The Pathogenic Implication of Abnormal Interaction Between Apolipoprotein E Isoforms, Amyloid-beta Peptides, and Sulfatides in Alzheimer’s Disease

Alzheimer’s disease (AD) is the most common cause of dementia in the aging population. Prior work has shown that the ε4 allele of apolipoprotein E (apoE4) is a major risk factor for “sporadic” AD, which accounts for >99% of AD cases without a defined underlying mechanism. Recently, we have demonstrated that sulfatides are substantially and specifically depleted at the very early stage of AD. To identify the mechanism(s) of sulfatide loss concurrent with AD onset, we have found that: (1) sulfatides are specifically associated with apoE-associated particles in cerebrospinal fluid (CSF); (2) apoE modulates cellular sulfatide levels; and (3) the modulation of sulfatide content is apoE isoform dependent. These findings not only lead to identification of the potential mechanisms underlying sulfatide depletion at the earliest stages of AD but also serve as mechanistic links to explain the genetic association of apoE4 with AD. Moreover, our recent studies further demonstrated that (1) apoE mediates sulfatide depletion in amyloid-β precursor protein transgenic mice; (2) sulfatides enhance amyloid β (Aβ) peptides binding to apoE-associated particles; (3) Aβ42 content notably correlates with sulfatide content in CSF; (4) sulfatides markedly enhance the uptake of Aβ peptides; and (5) abnormal sulfatide-facilitated Aβ uptake results in the accumulation of Aβ in lysosomes. Collectively, our studies clearly provide a link between apoE, Aβ, and sulfatides in AD and establish a foundation for the development of effective therapeutic interventions for AD.     

β-Amyloid Peptides Increase the Binding and Internalization of Apolipoprotein E to Hippocampal Neurons

Abstract: The frequency of the ε4 allele of apolipoprotein E(apoE) is increased in late-onset and sporadic forms of Alzheimer's disease (AD). ApoE also binds to β-amyloid (Aβ) and both proteins are found in AD plaques. To further investigate the potential interaction of apoE and Aβ in the pathogenesis of AD, we have determined the binding, internalization, and degradation of human apoE isoforms in the presence and absence of Aβ peptides to rat primary hippocampal neurons. We demonstrate that the lipophilic Aβ peptides, in particular Aβ_1–42, Aβ_1–40, and Aβ_25–35, increase significantly apoE-liposome binding to hippocampal neurons. For each Aβ peptide, the increase was significantly greater for the apoE4 isoform than for the apoE3 isoform. The most effective of the Aβ peptides to increase apoE binding, Aβ_25–35, was further shown to increase significantly the internalization of both apoE3- and apoE4-liposomes, without affecting apoE degradation. Conversely, Aβ_1–40 uptake by hippocampal neurons was shown to be increased in the presence of apoE-liposomes, more so in the presence of the apoE4 than the apoE3 isoform. These results provide evidence that Aβ peptides interact directly with apoE lipoproteins, which may then be transported together into neuronal cells through apoE receptors.     

Characterization of the Binding of Amyloid-β Peptide to Cell Culture-Derived Native Apolipoprotein E2, E3, and E4 Isoforms and to Isoforms from Human Plasma

