beta-Secretase cleavage is not required for generation of the intracellular C-terminal domain of the amyloid precursor family of proteins

Abstract

The amyloid precursor family of proteins are of considerable interest, both because of their role in Alzheimer's disease pathogenesis and because of their normal physiological functions. In mammals, the amyloid precursor protein (APP) has two homologs, amyloid precursor-like protein (APLP) 1 and APLP2. All three proteins undergo ectodomain shedding and regulated intramembrane proteolysis, and important functions have been attributed to the full-length proteins, shed ectodomains, C-terminal fragments and intracellular domains (ICDs). One of the proteases that is known to cleave APP and that is essential for generation of the amyloid beta-protein is the beta-site APP-cleaving enzyme 1 (BACE1). Here, we investigated the effects of genetic manipulation of BACE1 on the processing of the APP family of proteins. BACE1 expression regulated the levels and species of full-length APLP1, APP and APLP2, of their shed ectodomains, and of their membrane-bound C-terminal fragments. In particular, APP processing appears to be tightly regulated, with changes in beta-cleaved APPs (APPsbeta) being compensated for by changes in alpha-cleaved APPs (APPsalpha). In contrast, the total levels of soluble cleaved APLP1 and APLP2 species were less tightly regulated, and fluctuated with BACE1 expression. Importantly, the production of ICDs for all three proteins was not decreased by loss of BACE1 activity. These results indicate that BACE1 is involved in regulating ectodomain shedding, maturation and trafficking of the APP family of proteins. Consequently, whereas inhibition of BACE1 is unlikely to adversely affect potential ICD-mediated signaling, it may alter other important facets of amyloid precursor-like protein/APP biology.

Fig. 1. Levels of total APPs are unaffected by changes in BACE1 expression whereas APPsα levels are dependent on BACE1 activity

TBS homogenates of brains from wild type (WT), BACE1 knock-out (KO) and BACE1 transgenic (Tg) mice were electrophoresed on 10% tris-glycine polyacrylamide gels and western blotted with a panel of antibodies which allow detection of total APPs (22C11, A), APPsα (anti-Aβ rodent, C) and full-length and C-terminal fragments of APP (C8, E). Western blotting for GAPDH was included to check for equal protein loading (F). Lysates of a cell line over-expressing human wild type APP₆₉₅ (+), were included as a positive control and TBS homogenates of brains from APP knock-out mice (−) were included as a negative control. The levels of total APPs and of APPsα (B and D respectively) were quantitated by densitometry and values normalized versus WT control are presented as averages ± standard error of duplicate measurements of three animals of each genotype.

Fig. 2. BACE1 deletion decreases the levels of secreted APLP1 (APLP1s) and increases the levels of FLAPLP1

TBS homogenates of brains from wild type (WT), BACE1 knock-out (KO) and BACE1 transgenic (Tg) mice were electrophoresed on 10% tris-glycine polyacrylamide gels and western blotted with antibodies recognizing the N-terminus (W1NT, A) and C-terminus (W1CT, C) of APLP1. Western blotting for GAPDH was included to check for equal protein loading (E). Lysates of a cell line over-expressing human APLP1₆₅₀ (+), are included as a positive control and TBS homogenates of brains from APLP1 knock-out mice (−) are included as a negative control. FLAPLP1 and APLP1s bands detected by W1NT are indicated by arrows in A. APLP1s and of FLAPLP1 levels (B and D respectively) were quantitated by densitometry and values normalized relative to WT control are presented as averages ± standard error of duplicate measurements of three animals of each genotype.

