Xiao-Ping Shi

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.

LRRTM3 promotes processing of amyloid-precursor protein by BACE1 and is a positional candidate gene for late-onset Alzheimer's disease

Rare familial forms of Alzheimers disease (AD) are thought to be caused by elevated proteolytic production of the Aβ42 peptide from the β-amyloid-precursor protein (APP). Although the pathogenesis of the more common late-onset AD (LOAD) is not understood, BACE1, the protease that cleaves APP to generate the N terminus of Aβ42, is more active in patients with LOAD, suggesting that increased amyloid production processing might also contribute to the sporadic disease. Using high-throughput siRNA screening technology, we assessed 15,200 genes for their role in Aβ42 secretion and identified leucine-rich repeat transmembrane 3 (LRRTM3) as a neuronal gene that promotes APP processing by BACE1. siRNAs targeting LRRTM3 inhibit the secretion of Aβ40, Aβ42, and sAPPβ, the N-terminal APP fragment produced by BACE1 cleavage, from cultured cells and primary neurons by up to 60%, whereas overexpression increases Aβ secretion. LRRTM3 is expressed nearly exclusively in the nervous system, including regions affected during AD, such as the dentate gyrus. Furthermore, LRRTM3 maps to a region of chromosome 10 linked to both LOAD and elevated plasma Aβ42, and is structurally similar to a family of neuronal receptors that includes the NOGO receptor, an inhibitor of neuronal regeneration and APP processing. Thus, LRRTM3 is a functional and positional candidate gene for AD, and, given its receptor-like structure and restricted expression, a potential therapeutic target.

Human erythroleukemic (HEL) cells express a platelet P2T-like ADP receptor.

Treatment of Fura-2 loaded HEL cells, a human megakaryocyte-like cell line, with P2-purinoceptor nucleotide ligands (ADP, ATP, UTP, 2-methylthio-ATP) evoked a rise in cytosolic calcium. Homologous- and cross-desensitization studies using sequential addition of nucleotides showed that the ADP-induced calcium response in HEL cells was mediated mainly by purinoceptor(s) for which ADP but not ATP was an agonist. There were also minor contributions from purinoceptors for which ATP and ADP are both agonists (probably P2U and P2Y). ATP inhibited the ADP-induced calcium response in HEL cells. This inhibition was overcome by raising the ADP concentration, thus indicating that ATP was an antagonist for the HEL cell ADP receptor. AMP was also an antagonist, albeit weak, for the HEL cell ADP receptor. Antagonism of the ADP-induced calcium response by ATP was similarly observed in MEG-01 cells, another human megakaryocyte-like cell line, but not in 293 cells, a nonhematopoietic cell line. These studies suggest that HEL cells express an ADP receptor for which ATP and AMP are antagonists. This characteristic of the HEL cell ADP receptor is also displayed by the platelet P2T receptor. Thus, HEL cells appear to be a surrogate source of the platelet ADP receptor.

Aβpp secretases are co-expressed with Aβpp in the pancreatic islets

Amyloid beta protein precursor is cleaved by beta- and gamma-secretases to produce Abeta peptides which deposit in amyloid plaques in Alzheimers disease (AD) brain. A recently identified beta-site cleaving enzyme (BACE) appears to fulfill the requirements for beta-secretase, and presenilin-1 (PS1) appears to constitute the catalytic component of gamma-secretase. Each protein has a close homologue (BACE2 and PS2, respectively), whose roles in AbetaPP cleavage remain uncertain. All four of these genes have been reported to be expressed in the human pancreas, but the cell types expressing these genes remains unknown. We demonstrate here the cell-specific expression of AbetaPP, BACE, BACE2, PS1, and PS2 in the human pancreas. The insulin-producing betacells were found to express AbetaPP, BACE and PS2 at high levels, and PS1 at a lower level. The other islet cell types expressed none of these five genes. By contrast, the exocrine ductal cells of the pancreas expressed AbetaPP and BACE2 selectively. These results suggest that secretase inhibitors under development for the treatment of AD, particularly those that target BACE, may have potential for adverse effects on pancreatic beta cell function, and therefore glycemic control.

Activity profile-based siRNA screen to explore the functional genomics of Alzheimer's disease

Multiparametric assays generate biological activity profiles that provide valuable insight into complex disease models. The use of multiple assay measurements in RNA interference (RNAi) high-throughput screening (HTS) provides biological signatures produced by knocking down individual genes. This strategy has been applied to a genome-wide high-throughput small interfering RNA (siRNA) screen measuring proteolysis of β-amyloid precursor protein (APP) into amyloid β peptides, a critical step in the pathogenesis of Alzheimer’s disease. The assay measures amounts of secreted Aβ40, Aβ42, sAPPα, and sAPPβ from HEK 293 cells stably expressing an optimized APP construct following siRNA transfection. The effect of each siRNA on the four different APP products was simultaneously measured in order to identify human genes that regulate the amyloidogenic processing of APP. Genes with BACE-like and γ-secretase-like activity profiles were identified for further biological characterization.