Abstract: The ε4 allele of apolipoprotein E (apoE, protein; APOE, gene) is a major risk factor for Alzheimer's disease (AD). Genetically, the frequency of the ε4 allele is enriched in early-onset sporadic, late-onset familial, and common late-onset sporadic AD. ApoE is found in the extracellular amyloid-β (Aβ) deposits that are characteristic features of AD. In this study, we examined the interaction between Aβ and apoE isoforms. The apoE isoforms used in this study were either produced by stably transfected Chinese hamster ovary cells (CHO) or were from human plasma. We report that when similar concentrations of the apoE isoforms were used, native nonpurified apoE3 from recombinant CHO-derived sources bound Aβ, but apoE4 did not. In fact, in our system, binding of recombinant apoE4 to Aβ was never detectable, even after incubation for 4 days. Furthermore, using the same assay conditions, native apoE2, like apoE3, binds Aβ avidly. Furthermore, when human plasma apoE isoforms are tested in Aβ binding experiments, apoE3 bound Aβ more avidly than apoE4, and a major apoE/Aβ complex (the 40-kDa form) was observed with plasma apoE3 but not apoE4. These data extend our understanding of apoE isoform-dependent binding of Aβ by associating apoE2 with efficient apoE/Aβ complex formation and demonstrate that native apoE3 (whether recombinant or derived from human plasma) forms sodium dodecyl sulfate-stable apoE/Aβ complexes more readily than native apoE4. The different Aβ-binding properties of native apoE4 versus native apoE3 provide insight into the molecular mechanisms by which the APOE ε4 allele exerts its risk factor effects in AD.     

Endosomes and lysosomes play distinct roles in sulfatide-induced neuroblastoma apoptosis: potential mechanisms contributing to abnormal sulfatide metabolism in related neuronal diseases

Alterations in sulfatide metabolism, trafficking and homoeostasis are present at the earliest clinically recognizable stages of Alzheimer's disease and are associated with metachromatic leukodystrophy. However, the role of sulfatide in these disease states remains unknown. In the present study, we investigated the sequelae of NB (neuroblastoma) cells upon sulfatide supplementation and the biochemical mechanisms contributing to the sulfatide-induced changes. By using shotgun lipidomics, we showed dramatic accumulations of sulfatide, ceramide and sphingosine in NB cells in a time- and dose-dependent manner. Further studies utilizing subcellular fractionation and shotgun lipidomics analyses demonstrated that most of the increased ceramide content was generated in the endosomal compartment, whereas sulfatides predominantly accumulated in lysosomes. In addition, we determined that the sulfatide-mediated increase in endosomal ceramide content mainly resulted from beta-galactosidase activity, which directly hydrolyses sulfatide to ceramide without a prior desulfation step. Substantial cell apoptosis occurred in parallel with the accumulation of sulfatides and ceramides, as revealed by mitochondrial membrane depolarization, by phosphatidylserine translocation and by the TUNEL (terminal deoxynucleotidyl transferase-mediated dUTP nick-end labelling) assay. These findings were also demonstrated with primary neuron cultures. Collectively, our results demonstrate that abnormal sulfatide metabolism can induce cell apoptosis due to endosome-mediated ceramide generation and the accumulation of cytotoxic levels of sulfatides in lysosomes.     

β-Amyloid Peptide Interacts Specifically with the Carboxyl-Terminal Domain of Human Apolipoprotein E

Abstract : Growing evidence indicates the involvement of apolipoprotein E (apoE) in the development of late-onset and sporadic forms of Alzheimer's disease, although its exact role remains unclear. We previously demonstrated that β-amyloid peptide (Aβ) displays membrane-destabilizing properties and that only apoE2 and E3 isoforms inhibit these properties. In this study, we clearly demonstrate that the carboxy-terminal lipid-binding domain of apoE (e.g., residues 200-299) is responsible for the Aβ-binding activity of apoE and that this interaction involves pairs of apoE amphipathic α-helices. We further demonstrate that Aβ is able to inhibit the association of the C-terminal domain of apoE with lipids due to the formation of Aβ/apoE complexes resistant to sodium dodecyl sulfate-polyacrylamide gel electrophoresis. On the contrary, the amino-terminal receptor-binding domain of apoE (e.g., residues 129-169) is not able to form stable complexes with Aβ. These data extend our understanding of human apoE-dependent binding of Aβ by involving the C-terminal domain of apoE in the efficient formation of apoE/Aβ complex.     

