Characterization of Cerebrospinal Fluid BACE1 Species

Abstract

Background: The primary neuronal β-secretase in the brain is the β-site amyloid precursor protein cleaving enzyme 1 (BACE1). BACE1 is the main protease responsible for the amyloidogenic processing of the amyloid precursor protein (APP) leading to Aβ generation. Previous studies have suggested the potential of cerebrospinal fluid (CSF) β-secretase activity to be a diagnostic biomarker for Alzheimer's disease (AD), but these studies have not demonstrated consistent changes in AD CSF. Therefore, further biochemical characterization of BACE1 protein and β-secretase activity in AD CSF is needed. Methods: β-Secretase activity was determined in CSF from AD patients (n= 19) and age-matched non-AD controls (n= 19) using a fluorogenic substrate in the absence and presence of a BACE1 inhibitor. BACE1 protein was characterized by lectin-binding, immunoprecipitation, blue native-PAGE and western blotting using specific antibodies. Results: We show that the majority of the β-secretase activity in the CSF is not inhibited by a specific BACE1 inhibitor, and that β-secretase activity attributable to BACE1 is higher in AD subjects. BACE1 is present in human CSF, both as immature forms of ~50 kDa that retain the prodomain and mature forms of ~70 kDa that probably comprise truncated and full-length species. CSF-BACE1 forms large hetero-complexes containing distinct species. Immunoblotting with an antibody against the C-terminus of BACE1 revealed significantly higher levels of the 70-kDa full-length BACE1, while the 50 kDa immature form remained unaltered. When the 70-kDa band was probed with an antibody against the N-terminus of BACE1, which does not discriminate between truncated and full-length forms, no increase was observed, suggesting that truncated forms of BACE1 do not increase in AD. Lopez-Font et al. 3 Conclusions: The activity of β-secretase in the CSF cannot be attributed only to BACE1. Both mature and immature species of BACE1 occur as heteromers. Only mature full-length BACE1 shows increased levels in AD CSF. The complexity of BACE1 species in CSF has to be taken into consideration for the determination of BACE1 activity and protein levels in CSF as biomarkers of AD.

β-Secretase1 biological markers for Alzheimer’s disease: state-of-art of validation and qualification

“…It should be acknowledged, however, that several in vitro and animal data point to a non-linear relationship between the levels of gene expression and rates of enzyme activity that is highly influenced by post-translational modifications [55]. Indeed, experimental studies indicate that BACE1 activity significantly increases over time while its expression levels are less likely to be altered during cognitively healthy aging as well as in the presence of AD-related cognitive decline [3].…”

“…Moreover, recent research suggests that the existence of multiple enzyme isoforms could affect the correct estimation of BACE1 concentration. It is worth noting that some spliced forms do not have APPcleaving activity and that it is not known which (and whether) specific forms vary in AD [55]. The same study shows that other enzymes, detectable in CSF, such as cathepsin B, meprin β, and BACE2, could exert β-secretase activity.…”

Long Non-coding RNA: Insight Into Mechanisms of Alzheimer's Disease

“…LncRNAs are relatively stable, which indicates that the serum or CSF lncRNAs might be promising biomarkers and therapeutic targets for AD diagnosis and treatment (Table 1). The concentration of BACE1 in CSF and plasma shows a good diagnostic value in AD patients (Shen et al, 2018;Lopez-Font et al, 2019). Therapeutic strategies targeting BACE1 have been extensively developed but discontinued due to futility or safety reasons (Ghosh and Osswald, 2014;Hampel et al, 2021).…”

BACE2 beyond β‐processing of APP, its neuroprotective role in cerebrovascular endothelium

“…In this regard, BACE2 expression in human brain capillaries is higher than in neurons and higher than the expression of BACE1 (Yang et al, 2022). An early study indicated that, unlike BACE1, BACE2 activity was not related to Aβ concentration, and did not change significantly in the AD brain (Ahmed et al, 2010); but studies based on β‐secretase activity are problematic due to the large discrepancies in β‐secretase activity and expected BACE protein content (Lopez‐Font et al, 2019). More recently, by specifically analysing nuclei from brain vessels, it has been demonstrated that BACE2 mRNA levels are downregulated in cerebral vasculatures of AD patients (Yang et al, 2022).…”