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Overexpression of Nicastrin increases Aß production¹

^* Mayo Clinic Jacksonville, Laboratory of Molecular Neurobiology, Department of Neuroscience, Jacksonville, Florida, USA

³Correspondence: Mayo Clinic Jacksonville, Laboratory of Molecular Neurobiology, Department of Neuroscience, 4500 San Pablo Rd, Jacksonville, FL 32224, USA. E-mail: tgolde{at}mayo.edu

SPECIFIC AIMS

In this study, we examined the effects of the overexpression of the -secretase enzyme component Nicastrin on Aß production in whole cells and in a broken cell assay.

PRINCIPAL FINDINGS

1. Overexpression of wild-type human Nicastrin results in an increase in the secretion of the amyloid ß peptide (Aß) from intact cells
Cells were transfected with the pcDNA6 vector alone or pcDNA6 containing human wild-type (WT) Nicastrin or one of three mutants (MT) encompassing the conserved DYIGS region previously reported to alter Aß secretion (amino acid deletions of either 312 to 340 (340), 312 to 369 (369), or the double point mutation of D336A+Y337A (DYAA)). We derived cell lines stably coexpressing WT or MT Nicastrin and a Swedish amyloid ß protein precursor (APP) mutant (APP₆₉₅NL). We used this form of APP to maximize the amount of secreted Aß per unit of full-length APP and thus improve detection.

Although pooled stable lines have a short usable life span, they have the advantage of being relatively insensitive to the variation observed in clonally derived cell lines. Studies of pooled stable H4 lines expressing WT or MT Nicastrin indicated that neither affected APP maturation, APP carboxyl-terminal fragment (CTF) accumulation, or secreted APP production as analyzed by pulse-chase metabolic labeling. Unexpectedly, overexpression of WT Nicastrin increased the amount of secreted Aß relative to the pcDNA6 vector alone by fourfold when standardized to metabolically labeled, immunoprecipitated APP holoprotein (Fig. 1 A). As reported elsewhere, only WT Nicastrin overexpression resulted in the appearance of a higher molecular weight band believed to be the fully mature form of the protein (Fig. 1B ). Levels of presenilin 1 (PS1) did not appear to be altered. Examination of clonal H4 lines confirmed that WT Nicastrin overexpression resulted in increased Aß secretion, whereas MT overexpression did not show this effect (Fig. 1C ). Effects on Aß₄₀ and Aß₄₂ were nearly identical.

View larger version (23K): [in this window] [in a new window]   Figure 1. Overexpression of wild-type human Nicastrin increases Aß secretion in H4 cells. A) Nicastrin overexpression increases Aß secretion fourfold when standardized to metabolically labeling full-length APP holoprotein immunoprecipitated by CT-20 (N=3). B) Anti-V5 immunoblot showing expression of Nicastrin and mutants from H4 clonal lines. The higher MW band in the case of the WT cell line is the fully mature form absent in the case of the mutants. C) Standardization to APP expression in individual clonal lines indicated that WT human Nicastrin increased Aß secretion relative to those cell lines expressing either pcDNA6 vector or mutant Nicastrin; all 3 Nicastrin mutants were approximately equivalent to vector alone (number of clones analyzed is indicated).

2. Mature, wild-type Nicastrin is found in the active -secretase complex and causes an increase in Aß production
Our group has recently demonstrated that most -secretase activity localizes to buoyant, cholesterol-rich membrane microdomains in vitro. Since Nicastrin is hypothesized to be an important component of the active -secretase complex, it follows that it should be found in these fractions. Before undertaking in vitro studies, we examined Nicastrin in fractionated membrane preparations from mouse brain. Nicastrin cofractionated with PS1, APP, and APP CTFs in the buoyant fractions (4 and 5) along with the marker proteins TfR and Flotilin.

Additional Nicastrin clonal lines were derived using a CHO APP695NL,I line we have used successfully to measure in vitro -secretase activity. Expression of WT and mutant Nicastrin on a background of constant APP expression was necessary to facilitate the direct comparison of Aß production, since this system relies on endogenous APP CTF substrate. CHO cell endogenous Nicastrin could not be detected using our antibody. Buoyant, cholesterol-rich fractions were prepared from cells pretreated overnight with 50 µM of the reversible -secretase inhibitor z-IL-CHO. After washing, the cells were lysed in 2% CHAPSO/0.15 M sodium citrate buffer and layered onto a sucrose gradient (5%/35%/45%). The gradient was centrifuged for 19 h at 39,000 rpm in an SW-41 Ti rotor at 4°C and fractions collected from the top of the gradient. Aß production was determined by ELISA after incubation for 2 h at 37°C relative to time zero. Protein levels were measured using the Pierce BCA kit or standard Bradford assay. Cholesterol levels were assayed using the Amplex Red cholesterol assay kit (Molecular Probes, Eugene, OR, USA).

