Beta-site APP Cleaving Enzyme 1 Activity Research Articles

Serum Beta-Secretase 1 Activity Is a Potential Marker for the Differential Diagnosis between Alzheimer's Disease and Frontotemporal Dementia: A Pilot Study.

Alzheimer's disease (AD) and frontotemporal dementia (FTD) are the two major neurodegenerative diseases causing dementia. Due to similar clinical phenotypes, differential diagnosis is challenging without specific biomarkers. Beta-site Amyloid Precursor Protein cleaving enzyme 1 (BACE1) is a β-secretase pivotal in AD pathogenesis. In AD and mild cognitive impairment subjects, BACE1 activity is increased in brain/cerebrospinal fluid, and plasma levels appear to reflect those in the brain. In this study, we aim to evaluate serum BACE1 activity in FTD, since, to date, there is no evidence about its role. The serum of 30 FTD patients and 30 controls was analyzed to evaluate (i) BACE1 activity, using a fluorescent assay, and (ii) Glial Fibrillary Acid Protein (GFAP) and Neurofilament Light chain (NfL) levels, using a Simoa kit. As expected, a significant increase in GFAP and NfL levels was observed in FTD patients compared to controls. Serum BACE1 activity was not altered in FTD patients. A significant increase in serum BACE1 activity was shown in AD vs. FTD and controls. Our results support the hypothesis that serum BACE1 activity is a potential biomarker for the differential diagnosis between AD and FTD.

Beta-Site APP-Cleaving Enzyme-1 Inhibitory Role of Natural Flavonoids in the Treatment of Alzheimer's Disease.

Alzheimer's Disease (AD) is a devastating neurological condition characterized by a progressive decline in cognitive function, including memory loss, reasoning difficulties, and disorientation. Its hallmark features include the formation of neurofibrillary tangles and neuritic plaques in the brain, disrupting normal neuronal function. Neurofibrillary tangles, composed of phosphorylated tau protein and neuritic plaques, containing amyloid-β protein (Aβ) aggregates, contribute to the degenerative process. The discovery of the beta-site amyloid precursor protein cleaving enzyme 1 (BACE1) in 1999 revolutionized our understanding of AD pathogenesis. BACE1 plays a crucial role in the production of Aβ, the toxic protein implicated in AD progression. Elevated levels of BACE1 have been observed in AD brains and bodily fluids, underscoring its significance in disease onset and progression. Despite setbacks in clinical trials of BACE1 inhibitors due to efficacy and safety concerns, targeting BACE1 remains a promising therapeutic strategy for early-stage AD. Natural flavonoids have emerged as potential BACE1 inhibitors, demonstrating the ability to reduce Aβ production in neuronal cells and inhibit BACE1 activity. In our review, we delve into the pathophysiology of AD, highlighting the central role of BACE1 in Aβ production and disease progression. We explore the therapeutic potential of BACE1 inhibitors, including natural flavonoids, in controlling AD symptoms. Additionally, we provide insights into ongoing clinical trials and available patents in this field, shedding light on future directions for AD treatment research.

Electrochemiluminescence and electrochemical dual-mode detection of BACE1 activity based on the assembly of peptide and luminol co-functionalized silver nanoparticles induced by cucurbit[8]uril

A novel electrochemiluminescence (ECL) and electrochemical dual-mode sensor was developed for detecting the activity of beta-site amyloid precursor protein cleaving enzyme 1 (BACE1) and screening its inhibitor. Specifically, the adamantane (ADA)-functionalized peptide (P1), a designed substrate peptide for BACE1, was immobilized on the electrode surface via host-guest interaction between β-cyclodextrin (β-CD) and ADA. The aggregation of the peptide (P2) and luminol co-functionalized silver nanoparticles could be induced by cucurbit [8]uril (CB[8] due to the ability of CB[8] to accommodate two aromatic residues simultaneously. The obtained (CB[8]-P2-AgNPs-luminol)n aggregates with both ECL and electrochemical activity, used as the dual-mode signal probe, could be captured to the N-terminal of P1 through CB[8]. Once the substrate P1 was cleaved by BACE1, the probe-binding polypeptide fragment detached from the electrode surface, resulting in a remarkable decrease in the ECL and electrochemical signals. Taking advantage of the signal amplification function of the signal probe, the sensitive dual-mode assay for BACE1 activity can be achieved with the low detection limits of 33.11 pM for ECL and 53.19 pM for electrochemical mode. The superior analytical performance of this novel dual-mode sensor toward BACE1 activity suggested the promising application in early diagnosis of Alzheimer's disease (AD).

