ApoE and clusterin cooperatively suppress Abeta levels and deposition: evidence that ApoE regulates extracellular Abeta metabolism in vivo.

Apolipoprotein E (apoE) and clusterin can influence structure, toxicity, and accumulation of the amyloid-beta (Abeta) peptide in brain. Both molecules may also be involved in Abeta metabolism prior to its deposition. To assess this possibility, we compared PDAPP transgenic mice that develop age-dependent Abeta accumulation in the absence of apoE or clusterin as well as in the absence of both proteins. apoE(-/-) and clusterin(-/-) mice accumulated similar Abeta levels but much less fibrillar Abeta. In contrast, apoE(-/-)/clusterin(-/-) mice had both earlier onset and markedly increased Abeta and amyloid deposition. Both apoE(-/-) and apoE(-/-)/clusterin(-/-) mice had elevated CSF and brain interstitial fluid Abeta, as well as significant differences in the elimination half-life of interstitial fluid Abeta measured by in vivo microdialysis. These findings demonstrate additive effects of apoE and clusterin on influencing Abeta deposition and that apoE plays an important role in regulating extracellular CNS Abeta metabolism independent of Abeta synthesis.

In vivo assessment of brain interstitial fluid with microdialysis reveals plaque-associated changes in amyloid-beta metabolism and half-life.

Soluble amyloid-beta (Abeta) peptide converts to structures with high beta-sheet content in Alzheimer's disease (AD). Soluble Abeta is released by neurons into the brain interstitial fluid (ISF), in which it can convert into toxic aggregates. Because assessment of ISF Abeta levels may provide unique insights into Abeta metabolism and AD, an in vivo microdialysis technique was developed to measure it. Our Abeta microdialysis technique was validated ex vivo with human CSF and then in vivo in awake, freely moving mice. Using human amyloid precursor protein (APP) transgenic mice, we found that, before the onset of AD-like pathology, ISF Abeta in hippocampus and cortex correlated with levels of APP in those tissues. After the onset of Abeta deposition, significant changes in the ISF Abeta40/Abeta42 ratio developed without changes in Abeta1-x. These changes differed from changes seen in tissue lysates from the same animals. By rapidly inhibiting Abeta production, we found that ISF Abeta half-life was short ( approximately 2 hr) in young mice but was twofold longer in mice with Abeta deposits. This increase in half-life, without an increase in steady-state levels, suggests that inhibition of Abeta synthesis reveals a portion of the insoluble Abeta pool that is in dynamic equilibrium with ISF Abeta. This now measurable in vivo pool is a likely target for new diagnostic and therapeutic strategies.

The low density lipoprotein receptor regulates the level of central nervous system human and murine apolipoprotein E but does not modify amyloid plaque pathology in PDAPP mice.

Apolipoprotein E (apoE), a chaperone for the amyloid beta (Abeta) peptide, regulates the deposition and structure of Abeta that deposits in the brain in Alzheimer disease (AD). The primary apoE receptor that regulates levels of apoE in the brain is unknown. We report that the low density lipoprotein receptor (LDLR) regulates the cellular uptake and central nervous system levels of astrocyte-derived apoE. Cells lacking LDLR were unable to appreciably endocytose astrocyte-secreted apoE-containing lipoprotein particles. Moreover, cells overexpressing LDLR showed a dramatic increase in apoE endocytosis and degradation. We also found that LDLR knock-out (Ldlr-/-) mice had a significant, approximately 50% increase in the level of apoE in the cerebrospinal fluid and extracellular pools of the brain. However, when the PDAPP mouse model of AD was bred onto an Ldlr-/- background, we did not observe a significant change in brain Abeta levels either before or after the onset of Abeta deposition. Interestingly, human APOE3 or APOE4 (but not APOE2) knock-in mice bred on an Ldlr-/- background had a 210% and 380% increase, respectively, in the level of apoE in cerebrospinal fluid. These results demonstrate that central nervous system levels of both human and murine apoE are directly regulated by LDLR. Although the increase in murine apoE caused by LDLR deficiency was not sufficient to affect Abeta levels or deposition by 10 months of age in PDAPP mice, it remains a possibility that the increase in human apoE3 and apoE4 levels caused by LDLR deficiency will affect this process and could hold promise for therapeutic targets in AD.

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

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-beta. Under the same conditions, only a small fraction of A beta formed sodium dodecyl sulfate (SDS)-stable complexes with apoE (E3 > E4). These immortalized astrocytes will be useful for studying mechanisms underlying the isoform-specific effects of apoE in the CNS.

Plaque-associated disruption of CSF and plasma amyloid-beta (Abeta) equilibrium in a mouse model of Alzheimer's disease.

To better understand amyloid-beta (Abeta) metabolism in vivo, we assessed the concentration of Abeta in the CSF and plasma of APP(V717F) (PDAPP) transgenic mice, a model that develops age-dependent Alzheimer's disease (AD)-like pathology. In 3-month-old mice, prior to the development of Abeta deposition in the brain, there was a highly significant correlation between Abeta levels in CSF and plasma. In 9-month-old-mice, an age at which some but not all mice have developed Abeta deposition, there was also a significant correlation between CSF and plasma Abeta; however, the correlation was not as strong as that present in young mice. In further exploring CSF and plasma Abeta levels in 9-month-old mice, levels of CSF Abeta were found to correlate highly with Abeta burden. Analysis of the CSF: plasma Abeta ratio revealed a selective two-fold increase in plaque versus non-plaque bearing mice, strongly suggesting a plaque-mediated sequestration of soluble Abeta in brain. Interestingly, in 9-month-old mice, a significant correlation between CNS and plasma Abeta was limited to mice lacking Abeta deposition. These findings suggest that there is a dynamic equilibrium between CNS and plasma Abeta, and that plaques create a new equilibrium because soluble CNS Abeta not only enters the plasma but also deposits onto amyloid plaques in the CNS.

Clusterin promotes amyloid plaque formation and is critical for neuritic toxicity in a mouse model of Alzheimer's disease.

Studies have shown that clusterin (also called apolipoprotein J) can influence the structure and toxicity of amyloid-beta (Abeta) in vitro. To determine whether endogenous clusterin plays a role in influencing Abeta 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 Abeta deposition as did PDAPP, clusterin(+/+) mice. Although Abeta deposition was similar, PDAPP, clusterin(-/-) mice had significantly fewer fibrillar Abeta (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 Abeta structure and neuritic toxicity in vivo and is likely to play an important role in Alzheimer's disease pathogenesis.