Similar to mouse brain preparations, membrane fractionation studies indicated that WT Nicastrin was found in fractions 4 and 5. This was preferentially the mature, higher molecular weight form, although immature Nicastrin was also present. However, MT Nicastrin was entirely absent from these fractions (Fig. 2 A). Cholesterol (found primarily in fraction 4) and protein (found mostly in fractions 8+) levels and distribution were highly comparable. Analysis of Aß₄₀ production from the isolated fractions indicated that the majority of -secretase activity was also localized to fractions 4 and 5 (Aß₄₂ production was too low to assess reliably in this experiment). Further examination of these fractions indicated that, consistent with observations in whole cell assays, more Aß₄₀ was produced in fractions isolated from cells overexpressing human WT Nicastrin; overexpression of either the 340 or 369 Nicastrin mutants had no effect (Fig. 2B ).

View larger version (55K): [in this window] [in a new window]   Figure 2. Nicastrin cofractionates with APP and PS1 in buoyant membrane microdomains and increases Aß production in an in vitro -secretase assay. A) Human WT Nicastrin was expressed in CHO APPhisNL,I cells. Cell lines (N=2) stably overexpressing either WT Nicastrin or the 340 or 369 mutants were lysed, fractionated, separated by SDS-PAGE, and immunoblotted (see text). The fully mature, higher molecular weight WT Nicastrin is visible exclusively in fractions 4 and 5, whereas the majority of immature Nicastrin (both WT and MT) is found in the remaining fractions. B) -Secretase activity is primarily localized in fractions 4 and 5 in all cell lines analyzed; activity is increased in cells overexpressing WT but not MT Nicastrin.

CONCLUSIONS AND SIGNIFICANCE

The observation that WT Nicastrin overexpression increases Aß production confirms prior assertions that Nicastrin is an essential functional component of the -secretase complex. However, the apparent facilitation of -secretase cleavage by Nicastrin overexpression is distinct from what has been shown in previous reports. Thus, our data provide a distinct conceptual framework for how Nicastrin might function.

This study is the first report to demonstrate that overexpression of a component of the putative -secretase enzyme complex leads to an increase in -secretase activity. Marked overexpression of PS itself is not possible, likely due to the existence of a limiting cellular factor that tightly controls the levels of the active PS amino- and carboxyl-terminal fragments. Although recent reports have shown that a down-regulation of Nicastrin causes a reduction in PS1 and vice versa, overexpression of Nicastrin does not appear to noticeably increase the amount of PS1 or fragments. Although we cannot exclude small changes in PS1 levels, these would be unable to account for the two- to fourfold increase in Aß production we observe in intact cells. We find that mutation of the highly conserved DYIGS domain, which causes a loss of Nicastrin’s ability to bind PS, results in a form of Nicastrin that has little or no ability to modulate -secretase processing of APP in intact cells or isolated membrane preparations. Because MT Nicastrin appears to have minimal effect on -secretase activity, these observations help explain why expression of these constructs have no effect on APP CTFs levels. In contrast to the results of this study, WT Nicastrin overexpression was originally described as having no effect on Aß production while the DYAA mutant was shown to cause an increase. These discrepancies may be attributed to standardization, since a significant effect is consistently seen only if the data are corrected to APP expression. Alternatively, WT Nicastrin overexpression may have effects that vary between cell lines, dependent on, for example, the relative abundance of the different -secretase complex components.

The observation that WT but not MT Nicastrin cofractionates with -secretase activity in a broken cell assay suggests that the failure of MT Nicastrin to increase Aß is directly related to an inability to become incorporated into the mature enzyme complex. If the hypothesis that PS are the catalytic subunits of -secretase is correct, then one possible role for Nicastrin could be in positioning or transporting -secretase substrate(s) into the active enzyme complex. This possibility received additional support from a recent study in which C83 (CTF) and Nicastrin were coisolated from a broken cell assay that maintains -secretase activity. In this study, PS1, Nicastrin and CTF could be purified using a -secretase inhibitor column, which most likely captures the complex by binding to the active site. In this scenario, where Nicastrin is the substrate-presenting subunit, both MT and WT Nicastrin might interact correctly with -substrates such as APP CTFs, but MT Nicastrin might not enter the active enzyme complex. The increase in Aß production in the case of WT Nicastrin overexpression might then be related to an increase in the abundance of the Nicastrin:substrate component.

Alternatively, Nicastrin may be a vital structural component of the -secretase complex responsible for its proper assembly and/or subcellular localization. Recent studies demonstrating aberrations in presenilin subcellular localization and stability in Nicastrin-deficient Drosophila support this hypothesis. Nicastrin and PS do not mature correctly in the absence of each other, and deficiencies in Aph-1 and Pen-2 have similar effects. These two possibilities, Nicastrin as a substrate binding subunit and as a critical structural component, are not necessarily mutually exclusive. Since mammalian studies thus far have all been performed on a background of endogenous Nicastrin expression, the development of deficient mammalian models will greatly assist in defining its precise role.

View larger version (15K): [in this window] [in a new window]   Figure 3. Model of Nicastrin as the substrate presenting unit of -secretase. CTF substrate associates with Nicastrin (a), which then positions it (b) for cleavage by PS (c). Cleavage results in the secretion of Aß (d) and translocation of the APP intracellular domain (AICD; e) to the nucleus.

FOOTNOTES

¹ To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.02-1050fje; to cite this article, use FASEB J. (April 8, 2003) 10.1096/fj.02-1050fje

² These authors each contributed equally to this work.

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