Acute interleukin-6 modulates key enzymes involved in prefrontal cortex and hippocampal amyloid precursor protein processing.

Exercise is beneficial for individuals with Alzheimer's disease (AD). In rodent models of AD, exercise decreases the amyloidogenic processing of the amyloid precursor protein (APP). Although, it remains unclear how exercise is promoting this shift away from pathological APP processing, there is emerging evidence that exercise-induced factors released from peripheral tissues may facilitate these alterations in brain APP processing. Interleukin-6 (IL-6) is released from multiple organs into peripheral circulation during exercise and is among the most characterized exerkines. The purpose of this study is to examine if acute IL-6 can modulate key enzymes responsible for APP processing, namely a disintegrin and metalloproteinase 10 (ADAM10) and beta-site amyloid precursor protein cleaving enzyme 1 (BACE1), which initiate the non-amyloidogenic and amyloidogenic cascades respectively. Male 10-week-old C57BL/6J mice underwent and acute treadmill exercise bout or were either injected with IL-6 or a PBS control 15 min prior to tissue collection. ADAM10 and BACE1 enzyme activity, mRNA, and protein expression, as well as downstream markers of both cascades, including sAPPa and sAPPβ, were examined. Exercise increased circulating IL-6 and brain IL-6 signaling (pSTAT3 and Socs3 mRNA). This occurred alongside a reduction in BACE1 activity and an increase in ADAM10 activity. IL-6 injection reduced BACE1 activity and increased sAPPa protein content in the prefrontal cortex. In the hippocampus, IL-6 injection decreased BACE1 activity and sAPPβ protein content. Our results show that acute IL-6 injection increases markers of the non-amyloidogenic cascade and decreases markers of the amyloidogenic cascade in the cortex and hippocampus of the brain.

The BACE1-generated C-terminal fragment of the neural cell adhesion molecule 2 (NCAM2) promotes BACE1 targeting to Rab11-positive endosomes

Beta-site amyloid precursor protein cleaving enzyme 1 (BACE1), also known as β-secretase, is an aspartic protease. The sorting of this enzyme into Rab11-positive recycling endosomes regulates the BACE1-mediated cleavage of its substrates, however, the mechanisms underlying this targeting remain poorly understood. The neural cell adhesion molecule 2 (NCAM2) is a substrate of BACE1. We show that BACE1 cleaves NCAM2 in cultured hippocampal neurons and NCAM2-transfected CHO cells. The C-terminal fragment of NCAM2 that comprises the intracellular domain and a small portion of NCAM2’s extracellular domain, associates with BACE1. This association is not affected in cells with inhibited endocytosis, indicating that the interaction of NCAM2 and BACE1 precedes the targeting of BACE1 from the cell surface to endosomes. In neurons and CHO cells, this fragment and BACE1 co-localize in Rab11-positive endosomes. Overexpression of full-length NCAM2 or a recombinant NCAM2 fragment containing the transmembrane and intracellular domains but lacking the extracellular domain leads to an increase in BACE1 levels in these organelles. In NCAM2-deficient neurons, the levels of BACE1 are increased at the cell surface and reduced in intracellular organelles. These effects are correlated with increased levels of the soluble extracellular domain of BACE1 in the brains of NCAM2-deficient mice, suggesting increased shedding of BACE1 from the cell surface. Of note, shedding of the extracellular domain of Sez6, a protein cleaved exclusively by BACE1, is reduced in NCAM2-deficient animals. These results indicate that the BACE1-generated fragment of NCAM2 regulates BACE1 activity by promoting the targeting of BACE1 to Rab11-positive endosomes.

Examination of BDNF Treatment on BACE1 Activity and Acute Exercise on Brain BDNF Signaling.

