Nicastrin and presenilin are two major components of the γ-secretase complex, which executes the intramembrane proteolysis of type I integral membrane proteins such as the amyloid precursor protein (APP) and Notch. Nicastrin is synthesized in fibroblasts and neurons as an endoglycosidase-H-sensitive glycosylated precursor protein (immature nicastrin) and is then modified by complex glycosylation in the Golgi apparatus and by sialylation in the trans-Golgi network (mature nicastrin). These modifications are not observed with exogenously overexpressed nicastrin. Under normal cell culture conditions, only mature nicastrin is expressed at the cell surface and binds to the presenilin heterodimers. Mature nicastrin has a half-life of more than 24 hours. In the absence of presenilin 1 and 2,nicastrin remains entirely endoglycosidase H sensitive, is retained in the endoplasmic reticulum and is slowly degraded. Single presenilin 1 or presenilin 2 deficiency affects glycosylation of nicastrin to a lesser extent than the combined presenilin deficiencies, suggesting a correlation between either the transport of nicastrin out of the endoplasmic reticulum or the concomitant complex glycosylation of nicastrin, and γ-secretase activity. However, when complex glycosylation of nicastrin was inhibited using mannosidase I inhibitors, γ-secretase cleavage of APP or Notch was not inhibited and the immature nicastrin still associates with presenilin and appears at the cell surface. Complex glycosylation of nicastrin is therefore not needed for γ-secretase activity. Because the trafficking of nicastrin to the Golgi apparatus is dependent on presenilins, our data point to a central role of presenilin in nicastrin maturation/localization, which could help to partially resolve the `spatial paradox'.

Our aim was to investigate the effect of TiF₄ solutions on mineral loss on enamel and dentine in vitro. Samples were fluoridated 1 × 5 min per day with 1.64% w/v TiF₄ or 2.2% w/v NaF solutions, each with a pH of 1.2, and then subjected to a cyclic de- and remineralization procedure for 5 days. Demineralization was performed for 6 × 10 min per day with citric acid (pH 2.3). In controls no fluoridation was performed. Mineral content was determined by longitudinal microradiography. Enamel mineral loss was markedly reduced by both fluoride solutions, but TiF₄ was significantly more effective than NaF: cumulative mineral loss on day 3 was 61.7 ± 15.0 µm in the NaF and 34.2 ± 13.1 µm in the TiF₄ group (p ≤ 0.001) compared with 121.0 ± 27.0 µm in the control group. Dentine mineral loss ceased after both TiF₄ and NaF applications (cumulative mineral loss on day 5 in controls: 61.0 ± 17.0 µm, in the TiF₄ group: 15.4 ± 13.4 µm and in the NaF group: 21.8 ± 11.8 µm). Both TiF₄ and NaF application reduced mineral loss both on enamel and dentine, which could open new possibilities for a symptomatic therapy of erosions.

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The amyloid precursor protein (APP) is a member of a protein family in mammals that includes the APP-like proteins APLP1 and APLP2 [1]. All APP ⁄ APLP family members are type I integral membrane proteins with large extracellular ectodomains and short cytoplasmic tails. Compared with APP, both APLPs are highly homologous in their amino acid sequence (e.g. APLP2 ⁄ APP 52% identical, 71% similar) [2] and are proteolytically processed in a similar way. The N-terminal ectodomains are released by a shedding enzyme [2,3], whereas the C-termini remain in the membrane [2,4,5] and can be further processed to release a cytoplasmic fragment with signaling properties [4,6,7].Further elucidation of APLP2-processing is of relevance with regard to the outstanding function of this protein, which was derived from knockout experiments. Whereas a double knockout of APP and APLP1 did not show severe phenotypic changes in mice, the combined knockout of APLP2 with both of the other APP family members resulted in postnatal lethality [8,9]. This shows that APLP2 and ⁄ or one of its proteolytic fragments are essential for normal development and Cleavage of the amyloid precursor protein (APP) within the amyloid-beta (Ab) sequence by the a-secretase prevents the formation of toxic Ab peptides. It has been shown that the disintegrin-metalloproteinases ADAM10 and TACE (ADAM17) act as a-secretases and stimulate the generation of a soluble neuroprotective fragment of APP, APPsa. Here we demonstrate that the related APP-like protein 2 (APLP2), which has been shown to be essential for development and survival of mice, is also a substrate for both proteinases. Overexpression of either ADAM10 or TACE in HEK293 cells increased the release of neurotrophic soluble APLP2 severalfold. The strongest inhibition of APLP2 shedding in neuroblastoma cells was observed with an ADAM10-preferring inhibitor. Transgenic mice with neuron-specific overexpression of ADAM10 showed significantly increased levels of soluble APLP2 and its C-terminal fragments. To elucidate a possible regulatory mechanism of APLP2 shedding in the neuronal context, we examined retinoic acid-induced differentiation of neuroblastoma cells. Retinoic acid treatment of two neuroblastoma cell lines upregulated the expression of both APLP2 and ADAM10, thus leading to an increased release of soluble APLP2.Abbreviations ADAM, a disintegrin and metalloproteinase; ADAM10DN, catalytically inactive dominant negative mutant form of ADAM10; APLP1, APP-like protein 1; APLP2, APP-like protein 2; APLP2s, cleaved soluble APLP2; APP, amyloid precursor protein; BACE, b-site APP-cleaving enzyme; CS-GAG, chondroitin sulfate glycosaminoglycan; PKC, protein kinase C; PMA, phorbol-12-myristate-13-acetate; PVDF, poly(vinylidene difluoride); RA, retinoic acid; TACE, tumor necrosis factor-a converting enzyme.

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