Regulation of betaAPP and PrPc cleavage by alpha-secretase: mechanistic and therapeutic perspectives.

Alzheimer's disease (AD) is by far the most common form of dementia in the elderly and concerns one out of three individuals over 85. Like other neurodegenerative disorders such as Parkinson, Hungtington or prion diseases, AD is characterized by the formation of amyloid plaques in the central nervous system. In the brain of AD patients, the main component of these abnormal deposits is an aggregated form of the so-called amyloid beta-peptide (Abeta), which is produced from a large trans-membrane type-1 protein, the beta-amyloid precursor protein (betaAPP), by the sequential action of the beta- and gamma-secretases. Beside these two amyloidogenic proteolytic attacks, betaAPP is targeted by a third enzyme termed alpha-secretase. Of utmost importance, this cleavage, which can be of constitutive or regulated origin, occurs right in the middle of the Abeta sequence, thus precluding its production. For this reason, and because the sAPPalpha secreted fragment derived from this cleavage displays beneficial effects, tremendous efforts have been made recently in order to both identify the proteases involved and the way they are regulated. More recently, it emerged that alpha-secretase was also responsible for the physiological processing of the cellular prion protein (PrP(c)) in the middle of its toxic 106-126 sequence. This review will focus on the recent advances in the alpha-secretase pathways regulation and will discuss the putative therapeutic approaches that could be envisioned concerning the treatment of two apparently distinct diseases that share common denominators according to their metabolism.

The C-terminal products of cellular prion protein processing, C1 and C2, exert distinct influence on p53-dependent staurosporine-induced caspase-3 activation.

The cellular prion protein (PrP(c)) undergoes various endopro-teolytic attacks within its N-terminal domain, leading to the production of C-terminal fragments (C) tethered to the plasma membrane and soluble N-terminal peptides (N). One of these cleavages occurs at position 110/111, thereby generating C1 and N1 products. We have reported that disintegrins ADAM-10, -9, and -17 participate either directly or indirectly to this proteolytic event. An alternative proteolytic event taking place around residue 90 yields C2 and N2 fragments. The putative function of these proteolytic fragments remained to be established. We have set up two novel human embryonic kidney 293 cell lines stably overexpressing either C1 or C2. We show that C1 potentiates staurosporine-induced caspase-3 activation through a p53-dependent mechanism. Thus, C1 positively controls p53 transcription and mRNA levels and increases p53-like immunoreactivity and activity. C1-induced caspase-3 activation remained unaffected by the blockade of endocytosis in HEK 293 cells and was abolished in p53-deficient fibroblasts. Conversely, overexpression of the C2 fragment did not significantly sensitize HEK 293 cells to apoptotic stimuli and did not modify p53 mRNA levels or activity. Therefore, the nature of the proteolytic cleavage taking place on PrP(c) yielded C-terminal catabolites with distinct function and could be seen as a switch mechanism controlling the function of the PrP(c) in cell survival.

Design and characterization of a novel cellular prion-derived quenched fluorimetric substrate of alpha-secretase.

Under normal conditions, the cellular prion protein (PrP(c)) undergoes a proteolytic attack between amino acids 111 and 112 which gives rise to the N-terminal secreted N1 fragment and its C-terminal membrane-tethered counterpart C1. Importantly, this cleavage precludes the integrity of the neurotoxic 106-126 sequence. Here, we describe an original and reliable assay based on a quenched fluorimetric substrate (JMV2770) encompassing the 111/112 sequence of PrP(c). In whole brain homogenate, the JMV2770-hydrolysing activity is optimal at neutral pH and sensitive to the metalloprotease inhibitor BB3103 but not to acidic and serine protease blockers. JMV2770 is efficiently cleaved by intact HEK293 cells and fibroblasts in culture, consistent with an hydrolysis by a typical ectoprotease. Overexpressions of alpha-secretases a disintegrin and metalloprotease-9 (ADAM9), ADAM10 or TACE (ADAM17) in human cells increase BB3103-sensitive JMV2770 hydrolysis, while invalidation of ADAM10 and TACE or reduced expression of ADAM9 by an antisense approach significantly reduced its cleavage. Finally, analysis of JMV2770 hydrolysis following transient transfection of ADAM10 or ADAM9 cDNA in ADAM10(-/-) fibroblasts allowed to confirm our previous data establishing that ADAM9 does not behave as a genuine alpha-secretase but rather acts as an important upstream regulator of ADAM10 activity.

The disintegrin ADAM9 indirectly contributes to the physiological processing of cellular prion by modulating ADAM10 activity.

The cellular prion protein (PrP(c)) is physiologically cleaved in the middle of its 106-126 amino acid neurotoxic region at the 110/111 downward arrow112 peptidyl bond, yielding an N-terminal fragment referred to as N1. We recently demonstrated that two disintegrins, namely ADAM10 and ADAM17 (TACE, tumor necrosis factor alpha converting enzyme) participated in both constitutive and protein kinase C-regulated generation of N1, respectively. These proteolytic events were strikingly reminiscent of those involved in the so-called "alpha-secretase pathway" that leads to the production of secreted sAPPalpha from betaAPP. We show here, by transient and stable transfection analyses, that ADAM9 also participates in the constitutive secretion of N1 in HEK293 cells, TSM1 neurons, and mouse fibroblasts. Decreasing endogenous ADAM9 expression by an antisense approach drastically reduces both N1 and sAPPalpha recoveries. However, we establish that ADAM9 was unable to increase N1 and sAPPalpha productions after transient transfection in fibroblasts depleted of ADAM10. Accordingly, ADAM9 is unable to cleave a fluorimetric substrate of membrane-bound alpha-secretase activity in ADAM10(-/-) fibroblasts. However, we establish that co-expression of ADAM9 and ADAM10 in ADAM10-deficient fibroblasts leads to enhanced membrane-bound and released fluorimetric substrate hydrolyzing activity when compared with that observed after ADAM10 cDNA transfection alone in ADAM10(-/-) cells. Interestingly, we demonstrate that shedded ADAM10 displays the ability to cleave endogenous PrP(c) in fibroblasts. Altogether, these data provide evidence that ADAM9 is an important regulator of the physiological processing of PrP(c) and betaAPP but that this enzyme acts indirectly, likely by contributing to the shedding of ADAM10. ADAM9 could therefore represent, besides ADAM10, another potential therapeutic target to enhance the breakdown of the 106-126 and Abeta toxic domains of the prion and betaAPP proteins.