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N-terminally truncated amyloid-β (Aβ) peptides are present in early and diffuse plaques of individuals with Alzheimer's disease (AD), are overproduced in early onset familial AD and their amount seems to be directly correlated to the severity and the progression of the disease in AD and Down's syndrome (DS). The pyroglutamate-containing isoforms at position 3 [AβN3(pE)-40/42] represent the prominent form among the N-truncated species, and may account for more than 50% of Aβ accumulated in plaques. In this study, we compared the toxic properties, fibrillogenic capabilities, and in vitro degradation profile of Aβ1-40, Aβ1-42, AβN3(pE)-40 and AβN3(pE)-42. Our data show that fibre morphology of Aβ peptides is greatly influenced by the C-terminus while toxicity, interaction with cell membranes and degradation are influenced by the N-terminus. AβN3(pE)-40 induced significantly more cell loss than the other species both in neuronal and glial cell cultures. Aggregated AβN3(pE) peptides were heavily distributed on plasma membrane and within the cytoplasm of treated cells. AβN3(pE)-40/42 peptides showed a significant resistance to degradation by cultured astrocytes, while fulllength peptides resulted partially degraded. These findings suggest that formation of N-terminally modified peptides may enhance β-amyloid aggregation and toxicity, likely worsening the onset and progression of the disease. [ABSTRACT FROM AUTHOR]

The presenilin (PS)-dependent gamma-secretase activity refers to a high molecular mass-complex including, besides PS1 or PS2, three other proteins recently identified, namely nicastrin, Aph-1, and Pen-2. This proteolytic complex has been shown to contribute to both gamma- and epsilon-cleavages of the beta-amyloid precursor protein (betaAPP), thereby generating beta-amyloid peptides (Abeta) and the APP intracellular domain (AICD), respectively. TMP21, a member of the p24 cargo protein family, was recently shown to interact with PS complexes. Interestingly, TMP21 modulates gamma-secretase-mediated Abeta production but does not regulate epsilon-secretase-derived AICD formation [F. Chen, H. Hasegawa, G. Schmitt-ulms, T. Kawarai, C. Bohm, T. Katayama, Y. Gu, N. Sanjo, M. Glista, E. Rogaeva, Y. Wakutami, R. Pardossi-Piquard, X. Ruan, A. Tandon, F. Checler, P. Marambaud, K. Hansen, D. Westaway, P. St. George-Hyslop, P. Fraser, TMP21 is a presenilin complex component that modulates gamma- but not epsilon-secretase activities, Nature 440 (2006) 1208-1212]. Here we investigate the functional incidence of the over-expression or depletion of TMP21 on both intracellular and secreted Abeta recoveries and AICD-associated phenotypes. First we confirm that TMP21 depletion yields increased levels of secreted Abeta40. However, we demonstrate that both staurosporine-stimulated caspase-3 activation, p53 and neprilysin expression and activity were not affected by TMP21 over-expression or depletion. Overall, our functional data further reinforce the view that TMP21 behaves as a regulator of gamma- but not epsilon-cleavages generated by PS-dependent gamma-secretase complex.

The presenilin-dependent gamma-secretase activity, which is responsible for the generation of amyloid beta-peptide, is a high molecular weight complex composed of at least four components, namely, presenilin-1 (or presenilin-2), nicastrin, Aph-1, and Pen-2. Previous data indicated that presenilins, which are thought to harbor the catalytic core of the complex, also control p53-dependent cell death. Whether the other components of the gamma-secretase complex could also modulate the cell death process in mammalian neurons remained to be established. Here, we examined the putative contribution of Aph-1 and Pen-2 in the control of apoptosis in TSM1 cells from a neuronal origin. We show by terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling and DNA fragmentation analyses that the overexpression of Aph-1a, Aph-1b, or Pen-2 drastically lowered staurosporine-induced cellular toxicity. In support of an apoptosis rather than necrosis process, Aph-1 and Pen-2 also lower staurosporine- and etoposide-induced caspase-3 expression and diminished caspase-3 activity and poly(ADP-ribose) polymerase inactivation. The Aph-1 and Pen-2 anti-apoptotic phenotype was associated with a drastic reduction of p53 expression and activity and lowered p53 mRNA transcription. Furthermore, the Aph-1- and Pen-2-associated reduction of staurosporine-induced caspase-3 activation was fully abolished by p53 deficiency. Conversely, Aph-1a, Aph-1b, and Pen-2 gene inactivation increases both caspase-3 activity and p53 mRNA levels. Finally, we show that Aph-1 and Pen-2 did not trigger an anti-apoptotic response in cells devoid of presenilins or nicastrin, whereas the protective response was still observed in fibroblasts devoid of beta-amyloid precursor protein and amyloid precursor protein like-protein 2. Furthermore, Aph-1- and Pen-2-associated protection against staurosporine-induced caspase-3 activation was not affected by the gamma-secretase inhibitors N-[N-(3,5-difluorophenacetyl)-l-alanyl]-S-phenylglycine t-butyl ester and difluoromethylketone. Altogether, our study indicates that Aph-1 and Pen-2 trigger an anti-apoptotic response by lowering p53-dependent control of caspase-3. Our work also demonstrates that this phenotype is strictly dependent on the molecular integrity of the gamma-secretase complex but remains independent of the gamma-secretase catalytic activity.

