Effects of Neprilysin Chimeric Proteins Targeted to Subcellular Compartments on Amyloid β Peptide Clearance in Primary Neurons

Abstract

Neprilysin (NEP) is a rate-limiting amyloid ␤ peptide (A␤)-degrading enzyme in the brain. We demonstrated previously that overexpression of neprilysin in primary cortical neurons remarkably decreased not only extracellular but also intracellular A␤ levels. To investigate the subcellular compartments where neprilysin degrades A␤ most efficiently, we expressed neprilysin chimeric proteins containing various subcellular compartment-targeting domains in neurons. Sec12-NEP, ␤-galactoside ␣2,6-sialyltransferase-NEP, transferrin receptor-NEP, and growth-associated protein 43-NEP were successfully sorted to the endoplasmic reticulum, trans-Golgi network, early/recycling endosomes, and lipid rafts, respectively. We found that intracellularly, wild-type neprilysin and all the chimeras showed equivalent A␤40-degrading activities. A␤40 was more effectively cleared than A␤42, and this tendency was greater for intracellular A␤ than for extracellular A␤. Wild-type and trans-Golgi network-targeted ST-NEP cleared more intracellular A␤42 than the other chimeras. Wild-type neprilysin cleared extracellular A␤ more effectively than any of the chimeras, among which endoplasmic reticulum-targeted Sec12-NEP was the least effective. These observations indicate that different intracellular compartments may be involved in the metabolism of distinct pools of A␤ (A␤40 and A␤42) to be retained or recycled intracellularly and to be secreted extracellularly, and that the endogenous targeting signal in wildtype neprilysin is well optimized for the overall neuronal clearance of A␤.Accumulation of amyloid ␤ peptide (A␤) 1 in the brain is a triggering event leading to the pathological cascade of Alzheimer's disease (AD) (1). A␤ is a physiological peptide, the steadystate level of which is strictly determined through a balance between the anabolic and catabolic activities. Changes in the metabolic balance are closely associated with A␤ accumulation, because an increase in A␤ anabolic activity causes A␤ deposition in almost all cases of familial AD (2, 3). In sporadic AD brains, where elevation of anabolism seems to be rare, a reduction in catabolic activity involving A␤-degrading enzyme(s) has been a candidate cause that may account for A␤ accumulation. Recently, several proteases, such as neprilysin (4), insulindegrading enzyme (5, 6), and endothelin-converting enzymes 1 and 2 (7), have been identified as A␤-degrading enzymes in the brain by reverse genetic studies. Deficiencies of these proteases were associated with an elevation of brain A␤ levels in each case, demonstrating that a reduction in the catabolic activity will also contribute to AD development by promoting A␤ accumulation. Although these peptidases are likely to contribute to overall A␤ clearance in the brain by complementing each other in a subcellular, cell type-and/or brain region-specific manner, neprilysin seems to play the primary role among all the A␤-degrading enzymes thus far examined (8,9). It is also important that the in vivo action of neprilysin in the brain is quite sel...

Overexpression of Neprilysin Reduces Alzheimer Amyloid-β42 (Aβ42)-induced Neuron Loss and Intraneuronal Aβ42 Deposits but Causes a Reduction in cAMP-responsive Element-binding Protein-mediated Transcription, Age-dependent Axon Pathology, and Premature Death in Drosophila

“…NEP is a member of the neutral endopeptidase family, which exhibits maximum catalytic activity in neutral pH environments (33). not only extracellular but also intraneuronal A␤ levels are reduced by NEP overexpression in primary cortical neurons, presumably by degradation in the secretory pathway (62). Moreover, a potential intracellular role for NEP in A␤ catabolism in vivo has recently been suggested from analysis of the half-life of interstitial fluid A␤ in NEP knock-out mice (6).…”

Potent amyloidogenicity and pathogenicity of Aβ43

“…Overexpression of wild-type human APP (hAPPwt) in the above-stated MEFs using a semliki-forest virus vector 23 resulted in a significant increase in Aβ43 in the heterozygous R278I knockin cells (Supplementary Fig. 9).…”

Curcumin Inhibits the AKT/NF-κB Signaling via CpG Demethylation of the Promoter and Restoration of NEP in the N2a Cell Line

“…Several lines of evidence now demonstrate that aberrant DNA methylations of genes in AD are implicated in the initiation and progression of AD (32). Neprilysin (NEP), a zincdependent metallopeptidase, serves as the major physiological Aβ-degrading enzyme (9). However, both the expression and activity of NEP decrease with age in AD brains (13,33), and the transcriptional regulatory mechanisms that regulate NEP expression are relatively unknown.…”

“…Neprilysin (NEP, EP24.11), a zinc-dependent metallopeptidase expressed relatively low in the brain, has been characterized as capable of reducing neurotoxicity through degradation of Aβ in AD (9,10). Increasing evidence also indicates an important function of its inhibition in AKT activation (11,12).…”