Expression of C9orf72-related dipeptides impairs motor function in a vertebrate model.

Large expansions of hexanucleotide GGGGCC (G4C2) repeats (hundreds to thousands) in the first intron of the chromosome 9 open reading frame 72 (C9orf72) locus are the strongest known genetic factor associated with amyotrophic lateral sclerosis and frontotemporal lobar degeneration. Different hypotheses exist about the underlying disease mechanism including loss of function by haploinsufficiency, toxicity arising as a result of RNA or dipeptide repeats (DPRs). Five different DPRs are produced by repeat-associated non-ATG-initiated translation of the G4C2 repeats. Though earlier studies have indicated toxicity of the DPRs in worms, flies, primary cultured cells and cell lines, the effect of expressing DPRs of amyotrophic lateral sclerosis-relevant length has not been tested on motor behaviour in vertebrate models. In this study, by expressing constructs with alternate codons encoding different lengths of each DPR (40, 200 and 1000) in the vertebrate zebrafish model, the GR DPR was found to lead to the greatest developmental lethality and morphological defects, and GA, the least. However, expressing 1000 repeats of any DPR, including the 'non-toxic' GA DPR led to locomotor defects. Based on these observations, a transgenic line stably expressing 100 GR repeats was generated to allow specific regional and temporal expression of GR repeats in vivo. Expression of GR DPRs ubiquitously resulted in severe morphological defects and reduced swimming. However, when expressed specifically in motor neurons, the developmental defects were significantly reduced, but the swimming phenotype persisted, suggesting that GR DPRs have a toxic effect on motor neuron function. This was validated by the reduction in motor neuron length even in already formed motor neurons when GR was expressed in these. Hence, the expression of C9orf72-associated DPRs can cause significant motor deficits in vertebrates.

Unraveling the role of aurora A beyond centrosomes and spindle assembly: implications in muscle differentiation.

Aurora kinases are critical mitotic serine/threonine kinases and are often implicated in tumorigenesis. Recent studies of the interphase functions for aurora kinase (Aurk)A have considerably expanded our understanding of its role beyond mitosis. To identify the unknown targets of AurkA, we used peptide array-based screening and found E2F4 to be a novel substrate. Phosphorylation of E2F4 by AurkA at Ser75 regulates its DNA binding and subcellular localization. Because E2F4 plays an important role in skeletal muscle differentiation, we attempted to gain insight into E2F4 phosphorylation in this context. We observed that a block in E2F4 phosphorylation retained it better within the nucleus and inhibited muscle differentiation. RNA sequencing analysis revealed a perturbation of the gene network involved in the process of muscle differentiation and mitochondrial biogenesis. Collectively, our findings establish a novel role of AurkA in the process of skeletal muscle differentiation.-Dhanasekaran, K., Bose, A., Rao, V. J., Boopathi, R., Shankar, S. R., Rao, V. K., Swaminathan, A., Vasudevan, M., Taneja, R., Kundu, T. K. Unravelling the role of aurora A beyond centrosomes and spindle assembly: implications in muscle differentiation.

γ-Aminobutyric acid receptor alpha 1 subunit loss of function causes genetic generalized epilepsy by impairing inhibitory network neurodevelopment.

OBJECTIVE: In humans, mutations of the γ-aminobutyric acid receptor subunit 1 (GABRA1) cause either mild or severe generalized epilepsy. Although these epilepsy-causing mutations have been shown to disrupt the receptor activity in vitro, their in vivo consequences on brain development and activity are not known. Here, we aim at unraveling the epileptogenesis mechanisms of GABRA1 loss of function. METHODS: We generated a gabra1-/- zebrafish mutant line displaying highly penetrant epileptic seizures. We sought to identify the underlying molecular mechanisms through unbiased whole transcriptomic assay of gabra1-/- larval brains. RESULTS: Interestingly, mutant fish show fully penetrant seizures at juvenile stages that accurately mimic tonic-clonic generalized seizures observed in patients. Moreover, highly penetrant seizures can be induced by light stimulation, thus providing us with the first zebrafish model in which evident epileptic seizures can be induced by nonchemical agents. Our transcriptomic assay identified misregulated genes in several pathways essential for correct brain development. More specifically, we show that the early development of the brain inhibitory network is specifically affected. Although the number of GABAergic neurons is not altered, we observed a drastic reduction in the number of inhibitory synapses and a decreased complexity of the GABAergic network. This is consistent with the disruption in expression of many genes involved in axon guidance and synapse formation. SIGNIFICANCE: Together with the role of GABA in neurodevelopment, our data identify a novel aspect of epileptogenesis, suggesting that the substratum of GABRA1-deficiency epilepsy is a consequence of early brain neurodevelopmental defects, in particular at the level of inhibitory network wiring.

