Mol Neurobiol DOI 10.1007/s12035-015-9206-2

LSD1 is Required for Hair Cell Regeneration in Zebrafish Yingzi He 1 & Dongmei Tang 1 & Chengfu Cai 6 & Renjie Chai 5 & Huawei Li 1,2,3,4

Received: 15 December 2014 / Accepted: 1 May 2015 # Springer Science+Business Media New York 2015

Abstract Lysine-specific demethylase 1 (LSD1/KDM1A) plays an important role in complex cellular processes such as differentiation, proliferation, apoptosis, and cell cycle progression. It has recently been demonstrated that during development, downregulation of LSD1 inhibits cell proliferation, modulates the expression of cell cycle regulators, and reduces hair cell formation in the zebrafish lateral line, which suggests that LSD1-mediated epigenetic regulation plays a key role in the development of hair cells. However, the role of LSD1 in hair cell regeneration after hair cell loss remains poorly understood. Here, we demonstrate the effect of LSD1 on hair cell regeneration following neomycin-induced hair cell loss. We show that the LSD1 inhibitor trans-2-phenylcyclopropylamine (2-PCPA) significantly decreases the regeneration of hair cells in zebrafish after neomycin damage. In addition, immunofluorescent staining demonstrates that 2-PCPA administration suppresses supporting cell proliferation and alters cell cycle progression. Finally, in situ hybridization shows that 2-PCPA significantly

downregulates the expression of genes related to Wnt/βcatenin and Fgf activation. Altogether, our data suggest that downregulation of LSD1 significantly decreases hair cell regeneration after neomycin-induced hair cell loss through inactivation of the Wnt/β-catenin and Fgf signaling pathways. Thus, LSD1 plays a critical role in hair cell regeneration and might represent a novel biomarker and potential therapeutic approach for the treatment of hearing loss. Keywords LSD1 . Zebrafish . Hair cell . Regeneration . Fgf signaling pathway . Wnt signaling pathway

Introduction The sensory hair cells of the inner ear are highly metabolic and are sensitive to a wide variety of noxious insults. Known etiologies for the loss of hair cells include aging, trauma, disease,

Yingzi He and Dongmei Tang contributed equally to this work. Electronic supplementary material The online version of this article (doi:10.1007/s12035-015-9206-2) contains supplementary material, which is available to authorized users. * Huawei Li [email protected] 1

Department of Otorhinolaryngology, Affiliated Eye and ENT Hospital, Fudan University, Shanghai 200031, People’s Republic of China


State Key Laboratory of Medical Neurobiology, Fudan University, Shanghai, People’s Republic of China


Institute of Stem Cell and Regeneration Medicine, Institute of Biomedical Science, Fudan University, Shanghai, People’s Republic of China


Key Laboratory of Hearing Science, Ministry of Health, EENT Hospital, Fudan University, Shanghai, People’s Republic of China


Co-innovation Center of Neuroregeneration, Key Laboratory for Developmental Genes and Human Disease, Institute of Life Sciences, Southeast University, Nanjing, Jiangsu 210096, People’s Republic of China


Department of Otolaryngology Head and Neck Surgery, The First Affiliated Hospital of Xiamen University, Xiamen, Fujian, People’s Republic of China

Mol Neurobiol

noise exposure, genetic disorders, and ototoxic drugs. Normally, hair cells do not regenerate in adult mammals [1], but a limited degree of hair cell regeneration can occur in the vestibular sensory epithelium and in early postnatal mammalian cochleae [2–7]. Consequently, hearing loss caused by loss of hair cells is irreversible in humans. In contrast, spontaneous regeneration of hair cells has been observed in many nonmammalian vertebrates such as birds, amphibians, and fish following hair cell loss due to either ototoxic insults or acoustic trauma [8–11]. Thus, understanding the mechanisms of hair cell regeneration in nonmammalian vertebrates might shed light on the potential therapeutic targets in humans. The zebrafish lateral line system offers a convenient and efficient model to study hair cell development, survival, death, and regeneration [12–15]. Lateral line organs are made up of a set of rosette-like sensory organs called neuromasts, which are located on the surface of the head and along the body in species-specific patterns [16, 17]. The mature lateral line neuromast is made up of a group of hair cells in the center and supporting cells surrounding them. The hair cells in zebrafish lateral line share structural, functional, and molecular similarities with the hair cells in the mammalian inner ear [18, 19]. Neuromasts are located on the surface of zebrafish larvae, and this makes the hair cells susceptible to ototoxic insults such as aminoglycoside antibiotics and chemotherapy agents—just like their counterparts in mammalian and avian ears [10, 20–24]. Additionally, zebrafish have the capacity to regenerate lost hair cells following ototoxic drug-induced hair cell damage, and this makes the zebrafish lateral line a useful system for identifying genes involved in hair cell regeneration and survival [25]. It has been well documented that newly regenerated hair cells in the sensory epithelia usually arise through the proliferation and differentiation of supporting cells during the process of regeneration, which is known as mitotic regeneration [8, 26, 27]. Coordinated and strictly regulated gene expression is essential for the development, survival, and function of mechanosensory cells in vertebrates, and chromatin remodeling is an important epigenetic process that controls the regulation of gene transcription [28]. In most cases, chromatin regulation is achieved by posttranslational modification of the histone amino terminal tails, including methylation, acetylation, phosphorylation, and ubiquitination [29]. Histone methylation and demethylation are major covalent modifications of histones that have been linked to the regulation of gene transcription. Histone methylation is catalyzed by the histone methyltransferases, while the removal of methyl groups from histone lysine residues is catalyzed by the histone demethylases [30]. Recent studies have demonstrated the specific function of histone methyltransferases/demethylases in many cellular processes such as cell cycle regulation, differentiation, survival, death, and proliferation [31, 32]. However, little is known about the implication of histone methylation in determining the fate of hair

