Takahisa Kanekiyo

Apolipoprotein E and Alzheimer disease: risk, mechanisms and therapy

Apolipoprotein E (Apo-E) is a major cholesterol carrier that supports lipid transport and injury repair in the brain. APOE polymorphic alleles are the main genetic determinants of Alzheimer disease (AD) risk: individuals carrying the ε4 allele are at increased risk of AD compared with those carrying the more common ε3 allele, whereas the ε2 allele decreases risk. Presence of the APOE ε4 allele is also associated with increased risk of cerebral amyloid angiopathy and age-related cognitive decline during normal ageing. Apo-E–lipoproteins bind to several cell-surface receptors to deliver lipids, and also to hydrophobic amyloid-β (Aβ) peptide, which is thought to initiate toxic events that lead to synaptic dysfunction and neurodegeneration in AD. Apo-E isoforms differentially regulate Aβ aggregation and clearance in the brain, and have distinct functions in regulating brain lipid transport, glucose metabolism, neuronal signalling, neuroinflammation, and mitochondrial function. In this Review, we describe current knowledge on Apo-E in the CNS, with a particular emphasis on the clinical and pathological features associated with carriers of different Apo-E isoforms. We also discuss Aβ-dependent and Aβ-independent mechanisms that link Apo-E4 status with AD risk, and consider how to design effective strategies for AD therapy by targeting Apo-E.

ApoE and Aβ in Alzheimer’s disease: accidental encounters or partners?

Among the three human apolipoprotein E (apoE) isoforms, apoE4 increases the risk of Alzheimers disease (AD). While transporting cholesterol is a primary function, apoE also regulates amyloid-β (Aβ) metabolism, aggregation, and deposition. Although earlier work suggests that different affinities of apoE isoforms to Aβ might account for their effects on Aβ clearance, recent studies indicate that apoE also competes with Aβ for cellular uptake through apoE receptors. Thus, several factors probably determine the variable effects apoE has on Aβ. In this Review, we examine biochemical, structural, and functional studies and propose testable models that address the complex mechanisms underlying apoE-Aβ interaction and how apoE4 may increase AD risk and also serve as a target pathway for therapy.

Central role for PICALM in amyloid-β blood-brain barrier transcytosis and clearance

PICALM is a highly validated genetic risk factor for Alzheimers disease (AD). We found that reduced expression of PICALM in AD and murine brain endothelium correlated with amyloid-β (Aβ) pathology and cognitive impairment. Moreover, Picalm deficiency diminished Aβ clearance across the murine blood-brain barrier (BBB) and accelerated Aβ pathology in a manner that was reversible by endothelial PICALM re-expression. Using human brain endothelial monolayers, we found that PICALM regulated PICALM/clathrin-dependent internalization of Aβ bound to the low density lipoprotein receptor related protein-1, a key Aβ clearance receptor, and guided Aβ trafficking to Rab5 and Rab11, leading to Aβ endothelial transcytosis and clearance. PICALM levels and Aβ clearance were reduced in AD-derived endothelial monolayers, which was reversible by adenoviral-mediated PICALM transfer. Inducible pluripotent stem cell–derived human endothelial cells carrying the rs3851179 protective allele exhibited higher PICALM levels and enhanced Aβ clearance. Thus, PICALM regulates Aβ BBB transcytosis and clearance, which has implications for Aβ brain homeostasis and clearance therapy.

LRP1 in brain vascular smooth muscle cells mediates local clearance of Alzheimer's amyloid-β.

Impaired clearance of amyloid-β (Aβ) is a major pathogenic event for Alzheimers disease (AD). Aβ depositions in brain parenchyma as senile plaques and along cerebrovasculature as cerebral amyloid angiopathy (CAA) are hallmarks of AD. A major pathway that mediates brain Aβ clearance is the cerebrovascular system where Aβ is eliminated through the blood–brain barrier (BBB) and/or degraded by cerebrovascular cells along the interstitial fluid drainage pathway. An Aβ clearance receptor, the low-density lipoprotein receptor-related protein 1 (LRP1), is abundantly expressed in cerebrovasculature, in particular in vascular smooth muscle cells. Previous studies have indicated a role of LRP1 in endothelial cells in transcytosing Aβ out of the brain across the BBB; however, whether this represents a significant pathway for brain Aβ clearance remains controversial. Here, we demonstrate that Aβ can be cleared locally in the cerebrovasculature by an LRP1-dependent endocytic pathway in smooth muscle cells. The uptake and degradation of both endogenous and exogenous Aβ were significantly reduced in LRP1-suppressed human brain vascular smooth muscle cells. Conditional deletion of Lrp1 in vascular smooth muscle cell in amyloid model APP/PS1 mice accelerated brain Aβ accumulation and exacerbated Aβ deposition as amyloid plaques and CAA without affecting Aβ production. Our results demonstrate that LRP1 is a major Aβ clearance receptor in cerebral vascular smooth muscle cell and a disturbance of this pathway contributes to Aβ accumulation. These studies establish critical functions of the cerebrovasculature system in Aβ metabolism and identify a new pathway involved in the pathogenesis of both AD and CAA.

