第774回 千葉医学会例会・第二内科例会 64.

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

A novel role for the Notch effector RBPJ in tumorigenesis

The Notch signaling pathway, which converges on RBPJ, is deregulated in a number of malignancies. Following pathway activation, RBPJ, the DNA-binding component of the pathway, associates with Notch to activate transcription of target genes. In the absence of Notch activity, RBPJ acts as a transcriptional repressor by recruiting a co-repressor complex that must be displaced to reinitiate the cycle of activation. As RBPJ is a key regulator of Notch signaling and is constitutively expressed in normal cells, we set out to evaluate the effect of RBPJ loss in the context of human cancer. Frequent RBPJ loss was detected in human breast and lung tumors. Moreover, depletion of RBPJ in a human breast cancer cell line accelerated xenograft tumor growth, whereas over-expression of a mutated version of RBPJ (which allows retained function as a transcriptional repressor but prevents activation via Notch) reduced tumor growth in a mouse model. These findings were confirmed in a lymphoma knock-out cell line, where a complete loss of RBPJ strikingly increased tumor growth in mice. RBPJ-deficient tumor xenografts showed up-regulated expression of HEY family genes, which represent direct canonical RBPJ targets. Blockade of Notch activation had no effect on the magnitude of HEY gene derepression in the absence of RBPJ, indicating that Notch does not participate in deregulated signal activation resulting from loss of transcriptional repression. To identify other aberrantly induced genes that contribute to the oncogenic phenotype with RBPJ loss, we performed a global analysis. RBPJ removal led to enrichment of acetylated histone H4 at induced gene promoters. We therefore used this epigenetic mark as an indirect measure of promoter activity to identify processes that were differentially active in the RBPJ-depleted breast cancer cells compared to RBPJ-containing controls. RBPJ loss enriched for a Notch-like signal and increased acetyl marks at genes associated with cell survival. Indeed, resistance to cell death was observed in RBPJ-deficient breast cancer and lymphoma tumors both in vitro and in vivo. This work defines a new role for RBPJ as a tumor suppressor, the loss of which represents an alternate mechanism for deregulating Notch signaling in cancer.Medicine, Faculty ofMedicine, Department ofExperimental Medicine, Division ofGraduat

Loss of the Notch effector RBPJ promotes tumorigenesis

Aberrant Notch activity is oncogenic in several malignancies, but it is unclear how expression or function of downstream elements in the Notch pathway affects tumor growth. Transcriptional regulation by Notch is dependent on interaction with the DNA-binding transcriptional repressor, RBPJ, and consequent derepression or activation of associated gene promoters. We show here that RBPJ is frequently depleted in human tumors. Depletion of RBPJ in human cancer cell lines xenografted into immunodeficient mice resulted in activation of canonical Notch target genes, and accelerated tumor growth secondary to reduced cell death. Global analysis of activated regions of the genome, as defined by differential acetylation of histone H4 (H4ac), revealed that the cell death pathway was significantly dysregulated in RBPJ-depleted tumors. Analysis of transcription factor binding data identified several transcriptional activators that bind promoters with differential H4ac in RBPJ-depleted cells. Functional studies demonstrated that NF-κB and MYC were essential for survival of RBPJ-depleted cells. Thus, loss of RBPJ derepresses target gene promoters, allowing Notch-independent activation by alternate transcription factors that promote tumorigenesis

High-density lipoproteins suppress Aβ-induced PBMC adhesion to human endothelial cells in bioengineered vessels and in monoculture

Background: Alzheimer’s Disease (AD), characterized by accumulation of beta-amyloid (Aβ) plaques in the brain, can be caused by age-related failures to clear Aβ from the brain through pathways that involve the cerebrovasculature. Vascular risk factors are known to increase AD risk, but less is known about potential protective factors. We hypothesize that high-density lipoproteins (HDL) may protect against AD, as HDL have vasoprotective properties that are well described for peripheral vessels. Epidemiological studies suggest that HDL is associated with reduced AD risk, and animal model studies support a beneficial role for HDL in selectively reducing cerebrovascular amyloid deposition and neuroinflammation. However, the mechanism by which HDL may protect the cerebrovascular endothelium in the context of AD is not understood. Methods: We used peripheral blood mononuclear cell adhesion assays in both a highly novel three dimensional (3D) biomimetic model of the human vasculature composed of primary human endothelial cells (EC) and smooth muscle cells cultured under flow conditions, as well as in monolayer cultures of ECs, to study how HDL protects ECs from the detrimental effects of Aβ. Results: Following Aβ addition to the abluminal (brain) side of the vessel, we demonstrate that HDL circulated within the lumen attenuates monocyte adhesion to ECs in this biofidelic vascular model. The mechanism by which HDL suppresses Aβ-mediated monocyte adhesion to ECs was investigated using monotypic EC cultures. We show that HDL reduces Aβ-induced PBMC adhesion to ECs independent of nitric oxide (NO) production, miR-233 and changes in adhesion molecule expression. Rather, HDL acts through scavenger receptor (SR)-BI to block Aβ uptake into ECs and, in cell-free assays, can maintain Aβ in a soluble state. We confirm the role of SR-BI in our bioengineered human vessel. Conclusion: Our results define a novel activity of HDL that suppresses Aβ-mediated monocyte adhesion to the cerebrovascular endothelium.Medicine, Faculty ofScience, Faculty ofOther UBCMedicine, Department ofNeurology, Division ofPathology and Laboratory Medicine, Department ofZoology, Department ofReviewedFacult

