Role of the Low-density Lipoprotein Receptor–Related Protein in β-Amyloid Metabolism and Alzheimer Disease

Deposition of β-amyloid (Aβ), a metabolite of approximately 4 kd of the amyloid precursor protein, is a critical pathological feature in Alzheimer disease. We postulate that deposition reflects an imbalance of Aβ synthesis and clearance. Several pathways that impact Aβ converge on a single receptor molecule, the low-density lipoprotein receptor–related protein (LRP). This multifunctional receptor is the major neuronal receptor both for apolipoprotein E (apoE, protein; APOE, gene) and for α₂-macroglobulin (α₂M, protein; A2M, gene), and it mediates clearance of apoE/Aβ and α₂M/Aβ complexes. The LRP also interacts with the amyloid precursor protein itself. In this review, we highlight data that support a role for LRP in Aβ metabolism and hypothesize that LRP therefore plays a critical role in Alzheimer disease.

The neuropathological characteristics of Alzheimer disease (AD) include the development of neurofibrillary tangles and senile plaques throughout cortical and limbic brain regions, ultimately leading to marked neuronal and synaptic loss and cortical atrophy.^1,2 Senile plaques consist of β-amyloid (Aβ), a peptide made up of 40 or 42 amino acids derived from the amyloid precursor protein (APP).³ Early-onset autosomal dominant AD is caused by mutations in APP, presenilin 1, or presenilin 2,^4-6 all of which modify Aβ synthesis,^3,7,8 suggesting a central role for Aβ in causing AD.

The early-onset autosomal dominant forms of the disease, in which the key metabolic error is a presumed alteration in Aβ synthesis, are quite rare. We hypothesize that late-onset AD, which accounts for the vast majority of all AD (and indeed of all dementia), may be caused by alterations in Aβ clearance mechanisms. The idea that Aβ clearance occurs at all is based, in part, on the observation that the total amount of Aβ (called the Aβ burden) in AD brains remains fairly constant from early in the disease until the end stage, suggesting a steady state of Aβ deposition and clearance.⁹ Detailed examination of the geometry of Aβ deposits and computer modeling of aggregation and disaggregation models support this idea.^10,11 Moreover, in vitro, several molecular mechanisms for clearance of Aβ or small Aβ aggregates have been demonstrated, including microglial clearance¹² via the macrophage scavenger receptor¹³ or the receptor for advanced glycation end products.¹⁴

Herein, we postulate that a major clearance route for Aβ is by forming complexes with 2 proteins known to bind and clear a variety of molecules: apolipoprotein E (apoE) and α₂-macroglobulin (α₂M). Both of these molecules are internalized by a common receptor, the low-density lipoprotein receptor–related protein (LRP). Several lines of evidence suggest that LRP plays a prominent role in putative Aβ clearance pathways and support the hypothesis that LRP occupies a critical position in the complex metabolic cascades that influence the balance of Aβ clearance and synthesis. We first review the LRP structure and known functions and then examine in more detail the evidence implicating 3 different LRP ligands (apoE, α₂M, and APP itself) in the disease process.

The LRP is a multifunctional receptor greater than 600 kd (4454 amino acids in length) with a single transmembrane-spanning domain expressed on the cell surface (Figure 1). It is cleaved by furin in the trans-Golgi network to form a heterodimer.¹⁵ The extracellular domain (approximately 515 kd) contains multiple epidermal growth factor and growth factor repeats and 4 distinct ligand binding sites. An 85-kd carboxy terminal domain contains 2 intracellular NPXY sites that direct endocytosis of the receptor.^16,17 In the central nervous system, the LRP is strongly expressed on neurons and is also upregulated on activated astrocytes and microglia, placing it in an ideal location to clear a variety of bioactive substances. The LRP is also found on senile plaques¹⁸(Figure 2).

