Caloric restriction and SirT1 modulate APP metabolism in vitro and in vivo

Proteolytic processing of the β-amyloid precursor protein (APP) by α-, β-and γ-secretase enzymes generating the amyloid-beta (Aβ) peptide and the APP intracellular domain (AICD) is a central event in Alzheimer’s disease (AD). Herewe show that in vitro CR decreases Aβ, AICD and full-length APP levels in human cell lines without affecting APP transcription and that some of these effects can be recapitulated by over-expressing the NAD+ dependent deacetylase SirT1 in our cell lines. Resveratrol, a SirT1 agonist, also has similar effects on APP metabolism. SirT1 and resveratrol however, do not affect full-length APP levels. In our cell lines, SirT1and resveratrol reduces secreted Aβ levels by increasing the α-secretase cleavage of APP and also possibly by affecting γ-secretase activity. Extending these studies to an in vivo setting, an AICD reporter Drosophila model of AD, shows that caloric restriction, Sir2 gain-of-function and resveratrol treatment suppress AD-like rough-eye phenotype in the fly eyes. Finally to study the mechanism of CR and SirT1 mediated effects on APP metabolism in entire central nervous system, we created and characterized a novel Drosophila model of AD. We have shown that our model displays neuroanatomical and behavioral features that are characteristic of AD patients.Ph.D., Biology -- Drexel University, 201

An AICD-based functional screen to identify APP metabolism regulators

<p>Abstract</p> <p>Background</p> <p>A central event in Alzheimer's disease (AD) is the regulated intramembraneous proteolysis of the β-amyloid precursor protein (APP), to generate the β-amyloid (Aβ) peptide and the APP intracellular domain (AICD). Aβ is the major component of amyloid plaques and AICD displays transcriptional activation properties. We have taken advantage of AICD transactivation properties to develop a genetic screen to identify regulators of APP metabolism. This screen relies on an APP-Gal4 fusion protein, which upon normal proteolysis, produces AICD-Gal4. Production of AICD-Gal4 induces Gal4-UAS driven luciferase expression. Therefore, when regulators of APP metabolism are modulated, luciferase expression is altered.</p> <p>Results</p> <p>To validate this experimental approach we modulated α-, β-, and γ-secretase levels and activities. Changes in AICD-Gal4 levels as measured by Western blot analysis were strongly and significantly correlated to the observed changes in AICD-Gal4 mediated luciferase activity. To determine if a known regulator of APP trafficking/maturation and Presenilin1 endoproteolysis could be detected using the AICD-Gal4 mediated luciferase assay, we knocked-down Ubiquilin 1 and observed decreased luciferase activity. We confirmed that Ubiquilin 1 modulated AICD-Gal4 levels by Western blot analysis and also observed that Ubiquilin 1 modulated total APP levels, the ratio of mature to immature APP, as well as PS1 endoproteolysis.</p> <p>Conclusion</p> <p>Taken together, we have shown that this screen can identify known APP metabolism regulators that control proteolysis, intracellular trafficking, maturation and levels of APP and its proteolytic products. We demonstrate for the first time that Ubiquilin 1 regulates APP metabolism in the human neuroblastoma cell line, SH-SY5Y.</p

Characterization of a Drosophila Alzheimer's Disease Model: Pharmacological Rescue of Cognitive Defects

Transgenic models of Alzheimer's disease (AD) have made significant contributions to our understanding of AD pathogenesis, and are useful tools in the development of potential therapeutics. The fruit fly, Drosophila melanogaster, provides a genetically tractable, powerful system to study the biochemical, genetic, environmental, and behavioral aspects of complex human diseases, including AD. In an effort to model AD, we over-expressed human APP and BACE genes in the Drosophila central nervous system. Biochemical, neuroanatomical, and behavioral analyses indicate that these flies exhibit aspects of clinical AD neuropathology and symptomology. These include the generation of Aβ40 and Aβ42, the presence of amyloid aggregates, dramatic neuroanatomical changes, defects in motor reflex behavior, and defects in memory. In addition, these flies exhibit external morphological abnormalities. Treatment with a γ-secretase inhibitor suppressed these phenotypes. Further, all of these phenotypes are present within the first few days of adult fly life. Taken together these data demonstrate that this transgenic AD model can serve as a powerful tool for the identification of AD therapeutic interventions

An AICD-based functional screen to identify APP metabolism regulators-3

<p><b>Copyright information:</b></p><p>Taken from "An AICD-based functional screen to identify APP metabolism regulators"</p><p>http://www.molecularneurodegeneration.com/content/2/1/15</p><p>Molecular Neurodegeneration 2007;2():15-15.</p><p>Published online 24 Aug 2007</p><p>PMCID:PMC2071909.</p><p></p>83-Gal4, and sAPPα levels compared to cells transfected with empty vector. (B) Quantification of Western blot densitometry in panel A. (C) ADAM17 transient over-expression significantly increases ADAM17, AICD-Gal4, C83-Gal4, and sAPPα levels. (D) Quantification of Western blot densitometry in panel C. (E) Transient over-expression of individual secretase genes increases AICD-Gal4 mediated luciferase activity. Luciferase was normalized to transfection efficiency, by dividing by luciferase activity. Individual secretase over-expression plasmids were co-transfected with pRL-SV40 plasmid, expressing luciferase. Bars represent the mean normalized luciferase activity of four independent trials and error bars represent standard errors. Statistical significance was determined using two-sample, one-tail t-tests to compare each secretase gene with the empty vector, followed by sequential Bonferroni procedure to adjust for multiple comparisons. * indicates p < 0.05; ** indicates p < 0.01

An AICD-based functional screen to identify APP metabolism regulators-8

<p><b>Copyright information:</b></p><p>Taken from "An AICD-based functional screen to identify APP metabolism regulators"</p><p>http://www.molecularneurodegeneration.com/content/2/1/15</p><p>Molecular Neurodegeneration 2007;2():15-15.</p><p>Published online 24 Aug 2007</p><p>PMCID:PMC2071909.</p><p></p>lls. (B) Ubiquilin 1 over-expression increases PS1 CTF levels in SY5Y-APP-Gal4 cells