Perturbations in metabolism results in the accumulation of beta-amyloid peptides, which is a pathological feature of Alzheimer’s disease. Beta-site amyloid precursor protein cleaving enzyme 1 (BACE1) is the rate limiting enzyme responsible for beta-amyloid production. Obesogenic diets increase BACE1 while exercise reduces BACE1 activity, although the mechanisms are unknown. Brain-derived neurotropic factor (BDNF) is an exercise inducible neurotrophic factor, however, it is unknown if BDNF is related to the effects of exercise on BACE1. The purpose of this study was to determine the direct effect of BDNF on BACE1 activity and to examine neuronal pathways induced by exercise. C57BL/6J male mice were assigned to either a low (n = 36) or high fat diet (n = 36) for 10 weeks. To determine the direct effect of BDNF on BACE1, a subset of mice (low fat diet = 12 and high fat diet n = 12) were used for an explant experiment where the brain tissue was directly treated with BDNF (100 ng/ml) for 30 min. To examine neuronal pathways activated with exercise, mice remained sedentary (n = 12) or underwent an acute bout of treadmill running at 15 m/min with a 5% incline for 120 min (n = 12). The prefrontal cortex and hippocampus were collected 2-h post-exercise. Direct treatment with BDNF resulted in reductions in BACE1 activity in the prefrontal cortex (p < 0.05), but not the hippocampus. The high fat diet reduced BDNF content in the hippocampus; however, the acute bout of exercise increased BDNF in the prefrontal cortex (p < 0.05). These novel findings demonstrate the region specific differences in exercise induced BDNF in lean and obese mice and show that BDNF can reduce BACE1 activity, independent of other exercise-induced alterations. This work demonstrates a previously unknown link between BDNF and BACE1 regulation.

Identification of aplysinopsin as a blood-brain barrier permeable scaffold for anti-cholinesterase and anti-BACE-1 activity

Aplysinopsins are a group of marine-derived indole alkaloids that display diverse array of pharmacological effects. However, their effect on anti-Alzheimer targets has not been reported. Herein, we report the synthesis of aplysinopsin (1) and its effect on cholinesterases and beta-site amyloid-precursor protein cleaving enzyme 1 (BACE-1). It inhibits electric eel acetylcholinesterase (AChE), equine serum butyrylcholinesterase (BChE), and human BACE-1 with IC50 values of 33.9, 30.3, and 33.7 µM, respectively, and excellent BBB permeability (Pe 8.92 × 10−6 cm/s). To optimize its sub-micromolar activity, the first-generation analogs were prepared and screened. Two most active analogs 5b and (Z)-8g were found to effectively permeate the BBB (Pe > 5 × 10−6 cm/s). The N-sulphonamide derivative 5b display better cholinesterase inhibition, whereas the other analog (Z)-8g strongly inhibits BACE-1 (IC50 0.78 µM) activity. The analog 5b interacts primarily with PAS of AChE, and thus exhibit a mixed-type of inhibition. In addition, aplysinopsin along with new analogs inhibited the self-induced Aβ1-42 aggregation. The data presented herein indicate that the aplysinopsin-scaffold holds a potential for further investigation as a multi-targeted anti-Alzheimer agent.

Dimethyl fumarate exerts neuroprotection by modulating calcineurin/NFAT1 and NFκB dependent BACE1 activity in Aβ1-42 treated neuroblastoma SH-SY5Y cells

Ample studies indicate that calcineurin, a Ca2+/calmodulin-sensitive phosphatase, plays a key role in the initiation and/or clinical progression of Alzheimer's disease, and alteration of calcineurin has been confirmed in Alzheimer’s disease brain, impacting tau proteins and amyloid beta (Aβ) levels and resulting in neuronal cell death. As, it is sensible to deliberate the likelihood of calcineurin inhibition as a pharmacological target in the progress of novel Alzheimer’s disease therapies, we investigated the neuroprotective efficacy of dimethyl fumarate (DMF) via calcineurin dependent downstream targets in oligomeric Aβ1-42 treated neuroblastoma SH-SY5Y cells. DMF pre-treatment reduced LDH release, increased cell survival and decreased calcineurin activity in Aβ1-42-incubated cells. DMF was found to block calcineurin dependent apoptosis induced by Aβ1-42 through Bcl-2 linked death protein (BAD) and cAMP response element binding (CREB) dephosphorylation; moreover, rescued the opposing effect of Aβ1-42 on CREB-driven transcription in cells transfected with the CRE-SEAP reporter gene indicating its efficacy on long term potentiation and synaptic plasticity. DMF reversed Aβ1-42-induced enhancement in the active form of nuclear factor of activated T-cells (NFAT1) and further associated beta-site amyloid precursor protein cleaving enzyme-1 (BACE1) expression, BACE1 promoter activity and BACE1 enzymatic activity. DMF exhibited another neuroprotective mechanism against Aβ1-42 treatment by reducing nuclear factor kappa-light-chain-enhancer of activated B-cells (NFκB) dependent BACE1 gene transcription. DMF does not alter either beta amyloid precursor protein (βAPP) or its mRNA levels in SH-SY5Y cells, confirming that it does not possess amyloidogenic effect. Inhibiting molecular events related to Aβ1-42-induced calcineurin activity by DMF, may be an approach towards finding suitable treatment for Alzheimer’s disease.

