Purification and characterization of astrocyte-secreted apolipoprotein E and J-containing lipoproteins from wild-type and human apoE transgenic mice

The epsilon4 allele of apolipoprotein E (apoE) is a genetic risk factor for Alzheimer's disease (AD). In order to gain a better understanding of the m olecular mechanisms by which apoE and possibly other apolipoproteins produc ed in the central nervous system (CNS) influence AD pathogenesis, we have p urified and characterized the two most abundant apolipoproteins produced in the CNS, apoE and apoJ. We purified apoE and apoJ from primary cultures of mouse astrocytes, which were derived from transgenic mice expressing human apoE isoforms in the absence of mouse apoE. Utilizing antibody affinity co lumns, we were able to purify both human apoE3 and apoE4, as well as mouse apoJ-containing lipoproteins. Astrocyte-secreted human apoE was present in high density-like lipoproteins of three predominant sizes ranging from 8 to 15 nm in diameter. Mouse apoJ was in particles between 10 and 17 nm in dia meter with a peak size range of similar to 11 nm. ApoE and apoJ were in dis tinct lipoproteins. Utilization of quick-freeze, deep-etch electron microsc opy revealed the apoE particles were discs while the apoJ particles were sm aller and more irregular in appearance. The lipid composition of apoE parti cles was very different from those containing apoJ. ApoE-particles containe d a similar mass of apoE and lipid. with cholesterol and phospholipid being about equal in mass per particle. ApoJ-particles were relatively lipid poo r (three parts protein. one part lipid), with phospholipids being much more abundant than cholesterol. Detailed characterization of phospholipid compo sition by electrospray ionization mass spectrometry analysis revealed ethan olamine glycerophospholipids to be the most abundant phospholipid present i n both apoE and apoJ particles. Analysis of cerebrospinal fluid from apoE3 and apoE4 transgenic mice revealed that human and mouse apoE were in partic les the same size as those secreted by astrocytes. Further use of physiolog ical preparations of CNS-derived lipoproteins may allow for a detailed unde rstanding of the role of these molecules in the normal brain and in disease s such as AD. (C), 2001 Elsevier Science Ltd. All rights reserved.