Guo X et al. (2015), Subcellular Localization of Class I Histone Dea...

XB-ART-51792

Front Cell Neurosci 2015 Sep 23;9:510. doi: 10.3389/fncel.2015.00510.

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Subcellular Localization of Class I Histone Deacetylases in the Developing Xenopus tectum.

Guo X , Ruan H , Li X , Qin L , Tao Y , Qi X , Gao J , Gan L , Duan S , Shen W .

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Histone deacetylases (HDACs) are thought to localize in the nucleus to regulate gene transcription and play pivotal roles in neurogenesis, apoptosis, and plasticity. However, the subcellular distribution of class I HDACs in the developing brain remains unclear. Here, we show that HDAC1 and HDAC2 are located in both the mitochondria and the nucleus in the Xenopus laevis stage 34 tectum and are mainly restricted to the nucleus following further brain development. HDAC3 is widely present in the mitochondria, nucleus, and cytoplasm during early tectal development and is mainly distributed in the nucleus in stage 45 tectum. In contrast, HDAC8 is broadly located in the mitochondria, nucleus, and cytoplasm during tectal development. These data demonstrate that HDAC1, HDAC2, and HDAC3 are transiently localized in the mitochondria and that the subcellular distribution of class I HDACs in the Xenopus tectum is heterogeneous. Furthermore, we observed that spherical mitochondria accumulate in the cytoplasm at earlier stages, whereas elongated mitochondria are evenly distributed in the tectum at later stages. The activity of histone acetylation (H4K12) remains low in mitochondria during tectal development. Pharmacological blockades of HDACs using a broad spectrum HDAC inhibitor of Trichostatin A (TSA) or specific class I HDAC inhibitors of MS-275 and MGCD0103 decrease the number of mitochondria in the tectum at stage 34. These findings highlight a link between the subcellular distribution of class I HDACs and mitochondrial dynamics in the developing optic tectum of Xenopus laevis.

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Species referenced: Xenopus laevis
Genes referenced: hdac1 hdac2 hdac3 hdac8 tecta.2