The amyloid precursor protein (APP) is an ubiquitous receptor-like molecule involved in the pathogenesis of Alzheimer's disease (AD). APP and some of its C-terminal proteolytic fragments (CTFs) have been shown to be phosphorylated and to interact with cytosolic phosphotyrosine binding (PTB) domain containing proteins involved in cell signaling and vesicular transport. Among others, the interaction between tyrosine-phosphorylated CTFs and ShcA-Grb2 adaptors is highly enhanced in AD brain. Here we have identified in SH-SY5Y neuroblastoma cells an interaction between APP holoprotein and the adaptor Grb2. Upon activation of apoptotic cell death this interaction is rapidly degraded, APP is partially cleaved and the complex APP/Grb2 is replaced by a new complex between CTFs and ShcA that still involves Grb2. The formation of these complexes is regulated by beta-site APP-cleaving enzyme 1 and influences the phosphorylation of mitogen-activated protein kinase p44/42 extracellular signal-regulated kinase as well as the level of apoptotic death of the cells. These data suggest a dual role in cell signaling for APP and its CTFs in neuroblastoma cells, in a manner similar to that previously reported for other tyrosine kinase receptor, through a tightly regulated coupling with alternative intracellular adaptors to control the signaling of the cell.

The processing of the amyloid precursor protein (APP) through the formation of C-terminal fragments (CTFs) and the production of beta-amyloid, are events likely to influence the development and the progression of Alzheimer's disease (AD). APP is a transmembrane protein similar to a cell-surface receptor with the intraluminal NPTY motif in the cytosolic C terminus. Although APP holoprotein can be bound to intracellular proteins like Fe65, X11, and mDab, the ultimate function and the mechanisms through which this putative receptor transfers its message are unclear. Here it is shown that in human brain, a subset of tyrosine-phosphorylated CTFs represent docking sites for the adaptor protein ShcA. ShcA immunoreactivity is greatly enhanced in Alzheimer's patients; it is mainly localized to glial cells and occurs at reactive astrocytes surrounding cerebral vessels and amyloid plaques. Grb2 also is involved in complexes with ShcA and tyrosine-phosphorylated CTFs, and in AD brain the interaction between Grb2-ShcA and CTFs is enhanced. Also, a higher amount of phospho-ERK1,2 is present in AD brain in comparison with control cases, likely as a result of the ShcA activation. In vitro experiments show that the ShcA-CTFs interaction is strictly confined to glial cells when treated with thrombin, which is a well-known ShcA and ERK1,2 activator, mitogen, and regulator of APP cleavage. In untreated cells ShcA does not interact with either APP or CTFs, although they are normally produced. Altogether these data suggest that CTFs are implicated in cell signaling via Shc transduction machinery, likely influencing MAPK activity and glial reaction in AD patients.

The proteolytic processing of amyloid precursor protein (APP) through the formation of membrane-bound C-terminal fragments (CTFs) and of soluble beta-amyloid peptides likely influences the development of Alzheimer's disease (AD). We show that in human brain a subset of CTFs are tyrosine-phosphorylated and form stable complexes with the adaptor protein ShcA. Grb2 is also part of these complexes, which are present in higher amounts in AD than in control brains. ShcA immunoreactivity is also greatly enhanced in patients with AD and occurs at reactive astrocytes surrounding cerebral vessels and amyloid plaques. A higher amount of phospho-ERK1,2, likely as result of the ShcA activation, is present in AD brains. In vitro experiments show that the ShcA-CTFs interaction is strictly confined to glial cells when treated with thrombin, which is a well known ShcA and ERK1,2 activator and a regulator of APP cleavage. In untreated cells ShcA does not interact with either APP or CTFs, although they are normally generated. Altogether these data suggest that CTFs are implicated in cell signaling via Shc transduction machinery, likely influencing MAPK activity and glial reaction in AD patients.

