Estrogen Stimulates Degradation of ��-Amyloid Peptide by Up-regulating Neprilysin

Postmenopausal estrogen depletion is a characterized risk factor for Alzheimer disease (AD), a human disorder linked to high levels of β-amyloid peptide (Aβ) in brain tissue. Previous studies suggest that estrogen negatively regulates the level of Aβ in the brain, but the molecular mechanism is unknown. Here, we provide evidence that estrogen promotes Aβ degradation mainly through a principal Aβ degrading enzyme, neprilysin, in neuroblastoma SH-SY5Y cells. We also demonstrate that up-regulation of neprilysin by estrogen is dependent on both estrogen receptor α and β (ERα and ERβ), and ligand-activated ER regulates expression of neprilysin through physical interactions between ER and estrogen response elements (EREs) identified in the neprilysin gene. These results were confirmed by in vitro gel shift and in vivo chromatin immunoprecipitation analyses, which demonstrate specific binding of ERα and ERβ to two putative EREs in the neprilysin gene. The EREs also enhance ERα- and ERβ-dependent reporter gene expression in a yeast model system. Therefore, the study described here provides a putative mechanism by which estrogen positively regulates expression of neprilysin to promote degradation of Aβ, reducing risk for AD. These results may lead to novel approaches to prevent or treat AD.     

Cell Surface Expression of the Major Amyloid-β Peptide (Aβ)-degrading Enzyme, Neprilysin, Depends on Phosphorylation by Mitogen-activated Protein Kinase/Extracellular Signal-regulated Kinase Kinase (MEK) and Dephosphorylation by Protein Phosphatase 1a

Neprilysin is one of the major amyloid-β peptide (Aβ)-degrading enzymes, the expression of which declines in the brain during aging. The decrease in neprilysin leads to a metabolic Aβ imbalance, which can induce the amyloidosis underlying Alzheimer disease. Pharmacological activation of neprilysin during aging therefore represents a potential strategy to prevent the development of Alzheimer disease. However, the regulatory mechanisms mediating neprilysin activity in the brain remain unclear. To address this issue, we screened for pharmacological regulators of neprilysin activity and found that the neurotrophic factors brain-derived neurotrophic factor, nerve growth factor, and neurotrophins 3 and 4 reduce cell surface neprilysin activity. This decrease was mediated by MEK/ERK signaling, which enhanced phosphorylation at serine 6 in the neprilysin intracellular domain (S6-NEP-ICD). Increased phosphorylation of S6-NEP-ICD in primary neurons reduced the levels of cell surface neprilysin and led to a subsequent increase in extracellular Aβ levels. Furthermore, a specific inhibitor of protein phosphatase-1a, tautomycetin, induced extensive phosphorylation of the S6-NEP-ICD, resulting in reduced cell surface neprilysin activity. In contrast, activation of protein phosphatase-1a increased cell surface neprilysin activity and lowered Aβ levels. Taken together, these results indicate that the phosphorylation status of S6-NEP-ICD influences the localization of neprilysin and affects extracellular Aβ levels. Therefore, maintaining S6-NEP-ICD in a dephosphorylated state, either by inhibition of protein kinases involved in its phosphorylation or by activation of phosphatases catalyzing its dephosphorylation, may represent a new approach to prevent reduction of cell surface neprilysin activity during aging and to maintain physiological levels of Aβ in the brain.     

Neprilysin Impedes Islet Amyloid Formation by Inhibition of Fibril Formation Rather Than Peptide Degradation