cells. Lysine-specific demethylase 1 (LSD1, also referred to as KDM1A) was the first discovered histone demethylase and belongs to the superfamily of the flavin adenine dinucleotidedependent amine oxidases [33]. The enzyme is able to modulate gene expression through demethylation of lysines 4 and 9 of histone 3 by interacting with a variety of molecular partners [33–39]. LSD1-mediated epigenetic regulation has also been shown to play a central role in many of the pathological processes of cancer [40]. Numerous studies have shown that overexpression of LSD1 might promote cell phase transition and cell proliferation, suggesting that downregulation of LSD1 might promote the initiation and invasion of various cancers [41, 42]. LSD1 has also been recently reported to play crucial roles in maintaining the pluripotency of embryonic and cancer stem cells [43–46]. Both small-interfering RNA knockdown of LSD1 and pharmacological inhibition have been shown to lead to the suppression of proliferation in a variety of cancer cells and in both neural and embryonic stem cells [47, 48]. Our previous studies [49] have shown that maintenance of high levels of histone 3 lys4 dimethylation (H3K4me2) through the pharmacological inhibition of LSD1 attenuates neomycininduced hair cell apoptosis. We also reported that LSD1 inhibitor treatment could inhibit cell proliferation and decrease hair cell differentiation in the zebrafish lateral line during development [50]. However, whether LSD1 has any effect on hair cell regeneration remains unknown. In this study, we examined the role of LSD1 in hair cell regeneration after neomycin-induced hair cell loss in the zebrafish lateral line and investigated the possible mechanisms involved in this effect. We found that pharmacological inhibition of LSD1 with trans-2phenylcyclopropylamine (2-PCPA) during the hair cell regeneration period strongly suppressed supporting cell proliferation and decreased hair cell formation but did not lead to subsequent cell death in neuromasts. We further showed that 2-PCPA significantly downregulated the expression of genes related to Fgf and Wnt/β-catenin activation. These results suggest that LSD1 is required for hair cell regeneration in the zebrafish lateral line neuromasts. Furthermore, Fgf and Wnt signaling might be subject to regulation by this histone demethylase.

Materials and Methods Zebrafish Maintenance Zebrafish embryos were obtained from the natural spawning of wild-type adults and were maintained in our facility according to standard procedures. The Tg(brn3c:GFP)s356t transgenic line was obtained from the laboratory of Professor Zhengyi Chen, our collaborator at Harvard University. Zebrafish larvae were staged according to Kimmel et al. [51] and raised at 28.5 °C in Petri dishes. The ages of the embryos are given as days postfertilization (dpf).

