The histone demethylase LSD1 regulates inner ear progenitor differentiation through interactions with Pax2 and the NuRD repressor complex

The histone demethylase LSD1 plays a pivotal role in cellular differentiation, particularly in silencing lineage-specific genes. However, little is known about how LSD1 regulates neurosensory differentiation in the inner ear. Here we show that LSD1 interacts directly with the transcription factor Pax2 to form the NuRD co-repressor complex at the Pax2 target gene loci in a mouse otic neuronal progenitor cell line (VOT-N33). VOT-N33 cells expressing a Pax2-response element reporter were GFP-negative when untreated, but became GFP positive after forced differentiation or treatment with a potent LSD inhibitor. Pharmacological inhibition of LSD1 activity resulted in the enrichment of mono- and di-methylation of H3K4, upregulation of sensory neuronal genes and an increase in the number of sensory neurons in mouse inner ear organoids. Together, these results identify the LSD1/NuRD complex as a previously unrecognized modulator for Pax2-mediated neuronal differentiation in the inner ear

Identifying the Neural Circuit that Regulates Social Familiarity Induced Anxiolysis (SoFiA)

Mental health is crucially linked to social behavior. A crucial aspect of healthy social behavior involves learning to adapt emotional responses to social cues, for example learning to suppress anxiety through social familiarity, or social familiarity-induced anxiolysis (SoFiA). SoFiA is well documented; however, the neural mechanisms of SoFiA are unclear. SoFiA is modeled in rats by employing a social interaction habituation (SI-hab) protocol. Using SI-hab protocol it has been determined that SoFiA represents social safety learning, which requires both anxiogenic stimulus (Anx) and social familiarity (SF) during training sessions (5–6 daily SI sessions), and SoFiA expression is dependent on infralimbic cortex (IL). Based on these findings we hypothesize that Anx and SF are processed by unique neural systems, and repeated convergence of these signals interact within IL to induce plasticity, resulting in social safety learning and anxiolysis. Following SoFiA expression, rats were either sacrificed 30 minutes {for gene expression or Neural Activity Regulated Gene (NARG) analysis} or perfused 90 minutes (for cFos immunoreactivity analysis) after SI session on social training day 5. This led to gaining insights into regions of brain involved in SoFiA response as well as the underlying molecular mechanisms. We identified amygdala, specifically the central amygdala (CeA), basomedial amygdala (BMA) and basolateral amygdala (BLA) as potential candidate regions in SoFiA response. Next, we investigated the role of IL and its efferent pathways in SoFiA expression using inhibitory DREADDs and intersectional chemogenetics to inhibit IL projection neurons and/or axons. We identified that specific projection neurons within the IL are pivotal for SoFiA expression, and that within these projections, the ones that specifically projected to the amygdala are most crucial for expression of SoFiA

The Forkhead Transcription Factor, FOXO1, is Present in Quiescent Pituitary Cells During Development and in Adulthood

The present study revealed that FOXO1 is present in the nuclei of non-dividing pituitary cells and in a subset of differentiated cells with highest level of expression in somatotrophs, followed by corticotrophs, thyrotrophs and gonadotrophs throughout development and in adulthood stage. A significant difference in Foxo1 transcript between age-matched males and females at 8-9 weeks of age was demonstrated in the anterior pituitary for the first time. IHC data demonstrating (i) FOXO1 co-localization with p27kip1 (ii) an increase in FOXO1 immunopositive cells within anterior pituitary in p27KO embryos compared to WT (iii) absence of FOXO1 in the nucleus of BrdU positive cells suggested that in absence of p27Kip1 FOXO1 might be important for preventing unbridled cell proliferation. Data suggested that FOXO1 might not be important for initiating pituitary cell differentiation but might be involved with p27kip1 in maintaining pituitary cell quiescence. Increase in nuclear localization of FOXO1 in the pituitary of Foxp3 mutant (lacking insulin signaling) suggested that it might be a down-stream target of insulin/PI3K/PKB pathway in the pituitary as it is in several other tissues

Forkhead Box O1 is present in quiescent pituitary cells during development and is increased in the absence of p27 Kip1.