Deposition of islet amyloid polypeptide (IAPP) as islet amyloid in type 2 diabetes contributes to loss of β-cell function and mass, yet the mechanism for its occurrence is unclear. Neprilysin is a metallopeptidase known to degrade amyloid in Alzheimer disease. We previously demonstrated neprilysin to be present in pancreatic islets and now sought to determine whether it plays a role in degrading islet amyloid. We used an in vitro model where cultured human IAPP (hIAPP) transgenic mouse islets develop amyloid and thereby have increased β-cell apoptosis. Islet neprilysin activity was inhibited or up-regulated using a specific inhibitor or adenovirus encoding neprilysin, respectively. Following neprilysin inhibition, islet amyloid deposition and β-cell apoptosis increased by 54 and 75%, respectively, whereas when neprilysin was up-regulated islet amyloid deposition and β-cell apoptosis both decreased by 79%. To determine if neprilysin modulated amyloid deposition by cleaving hIAPP, analysis of hIAPP incubated with neprilysin was performed by mass spectrometry, which failed to demonstrate neprilysin-induced cleavage. Rather, neprilysin may act by reducing hIAPP fibrillogenesis, which we showed to be the case by fluorescence-based thioflavin T binding studies and electron microscopy. In summary, neprilysin decreases islet amyloid deposition by inhibiting hIAPP fibril formation, rather than degrading hIAPP. These findings suggest that targeting the role of neprilysin in IAPP fibril assembly, in addition to IAPP cleavage by other peptidases, may provide a novel approach to reduce and/or prevent islet amyloid deposition in type 2 diabetes.     

Comparative studies for amyloid beta degradation: “Neprilysin vs insulysin”, “monomeric vs aggregate”, and “whole Aβ40 vs its peptide fragments”

Amyloid beta (Aβ) proteins are produced from amyloid precursor protein cleaved by β- and γ-secretases, and are the main components of senile plaques pathologically found in Alzheimer's disease (AD) patient brains. Therefore, the relationship between AD and Aβs has been well studied for both therapeutic and diagnostic purposes. Several enzymes have been reported to degrade Aβs in vivo, with neprilysin (NEP) and insulysin (insulin-degrading enzyme, IDE) being the most prominent. In this article, we describe the mass spectrometric characterization of peptide fragments generated using NEP and IDE, and clarify the differences in digestion specificities between these two enzymes for non-aggregated Aβ₄₀, aggregated Aβ₄₀, and Aβ₄₀ peptide fragments, including Aβ₁₆. Our results allowed identification of all the peptide fragments from non-aggregated Aβ₄₀: NEP, 23 peptide fragments consisting of 2–11 amino-acid residues, 17 cleavage sites; IDE, 23 peptide fragments consisting of 6–33 amino-acid residues, 15 cleavage sites. Also, we confirmed that IDE can digest only whole Aβ₄₀, whereas NEP can digest both Aβ₄₀ and partial structures such as Aβ₁₆ and peptide fragments generated by the digestion of Aβ₄₀ by IDE. Furthermore, we confirmed that IDE and NEP are unable to digest aggregated Aβ₄₀.     

Loss of neprilysin alters protein expression in the brain of Alzheimer's disease model mice

Alzheimer's disease (AD) is a neurodegenerative disease displaying extracellular plaques formed by the neurotoxic amyloid β‐peptide (Aβ), and intracellular neurofibrillary tangles consisting of protein tau. However, how these pathologies relate to the massive neuronal death that occurs in AD brains remain elusive. Neprilysin is the major Aβ‐degrading enzyme and a lack thereof increases Aβ levels in the brain twofold. To identify altered protein expression levels induced by increased Aβ levels, we performed a proteomic analysis of the brain of the AD mouse model APPsw and compared it to that of APPsw mice lacking neprilysin. To this end we established an LC‐MS/MS method to analyze brain homogenate, using an ¹⁸O‐labeled internal standard to accurately quantify the protein levels. To distinguish between alterations in protein levels caused by increased Aβ levels and those induced by neprilysin deficiency independently of Aβ, the brain proteome of neprilysin deficient APPsw mice was also compared to that of neprilysin deficient mice. By this approach we identified approximately 600 proteins and the levels of 300 of these were quantified. Pathway analysis showed that many of the proteins with altered expression were involved in neurological disorders, and that tau, presenilin and APP were key regulators in the identified networks. The data have been deposited to the ProteomeXchange Consortium with identifiers PXD000968 and PXD001786 ( http://proteomecentral.proteomexchange.org/dataset/PXD000968 and ( http://proteomecentral.proteomexchange.org/dataset/PXD001786 ). Interestingly, the levels of several proteins, including some not previously reported to be linked to AD, were associated with increased Aβ levels.     

