The Presenilin 1 C92S Mutation Increases A 42 Production Patrick A. Lewis,* Jordi Perez-Tur,* , † Todd E. Golde,* ,1 and John Hardy* *Mayo Clinic Jacksonville, 4500 San Pablo Road, Jacksonville, Florida 32224; and †Unitat de Genetica Molecular, Institut de Biomedicina de Valencia (CSIC), C/. Jaume Roig, 11, Valencia E-46010, Spain Received September 1, 2000 Although wild-type human presenilin 1 (PS1) res- cues the C. elegans egg-laying (egl) phenotype that is caused by a loss of function mutation in the C. elegans presenilin homologue sel12, most familial Alzheimer’s disease (FAD)-linked PS1 mutants only partially res- cue this phenotype. To investigate the effects of the loss of function sel12 mutation on A production in mammalian cells, we analyzed A production in trans- fected H4 neuroglioma cells expressing the PS1 homo- logue of the sel12 C60S mutant, PS1 C92S. This analy- sis revealed that PS1 C92S increased A42 levels in a similar fashion to other pathogenic Alzheimer’s dis- ease (AD) PS1 mutations. Significantly, the PS1 C92S mutation has recently been identified as the patho- genic mutation in an Italian family with FAD. Thus, placing a mutation that results in loss of function in C. elegans into a context whereby its effect on mamma- lian cells can be evaluated suggests that all FAD- linked PS1 mutants result in increased A42 produc- tion through a partial loss of function mechanism. © 2000 Academic Press Key Words: presenilin; Alzheimer’s disease; sel12; C. elegans; A; amyloid protein precursor. Mutations in the human presenilin 1 and 2 (PS1, PS2) genes have been shown to cause up to 60% of early onset familial Alzheimer’s disease (FAD), and evidence from a number of studies indicates that the FAD-linked PS mutations cause AD by increasing pro- duction of A42 from the amyloid precursor protein (APP) (reviewed in (2)). The C. elegans homologue of PS1 is sel12, and it has been show that mutations disrupting this locus cause an egg-laying phenotype (egl) through a loss of function mechanism, with one of these mutations being a missense variant, C60S (3). While wild-type human PS1 can rescue the egl pheno- type, all pathogenic missense PS1 mutations tested only partially rescue the phenotype suggesting that they have reduced activity in this assay (1). Sel12 and PS1 have about 50% sequence identity and the sel12 residue C60 is conserved with a homologous residue occurring at position 92 in PS1 (3). Therefore, we have sought to assess whether the PS1 homologue of the sel12 loss of function mutation (PS1 C92S) had any affects on A production in order to gain some insight into the genetic mechanisms underlying the patho- genic functions of PS1. Interestingly, after carrying out these studies, linkage of this mutation to an Italian family with FAD was reported suggesting that expres- sion of this mutation was likely to alter A production. MATERIALS AND METHODS In these experiments we assessed the effects of expression of PS1-wild-type, the FAD-linked mutant PS1 M139V; and PS1 C92S (the human homologue of the sel12 egl mutation) on A production. Stable H4 human neuroglioma derived cells expressing these con- structs were generated as previously described (4). To facilitate measurement of A, these stable lines were then transiently trans- fected with pcDNA3 APP 695NL, and total A,A40 A42, and sAPP analyzed in the conditioned media by ELISA as previously described (4). For Western blot analysis, 48 h after transfection the cells were lysed and processed for immunodetection of PS1 as previ- ously described (4). RESULTS Expression of both PS1 M139V and PS1 C92S mu- tation significantly increased the relative percentage of A42 in the conditioned media by approximately two- fold as compared to cells stably overexpressing PS1 wt (Fig. 1). The increase seen in the C92S mutant was slightly less than the increase in the M139V, an FAD- linked PS1 point mutant that has a very strong effect on A42 production. Western blot analysis of these stable cell lines indicates that the level of expression of PS1wt, M139V, and C92S were quite similar (Fig. 2). Although attempts were made to directly express epitope tagged versions of sel12 and sel12 C60S in H4 cells, expression of the C. elegans PS1 homologue could 1 To whom correspondence should be addressed. Fax: (904) 953- 7370. E-mail: tgolde@mayo.edu. Biochemical and Biophysical Research Communications 277, 261–263 (2000) doi:10.1006/bbrc.2000.3646, available online at http://www.idealibrary.com on 261 0006-291X/00 $35.00 Copyright © 2000 by Academic Press All rights of reproduction in any form reserved.