ABSTRACT
The varepsilon4 allele of apolipoprotein E (apoE) is a genetic risk factor for Alzheimer's disease (AD). In order to gain a better understanding of the molecular mechanisms by which apoE and possibly other apolipoproteins produced in the central nervous system (CNS) influence AD pathogenesis, we have purified 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 columns, 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 diameter with a peak size range of approximately 11 nm. ApoE and apoJ were in distinct lipoproteins. Utilization of quick-freeze, deep-etch electron microscopy revealed the apoE particles were discs while the apoJ particles were smaller and more irregular in appearance. The lipid composition of apoE particles was very different from those containing apoJ. ApoE-particles contained a similar mass of apoE and lipid, with cholesterol and phospholipid being about equal in mass per particle. ApoJ-particles were relatively lipid poor (three parts protein, one part lipid), with phospholipids being much more abundant than cholesterol. Detailed characterization of phospholipid composition by electrospray ionization mass spectrometry analysis revealed ethanolamine glycerophospholipids to be the most abundant phospholipid present in both apoE and apoJ particles. Analysis of cerebrospinal fluid from apoE3 and apoE4 transgenic mice revealed that human and mouse apoE were in particles the same size as those secreted by astrocytes. Further use of physiological preparations of CNS-derived lipoproteins may allow for a detailed understanding of the role of these molecules in the normal brain and in diseases such as AD.
Subject(s)
Apolipoproteins E/analysis , Astrocytes/metabolism , Glycoproteins/analysis , Lipoproteins/chemistry , Lipoproteins/isolation & purification , Molecular Chaperones/analysis , Animals , Apolipoproteins E/cerebrospinal fluid , Apolipoproteins E/chemistry , Cells, Cultured , Clusterin , Glycoproteins/cerebrospinal fluid , Glycoproteins/chemistry , Humans , Lipids/analysis , Lipoproteins, HDL/chemistry , Mice , Mice, Transgenic , Molecular Chaperones/cerebrospinal fluid , Molecular Chaperones/chemistry , Particle Size , Phospholipids/analysis , Reference ValuesABSTRACT
Clusterin, also known as apolipoprotein J, is a ubiquitously expressed molecule thought to influence a variety of processes including cell death. In the brain, it accumulates in dying neurons following seizures and hypoxic-ischemic (H-I) injury. Despite this, in vivo evidence that clusterin directly influences cell death is lacking. Following neonatal H-I brain injury in mice (a model of cerebral palsy), there was evidence of apoptotic changes (neuronal caspase-3 activation), as well as accumulation of clusterin in dying neurons. Clusterin-deficient mice had 50% less brain injury following neonatal H-I. Surprisingly, the absence of clusterin had no effect on caspase-3 activation, and clusterin accumulation and caspase-3 activation did not colocalize to the same cells. Studies with cultured cortical neurons demonstrated that exogenous purified astrocyte-secreted clusterin exacerbated oxygen/glucose-deprivation-induced necrotic death. These results indicate that clusterin may be a new therapeutic target to modulate non-caspase-dependent neuronal death following acute brain injury.