The LRP has more than 20 identified ligands, many of them of import in the central nervous system. The ligands fall into several broad categories: apoE and lipid-related ligands; proteinase and proteinase inhibitor complexes (including APP containing Kunitz proteinase inhibitor, α₂M, and tissue plasminogen activator and plasminogen activator inhibitor 1 complexes); and others (eg, lactoferrin). The binding of ligands to LRP leads to endocytosis and degradation, which can be blocked by the receptor-associated protein. The receptor-associated protein is a 39-kd protein that was initially isolated with LRP and has a very high affinity for LRP.¹⁷ Used pharmacologically, the receptor-associated protein blocks binding of all known LRP ligands.^19-21

Apolipoprotein E is a small protein that contains 2 major domains: an amphipathic helical domain that binds to hydrophobic substances, and a receptor binding domain that binds members of the low-density lipoprotein receptor family,²² including LRP.²³ Immunohistochemical results showed that antibodies to apoE stained senile plaques^24,25 when data from genetic studies implicated it in the pathogenesis of AD. The APOE gene is inherited in 3 common alleles (ϵ 2, ϵ3, and ϵ4). Inheritance of the APOE4 allele is a genetic risk factor for late-onset AD (age 60 years or older) both in individuals with a family history of AD²⁶ and in the general population.^18,27-29 Heterozygosity for APOE4 increases the risk for AD compared with the common APOEϵ3/ϵ3 genotype by approximately 3-fold; APOE2 decreases the risk for AD by about half, and homozygosity changes the odds ratios to an even greater extent (see Hyman³⁰ for review).

Several hypotheses have been proposed for the role of apoE in AD. It has been implicated in clearance of debris after neuronal injury.^22,31 There are isoform-specific effects on neurite outgrowth, mediated through LRP,^32,33 and on in vitro apoE-τ complex formation.³⁴ Apolipoprotein E also can modulate Aβ fibrillogenesis, although under different conditions apoE4 produces either a promoting or inhibiting effect.^35-38 We postulated that apoE may be involved in an Aβ clearance mechanism.¹⁸ Consistent with this idea, there is a clear effect of the APOE genotype on Aβ deposition. Inheritance of APOE4 is associated with increased deposition of Aβ in plaques^18,39-41 and in congophilic amyloid angiopathy.⁴² The apoE forms a complex with Aβ both in vitro and in vivo,^26,43-45 and both apoE and LRP are associated with Aβ deposits in the AD brain (Figure 2). Importantly, cellular uptake of Aβ/apoE complexes has been demonstrated to occur through LRP in several systems.^46,47

α₂-Macroglobulin is a tetrameric complex that acts as a pan-protease inhibitor through a unique trapping mechanism. When a protease cleaves one of several amino acids in a bait region, a marked conformational alteration occurs that sterically traps the protease and makes α₂M a ligand (referred to as α₂M*) for LRP endocytosis and clearance. It has been demonstrated that α₂M* binds Aβ⁴⁸ and alters the likelihood of Aβ to cause fibrillogenesis or to display in vitro toxic effects.^49,50 It also has been shown that Aβ/α₂M* complexes can be metabolized⁵¹ or cleared via an LRP-mediated process.^52,53 One other aspect of α₂M/LRP function warrants mention. There is evidence⁵⁴ that LRP can act to enhance antigen presentation by monocytes after α₂M complexes are internalized by LRP. It is possible that LRP plays a role in presenting α₂M/Aβ complexes to the immune system. Recent data⁵⁵ suggesting that immune responses to Aβ can modulate Aβ deposition in transgenic models make this an intriguing speculation.

Recently, 2 different polymorphisms in the A2M gene have been genetically linked to increased risk for AD.^56,57 The first polymorphism is a pentanucleotide deletion near an intronic splice site postulated to alter splicing near the critical bait region. The deletion was found to be a risk factor in an analysis of a large number of sibships and small families selected for late-onset AD.⁵⁶ The second polymorphism, an Ile-Val interchange at position 1000, is near the active site. Homozygosity for the rare Val allele was associated with a modestly increased risk for AD in a large case-control study⁵⁷ using a hypothesis forming–hypothesis testing study design with 2 separate populations. The polymorphisms are not in linkage disequilibrium with one another. Other studies^58-61 have provided conflicting results, and, unlike APOE, neither polymorphism appears to be a strong risk factor for AD in the general population. We believe that the importance of these genetic associations is to reinforce the potential role of α₂M in AD pathobiology.

