Age-Dependent Effects of apoE Reduction Using Antisense Oligonucleotides in a Model of ß-amyloidosis.

The apolipoprotein E (APOE) gene is the strongest genetic risk factor for late-onset Alzheimer disease. Previous studies suggest that reduction of apoE levels through genetic manipulation can reduce Aß pathology. However, it is not clear how reduction of apoE levels after birth would affect amyloid deposition. We utilize an antisense oligonucleotide (ASO) to reduce apoE expression in the brains of APP/PS1-21 mice homozygous for the APOE-ε4 or APOE-ε3 allele. ASO treatment starting after birth led to a significant decrease in Aß pathology when assessed at 4 months. Interestingly, ASO treatment starting at the onset of amyloid deposition led to an increase in Aß plaque size and a reduction in plaque-associated neuritic dystrophy with no change in overall plaque load. These results suggest that lowering apoE levels prior to plaque deposition can strongly affect the initiation of Aß pathology while lowering apoE after Aß seeding modulates plaque size and toxicity.

Anti-apoE immunotherapy inhibits amyloid accumulation in a transgenic mouse model of Aß amyloidosis.

The apolipoprotein E (APOE) ε4 allele is the strongest genetic risk factor for Alzheimer's disease (AD). The influence of apoE on amyloid ß (Aß) accumulation may be the major mechanism by which apoE affects AD. ApoE interacts with Aß and facilitates Aß fibrillogenesis in vitro. In addition, apoE is one of the protein components in plaques. We hypothesized that certain anti-apoE antibodies, similar to certain anti-Aß antibodies, may have antiamyloidogenic effects by binding to apoE in the plaques and activating microglia-mediated amyloid clearance. To test this hypothesis, we developed several monoclonal anti-apoE antibodies. Among them, we administered HJ6.3 antibody intraperitoneally to 4-mo-old male APPswe/PS1ΔE9 mice weekly for 14 wk. HJ6.3 dramatically decreased amyloid deposition by 60-80% and significantly reduced insoluble Aß40 and Aß42 levels. Short-term treatment with HJ6.3 resulted in strong changes in microglial responses around Aß plaques. Collectively, these results suggest that anti-apoE immunization may represent a novel AD therapeutic strategy and that other proteins involved in Aß binding and aggregation might also be a target for immunotherapy. Our data also have important broader implications for other amyloidosis. Immunotherapy to proteins tightly associated with misfolded proteins might open up a new treatment option for many protein misfolding diseases.

Ultrastructural studies in APP/PS1 mice expressing human ApoE isoforms: implications for Alzheimer's disease.

Alzheimer's disease is characterized in part by extracellular aggregation of the amyloid-ß peptide in the form of diffuse and fibrillar plaques in the brain. Electron microscopy (EM) has made an important contribution in understanding of the structure of amyloid plaques in humans. Classical EM studies have revealed the architecture of the fibrillar core, characterized the progression of neuritic changes, and have identified the neurofibrillary tangles formed by paired helical filaments (PHF) in degenerating neurons. Clinical data has strongly correlated cognitive impairment in AD with the substantial synapse loss observed in these early ultrastructural studies. Animal models of AD-type brain amyloidosis have provided excellent opportunities to study amyloid and neuritic pathology in detail and establish the role of neurons and glia in plaque formation. Transgenic mice overexpressing mutant amyloid precursor protein (APP) alone with or without mutant presenilin 1 (PS1), have shown that brain amyloid plaque development and structure grossly recapitulate classical findings in humans. Transgenic APP/PS1 mice expressing human apolioprotein E isoforms also develop amyloid plaque deposition. However no ultrastructural data has been reported for these animals. Here we show results from detailed EM analysis of amyloid plaques in APP/PS1 mice expressing human isoforms of ApoE and compare these findings with EM data in other transgenic models and in human AD. Our results show that similar to other transgenic animals, APP/PS1 mice expressing human ApoE isoforms share all major cellular and subcellular degenerative features and highlight the identity of the cellular elements involved in Aß deposition and neuronal degeneration.

Low-density lipoprotein receptor overexpression enhances the rate of brain-to-blood Aß clearance in a mouse model of ß-amyloidosis.

The apolipoprotein E (APOE)-ε4 allele is the strongest genetic risk factor for late-onset, sporadic Alzheimer's disease, likely increasing risk by altering amyloid-ß (Aß) accumulation. We recently demonstrated that the low-density lipoprotein receptor (LDLR) is a major apoE receptor in the brain that strongly regulates amyloid plaque deposition. In the current study, we sought to understand the mechanism by which LDLR regulates Aß accumulation by altering Aß clearance from brain interstitial fluid. We hypothesized that increasing LDLR levels enhances blood-brain barrier-mediated Aß clearance, thus leading to reduced Aß accumulation. Using the brain Aß efflux index method, we found that blood-brain barrier-mediated clearance of exogenously administered Aß is enhanced with LDLR overexpression. We next developed a method to directly assess the elimination of centrally derived, endogenous Aß into the plasma of mice using an anti-Aß antibody that prevents degradation of plasma Aß, allowing its rate of appearance from the brain to be measured. Using this plasma Aß accumulation technique, we found that LDLR overexpression enhances brain-to-blood Aß transport. Together, our results suggest a unique mechanism by which LDLR regulates brain-to-blood Aß clearance, which may serve as a useful therapeutic avenue in targeting Aß clearance from the brain.

