Bexarotene-Activated Retinoid X Receptors Regulate Neuronal Differentiation and Dendritic Complexity

Abstract

Bexarotene-activated retinoid X receptors (RXRs) ameliorate memory deficits in Alzheimer's disease mouse models, including mice expressing human apolipoprotein E (APOE) isoforms. The goal of this study was to gain further insight into molecular mechanisms whereby ligand-activated RXR can affect or restore cognitive functions. We used an unbiased approach to discover genome-wide changes in RXR cistrome (ChIP-Seq) and gene expression profile (RNA-Seq) in response to bexarotene in the cortex of APOE4 mice. Functional categories enriched in both datasets revealed that bexarotene-liganded RXR affected signaling pathways associated with neurogenesis and neuron projection development. To further validate the significance of RXR for these functions, we used mouse embryonic stem (ES) cells, primary neurons, and APOE3 and APOE4 mice treated with bexarotene. In vitro data from ES cells confirmed that bexarotene-activated RXR affected neuronal development at different levels, including proliferation of neural progenitors and neuronal differentiation, and stimulated neurite outgrowth. This effect was validated in vivo by demonstrating an increased number of neuronal progenitors after bexarotene treatment in the dentate gyrus of APOE3 and APOE4 mice. In primary neurons, bexarotene enhanced the dendritic complexity characterized by increased branching, intersections, and bifurcations. This effect was confirmed by in vivo studies demonstrating that bexarotene significantly improved the compromised dendritic structure in the hippocampus of APOE4 mice. We conclude that bexarotene-activated RXRs promote genetic programs involved in the neurogenesis and development of neuronal projections and these results have significance for the improvement of cognitive deficits.

Significance statement: Bexarotene-activated retinoid X receptors (RXRs) ameliorate memory deficits in Alzheimer's disease mouse models, including mice expressing human apolipoprotein E (APOE) isoforms. The goal of this study was to gain further insight into molecular mechanisms whereby ligand-activated RXR can affect or restore cognitive functions. We used an unbiased approach to discover genome-wide changes in RXR cistrome (ChIP-Seq) and gene expression profile (RNA-Seq) in response to bexarotene in the cortex of APOE4 mice. Functional categories enriched in both datasets revealed that liganded RXR affected signaling pathways associated with neurogenesis and neuron projection development. The significance of RXR for these functions was validated in mouse embryonic stem cells, primary neurons, and APOE3 and APOE4 mice treated with bexarotene.

Keywords: APOE4 and APOE3; ChIP-Seq/RNA-Seq; adult neurogenesis; bexarotene; neuronal differentiation; retinoid X receptor.

Figure 1.

Outline of the protocol for mouse ES cell treatment and differentiation.

Figure 2.

ChIP-Seq data revealing that bexarotene-activated RXR binds genes associated with neuronal differentiation in brain of APOE4 mice. Six-month-old APOE4 mice were treated with bexarotene for 10 d and ChIP-Seq was performed on DNA immunoprecipitated with an antibody against RXR. A, Venn diagram showing the number of overlapping peaks in bexarotene- and vehicle-treated APOE4 mice. B, Binding sites in a genome browser identified in both bexarotene- and vehicle-treated APOE4 mice. The corresponding ChIP-qPCR validation data of these targets performed on cortices from WT mice treated 24 h with bexarotene are shown on the right. C, Most significant functional annotation categories (GOTERM Biological Process) of direct RXR target genes. In red are categories identical in ChIP-Seq and RNA-Seq datasets. D, Genome browser view of RXR binding to sites in the proximal promoters of or nearby genes related to neuronal differentiation (BP category “GO:0030182 neuron differentiation”) and unique for bexarotene-treated APOE4 mice. E, RXR-binding sites of the indicated individual genes associated with neuronal differentiation were confirmed by ChIP-qPCR in P19 cells treated with 100 nm bexarotene for 6 h. Values are mean ± SEM. For B and E, n = 3–4. **p < 0.01; *p < 0.05. Blue color denotes vehicle samples and red bexarotene in all panels.

Figure 3.

RNA-Seq results demonstrating that bexarotene significantly affects genes related to neuronal differentiation and neuron projection. Six-month-old APOE4 mice treated with bexarotene or vehicle (n = 3, males) were used for RNA-Seq. Differential gene expression analysis using edgeR identified 551 upregulated and 227 downregulated genes at FDR < 0.01. A, Statistically significant functional annotation categories (GOTERM Biological Process) were determined by DAVID using a custom gene list generated at FDR < 0.01. In red are depicted GOTERM categories identical in ChIP-Seq and RNA-Seq datasets. n = 3 mice per group. B, RNA expression level of significantly changed genes identified by RNA-Seq in categories “neuronal differentiation” and “neuron projection development.” Plotted are log2-normalized reads per million (RPM) of genes with a fold change of at least 2-fold. Abca1 is shown to confirm a target engagement. Statistic is by edgeR, FDR < 0.01, n = 3. C, Validation of RNA-Seq results by qPCR using total RNA obtained from cortices of APOE4 mice. n = 5 male and 4 female mice per group. D, Validation of the RNA-Seq results by qPCR using ES cells. ES cells at the EB stage (Fig. 1) were treated with 5 μm bexarotene and vehicle for 24 h. For C and D, statistics is by t test; n = 4, *p < 0.05 **p < 0.001 and ***p < 0.0001.

Figure 4.