ROLE OF ACUTE EXERCISE-INDUCED BRAIN BDNF ON APP PROCESSING AND BACE1

Perturbations in metabolism results in the accumulation of beta-amyloid, which is a pathological feature of Alzheimer's Disease (AD) (1). Beta-site amyloid precursor protein cleaving enzyme 1 (BACE1) is the rate limiting enzyme in the pathway responsible for beta-amyloid production (2). Exercise has been shown to reduce BACE1 activity, although the exact neurobiological mechanisms responsible are unknown (3). Exercise has also been shown to increase brain-derived neurotropic factor (BDNF) content and signaling (4), however whether this neurotrophic factor mediates the effects of exercise on BACE1 regulation requires further investigation. C57BL6 male mice were placed on a low (LFD; n=24) or high fat diet (HFD; n=24) for 10-weeks. Following the intervention, the mice either remained sedentary (n=12) or underwent an acute bout of treadmill running (n=12), proceeded by glucose/insulin tolerance tests and a Barnes maze test (spatial memory). Mice were euthanized and the prefrontal cortex was collected for protein quantification. Markers of the amyloidogenic pathway, along with targets of the BDNF signaling pathway, were examined. The 10-week HFD intervention led to increased body mass, inguinal and epididymal white adipose tissue mass, and liver mass (p < 0.05). HFD groups had impairments in glucose and insulin tolerance, as well as spatial memory (time in incorrect zones) (p < 0.05). A HFD led to increases in Akt phosphorylation (insulin signalling) and decreases in ERK phosphorylation (downstream of BDNF) (p < 0.05). An acute bout of exercise was able to recover ERK phosphorylation in the HFD group (p < 0.05) and significantly increase BDNF protein content in both LFD and HFD groups (p < 0.05). However, there were no changes in BACE1 protein content with diet or exercise (p > 0.05). Results from this study suggest that exercise can increase BDNF protein content in an obesogenic rodent model. Furthermore, a HFD potentially contributes to memory impairments by aberrant insulin signalling and impairments in BDNF signalling, which can be recovered through exercise. Although BACE1 protein content did not change, future analysis will examine BACE1 activity. Through these mechanisms, we gain further insight into using BDNF, mediated through exercise, as a therapeutic intervention.

A\u03b234 is a BACE1-derived degradation intermediate associated with amyloid clearance and Alzheimer\u2019s disease progression

The beta-site APP cleaving enzyme 1 (BACE1) is known primarily for its initial cleavage of the amyloid precursor protein (APP), which ultimately leads to the generation of Aβ peptides. Here, we provide evidence that altered BACE1 levels and activity impact the degradation of Aβ40 and Aβ42 into a common Aβ34 intermediate. Using human cerebrospinal fluid (CSF) samples from the Amsterdam Dementia Cohort, we show that Aβ34 is elevated in individuals with mild cognitive impairment who later progressed to dementia. Furthermore, Aβ34 levels correlate with the overall Aβ clearance rates in amyloid positive individuals. Using CSF samples from the PREVENT-AD cohort (cognitively normal individuals at risk for Alzheimer’s disease), we further demonstrate that the Aβ34/Aβ42 ratio, representing Aβ degradation and cortical deposition, associates with pre-clinical markers of neurodegeneration. We propose that Aβ34 represents a marker of amyloid clearance and may be helpful for the characterization of Aβ turnover in clinical samples.