Recent data suggest that, in addition to the relationship between LRP and putative Aβ clearance mechanisms, there may be direct interactions between LRP and APP itself. Isoforms of APP containing Kunitz protease inhibitor (APP751 and APP770) are ligands for LRP, and APP is bound and internalized by LRP.^62,63 Whether or not this directly impacts Aβ production is not yet clear, but since recent data⁶⁴ suggest that endocytosis of APP is an important step in Aβ synthesis in some systems, it is plausible that LRP-APP interactions could affect Aβ generation. Indeed, prolonged treatment of cells with receptor-associated protein, which blocks LRP, dramatically reduces Aβ production in culture, while increasing LRP increases Aβ production.⁶⁵ Additional data implicate an indirect interaction between LRP and APP via intracellular adapter proteins. The carboxy terminal of LRP interacts with an adaptor protein, Fe65, in isolated protein coprecipitation experiments⁶⁶; Fe65 contains 2 distinct protein interaction domains; one interacts with LRP and the other is known to interact with APP.^67-71 An analogous situation has been suggested for another adapter protein called disabled.^66,72 These observations raise the possibility that LRP can modulate the intracellular trafficking of APP.

Because of the multiple relationships between LRP and AD pathobiological findings, the LRP gene has been tested as a "candidate gene" in late-onset AD. Two common polymorphisms in the LRP gene have been studied, neither of which alters the coding region of the protein: a tetranucleotide repeat in the 5′ region and a silent polymorphism in exon 3. Although data on the tetranucleotide repeat in the 5′ region of LRP are not consistent among studies,^73-76 Kang and colleagues⁷⁷ found an association between a risk for AD and a silent polymorphism in exon 3 of LRP that has been confirmed by our studies⁷⁸ and others.⁷⁹

In summary, our overarching hypothesis is that LRP plays an important role in determining the balance between Aβ synthesis and clearance mechanisms (Figure 3). A remarkable convergence of data relating AD pathophysiology and LRP supports this hypothesis: (1) LRP^18,80,81 and multiple LRP ligands are associated with senile plaques^24,81-83; (2) LRP is the primary apoE receptor in neurons, APOE is a genetic risk factor for AD, and apoE is present on senile plaques^18,25; (3) LRP can bind and clear apoE/Aβ complexes^46,47; (4) LRP is the major α₂M receptor in the brain, and A2M may be a genetic risk factor for AD^56,57; (5) LRP can bind and clear α₂M/Aβ complexes^48,49,52,53; (6) LRP interacts with isoforms of APP that contain the Kunitz protease inhibitor domain,^62,63 and blocking LRP in vitro decreases Aβ production⁶⁵; (7) the C-terminus of LRP interacts with intracellular adaptor proteins that also bind the C-terminus of APP^66,72; (8) LRP mediates the neurite outgrowth response of neurons to apoE^32,33 and α₂M⁸³; and (9) the LRP gene on chromosome 12 has been suggested as a genetic risk factor for AD.^77,78 We suggest that strategies aimed at manipulating LRP activity in the central nervous system may prove beneficial in enhancing Aβ clearance and hence altering the imbalance of Aβ synthesis and clearance that leads to Aβ deposition in late-onset AD.

Accepted for publication October 6, 1999.

Supported by grant AG12406 from the National Institutes of Health, Bethesda, Md, and a grant from the American Health Assistance Foundation, Rockville, Md.

Corresponding author: Bradley T. Hyman MD, PhD, Alzheimer Disease Research Laboratory, Massachusetts General Hospital, 149 13th St, Room 6405, Charlestown, MA 02129 (e-mail: B_Hyman@helix.mgh.harvard.edu).