Article Figures & Data
Figures
- Figure 1.
Development of a linear dose–response assay for γ-secretase activity. A, Psen-null MEFs were transfected with increasing amounts of vector encoding WT PS1, and Western analysis was performed with antibody recognizing the PS1 N terminus. untrans, Untransfected MEFs. B, Quantification of PS1-NTF levels demonstrates linear nonsaturating expression as a function of increasing vector transfected. C–F, Psen-null MEFs were cotransfected with increasing amounts of vector expressing WT PS1 and a constant amount of vector expressing APP C99-myc (C,D) or NotchΔE-myc (E,F). Western analysis was performed with antibody recognizing the C-terminal myc epitope (C) or the cleaved N terminus of NICD (E). *γ-Secretase-independent band. Quantification of AICD (D) and NICD (F) production demonstrates that the activity assays saturated with respect to PS1 expression at >25 ng of PS1 vector transfected. Data are expressed as percentage of maximum NTF level ± SEM (n = 3 independent experiments).
- Figure 2.
FAD PSEN1 mutations inhibit presenilinase activity of mutant PS1 in cis and WT PS1 in trans. A, B, To assess the cis effects of PSEN1 mutations on presenilinase activity, Psen-null MEFs were transfected with vector encoding WT or mutant PS1. PS1 endoproteolysis was assessed by Western analysis with antibodies recognizing PS1 holoprotein and NTF, or CTF. Quantification of PS1-NTF levels demonstrates significantly reduced presenilinase activity with all mutations. untrans, Untransfected MEFs. C, D, To assess the trans effects of PSEN1 mutations on presenilinase activity, Psen-null MEFs were transiently transfected with vector encoding WT and mutant PS1 in a 1:1 ratio. PS1 endoproteolysis was assessed by Western analysis. Coexpression of several PS1 mutants (R278I, L435F, Δex9) with WT PS1 significantly reduced PS1-NTF levels compared with the level observed for WT PS1 alone, indicating dominant-negative inhibition of WT endoproteolysis, whereas coexpression of the remaining PS1 mutants did not interfere with WT endoproteolysis. Data are expressed as percentage of WT level ± SEM (n = 3 independent experiments). *p < 0.05 relative to WT.
- Figure 3.
FAD PSEN1 mutations inhibit APP processing by mutant PS1 in cis and by WT PS1 in trans in a dominant-negative manner. A, B, To assess the cis effects of PSEN1 mutations on APP processing, Psen-null MEFs were transfected with vector encoding WT or mutant PS1 together with vector encoding APP C99-myc substrate. C99 and AICD were detected by Western analysis with anti-myc antibody. untrans, Untransfected MEFs. *γ-Secretase-independent band. Quantification of AICD production shows that all mutations tested significantly reduced intrinsic PS1 activity. C, D, To assess the trans effects of PSEN1 mutations on APP processing, Psen-null MEFs were transfected with vectors encoding WT and mutant PS1 in a 1:1 ratio together with vector encoding APP C99-myc substrate. Quantification of AICD levels shows that mutant PS1 antagonized WT PS1 activity, indicated most clearly by suppression of AICD production below the level displayed by WT alone in the case of several mutations (L166P, R278I, C410Y, L435F). E, F, Psen-null MEFs were cotransfected with a constant amount of vector encoding WT PS1 and increasing amounts of vector encoding either L435F or C410Y mutant PS1, together with constant amount of vector encoding APP C99-myc. PS1 was detected by Western analysis with antibody recognizing the C terminus; C99 and AICD were detected with anti-myc antibody. Quantification of AICD production shows that PS1 bearing the L435F or C410Y mutations produced similar gene dosage-dependent suppression of AICD production by WT PS1, consistent with dominant-negative inhibition of γ-secretase activity. Data are expressed as percentage of WT level ± SEM (n = 3–5 independent experiments). *p < 0.05 relative to WT.
- Figure 4.