Aβ aggregation and possible implications in Alzheimer's disease pathogenesis

Amyloid β protein (Aβ) has been associated with Alzheimer's disease (AD) because it is a major component of the extracellular plaque found in AD brains. Increased Aβ levels correlate with the cognitive decline observed in AD. Sporadic AD cases are thought to be chiefly associated with lack of Aβ clearance from the brain, unlike familial AD which shows increased Aβ production. Aβ aggregation leading to deposition is an essential event in AD. However, the factors involved in Aβ aggregation and accumulation in sporadic AD have not been completely characterized. This review summarizes studies that have examined the factors that affect Aβ aggregation and toxicity. By necessity these are studies that are performed with recombinant-derived or chemically synthesized Aβ. The studies therefore are not done in animals but in cell culture, which includes neuronal cells, other mammalian cells and, in some cases, non-mammalian cells that also appear susceptible to Aβ toxicity. An understanding of Aβ oligomerization may lead to better strategies to prevent AD.     

Effect of Apolipoprotein E on Neurite Outgrowth and β-Amyloid-Induced Toxicity in Developing Rat Primary Hippocampal Cultures

Abstract: The correlation between the ε4 allele of apolipoprotein E (apoE) and Alzheimer's disease is well established. However, the role of apoE in normal as well as pathological brain processes remains unclear. We evaluated the effect of apoE treatment on development and β-amyloid (Aβ)-induced toxicity using primary cultures of developing rat hippocampal neurons. The source of apoE was conditioned media from HEK cells stably transfected with human apoE3 or apoE4 cDNA, a preparation where apoE is lipid-associated. Morphological and biochemical changes in the cultures were assessed at 1 and 3 days following low- and high-density plating with either apoE3 or E4 with or without Aβ. Both apoE isoforms were neurotrophic, as measured by increased neurite length. Aged Aβ(1–42), a peptide preparation exhibiting extensive fibril and aggregate formation, is toxic to these cultures. Addition of apoE3 and E4 significantly and comparably attenuated the Aβ-induced reduction in both neurite length and cell viability. The level of protection against this toxicity was proportional to the neurotrophic actions of the two apoE isoforms. Thus, apoE acts as a potent growth factor in both the absence and the presence of Aβ, supporting a potentially important role for apoE in neurobiology.     

Apolipoprotein E: a major piece in the Alzheimer's disease puzzle

Alzheimer's disease (AD) is a complex neurodegenerative disorder with multiple etiologies. The presence of the E4 isoform of apolipoprotein E (apoE) has been shown to increase the risk and to decrease the age of onset for AD and is the major susceptibility factor known for the disease. ApoE4 has been shown to intensify all the biochemical distrubances characteristic of AD, including beta amyloid (Aβ) deposition, tangle formation, neuronal cell death, oxidative stress, synaptic plasticity and dysfunctions of lipid homeostasis and cholinergic signalling. In contrast, other apoE isoforms are protective. Here we review and discuss these major hypotheses of the apoE4-AD association.     

Apolipoprotein E Attenuates β-Amyloid-Induced Astrocyte Activation

Abstract: A common feature of Alzheimer's disease pathology is an abundance of activated glia, indicative of an inflammatory reaction in the brain. The relationship between glial activation and neurodegeneration is not known, although several cytokines and inflammatory mediators produced by activated glia have the potential to initiate or exacerbate the progression of neuropathology. As β-amyloid (Aβ) is one of several stimuli that can activate glia, it is important to determine how Aβ-induced glial activation is influenced by other proteins present in the plaque, such as apolipoprotein E (apoE). We examined the effect of native preparations of apoE on activation of rat cortical astrocyte cultures by Aβ1–42. The apoE source was conditioned medium from human embryonic kidney 293 cells stably transfected with human apoE3 or apoE4 cDNA. By morphological criteria, apoE inhibited Aβ-induced astrocyte activation in three experimental paradigms: apoE pretreatment blocked subsequent Aβ-induced activation, Aβ aged in the presence of apoE did not activate astrocytes, and apoE addition to activated astrocytes transiently reversed the activated phenotype. No apoE isoform selectivity was observed. The effect of apoE appears to be specific to Aβ, as apoE did not attenuate cyclic AMP-induced astrocyte activation. These data suggest that apoE may modulate the ability of Aβ to induce inflammatory responses in the brain.     