Transcriptional Coactivator and Chromatin Protein PC4 Is Involved in Hippocampal Neurogenesis and Spatial Memory Extinction.

Although the elaborate combination of histone and non-histone protein complexes defines chromatin organization and hence regulates numerous nuclear processes, the role of chromatin organizing proteins remains unexplored at the organismal level. The highly abundant, multifunctional, chromatin-associated protein and transcriptional coactivator positive coactivator 4 (PC4/Sub1) is absolutely critical for life, because its absence leads to embryonic lethality. Here, we report results obtained with conditional PC4 knock-out (PC4(f/f) Nestin-Cre) mice where PC4 is knocked out specifically in the brain. Compared with the control (PC4(+/+) Nestin-Cre) mice, PC4(f/f) Nestin-Cre mice are smaller with decreased nocturnal activity but are fertile and show no motor dysfunction. Neurons in different areas of the brains of these mice show sensitivity to hypoxia/anoxia, and decreased adult neurogenesis was observed in the dentate gyrus. Interestingly, PC4(f/f) Nestin-Cre mice exhibit a severe deficit in spatial memory extinction, whereas acquisition and long term retention were unaffected. Gene expression analysis of the dorsal hippocampus of PC4(f/f) Nestin-Cre mice revealed dysregulated expression of several neural function-associated genes, and PC4 was consistently found to localize on the promoters of these genes, indicating that PC4 regulates their expression. These observations indicate that non-histone chromatin-associated proteins like PC4 play a significant role in neuronal plasticity.

Chromatin organization, epigenetics and differentiation: an evolutionary perspective.

Genome packaging is a universal phenomenon from prokaryotes to higher mammals. Genomic constituents and forces have however, travelled a long evolutionary route. Both DNA and protein elements constitute the genome and also aid in its dynamicity. With the evolution of organisms, these have experienced several structural and functional changes. These evolutionary changes were made to meet the challenging scenario of evolving organisms. This review discusses in detail the evolutionary perspective and functionality gain in the phenomena of genome organization and epigenetics.

Glucocorticoid-sensitive period of corticotroph development-Implications for mechanisms of early life stress.

Corticotrophs are intermediaries in the hypothalamic-pituitary-adrenal (HPA) axis, which plays a crucial role in stress response in vertebrates. The HPA axis displays an intricate mode of negative feedback regulation, whereby the peripheral effector, cortisol inhibits the secretion of its upstream regulator, adrenocorticotropic hormone (ACTH) from proopiomelanocortin (POMC)-expressing cells in the pituitary. While the feedback regulation of the HPA axis is well characterized in the adult organism, the effect of feedback regulation on the development of corticotrophs is poorly understood. Here, we studied the effect of glucocorticoids on the development of POMC-expressing cells in the zebrafish pituitary. The development of POMC cells showed a steady increase in numbers between 2-6 days post fertilization. Inhibition of endogenous glucocorticoid synthesis resulted in an increase in POMC cell number due to reduced developmental feedback inhibition of cortisol on POMC cells. Conversely, addition of exogenous dexamethasone at a critical developmental window led to a decrease in corticotroph cell number, mimicking greater feedback control due to increased cortisol levels. Finally, developmental dysregulation of ACTH levels resulted in impaired anxiety-like and stress-coping behaviours. Hence, we identified a sensitive developmental window for the effect of glucocorticoids on corticotrophs and demonstrate the downstream effect on stress-responsive behaviour.

Stress resilience is established during development and is regulated by complement factors.

