Identification of a Chrysanthemic Ester as an Apolipoprotein E Inducer in Astrocytes

Abstract

The apolipoprotein E (APOE) gene is the most highly associated susceptibility locus for late onset Alzheimer’s Disease (AD), and augmenting the beneficial physiological functions of apoE is a proposed therapeutic strategy. In a high throughput phenotypic screen for small molecules that enhance apoE secretion from human CCF-STTG1 astrocytoma cells, we show the chrysanthemic ester 82879 robustly increases expressed apoE up to 9.4-fold and secreted apoE up to 6-fold and is associated with increased total cholesterol in conditioned media. Compound 82879 is unique as structural analogues, including pyrethroid esters, show no effect on apoE expression or secretion. 82879 also stimulates liver x receptor (LXR) target genes including ATP binding cassette A1 (ABCA1), LXRα and inducible degrader of low density lipoprotein receptor (IDOL) at both mRNA and protein levels. In particular, the lipid transporter ABCA1 was increased by up to 10.6-fold upon 82879 treatment. The findings from CCF-STTG1 cells were confirmed in primary human astrocytes from three donors, where increased apoE and ABCA1 was observed along with elevated secretion of high-density lipoprotein (HDL)-like apoE particles. Nuclear receptor transactivation assays revealed modest direct LXR agonism by compound 82879, yet 10 μM of 82879 significantly upregulated apoE mRNA in mouse embryonic fibroblasts (MEFs) depleted of both LXRα and LXRβ, demonstrating that 82879 can also induce apoE expression independent of LXR transactivation. By contrast, deletion of LXRs in MEFs completely blocked mRNA changes in ABCA1 even at 10 μM of 82879, indicating the ability of 82879 to stimulate ABCA1 expression is entirely dependent on LXR transactivation. Taken together, compound 82879 is a novel chrysanthemic ester capable of modulating apoE secretion as well as apoE-associated lipid metabolic pathways in astrocytes, which is structurally and mechanistically distinct from known LXR agonists.

Plasminogen binding inhibitors demonstrate unwanted activities on GABA A and glycine receptors in human iPSC derived neurons

“…This indicate that hit-finding in HTS for APOE enhancer will be dependent on cellular context. Model bias was also demonstrated by Finan et al which used primary fetal astrocytes in the primary screen (269), more commonly used in confirmation/secondary screens (322). The strategy led to the identification of new APOE enhancers not stimulating CCF (269), which otherwise would have been missed if the CCF was used in the primary screening.…”

“…Moreover, for drug induced effects of astrocytic models, several studies have focused on phenotypic readouts of reduced oxidative stress (323), neurotoxicity (23), APOE secretion, (269, 322), and AD (264). A larger number of evaluated compounds is observed in a few studies using hiPSC derived or human primary astrocytes (23,269,322,324).…”

“…Screening for APOE enhancing agents using human astrocytoma cell line CCF-STTG1 and human fetal astrocytes confirm upregulated expression of APOE during treatment with LXR agonists GW3965 and T0901317 (Tularik) (269, 322). However, as these agonists cause unwanted effects such as hypertriglyceridemia (347) Fan et al could identify a LXR transactivating compound with lower SREBP-1c induction but still increased ABCA1 and APOE expression, thereby likely reducing hepatotoxic effects observed with traditional LXR/RXR agonists (322). Further development identified APOE and ABCA1 enhancing P2X7 annotated compounds with low SREBP-1c induction through an indirect LXR mechanism (348).…”

“…Notably, an important aspect is that the pharmaceutical industry due to ethical considerations, is very restricted in the use of fetal derived models. This means that the most logical comparison between NES-Astro and fetal astrocytes, commonly used in the research field (20,269,322), is not included among the evaluated models. However, to identify neural and astrocyte associated characteristics we included ltNES (neural representation) and HEK293 cells (non-neural representation).…”

An in vitro bioengineered model of the human arterial neurovascular unit to study neurodegenerative diseases

“…Secreted apoE levels in both the tissue chamber and circulation media were quantified by an apoE ELISA protocol as described previously 22 . 1 µM of the liver-X-receptor agonist GW3965 or dimethyl sulfoxide (DMSO) vehicle control were circulated through the lumen for 96 h before collecting media.…”