Murphy, M. Paul

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.

ErbB-4 is a transmembrane receptor tyrosine kinase that regulates cell proliferation and differentiation. After binding of its ligand heregulin (HRG) or activation of protein kinase C (PKC) by 12- O -tetradecanoylphorbol-13-acetate (TPA), the ErbB-4 ectodomain is cleaved by a metalloprotease. We now report a subsequent cleavage by γ-secretase that releases the ErbB-4 intracellular domain from the membrane and facilitates its translocation to the nucleus. γ-Secretase cleavage was prevented by chemical inhibitors or a dominant negative presenilin. Inhibition of γ-secretase also prevented growth inhibition by HRG. γ-Secretase cleavage of ErbB-4 may represent another mechanism for receptor tyrosine kinase–mediated signaling.

ABSTRACT The amyloid β‐protein (Aβ) deposited in Alzheimer's disease (AD) is a normally secreted proteolytic product of the amyloid β‐protein precursor (APP). Generation of Aβ from the APP requires two sequential proteolytic events: an initial β‐secretase cleavage at the amino terminus of the Aβ sequence followed by γ ‐secretase cleavage at the carboxyl terminus of Aβ. We describe the development of a robust in vitro assay for γ ‐secretase cleavage by showing de novo Aβ production in vitro and establish that this assay monitors authentic gamma‐secretase activity by documenting the production of a cognate γ ‐CTF, confirming the size of the Aβ produced by mass spectrometry, and inhibiting cleavage in this system with multiple inhibitors that alter γ‐ secretase activity in living cells. Using this assay, we demonstrate that the γ‐secretase activity 1 ) is tightly associated with the membrane, 2 ) can be solubilized, 3 ) has a pH optimum of 6.8 but is active from pH 6.0 to pH >8.4, and 4 ) ascertain that activities of the γ ‐40 and γ ‐42 are indeed pharmacologically distinct. These studies should facilitate the purification of the protease or proteases that are responsible for this unusual activity, which is a major therapeutic target for the treatment of AD.

Inclusion body myositis (IBM), the most common age-related muscle disease in the elderly population, is an incurable disorder leading to severe disability. Sporadic IBM has an unknown etiology, although affected muscle fibers are characterized by many of the pathobiochemical alterations traditionally associated with neurodegenerative brain disorders such as Alzheimer's disease. Accumulation of the amyloid-β peptide, which is derived from proteolysis of the larger amyloid-β precursor protein (βAPP), seems to be an early pathological event in Alzheimer's disease and also in IBM, where in the latter, it predominantly occurs intracellularly within affected myofibers. To elucidate the possible role of βAPP mismetabolism in the pathogenesis of IBM, transgenic mice were derived in which we selectively targeted βAPP overexpression to skeletal muscle by using the muscle creatine kinase promoter. Here we report that older (>10 months) transgenic mice exhibit intracellular immunoreactivity to βAPP and its proteolytic derivatives in skeletal muscle. In this transgenic model, selective overexpression of βAPP leads to the development of a subset of other histopathological and clinical features characteristic of IBM, including centric nuclei, inflammation, and deficiencies in motor performance. These results are consistent with a pathogenic role for βAPP mismetabolism in human IBM.

Presenilins mediate an unusual intramembranous proteolytic activity known as γ-secretase, two substrates of which are the Notch receptor (Notch) and the β-amyloid precursor protein (APP). γ-Secretase-mediated cleavage of APP, like that of Notch, yields an intracellular fragment [ A PP i ntra c ellular d omain (AICD)] that forms a transcriptively active complex. We now demonstrate a functional role for AICD in regulating phosphoinositide-mediated calcium signaling. Genetic ablation of the presenilins or pharmacological inhibition of γ-secretase activity (and thereby AICD production) attenuated calcium signaling in a dose-dependent and reversible manner through a mechanism involving the modulation of endoplasmic reticulum calcium stores. Cells lacking APP (and hence AICD) exhibited similar calcium signaling deficits, and—notably—these disturbances could be reversed by transfection with APP constructs containing an intact AICD, but not by constructs lacking this domain. Our findings indicate that the AICD regulates phosphoinositide-mediated calcium signaling through a γ-secretase-dependent signaling pathway, suggesting that the intramembranous proteolysis of APP may play a signaling role analogous to that of Notch.