Congenital pituitary hormone deficiencies have been reported in approximately one in 4,000 live births, however studies reporting mutations in some widely studied transcription factors account for only a fraction of congenital hormone deficiencies in humans. Anterior pituitary hormones are required for development and function of several glands including gonads, adrenals, and thyroid. In order to identify additional factors that contribute to human congenital hormone deficiencies, we are investigating the forkhead transcription factor, FOXO1, which has been implicated in development of several organs including ovary, testis, and brain. We find that FOXO1 is present in the nuclei of non-dividing pituitary cells during embryonic development, consistent with a role in limiting proliferation and/or promoting differentiation. FOXO1 is present in a subset of differentiated cells at e18.5 and in adult with highest level of expression in somatotrope cells. We detected FOXO1 in p27(Kip1)-positive cells at e14.5. In the absence of p27(Kip1) the number of pituitary cells containing FOXO1 is significantly increased at e14.5 suggesting that a feedback loop regulates the interplay between FOXO1 and p27(Kip1)

Tumor Necrosis Factor Alpha (TNF-α) Disrupts Kir4.1 Channel Expression Resulting in Müller Cell Dysfunction in the RetinaDiurnal Rhythm of Kir4.1 in the Retina

Purpose: Diabetic patients often are affected by vision problems. We previously identified diabetic retinopathy (DR) as a disease of clock gene dysregulation. TNF-α, a proinflammatory cytokine, is known to be elevated in DR. Müller cells maintain retinal water homeostasis and K+ concentration via Kir4.1 channels. Notably, Kir4.1 expression is reduced in diabetes; however, the interplay of TNF-α, Kir4.1, and clock genes in Müller cells remains unknown. We hypothesize that the Kir4.1 in Müller cells is under clock regulation, and increase in TNF-α is detrimental to Kir4.1. Methods: Long-Evans rats were made diabetic using streptozotocin (STZ). Retinal Kir4.1 expression was determined at different time intervals. Rat Müller (rMC-1) cells were transfected with siRNA for Per2 or Bmal1 and in parallel treated with TNF-α (5–5000 pM) to determine Kir4.1 expression. Results: Kir4.1 expression exhibited a diurnal rhythm in the retina; however, with STZ-induced diabetes, Kir4.1 was reduced overall. Kir4.1 rhythm was maintained in vitro in clock synchronized rMC-1 cells. Clock gene siRNA-treated rMC-1 exhibited a decrease in Kir4.1 expression. TNF-α treatment of rMCs lead to a profound decrease in Kir4.1 due to reduced colocalization of Kir4.1 channels with synapse-associated protein (SAP97) and disorganization of the actin cytoskeleton. Conclusions: Our findings demonstrate that Kir4.1 channels possess a diurnal rhythm, and this rhythm is dampened with diabetes, thereby suggesting that the increase in TNF-α is detrimental to normal Kir4.1 rhythm and expression

Generation and characterization of PRS4-EGFP N33 (PE) stable cells.

<p>(A) A linearized PRS4-EGFP construct was transfected into N33 cells. G418 antibiotic at 500 μg/ml was used as a negative selection for stable clones. The upper panel indicates cells after 24 hours of transfection. As indicated in the lower panel, almost all non-transformed cells died by day 9, but cells with stable incorporation of the PRS4-EGFP cassette (PE) survived. (B) FACS sorting of PE positive and negative cells based upon GFP expression. N33 cells were used as a negative control for gate setup during separation of PE negative cells. The lower panel in the figure shows histogram representation of the upper panel. (C) FACS sorted PE positive (PE + ve) and PE negative (PE—ve) cells were separately grown and analyzed for GFP expression by fluorescence microscopy. (D) GFP expression was determined by Western blot analysis using GFP antibodies. Upper panel in the Western blot shows the absence of GFP expression in the PE—ve cells. β-ACTIN protein was used as a loading control in the lower panel. (E) PCR analysis was performed to confirm the presence of the PE cassette integration in DNA isolated from PE negative cells using PRS4 sequence specific primers (upper panel). PCR primers designed in the promoter region of <i>Gapdh</i> were used as an equal loading control for PCR products (lower panel). Chromatin isolated from normal N33 or PE positive cells were used as negative and positive controls, respectively. Scale bars, 100 μm.</p

