It is widely accepted that oxidative stress is one of the earliest changes that occurs in the pathogenesis of AD, arising from the imbalance between increased production of reactive oxygen and nitrogen species and impaired antioxidant defenses, as reflected in the accumulation of oxidative damage to macromolecules detected in MCI and AD brains [39
]. In this work, we present in vitro
and in vivo
evidence of NO-mediated regulation of BACE1. We are the first to demonstrate that NO, at different levels, can exert differential regulation on BACE1: at low levels, NO suppresses BACE1 transcription while at modest to high levels, NO induces S-nitrosylation of BACE1 and inactivates the enzyme. Furthermore, we show that S-nitrosylation of BACE1 occurs in normal aging and MCI brains but is significantly diminished in late stage AD brains. Given the central role of Aβ in AD pathogenesis and the fact that BACE1 is the rate-limiting enzyme in APP processing and Aβ generation, the redox regulation of BACE1 identified herein may represent a novel and crucial mechanism for keeping BACE1 at physiological levels/activity.
The multifaceted actions of the NO group can be classified into two categories: classic NO-mediated/cGMP-dependent actions and reactive nitrogen species-mediated/cGMP-independent actions. The cGMP-dependent actions often play critical roles in a variety of physiological processes, including NO-mediated vasodilation. In contrast, cGMP-independent actions are more frequently postulated to be involved in the pathological responses which are primarily effected by nitrosative post-translational modifications of proteins such as S-nitrosylation and tyrosine nitration [41
]. Although low nanomolar concentrations of NO donors are sufficient to elicit cGMP-dependent signals, 50-100 μM of NO donors is required for S-nitrosylation-mediated alterations of protein function in cultured cells [37
Based on these ideas, we speculate that the NO generated by different NOSs exerts differential modulations of BACE1. For example, the low levels of NO that result in suppression of BACE1 transcription may represent the NO released from vascular eNOS. Although it is difficult to measure the precise concentration of the bioreactive NO in the blood circulation of a healthy vertebrate, the decreased BACE1 transcription induced at the 10-100 nM range of NO donors may be related to the protective actions caused by the release of NO from vascular eNOS. In fact, this is collaborated by the recent finding that BACE1 expression was elevated in mice deficient in eNOS [55
]. Our data also suggest that PGC-1α, a crucial PPARγ coactivator in the transcriptional controls of gluconeogenesis and energy metabolism [56
], may be the key factor executing the effects of low NO, through the activated cGMP-PKG signaling pathway. Since PGC-1α is the most critical regulator in response to metabolic stress, it is believed to play a key role in AD pathogenesis. Our finding that PGC-1α is likely involved in BACE1 transcriptional control provides the first molecular basis of a metabolic signal/factor regulating an AD gene. In support of this, PGC-1α expression was found to be reduced in AD brains [57
] and it was recently reported that PGC-1α facilitates BACE1 protein degradation via the UPS [58
]. Further characterization of the transcriptional network involving PGC-1α, directly or indirectly, on BACE1 promoter, will draw a fuller picture of the metabolic factors regulating AD genes, which likely involve the entire AMPK-SIRT1-PGC-1α pathway, in which NO signaling plays an important role.
On the other hand, the high levels of NO-mediated BACE1 inactivation via post-translational modification in cultured neurons likely reflects the NO generated by iNOS, known to elicit much higher NO production (over 1,000-fold) compared with that generated by the constitutive NOSs [37
]. Inducible iNOS, shown to be involved in the pathogenesis of AD, is activated under inflammatory conditions and may upregulate BACE1 as a result of activated NF-κB and toxic peroxynitrite formed by NO and superoxide anions. Although we show that SNO-BACE1 is associated with reduced enzymatic activity, the elevated protein expression and enzymatic activity of BACE1 in late stages of AD may reflect the dominant effect of severe oxidation by H2
and peroxynitrite; it has been shown previously that lipid oxidative products, such as 4-HNE, can upregulate BACE1 transcriptionally [30
-induced modification and S-nitrosylation represent the two dominant oxidative events modifying critical Cys residues in proteins. Interestingly, we observed opposite effects from these two oxidative modifications in BACE1 expression and activity. The interplay between S-nitrosylation and H2
-type oxidation of BACE1 at the molecular level is not yet clear, albeit both occur at certain critical Cys residues. Based on our finding that NO supresses BACE1, we speculate that S-nitrosylation and H2
-type oxidation occur on overlapping Cys residues on BACE1; nitrosylation precludes further oxidation for enzymatic activation and thus represents a self-defensive or house-keeping mechanism. Among the 11 Cys residues on BACE1, it is known that six Cys residues form three pairs of intramolecular disulfide bonds in mature BACE1 (Cys216-420
; Figure ) which is essential for its membrane-association and protein maturation but is not required for its enzymatic activity [60
]. Since the pattern of BACE1 S-nitrosylation shows that it occurs predominantly on the mature form of BACE1 (Figure ), we reason that the three pairs of Cys in the enzymatic pocket are not likely sites for nitrosylation. Indeed, our preliminary study used site-directed mutagenesis to analyze individual BACE1 mutants with each Cys mutated to Ala and showed that Cys mutation at the positions 216, 278, 330, 380, 420, 443, or 466 (the membrane-proximal ones) resulted in lack of mature BACE1 proteins (Figure ). Substitution of each of the four Cys residues in the cytoplasmic tails, which are also the S-palmitoylation sites, had different results; the C483A mutant abolished mature BACE1, making it difficult to assess its contribution as a nitrosylation site; Cys478A and Cys482A mutants appeared to show reduced BACE1 nitrosylation and likely represent S-nitrosylation sites under physiological conditions. It should be pointed out that the results of this type of semi-quantitative analysis are not sufficient to determine the molecular basis of nitrosylation sites unambiguously. In particular, the cytoplasmic tail has been reported to be non-essential for the enzymatic activity of BACE1 [61
], it remains unclear how the nitrosylation affects BACE1 activity. Additional research to determine the molecular basis of the nitrosylation and oxidation events by quantitative mass spectrometry, as well as further analysis of BACE1 and subcellular trafficking, localization and distribution in lipid rafts using cell biology approaches, are necessary to clarify the mechanism.
Figure 6 Mapping crucial cysteine residues for SNO-BACE1. (A) Schematic diagram of BACE1. BACE1 is a 501 amino-acid transmembrane protein. Signal peptide (1-21 aa) and pro-peptide (22-45 aa) are cleaved during protein maturation. Within the lumenal domain there (more ...)