Neuronal Clearance of Amyloid-β by Endocytic Receptor LRP1

Alzheimers disease (AD) is the most prevalent form of dementia in the elderly population. Accumulation, aggregation, and deposition of amyloid-β (Aβ) peptides generated through proteolytic cleavage of amyloid precursor protein (APP) are likely initiating events in the pathogenesis of AD. While Aβ production is accelerated in familial AD, increasing evidence indicates that impaired clearance of Aβ is responsible for late-onset AD. Because Aβ is mainly generated in neurons, these cells are predicted to have the highest risk of encountering Aβ among all cell types in the brain. However, it is still unclear whether they are also involved in Aβ clearance. Here we show that receptor-mediated endocytosis in neurons by the low-density lipoprotein receptor-related protein 1 (LRP1) plays a critical role in brain Aβ clearance. LRP1 is known to be an endocytic receptor for multiple ligands including Aβ. Conditional knock-out of Lrp1 in mouse forebrain neurons leads to increased brain Aβ levels and exacerbated amyloid plaque deposition selectively in the cortex of amyloid model APP/PS1 mice without affecting Aβ production. In vivo microdialysis studies demonstrated that Aβ clearance in brain interstitial fluid is impaired in neuronal Lrp1 knock-out mice. Because the neuronal LRP1-deletion did not affect the mRNA levels of major Aβ degrading enzymes, neprilysin and insulin-degrading enzyme, the disturbed Aβ clearance is likely due to the suppression of LRP1-mediated neuronal Aβ uptake and degradation. Together, our results demonstrate that LRP1 plays an important role in receptor-mediated clearance of Aβ and indicate that neurons not only produce but also clear Aβ.

APOE4-specific changes in Aβ accumulation in a new transgenic mouse model of Alzheimer disease.

Background: APOE genotype effects on Aβ accumulation were determined using new EFAD transgenic mice. Results: In E4FAD mice, compact plaques are greater, total apoE4 is lower, less apoE4 is lipoprotein-associated, and oligomeric Aβ is higher compared with E2FAD/E3FAD, while intraneuronal Aβ is unaffected. Conclusion: APOE4 uniquely effects Aβ accumulation. Significance: These data provide a basis for APOE-induced AD risk. APOE4 is the greatest risk factor for Alzheimer disease (AD) and synergistic effects with amyloid-β peptide (Aβ) suggest interactions among apoE isoforms and different forms of Aβ accumulation. However, it remains unclear how the APOE genotype affects plaque morphology, intraneuronal Aβ, soluble Aβ42, and oligomeric Aβ (oAβ), particularly in vivo. As the introduction of human APOE significantly delays amyloid deposition in transgenic mice expressing familial AD (FAD) mutations (FAD-Tg), 5xFAD-Tg mice, which exhibit amyloid deposition by age 2 months, were crossed with apoE-targeted replacement mice to produce the new EFAD-Tg mice. Compared with 5xFAD mice, Aβ deposition was delayed by ∼4 months in the EFAD mice, allowing detection of early changes in Aβ accumulation from 2–6 months. Although plaque deposition is generally greater in E4FAD mice, E2/E3FAD mice have significantly more diffuse and E4FAD more compact plaques. As a first report in FAD-Tg mice, the APOE genotypes had no effect on intraneuronal Aβ accumulation in EFAD mice. In E4FAD mice, total apoE levels were lower and total Aβ levels higher than in E2FAD and E3FAD mice. Profiles from sequential three-step extractions (TBS, detergent, and formic acid) demonstrated that the lower level of total apoE4 is reflected only in the detergent-soluble fraction, indicating that less apoE4 is lipoprotein-associated, and perhaps less lipidated, compared with apoE2 and apoE3. Soluble Aβ42 and oAβ levels were highest in E4FAD mice, although soluble apoE2, apoE3, and apoE4 levels were comparable, suggesting that the differences in soluble Aβ42 and oAβ result from functional differences among the apoE isoforms. Thus, APOE differentially regulates multiple aspects of Aβ accumulation.

