Our findings suggest that sAPP-α can promote non-amyloidogenic APP processing. Our initial indication of this was evidenced in cells overexpressing APPswe and PS1wt (CHO/APPswe/PS1wt cells) where exogenous hsAPP-α inhibited Aβ40, 42 and β-CTF production (); a pattern indicative of suppression of β-secretase APP cleavage ().
Underlying this, we found a physical association between sAPP-α with BACE1 in vitro
(), and in both TgsAPP-α mice and wild-type littermates and at endogenous levels (); suggesting a physiologically relevant phenomenon. Indeed, immunoprecipitaton (IP) with an anti-BACE1 antibody and immunoblot (IB) with an anti-C-terminal sAPP-α specific 2B3 antibody exhibited strong co-IP of sAPP-α in both TgsAPP-α mice and wild-type littermates (). These results strongly support a physiologically relevant BACE1 and sAPP-α interaction at endogenous levels. Furthermore, others have already shown that APP itself contains a unique C-terminal sequence found to decrease BACE1 activity44
lending further credence to our data and conclusions as this region is also present in sAPP-α but not in sAPP-β. In further support, BACE1 has been shown to be secreted in conditioned media45–48
and thus binding of sAPP-α to BACE1 () could occur in either the extracellular compartment, at the membrane, or presumably during endocytosis.
Reduced interaction of BACE1 with full-length APP would be expected if sAPP-α could interfere with their association. Indeed, we found that PSAPP mice overexpressing sAPP-α show decreased BACE1 and full-length APP interaction (), whereas PSAPP mice exhibited the opposite (). Alternatively, suppression of sAPP-α function via antibody immunodepletion of exogenous (), and immunoneutralization of endogenously generated sAPP-α () enhanced Aβ production. These findings demonstrate for the first time, that sAPP-α may be involved in a positive feedback loop whereby it promotes its own production by inhibiting BACE1’s ability to interact with and proteolyze full-length APP at physiological levels.
To confirm this physiological relevance, we conducted the co-IP experiments both in conditioned media and brain homogenates. Specifically, we performed co-IP of CHO/APPwt
cell lysates utilizing the specific sAPP-α antibody (2B3) that neither recognizes Aβ nor full-length APP. These co-IP data suggested that sAPP-α interacts with BACE1 in some manner in these cell culture systems and this interaction is associated with decreased Aβ generation. However, these data do not completely rule out the possibility that intermediate factors are required for inhibition of BACE1’s association and proteolysis of APP. In light of the attenuation of sAPP-α’s Aβ lowering effects by immunoneutralization utilizing 2B3 antibody (), as well as, similar findings with utilizing an antibody against Aβ1-17
peptide (6E10 antibody) (Fig. S3
), intermediate factors presumably still present in this immunoneutralization paradigm did not appear to mitigate the effects of directly targeting sAPP-α.
The possibility of the immunodepletion antibody, 6E10, interfering with either Aβ generation or its measurement by ELISA exists, but at an extremely low likelihood given that we found similar results with the most specific sAPP-α 2B3 antibody by both IP and IB analyses (). In this regard, one may also speculate that contaminating sAPP-β may be present and confounding our data. However, this is also likely not occurring because this peptide would not be removed through immunodepletion of sAPP-α with the specific 2B3 antibody and thus suppression of Aβ peptides should still be observed; yet the results support the opposite conclusion ( & Fig. S2
The possibility of another receptor, in addition or alternative to BACE1, being responsible for sAPP-α’s Aβ lowering effects exists since APP and its metabolites, sAPPs are known to exhibit multimodal effects and multiple binding partners. Importantly, however, the immunoneutralization results using sAPP-α specific antibody () suggested that no regions, other than the region at or near the target epitope at the C-terminal of sAPP-α, appear required for sAPP-α’s anti-amyloidogenic effects. Further, the regions bound by the immunoneutralization antibodies are regions of sAPP-α characterized to have a unique BACE1 inhibition sequence44
. In contrast IgG2b
immunoneutralization or immunodepletion of endogenous or exogenous sAPP-α, respectively, yielded no changes in APP processing essentially ruling out the possibility of off-target antibody effects in our model systems. Importantly, 2B3 antibody characterization (Fig. S1
) indicated no detectable binding to endogenous murine APP, Aβ, and CTF species, further confirming specificity of this antibody. Prior reports, in combination with these data presented here, altogether supported our hypothesis that sAPP-α directly mediates suppression of Aβ generation.
Based on our findings that sAPP-α binds and interferes with BACE1-mediated APP processing, suppression of sAPP-α likely results in BACE1 disinhibition. These results implicate decreased sAPP-α as a potential pathologic determinant in AD pathogenesis. These properties of sAPP-α appeared to depend in part on the secondary and tertiary structure of sAPP-α given that heat-inactivated sAPP-α lost its ability to suppress Aβ production or interfere with BACE1/APP interaction (Fig. S4
). Additionally, the possibility that sAPP-α is binding to and interfering with full-length APP32–34
or β-CTF rather than BACE1 exists but in our model systems is unlikely given negative IP/IB data suggesting that sAPP-α did not interact well with full-length APP or β-CTF (Fig. S4c & S4d
Perhaps most importantly, in PSAPP mouse model (human mutant APPK595N/M596L [APPswe] and PS1ΔE9 overexpressing mice), sAPP-α overexpression led to reduced levels of detergent soluble and insoluble Aβ (), decreased β-CTF production (), and ameliorated brain β-amyloid plaques (), as well as, in our cell systems where sAPP-α bound BACE1 and reduced BACE1/APP binding indicating that in vivo sAPP-α can regulate APP processing (). Finally, i.c.v. administration of an anti-C-terminal sAPP-α specific 2B3 antibody in PSAPP mice resulted in exacerbation of amyloidogenic APP processing compared with i.c.v. injection using isotype-matched IgG2b control antibody ().
The results of the present study implicated that inadequate sAPP-α levels are likely sufficient to polarize APP processing toward the amyloidogenic, Aβ producing route due to dishinhibition of BACE1. β-secretase cleavage is considered to be the rate-limiting step in the generation of Aβ. While direct inhibition of BACE shows promise for the treatment of AD, other BACE substrates, including the proteins involved in control of myelination49–55
, could be affected by non-selectively inhibiting BACE1 activity.
In sum, we suggested a novel mechanism of APP autoregulation via sAPP-α, which appears to attenuate Aβ formation under the physiological condition. If this endogenous sAPP-α-mediated BACE1 inhibition is disturbed, pathologic Aβ generation likely responsible for subsets of sporadic AD may ensue. We have confirmed this mechanism through multiple modalities both in vitro and in three mouse models whereby there is a physical interaction between sAPP-α and BACE1; an event that in AD patients may precede the generation of toxic Aβ species. Development of selective BACE modulators, interventions to restore sAPP-α’s BACE modulation functions, or sAPP-α mimetics, could hold therapeutic value as AD prophylactics or therapeutics.