The presenilin 2 mutation (N141I) linked to familial Alzheimer disease (Volga German families) increases the secretion of amyloid beta protein ending at the 42nd (or 43rd) residue

Abstract

To gain insights into the significance of presenilins (PS) in the pathogenetic mechanisms of early-onset familial Alzheimer disease (FAD), we expressed cDNAs for wild-type PS2 and PS2 with the Volga German (N141I) mutation in cultured cells and then examined the metabolism of the transfected proteins and their effect on the C-terminal properties of secreted amyloid beta protein (A beta). PS2 was identified as a 50- to 55-kDa protein, which was cleaved to produce N-terminal fragments of 35-40 kDa and C-terminal fragments of 19-23 kDa. The Volga German (N141I) mutation did not cause any significant change in the metabolism of PS2. COS-1 cells doubly transfected with cDNAs for N141I mutant PS2 and human beta-amyloid precursor protein (betaAPP) or a C-terminal fragment thereof, as well as mouse Neuro2a neuroblastoma cells stably transfected with N141I mutant PS2 alone, secreted 1.5- to 10-fold more A beta ending at residues 42 (or 43) [A beta42(43)] compared with those expressing the wild-type PS2. These results strongly suggest that the PS2 mutation (N141I) linked to FAD alters the metabolism of A beta/betaAPP to foster the production of the form of A beta that most readily deposits in amyloid plaques. Thus, mutant PS2 may lead to AD by altering the metabolism of A beta/betaAPP.

Figure 1

PS2 expression and metabolism in transiently transfected COS-1 cells. (A) Western blot analysis of human PS2 expression in comparison with that of PS1 in transiently transfected COS-1 cells. Cell lysates (10 μg protein) from COS-1 cells transfected with empty pcDNA3 vector (lanes 1, 4, 7, 10), with wild-type human PS1 (lanes 2, 5, 8, and 11), or PS2 (lanes 3, 6, 9, and 12) cDNAs were fractionated by SDS/PAGE and analyzed by immunoblotting with 2972 (raised against the N terminus of PS2) (lanes 1–3), anti-PSC1/2 (lanes 4–6), anti-PS1N (lanes 7–9), and anti-PS1L (lanes 10–12) antibodies. Full-length PS proteins are marked by arrows, and fragments thereof by arrowheads. Molecular mass standards are shown in kilodaltons. (B) Comparison of proteolytic processing of wild-type and N141I mutant PS2. Cell lysates from nontransfected COS-1 cells (lane 1), cells transfected with wild-type human PS2 (lane 2), or N141I mutant PS2 (lane 3) cDNAs were probed with anti-PSC1/2.

Figure 2

Expression of PS2 and βAPP in stable N2a cells. Western blot analysis of stable N2a cells transfected with human PS2 cDNAs. Triton X-100 soluble fractions from lysates of N2a cells (10 μg protein) transfected with an empty vector (lane 1), cDNAs encoding wild-type (lanes 2–4, corresponding to clones PS2.a–c in Fig. 3B, respectively), or N141I mutant human PS2 (lanes 5–7, corresponding to clones PS2_N141I.a–c in Fig. 3B, respectively) were fractionated by SDS/PAGE and analyzed by immunoblotting with 2972 (raised against the N terminus of PS2) (A), anti-PS2L (B), and 22C11 (C). Immunobands representing full-length PS2 and fragments thereof are marked by arrows and arrowheads, respectively, in A and B. Molecular mass standards are shown in kilodaltons.

Figure 3

Secreted Aβ40 and Aβ42(43) from cells expressing wild-type or N141I mutant PS2 genes. (A) Levels of Aβx-40, Aβx-42(43), Aβ1–40, and Aβ1–42(43) secreted from COS-1 cells doubly transfected with human βAPP and PS2 genes quantitated by two-site ELISAs. Mean values ± SE in three independent experiments are shown. Combinations of the transfected cDNAs are indicated below the columns: PS2, wild-type PS2; PS2_N141I, N141I mutant PS2; APP, wild-type human βAPP695; APP_NL, 595/596 (KM-NL) mutant human βAPP695; C100, C-terminal fragment of human βAPP. (B) Levels of Aβx-40 and Aβx-42(43) secreted from N2a cells stably transfected with PS2 cDNAs. Mean values ± SE (n = 4) from three independent cell lines (PS2.a–c transfected with wild-type PS2 and PS2_N141I.a–c with N141I mutant PS2 cDNAs) are shown together with those from two nontransfected cell lines (Null.a and b) and cells transfected with empty vector (Vector.a and b).