthcare, Amersham, Piscataway Township, NJ) were used.

Techniques: Isolation, Mutagenesis, Staining, Membrane

Journal: PLoS ONE

Article Title: Pulmonary Abnormalities in Animal Models Due to Niemann-Pick Type C1 (NPC1) or C2 (NPC2) Disease

doi: 10.1371/journal.pone.0067084

Figure Lengend Snippet: Alveolar macrophages were isolated and cultured as in Fig. 7. The cells were harvested and the phospholipid and cholesterol content analyzed. W1,W2. Wild type littermates from NPC1 or NPC2 mutant mice, respectively. C1, NPC1; C2, NPC2. The data are mean±SE, n = 4 separate mice. *Statistically significant difference from corresponding wild type littermates. # Significant difference between NPC1 and NPC2 mutant macrophages, P<0.05.

Article Snippet: Primary antibody against rabbit anti-NPC1 (diluted 1∶200 Novus Biologicals, Littleton, CO), NPC2 (diluted 1∶750, Sigma, St. Louis, MO, Cat# HPA000835), secondary antibody goat anti-rabbit IgG horseradish peroxidase-conjugated (diluted 1∶3,000, Upstate Millipore, Billerica, MA), and ECL Plus Western Blotting Detection Reagent (GE Healthcare, Amersham, Piscataway Township, NJ) were used.

Techniques: Isolation, Cell Culture, Mutagenesis

Journal: PLoS ONE

Article Title: Pulmonary Abnormalities in Animal Models Due to Niemann-Pick Type C1 (NPC1) or C2 (NPC2) Disease

doi: 10.1371/journal.pone.0067084

Figure Lengend Snippet: A. Histology of feline lung. Wild type (WT) and NPC1 mutant (NPC1) feline lungs (25 weeks old) stained with hematoxylin and eosin. NPC1 mutant feline show thickened septae, enlarged “foamy” macrophages in capillaries and alveolar macrophages in the alveolar space. B. Electron micrographs (EM) of typical type II cells from wild type (WT) and NPC1 mutant (NPC1) feline indicating that the size of NPC1 mutant feline lamellar bodies is enlarged. C . Histogram of the lamellar bodies from wild type (white triangles) and NPC1 (gray circles) type II cells. Using electron microscopic photographs of type II cells from wild type and NPC1 mutant felines, the size of the lamellar bodies was analyzed using ImageJ. C , Inset: size of lamellar bodies in grouped bins in ranges of areas. Frequency of each lamellar body size in micron 2 is expressed as a % of the total numbers of lamellar bodies. Wild type (WT): 28 type II cells, 250 LBs, 3 feline; NPC1 mutant: 35 type II cells, 323 LBs, 3 feline.

Article Snippet: Primary antibody against rabbit anti-NPC1 (diluted 1∶200 Novus Biologicals, Littleton, CO), NPC2 (diluted 1∶750, Sigma, St. Louis, MO, Cat# HPA000835), secondary antibody goat anti-rabbit IgG horseradish peroxidase-conjugated (diluted 1∶3,000, Upstate Millipore, Billerica, MA), and ECL Plus Western Blotting Detection Reagent (GE Healthcare, Amersham, Piscataway Township, NJ) were used.

Techniques: Mutagenesis, Staining

Journal: PLoS ONE

Article Title: Pulmonary Abnormalities in Animal Models Due to Niemann-Pick Type C1 (NPC1) or C2 (NPC2) Disease

doi: 10.1371/journal.pone.0067084

Figure Lengend Snippet: A. Lipid content of the lung. Data are mean±SE, n = 3 feline. B. Lipid content of lamellar bodies isolated from the lungs of the wild type and NPC1 mutant feline. Data are the mean±SE and range of 5 (WT) or 4 (NPC1 mut) lamellar body preparations isolated from feline lungs analyzed in triplicate. *Significant different from WT.

Article Snippet: Primary antibody against rabbit anti-NPC1 (diluted 1∶200 Novus Biologicals, Littleton, CO), NPC2 (diluted 1∶750, Sigma, St. Louis, MO, Cat# HPA000835), secondary antibody goat anti-rabbit IgG horseradish peroxidase-conjugated (diluted 1∶3,000, Upstate Millipore, Billerica, MA), and ECL Plus Western Blotting Detection Reagent (GE Healthcare, Amersham, Piscataway Township, NJ) were used.