Individuals in a population respond differently to stressful situations. While resilient individuals recover efficiently, others are susceptible to the same stressors. However, it remains challenging to determine if resilience is established as a trait during development or acquired later in life. Using a behavioral paradigm in zebrafish larvae, we show that resilience is a stable and heritable trait, which is determined and exhibited early in life. Resilient larvae show unique stress-induced transcriptional response, and larvae with mutations in resilience-associated genes, such as neuropeptide Y and miR218, are less resilient. Transcriptome analysis shows that resilient larvae downregulate multiple factors of the innate immune complement cascade in response to stress. Perturbation of critical complement factors leads to an increase in resilience. We conclude that resilience is established as a stable trait early during development and that neuropeptides and the complement pathway play positive and negative roles in determining resilience, respectively.

Publisher Correction: Splice-specific deficiency of the PTSD-associated gene PAC1 leads to a paradoxical age-dependent stress behavior.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Splice-specific deficiency of the PTSD-associated gene PAC1 leads to a paradoxical age-dependent stress behavior.

The pituitary adenylate cyclase-activating polypeptide receptor (PAC1, also known as ADCYAP1R1) is associated with post-traumatic stress disorder and modulation of stress response in general. Alternative splicing of PAC1 results in multiple gene products, which differ in their mode of signalling and tissue distribution. However, the roles of distinct splice variants in the regulation of stress behavior is poorly understood. Alternative splicing of a short exon, which is known as the "hop cassette", occurs during brain development and in response to stressful challenges. To examine the function of this variant, we generated a splice-specific zebrafish mutant lacking the hop cassette, which we designated 'hopless'. We show that hopless mutant larvae display increased anxiety-like behavior, including reduced dark exploration and impaired habituation to dark exposure. Conversely, adult hopless mutants displayed superior ability to rebound from an acute stressor, as they exhibited reduced anxiety-like responses to an ensuing novelty stress. We propose that the developmental loss of a specific PAC1 splice variant mimics prolonged mild stress exposure, which in the long term, predisposes the organism's stress response towards a resilient phenotype. Our study presents a unique genetic model demonstrating how early-life state of anxiety paradoxically correlates with reduced stress susceptibility in adulthood.

Chromatin Protein PC4 Orchestrates B Cell Differentiation by Collaborating with IKAROS and IRF4.

The chromatin protein positive coactivator 4 (PC4) has multiple functions, including chromatin compaction. However, its role in immune cells is largely unknown. We show that PC4 orchestrates chromatin structure and gene expression in mature B cells. B-cell-specific PC4-deficient mice show impaired production of antibody upon antigen stimulation. The PC4 complex purified from B cells contains the transcription factors (TFs) IKAROS and IRF4. IKAROS protein is reduced in PC4-deficient mature B cells, resulting in de-repression of their target genes in part by diminished interactions with gene-silencing components. Upon activation, the amount of IRF4 protein is not increased in PC4-deficient B cells, resulting in reduction of plasma cells. Importantly, IRF4 reciprocally induces PC4 expression via a super-enhancer. PC4 knockdown in human B cell lymphoma and myeloma cells reduces IKAROS protein as an anticancer drug, lenalidomide. Our findings establish PC4 as a chromatin regulator of B cells and a possible therapeutic target adjoining IKAROS in B cell malignancies.

The Dietary Flavonoid, Luteolin, Negatively Affects Neuronal Differentiation.

Luteolin, a polyphenolic plant flavonoid, has been attributed with numerous beneficial properties like anti-cancer, antioxidant, and anti-inflammatory action. Luteolin has been reported earlier to be neuroprotective in models of spinal cord injury and traumatic brain injury and also induces neurite outgrowth in PC12 cells. However, the effect of luteolin on early differentiation, which might be important for its beneficial effects, is unknown. In this report, we show that luteolin negatively affects early differentiation of embryonic stem cells, hampering the formation of embryoid bodies. At later stages of differentiation, luteolin specifically inhibits neuronal differentiation, where the expression of early neuronal markers is suppressed, whereas luteolin treatment does not inhibit expression of meso- and endodermal markers. Further, in a developing zebrafish model, luteolin treatment leads to fewer numbers of mitotic cells in the brain. These specific effects of luteolin on neuronal differentiation could possibly be due to its ability to inhibit the lysine acetyltransferase, p300, since the structurally closely related p300 non-inhibitor flavonoid, apigenin, does not inhibit neuronal differentiation. These results show that luteolin perturbs neuronal differentiation of embryonic stem cells.