Aβ43 is more frequent than Aβ40 in amyloid plaque cores from Alzheimer disease brains

One hallmark of Alzheimer disease (AD) is the extracellular deposition of the amyloid β-peptide (Aβ) in senile plaques. Two major forms of Aβ are produced, 40 (Aβ40) and 42 (Aβ42) residues long. The most abundant form of Aβ is Aβ40, while Aβ42 is more hydrophobic and more prone to form toxic oligomers and the species of particular importance in early plaque formation. Thus, the length of the hydrophobic C-terminal seems to be very important for the oligomerization and neurotoxicity of the Aβ peptide. Here we investigated which Aβ species are deposited in AD brain. We analyzed plaque cores, prepared from occipital and frontal cortex, from sporadic and familial AD cases and performed a quantitative study using Aβ standard peptides. Cyanogen bromide was used to generate C-terminal Aβ fragments, which were analyzed by HPLC coupled to an electrospray ionisation ion trap mass spectrometer. We found a longer peptide, Aβ43, to be more frequent than Aβ40. No variants longer than Aβ43 could be observed in any of the brains. Immunohistochemistry was performed and was found to be in line with our findings. Aβ1-43 polymerizes rapidly and we suggest that this variant may be of importance for AD.     

β-Amyloid-Induced Cell Toxicity: Enhancement of 3-(4,5-Dimethylthiazol-2-yl)-2,5-Diphenyltetrazolium Bromide-Dependent Cell Death

Abstract: In an attempt to understand the cause of neurodegeneration in Alzheimer's disease, the toxic effects of β-amyloid (Aβ) peptides have been widely studied. At high micromolar concentrations Aβ peptides have been demonstrated to be acutely toxic to various cell types. At submicromolar concentrations, Aβ peptides have been suggested to inhibit cellular metabolic activity, due to their inhibition of the ability of cells to metabolize the oxidoreductase substrate 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT). Here we show, first, that MTT reduction surprisingly leads to a breakdown in PC12 cell membrane integrity and cell death, presumably through the formation of a crystalline formazan product, and, second, that pretreatment of PC12 cells with nanomolar concentrations of Aβ peptide, rather than inhibiting their metabolic activity, increases the susceptibility of these cells to the secondary toxic effect of formazan crystal formation. These results suggest that low nanomolar concentrations of Aβ render membranes more susceptible to damage by a secondary insult, in this case, MTT reduction. It is plausible that such an effect, when combined with additional risk factors, could contribute to the neurodegeneration that occurs in Alzheimer's disease.     

Novel role for apolipoprotein E in the central nervous system. Modulation of sulfatide content

It has long been postulated that apolipoprotein E (apoE) may play a role in lipid metabolism in the brain. However, direct evidence that apoE plays such a role is lacking. We investigated whether apoE isoforms influence lipid content in the brain. We compared the brains of wild-type mice to apoE knockout (-/-) and human apoE3 and apoE4 transgenic mice and compared cerebrospinal fluid (CSF) of humans with different apoE isoforms. We found that there was no effect of apoE on the content of multiple phospholipids, sphingolipids, and cholesterol. There was, however, a marked effect of apoE on the sulfatide (ST) content in both the brain and CSF. The sulfatide mass in hippocampus and cortex of apoE knockout mice was found to be 61 and 114 mol% higher than wild-type mice counterparts at 12 months of age. In contrast, the sulfatide content in brain tissues from human apoE4-expressing mice was approximately 60% less than those found in wild-type mice of the same age. The ST mass in human CSF was significantly dependent on the APOE genotypes of the subjects. Examination of potential sulfatide carrier(s) in human CSF demonstrated that sulfatides are specifically associated with apoE-containing high density lipoproteins, suggesting that sulfatide levels in the central nervous system (CNS) are likely to be directly modulated by the same metabolic pathways that regulate levels of apoE-containing CNS lipoproteins. This novel role for apoE in the CNS may provide new insights into the connection of apoE with Alzheimer's disease and poor recovery after brain injury.     