Techniques: Isolation, Mutagenesis

Journal: bioRxiv

Article Title: Impaired cholesterol transport from aged astrocytes to neurons can be rescued by cannabinoids

doi: 10.1101/2023.07.24.550299

Figure Lengend Snippet: A) Western blot analysis, along with quantification showing decreased NPC1 protein levels in aged primary hippocampal astrocytes compared to control cultures. B) RT-qPCR analysis showing decreased NPC1 mRNA levels aged rat primary hippocampal astrocytes. C) Western blot analysis, along with quantification showing decreased NPC1 protein levels in the hippocampus of old (20 months-old) mice compared to young (2 months-old) C57BL/6 mice (YOUNG = 1.0630 + 0.0364 SEM, OLD = 0.8919 + 0.0494 SEM, p = 0.0303). D) RT-qPCR analysis showing decreased NPC1 mRNA levels in the hippocampus of old C57BL/6 mice compared to young individuals (YOUNG = 1.0635 + 0.1290 SEM, OLD = 0.5290 + 0.1213 SEM, p = 0.0242). E) Quantification of miR-33 levels by RT-qPCR in control, aged, or H2O 2 -treated hippocampal astrocytes, indicating increased expression of miR-33 in these cells due to aging or oxidative stress. F) RT-qPCR analysis showing decreased NPC1 mRNA levels in hippocampal astrocytes treated with H 2 O 2 compared to untreated controls. G) RT-qPCR quantification of ABCA1 mRNA levels in control, aged, or H 2 O 2 -treated hippocampal astrocytes, demonstrating downregulation of ABCA1 in primary astrocytes due to aging or oxidative stress. H) Fluorescence microscopy images of rat primary hippocampal astrocytes treated with Probucol 10µM (ABCA1 inhibitor), U18666A 4µg/mL (NPC1 inhibitor), and the combination of both, labeled with Bodipy-cholesterol. I) Quantification of images including those shown in H, indicating that NPC1 or ABCA1 inhibition leads to Bodipy-cholesterol accumulation in astrocytes, with enhanced effects observed when both proteins are inhibited. J) Treatment of aged astrocytes with the ABCA1 agonist evodiamine leads to a significant decrease in the levels of accumulated Bodipy-cholesterol. Data are represented as Mean ± SEM. ****P-value < 0.0001, *P-value < 0.05.

Article Snippet: Membranes were blocked for 1 hour and incubated overnight at 4°C with the NPC1 primary antibody (rabbit polyclonal, Novus Biologicals, UK, NB 400-148).

Techniques: Western Blot, Control, Quantitative RT-PCR, Expressing, Fluorescence, Microscopy, Labeling, Inhibition

Journal: bioRxiv

Article Title: Impaired cholesterol transport from aged astrocytes to neurons can be rescued by cannabinoids

doi: 10.1101/2023.07.24.550299

Figure Lengend Snippet: A) Fluorescence microscopy images of aged rat hippocampal astrocytes labeled with Bodipy-cholesterol and treated with the cannabinoids Anandamide (AEA 2µM), 2-Arachidonoylglycerol (2AG 2µM) and cannabidiol (CBD 5µM). B) Quantification of Bodipy-cholesterol intensity from images as shown in A demonstrates that treatment of aged astrocytes with AEA, 2AG, or CBD significantly reduces the Bodipy-cholesterol accumulation characteristic of aged astrocytes. C) Immunofluorescence images showing the levels of Bodipy-cholesterol incorporated in 7DIV hippocampal neurons co- cultured with either control, aged, or aged astrocytes in the presence of AEA for 6 hours. Neuronal cells were labeled for β3-Tubulin. D) Quantification of Bodipy-cholesterol intensity from images including those in C demonstrates that the cholesterol uptake by neurons co-cultured with aged astrocytes is improved by addition of AEA. E) Fluorescence microscopy images showing the levels of Bodipy-cholesterol accumulated in control, U18666A+Probucol, and U18666A+Probucol+AEA treated primary astrocytes. F) Bodipy-cholesterol quantification of images as shown in E demonstrate that the cholesterol buildup triggered by the inhibition of NPC1 and ABCA1 in primary astrocytes is alleviated by either AEA, 2AG, or CBD addition even in the presence of the inhibitors (U18666A+Probucol). These data suggest that cannabinoids act through NPC1 and ABCA1-independent pathways. Data are represented as Mean ± SEM. ****P-value < 0.0001.

Article Snippet: Membranes were blocked for 1 hour and incubated overnight at 4°C with the NPC1 primary antibody (rabbit polyclonal, Novus Biologicals, UK, NB 400-148).

Techniques: Fluorescence, Microscopy, Labeling, Immunofluorescence, Cell Culture, Control, Inhibition