Depletion of GGA3 stabilizes BACE and enhances beta-secretase activity

Beta-site APP-cleaving enzyme (BACE) is required for production of the Alzheimer's disease (AD)-associated Abeta protein. BACE levels are elevated in AD brain, and increasing evidence reveals BACE as a stress-related protease that is upregulated following cerebral ischemia. However, the molecular mechanism responsible is unknown. We show that increases in BACE and beta-secretase activity are due to posttranslational stabilization following caspase activation. We also found that during cerebral ischemia, levels of GGA3, an adaptor protein involved in BACE trafficking, are reduced, while BACE levels are increased. RNAi silencing of GGA3 also elevated levels of BACE and Abeta. Finally, in AD brain samples, GGA3 protein levels were significantly decreased and inversely correlated with increased levels of BACE. In summary, we have elucidated a GGA3-dependent mechanism regulating BACE levels and beta-secretase activity. This mechanism may explain increased cerebral levels of BACE and Abeta following cerebral ischemia and existing in AD.     

3-Hydroxykynurenine and 3-hydroxyanthranilic acid generate hydrogen peroxide and promote alpha-crystallin cross-linking by metal ion reduction

The kynurenine pathway catabolite 3-hydroxykynurenine (3HK) and redox-active metals such as copper and iron are implicated in cataractogenesis. Here we investigate the reaction of kynurenine pathway catabolites with copper and iron, as well as interactions with the major lenticular structural proteins, the alpha-crystallins. The o-aminophenol kynurenine catabolites 3HK and 3-hydroxyanthranilic acid (3HAA) reduced Cu(II)>Fe(III) to Cu(I) and Fe(II), respectively, whereas quinolinic acid and the nonphenolic kynurenine catabolites kynurenine and anthranilic acid did not reduce either metal. Both 3HK and 3HAA generated superoxide and hydrogen peroxide in a copper-dependent manner. In addition, 3HK and 3HAA fostered copper-dependent alpha-crystallin cross-linking. 3HK- or 3HAA-modifed alpha-crystallin showed enhanced redox activity in comparison to unmodified alpha-crystallin or ascorbate-modified alpha-crystallin. These data support the possibility that 3HK and 3HAA may be cofactors in the oxidative damage of proteins, such as alpha-crystallin, through interactions with redox-active metals and especially copper. These findings may have relevance for understanding cataractogenesis and other degenerative conditions in which the kynurenine pathway is activated.     

Reduced amyloidogenic processing of the amyloid β-protein precursor by the small-molecule Differentiation Inducing Factor-1

The detection of cell cycle proteins in Alzheimer's disease (AD) brains may represent an early event leading to neurodegeneration. To identify cell cycle modifiers with anti-Aβ properties, we assessed the effect of Differentiation-Inducing Factor-1 (DIF-1), a unique, small-molecule from Dictyostelium discoideum, on the proteolysis of the amyloid β-protein precursor (APP) in a variety of different cell types. We show that DIF-1 slows cell cycle progression through G0/G1 that correlates with a reduction in cyclin D1 protein levels. Western blot analysis of DIF-treated cells and conditioned medium revealed decreases in the levels of secreted APP, mature APP, and C-terminal fragments. Assessment of conditioned media by sandwich ELISA showed reduced levels of Aβ40 and Aβ42, also demonstrating that treatment with DIF-1 effectively decreases the ratio of Aβ42 to Aβ40. In addition, DIF-1 significantly diminished APP phosphorylation at residue T668. Interestingly, site-directed mutagenesis of APP residue Thr668 to alanine or glutamic acid abolished the effect of DIF-1 on APP proteolysis and restored secreted levels of Aβ. Finally, DIF-1 prevented the accumulation of APP C-terminal fragments induced by the proteasome inhibitor lactacystin, and calpain inhibitor N-acetyl-leucyl-leucyl-norleucinal (ALLN). Our findings suggest that DIF-1 affects G0/G1-associated amyloidogenic processing of APP by a γ-secretase-, proteasome- and calpain-insensitive pathway, and that this effect requires the presence of residue Thr668.     

Electrophysiological study of the frog egg at various stages of development. IV. Changes associated with the maturation process

The I/V relationship of frog oocytes is markedly modified following maturation, lacking the mature unfertilized egg cell membrane of K-selective channels and the leakage being strongly reduced. The only kind of voltage-dependent channels still present in this membrane are activated by depolarizing steps to potentials more positive than 30-40 mV; the current flowing through these channels is carried at least partly by Na+. At difference with the immature oocyte, where several ionic mechanisms are oriented to maintain the membrane potential at a constant level, the mature egg-cell admits considerable voltage deflections. The observed variations in membrane properties could be of importance in allowing the maturation process and/or in preparing the mature egg-cell to successful fertilization.     

The Influence of Plant-Growth Factors on the Initiation and Growth of Crown-Gall Tumours on Primary Pinto Bean Leaves

The number of tumours developing on primary pinto bean leaves(Phaseolus vulgaris L. variety Pinto) inoculated with Agrobacteriumtumefaciens (Smith and Town.) Conn, strain B6, was increasedby as much as 100 per cent through the addition of several plant-growthfactors and antagonists of some of these factors to the freshlyinoculated leaves. Naphthylacetic acid, gibberellic acid, (2-chloroethyl)trimethylammonium chloride, and adenine gave a biphasic responsewith optimal promotions of tumour initiation at 10^-5 to 10^-4mg/ml. Tri-iodobenzoic acid and 4-chlorophenoxyisobutyric acidwere most active at 10^-1 mg/ml. Mean tumour diameter showeda direct correlation with tumour number in these experiments.The results show tumour initiation to be sensitive to growthfactor changes, possibly through a heightened traumatic responseof the wounded leaves. The growth of the tumours was unaffectedby these additions except as they altered tumour number andthis secondarily affected tumour growth.