Corrigendum: The Dietary Flavonoid, Luteolin, Negatively Affects Neuronal Differentiation.

[This corrects the article DOI: 10.3389/fnmol.2019.00041.].

Non-canonical mTOR-Independent Role of DEPDC5 in Regulating GABAergic Network Development.

Mutations in DEPDC5 are causal factors for a broad spectrum of focal epilepsies, but the underlying pathogenic mechanisms are still largely unknown. To address this question, a zebrafish depdc5 knockout model showing spontaneous epileptiform events in the brain, increased drug-induced seizure susceptibility, general hypoactivity, premature death at 2-3 weeks post-fertilization, as well as the expected hyperactivation of mTOR signaling was developed. Using this model, the role of DEPDC5 in brain development was investigated using an unbiased whole-transcriptomic approach. Surprisingly, in addition to mTOR-associated genes, many genes involved in synaptic function, neurogenesis, axonogenesis, and GABA network activity were found to be dysregulated in larval brains. Although no gross defects in brain morphology or neuron loss were observed, immunostaining of depdc5-/- brains for several GABAergic markers revealed specific defects in the fine branching of the GABAergic network. Consistently, some defects in depdc5-/- could be compensated for by treatment with GABA, corroborating that GABA signaling is indeed involved in DEPDC5 pathogenicity. Further, the mTOR-independent nature of these neurodevelopmental defects was demonstrated by the inability of rapamycin to rescue the GABAergic network defects observed in depdc5-/- brains and, conversely, the inability of GABA to rescue the hypoactivity in another genetic model showing mTOR hyperactivation. This study hence provides the first in vivo evidence that DEPDC5 plays previously unknown roles apart from its canonical function as an mTOR inhibitor. Moreover, these results propose that defective neurodevelopment of GABAergic networks could be a key factor in epileptogenesis when DEPDC5 is mutated.

Multifunctional human transcriptional coactivator protein PC4 is a substrate of Aurora kinases and activates the Aurora enzymes.

Positive coactivator 4 (PC4), a human transcriptional coactivator, is involved in diverse processes like chromatin organization and transcription regulation. It is hyperphosphorylated during mitosis, with unknown significance. For the first time, we demonstrate the function of PC4 outside the nucleus upon nuclear envelope breakdown. A fraction of PC4 associates with Aurora A and Aurora B and undergoes phosphorylation, following which PC4 activates both Aurora A and B to sustain optimal kinase activity to maintain the phosphorylation gradient for the proper functioning of the mitotic machinery. This mitotic role is evident in PC4 knockdown cells where the defects are rescued only by the catalytically active Aurora kinases, but not the kinase-dead mutants. Similarly, the PC4 phosphodeficient mutant failed to rescue such defects. Hence, our observations establish a novel mitotic function of PC4 that might be dependent on Aurora kinase-mediated phosphorylation.

CARM1 regulates astroglial lineage through transcriptional regulation of Nanog and posttranscriptional regulation by miR92a.

Coactivator-associated arginine methyltransferase (CARM1/PRMT4)-mediated transcriptional coactivation and arginine methylation is known to regulate various tissue-specific differentiation events. Although CARM1 is expressed in the neural crest region in early development, coinciding with early neuronal progenitor specification, the role of CARM1 in any neuronal developmental pathways has been unexplored. Using a specific small-molecule inhibitor of CARM1-mediated H3R17 methylation in human embryonic stem cell line, we find that H3R17 methylation contributes to the maintenance of the astroglial cell population. A network of regulation was observed on the miR92a promoter by which H3R17-responsive Nanog bound to the miR92a promoter decreased upon inhibition, resulting in an abnormal gene expression program influencing the glial lineage. This was also true in zebrafish, in which, with the help of CARM1 inhibitor and CARM1 morpholinos, we show that inhibition of H3R17 methylation results in defective glial cell morphology and a sensory defect in a subpopulation. A gain-of-function strategy in which mCARM1 was introduced in the morpholino-treated embryos exhibited recovery of the sensory defect phenotype. This study thus establishes the functional cooperation between arginine methylation and microRNA expression in the neuronal developmental process, with potential implications in sensory development pathways.