A Neuroprotective Brain-penetrating Endopeptidase Fusion Protein Ameliorates Alzheimer Disease Pathology and Restores Neurogenesis

Alzheimer disease (AD) is characterized by widespread neurodegeneration throughout the association cortex and limbic system, deposition of amyloid-β peptide (Aβ) in the neuropil and around the blood vessels, and formation of neurofibrillary tangles. The endopeptidase neprilysin has been successfully used to reduce the accumulation of Aβ following intracranial viral vector delivery or ex vivo manipulated intracranial delivery. These therapies have relied on direct injections into the brain, whereas a clinically desirable therapy would involve i.v. infusion of a recombinant enzyme. We previously characterized a recombinant neprilysin that contained a 38-amino acid brain-targeting domain. Recombinant cell lines have been generated expressing this brain-targeted enzyme (ASN12). In this report, we characterize the ASN12 recombinant protein for pharmacology in a mouse as well as efficacy in two APPtg mouse models of AD. The recombinant ASN12 transited to the brain with a t_½ of 24 h and accumulated to 1.7% of injected dose at 24 h following i.v. delivery. We examined pharmacodynamics in the tg2576 APPtg mouse with the prion promoter APP695 SWE mutation and in the Line41 mThy1 APP751 mutation mouse. Treatment of either APPtg mouse resulted in reduced Aβ, increased neuronal synapses, and improved learning and memory. In addition, the Line41 APPtg mice showed increased levels of C-terminal neuropeptide Y fragments and increased neurogenesis. These results suggest that the recombinant brain-targeted neprilysin, ASN12, may be an effective treatment for AD and warrant further investigation in clinical trials.     

The epigenetic effects of amyloid-β1-40 on global DNA and neprilysin genes in murine cerebral endothelial cells

Amyloid-β (Aβ) is the core component of senile plaques, which are the pathological markers for Alzheimer’s disease and cerebral amyloid angiopathy. DNA methylation/demethylation plays a crucial role in gene regulation and could also be responsible for presentation of senescence. Oxidative stress, which may be induced by Aβ, is thought to be an important contributor of DNA hyper-methylation; however, contradicting this is the fact that global DNA hypo-methylation has been found in aging brains. It therefore remains largely unknown as to whether Aβ does in fact cause DNA methylation/demethylation. Neprilysin (NEP) is one of the enzymes responsible for Aβ degradation, with its expression decreasing in both Alzheimer and aging brains. Using high-performance liquid chromatography (HPLC), we explore whether Aβ is responsible for alteration of the global DNA methylation status on a murine cerebral endothelial cells model, and also use methylation-specific PCR (MSPCR) to examine whether DNA methylation status is altered on the NEP promoter region. We find that Aβ reduces global DNA methylation whilst increasing NEP DNA methylation and further suppressing the NEP expression in mRNA and protein levels. Our results support that Aβ induces epigenetic effects, implying that DNA methylation may be part of a vicious cycle involving the reduction in NEP expression along with a resultant increase in Aβ accumulation, and that Aβ may induce global DNA hypo-methylation.     

Oxidized neprilysin in aging and Alzheimer's disease brains

Deposition of amyloid in the brain is important in the pathogenesis of Alzheimer's disease (AD), but it remains to be determined if deposition is due to increased production or decreased clearance of fibrillogenic forms of beta-amyloid (Abeta). Except for rare genetic forms of AD, there is little evidence for increased production of Abeta, but decreases in enzymes involved in the clearance of Abeta are increasingly being investigated. Neprilysin (NEP) is a major enzyme for degradation of Abeta and changes in amount or activity of NEP may play a role in Abeta deposition in AD. Since oxidative damage to proteins, including formation of adducts such as 4-hydroxynonenal (HNE), has been reported in AD, it was of interest to determine if NEP might be susceptible to oxidative modification. To address this question, monoclonal antibody immunoprecipitates of NEP were probed with polyclonal antibodies to NEP and HNE. The results showed decreased NEP in AD compared to normal controls. NEP in both AD and controls had HNE-modification and the ratio of oxidized to total NEP was greater in AD than in controls. These findings suggest that decreased NEP may contribute to Abeta deposition in AD and that age-related oxidative damage to NEP may play a role in age-related cerebral amyloidosis that is exacerbated in AD.