Impaired Cerebrospinal Fluid Lipoprotein-Mediated Cholesterol Delivery to Neurons in Alzheimer's Disease

Abstract

In the central nervous system, apolipoprotein (APO) E-containing high-density lipoprotein (HDL)-like particles mediate the transport of glial-derived cholesterol to neurons, which is essential for neuronal membrane remodeling and maintenance of the myelin sheath. Despite this, the role of HDL-like cholesterol trafficking on Alzheimer's disease (AD) pathogenesis remains poorly understood. We aimed to examine cholesterol transport via HDL-like particles in cerebrospinal fluid (CSF) of AD patients compared to control individuals. Additionally, we explored the ability of reconstituted HDL containing different APOE isoforms to regulate cholesterol transport. We evaluated the capacity of CSF HDL-like particles to facilitate radiolabeled unesterified cholesterol efflux from A172 human glioblastoma astrocytes and to deliver cholesterol to SH-SY5Y human neuronal cells. The HDL-like proteome in the AD and control groups was analyzed by liquid chromatography-mass spectrometry (LC-MS/MS). Reconstituted HDL nanoparticles were prepared by combining phospholipids and cholesterol with human APOE3 or APOE4, followed by radiolabeling with unesterified cholesterol. Our results showed that cholesterol efflux from astrocytes to CSF were similar between AD patients and controls, both under baseline conditions and after activation of ATP-binding cassette transporters A1 and G1. However, CSF HDL-like particle-mediated neuronal cholesterol uptake was significantly reduced in the AD group. LC-MS/MS analysis identified 775 proteins associated with HDL-like particles in both groups, with no major alterations in proteins linked to cholesterol metabolism. However, 27 proteins involved in non-cholesterol-related processes were differentially expressed. Notably, synthetic reconstituted HDL particles containing APOE4 exhibited reduced capacity to deliver cholesterol to neurons compared to those with APOE3. These findings indicate that CSF HDL-like particles from patients with AD demonstrate impaired cholesterol delivery to neurons. Our study highlights APOE4 as a critical contributor to abnormal neuronal cholesterol uptake in AD pathophysiology.

Keywords: APOE; Alzheimer's Disease; Cholesterol; HDL-like lipoprotein.

Figure 1. Astrocyte Cholesterol Efflux to CSF Remains Similar in AD and Control Groups, whereas CSF HDL-like-mediated Cholesterol Delivery to Neurons is Impaired in AD.

(a) Astrocyte Cholesterol Efflux Assay. Human glioblastoma astrocytes were cultured for 24 hours, followed by a 48-hour incubation with radiolabeled cholesterol. Cells were then treated for 18 hours with or without T0901317 to activate ABCA1/G1 pathways. Serum-free medium containing CSF was added for 4 hours. Both the medium and cell fractions were processed to quantify radiolabeled cholesterol. (b) Astrocyte Cholesterol Efflux Results: Left panel - Cholesterol efflux from human glioblastoma astrocytes to CSF (30% v/v) is shown for both control and AD samples, under baseline conditions and following T0901317 pre-treatment. Right panel - Specific ABCA1/G1-dependent cholesterol efflux was calculated subtracting baseline levels from those observed in ABCA1/G1-expressing cells. (c) CSF HDL-like-Mediated Cholesterol Uptake Assay: Human neuroblastoma cells were seeded and differentiated into neurons in a low-serum medium containing retinoic acid. After 24 hours, radiolabeled CSF HDL-like particles containing unesterified cholesterol were added (10% v/v). The cells were incubated with serum-free medium containing CSF for 4 hours, and both the medium and cell lysates were processed for radiolabeled cholesterol quantification. (d) Cholesterol Uptake results: CSF HDL-like-mediated cholesterol uptake was measured in human neurons exposed to CSF from both control individuals and patients with AD. (e and f) Influence of Tau and Aβ_1–42 on Neuronal Cholesterol Uptake: Cholesterol uptake in SH-SY5Y neurons mediated by control CSF HDL-like particles was assessed in the presence of Tau or Aβ_1–42 added to the culture media at concentrations up to 1,000 and 1,500 pg/mL, respectively. Values are shown as the mean ± SD for 10 subjects per group in panels (b) and (d). The Student’s t-test was used to compare the CSF HDL-like-mediated cholesterol uptake by neurons, as well as the astrocyte cholesterol efflux under various conditions between the control and AD groups. One-way ANOVA with a post-test for linear trend was used in panels (e) and (f). Three separate experiments were carried out for each condition.

Figure 2. Altered Proteome of CSF HDL-like Particles in AD.