Inhibition of p300 lysine acetyltransferase activity by luteolin reduces tumor growth in head and neck squamous cell carcinoma (HNSCC) xenograft mouse model.

Chromatin acetylation is attributed with distinct functional relevance with respect to gene expression in normal and diseased conditions thereby leading to a topical interest in the concept of epigenetic modulators and therapy. We report here the identification and characterization of the acetylation inhibitory potential of an important dietary flavonoid, luteolin. Luteolin was found to inhibit p300 acetyltransferase with competitive binding to the acetyl CoA binding site. Luteolin treatment in a xenografted tumor model of head and neck squamous cell carcinoma (HNSCC), led to a dramatic reduction in tumor growth within 4 weeks corresponding to a decrease in histone acetylation. Cells treated with luteolin exhibit cell cycle arrest and decreased cell migration. Luteolin treatment led to an alteration in gene expression and miRNA profile including up-regulation of p53 induced miR-195/215, let7C; potentially translating into a tumor suppressor function. It also led to down-regulation of oncomiRNAs such as miR-135a, thereby reflecting global changes in the microRNA network. Furthermore, a direct correlation between the inhibition of histone acetylation and gene expression was established using chromatin immunoprecipitation on promoters of differentially expressed genes. A network of dysregulated genes and miRNAs was mapped along with the gene ontology categories, and the effects of luteolin were observed to be potentially at multiple levels: at the level of gene expression, miRNA expression and miRNA processing.

Modulation of neurogenesis by targeting epigenetic enzymes using small molecules: an overview.

Neurogenesis consists of a plethora of complex cellular processes including neural stem cell (NSC) proliferation, migration, maturation or differentiation to neurons, and finally integration into the pre-existing neural circuits in the brain, which are temporally regulated and coordinated sequentially. Mammalian neurogenesis begins during embryonic development and continues in postnatal brain (adult neurogenesis). It is now evident that adult neurogenesis is driven by extracellular and intracellular signaling pathways, where epigenetic modifications like reversible histone acetylation, methylation, as well as DNA methylation play a vital role. Epigenetic regulation of gene expression during neural development is governed mainly by histone acetyltransferases (HATs), histone methyltransferase (HMTs), DNA methyltransferases (DNMTs), and also the enzymes for reversal, like histone deacetylases (HDACs), and many of these have also been shown to be involved in the regulation of adult neurogenesis. The contribution of these epigenetic marks to neurogenesis is increasingly being recognized, through knockout studies and small molecule modulator based studies. These small molecules are directly involved in regeneration and repair of neurons, and not only have applications from a therapeutic point of view, but also provide a tool to study the process of neurogenesis itself. In the present Review, we will focus on small molecules that act predominantly on epigenetic enzymes to enhance neurogenesis and neuroprotection and discuss the mechanism and recent advancements in their synthesis, targeting, and biology.

Acetyltransferases (HATs) as targets for neurological therapeutics.

The acetylation of histone and non-histone proteins controls a great deal of cellular functions, thereby affecting the entire organism, including the brain. Acetylation modifications are mediated through histone acetyltransferases (HAT) and deacetylases (HDAC), and the balance of these enzymes regulates neuronal homeostasis, maintaining the pre-existing acetyl marks responsible for the global chromatin structure, as well as regulating specific dynamic acetyl marks that respond to changes and facilitate neurons to encode and strengthen long-term events in the brain circuitry (e.g., memory formation). Unfortunately, the dysfunction of these finely-tuned regulations might lead to pathological conditions, and the deregulation of the HAT/HDAC balance has been implicated in neurological disorders. During the last decade, research has focused on HDAC inhibitors that induce a histone hyperacetylated state to compensate acetylation deficits. The use of these inhibitors as a therapeutic option was efficient in several animal models of neurological disorders. The elaboration of new cell-permeant HAT activators opens a new era of research on acetylation regulation. Although pathological animal models have not been tested yet, HAT activator molecules have already proven to be beneficial in ameliorating brain functions associated with learning and memory, and adult neurogenesis in wild-type animals. Thus, HAT activator molecules contribute to an exciting area of research.