FAD PSEN1 mutations inhibit Notch processing by mutant PS1 in cis and by WT PS1 in trans in a dominant-negative manner. A, B, To assess the cis effects of PSEN1 mutations on Notch processing, Psen-null MEFs were transfected with vector encoding WT or mutant PS1 together with vector encoding NotchΔE-myc substrate. NICD was detected by Western analysis with antibody recognizing the cleaved N terminus (V1744), and both full-length NotchΔE and NICD were detected with anti-myc antibody. Quantification of NICD production shows that all mutations tested significantly reduced intrinsic PS1 activity. untrans, Untransfected MEFs C, D, To assess the trans effects of PSEN1 mutations on Notch processing, Psen-null MEFs were transfected with vectors encoding WT and mutant PS1 in a 1:1 ratio together with vector encoding NotchΔE-myc substrate. Quantification of NICD levels shows none of the mutations was able to elevate NICD production above the level displayed by WT PS1 alone, with some mutations significantly suppressing NICD production below this level (e.g., C410Y, L435F), consistent with dominant-negative inhibition of Notch processing. Data are expressed as percentage of WT level ± SEM (n = 5–6 independent experiments). *p < 0.05 relative to WT.
- Figure 5.
FAD PSEN1 mutations inhibit Aβ production by mutant PS1 in cis and modulate Aβ production by WT PS1 in trans. A, B, To assess the cis effects of PSEN1 mutations on Aβ production, Psen-null MEFs were transfected with vector encoding WT or mutant PS1 together with vector encoding APP C99-myc. Production of Aβ40 and Aβ42 was assayed by sandwich ELISA with isoform-specific antibodies. All PS1 mutants exhibited significantly impaired production of Aβ40. PS1 bearing the L166P, R278I, C410Y, and L435F mutations displayed significantly reduced Aβ42 production, whereas PS1 carrying the L392V and Δex9 mutations showed elevated Aβ42 production. C, D, To assess the trans effects of PSEN1 mutations on Aβ production, Psen-null MEFs were transfected with vectors encoding WT and mutant PS1 in a 1:1 ratio together with vector encoding APP C99-myc. Coexpression of mutant PS1 antagonized Aβ40 production by WT PS1, illustrated most clearly by significant reduction of Aβ40 production below the level produced by WT PS1 alone in the case of several mutations (R278I, C410Y, L435F). In contrast, several PS1 mutants stimulated Aβ42 production by WT PS1, as indicated by their supra-additive effect on Aβ42 production (e.g., L166P, C410Y, L435F). E, To assess the cis effects of PSEN1 mutations on total Aβ production, Psen-null MEFs were transfected with vector encoding WT or mutant PS1 together with vector encoding APP C99-myc. Total secreted Aβ levels were measured in culture supernatants by sandwich ELISA detecting all Aβ species ranging from Aβ38 through Aβ46. All PSEN1 mutations tested significantly impaired total Aβ production by the mutant protein, with several pathogenic mutants producing <10% of WT levels. F, To assess the trans effects of PSEN1 mutations on total Aβ production, Psen-null MEFs were transfected with vectors encoding WT and mutant PS1 in a 1:1 ratio together with vector encoding APP C99-myc. The effects of mutant and WT PS1 coexpression on total Aβ production were qualitatively similar to those observed for Aβ40 production (C), indicating that Aβ40 is the major Aβ species produced by WT and mutant PS1 under these conditions. Data are expressed as percentage of WT level ± SEM (n = 4–6 independent experiments). *p < 0.05 relative to WT.
- Figure 6.
