Production and characterization of astrocyte-derived human apolipoprotein E isoforms from immortalized astrocytes and their interactions with amyloid-β

Abstract

The apolipoprotein E (apoE) genotype is an important genetic risk factor for Alzheimer's disease (AD). In the central nervous system (CNS), most apoE is produced by astrocytes and is present in unique high-density lipoprotein (HDL)-like particles that have distinct properties from apoE derived from other sources. To develop an efficient system to produce astrocyte-derived apoE in large quantities, we produced and characterized immortalized cell lines from primary astrocyte cultures derived from human APOE knock-in mice. APOE2, APOE3, and APOE4 expressing cell lines were established that secrete apoE in HDL-like particles at similar levels, cholesterol composition, and size as those produced by primary astrocytes. In physiological buffers, astrocyte-secreted apoE3 and E4 associated equally well with amyloid-B. Under the same conditions, only a small fraction of AB formed sodium dodecyl sulfate (SDS)-stable complexes with apoE (E3 N E4). These immortalized astrocytes will be useful for studying mechanisms underlying the isoform-specific effects of apoE in the CNS. D 2004 Elsevier Inc. All rights reserved.

Levels of Soluble Apolipoprotein E/Amyloid-β (Aβ) Complex Are Reduced and Oligomeric Aβ Increased with APOE4 and Alzheimer Disease in a Transgenic Mouse Model and Human Samples*

“…However, a corresponding value for total complex levels was not possible by Western analysis. With this new ELISA, the apoE isoforms exhibit a comparable affinity for A␤ in the absence of SDS, defined here as total apoE/A␤, consistent with previous reports using nonstringent conditions to measure the complex (31). The mechanism by which the apoE4/A␤ complex is less stable is unclear.…”

“…Second, the definition of an apoE/A␤ is primarily operational, with assay stringency the primary variable (7,(17)(18)(19)(20)(21)(22)(23)(24)(25)(26)(27)30). For example, apoE3/A␤ levels are greater than apoE4/A␤ as determined by Western analysis of SDS-PAGE (21), but by nondenaturing gel electrophoresis, the levels of the apoE3/A␤ complex are comparable with apoE4/A␤ (31). Although these data are consistent with an SDS-stable apoE3/A␤ complex (32), and an apoE4/A␤ complex that is disrupted by SDS, the total amount of apoE/A␤ cannot be quantified by Western analysis of SDS-PAGE.…”

Apolipoprotein E Inhibits Cerebrovascular Pericyte Mobility through a RhoA Protein-mediated Pathway

“…Immortalized apoE3 astrocytes were a gift from Dr. David M. Holtzman (Washington University). The cell line was generated from primary astrocyte cultures derived from human APOE-targeted replacement (TR) mice in which human APOE3 is knocked into the mouse endogenous Apoe gene locus (9). Immortalized apoE3 astrocytes were cultured in DMEM: F12 (Invitrogen) containing 20% FBS (Gemini Bio), 1 mM sodium pyruvate, 1% penicillin-streptomycin, 0.5% amphotericin B and 0.1% EGF.…”

“…Several types of cell can produce apoE, with hepatocytes and macrophages in the peripheral tissues and astrocytes in the CNS being the major sources, respectively (9). ApoE derived from astrocytes is a major apolipoprotein present in the brain, exhibiting a primary function in lipid transport and supporting synapses and dendritic spines (2,4,7).…”

Retinoic Acid Isomers Facilitate Apolipoprotein E Production and Lipidation in Astrocytes through the Retinoid X Receptor/Retinoic Acid Receptor Pathway

“…This cell line was generated from primary astrocytes derived from human APOE-TR mice, in which human APOE3 gene is knocked in the mouse Apoe locus (23). All-trans-RA, 9-cis-RA, 13-cis-RA, bexarotene, all-trans-retinal, 9-cis-retinal, 13-cis-retinal, TO901317, HX531, AGN193109, and geranylgeranyl pyrophosphate (GGPP) were purchased from Sigma.…”