Differential Regulation of Amyloid-β Endocytic Trafficking and Lysosomal Degradation by Apolipoprotein E Isoforms

Background: Apolipoprotein E (apoE) regulates amyloid-β (Aβ) clearance in an isoform-dependent manner. Results: Internalized Aβ traffics to lysosomal and recycling pathways. ApoE3 more efficiently promotes Aβ lysosomal trafficking and degradation than apoE4. Conclusion: ApoE isoforms differentially affect Aβ lysosomal trafficking and degradation. Significance: Differential effects of apoE isoforms on Aβ cellular degradation may explain why apoE4 is a risk factor for Alzheimer disease. Aggregation of amyloid-β (Aβ) peptides leads to synaptic disruption and neurodegeneration in Alzheimer disease (AD). A major Aβ clearance pathway in the brain is cellular uptake and degradation. However, how Aβ traffics through the endocytic pathway and how AD risk factors regulate this event is unclear. Here we show that the majority of endocytosed Aβ in neurons traffics through early and late endosomes to the lysosomes for degradation. Overexpression of Rab5 or Rab7, small GTPases that function in vesicle fusion for early and late endosomes, respectively, significantly accelerates Aβ endocytic trafficking to the lysosomes. We also found that a portion of endocytosed Aβ traffics through Rab11-positive recycling vesicles. A blockage of this Aβ recycling pathway with a constitutively active Rab11 mutant significantly accelerates cellular Aβ accumulation. Inhibition of lysosomal enzymes results in Aβ accumulation and aggregation. Importantly, apolipoprotein E (apoE) accelerates neuronal Aβ uptake, lysosomal trafficking, and degradation in an isoform-dependent manner with apoE3 more efficiently facilitating Aβ trafficking and degradation than apoE4, a risk factor for AD. Taken together, our results demonstrate that Aβ endocytic trafficking to lysosomes for degradation is a major Aβ clearance pathway that is differentially regulated by apoE isoforms. A disturbance of this pathway can lead to accumulation and aggregation of cellular Aβ capable of causing neurotoxicity and seeding amyloid.

Intraneuronal Aβ detection in 5xFAD mice by a new Aβ-specific antibody

BackgroundThe form(s) of amyloid-β peptide (Aβ) associated with the pathology characteristic of Alzheimers disease (AD) remains unclear. In particular, the neurotoxicity of intraneuronal Aβ accumulation is an issue of considerable controversy; even the existence of Aβ deposits within neurons has recently been challenged by Winton and co-workers. These authors purport that it is actually intraneuronal APP that is being detected by antibodies thought to be specific for Aβ. To further address this issue, an anti-Aβ antibody was developed (MOAB-2) that specifically detects Aβ, but not APP. This antibody allows for the further evaluation of the early accumulation of intraneuronal Aβ in transgenic mice with increased levels of human Aβ in 5xFAD and 3xTg mice.ResultsMOAB-2 (mouse IgG2b) is a pan-specific, high-titer antibody to Aβ residues 1-4 as demonstrated by biochemical and immunohistochemical analyses (IHC), particularly compared to 6E10 (a commonly used commercial antibody to Aβ residues 3-8). MOAB-2 did not detect APP or APP-CTFs in cell culture media/lysates (HEK-APPSwe or HEK-APPSwe/BACE1) or in brain homogenates from transgenic mice expressing 5 familial AD (FAD) mutation (5xFAD mice). Using IHC on 5xFAD brain tissue, MOAB-2 immunoreactivity co-localized with C-terminal antibodies specific for Aβ40 and Aβ42. MOAB-2 did not co-localize with either N- or C-terminal antibodies to APP. In addition, no MOAB-2-immunreactivity was observed in the brains of 5xFAD/BACE-/- mice, although significant amounts of APP were detected by N- and C-terminal antibodies to APP, as well as by 6E10. In both 5xFAD and 3xTg mouse brain tissue, MOAB-2 co-localized with cathepsin-D, a marker for acidic organelles, further evidence for intraneuronal Aβ, distinct from Aβ associated with the cell membrane. MOAB-2 demonstrated strong intraneuronal and extra-cellular immunoreactivity in 5xFAD and 3xTg mouse brain tissues.ConclusionsBoth intraneuronal Aβ accumulation and extracellular Aβ deposition was demonstrated in 5xFAD mice and 3xTg mice with MOAB-2, an antibody that will help differentiate intracellular Aβ from APP. However, further investigation is required to determine whether a molecular mechanism links the presence of intraneuronal Aβ with neurotoxicity. As well, understanding the relevance of these observations to human AD patients is critical.