PCR primers used for ChIP assays.

<p>PCR primers used for ChIP assays.</p

Blockade of LSD1/NuRD activity derepresses <i>Egfp</i> expression in PE negative cells.

<p>(A) PE negative cells were treated with DMSO (control) or 0.25 mM TCP for 48 hours and DIC and fluorescence images were captured. (B) PE negative cells were treated with DMSO (control) or TCP (48 hours) to inhibit LSD1. ChIP analyses were performed using H3K4 me1, me2 or me3 methyl marks specific antibodies as indicated. Rabbit IgG was used as a negative control. The immunoprecipitated chromatin was quantified by quantitative PCR using primers flanking to the Pax2 response element. The relative amounts of PCR products are expressed as a percent of input chromatin. All values are expressed as the mean of 3 replicates; error bars are one standard deviation. Statistically significant differences are indicated (*P<0.05). (C) PE negative cells were treated with DMSO (control), TSA, and TCP or alternatively differentiated by temperature induction for 48 hours followed by Western blot analysis of GFP protein expression. β-ACTIN was used as a protein loading control. (D) Schematic figure showing how GFP expression is activated in PE N33 cells upon differentiation.</p

Pax2 interacts with LSD1 and is associated with the NuRD complex.

<p>(A, B) Physical interaction between endogenously expressed Pax2 and LSD1 proteins in N33 cells. Nuclear cell lysates were prepared from undifferentiated N33 cells and immunoprecipitations with either anti-LSD1 or anti-Pax2 antibody were performed. (C, D) Pax2 and LSD1 immunoprecipitated complexes were probed against various members of the NuRD complex as indicated. Immunoprecipitated complexes were resolved by SDS-PAGE followed by immunoblot analysis using indicated antibodies. Rabbit IgG was used as a negative control for non-specific immunoprecipitation. Cell lysates before co-immunoprecipitation were used as input controls.</p

PAX2/LSD1/NuRD complex suppresses neuronal differentiation of N33 cells.

<p>(A, B) N33 cells were cultured under proliferative conditions. Cells were washed once the following day with PBS and treated with DMSO (control) or TSA, LSD1-C12 for 48 hours in the absence of gamma interferon. Quantitative real-time RT-PCR analysis was used to examine changes in the mRNA expression of the proneural markers <i>NeuroD</i> and <i>Ngn1</i>, the migrating neuroblast marker <i>Phox2b</i> and the inner ear sensory neural markers <i>Tlx3</i> and <i>Brn3a</i>. Expression levels were normalized to those of the housekeeping gene <i>L27</i>. All values are expressed as the mean of 3 replicates; error bars indicate one standard deviation. Statistically significant differences are indicated (*P<0.05). (C) N33 cells were cultured and treated under similar conditions to those described in A and B. Cells were washed once with PBS and fixed in 4% PFA at room temperature. Immunostaining was performed for analysis of NeuroD protein expression. Nuclei were counter stained using DAPI (blue). Scale bar, 100 μm. (D) N33 cells were treated with DMSO control or TSA for 48 or 72 hours. Western blot analysis was performed to confirm a significant up-regulation of NeuroD. β-ACTIN was used as a loading control. Histone 3 Acetyl mark specific (Ac-H3) antibody was used to show an increase in acetylation, as a positive control to TSA treatment.</p