Class I HDAC inhibition is a novel pathway for regulating astrocytic apoE secretion

Abstract

Despite the important role of apolipoprotein E (apoE) secretion from astrocytes in brain lipid metabolism and the strong association of apoE4, one of the human apoE isoforms, with sporadic and late onset forms of Alzheimer's disease (AD) little is known about the regulation of astrocytic apoE. Utilizing annotated chemical libraries and a phenotypic screening strategy that measured apoE secretion from a human astrocytoma cell line, inhibition of pan class I histone deacetylases (HDACs) was identified as a mechanism to increase apoE secretion. Knocking down select HDAC family members alone or in combination revealed that inhibition of the class I HDAC family was responsible for enhancing apoE secretion. Knocking down LXRα and LXRβ genes revealed that the increase in astrocytic apoE in response to HDAC inhibition occurred via an LXR-independent pathway. Collectively, these data suggest that pan class I HDAC inhibition is a novel pathway for regulating astrocytic apoE secretion.

Fig 1. Pan class I HDAC inhibition increases astrocytic apoE levels.

(A) The number of targets per class and (B) compounds per target class in Pfizer’s annotated chemogenomics library. (C) Number of library compounds resulting in apoE secretion from the CCF-STTG1 human astrocytoma cells following 10 μM treatment for 48 hours. ApoE levels were normalized to those that were obtained following exposure to 2 μM of TO901317. (D) Concentration-dependent increase in apoE secretion from CCF-STTG1 cells following treatment with HDAC inhibitors: MS275 (o), CI994 (●), SAHA (□) and BML-281(■) and TP38 (▲). Data are normalized to vehicle treated cells. (E) Concentration-dependent increase in apoE mRNA levels in CCF-STTG1 cells following exposure to MS275 and CI994. (F) Concentration-dependent increase in ABCA1 mRNA levels following exposure to MS275 and CI994. (See S3 Table for gene expression assays) (G) Concentration-dependent increase in apoE protein secretion from CCF-STTG1 cells following exposure to MS275 and CI994. (H) Concentration-dependent increase in apoE mRNA levels in primary human astrocytes following exposure to MS275 and CI994. (I) Concentration-dependent increase in apoE protein secretion in primary human astrocytes, expressing apoE3/E3 genotype (lot number 18709), following exposure to MS275 and CI994. Data were normalized to vehicle treated cells. Compounds for Fig 1A-D were synthesized at Pfizer. All data are represented as values ± SEM with n = 3, * p<0.05, ** p<0.01, *** p<0.001, **** p<0.0001.

Fig 2. Pan class I HDAC knockdown increases apoE secretion by astrocytes.

Relative mRNA expression of the zinc-dependent HDACs normalized to relative HDAC11 expression in total RNA isolated from CCF-STTG1 cells (A) and primary human astrocytes (B) (n = 4 per box). Effect of knocking down each of the four classes of zinc-dependent HDACs separately on relative apoE mRNA (C), relative ABCA1 mRNA (D) and secreted apoE protein levels (E) in CCF-STTG1 cells (n = 3 per condition). Relative apoE and ABCA1 mRNA levels obtained after knocking down HDACs were normalized to vehicle treated cells. (F) Effect of knocking down pan class I HDAC and each of the four class I HDAC members on relative apoE mRNA levels in CCF-STTG1 cells. Relative apoE mRNA levels obtained after knocking down HDACs were normalized to lipid only transfected cells. S3 and S4 Figs show siRNA knockdown selectivity and specificity for each member of the HDACs (see S2 and S3 Tables for siRNA oligonucleotides and gene expression assays, respectively). All data are represented as values ±SEM with n = 3; * p<0.05, **p<0.01, **** p<0.0001.

Fig 3. A pan class I HDAC inhibitor increases astrocytic apoE expression and secretion differently from a prototypic pan LXR agonist.

Concentration-dependent effect of MS275 (o) and TO901317 (●), on secreted apoE protein (A) and relative levels of apoE mRNA (B), ABCA1 mRNA (C), LXRα mRNA (D) and LXRβ mRNA (E) in CCF-STTG1 cells. The amount of apoE protein secreted into the media and the relative mRNA levels of apoE, ABCA1, LXRα and LXRβ after compound treatment were normalized to those obtained from vehicle treated controls. The average of GAPDH and 18S mRNA were used as endogenous controls for mRNA normalization. See S3 Table for gene expression assays. TO901317 and MS275 were synthesized at Pfizer and their binding to LXRα (F) and LXRβ (G) was determined using the Lanthascreen assay (Life Technologies). All data are represented as values ±SEM.

Fig 4. A pan class I HDAC inhibitor works synergistically with a pan LXR agonist on the expression of apoE but not ABCA1 or LXR.

Treatment of TO901317 (●), MS275 (▲) or co-treatment of the compounds (TO901317 + 100 nM MS275 (o) or MS275 + 20 nM TO901317 (Δ)) on the levels of secreted apoE protein (A), and the levels of apoE mRNA (B), ABCA1 mRNA (C), and LXRα mRNA (D) in CCF-STTG1 cells. For co-treatment, the amount of apoE protein and apoE, ABCA1, and LXRα mRNA levels were normalized to vehicle treated controls (Veh1) and the effect of the constant compound (100 nM MS275 or 20 nM TO901317, denoted Veh2) was subtracted out after normalization. The average of GAPDH and 18S mRNA were used as endogenous controls for normalization of mRNA levels. See S3 Table for gene expression assays. All data are represented as values ±SEM.

Fig 5. Pan class I HDAC inhibition increases astrocytic apoE secretion independent of LXR activation.

Specific siRNA oligonucleotides were used to knock down LXRα (αKD) and LXRβ (βKD) either separately or in combination (αβKD) in CCF-STTG1 cells (see S2 Table for siRNA oligonucleotides). Concentration-dependent effect of exposure to TO901317 on apoE protein secreted into the media (A); and on apoE mRNA levels (B) upon knocking down LXRα and LXRβ separately or in combination. Concentration-dependent effect of exposure to MS275 on relative apoE protein secreted into the media (C); and on apoE mRNA levels (D) upon knocking down LXRα and LXRβ separately or in combination. Concentration-dependent effect of exposure to TO901317 (E) and MS275 (F) on relative ABCA1 mRNA level upon knocking down LXRα and LXRβ separately and in combination. (G) Concentration-dependent response of apoE protein secreted from primary human astrocytes (lot number 06589) after exposure to MS275 upon knocking down LXRα and LXRβ separately or in combination. For all, levels were normalized to the respective vehicle treated control. See S6 Fig for knock-down efficiencies of LXRα and LXRβ genes and S3 Table for gene expression assays. All data are represented as values ±SEM.