Eric A. Price

A novel pathway for amyloid precursor protein processing

Amyloid precursor protein (APP) can be proteolytically processed along two pathways, the amyloidogenic that leads to the formation of the 40-42 amino acid long Alzheimer-associated amyloid β (Aβ) peptide and the non-amyloidogenic in which APP is cut in the middle of the Aβ domain thus precluding Aβ formation. Using immunoprecipitation and mass spectrometry we have shown that Aβ is present in cerebrospinal fluid (CSF) as several shorter isoforms in addition to Aβ1-40 and Aβ1-42. To address the question by which processing pathways these shorter isoforms arise, we have developed a cell model that accurately reflects the Aβ isoform pattern in CSF. Using this model, we determined changes in the Aβ isoform pattern induced by α-, β-, and γ-secretase inhibitor treatment. All isoforms longer than and including Aβ1-17 were γ-secretase dependent whereas shorter isoforms were γ-secretase independent. These shorter isoforms, including Aβ1-14 and Aβ1-15, were reduced by treatment with α- and β-secretase inhibitors, which suggests the existence of a third and previously unknown APP processing pathway involving concerted cleavages of APP by α- and β-secretase.

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.

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.

Rapid P1 SAR of brain penetrant tertiary carbinamine derived BACE inhibitors

This Letter describes the one pot synthesis of tertiary carbinamine 3 and related analogs of brain penetrant BACE-1 inhibitors via the alkylation of the Schiff base intermediate 2. The methodology developed for this study provided a convenient and rapid means to explore the P1 region of these types of inhibitors, where the P1 group is installed in the final step using a one-pot two-step protocol. Further SAR studies led to the identification of 10 which is twofold more potent in vitro as compared to the lead compound. This inhibitor was characterized in a cisterna magna ported rhesus monkey model, where significant lowering of CSF Abeta40 was observed.

High throughput monitoring of amyloid-β(42) assembly into soluble oligomers achieved by sensitive conformation state-dependent immunoassays.

Accumulation of small soluble assemblies of amyloid-β (Aβ)(42) in the brain is thought to play a key role in the pathogenesis of Alzheimers disease. As a result, there has been much interest in finding small molecules that inhibit the formation of synaptotoxic Aβ(42) oligomers that necessitates sensitive methods for detecting the initial steps in the oligomerization of Aβ(42). Modeling suggests that oligomerized Aβ(42) adopts a conformation in which the C-terminus is embedded in the center, whereas the N-terminus is exposed at the periphery of the oligomer. Here we report that an inverse change in Aβ(42) C-terminal and N-terminal epitope accessibility provides the basis of a sensitive method for assessing early steps in Aβ(42) oligomerization. Using ELISA and AlphaLISA, we found that Aβ(42) C-terminal immunoreactivity decreased in a time- and concentration-dependent manner under conditions favoring oligomerization. This reduction was accompanied by an increase in the N-terminal immunoreactivity, suggesting that assemblies with multiple exposed N-terminal epitopes were detected. Importantly the assay generates a robust window between monomers and oligomers at as low as 1 nM Aβ(42). Using this assay, known oligomerization inhibitors produced a dose-dependent unmasking of the Aβ(42) C-terminal epitope. After automation, the assay proved to be highly reproducible and effective for high throughput screening of small molecules that inhibit Aβ(42) oligomerization.

P3-094: Three novel CSF assays expand a core panel of CSF biomarkers: BACE activity, oligomeric Aβ and homocysteine assessed in pathologically confirmed Alzheimer subjects within the Oxford project to investigate memory and aging (Optima) cohort

Background: A key goal within the Alzheimer’s disease (AD) research community is the early clinical detection and diagnosis of AD in patients who are at risk, or present with symptoms, of the disease. In addition to the standard clinical criteria, much effort in the field is aimed at identifying biochemical and imaging biomarkers. Methods: In a collaborative effort to discover early biomarkers of AD between OPTIMA and Merck Research Laboratories, we have expanded a panel of six previously validated and commercially available CSF biochemical assays for APP catabolites (A 40, A 42, sAPP , sAPP ) and tau (tTau, pTau-181). Three new assays were developed, optimized and analytically validated for (1) CSF BACE activity, (2) CSF oligomeric A , and (3) CSF Homocysteine (HCY) levels. These biochemical markers were measured in the CSF of pathologically confirmed AD (N 27-30) and clinically evaluated control cases (N 29). Results: Uni variate results exhibited an age-dependent increase in CSF BACE activity ( 1.0 pM/yr, p 0.05). In AD subjects, a modest but significant decline in age-adjusted CSF BACE activity was observed compared to controls (50% reduction, p 0.02). Regarding oligomeric A , in a commercially purchased sample set of human CSF, we found AD patients have 82 % higher oligomeric A levels than their age-matched non-demented controls (p 0.01). When oligomeric A was assessed in the post-mortem confirmed OPTIMA samples, there did not appear to be a significant difference between AD and CTL. One can’t be sure if there is an assay sensitivity issue or the demographics of the sample sets were different. Finally regarding CSF Homocysteine, we observed meaningful age dependence in CSF Homocysteine levels (slope 0.016 per year, p 0.0003). Differences between AD and CTL after age-adjustment are significant in females (11.2% greater in AD v CTL females, p 0.049). Conclusions: Taken together, these findings obtained from a post-mortem confirmed case analysis, enhance the putative value and use of specific CSF markers in support of the detection and diagnosis of Alzheimer’s disease. P3-095 LEVELS OF CSF BIOMARKERS PREDICT RATE OF COGNITIVE DECLINE IN INDIVIDUALS WITH VERY MILD DEMENTIA OF THE ALZHEIMER’S TYPE