Identification of a histone deacetylase inhibitor as a therapeutic candidate for congenital central hypoventilation syndrome

Abstract

Congenital central hypoventilation syndrome (CCHS), a rare genetic disease caused by heterozygous PHOX2B mutations, is characterized by life-threatening breathing deficiencies. PHOX2B is a transcription factor required for the specification of the autonomic nervous system, which contains, in particular, brainstem respiratory centers. In CCHS, PHOX2B mutations lead to cytoplasmic PHOX2B protein aggregations, thus compromising its transcriptional capability. Currently, the only available treatment for CCHS is assisted mechanical ventilation. Therefore, identifying molecules with alleviating effects on CCHS-related breathing impairments is of primary importance. A transcriptomic analysis of cells transfected with different PHOX2B constructs was used to identify compounds of interest with the CMap tool. Using fluorescence microscopy and luciferase assay, the selected molecules were further tested in vitro for their ability to restore the nuclear location and function of PHOX2B. Finally, an electrophysiological approach was used to investigate ex vivo the effects of the most promising molecule on respiratory activities of PHOX2B-mutant mouse isolated brainstem. The histone deacetylase inhibitor SAHA was found to have low toxicity in vitro, to restore the proper location and function of PHOX2B protein, and to improve respiratory rhythm-related parameters ex vivo. Thus, our results identify SAHA as a promising agent to treat CCHS-associated breathing deficiencies.

Keywords: CCHS; MT: Bioinformatics; PHOX2B; aggregates; breathing; pharmacological treatment.

Graphical abstract

Figure 1

Cytoscape ClueGO pairwise analysis Differentially expressed genes (DEGs) were analyzed for their enrichment in specific pathways by using the Cytoscape app ClueGO v.2.5.9. Images show the representation of nodes connecting the terms of enriched pathways (A, C, and E). The corresponding bar diagrams of the detailed gene ontology (GO) terms are ranked according to their p values (B, D, and F) in the following pairwise comparisons: WT vs. +13Ala, +13Ala(17AAG) vs. +13Ala, and +13Ala(17AAG) vs. WT. Functionally grouped networks are shown, with terms as nodes linked based on their kappa score level (≥0.3), where only the label of the most significant term per group is shown. GO levels 1–20 were considered for analysis. Each pathway is represented by a different color ascribed to each GO regulatory action in both the schematics (A, C, and E) and the bar diagrams (B, D, and F). The node dimension represents the enrichment expressed as the ratio between the number of DEGs and the total genes in the corresponding GO term.

Figure 2

Connectivity Map data for compounds with strongest to weakest connections with the DEG in PHOX2B+13Ala cells treated with 17AAG compared to untreated control cells The name, action, and median tau score for each compound are indicated. The cell-line identifier is also shown for each column. The median tau score represents the strength of the connection between a query signature and a drug across multiple cell types. Compounds were considered significantly connected with the input signature when the median tau score was above 90.

Figure 3

Drug effects on WT and PHOX2B+13Ala cellular localization and activity (A) The subcellular localization of WT and PHOX2B+13Ala proteins after pharmacological treatments was analyzed using fluorescence microscopy. Forty thousand COS-7 cells were plated and transfected with plasmids (pcDNA 3.1 CT-GFP PHOX2B WT and pcDNA 3.1 CT-GFP PHOX2B+13Ala). Molecules tested were geldanamycin (GA), SAHA (S), parthenolide (P), trichostatin A (T), guggulsterone (G), CGP-57380 (CGP), and TPCA-1. Fifty COS-7 (PHOX2B-GFP+) transfected cells were counted and classified for each condition in terms of nuclear, cytoplasmic, or nuclear + cytoplasmic PHOX2B localization. Representative images of cells in the three subcellular localization groups are shown on the left, where the blue DAPI is for nuclear staining and the green GFP for PHOX2B protein, and their overlays are also shown. Pictures were taken using a 63× objective microscope. Scale bars, 7 μm. On the right, percentage values are presented in histograms as means ± SEM from three independent experiments. (B) Analysis of luciferase activity on HeLa cells induced by pharmacological treatments and obtained after cell co-transfection of WT or PHOX2B+13Ala with a reporter construct containing the DBH promoter upstream of the luciferase gene. The Renilla luciferase gene was used as an internal control. Raw data were first normalized against the empty vector. Bars represent the increase in luciferase activity under different conditions, normalized to untreated PHOX2B+13Ala. The WT construct (gray bar) shows greater activity on the DBH promoter than the luciferase value obtained in the presence of the expanded polyAla proteins. GA was used as a positive treatment control. Values are represented as means ± SEM of six to eight independent replicate experiments. P at 5 μM, S at 500 nM, and, in particular, S at 10 nM show similar levels of transcriptional activity compared to the WT condition. All the other treatments were less effective, as indicated by statistical comparison with PHOX2B WT (Welch’s t test, ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, and ∗∗∗∗p < 0.0001).