Systemic treatment with liver X receptor agonists raises apolipoprotein E,cholesterol, and amyloid-β peptides in the cerebral spinal fluid of rats

Background

Apolipoprotein E (apoE) is a major cholesterol transport protein found in association with brain amyloid from Alzheimer's disease (AD) patients and the ε4 allele of apoE is a genetic risk factor for AD. Previous studies have shown that apoE forms a stable complex with amyloid β (Aβ) peptides in vitro and that the state of apoE lipidation influences the fate of brain Aβ, i.e., lipid poor apoE promotes Aβ aggregation/deposition while fully lipidated apoE favors Aβ degradation/clearance. In the brain, apoE levels and apoE lipidation are regulated by the liver X receptors (LXRs).

Results

We investigated the hypothesis that increased apoE levels and lipidation induced by LXR agonists facilitates Aβ efflux from the brain to the cerebral spinal fluid (CSF). We also examined if the brain expression of major apoE receptors potentially involved in apoE-mediated Aβ clearance was altered by LXR agonists. ApoE, cholesterol, Aβ40, and Aβ42 levels were all significantly elevated in the CSF of rats after only 3 days of treatment with LXR agonists. A significant reduction in soluble brain Aβ40 levels was also detected after 6 days of LXR agonist treatment.

Conclusions

Our novel findings suggest that central Aβ lowering caused by LXR agonists appears to involve an apoE/cholesterol-mediated transport of Aβ to the CSF and that differences between the apoE isoforms in mediating this clearance pathway may explain why individuals carrying one or two copies of APOE ε4 have increased risk for AD.

     

Alzheimer Aβ Amyloid Forms an Inhibitory Neuronal Substrate

Abstract: Alzheimer's disease (AD) is identified by the accumulation of amyloid plaques, neurofibrillary degeneration, and the accompanying neuronal loss. AD amyloid assembles into compact fibrous deposits from the amyloid β(Aβ) protein, which is a proteo-lytic fragment of the membrane-associated amyloid precursor protein. To examine the effects of amyloid on neuron growth, a hybrid mouse motoneuron cell line (NSC34) exhibiting spontaneous process formation was exposed to artificial "plaques" created from aggregated synthetic Aβ peptides. These correspond to full-length Aβ residues 1–40 (Aβ1–40), an internal β-sheet region comprising residues 11–28 (Aβ11–28), and a proposed toxic fragment comprising residues 25–35 (Aβ25–35). Fibers were immobilized onto culture dishes, and addition of cells to these in vitro plaques revealed that Aβ was not a permissive substrate for cell adhesion. Neurites in close contact with these deposits displayed abnormal swelling and a tendency to avoid contact with the Aβ fibers. In contrast, Aβ did not affect the adhesion or growth of rat astrocytes, implicating a specific Aβ-neuron relationship. The inhibitory effects were also unique to Aβ as no response was observed to deposits of pancreatic islet amyloid poly-peptide fibers. Considering the importance of cell adhesion in neurite elongation and axonal guidance, the antiadhesive properties of Aβ amyloid plaques found in vivo may contribute to the neuronal loss responsible for the clinical manifestations of AD.     

