Rochette, Marjorie J.

Background: Beta‐secretase (BACE, types 1 and 2) has been identified as the enzyme that cleaves the β‐amyloid Precursor Protein to form the amyloid‐β peptide (Aβ). As the Aβ peptide is considered a primary causal factor in Alzheimer's disease (AD) pathogenesis, reducing Aβ production is currently a major therapeutic goal. In several disease states, as with normal aging, BACE protein and activity is increased in affected tissues. Therefore, determination of what regulates BACE is of critical importance. We sought to characterize one possible candidate. Objective: To determine the role of BACE in disease pathology, as it relates to levels of cellular nucleic acid binding protein (CNBP), a candidate regulatory factor. Methods: A fluorometric BACE assay was developed, validated, and optimized. Using this assay and Western Blot, BACE enzymatic activity and protein levels were measured in aging mice, and in human AD and age‐matched control tissue. Results: Cell culture studies demonstrate that CNBP regulates BACE protein and activity at the mRNA level. A BACE assay was developed to measure BACE1/2 activation in vivo as related to CNBP levels. CNBP levels and BACE activity were then measured in aging and diseased tissue, which showed a tendency for CNBP to increase with age. These data suggest CNBP may regulate BACE in vivo and in vitro , and may contribute to the development of AD and other age‐related degenerative diseases.

ABSTRACT γ‐Secretase cleavage is the final proteolytic step that releases the amyloid β‐peptide (Aβ) from the amyloid β‐protein precursor (APP). Significant evidence indicates that the presenilins (PS) are catalytic components of a high molecular weight γ‐secretase complex. The glycoprotein nicastrin was recently identified as a functional unit of this complex based on 1) binding to PS and 2) the ability to modulate Aβ production following mutation of a conserved DYIGS region. In contrast to the initial report, we find that overexpression of wild‐type (WT) nicastrin increases Aβ production, whereas DYIGS mutations (MT) have little or no effect. The increase in Aβ production is associated with an increase in γ‐secretase activity but not with a detectable increase in PS1 levels. Subcellular fractionation studies show that WT but not MT nicastrin matures into buoyant membrane fractions enriched in γ‐secretase activity. These data support the hypothesis that nicastrin is an essential component of the γ‐secretase complex. The finding that WT nicastrin overexpression can increase γ‐secretase activity without altering levels of the presumed catalytic component (PS) of the enzyme may point to a role for nicastrin in facilitating cleavage by regulating substrate interactions with the γ‐secretase complex.