Figure 4

Subcellular localization analysis of WT and PHOX2B+13Ala proteins after SAHA treatment COS-7 cells were transfected with pcDNA3.1-CT/GFP PHOX2B WT and pcDNA3.1-CT/GFP PHOX2B+13Ala plasmids and treated with 10 nM SAHA. ImageStream X Mark II was used to analyze PHOX2B nuclear relocalization after the drug treatment. On the left three types of PHOX2B subcellular localization are shown: (A) nuclear localization, (B) nuclear localization with cytoplasmic retention, and (C) cytoplasmic localization alone. PHOX2B was fused to GFP (green), nuclei were stained with Hoechst 33342 (purple), and their co-localization is shown at right (overlay). Approximately 5,000 PHOX2B-GFP-positive cells were acquired for each condition (i.e., WT, WT + SAHA, +13Ala, and +13Ala + SAHA) in four independent experiments. All images in (A)–(C) were taken at 60× magnification. (D) Bar graph representing the percentage of cells (means ± SD) exhibiting the different PHOX2B protein localizations. A paired t test was performed in each group of PHOX2B subcellular compartment localization (nuclear, cytoplasmic, nuclear + cyto) comparing WT vs. +13Ala, WT vs. +13Ala + SAHA, and +13Ala vs. +13Ala + SAHA (paired t test, ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, and ∗∗∗∗p < 0.0001). Scale bars, 7 μm.

Figure 5

Subcellular localization and activity of different expanded PHOX2B proteins and SAHA-mediated recovery (A) Subcellular localization of PHOX2B proteins in COS-7 cells expressing PHOX2B WT, +7Ala, and +13Ala before and after treatment with 10 nM SAHA. The immunofluorescence assay was performed with an antibody against the PHOX2B protein. Representative images of cells in the three subcellular localization groups (cells with PHOX2B in the nucleus, nucleus and cytoplasm, or cytoplasm alone) are shown on the left, where the blue DAPI is for nuclear staining and the green GFP for PHOX2B protein, and their overlays are also shown. Pictures were taken using a Leica SP5 confocal microscope (63× magnification). Scale bars, 7 μm. The histogram on the right represents the mean ± SEM of three independent experiments and shows the percentage values of cells for the different conditions. The nuclear relocalization of the PHOX2B+13Ala protein induced by drug treatment is statistically significant, but the positive effect of SAHA on the subcellular localization of PHOX2B+7Ala is also evident (p = 0.06). (B) Effect of 10 nM SAHA on the transactivation activity of the DBH promoter by expanded polyalanine PHOX2B. HeLa cells were co-transfected with PHOX2B WT, +7Ala, and +13Ala and the DBH promoter carrying a luciferase gene. A Renilla luciferase genetic construct was used as an internal control. The histogram shows the luciferase activity of the different conditions with or without SAHA treatment, normalized compared to PHOX2B WT. The mutated forms appear to lose the ability to activate the DBH promoter, which can be partially rescued by SAHA treatment. Values are expressed as the mean ± SEM of three independent experiments (paired t test, ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, and ∗∗∗∗p < 0.0001).

Figure 6

Effects of SAHA on fictive respiratory activity in isolated embryonic mouse brainstem preparations (A) Left: schematic representation of a brainstem preparation obtained at E18.5 from WT (Phox2b^+/+, top) and mutant (Phox2b^27Ala/+, bottom) embryos. Such a preparation hosts the main respiratory networks: the embryonic parafacial respiratory group (epF) and the pre-Bötzinger complex (preBötC). Spontaneous respiratory-related motor activity was monitored by an extracellular electrode placed on the phrenic (C4) motor root. Right: integrated phrenic nerve discharge (Int C4) obtained from Phox2b^+/+ (two top traces) and Phox2b^27Ala/+ (two bottom traces) preparations under normal aCSF (pH 7.4, traces above) and after acidification (pH 7.2, traces below). (B) Quantification of the mean fictive breathing burst frequency over time (measured at 2 min intervals) for 16 Phox2b^+/+ (green dots) and 15 Phox2b^27Ala/+ (blue dots) preparations at time 0 (T0) and after 7 h 30 (T7h30) in control aCSF conditions (pH 7.4, light gray) and after acidification (pH 7.2, dark gray). Note that mutant preparations exhibited a lower mean respiratory frequency compared to Phox2b^+/+ preparations and that acidification induced a significant increase in respiratory frequency solely in the Phox2b^+/+ preparations. (C) Same layout as in (B) for preparations (13 Phox2b^+/+ and 7 Phox2b^27Ala/+) exposed to 1 μM SAHA. Note the increased burst frequency in mutant preparations and a partial restoration of the response to acidosis at T7h30. (D) Bar graphs representing the mean respiratory frequency values measured over 4 min at steady state in pH 7.4 and during the maximal effect at pH 7.2 for all preparations in each condition, at T0 and T7h30. Graphs in B, C, and D are frequency mean values ± SEM. Two-way ANOVA, followed by a post hoc Sidak’s comparison test; ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, and ∗∗∗∗p < 0.0001.