Comparative pathway analysis of ChIP-Seq and RNA-Seq data. Shown are IPA analyses of RXR ChIP-Seq (A) and RNA-Seq (B) data identifying a network of genes associated with differentiation of neurons, neurite growth, and neuritogenesis regulated by bexarotene in the cortex of APOE4 mice. For ChIP-Seq, genomic sites uniquely identified only in bexarotene-treated mice were submitted, and for RNA-seq, only differentially expressed genes at an FDR of ≤0.01 were submitted. RXR binding to target genes identified by ChIP-Seq is represented by thick lines and genes identified only by RNA-Seq by dotted lines. Red color depicts genes overlapping in both sets; pink indicates upregulated genes and green downregulated genes; the genes that did not meet the FDR = 0.01 cutoff in the RNA-Seq data are colorless. The picture was generated using the IPA web tool.

Figure 5.

Bexarotene facilitates the commitment of ES cells to neuronal lineage and increases neuronal differentiation. ES cells were treated with bexarotene (0.1–1 μm) as shown in Figure 1. A, Bexarotene increases the expression of neuronal precursor markers. ES cells were treated for 4 d with increasing concentrations of bexarotene and the expression of the specific neuronal progenitor markers Nes, Pax6, and Fabp7 was examined by qPCR. Transcript levels were normalized to Gapdh. Analysis is by one-way ANOVA (p < 0.0001 for each gene). Results for Tukey's posttest are shown on the graph: **p < 0.001 and ***p < 0.0001 versus vehicle. B–D, Bexarotene increases the expression level of markers for neuronal differentiation. B, C, mRNA expression was examined on days 4 and 10. Markers for differentiation were Tubb3, Syp, vGlut1, and APP. Transcript levels were normalized to Gapdh. The level of expression of each marker is presented as fold of day 4. One-way ANOVA p < 0.001 for each time point except for APP at day 4. Results for Tukey's posttest are shown on the graph. *p < 0.05, **p < 0.001, and ***p < 0.0001 versus vehicle. D, Bexarotene increases SYP protein level. ES cells were treated with 0.5 or 5 μm bexarotene and SYP protein expression was determined at day 10 by Western blotting. The image is a representative of three samples per condition. The graph represents the SYP protein level as fold of vehicle control.

Figure 6.

Bexarotene increases neuronal differentiation in a dose-dependent manner in ES cells. ES cell were treated with 1 or 5 μm bexarotene for 4 d and neuronal differentiation was examined 6 d after plating (day 10). Control cells were treated with vehicle and processed in the same way. A, Cells were immunostained with an antibodies against DCX (Ac, Af, Ai, red) and MAP2 (Ab, Ae, Ah, green). The nuclei were stained with H33342 (in blue). Aa, Ad, and Ag represent merged images of DCX, MAP2, and staining for nuclei, respectively. Scale bar, 100 μm. B, Image of a growth cone. Scale bar, 25 μm. C, Graphs showing the percentage of neurons in each condition calculated as described in the Materials and Methods. Statistical analyses were by one-way ANOVA (p < 0.0001 for MAP2 and DCX) Tukey's posttest values are shown on the graph. The percentage of neurons in vehicle ((DCX, MAP2: 1.8%); 1 μm bexarotene (DCX: 22.9%, MAP2: 22.1%), and 5 μm bexarotene (DCX: 35.7%, MAP2: 35.8%) are shown.

Figure 7.

Bexarotene increases the number of BrdU+ cells in the DG of APOE3 and APOE4 mice. Two-month-old APOE3 and APOE4 mice were treated with bexarotene or vehicle for 7 d and injected with BrdU on the last day of the treatment. The number of BrdU+ cells was counted 1 week after BrdU injection. A, Representative images from APOE3 and APOE4 mice treated with bexarotene or vehicle. BrdU+ cells are stained in green. Images were taken using the EDF module in NIS elements (Nikon). Scale bars, 50 μm. B, Analysis by two-way ANOVA with bexarotene treatment and APOE genotype as variables. There was no interaction between treatment and genotype (F_(1,24) = 0.33; p = 0.57), a significant main effect of bexarotene treatment (F_(1.24) = 5.95, p = 0.022), and no main effect of APOE genotype (F_(1,24) = 0.6; p = 0.42). APOE3, n = 6–7 male and female mice per group; APOE4, n = 7–8 male and female mice per group.

Figure 8.

Bexarotene enhances neurite branching and affects dendrite complexity in primary neurons. Primary rat neurons were infected with GFP expressing lentivirus at DIV0 and treated with bexarotene or vehicle at DIV4 for 24 h. Sholl analysis was performed 6 d later at DIV11. A, B, Photomicrographs showing the morphology of DIV11 GFP+ neurons treated either with vehicle (A) or 1 μm bexarotene (B). C, D, Digital reconstructions of the cells in A and B are shown in C and D, respectively. E, Bexarotene-treated neurons exhibit distinct Sholl profiles with an increased number of branch intersections far from the soma. F, Clearly visible trend toward an increased total neurite length, which did not reach statistical significance. G, H, Consistent with the differing Sholl profile, both the number of bifurcations (G) and the number of branches (H) were increased in the neurons treated with bexarotene. For each treatment group, images were taken from at least nine wells and more than two GFP+ neurons per well.

Figure 9.

Bexarotene restores dendritic morphology of the CA1 region of the hippocampus of APOE4 mice. A, Representative images of MAP2 staining and dendritic tree reconstruction in the hippocampal CA1 region using Imaris filament tracing software (60× confocal imaging). A total of four images are shown for each of the four sections for each mouse. n = 3 male and female mice per group. The total dendritic length (B), branch points (C), and segments (D) were quantified using Imaris filament tracing macros and normalized to the total H33342+ nuclei of the CA1 region. Note that APOE4 vehicle-treated mice display significantly diminished dendritic length, branch points, and segments compared with APOE3 vehicle-treated mice and this characteristic was reversed by bexarotene treatment. Analysis by one-way ANOVA with Tukey's posttest, p < 0.05.