Studies have shown that clusterin (also called apolipoprotein J) can influence the structure and toxicity of amyloid-␤ (A␤) in vitro. To determine whether endogenous clusterin plays a role in influencing A␤ deposition, structure, and toxicity in vivo, we bred PDAPP mice, a transgenic mouse model of Alzheimer's disease, to clusterin ؊/؊ mice. By 12 months of age, PDAPP, clusterin ؊/؊ mice had similar levels of brain A␤ deposition as did PDAPP, clusterin ؉/؉ mice. Although A␤ deposition was similar, PDAPP, clusterin ؊/؊ mice had significantly fewer fibrillar A␤ (amyloid) deposits than PDAPP mice expressing clusterin. In the absence of clusterin, neuritic dystrophy associated with the deposited amyloid was markedly reduced, resulting in a dissociation between fibrillar amyloid formation and neuritic dystrophy. These findings demonstrate that clusterin markedly influences A␤ structure and neuritic toxicity in vivo and is likely to play an important role in Alzheimer's disease pathogenesis.A myloid-␤ (A␤) peptides are predominantly 39-43 aa in length and are derived from the amyloid precursor protein (APP) through endoproteolytic cleavage. Abundant evidence suggests that the conversion of A␤ from soluble to insoluble forms in the brain is a key event in the pathogenesis of Alzheimer's disease (AD). Genetic and biochemical evidence supporting this idea is that all known mutations that cause early-onset forms of familial AD or 〈␤-related cerebral amyloid angiopathy map to three genes [APP, presenilin-1 (PS-1), and PS-2] (1, 2). Most of these mutations result in relative overproduction of A␤ 42 , a particularly amyloidogenic form of A␤, which over time increases the probability of A␤ aggregation. Some mutations in APP within the A␤-coding region that result in familial cerebral amyloid angiopathy do not seem to increase A␤ production but increase its propensity for aggregation and toxicity (3, 4). Although these mutations have given insight into a central role for A␤ in both AD and cerebral amyloid angiopathy, cases of ''sporadic,'' late-onset AD (age Ͼ60 years), which accounts for most AD cases, are not associated with increased A␤ production or altered A␤ sequence. The probability that A␤ will aggregate into different insoluble forms in the brain can be influenced by A␤-binding proteins, a process which occurs after A␤ generation. One example of a protein that seems to influence A␤ in this way is apolipoprotein E (apoE). The apoE4 isoform of apoE is the only proven genetic risk factor for both late-onset AD and cerebral amyloid angiopathy, and studies suggest that apoE influences A␤ structure, clearance, and neuritic toxicity both in vitro and in vivo with no clear effect on A␤ production (5-7). Whether apolipoproteins other than apoE influence A␤ aggregation and toxicity in vivo is unknown, although a good candidate for such effects is apolipoprotein J, also known as clusterin.The two most abundantly expressed apolipoproteins in the central nervous system that are present at similar concentrations are apoE and cluste...

The primary structure of apolipoprotein J (apoJ) was deduced by the combined strategies of protein sequencing and cDNA cloning and sequencing. ApoJ, an apolipoprotein associated with discrete subclasses of high-density lipoproteins, is encoded by a single gene in both the human and mouse genomes. ApoJ is synthesized as a 427 amino acid polypeptide that is posttranslationally cleaved at an internal bond between Arg-205 and Ser-206. The subunits of apoJ are designated apoJa, corresponding to residues 1-205, and apoJ/3, corresponding to residues 206-427. The subunits are associated through disulfide bonds.

Glycoprotein 330 (gp330) is a member of a family of endocytic receptors related to the low density lipoprotein receptor. gp330 has previously been shown to bind a number of ligands in common with its family member, the low density lipoprotein receptor-related protein (LRP). To identify ligands specific for gp330 and relevant to its localization on epithelia such as in the mammary gland, gp330-Sepharose affinity chromatography was performed. As a result, a 70-kDa protein was selected from human milk and identified by protein sequencing to be apolipoprotein J/clusterin (apoJ). Solid-phase binding assays confirmed that gp330 bound to apoJ with high affinity (Kd = 14.2 nM). Similarly, gp330 bound to apoJ transferred to nitrocellulose after SDS-polyacrylamide gel electrophoresis. LRP, however, showed no binding to apoJ in either type of assay. The binding of gp330 to apoJ could be competitively inhibited with excess apoJ as well as with the gp330 ligands apolipoprotein E, lipoprotein lipase, and the receptor-associated protein, a 39-kDa protein that acts to antagonize binding of all known ligands for gp330 and LRP. Several cultured cell lines that express gp330 and ones that do not express the receptor were examined for their ability to bind and internalize 125I-apoJ. Only cells that expressed gp330 endocytosed and degraded radiolabeled apoJ. Furthermore, F9 cells treated with retinoic acid and dibutyryl cyclic AMP to increase expression levels of gp330 displayed an increased capacity to internalize and degrade apoJ. Cellular internalization and degradation of radiolabeled apoJ could be inhibited with unlabeled apoJ, receptor-associated protein, and gp330 antibodies. The results indicate that gp330 but not LRP can bind to apoJ in vitro and that gp330 expressed by cells can mediate apoJ endocytosis leading to lysosomal degradation.

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.

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