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	Figure 1. HDAC1 distribution in tectal cells in the developing Xenopus. (A) A cartoon showing the optic tectum at stage 45. The tectum is outlined in red. The brain was fixed and cryosectioned for immunostaining. NP, neuropil; OT, optic tectum; L, lateral. (B) The tectum was sectioned and stained with anti-HDAC1 antibody (green) at stage 34 (Ba–Bc). Scale bar: 50 μm. A higher-magnification image of the tectum is shown below. Scale bar, high magnification: 5 μm. HDAC1 was clearly localized in the organelle showing weak DAPI staining (Bd–Bf, red circles), whereas little HDAC1 was distributed in the DAPI-positive cell nuclei (arrowhead). The subcellular localization of HDAC1 in the organelle was almost absent from the tectum at stage 45 (Bg-Bl). Arrowheads indicate cell nuclei counterstained with DAPI (Bg-Bl).
	Figure 2. Subcellular distribution of HDAC1 in the developing Xenopus tectum. (A) Representative immunofluorescent images showing merged HDAC1 (green)/COX IV (red)/DAPI (blue) staining in cells of the developing tectum at stages 34 (Aa), 37 (Ab), 40 (Ac), and 45 (Ad). Scale bar: 100 μm. High-magnification images (Ae–At, scale bar: 5 μm) are demarked by white lines and connected to the original figures (Aa–Ad). Arrowheads indicate cell nuclei stained with DAPI, and arrows indicate mitochondria stained for COX IV. HDAC1 distribution is represented by anti-HDAC1 staining. (B) Summary showing that area of mitochondria stained by anti-COX IV antibody was decreased in tadpoles at stages 37, 40, and 45, during the development of the tectum, compared to stage 34 tadpoles. (C) Quantification showing that the number of mitochondria in the tectum was significantly increased at stage 45 compared to stages 34, 37, and 40. N = 3, 3, 3, 3 for stage 34, 37, 40, and 45. p < 0.05, *p < 0.01.
	Figure 3. Co-localization of HDAC1 and MitoTracker in developing tectum. (A) Representative co-staining images showing the anti-HDAC1 antibody (red) and MitoTracker (MTracker, green) in stage 34 (Aa), 37 (Ab), 40 (Ac), and 45 (Ad) tecta. Scale bar: 100 μm. High-magnification images (Ae–At, scale bar: 5 μm) are demarked by white lines from the original figures (Aa-Ad). Arrowheads indicate cell nuclei stained with DAPI, whereas arrows indicate mitochondria stained with MitoTracker. Scale bar: 5 μm.
	Figure 4. Mitochondrial morphology changes during the development of tectal cells. (A) Tecta at stages 34 (left) and 45 (right) were fixed for electron microscopy. White circle indicate aggregated mitochondria. Arrows indicate elongated mitochondria, whereas arrowheads indicate round/spherical mitochondria. Scale bar: 0.5 μm. M, mitochondria; N, Nucleus. (B) Quantification showing that the area of mitochondria in tecta was significantly decreased at stage 45 compared to stage 34 (Stage 34: 1.82 ± 0.20 μm2, Stage 45: 0.75 ± 0.15 μm2; N = 161, 224). (C) The ratio of mitochondrial length to width was dramatically increased at stage 45 compared to stage 34 (Stage 34: 1.30 ± 0.03, Stage 45: 1.65 ± 0.04; N = 161, 224). ***p < 0.001.
	Figure 5. Subcellular distribution of HDAC2 in developing tectum. (A) Immunostaining for HDAC2 (green)/COX IV (red)/DAPI (blue) in the developing tectum at stages 34 (Aa), 37 (Ab), 40 (Ac), and 45 (Ad). Scale bar: 100 μm. High-magnification images (Ae–At, Scale: 5 μm) are demarked by white lines and are connected to the original figures (Aa-Ad). Arrowheads indicate cell nuclei stained with DAPI, whereas arrows indicate mitochondria stained for COX IV. (B) Tectal cells were stained for HDAC2 (red) with an anti-HDAC2 antibody and counterstained with MitoTracker green at stages 34 (Ba), 37 (Bb), 40 (Bc), and 45 (Bd). Scale bar: 100 μm. High-magnification images (Be–Bt, scale bar: 5 μm) are demarked by white lines and are connected to the original figures (Ba–Bd). Arrowheads indicate cell nuclei stained with DAPI, whereas arrows indicate mitochondria stained with MitoTracker.
	Figure 6. Subcellular distribution of HDAC3 in the developing tectum. (A) Representative immunofluorescent images showing merged HDAC3 (green)/COX IV (red)/DAPI (blue) staining in cells of the developing tectum at stages 34 (Aa), 37 (Ab), 40 (Ac), and 45 (Ad). Scale bar: 100 μm. High-magnification images (Ae–At, scale bar: 10 μm) are demarked by white lines and are connected to the original figures (Aa-Ad). (B) Tecta at stages 34 (Ba), 37 (Bb), 40 (Bc), and 45 (Bd) were stained for HDAC3 (red) with an anti-HDAC3 antibody and counterstained with MitoTracker green. Scale bar: 100 μm. High-magnification images (Be–Bt, scale bar: 5 μm) are demarked by white lines. Arrowheads indicate cell nuclei counterstained with DAPI, whereas arrows indicate mitochondria stained with MitoTracker.
	Figure 7. Subcellular distribution of HDAC8 in the developing tectum. (A) Sections of tectum were immunostained for HDAC8 (green), COX IV (red), and DAPI (blue) at stages 34 (Aa), 37 (Ab), 40 (Ac), and 45 (Ad). Scale bar: 50 μm. High-magnification images (Ae–At, scale bar: 5 μm) are demarked by white lines. Arrowheads indicate DAPI-stained cell nuclei, and arrows indicate COX IV-positive mitochondria.
	Figure 8. Activity of acetylation in the mitochondria. (A) Tecta at stages 37 (Aa–Ah) and 45 (Ai–Ap) were stained with antibodies against Histone H4 (acetyl K12, H4K12Ac) (green) and the mitochondrial marker COX IV (red). Scale bar: 100 μm. High-magnification images (Ae–Ah, Am–Ap, Scale bar: 10 μm) are demarked by white lines. Arrowheads indicate cell nuclei, and arrows indicate mitochondria.
	Figure 9. Mitochondrial dynamics are regulated by HDAC inhibitors. (A) Representative images showing changes in mitochondrial dynamics at stage 37 tadpoles. Tecta in the control (Aa), TSA 25 nM (Ab), and TSA 50 nM (Ac) treatment groups were stained for HDAC1 (green), COX IV (red) and DAPI (blue). Scale bar: 50 μm. High-magnification images (Ad–Al, scale bar: 5 μm) are demarked by white lines and are connected to the original figures (Aa–Ac). Arrowheads indicate mitochondria stained for COX IV and HDAC1. (B) Summary showing that the number of mitochondria was dramatically decreased in TSA (25 and 50 nM)-treated animals compared to control tadpoles. N = 6, 5, 4 for Control, TSA 25 nM, and TSA 50 nM. (C) Representative co-staining images showing the anti-HDAC1 antibody (red) and MitoTracker (MTracker, green) after tadpoles were treated with TSA (50 nM, Cb), MS-275 (10 μM, Cc) and MGCD0103 (25 μM, Cd). Scale bar: 50 μm. High-magnification images (Ce–Cp, scale bar: 5 μm) are demarked by white lines from the original figures (Ca–Cd). Arrowheads indicate mitochondria stained with MitoTracker and HDAC1. Scale bar: 5 μm. (D) Quantification showing that the number of mitochondria was dramatically decreased in TSA-, MS-275-, and MGCD0103-treated animals compared to control tadpoles. N = 4, 5, 5, 4 for Control, TSA, MS-275, and MGCD0103. p < 0.05, *p < 0.01.

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