Guoxin Wu

Elevated cerebrospinal fluid BACE1 activity in incipient Alzheimer disease.

BACKGROUND We used a sensitive and specific beta-site amyloid precursor protein (APP)-cleaving enzyme 1 (BACE1) assay to determine the relationship between BACE1 activity in cerebrospinal fluid (CSF) and markers of APP metabolism and axonal degeneration in early and late stages of Alzheimer disease (AD). OBJECTIVE To assess CSF BACE1 activity in AD. DESIGN Case-control and longitudinal follow-up study. SETTING Specialized memory clinic. Patients Eighty-seven subjects with AD, 33 cognitively normal control subjects, and 113 subjects with mild cognitive impairment (MCI), who were followed up for 3 to 6 years. MAIN OUTCOME MEASURES Cerebrospinal fluid BACE1 activity in relation to diagnosis and CSF levels of secreted APP and amyloid beta protein (Abeta) isoforms and the axonal degeneration marker total tau. RESULTS Subjects with AD had higher CSF BACE1 activity (median, 30 pM [range, 11-96 pM]) than controls (median, 23 pM [range, 8-43 pM]) (P =.02). Subjects with MCI who progressed to AD during the follow-up period had higher baseline BACE1 activity (median, 35 pM [range, 18-71 pM]) than subjects with MCI who remained stable (median, 29 pM [range, 14-83 pM]) (P < .001) and subjects with MCI who developed other forms of dementia (median, 20 pM [range, 10-56 pM]) (P <.001). BACE1 activity correlated positively with CSF levels of secreted APP isoforms and Abeta(40) in the AD and control groups and in all MCI subgroups (P < .05) except the MCI subgroup that developed AD. Strong positive correlations were found between CSF BACE1 activity and total tau levels in all MCI subgroups (r >or= 0.57, P <or= .009). CONCLUSION Elevated BACE1 activity may contribute to the amyloidogenic process in sporadic AD and is associated with the intensity of axonal degeneration.

First Demonstration of Cerebrospinal Fluid and Plasma Aβ Lowering with Oral Administration of a β-Site Amyloid Precursor Protein-Cleaving Enzyme 1 Inhibitor in Nonhuman Primates

β-Site amyloid precursor protein (APP)-cleaving enzyme (BACE) 1 cleavage of amyloid precursor protein is an essential step in the generation of the potentially neurotoxic and amyloidogenic Aβ42 peptides in Alzheimers disease. Although previous mouse studies have shown brain Aβ lowering after BACE1 inhibition, extension of such studies to nonhuman primates or man was precluded by poor potency, brain penetration, and pharmacokinetics of available inhibitors. In this study, a novel tertiary carbinamine BACE1 inhibitor, tertiary carbinamine (TC)-1, was assessed in a unique cisterna magna ported rhesus monkey model, where the temporal dynamics of Aβ in cerebrospinal fluid (CSF) and plasma could be evaluated. TC-1, a potent inhibitor (IC50 ∼ 0.4 nM), has excellent passive membrane permeability, low susceptibility to P-glycoprotein transport, and lowered brain Aβ levels in a mouse model. Intravenous infusion of TC-1 led to a significant but transient lowering of CSF and plasma Aβ levels in conscious rhesus monkeys because it underwent CYP3A4-mediated metabolism. Oral codosing of TC-1 with ritonavir, a potent CYP3A4 inhibitor, twice daily over 3.5 days in rhesus monkeys led to sustained plasma TC-1 exposure and a significant and sustained reduction in CSF sAPPβ, Aβ40, Aβ42, and plasma Aβ40 levels. CSF Aβ42 lowering showed an EC50 of ∼20 nM with respect to the CSF [TC-1] levels, demonstrating excellent concordance with its potency in a cell-based assay. These results demonstrate the first in vivo proof of concept of CSF Aβ lowering after oral administration of a BACE1 inhibitor in a nonhuman primate.