DNA Aptamers Targeting BACE1 Reduce Amyloid Levels and Rescue Neuronal Deficiency in Cultured Cells

β-amyloid (Aβ) plays an essential role in the pathogenesis of Alzheimer’s disease (AD). Beta-site amyloid precursor protein cleaving enzyme 1 (BACE1) is indispensable for Aβ production, and knockout of BACE1 has no overt phenotypes in mouse. Thus, fine modulation of BACE1 may be a safe and effective treatment for AD patients. However, the large active site of BACE1 makes it challenging to target BACE1 with classical small-molecule inhibitors. DNA aptamer can have high affinity and specificity against diverse targets, and it provides an alternative strategy to target BACE1. In this study, we used a novel cell-systematic evolution of ligands by exponential enrichment (SELEX) strategy to select specific DNA aptamers optimized to target BACE1 under physiological status. After 17 rounds of selection, we identified two DNA aptamers against BACE1: BI1 and BI2. The identified aptamers interacted with BACE1 in pull-down assay, inhibited BACE1 activity in in vitro fluorescence resonance energy transfer (FRET) assay and HEK293-APP stable cell line, reduced Aβ in the culture medium of HEK293-amyloid protein precursor (APP) stable cell line and APP-PS1 primary cultured neurons, and rescued Aβ-induced neuronal deficiency in APP-PS1 primary cultured neurons. In contrast, the identified aptamers had no effect on α- or γ-secretase. In addition, cholesteryl tetraetylene glycol (TEG) modification further improved the potency of the identified aptamers. Our study suggests that it is feasible and effective to target BACE1 with DNA aptamers, and the therapeutic potential of the identified aptamers deserves further investigation.

BACE1 Inhibitor MK-8931 Alters Formation but Not Stability of Dendritic Spines.

Beta-site amyloid-precursor-protein cleaving enzyme 1 (BACE1) is the rate limiting protease in the production of the amyloid-beta peptide (Aβ), which is considered to be the causative agent in the pathogenesis of Alzheimer’s Disease (AD). Therefore, the therapeutic potential of pharmacological BACE1 inhibitors is currently tested in clinical trials for AD treatment. To ensure a positive clinical outcome it is crucial to identify and evaluate adverse effects associated with BACE1 inhibition. Preclinical studies show that chronic blockade of BACE1 activity alters synaptic functions and leads to loss of dendritic spines. To assess the mechanism of synapse loss, dendritic spine dynamics of pyramidal layer V cells were monitored by in vivo two-photon microscopy in the somatosensory cortex of mice, treated with the BACE1 inhibitor MK-8931. MK-8931 treatment significantly reduced levels of Aβ40 and density of dendritic spines in the brain. However, the steady decline in dendritic spine density specifically resulted from a diminished formation of new spines and not from a loss of stable spines. Furthermore, the described effects on spine formation were transient and recovered after inhibitor withdrawal. Since MK-8931 inhibition did not completely abolish spine formation, our findings suggest that carefully dosed inhibitors might be therapeutically effective without affecting the structural integrity of excitatory synapses if given at an early disease stage.

PALMITATE INDUCES BACE1 EXPRESSION AND ACTIVITY BY INDUCING STEROL RESPONSE ELEMENT BINDING PROTEIN 1 EXPRESSION AND ACTIVATION IN THE MOUSE HIPPOCAMPUS AND HUMAN SH-SY5Y NEUROBLASTOMA CELLS