Deficiency in LRP6-Mediated Wnt Signaling Contributes to Synaptic Abnormalities and Amyloid Pathology in Alzheimer’s Disease

Alzheimers disease (AD) is an age-related neurological disorder characterized by synaptic loss and dementia. The low-density lipoprotein receptor-related protein 6 (LRP6) is an essential coreceptor for Wnt signaling, and its genetic variants have been linked to AD risk. Here we report that neuronal LRP6-mediated Wnt signaling is critical for synaptic function and cognition. Conditional deletion of Lrp6 gene in mouse forebrain neurons leads to age-dependent deficits in synaptic integrity and memory. Neuronal LRP6 deficiency in an amyloid mouse model also leads to exacerbated amyloid pathology due to increased APP processing to amyloid-β. In humans, LRP6 and Wnt signaling are significantly downregulated in AD brains, likely by a mechanism that depends on amyloid-β. Our results define a critical pathway in which decreased LRP6-mediated Wnt signaling, synaptic dysfunction, and elevated Aβ synergistically accelerate AD progression and suggest that restoring LRP6-mediated Wnt signaling can be explored as a viable strategy for AD therapy.

ABCA7 Deficiency Accelerates Amyloid-β Generation and Alzheimer's Neuronal Pathology

In Alzheimers disease (AD), the accumulation and deposition of amyloid-β (Aβ) peptides in the brain is a central event. Aβ is cleaved from amyloid precursor protein (APP) by β-secretase and γ-secretase mainly in neurons. Although mutations in APP, PS1, or PS2 cause early-onset familial AD, ABCA7 encoding ATP-binding cassette transporter A7 is one of the susceptibility genes for late-onset AD (LOAD), in which its loss-of-function variants increase the disease risk. ABCA7 is homologous to a major lipid transporter ABCA1 and is highly expressed in neurons and microglia in the brain. Here, we show that ABCA7 deficiency altered brain lipid profile and impaired memory in ABCA7 knock-out (Abca7−/−) mice. When bred to amyloid model APP/PS1 mice, plaque burden was exacerbated by ABCA7 deficit. In vivo microdialysis studies indicated that the clearance rate of Aβ was unaltered. Interestingly, ABCA7 deletion facilitated the processing of APP to Aβ by increasing the levels of β-site APP cleaving enzyme 1 (BACE1) and sterol regulatory element-binding protein 2 (SREBP2) in primary neurons and mouse brains. Knock-down of ABCA7 expression in neurons caused endoplasmic reticulum stress highlighted by increased level of protein kinase R-like endoplasmic reticulum kinase (PERK) and increased phosphorylation of eukaryotic initiation factor 2α (eIF2α). In the brains of APP/PS1;Abca7−/− mice, the level of phosphorylated extracellular regulated kinase (ERK) was also significantly elevated. Together, our results reveal novel pathways underlying the association of ABCA7 dysfunction and LOAD pathogenesis. SIGNIFICANCE STATEMENT Gene variants in ABCA7 encoding ATP-binding cassette transporter A7 are associated with the increased risk for late-onset Alzheimers disease (AD). Importantly, we found the altered brain lipid profile and impaired memory in ABCA7 knock-out mice. The accumulation of amyloid-β (Aβ) peptides cleaved from amyloid precursor protein (APP) in the brain is a key event in AD pathogenesis and we also found that ABCA7 deficit exacerbated brain Aβ deposition in amyloid AD model APP/PS1 mice. Mechanistically, we found that ABCA7 deletion facilitated the processing of APP and Aβ production by increasing the levels of β-secretase 1 (BACE1) in primary neurons and mouse brains without affecting the Aβ clearance rate in APP/PS1 mice. Our study demonstrates a novel mechanism underlying how dysfunctions of ABCA7 contribute to the risk for AD.