Mechanism of cytotoxicity mediated by the C31 fragment of the amyloid precursor protein

Abstract

The cytoplasmic tail of the amyloid precursor protein (APP) contains two putatively cytotoxic peptides, Jcasp and C31, derived by caspase cleavage of APP. Jcasp is a fragment starting from the epsilon-secretase site to position 664, while C31 is a fragment from position 665 to the C-terminus. Our studies now showed that compared to C31, Jcasp appeared to play a minor role in cytotoxicity. In particular, inhibition of Jcasp generation by treatment of gamma-secretase inhibitor did not lead to any attenuation of C31-induced toxicity. Secondly, because C31 toxicity is largely absent in cells lacking endogenous APP, we determined, using a split beta-galactosidase complementary assay to monitor protein-protein interactions, the presence of APP associated complexes. Our results demonstrated that both APP homomeric and C31/APP heteromeric complexes were correlated with cell death, indicating that C31 complexes with APP to recruit the interacting partners that initiate the signals related to cellular toxicity.

Fig. 1

C31-induced toxicity in B103 cells is correlated with C31/C99 dimer formation. B103 cells were transiently transfected with control vector or APP-β-gal chimeric constructs based on C99, C31, C99-D664A, or C99-ΔC31. 24 hours after transfection, cells were assayed in parallel samples for cell death and β-gal activity. (A, C) Cell death was determined by ethidium homodimer (EthD-1, Live Dead Assay) staining. (B, D) Dimerization was determined by β-gal activity. Results showed that toxicity was limited to C99 or C99/C31 co-expression but not when C99-D664A or C99ΔC31 was expressed. However, elevated β-gal activity was seen with all C99 and C99-derived constructs even in the absence of cell death (*p < 0.001 one way ANOVA, post-hoc Tukey).

Fig. 1

C31-induced toxicity in B103 cells is correlated with C31/C99 dimer formation. B103 cells were transiently transfected with control vector or APP-β-gal chimeric constructs based on C99, C31, C99-D664A, or C99-ΔC31. 24 hours after transfection, cells were assayed in parallel samples for cell death and β-gal activity. (A, C) Cell death was determined by ethidium homodimer (EthD-1, Live Dead Assay) staining. (B, D) Dimerization was determined by β-gal activity. Results showed that toxicity was limited to C99 or C99/C31 co-expression but not when C99-D664A or C99ΔC31 was expressed. However, elevated β-gal activity was seen with all C99 and C99-derived constructs even in the absence of cell death (*p < 0.001 one way ANOVA, post-hoc Tukey).

Fig. 2

(A, B) Cytotoxicity following Jcasp and C31 expression. GFP-stabilized Jcasp or C31 fragments were expressed in N2A cells and assessed for cell death by Hoechst staining. (A) Western blot of transfected cell lysates using GFP antibody. (B) C31 showed the highest level of cell death, with Jcasp and Jcasp-Y653D peptides showing significant but lower levels of toxicity (*p ≤ 0.001). Cytotoxicity of APP C-terminal fragment was due primarily to C31. (C, D) N2A cells were transiently transfected with GFP-stabilized C31, C50, and C50-D664A mutant. (C) Western blots of transfected cell lysates using GFP antibody. After treatment with staurosporine (STS), C50 peptide, but not the C50-D664A mutant, was cleaved into a shorter fragment (arrowhead) that co-migrated with C31 (lower panel). The Hoechst staining (D) showed cytotoxicity following C31 and C50 expression. However, toxicity was lost in C50-D664A transfected cells (*p < 0.001).

Fig. 2

(A, B) Cytotoxicity following Jcasp and C31 expression. GFP-stabilized Jcasp or C31 fragments were expressed in N2A cells and assessed for cell death by Hoechst staining. (A) Western blot of transfected cell lysates using GFP antibody. (B) C31 showed the highest level of cell death, with Jcasp and Jcasp-Y653D peptides showing significant but lower levels of toxicity (*p ≤ 0.001). Cytotoxicity of APP C-terminal fragment was due primarily to C31. (C, D) N2A cells were transiently transfected with GFP-stabilized C31, C50, and C50-D664A mutant. (C) Western blots of transfected cell lysates using GFP antibody. After treatment with staurosporine (STS), C50 peptide, but not the C50-D664A mutant, was cleaved into a shorter fragment (arrowhead) that co-migrated with C31 (lower panel). The Hoechst staining (D) showed cytotoxicity following C31 and C50 expression. However, toxicity was lost in C50-D664A transfected cells (*p < 0.001).

Fig. 3

Toxicity of APP-C31 co-transfection was lost in APP deleted of Jcasp domain. (A) APPΔJ1 is a construct with a 13 amino acid deletion consisting of the entire Jcasp region while APPΔJ2 was a 9 amino acid deletion construct preserving the VEVD caspase cleavage motif. (B) Western blot using an end-specific antibody, APP-α664 (upper most). APPΔC31 and APP695ΔJ2_VEVD_ΔC31 transfected cells, but not wild type APP (APPwt) were immunoreactive to APP-α664 antibody. Following staurosporine treatment (STS), Asp-664 cleavage was detected in APP695wt transfected cells, but not in APP695ΔJ1, APP695ΔJ2 or APPD664A transfected cells. Expression of all constructs was comparable as determined by immunoblotting with 6E10 antibody. Cell death assay by ethidium homodimer staining (C) showed significantly increased cytotoxicity only for C31 cotransfected with APPwt. Neither Jcasp deleted constructs was able to augment C31 induced cytotoxicity (*p ≤ 0.001).

Fig. 3

Toxicity of APP-C31 co-transfection was lost in APP deleted of Jcasp domain. (A) APPΔJ1 is a construct with a 13 amino acid deletion consisting of the entire Jcasp region while APPΔJ2 was a 9 amino acid deletion construct preserving the VEVD caspase cleavage motif. (B) Western blot using an end-specific antibody, APP-α664 (upper most). APPΔC31 and APP695ΔJ2_VEVD_ΔC31 transfected cells, but not wild type APP (APPwt) were immunoreactive to APP-α664 antibody. Following staurosporine treatment (STS), Asp-664 cleavage was detected in APP695wt transfected cells, but not in APP695ΔJ1, APP695ΔJ2 or APPD664A transfected cells. Expression of all constructs was comparable as determined by immunoblotting with 6E10 antibody. Cell death assay by ethidium homodimer staining (C) showed significantly increased cytotoxicity only for C31 cotransfected with APPwt. Neither Jcasp deleted constructs was able to augment C31 induced cytotoxicity (*p ≤ 0.001).

Fig. 4

The γ-secretase inhibitor DAPT did not affect cell death induced by C31 expression. (A) Western blot showing no substantive change in APP processing as seen by immunoblotting with 6E10 and CT 15 antibodies. The levels of C-terminal fragment markedly increased following exposure to DAPT. (B) The ethidium homodimer staining showed substantial cell death after co-expression of C31 and APP that were unchanged after DAPT treatment. Expression of APP-D664A in place of wild type APP resulted in an absence of cytotoxicity that was not affected by DAPT treatment. (*p < 0.001).