In Vitro Induction of Apoptosis or Differentiation by Dopamine in an Immortalized Olfactory Neuronal Cell Line

Abstract: Amyloid β-peptide (Aβ) is deposited as insoluble fibrils in the brain parenchyma and cerebral blood vessels in Alzheimer's disease (AD). In addition to neuronal degeneration, cerebral vascular alterations indicative of damage to vascular endothelial cells and disruption of the blood-brain barrier occur in AD. Here we report that Aβ25-35 can impair regulatory functions of endothelial cells (ECs) from porcine pulmonary artery and induce their death. Subtoxic exposures to Aβ25-35 induced albumin transfer across EC monolayers and impaired glucose transport into ECs. Cell death induced by Aβ25-35 was of an apoptotic form, characterized by DNA condensation and fragmentation, and prevented by inhibitors of macromolecular synthesis and endonucleases. The effects of Aβ25-35 were specific because Aβ1-40 also induced apoptosis in ECs with the apoptotic cells localized to the microenvironment of Aβ1-40 aggregates and because astrocytes did not undergo similar changes after exposure to Aβ25-35. Damage and death of ECs induced by Aβ25-35 were attenuated by antioxidants, a calcium channel blocker, and a chelator of intracellular calcium, indicating the involvement of free radicals and dysregulation of calcium homeostasis. The data show that Aβ induces increased permeability of EC monolayers to macromolecules, impairs glucose transport, and induces apoptosis. If similar mechanisms are operative in vivo, then Aβ and other amyloidogenic peptides may be directly involved in vascular EC damage documented in AD and other disorders that involve vascular amyloid accumulation.     

Apolipoprotein E genotype and LRP1 polymorphisms in patients with different clinical types of metachromatic leukodystrophy

Metachromatic leukodystrophy (MLD) is a severe, neurodegenerative, metabolic disease which is caused by deficient activity of arylsulfatase A (ARSA). Sulfatides and other substrates of ARSA are stored in central and peripheral nervous systems, and in some other organs. Accumulated sulfatides are especially toxic to oligodendrocytes and Schwann cells leading to progressive demyelination. The kind of apolipoprotein E (apoE) isoform is of essential significance for the modulation of sulfatide quantity in the brain as apoE4 contains more sulfatides than apoE3. Taking into consideration the fact that apoE4 leads to the loss of sulfatides in CSF of Alzheimer's disease patients, we examined if apoE isoforms display any impact on clinical outcome in patients with different forms of MLD in whom sulfatides accumulate. The significant association of age at the onset of MLD symptoms with APOE ε2/ε3/ε4 and LRP1 c.766C>T polymorphisms was shown in multivariate stepwise regression analysis, in which other factors known to affect age at onset of the disease, i.e. clinical type of MLD, family connection of the patient and sex were also analyzed. As expected, the clinical type of MLD explained about 80% of the variance of the dependent variable. The impact of both polymorphisms on age of onset of the disease was considerably lower: 2.0% in the case of APOE polymorphism and 1.0% in the case of LRP1 polymorphism. Thus, the clinical outcome in MLD patients is related principally to the genotype of the ARSA gene, while the polymorphisms in the APOE and LRP1 genes are only slightly modifying factors.     

Processing of Amyloid Precursor Protein in Human Primary Neuron and Astrocyte Cultures

Abstract: Increased production of amyloid β peptide (Aβ) is highly suspected to play a major role in Alzheimer's disease (AD) pathogenesis. Because Aβ deposits in AD senile plaques appear uniquely in the brain and are fairly restricted to humans, we assessed amyloid precursor protein (APP) metabolism in primary cultures of the cell types associated with AD senile plaques: neurons, astrocytes, and microglia. We find that neurons secrete 40% of newly synthesized APP, whereas glia secrete only 10%. Neuronal and astrocytic APP processing generates five C-terminal fragments similar to those observed in human adult brain, of which the most amyloidogenic higher-molecular-weight fragments are more abundant. The level of amyloidogenic 4-kDa Aβ exceeds that of nonamyloidogenic 3-kDa Aβ in both neurons and astrocytes. In contrast, microglia make more of the smallest C-terminal fragment and no detectable Aβ. We conclude that human neurons and astrocytes generate higher levels of amyloidogenic fragments than microglia and favor amyloidogenic processing compared with previously studied culture systems. Therefore, we propose that the higher amyloidogenic processing of APP in neurons and astrocytes, combined with the extended lifespan of individuals, likely promotes AD pathology in aging humans.     

