BACE1 physiological function and its relevance for Alzheimer's Diseasetherapy and diagnosis

Alzheimer’s disease (AD) is the most common form of dementia clinically characterized by a significant loss of cognition and memory and changes in behavior and personality. The major pathological hallmarks in brain are extracellular deposits of amyloid-β peptide (Aβ) in the form of plaques and intracellular deposits of hyperphosphorylated tau protein in the form of neurofibrillary tangles. The accumulation and deposition of Aβ in brain tissue of AD patients is the major and common feature of both sporadic and familial forms of AD and Aβ has then been suggested to have a central role in AD pathogenesis. Aβ is generated by the sequential cleavage of the amyloid precursor protein (APP) by β- and γ-secretases. In 1999, β-site APP-cleaving enzyme (BACE1) was identified as the rate limiting enzyme in the generation of Aβ from APP and therefore it is currently considered as a very attractive therapeutic target for AD. Despite BACE1 potential as a therapeutic target, its physiological functions remain largely unknown. BACE1 physiological functions – Identification and validation of Bace1 substrates Despite the fact that Aβ is absent in the brain of BACE1 knockout mice, these animals display a complex phenotype suggesting that BACE1 is involved in the processing of other substrates besides APP that are crucial for brain development and function. In the first part of this work, we took a quantitative proteomic approach to analyze the secretome of primary neurons after acute BACE1 inhibition and identified several novel substrate candidates for BACE1. Many of these molecules are involved in neuronal network formation in the developing nervous system. We selected the adhesion molecules L1 and close homolog of L1 (CHL1), which are crucial for axonal guidance and maintenance of neural circuits, for further validation as BACE1 substrates. Using both BACE1 knockout mice and acute pharmacological BACE1 inhibition in primary neuronal cultures and mouse models, we show that L1 and CHL1 are cleaved by BACE1 under physiological conditions. Additionally, we determined the BACE1 cleavage sites at the membrane-proximal regions of L1 (between Y1086 and E1087) and CHL1 (between Q1061 and D1062) by mass spectrometry. This work provided molecular insight into the pathways regulated by BACE1 protease activity, and will help to predict possible side-effects of BACE1 inhibitor drugs in current AD clinical trials. BACE1 physiological functions – Physiological relevance of CHL1 as a BACE1 substrate After the identification and validation of neuronal adhesion molecules as BACE1 substrates in vivo, we studied the functional relevance of CHL1 processing by BACE1 for Semaphorin3A (Sema3A)-dependent axonal guidance. Cycles of growth cone collapse and restoration are crucial for axons to find their correct trajectory in the brain. Although axonal guidance cues and their receptors are well studied, the molecular switches underlying the alternating behavior of the growth cone are poorly understood. We show that regulated proteolysis of CHL1 by the AD-related proteases, BACE1 and γ-secretase, is critical for Sema3A-mediated axonal guidance processes. An active membrane bound proteolytic CHL1 fragment is generated by BACE1 (CHL1CTFβ) to relay the Sema3A signal via ezrin-radixin-moesin (ERM) proteins to the neuronal cytoskeleton. γ-Secretase-mediated degradation of this fragment stops the Sema3A-induced collapse and sensitizes the growth cone for the next round of axonal extension. This work revealed that a cycle of BACE1 and γ-secretase dependent proteolytic activity underlying growth cone collapse and restoration is crucial for axons to find their correct trajectory in the brain. BACE1 as a biomarker for AD Previous studies have investigated the activity and protein levels of BACE1 in the brain and cerebrospinal fluid (CSF) of AD patients; however, the results remain contradictory. In the last part of this work, we generated a highly specific and sensitive BACE1 ELISA, which allows the accurate measurement of BACE1 levels in human samples. We found that BACE1 levels in CSF of AD patients and other neurological disorder patients are slightly increased when compared to those of the non-neurological disorder control group. BACE1 levels in CSF were well correlated with total-tau and hyperphosphorylated tau levels in the CSF suggesting that the recorded alterations in BACE1 levels correlate with cell death and neurodegeneration. This assay will be used in follow up analysis of BACE1 CSF levels in bigger cohorts of patients and studies to monitor peripheral BACE1 levels after administration of BACE1 inhibitors. General conclusion In this work we validate L1 and CHL1 as BACE1 substrates in vivo. Moreover, we revealed a new function for BACE1 and γ-secretase in the regulation of growth cone collapse and recovery, a process crucial for axonal guidance. Nevertheless, it remains unclear how essential BACE1 is in the different developmental stages and especially in adult brain. Thus, more studies are needed to determine the actual function of this protease and the safety of an AD therapy based on BACE1 inhibition. In the near future, the ongoing biomarker studies in combination with the clinical trials should also help to clarify the level of BACE1 inhibition and the stage of AD progression that allows efficient treatment.status: publishe