In Vivo β-Secretase 1 Inhibition Leads to Brain Aβ Lowering and Increased α-Secretase Processing of Amyloid Precursor Protein without Effect on Neuregulin-1

β-Secretase (BACE) cleavage of amyloid precursor protein (APP) is one of the first steps in the production of amyloid β peptide Aβ42, the putative neurotoxic species in Alzheimers disease. Recent studies have shown that BACE1 knockdown leads to hypomyelination, putatively caused by a decline in neuregulin (NRG)-1 processing. In this study, we have tested a potent cell-permeable BACE1 inhibitor (IC50 ∼ 30 nM) by administering it directly into the lateral ventricles of mice, expressing human wild-type (WT)-APP, to determine the consequences of BACE1 inhibition on brain APP and NRG-1 processing. BACE1 inhibition, in vivo, led to a significant dose- and time-dependent lowering of brain Aβ40 and Aβ42. BACE1 inhibition also led to a robust brain secreted (s)APPβ lowering that was accompanied by an increase in brain sAPPα levels. Although an increase in full-length NRG-1 levels was evident in 15-day-old BACE1 homozygous knockout (KO) (–/–) mice, in agreement with previous studies, this effect was also observed in 15-day-old heterozygous (+/–) mice, but it was not evident in 30-day-old and 2-year-old BACE1 KO (–/–) mice. Thus, BACE1 knockdown led to a transient decrease in NRG-1 processing in mice. Pharmacological inhibition of BACE1 in adult mice, which led to significant Aβ lowering, was without any significant effect on brain NRG-1 processing. Taken together, these results suggest that BACE1 is the major β-site cleavage enzyme for APP and that its inhibition can lower brain Aβ and redirect APP processing via the potentially nonamyloidogenic α-secretase pathway, without significantly altering NRG-1 processing.

PCSK9 is not involved in the degradation of LDL receptors and BACE1 in the adult mouse brain

Proprotein convertase subtilisin/kexin type 9 (PCSK9) is a secreted protein that regulates hepatic low-density lipoprotein receptor (LDLR) levels in humans. PCSK9 has also been shown to regulate the levels of additional membrane-bound proteins in vitro, including the very low-density lipoprotein receptor (VLDLR), apolipoprotein E receptor 2 (ApoER2) and the β-site amyloid precursor protein (APP)-cleaving enzyme 1 (BACE1), which are all highly expressed in the CNS and have been implicated in Alzheimers disease. To better understand the role of PCSK9 in regulating these additional target proteins in vivo, their steady-state levels were measured in the brain of wild-type, PCSK9-deficient, and human PCSK9 overexpressing transgenic mice. We found that while PCSK9 directly bound to recombinant LDLR, VLDLR, and apoER2 protein in vitro, changes in PCSK9 expression did not alter the level of these receptors in the mouse brain. In addition, we found no evidence that PCSK9 regulates BACE1 levels or APP processing in the mouse brain. In conclusion, our results suggest that while PCSK9 plays an important role in regulating circulating LDL cholesterol levels by reducing the number of hepatic LDLRs, it does not appear to modulate the levels of LDLR and other membrane-bound proteins in the adult mouse brain.

Acute gamma-secretase inhibition of nonhuman primate CNS shifts amyloid precursor protein (APP) metabolism from amyloid-beta production to alternative APP fragments without amyloid-beta rebound

The accumulation of amyloid β (Aβ) in Alzheimers disease is caused by an imbalance of production and clearance, which leads to increased soluble Aβ species and extracellular plaque formation in the brain. Multiple Aβ-lowering therapies are currently in development: an important goal is to characterize the molecular mechanisms of action and effects on physiological processing of Aβ, as well as other amyloid precursor protein (APP) metabolites, in models which approximate human Aβ physiology. To this end, we report the translation of the human in vivo stable-isotope-labeling kinetics (SILK) method to a rhesus monkey cisterna magna ported (CMP) nonhuman primate model, and use the model to test the mechanisms of action of a γ-secretase inhibitor (GSI). A major concern of inhibiting the enzymes which produce Aβ (β- and γ-secretase) is that precursors of Aβ may accumulate and cause a rapid increase in Aβ production when enzyme inhibition discontinues. In this study, the GSI MK-0752 was administered to conscious CMP rhesus monkeys in conjunction with in vivo stable-isotope-labeling, and dose-dependently reduced newly generated CNS Aβ. In contrast to systemic Aβ metabolism, CNS Aβ production was not increased after the GSI was cleared. These results indicate that most of the CNS APP was metabolized to products other than Aβ, including C-terminal truncated forms of Aβ: 1-14, 1-15 and 1-16; this demonstrates an alternative degradation pathway for CNS amyloid precursor protein during γ-secretase inhibition.

