Trans-dominant negative effects of pathogenic PSEN1 mutations on γ-secretase activity and Aß production.

Mutations in the PSEN1 gene encoding Presenilin-1 (PS1) are the predominant cause of familial Alzheimer's disease (FAD), but the underlying mechanisms remain unresolved. To reconcile the dominant action of pathogenic PSEN1 mutations with evidence that they confer a loss of mutant protein function, we tested the hypothesis that PSEN1 mutations interfere with γ-secretase activity in a dominant-negative manner. Here, we show that pathogenic PSEN1 mutations act in cis to impair mutant PS1 function and act in trans to inhibit wild-type PS1 function. Coexpression of mutant and wild-type PS1 at equal gene dosage in presenilin-deficient mouse embryo fibroblasts resulted in trans-dominant-negative inhibition of wild-type PS1 activity, suppressing γ-secretase-dependent cleavage of APP and Notch. Surprisingly, mutant PS1 could stimulate production of Aß42 by wild-type PS1 while decreasing its production of Aß40. Mutant and wild-type PS1 efficiently coimmunoprecipitated, suggesting that mutant PS1 interferes with wild-type PS1 activity via physical interaction. These results support the conclusion that mutant PS1 causes wild-type PS1 to adopt an altered conformation with impaired catalytic activity and substrate specificity. Our findings reveal a novel mechanism of action for pathogenic PSEN1 mutations and suggest that dominant-negative inhibition of presenilin activity plays an important role in FAD pathogenesis.

A presenilin-1 mutation identified in familial Alzheimer disease with cotton wool plaques causes a nearly complete loss of gamma-secretase activity.

Mutations in presenilin-1 and presenilin-2 (PS1 and PS2) are the most common cause of familial Alzheimer disease. PS1 and PS2 are the presumptive catalytic components of the multisubunit gamma-secretase complex, which proteolyzes a number of type I transmembrane proteins, including the amyloid precursor protein (APP) and Notch. APP processing by gamma-secretase produces beta-amyloid peptides (Abeta40 and Abeta42) that accumulate in the Alzheimer disease brain. Here we identify a pathogenic L435F mutation in PS1 in two affected siblings with early-onset familial Alzheimer disease characterized by deposition of cerebral cotton wool plaques. The L435F mutation resides in a conserved C-terminal PAL sequence implicated in active site conformation and catalytic activity. The impact of PS1 mutations in and around the PAL motif on gamma-secretase activity was assessed by expression of mutant PS1 in mouse embryo fibroblasts lacking endogenous PS1 and PS2. Surprisingly, the L435F mutation caused a nearly complete loss of gamma-secretase activity, including >90% reductions in the generation of Abeta40, Abeta42, and the APP and Notch intracellular domains. Two nonpathogenic PS1 mutations, P433L and L435R, caused essentially complete loss of gamma-secretase activity, whereas two previously identified pathogenic PS1 mutations, P436Q and P436S, caused partial loss of function with substantial reductions in production of Abeta40, Abeta42, and the APP and Notch intracellular domains. These results argue against overproduction of Abeta42 as an essential property of presenilin proteins bearing pathogenic mutations. Rather, our findings provide support for the hypothesis that pathogenic mutations cause a general loss of presenilin function.

Manganese and iron transport across pulmonary epithelium.

Pathways mediating pulmonary metal uptake remain unknown. Because absorption of iron and manganese could involve similar mechanisms, transferrin (Tf) and transferrin receptor (TfR) expression in rat lungs was examined. Tf mRNA was detected in bronchial epithelium, type II alveolar cells, macrophages, and bronchus-associated lymphoid tissue (BALT). Tf protein levels in lung and bronchoalveolar lavage fluid did not change in iron deficiency despite increased plasma levels, suggesting that lung Tf concentrations are regulated by local synthesis in a manner independent of body iron status. Iron oxide exposure upregulated Tf mRNA in bronchial and alveolar epithelium, macrophages, and BALT, but protein was not significantly increased. In contrast, TfR mRNA and protein were both upregulated by iron deficiency. To examine potential interactions with lung Tf, rats were intratracheally instilled with (54)Mn or (59)Fe. Unlike (59)Fe, interactions between (54)Mn and Tf in lung fluid were not detected. Absorption of intratracheally instilled (54)Mn from the lungs to the blood was unimpaired in Belgrade rats homozygous for the functionally defective G185R allele of divalent metal transporter-1, indicating that this transporter is also not involved in pulmonary manganese absorption. Pharmacological studies of (54)Mn uptake by A549 cells suggest that metal uptake by type II alveolar epithelial cells is associated with activities of both L-type Ca(2+) channels and TRPM7, a member of the transient receptor potential melastatin subfamily. These results demonstrate that iron and manganese are absorbed by the pulmonary epithelium through different pathways and reveal the potential role for nonselective calcium channels in lung metal clearance.