Physical interaction between mutant PS1 and WT PS1. A, To confirm that the epitope tags did not compromise γ-secretase activity, the ability of HA- and FLAG-tagged WT PS1 to reconstitute APP and Notch processing was assessed relative to untagged WT PS1. Psen-null MEFs were cotransfected with vector encoding untagged or tagged PS1 and vector encoding APP C99-myc or NotchΔE-myc substrate. AICD (left) and NICD (right) were detected by Western analysis with antibodies directed against the myc epitope or the cleaved N terminus of NICD. No differences in APP or Notch processing activity were observed between tagged and untagged PS1. B, WT and mutant PS1 bearing N-terminal FLAG or HA epitope tags were coexpressed in 293T cells. Expression levels of epitope-tagged PS1, PS1-CTF, and the endogenous γ-secretase components Nicastrin (Nct) and Pen2 were evaluated by Western analysis of cell lysates (left). Immunoprecipitation was performed on cell lysates with mouse anti-FLAG antibody. Western analysis of immunoprecipitates from cells coexpressing HA-PS1-WT and FLAG-PS1-WT (right) revealed coimmunoprecipitation of HA-PS1 holoprotein and NTF, PS1-CTF, Nct, and Pen2 with FLAG-PS1-WT (lane 2). Coimmunoprecipitation of HA-PS1 or γ-secretase components with anti-FLAG antibody was not observed in the absence of FLAG-PS1 (lanes 1 and 3). *Background bands resulting from interaction of primary antibodies with mouse IgG in immunoprecipitates. Analysis of immunoprecipitates from cells coexpressing FLAG-PS1-WT and HA-PS1-L435F (lane 4), or conversely HA-PS1-WT and FLAG-PS1-L435F (lane 5), revealed coimmunoprecipitation of mutant and WT PS1 in association with endogenous Nct and Pen-2. Epitope-tagged NTF is not detected for the PS1-L435F mutant because of its impaired presenilinase cleavage. C, To confirm the specificity of the interaction between WT and mutant PS1, the reciprocal experiment was performed with the epitopes used for immunoprecipitation and Western analysis reversed. Left, Expression of HA- and FLAG-tagged PS1 in cell lysates used for immunoprecipitation was evaluated by Western analysis. Right top, Immunoprecipitation was performed with chicken anti-HA antibody and confirmed by Western analysis with mouse anti-HA antibody. Right bottom, Western analysis with mouse anti-FLAG antibody revealed coimmunoprecipitation of HA-PS1-WT with either FLAG-PS1-WT holoprotein and NTF or FLAG-PS1-L435F holoprotein. FLAG-holo and HA-holo, PS1 holoprotein appended with the designated epitope tags; FLAG-NTF and HA-NTF, PS1 NTF appended with the designated epitope tags; PS1-C, antibody recognizing the PS1 C terminus; Nct-m and Nct-imm, the mature and immature forms of Nct, respectively; untrans, lysate from untransfected cells.
- Figure 7.
Dominant-negative model for inhibition of PS1 activity and stimulation of Aβ42 production by PSEN1 mutations and γ-secretase inhibitors. Schematic representation of possible interactions between WT and mutant or inhibitor-bound PS1 is shown. If PSEN1 mutations caused a simple loss of mutant protein function (i.e., a hypomorphic or amorphic mechanism), coexpression of WT and mutant PS1 as in FAD patients would be expected to result in additive summation of their individual activities, yielding total PS1 activity ≥ 100% of the level contributed by WT PS1 alone (left). In contrast, a reduction in total PS1 activity below the level displayed by WT PS1 alone would indicate dominant-negative inhibition of WT PS1 by mutant PS1 (i.e., an antimorphic mechanism) (middle). Our data suggest that mutant PS1 antagonizes WT PS1 activity through an allosteric physical interaction and that dominant-negative inhibition of WT PS1 can alter its substrate specificity to enhance its production of Aβ42. At low to moderate concentrations, active site-directed γ-secretase inhibitors can decrease Aβ40 production yet increase Aβ42 production, analogous to the effect of dominant-negative PSEN1 mutations (right). We suggest that inhibitor-bound (and thus catalytically inactive) PS1 can interact allosterically with unbound PS1, altering its substrate specificity to favor production of Aβ42.