A Sensitive Aβ Oligomer Assay Discriminates Alzheimer's and Aged Control Cerebrospinal Fluid

A hallmark of Alzheimers disease (AD) brain is the amyloid β (Aβ) plaque, which is comprised of Aβ peptides. Multiple lines of evidence suggest that Aβ oligomers are more toxic than other peptide forms. We sought to develop a robust assay to quantify oligomers from CSF. Antibody 19.3 was compared in one-site and competitive ELISAs for oligomer binding specificity. A two-site ELISA for oligomers was developed using 19.3 coupled to a sensitive, bead-based fluorescent platform able to detect single photons of emitted light. The two-site ELISA was >2500× selective for Aβ oligomers over Aβ monomers with a limit of detection ∼0.09 pg/ml in human CSF. The lower limit of reliable quantification of the assay was 0.18 pg/ml and the antibody pairs recognized Aβ multimers comprised of either synthetic standards, or endogenous oligomers isolated from confirmed human AD and healthy control brain. Using the assay, a significant 3- to 5-fold increase in Aβ oligomers in human AD CSF compared with comparably aged controls was demonstrated. The increase was seen in three separate human cohorts, totaling 63 AD and 54 controls. CSF oligomers ranged between 0.1 and 10 pg/ml. Aβ oligomer levels did not strongly associate with age or gender, but had an inverse correlation with MMSE score. The C statistic for the Aβ oligomer ROC curve was 0.86, with 80% sensitivity and 88% specificity to detect AD, suggesting reasonable discriminatory power for the AD state and the potential for utility as a diagnostic marker.

CNS Amyloid-β, Soluble APP-α and -β Kinetics during BACE Inhibition

BACE, a β-secretase, is an attractive potential disease-modifying therapeutic strategy for Alzheimers disease (AD) as it results directly in the decrease of amyloid precursor protein (APP) processing through the β-secretase pathway and a lowering of CNS amyloid-β (Aβ) levels. The interaction of the β-secretase and α-secretase pathway-mediated processing of APP in the rhesus monkey (nonhuman primate; NHP) CNS is not understood. We hypothesized that CNS inhibition of BACE would result in decreased newly generated Aβ and soluble APPβ (sAPPβ), with increased newly generated sAPPα. A stable isotope labeling kinetics experiment in NHPs was performed with a 13C6-leucine infusion protocol to evaluate effects of BACE inhibition on CNS APP processing by measuring the kinetics of sAPPα, sAPPβ, and Aβ in CSF. Each NHP received a low, medium, or high dose of MBI-5 (BACE inhibitor) or vehicle in a four-way crossover design. CSF sAPPα, sAPPβ, and Aβ were measured by ELISA and newly incorporated label following immunoprecipitation and liquid chromatography-mass spectrometry. Concentrations, kinetics, and amount of newly generated APP fragments were calculated. sAPPβ and sAPPα kinetics were similar, but both significantly slower than Aβ. BACE inhibition resulted in decreased labeled sAPPβ and Aβ in CSF, without observable changes in labeled CSF sAPPα. ELISA concentrations of sAPPβ and Aβ both decreased and sAPPα increased. sAPPα increased by ELISA, with no difference by labeled sAPPα kinetics indicating increases in product may be due to APP shunting from the β-secretase to the α-secretase pathway. These results provide a quantitative understanding of pharmacodynamic effects of BACE inhibition on NHP CNS, which can inform about target development.