Axl receptor tyrosine kinase is a regulator of apolipoprotein E

Alzheimer’s disease (AD), the leading cause of dementia, is a chronic neurodegenerative disease. Apolipoprotein E (apoE), which carries lipids in the brain in the form of lipoproteins, plays an undisputed role in AD pathophysiology. A high-throughput phenotypic screen was conducted using a CCF-STTG1 human astrocytoma cell line to identify small molecules that could upregulate apoE secretion. AZ7235, a previously discovered Axl kinase inhibitor, was identified to have robust apoE activity in brain microglia, astrocytes and pericytes. AZ7235 also increased expression of ATP-binding cassette protein A1 (ABCA1), which is involved in the lipidation and secretion of apoE. Moreover, AZ7235 did not exhibit Liver-X-Receptor (LXR) activity and stimulated apoE and ABCA1 expression in the absence of LXR. Target validation studies using AXL−/− CCF-STTG1 cells showed that Axl is required to mediate AZ7235 upregulation of apoE and ABCA1. Intriguingly, apoE expression and secretion was significantly attenuated in AXL-deficient CCF-STTG1 cells and reconstitution of Axl or kinase-dead Axl significantly restored apoE baseline levels, demonstrating that Axl also plays a role in maintaining apoE homeostasis in astrocytes independent of its kinase activity. Lastly, these effects may require human apoE regulatory sequences, as AZ7235 exhibited little stimulatory activity toward apoE and ABCA1 in primary murine glia derived from neonatal human APOE3 targeted-replacement mice. Collectively, we identified a small molecule that exhibits robust apoE and ABCA1 activity independent of the LXR pathway in human cells and elucidated a novel relationship between Axl and apoE homeostasis in human astrocytes.Medicine, Faculty ofOther UBCNon UBCPathology and Laboratory Medicine, Department ofReviewedFacult

Compound 82879 upregulates apoE and ABCA1 mRNA in primary human astrocytes.

<p>Primary human astrocytes from three donors genotyped for <i>APOE</i> (D1: <i>E3/E4</i>, D2: <i>E3/E4</i>, D3: <i>E3/E3</i>) were treated with DMSO alone, GW3965, or compound 82879 for 96 h in two independent experiments. (<i>A-C</i>) ApoE mRNA and (<i>D-F</i>) ABCA1 mRNA levels were measured. Graphs represent mean and 95% CI of fold change relative to DMSO treatment (which corresponds to 1 on the Y-axis, dashed line). Statistics were determined by a one-way ANOVA with a blocking factor (Experiment) and a Dunnett’s post-test (* p<0.05, ** p<0.01, *** p< 0.001).</p

82879 induces LXRα-dependent upregulation of ABCA1 mRNA in MEFs whereas apoE expression is stimulated through LXR-dependent and LXR-independent activities.

<p>LXRα-/β-, and LXRα expressing (LXRα+/β-) MEF cells were treated for 24 h withDMSO alone, GW3965, or compound 82879 at the indicated concentrations. Differential mRNA expression levels of (<i>A</i>) apoE and (<i>B</i>) ABCA1 were measured. Data are expressed as fold-change relative to the respective control group (dashed line) within each genotype. The responses to drug treatment compared to the DMSO control within each genotype (***p <0.001) were compared using a Linear Mixed Model with a Sidak's multiple-comparison test on ΔCT values obtained by qRT-PCR. The magnitudes of drug effect between two genotypes were compared using one-way ANOVA with a blocking factor (Experiment) on ΔΔCT values (# p<0.05, ## p<0.01, ### p<0.001). Data are expressed as mean and 95% CI from at least 6 independent experiments for GW3965 treatment and 3 experiments for 82879 treatments.</p