Subcellular Compartment and Molecular Subdomain of β-Amyloid Precursor Protein Relevant to the Aβ42-promoting Effects of Alzheimer Mutant Presenilin 2

The final step in A␤ generation is the cleavage of the C-terminal 99 amino acid residues of the amyloid precursor protein by ␥-secretase. ␥-Secretase activity is closely linked to the multi-transmembrane-spanning proteins presenilin 1 and presenilin 2. To elucidate whether the cleavage site specificities of ␥-secretase leading to the formation of secreted and intracellular A␤ are identical, we made use of point mutations close to the ␥-cleavage site, known to have a dramatic effect on the 42/40 ratio of secreted A␤. We found that the selected point mutations only marginally influenced the 42/40 ratio of intracellular A␤, suggesting differences in the ␥-secretase cleavage site specificity for the generation of secreted and intracellular A␤. The analysis of the subcellular compartments involved in the generation of intracellular A␤ revealed that A␤ is not generated in the early secretory pathway in the human SH-SY5Y neuroblastoma cell line. In this study we identified late Golgi compartments to be involved in the generation of intracellular A␤. Moreover, we demonstrate that the presence of processed PS1 is not sufficient to obtain ␥-secretase processing of the truncated amyloid precursor protein construct C99, proposing the existence of an additional factor downstream of the endoplasmic reticulum and early Golgi required for the formation of an active ␥-secretase complex.

Section: Discussionsupporting

confidence: 82%

Section: Generation Of A␤ By C99 Proteins Bearing a Tgn-sortingmentioning

confidence: 99%

mentioning

confidence: 99%

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γ-Secretase Cleavage Site Specificity Differs for Intracellular and Secretory Amyloid β

Grimm

Beher

Lichtenthaler

et al. 2003

“…Membrane fractions were prepared as described previously (9,28,29). The membrane pellets were resuspended in HEPES buffer to yield a protein concentration of 5-10 mg/ml and were stored at Ϫ70°C.…”

Section: Compounds and Peptides-synthesis Of Daptmentioning

confidence: 99%

Sulindac Sulfide Is a Noncompetitive γ-Secretase Inhibitor That Preferentially Reduces Aβ42 Generation

Takahashi

Hayashi

Tominari

et al. 2003

Self Cite

185

189

Nonsteroidal anti-inflammatory drugs (NSAIDs) have been known to reduce risk for Alzheimer's disease. In addition to the anti-inflammatory effects of NSAIDs to block cylooxygenase, it has been shown recently that a subset of NSAIDs selectively inhibits the secretion of highly amyloidogenic A␤42 from cultured cells, although the molecular target(s) of NSAIDs in reducing the activity of ␥-secretase for A␤42 generation (␥ 42 -secretase) still remain unknown. Here we show that sulindac sulfide (SSide) directly acts on ␥-secretase and preferentially inhibits the ␥ 42 -secretase activity derived from the 3-[(3-cholamidopropyl)dimethylammonio]-2-hydroxy-1-propanesulfonate-solubilized membrane fractions of HeLa cells, in an in vitro ␥-secretase assay using recombinant amyloid ␤ precursor protein C100 as a substrate. SSide also inhibits activities for the generation of A␤40 as well as for Notch intracellular domain at higher concentrations. Notably, SSide displayed linear noncompetitive inhibition profiles for ␥ 42 -secretase in vitro. Our data suggest that SSide is a direct inhibitor of ␥-secretase that preferentially affects the ␥ 42 -secretase activity. Alzheimer's disease (AD)1 is a dementing neurodegenerative disorder of the elderly characterized pathologically by neuronal loss in the cerebral cortex accompanied by massive deposition of amyloid ␤ peptides (A␤) as senile plaques (1). A␤ is produced by sequential proteolytic cleavages of the amyloid ␤ precursor protein (␤APP) by a set of membrane-bound proteases termed ␤-and ␥-secretases. The C-terminal length of A␤ generated by ␥-secretase is heterogeneous; A␤42 is a relatively minor molecular species of the A␤ secreted from cells, but it has a much higher propensity to aggregate and form amyloid compared with other A␤ species. These findings provide strong support for the hypothesis that the deposition of A␤42 is closely related to the pathogenesis of AD, implicating ␥-secretase as an important therapeutic target.Mutations in PS1 or PS2 genes account for the majority of early onset familial AD, and these mutations cause an increase in the ratio or levels of production of A␤42 (1). It is known that PS is essential for the ␥-secretase-mediated intramembranous cleavage not only for ␤APP but for other type I transmembrane proteins (e.g. Notch, ErbB4, E-cadherin, low density lipoprotein receptor-related protein, and CD44) (2). PS proteins undergo endoproteolysis to generate N-and C-terminal fragments and interact with other proteins (i.e. nicastrin, APH-1, and PEN-2) to form a high molecular weight (HMW) protein complex (3). The functional role of PS complex in ␥-secretase activity still remains unknown. However, aspartyl protease transition state analogue inhibitors of ␥-secretase, which harbors a hydroxyl ethylene isostere or a difluoro alcohol moiety, directly label PS fragments (4 -6). In addition, a systematic analysis using a variety of PS mutants revealed that HMW complex formation of PS as well as conserved aspartyl residues within the transmembrane domain are es...

“…This indicates that the relatively small activity of neprilysin expressed in ER was sufficient to degrade intracellular A␤40, because wild-type neprilysin and all the other chimeras reside at least transiently in the ER after synthesis. Considering the previous reports describing A␤ generation in ER, TGN, and endosome (14,15,35), our observation implies that a certain pool of intracellular A␤40 is retained in or recycled back to the ER in cultured neurons, as it is unlikely that the ER is a major site of A␤ generation from APP (36). If aging causes such a pool of intracellular A␤ to remain abnormally unmetabolized, pathological intraneuronal A␤ accumulation (37-39) may arise, possibly leading to ER stresses.…”

Section: Discussionmentioning

confidence: 97%

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

Hama

Shirotani

Iwata

et al. 2004

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...