Pyroglutamate-Modified Amyloid-β Protein Demonstrates Similar Properties in an Alzheimer's Disease Familial Mutant Knock-In Mouse and Alzheimer's Disease Brain

Background: N-terminally truncated, pyroglutamate-modified amyloid-β (Aβ) peptides are major constituents of amyloid deposits in Alzheimers disease (AD). Methods: Using a newly developed ELISA for Aβ modified at glutamate 3 with a pyroglutamate (pE3Aβ), brain pE3Aβ was characterized in human AD in an AD mouse model harboring double knock-in amyloid precursor protein (APP)-KM670/671NL and presenilin 1 (PS1)-P264L (APP/PS1-dKI) mutations, and in a second mouse model with transgenic overexpression of human APP695 with APP-KM670/671NL (Tg2576). Results: pE3Aβ increased in the AD brain versus age-matched controls, with pE3Aβ/total Aβ at 45 and 10%, respectively. Compared to controls, the AD brain demonstrated 8.5-fold increased pE3Aβ compared to non-pE3Aβ species, which increased 2.7-fold. In the APP/PS1-dKI brain, pE3Aβ/total Aβ increased from 7% at 3 months to 16 and 19% at 15 and 19 months, respectively. In Tg2576, pE3Aβ/total Aβ was only 1.5% at 19 months, suggesting that APP/PS1-dKI, despite less total Aβ compared to Tg2576 at comparable ages, more closely mimics AD brain pathology. Conclusion: This report supports a significant role for pE3Aβ in AD pathogenesis by confirming that pE3Aβ represents a large fraction of Aβ within the AD brain. Compared to the age-matched control brain, pE3Aβ increased to a greater extent compared to Aβ species without this N-terminal modification. Further, the APP/PS1-dKI model more closely resembles the AD brain in this regard, compared to the Tg2576 model.

Cisterna magna cannulated repeated CSF sampling rat model--effects of a gamma-secretase inhibitor on Aβ levels.

Cerebrospinal fluid (CSF) provides a window into central nervous system (CNS) physiology and pathophysiology in human neurodegenerative conditions such as Alzheimers disease. Changes in CSF bioanalytes also provide a direct readout of target engagement in the CNS following pharmacological interventions in clinical trials. Given the importance of tracking CNS bioanalytes in drug discovery, we have developed a novel cisterna magna cannulated rat model for repeated CSF sampling and used it to assess an amyloid beta (Aβ) lowering agent. The surgically implanted cisterna magna cannula was patent over a period of 1-2 weeks and enabled repeated sampling of CSF (volume of ∼30-50μL/sample) from each rat. CSF Aβ40 levels showed good intra-animal variability across time points and inter-animal variability within a time point. Peripheral treatment with a gamma-secretase inhibitor (GSI) led to a rapid and robust decline in CSF Aβ40 levels that returned to baseline over 24-96h after dosing. Terminal brain, CSF and plasma Aβ levels measured at 24h after dosing demonstrated robust Aβ lowering and showed excellent correlation across these compartments. These results are the first pharmacological validation of the repeated CSF sampling rat model for Aβ lowering agents. This model can have broad applicability in pharmacological evaluation for diverse CNS targets.

Soluble BACE-1 Activity and sAβPPβ Concentrations in Alzheimer’s Disease and Age-Matched Healthy Control Cerebrospinal Fluid from the Alzheimer’s Disease Neuroimaging Initiative-1 Baseline Cohort

β-site amyloid precursor protein-cleaving enzyme 1 (BACE1) plays an important role in the development of Alzheimers disease (AD), freeing the amyloid-β (Aβ) N-terminus from the amyloid-β protein precursor (AβPP), the first step in Aβ formation. Increased BACE1 activity in AD brain or cerebrospinal fluid (CSF) has been reported. Other studies, however, found either no change or a decrease with AD diagnosis in either BACE1 activity or sAβPPβ, the N-terminal secreted product of BACE1 (sBACE1) activity on AβPP. Here, sBACE1 enzymatic activity and secreted AβPPβ (sAβPPβ) were measured in Alzheimers Disease Neuroimaging Initiative-1 (ADNI-1) baseline CSF samples and no statistically significant changes were found in either measure comparing healthy control, mild cognitively impaired, or AD individual samples. While CSF sBACE1 activity and sAβPPβ demonstrated a moderate yet significant degree of correlation with each other, there was no correlation of either analyte to CSF Aβ peptide ending at residue 42. Surprisingly, a stronger correlation was demonstrated between CSF sBACE1 activity and tau, which was comparable to that between CSF Aβ₄₂ and tau. Unlike for these latter two analytes, receiver-operator characteristic curves demonstrate that neither CSF sBACE1 activity nor sAβPPβ concentrations can be used to differentiate between healthy elderly and AD individuals.