Mol Neurobiol

Neomycin Treatment and Pharmacological Administration At 5 dpf, larvae were treated with 400 μM neomycin (SigmaAldrich, Inc., St. Louis, MO, USA) for 1 h, rinsed three times in fresh water, and allowed to recover at 28.5 °C. 2-PCPA (Sigma-Aldrich, Inc) was dissolved in double-distilled water (ddH2O) at stock concentrations of 200 mM and then diluted to the indicated concentrations in fish water. Dose-response data were obtained by treating larvae with 50 or 100 μM 2PCPA after neomycin exposure. Larvae were anesthetized with 0.02 % MS-222 (ethyl 3-aminobenzoate methanesulfonate; Sigma-Aldrich, Inc.) for 5 min before observation and scoring. In all experimental conditions, control groups were maintained in parallel under the same conditions but without 2-PCPA treatment. Immunohistochemistry For immunohistochemistry analysis, larvae were fixed in 4 % paraformaldehyde (PFA) and were permeabilized with phosphate-buffered saline (PBS) containing 0.5 % Triton X-100 (PBT-2) for 30 min followed by incubation in blocking solution for 1 h. The primary antibodies were anti-dimethyl H3K4 (1:500 dilution; Abcam, Cambridge, UK), anti-GFP (green fluorescent protein) (1:1000 dilution; Abcam), and anti-Sox2 (1:200 dilution; Abcam). The embryos were washed three times with PBS and incubated with secondary antibodies to detect primary antibodies. Nuclei were labeled with 4,6-diamidino-2-phenylindole (DAPI; Invitrogen, Carlsbad, CA, USA) for 20 min at room temperature. FM1-43FX Labeling To visualize and image the functional hair cells in lateral line neuromasts, the vital dye FM1-43FX (Molecular Probes, Eugene, OR, USA)—which enters mature hair cells through mechanotransduction-dependent activity—was applied at a concentration of 3 μM to live 5-dpf larvae for 45 s in the dark. After quickly rinsing three times with fresh water, the larvae were anesthetized in 0.02 % MS-222 and fixed with 4 % PFA in PBS for 2 h at room temperature or overnight at 4 °C. Cell Proliferation Assay To assess the mitotic events in the neuromasts of the lateral line during the regeneration period, larvae were incubated in fresh water containing 10 mM BrdU (Sigma) for 24 or 48 h with and without 2-PCPA at 28.5 °C starting at 1 h after neomycin exposure. Larvae were then fixed with 4 % PFA overnight at 4 °C. BrdU incorporation was detected by immunocytochemistry. The fixed larvae were washed three times in PBT-2 and placed in 2 N HCl for 0.5 h at 37 °C. Larvae were

then placed in blocking solution (10 % normal goat serum) for 1 h at room temperature and incubated with the monoclonal primary anti-BrdU antibody (1:200 dilution; Santa Cruz Biotechnology, Inc., CA, USA) overnight at 4 °C. The next day, the larvae were washed three times for 10 min each with PBT2 and then incubated with the secondary antibody for 1 h at 37 °C. Samples were examined with a Leica confocal fluorescence microscope (TCS SP5; Leica, Wetzlar, Germany). Western Blot Analysis Total protein was isolated with the AllPrep DNA/RNA/Protein Mini Kit (QIAGEN, Hilden, Germany) according to the manufacturer’s instructions. Protein concentrations were measured using a bicinchoninic acid (BCA) protein kit (Thermo Fisher Scientific, Rockford, IL), and proteins were separated on sodium dodecyl sulfate (SDS)-polyacrylamide gels and transferred onto polyvinylidene difluoride (PVDF) membranes (Immobilon-P; Millipore, Bedford, MA, USA). The membranes were blocked with 5 % nonfat dried milk in Tris-buffered saline with Tween 20 (TBST) (50 mM TrisHCl (pH 7.4), 150 mM NaCl, and 0.1 % Tween-20) for 1 h at room temperature and then blotted overnight with primary antibodies at 4 °C. The following antibodies were used as primary antibodies: anti-dimethyl H3K4 (1:1000 dilution; Abcam), anti-cleaved caspase-3 (1:1000 dilution; Cell Signaling Technology Inc., Danvers, MA, USA), anti-p21Cip1 C-19 (1:1000 dilution; Santa Cruz Biotechnology, Inc.), antip27Kip1 (1:500 dilution; Santa Cruz Biotechnology), anticyclin E1 (1:500 dilution), anti-cyclin D1 (1:500 dilution), anti-CDK2 (1:500 dilution), anti-CDK4 (1:500 dilution), and anti-CDK6 (1:500 dilution). The last five antibodies were purchased from ProteinTech Group, Inc. (Chicago, IL, USA). Whole-Mount In Situ Hybridization Regular whole-mount in situ hybridization of zebrafish embryos was performed as previously described [52]. All primers for probe synthesis are listed in Supplemental table. Cell Counts and Statistical Analysis Cells in the first five posterior lateral line (PLL) neuromasts (L1–L5) were counted, and statistical analysis was performed with SigmaPlot (version 12.0 for Windows). Prior to analysis, all data were first examined for normality and homogeneity of variances by the Shapiro-Wilk test and Levene’s test, respectively. Data were analyzed using either t tests or one-way analysis of variance for multiple comparisons. All data are presented as the mean±SEM. A p value

LSD1 is Required for Hair Cell Regeneration in Zebrafish.

Lysine-specific demethylase 1 (LSD1/KDM1A) plays an important role in complex cellular processes such as differentiation, proliferation, apoptosis, an...
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