N-terminally truncated amyloid-beta (Abeta) peptides are present in early and diffuse plaques of individuals with Alzheimer's disease (AD), are overproduced in early onset familial AD and their amount seems to be directly correlated to the severity and the progression of the disease in AD and Down's syndrome (DS). The pyroglutamate-containing isoforms at position 3 [AbetaN3(pE)-40/42] represent the prominent form among the N-truncated species, and may account for more than 50% of Abeta accumulated in plaques. In this study, we compared the toxic properties, fibrillogenic capabilities, and in vitro degradation profile of Abeta1-40, Abeta1-42, AbetaN3(pE)-40 and AbetaN3(pE)-42. Our data show that fibre morphology of Abeta peptides is greatly influenced by the C-terminus while toxicity, interaction with cell membranes and degradation are influenced by the N-terminus. AbetaN3(pE)-40 induced significantly more cell loss than the other species both in neuronal and glial cell cultures. Aggregated AbetaN3(pE) peptides were heavily distributed on plasma membrane and within the cytoplasm of treated cells. AbetaN3(pE)-40/42 peptides showed a significant resistance to degradation by cultured astrocytes, while full-length peptides resulted partially degraded. These findings suggest that formation of N-terminally modified peptides may enhance beta-amyloid aggregation and toxicity, likely worsening the onset and progression of the disease.

Alzheimer's disease (AD) is a degenerative disease of the brain, and the most common form of dementia. It is estimated that more than 22 million individuals worldwide will have AD by 2025. The causes of the disease are still unknown and recent hypotheses suggest that an aberrant protein processing initiates the neurodegeneration. Several lines of research are centered on the study of proteins that are genetically associated with this syndrome, such as amyloid precursor protein (APP) and presenilins. This review focuses on recent advances in the processing of APP and on the neuropathological role of its amyloidogenic fragments, which have been shown to be directly involved in neurodegeneration and glial inflammation and which likely influence the development of AD.

The carboxy-terminal fragments (CTFs) of the amyloid precursor protein (APP) are considered beta-amyloid (Abeta) precursors as well as molecular species possibly amyloidogenic and neurotoxic by [corrected] in vitro or in animal models. The CTF's role in the pathogenesis of Alzheimer's disease (AD) is however relatively unexplored in human brain. In this study, we analyzed brain extracted CTFs in subjects with AD, non-AD control, and Down's syndrome (DS) cases. Our data indicate that: (i) In fetal DS subjects CTFs levels are increased in comparison to age-matched control, suggesting that the enhanced CTFs formation is important for the early occurrence of plaques deposition in DS. No significant difference in CTFs level [corrected] between AD and age-matched control cases. (ii) CTFs modified at their N-terminus are the direct precursors of similarly N-terminally modified Abeta peptides, which constitute the most abundant species in AD and DS plaques. This observation suggests that N-truncated Abeta peptides are formed directly at beta-secretase level and not through a progressive proteolysis of full-length Abeta1-40/42. (iii) Among the differently cleaved CTFs, only the 22- and 12.5-kDa CTF polypeptides are tyrosine phosphorylated in both AD and control brain while the full-length APP and the CTFs migrating below the 12.5-kDa marker are not phosphorylated, suggesting that APP and CTFs may be involved in different pathways depending on their length and sequences. This study provides evidence that CTFs constitute in human brain a molecular species directly involved in AD pathogenesis and in the development of the AD-like pathology in DS subjects., (Copyright 2001 Academic Press.)

Caulerpenyne (Cyn), the major secondary metabolite synthesized by the green alga Caulerpa taxifolia proliferating in the Mediterranean Sea, is a cytotoxic sesquiterpene. As this compound has an antiproliferative potency by inhibiting division of many types of cells, we examined the precise effects of Cyn during the early development of the sea urchin Paracentrotus lividus. Whereas Cyn (60 microM) had no effect on fertilization, it blocked the first cell division in the same manner whether added before or after fertilization, provided the drug was added before or during metaphase. Immunofluorescence localization revealed that Cyn had no effect on the microtubular sperm aster formation, pronuclei migration and fusion, chromosome condensation, nuclear envelope breakdown, and bipolar mitotic spindle assembly. However, mitosis was blocked in a metaphase-like stage at which most chromosomes were aligned at the equatorial plate, while a few of them had not even migrated towards the metaphase plate. When added after the metaphase-anaphase transition, the first division occurred normally but the second division was inhibited with the same phenotype as described above. We previously showed that Cyn did not affect protein synthesis or H1 kinase activation or deactivation (Pesando et al., 1996, Aquat. Toxicol. 35, 139), but that it partially inhibited DNA synthesis. Our results establish that Cyn does not affect the microfilament-dependent processes of fertilization and cytokinesis and allows the beginning of mitosis, but prevents normal DNA replication and results in metaphase-like arrest of sea urchin embryos.