The etiology of Alzheimer's disease (AD) is multifactorial and poorly defined and characterized by a dynamic interplay of a myriad of risk variables that include dietary and environmental factors. Epidemiological studies have implicated saturated fat-enriched diets in the etio-pathogenesis of AD. The aspartyl protease beta-site amyloid precursor protein cleaving enzyme 1 (BACE1) catalyzes the rate limiting step in amyloid-beta (Aβ) production, a peptide that serves as a core instigator of pathophysiological neurodegenrative processes in AD. Emerging evidence from our laboratory studies and other contemporary studies have shown that saturated fat-enriched diets, such as palmitate-enriched diets, increase BACE1 activity and enhance Aβ production. Our preliminary studies have shown that a palmitate-enriched diet increases the expression and activity of Sterol Response Element binding protein 1 (SREBP1) in the mouse hippocampus. In this study, we determined the role of SREBP1 activation in the palmitate-induced increase in BACE1 expression, activity, and subsequent Aβ genesis. We fed nine-month old C57BL/6J mice a palmitate-enriched diet or the respective control-chow diet for three months and determined the extent to which increased SREBP1 expression and activity plays a role in the palmitate-enriched diet-induced increase in BACE1 expression, activity, and Aβ genesis in the hippocampus. We also determined the role SREBP1 activation in the induction of BACE1 expression, activity, and Aβ genesis in exogenous palmitate-treated human SH-SY5Y neuroblastoma cells and delineated the underlying mechanisms involved. Our ChIP analysis and luciferase reporter assays show that palmitate-induced SREBP1 activation directly regulates BACE1 expression at the transcriptional level. Knocking-down or ectopic expression of the dominant negative SREBP1 mutant significantly mitigated the palmitate-induced BACE1 expression and subsequent Aβ genesis. Our study elucidates a novel signaling pathway and SREBP1 as a unique downstream effector of palmitate-induced up-regulation in BACE1 expression and subsequent Aβ genesis.

PPAR-α agonist regulates amyloid-β generation via inhibiting BACE-1 activity in human neuroblastoma SH-SY5Y cells transfected with APPswe gene.

Alzheimer's disease is a neuroinflammatory disease and is the most common cause of dementia in the elderly. Studies have shown the beneficial effects of the peroxisome proliferator-activated receptor alpha (PPAR-α) agonists on the treatment of neuroinflammatory diseases. The aim of the present study is to examine the ability of GW7647 (a PPAR-α agonist) to regulate amyloid precursor protein (APP) amyloidogenic processing in human neuroblastoma SH-SY5Y cells transfected with APPswe gene. After administration of GW7647 for 24h, the levels of APP, soluble APPβ (sAPPβ), and presenilin 1 (PS-1) were assessed by Western blot. Cellular culture medium levels of amyloid-β 42 (Aβ42) were analyzed by ELISA, and the activity of beta-site APP cleaving enzyme 1 (BACE-1) was measured by fluorometric assay. We found that GW7647 decreased the expression of sAPPβ and the activity of BACE-1, and also reduced Aβ42 release. However, GW7647 did not modify the levels of APP and PS-1. Furthermore, LY294002, the phosphoinositide 3-kinase (PI3-K) inhibitor, reversed the effects of GW7647 on the BACE-1 activity and the levels of sAPPβ and Aβ42. Our data demonstrate that GW7647 may reduce Aβ production via inhibiting BACE-1 activity, and this may involve in PI3-K pathway.

A simple and label-free electrochemical method for detection of beta-site amyloid precursor protein cleaving enzyme and screening of its inhibitor

Beta-site amyloid precursor protein cleaving enzyme 1 (BACE1) is an aspartic protease that promotes the production of beta-amyloid (Aβ) peptides from beta-site amyloid precursor protein. Pharmacological reduction of Aβ production by inhibiting BACE1 has emerged as a promising target for the treatment of Alzheimer's disease (AD). In this work, we reported a simple electrochemical method for detection of BACE1 and screening of its inhibitor. This method made use of an unlabeled peptide that comprises a segment of beta-site amyloid precursor protein with the Swiss mutation. The peptide–heme complex formed on the electrode surface showed strong electrocatalytic O2 reduction. Cleavage of the peptide by BACE1 detached the heme-binding fragment from the electrode surface, leading to a remarkable decrease in the electrochemical signal. Suppression of BACE1 by the inhibitor yielded a larger voltammetric signal. The IC50 of the inhibitor tested by this method (15.7nM) was in agreement with that reported. The proposed method could serve as a promising alternative to the traditional methods for probing of BACE1 activity and screening of BACE1 inhibitor owing to its simplicity, high sensitivity and low assay cost.

