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

Journal: EMBO Reports

Article Title: NPC 1 enables cholesterol mobilization during long‐term potentiation that can be restored in Niemann–Pick disease type C by CYP 46A1 activation

doi: 10.15252/embr.201948143

Figure Lengend Snippet: A Single greyscale and coloured merged images of immunocytochemical staining against PSD‐95, synaptophysin‐1 (SYP1) and NPC1 proteins in a cultured hippocampal neuron from a wt mouse. Graphs show mean ± SEM of percentage of co‐localization of NPC1 with PSD95 or SYP1 ( n = 12 neurons from three different cultures). B Electron microscopy image of immunogold labelling against NPC1 in the CA1 hippocampal region of a wt mouse. Presynaptic compartments are indicated in pink while postsynaptic in blue. White arrows show the 15‐nm gold particles linked to NPC1 (d—dendrite, sv—synaptic vesicles, m—mitochondria). C, D Western blots against NPC1 and actin‐β (ACTB) in total brain (C) and synaptosomes (D) from wt and NPC1 nmf164 mice. Graphs show mean ± SEM NPC1 level normalized to ACTB as a percentage of control (wt) values ( n = 4 mice, 3 months old, unpaired Student's t ‐test, ** P synaptosomes = 0.0037). E Western blots against NPC1 and actin‐β (ACTB) in total brain extracts from wt and Npc1‐null mice (NPC1KO) containing the same amount of protein ( n = 2 mice, 6 weeks old). F Graph shows mean ± SEM mRNA levels of NPC1 in total extracts and in synaptosomes from wt and NPC1 nmf164 mice ( n = 4 mice, 3 months old). G Western blots against NPC1 and actin‐β (ACTB) in synaptosomes from wt and NPC1 nmf164 mice in which cLTP was induced or not in the presence or absence of the protein synthesis inhibitor cycloheximide (CHX). Graphs show mean ± SEM NPC1 level normalized to ACTB as a percentage of control (wt non‐cLTP‐induced) values ( n = 3 mice, 3 months old, 2‐way ANOVA, *** P < 0.001). H To the left Western blots against NPC1 and actin‐β (ACTB) in synaptosomal extracts from wt and NPC1 nmf164 mice used as input for the immunoprecipitation assays shown in the right. Immunoprecipitates were pulled down with an anti‐ubiquitin antibody and without it for negative control and analysed by Western blot against NPC1. MG132 was used as proteasome inhibitor. Graphs show mean ± SEM levels of NPC1 normalized to ACTB in the inputs (left) ( n = 3, one‐way ANOVA, * P WT+MG‐132 = 0.0496, * P NPC1nmf1+MG‐132 = 0.0358) and of NPC1 associated with ubiquitin (right) ( n = 3 mice, 3 months old, one‐way ANOVA, * P NPC1nmf164 = 0.0237, * P NPC1nmf164+MG‐132 = 0.0386).

Article Snippet: Antibodies against the following proteins were used for Western blots and immunofluorescence: NPC1 (rabbit polyclonal, Novus Biologicals, UK, NB 400‐148; rabbit monoclonal, Abcam, #ab134113), PSD‐95 (mouse monoclonal, NeuroMab, Ca, USA, 75‐028), synaptophysin‐1 (polyclonal guinea pig, Synaptic Systems, Germany, 101004), CYP46A1 (rabbit polyclonal, Proteintech, USA, 12486‐1‐AP), GluR1 C terminus (rabbit polyclonal (Abcam, ab31232), GluR1N terminus (mouse monoclonal, Merck‐Millipore, MAB2263), LAMP1 (rat monoclonal, DSHB) and anti‐β‐actin (mouse monoclonal Sigma‐Aldrich, A2228).

Techniques: Staining, Cell Culture, Electron Microscopy, Western Blot, Control, Immunoprecipitation, Ubiquitin Proteomics, Negative Control

Journal: EMBO reports

Article Title: NPC1 enables cholesterol mobilization during long-term potentiation that can be restored in Niemann-Pick disease type C by CYP46A1 activation.

doi: 10.15252/embr.201948143

Figure Lengend Snippet: Figure 6. Effects of CYP46A1 activation by EFV on cLTP-induced GluA1 surface delivery in wt- and NPC1-deficient conditions.

Article Snippet: Antibodies against the following proteins were used for Western blots and immunofluorescence: NPC1 (rabbit polyclonal, Novus Biologicals, UK, NB 400-148; rabbit monoclonal, Abcam, #ab134113), PSD-95 (mouse monoclonal, NeuroMab, Ca, USA, 75- 028), synaptophysin-1 (polyclonal guinea pig, Synaptic Systems, Germany, 101004), CYP46A1 (rabbit polyclonal, Proteintech, USA, 12486-1-AP), GluR1 C terminus (rabbit polyclonal (Abcam, ab31232), GluR1N terminus (mouse monoclonal, Merck-Millipore, MAB2263), LAMP1 (rat monoclonal, DSHB) and anti-b-actin (mouse monoclonal Sigma-Aldrich, A2228).

Techniques: Activation Assay