Person:

Tanzi, Rudolph

Background: 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. Results: 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. Conclusion: 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.

Background. Dementia and delirium have been postulated to share common pathophysiologic mechanisms; however, identification of these unifying mechanisms has remained elusive. The inhalation anesthetic isoflurane has been shown to enhance β-amyloid protein (Aβ) oligomerization and generation, to potentiate the cytotoxicity of Aβ, and to induce apoptosis. To address the molecular mechanisms of dementia and delirium associated with anesthesia and surgery, we assessed whether the Aβ fibrillar aggregation inhibitor Congo red can attenuate isoflurane-induced caspase-3 activation in H4 human neuroglioma cells overexpressing human β-amyloid precursor protein (APP).

Methods. H4 human neuroglioma cells stably transfected to express human full-length wild-type APP were exposed to 2% isoflurane for 6 hours. The cells were harvested at the end of the treatment. Caspase-3 activation was measured with quantitative Western blotting.

Results. We found that isoflurane induces cellular apoptosis in a dose-dependent manner, and that Congo red inhibits isoflurane-induced apoptosis in H4 human neuroglioma cells overexpressing APP. Interestingly, Congo red also inhibits staurosporine-induced apoptosis.

Conclusion. The demonstration that isoflurane contributes to well-described mechanisms of Alzheimer's neuropathogenesis provides a plausible link between the acute effects of anesthesia, a well-described risk factor for delirium, and the more long-term sequelae of dementia. These findings suggest that isoflurane-induced Aβ oligomerization and apoptosis may contribute to the risk of postoperative cognitive dysfunction and provide a potential pathogenic link between delirium and dementia.