(a) Native Gel Electrophoresis of CSF Samples. Representative native polyacrylamide gel electrophoresis of CSF from patients with AD and control individuals. Left panel - HDL-like particles and albumin bands following Coomassie blue staining. Right panel - Corresponding nitrocellulose blot probed with a polyclonal anti-APOE antibody, identifying the HDL-like band. (b) Proteins from Cholesterol Metabolism Quantified in HDL-like Particles from CSF. Increased (red) or decreased (blue) abundances are shown according to the indicated Zq scale. APOM: Apolipoprotein M, APOA1: Apolipoprotein A-I, APOD: Apolipoprotein D, LRP1: Low-density lipoprotein receptor-related protein 1, PLTP: Phospholipid transfer protein, PON1: Serum paraoxonase/arylesterase 1, APOH: Beta-2-glycoprotein 1, APOE: Apolipoprotein E, APOA2: Apolipoprotein A-II. (c) Differentially Regulated Proteins in HDL-like Particles from the CSF of Patients with AD. Heat map depicting significant protein abundance changes (p < 0.05) in AD and control groups. Increased (red) or decreased (blue) abundances are shown according to the indicated Zq scale. Differential protein expression analysis was performed using moderated t-statistics (limma test). ALDOA: Fructose-bisphosphate aldolase A, ACHL1: Neural cell adhesion molecule L1-like protein, ATRN: Attractin, LTBP4: Latent-transforming growth factor beta-binding protein 4, NRCAM: Neuronal cell adhesion molecule, IGLC2: Immunoglobulin lambda constant 2, GRIA4: Glutamate receptor 4, SEZ6: Seizure protein 6 homolog, IGLL5: Immunoglobulin lambda-like polypeptide 5, C5: Complement C5, GELS: Gelsolin, B4GAT1: Beta-1,4-glucoronyltransferase 1, CLEC11A: C-type lectin domain family 11 member A, FUCA2: Plasma alpha-L-fucosidase, MCAM: Cell surface glycoprotein MUC18, HPX: Hemopexin, C8B: Complement component C8 beta chain, ENPP2: Ectonucleotide pyrophosphatase/phosphodiesterase family member 2, AGT: Angiotensinogen, PLXDC2: Plexin domain-containing protein 2, BCAM: Basal cell adhesion molecule, LUM: Lumican, OLFML3: Isoform 2 of Olfactomedin-like protein 3, VCAM1: Vascular cell adhesion protein 1, CADM4: Cell adhesion molecule 4, CTSD: Cathepsin D, FBLN1: Fibulin-1.

Figure 3. Synthetic Reconstituted HDL Nanoparticles Containing Apolipoprotein E4 Exhibit Impaired CholesterolDelivery to Neurons.

(a) Schematic Representation of the Synthesis of Reconstituted (r)HDL-APOE Nanoparticles: Recombinant APOE3 or APOE4 were combined with DMPC and cholesterol in a molar ratio of 59:7:1. The mixture underwent three cycles of vortexing and temperature modulation, alternating between 37°C and 4°C, to optimize APOE-lipid interactions. (b) Native Gel Electrophoresis of Synthetic rHDL-APOE3 and HDL-APOE4 Nanoparticles: Representative native polyacrylamide gel electrophoresis image of synthetic rHDL-APOE3 and rHDL-APOE4 nanoparticles, visualized with Coomassie Blue staining. (c) Characterization of Synthetic rHDL-APOE3 and HDL-APOE4 Nanoparticles: The particle size distributions of purified synthetic rHDL-APOE3 and rHDL-APOE4 nanoparticles were analyzed using dynamic light scattering. Representative images from TEM are also shown as insets. (d) Astrocyte Cholesterol Efflux to Synthetic rHDL-APOE3 and rHDL-APOE4: Cholesterol efflux from human glioblastoma astrocytes to synthetic rHDL-APOE3 and rHDL-APOE4 (5 μg/mL) was measured under baseline conditions and after T0901317 pre-treatment, as described in Fig. 1A. (e) Neuronal Cholesterol Uptake Mediated by Synthetic rHDL-APOE3 and rHDL-APOE4: synthetic rHDL-APOE3 and rHDL-APOE4 (5 μg/mL) were loaded with radiolabeled unesterified cholesterol, and their capacity to facilitate cholesterol uptake in human neuroblastoma cells was assessed as described in Fig. 1c. Mean ± SD is used to express values. Student t-tests were used to compare HDL-mediated neuronal cholesterol delivery between synthetic rHDL-APOE3 and rHDL-APOE4, as well as astrocyte cholesterol efflux under various conditions. Five separate experiments were conducted to evaluate each condition.