Fig. 3. BACE1 deletion decreases APLP2s levels, whereas BACE1 over-expression increases APLP2s levels

TBS homogenates of brains from wild type (WT), BACE1 knock-out (KO) and BACE1 transgenic (Tg) mice were electrophoresed on 10% tris-glycine polyacrylamide gels and western blotted with antibodies recognizing either FLAPLP2 (D2-II, A) or the extreme C-terminus of APLP2 (W2CT, C). Western blotting for GAPDH was included to check for equal protein loading (D). Lysates of cell lines over-expressing human wild type APLP2₇₅₁ (+), are included as positive control and TBS homogenates of brains from APLP2 knock-out mice (−) are included as negative control. APLP2s bands (indicated by arrows, A) were quantitated by densitometry and values normalized versus the WT control are presented as averages ± standard error of duplicate measurements of three animals of each genotype (B).

Fig. 4. BACE1 expression decreases FLAPP steady state levels and gives rise to a ~14.3 kDa APP CTF

TBST homogenates of wild type (WT), BACE1 knock-out (KO) and BACE1 transgenic (Tg) mouse brains were electrophoresed on 10–20% tris-tricine polyacrylamide gels and western blotted with the anti-APP C-terminus specific antibody C8 (A, C). Lysates of a cell line over-expressing human wild type APP₆₉₅ (+) are included as a positive control; while TBST homogenates of brains from APP knock-out mice (−) are included as a negative control. The asterisk in C indicates a specific band detected in certain WT and Tg samples. FL and CTF bands (indicated by arrows in B and D respectively) were quantified by densitometry and normalized versus the WT control. Results are presented as averages ± standard error of duplicate measurements of three animals for each condition.

Fig. 5. BACE1 expression decreases FLAPLP1 levels and gives rise to a ~7.5 kDa APLP1 CTF

TBST homogenates of wild type (WT), BACE1 knock-out (KO) and BACE1 transgenic (Tg) mouse brains were electrophoresed on 10–20% tris-tricine polyacrylamide gels and western blotted with the anti-APLP1 C-terminus specific antibody W1CT (A, C). Lysates of a cell line over-expressing human wild type APLP1₆₅₀ (+) are included as a positive control; while TBST homogenates of brains from APLP1 knock-out mice (−) are included as a negative control. The FL and CTFs species identified (indicated by arrows in A and C respectively) were quantified by densitometry and normalized versus the WT control and results are presented as averages ± standard error of duplicate measurements of three animals for each condition (FLAPLP1: B; APLP1 CTFs: D).

Fig. 6. BACE1 expression decreases FLAPLP2 protein levels and gives rise to a ~14.8 kDa APLP2 CTF

TBST homogenates of wild type (WT), BACE1 knock-out (KO) and BACE1 transgenic (Tg) mouse brains were electrophoresed on 10–20% tris-tricine polyacrylamide gels and western blotted with the anti-APLP2 C-terminus specific antibody W2CT (A, C). Lysates of a cell line over-expressing human wild type APLP2₇₅₁ (+) are included as positive controls; while TBST homogenates of hemibrains of APLP2 knock-out mice (−) are included as negative controls. The FL and CTFs species identified (indicated by arrows in A and C respectively) were quantified by densitometry and normalized versus the WT control. Results are presented as averages ± standard error of duplicate measurements of three animals for each condition (FLAPLP2: B; APLP2 CTFs: D).

Fig. 7. BACE1 deletion does not compromise APP, APLP1 and APLP2 ICD generation

Microsomes prepared from BACE1 knock-out (KO) or wild type (WT) mouse brains were incubated at 37°C for 2 hours to allow de novo in vitro ICD production (A-C). ICDs were detected by western blot using specific antibodies for APP (C8 antibody, A), APLP1 (W1CT antibody, B) and APLP2 (W2CT antibody, C). Western blots shown in A-C are representative of three different experiments. Generation of ICDs was conducted either in presence (+) or absence (−) of protease inhibitors and insulin (PI mix). Endogenous ICDs were immunoprecipitated from mouse brains with C8, W1CT or W2CT, and immunoprecipitates analysed by western blotting with the same antibodies (D, E, F). Western blots shown in D-F are representative of two different experiments. For comparison, in vitro generated ICDs were electrophoresed alongside endogenous ICDs (D-F).