The mammalian amyloid precursor protein (APP) protein family consists of the APP and the amyloid precursor-like proteins 1 and 2 (APLP1 and APLP2). The neurotoxic amyloid ␤-peptide (A␤) originates from APP, which is the only member of this protein family implicated in Alzheimer disease. However, the three homologous proteins have been proposed to be processed in similar ways and to have essential and overlapping functions. Therefore, it is also important to take into account the effects on the processing and function of the APP-like proteins in the development of therapeutic drugs aimed at decreasing the production of A␤. Insulin and insulin-like growth factor-1 (IGF-1) have been shown to regulate APP processing and the levels of A␤ in the brain. In the present study, we show that IGF-1 increases ␣-secretase processing of endogenous APP and also increases ectodomain shedding of APLP1 and APLP2 in human SH-SY5Y neuroblastoma cells. We also investigated the role of different IGF-1-induced signaling pathways, using specific inhibitors for phosphatidylinositol 3-kinase and mitogenactivated protein kinase (MAPK). Our results indicate that phosphatidylinositol 3-kinase is involved in ectodomain shedding of APP and APLP1, but not APLP2, and that MAPK is involved only in the ectodomain shedding of APLP1.

The amyloid precursor protein (APP) belongs to a conserved gene family, also including the amyloid precursor-like proteins, APLP1 and APLP2. We have previously shown that all members of the APP protein family are up-regulated upon retinoic acid (RA)-induced neuronal differentiation of SH-SY5Y neuroblastoma cells. Here, we demonstrate that RA also affects the processing of APLP2 and APP, as shown by increased shedding of both sAPLP2 and sAPPa, as well as elevated levels of the APP intracellular domains (AICDs). Brain-derived neurotrophic factor (BDNF) has been reported to induce APP promoter activity and RA induces expression of the tyrosine kinase receptor B (TrkB) in neuroblastoma cells. We show that the increase in shedding of both APLP2 and APP in response to RA is not mediated through the TrkB receptor. However, BDNF concomitant with RA increased the expression of APP even further. In addition, the secretion of sAPLP2 and sAPPa as well as the levels of AICDs were increased in response to BDNF. In contrast, the levels of membrane-bound APP C-terminal fragment C99 significantly decreased. Our results suggest that RA and BDNF shifts APP processing towards the a-secretase pathway. In addition, we show that RA and BDNF regulate N-linked glycosylation of APLP1. Keywords: Alzheimer's disease, amyloid precursor protein, amyloid precursor-like proteins 1 and 2, amyloid precursor protein intracellular domain, processing.

␣-Secretase cleavage of the amyloid precursor protein (APP) is of great interest because it prevents the formation of the Alzheimer-linked amyloid-␤ peptide. APP belongs to a conserved gene family including the two paralogues APP-like protein (APLP) 1 and 2. Insulin-like growth factor-1 (IGF-1) stimulates the shedding of all three proteins. IGF-1-induced shedding of both APP and APLP1 is dependent on phosphatidylinositol 3-kinase (PI3-K), whereas APLP2 shedding is independent of this signaling pathway. Here, we used human neuroblastoma SH-SY5Y cells to investigate the involvement of protein kinase C (PKC) in the proteolytic processing of endogenously expressed members of the APP family. Processing was induced by IGF-1 or retinoic acid, another known stimulator of APP ␣-secretase shedding. Our results show that stimulation of APP and APLP1 processing involves multiple signaling pathways, whereas APLP2 processing is mainly dependent on PKC. Next, we wanted to investigate whether the difference in the regulation of APLP2 shedding compared with APP shedding could be due to involvement of different processing enzymes. We focused on the two major ␣-secretase candidates ADAM10 and TACE, which both are members of the ADAM (a disintegrin and metalloprotease) family. Shedding was analyzed in the presence of the ADAM10 inhibitor GI254023X, or after transfection with small interfering RNAs targeted against TACE. The results clearly demonstrate that different ␣-secretases are involved in IGF-1-induced processing. APP is mainly cleaved by ADAM10, whereas APLP2 processing is mediated by TACE. Finally, we also show that IGF-1 induces PKC-dependent phosphorylation of TACE. Alzheimer disease (AD)2 is histopathologically characterized by the presence of amyloid plaques in the brain parenchyma. The major constituent of the plaques is the amyloid-␤ (A␤) peptide, which is derived from the A␤ precursor protein (APP) by ␤-and ␥-secretase cleavage in the amyloidogenic pathway (1). In addition to A␤, a larger secreted fragment, sAPP␤, and an intracellular fragment (APP intracellular domain) are formed. Proteolytic processing of APP occurs mainly by an alternative pathway. In this nonamyloidogenic pathway, A␤ formation is precluded because ␣-secretase cleaves APP in the middle of the A␤ region. In addition to sAPP␣, a small secreted peptide, p3, and APP intracellular domain are generated by subsequent ␥-secretase cleavage.APP belongs to a conserved gene family including the two mammalian paralogues amyloid precursor-like protein 1 and 2 (APLP1 and APLP2). The exact biological function of APP and its paralogues is still unknown, although several different studies have shown involvement of APP in cell adhesion, neurite outgrowth, synaptogenesis, modulation of synaptic plasticity, and neuroprotection (reviewed in Ref. 2). Homo-and heterotypic cis interactions of APP family members have been detected (3). APLP1 could also form trans interactions, suggesting a specific role of APLP1 in cell adhesion. Importantly, double knock-out studies in mi...

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WNT signaling pathways play an important role in both development and disease. By analyzing the signaling capabilities of commercially available WNT3a preparations towards the PI3K/AKT/GSK3 signaling pathway, we discovered unexpected inconsistencies from lot to lot of recombinant WNT3a. We provide evidence that: (1) The ability to trigger AKT/GSK3 signaling varies dramatically between different lots of WNT3a, without any variation in their ability to activate the canonical WNT/b-catenin signaling. (2) sFRP1, a WNT signaling inhibitor, is unable to interfere with the activation of AKT/GSK3 signaling induced by some of the WNT3a lots. (3) Pharmacological inhibition of AKT/ GSK3 phosphorylation by PI3K inhibitors fails to affect the stabilization of b-catenin, the central effector of the canonical WNT/b-catenin signaling pathway. In summary, while all tested lots of recombinant WNT3a activated WNT/b-catenin pathway, our results suggest that individual lots of recombinant WNT3a activate the PI3K/AKT/GSK3 pathway in a WNT-independent manner, hampering thus the analysis of regulation of PI3K/AKT/GSK3 by WNT ligand.