ATP-binding cassette transporter A7 regulates processing of amyloid precursor protein in vitro

ATP-binding cassette transporter A7 (ABCA7) is expressed in the brain and, like its closest homolog ABCA1, belongs to the ABCA subfamily of full-length ABC transporters. ABCA1 promotes cellular cholesterol efflux to lipid-free apolipoprotein acceptors and also inhibits the production of neurotoxic β-amyloid (Aβ) peptides in vitro . The potential functions of ABCA7 in the brain are unknown. This study investigated the ability of ABCA7 to regulate cholesterol efflux to extracellular apolipoprotein acceptors and to modulate Aβ production. The transient expression of ABCA7 in human embryonic kidney cells significantly stimulated cholesterol efflux (fourfold) to apolipoprotein E (apoE) discoidal lipid complexes but not to lipid-free apoE or apoA-I. ABCA7 also significantly inhibited Aβ secretion from Chinese hamster ovary cells stably expressing human amyloid precursor protein (APP) or APP containing the Swedish K670M671→N670L671 mutations when compared with mock-transfected cells. Studies with fluorogenic substrates indicated that ABCA7 had no impact on α-, β-, or γ-secretase activities. Live cell imaging of Chinese hamster ovary cells expressing APP-GFP indicated an apparent retention of APP in a perinuclear location in ABCA7 co-transfected cells. These studies indicate that ABCA7 has the capacity to stimulate cellular cholesterol efflux to apoE discs and regulate APP processing resulting in an inhibition of Aβ production.     

Membrane-targeted strategies for modulating APP and Aβ-mediated toxicity

Alzheimer's disease (AD) is a progressive neurodegenerative disease characterized by numerous pathological features including the accumulation of neurotoxic amyloid-β (Aβ) peptide. There is currently no effective therapy for AD, but the development of therapeutic strategies that target the cell membrane is gaining increased interest. The amyloid precursor protein (APP) from which Aβ is formed is a membrane-bound protein, and Aβ production and toxicity are both membrane mediated events. This review describes the critical role of cell membranes in AD with particular emphasis on how the composition and structure of the membrane and its specialized regions may influence toxic or benign Aβ/APP pathways in AD. The putative role of copper (Cu) in AD is also discussed, and we highlight how targeting the cell membrane with Cu complexes has therapeutic potential in AD.     

Degradation of the Alzheimer's amyloid beta peptide by endothelin converting enzyme

The deposition of β-amyloid (Aβ) peptides in the brain is an early and invariant feature of all forms of Alzheimer's disease (AD). As such, a major focus of AD research has been the elucidation of the mechanisms responsible for the generation of Aβ. As with any peptide, however, the degree of Aβ accumulation is dependent not only on its production, but also on the mechanisms responsible for its removal. In cell-based and in vitro assays we have identified endothelin-converting enzymes (ECEs) as novel Aβ-degrading enzymes that appear to cleave predominately in an intracellular compartment. Overexpression of ECE-1 in cells that lack endogenous ECE activity reduces Aβ accumulation by up to 90%, and this effect is completely reversed by treatment of the cells with phosphoramidon. Additionally, we have shown that recombinant soluble ECE-1 is capable of hydrolyzing synthetic Aβ40 and Aβ42 in vitro at multiple sites, with a favorable kinetic profile. While several enzymes have been identified that can degrade Aβ in vitro , only neprilysin has thus far been reported to influence Aβ accumulation in the brains of knock-out mice. To examine the physiological role of ECE activity on Aβ accumulation in the brain we compared the amount of Aβ in wild-type and ECE-2 null mice. A significant elevation in both Aβ40 and Aβ42 was observed in the ECE-2 null animals compared to their wild-type littermates. These data provide direct evidence of a physiological role for this enzyme in limiting Aβ accumulation in the brain.
Acknowledgements: Supported by Smith Fellowships to C.E. and E.E., a Bursak Fellowship to E.E., and by the Mayo Foundation for Medical Education and Research.