Bidirectional relationship between functional connectivity and amyloid-ß deposition in mouse brain.

Brain region-specific deposition of extracellular amyloid plaques principally composed of aggregated amyloid-ß (Aß) peptide is a pathological signature of Alzheimer's disease (AD). Recent human neuroimaging data suggest that resting-state functional connectivity strength is reduced in patients with AD, cognitively normal elderly harboring elevated amyloid burden, and in advanced aging. Interestingly, there exists a striking spatial correlation between functional connectivity strength in cognitively normal adults and the location of Aß plaque deposition in AD. However, technical limitations have heretofore precluded examination of the relationship between functional connectivity, Aß deposition, and normal aging in mouse models. Using a novel functional connectivity optical intrinsic signal (fcOIS) imaging technique, we demonstrate that Aß deposition is associated with significantly reduced bilateral functional connectivity in multiple brain regions of older APP/PS1 transgenic mice. The amount of Aß deposition in each brain region was associated with the degree of local, age-related bilateral functional connectivity decline. Normal aging was associated with reduced bilateral functional connectivity specifically in retrosplenial cortex. Furthermore, we found that the magnitude of regional bilateral functional correlation in young APP/PS1 mice before Aß plaque formation was proportional to the amount of region-specific plaque deposition seen later in older APP/PS1 mice. Together, these findings suggest that Aß deposition and normal aging are associated with region-specific disruption of functional connectivity and that the magnitude of local bilateral functional connectivity predicts regional vulnerability to subsequent Aß deposition in mouse brain.

Haploinsufficiency of human APOE reduces amyloid deposition in a mouse model of amyloid-ß amyloidosis.

The ε4 allele of the apolipoprotein E (APOE) gene is the strongest genetic risk factor for Alzheimer's disease (AD). Evidence suggests that the effect of apoE isoforms on amyloid-ß (Aß) accumulation in the brain plays a critical role in AD pathogenesis. Like in humans, apoE4 expression in animal models that develop Aß amyloidosis results in greater Aß and amyloid deposition than with apoE3 expression. However, whether decreasing levels of apoE3 or apoE4 would promote or attenuate Aß-related pathology has not been directly addressed. To determine the effect of decreasing human apoE levels on Aß accumulation in vivo, we generated human APOE isoform haploinsufficient mouse models by crossing APPPS1-21 mice with APOE isoform knock-in mice. By genetically manipulating APOE gene dosage, we demonstrate that decreasing human apoE levels, regardless of isoform status, results in significantly decreased amyloid plaque deposition and microglial activation. These differences in amyloid load between apoE3- and apoE4-expressing mice were not due to apoE4 protein being present at lower levels than apoE3. These data suggest that current therapeutic strategies to increase apoE levels without altering its lipidation state may actually worsen Aß amyloidosis, while increasing apoE degradation or inhibiting its synthesis may be a more effective treatment approach.

Human apoE isoforms differentially regulate brain amyloid-ß peptide clearance.

The apolipoprotein E (APOE) ε4 allele is the strongest genetic risk factor for late-onset, sporadic Alzheimer's disease (AD). The APOE ε4 allele markedly increases AD risk and decreases age of onset, likely through its strong effect on the accumulation of amyloid-ß (Aß) peptide. In contrast, the APOE ε2 allele appears to decrease AD risk. Most rare, early-onset forms of familial AD are caused by autosomal dominant mutations that often lead to overproduction of Aß(42) peptide. However, the mechanism by which APOE alleles differentially modulate Aß accumulation in sporadic, late-onset AD is less clear. In a cohort of cognitively normal individuals, we report that reliable molecular and neuroimaging biomarkers of cerebral Aß deposition vary in an apoE isoform-dependent manner. We hypothesized that human apoE isoforms differentially affect Aß clearance or synthesis in vivo, resulting in an apoE isoform-dependent pattern of Aß accumulation later in life. Performing in vivo microdialysis in a mouse model of Aß-amyloidosis expressing human apoE isoforms (PDAPP/TRE), we find that the concentration and clearance of soluble Aß in the brain interstitial fluid depends on the isoform of apoE expressed. This pattern parallels the extent of Aß deposition observed in aged PDAPP/TRE mice. ApoE isoform-dependent differences in soluble Aß metabolism are observed not only in aged but also in young PDAPP/TRE mice well before the onset of Aß deposition in amyloid plaques in the brain. Additionally, amyloidogenic processing of amyloid precursor protein and Aß synthesis, as assessed by in vivo stable isotopic labeling kinetics, do not vary according to apoE isoform in young PDAPP/TRE mice. Our results suggest that APOE alleles contribute to AD risk by differentially regulating clearance of Aß from the brain, suggesting that Aß clearance pathways may be useful therapeutic targets for AD prevention.