From Fragment Screening to In Vivo Efficacy: Optimization of a Series of 2-Aminoquinolines as Potent Inhibitors of Beta-Site Amyloid Precursor Protein Cleaving Enzyme 1 (BACE1)

Using fragment-based screening of a focused fragment library, 2-aminoquinoline 1 was identified as an initial hit for BACE1. Further SAR development was supported by X-ray structures of BACE1 cocrystallized with various ligands and molecular modeling studies to expedite the discovery of potent compounds. These strategies enabled us to integrate the C-3 side chain on 2-aminoquinoline 1 extending deep into the P2' binding pocket of BACE1 and enhancing the ligand's potency. We were able to improve the BACE1 potency to subnanomolar range, over 10(6)-fold more potent than the initial hit (900 μM). Further elaboration of the physical properties of the lead compounds to those more consistent with good blood-brain barrier permeability led to inhibitors with greatly improved cellular activity and permeability. Compound 59 showed an IC(50) value of 11 nM on BACE1 and cellular activity of 80 nM. This compound was advanced into rat pharmacokinetic and pharmacodynamic studies and demonstrated significant reduction of Aβ levels in cerebrospinal fluid (CSF).

Ubiquitin Regulates GGA3-mediated Degradation of BACE1

BACE1 (beta-site amyloid precursor protein-cleaving enzyme 1) is a membrane-tethered member of the aspartyl proteases, essential for the production of beta-amyloid, a toxic peptide that accumulates in the brain of subjects affected by Alzheimer disease. The BACE1 C-terminal fragment contains a DXXLL motif that has been shown to bind the VHS (VPS27, Hrs, and STAM) domain of GGA1-3 (Golgi-localized gamma-ear-containing ARF-binding proteins). GGAs are trafficking molecules involved in the transport of proteins containing the DXXLL signal from the Golgi complex to endosomes. Moreover, GGAs bind ubiquitin and traffic synthetic and endosomal ubiquitinated cargoes to lysosomes. We have previously shown that depletion of GGA3 results in increased BACE1 levels and activity because of impaired lysosomal degradation. Here, we report that the accumulation of BACE1 is rescued by the ectopic expression of GGA3 in H4 neuroglioma cells depleted of GGA3. Accordingly, the overexpression of GGA3 reduces the levels of BACE1 and beta-amyloid. We then established that mutations in the GGA3 VPS27, Hrs, and STAM domain (N91A) or in BACE1 di-leucine motif (L499A/L500A), able to abrogate their binding, did not affect the ability of ectopically expressed GGA3 to rescue BACE1 accumulation in cells depleted of GGA3. Instead, we found that BACE1 is ubiquitinated at lysine 501 and is mainly monoubiquitinated and Lys-63-linked polyubiquitinated. Finally, a GGA3 mutant with reduced ability to bind ubiquitin (GGA3L276A) was unable to regulate BACE1 levels both in rescue and overexpression experiments. These findings indicate that levels of GGA3 tightly and inversely regulate BACE1 levels via interaction with ubiquitin sorting machinery.

P3-239: Heparin activates βeta-secretase(BACE1) of Alzheimer’s disease and increases autocatalysis of the enzyme

The beta–site APP cleaving enzyme 1 (BACE1) is an aspartic protease that generates the N–terminus of the beta–amyloid protein from the beta–amyloid precursor protein (APP). BACE1 is a key target for Alzheimer drug development. However, little is known about the physiological regulation of the enzyme. Heparin can promote beta–secretase cleavage of endogenous APP in neuroblastoma cells. However, heparin has also been reported to directly inhibit BACE1 activity in vitro. To clarify the role of heparin in regulating BACE1. We examined the effect of heparin on the activity of recombinant human BACE1 (rBACE1) in vitro. Low concentrations (1 microg/mL) of heparin were found to stimulate rBACE1, whereas higher concentrations (10 or 100 microg/mL) were inhibitory. Heparin affinity chromatography demonstrated that heparin interacted strongly with the partially active pro–enzyme form of rBACE1 and bound to a peptide homologous to the N–terminal pro–sequence of BACE1. Low concentrations of heparin promoted autocatalytic cleavage of rBACE1, resulting in a decrease in the concentration of the pro–enzyme form and an increase in lower molecular mass forms. Furthermore, mature (pro–sequence cleaved) enzyme appeared to lack the capacity to be stimulated by heparin providing support for the view that activation by heparin was through pro–domain cleavage. The results indicate that high affinity binding of heparin to the pro–domain of BACE1 activates the enzyme and increases autocatalysis of BACE1 in vitro. Our study provides evidence that heparan sulfate proteoglycans could regulate the rate of Abeta production in vivo.