Regulation Of Neural Differentiation Research Articles

Identification and Biochemical Characterization of High Mobility Group Protein 20A as a Novel Ca2+/S100A6 Target.

During screening of protein-protein interactions, using human protein arrays carrying 19,676 recombinant glutathione s-transferase (GST)-fused human proteins, we identified the high-mobility protein group 20A (HMG20A) as a novel S100A6 binding partner. We confirmed the Ca2+-dependent interaction of HMG20A with S100A6 by the protein array method, biotinylated S100A6 overlay, and GST-pulldown assay in vitro and in transfected COS-7 cells. Co-immunoprecipitation of S100A6 with HMG20A from HeLa cells in a Ca2+-dependent manner revealed the physiological relevance of the S100A6/HMG20A interaction. In addition, HMG20A has the ability to interact with S100A1, S100A2, and S100B in a Ca2+-dependent manner, but not with S100A4, A11, A12, and calmodulin. S100A6 binding experiments using various HMG20A mutants revealed that Ca2+/S100A6 interacts with the C-terminal region (residues 311–342) of HMG20A with stoichiometric binding (HMG20A:S100A6 dimer = 1:1). This was confirmed by the fact that a GST-HMG20A mutant lacking the S100A6 binding region (residues 311–347, HMG20A-ΔC) failed to interact with endogenous S100A6 in transfected COS-7 cells, unlike wild-type HMG20A. Taken together, these results identify, for the first time, HMG20A as a target of Ca2+/S100 proteins, and may suggest a novel linkage between Ca2+/S100 protein signaling and HMG20A function, including in the regulation of neural differentiation.

Abstract LB-173: DNA methylation analysis reveals epigenetic regulation of neural differentiation in AT/RTs

Abstract DNA methylation has proven to be powerful for brain tumor characterization and diagnostic classification. To obtain information about the oncogenic role of DNA methylation, we analyzed medulloblastoma, choroid plexus, and atypical teratoid/rhabdoid tumors (AT/RTs) with public data from 450K-methylation arrays (N=584) and gene-expression arrays (N=110). In addition, two AT/RTs, five choroid plexus tumors and three medulloblastomas were analyzed by using reduced representation bisulfite sequencing, exome sequencing, and RNA-sequencing of matched samples. Only few somatic alterations in addition to SMARCB1 deletion were present in our AT/RTs. DNA methylation analysis generated 2325-5739 and 17175-25187 differentially methylated regions (DMRs) between tumor types in 450K array and RRBS sequencing data, respectively. AT/RTs harbored generally higher DNA methylation levels than the other tumor types. Next, DNA methylation differences were integrated with gene expression data. Surprisingly, only eight genes showed cancer-specific association between differential DNA methylation and an opposite expression change at promoter or linked enhancer in both public and in-house data. There were 44 cancer-specific genes with expression-methylation association when DNA methylation analysis was extended to genomic neighborhoods. To gain information about changes in epigenetic regulation between tumor types, we studied which previously experimentally validated transcription factor (TF) binding sites are enriched in cancer specific DMRs. Several TFs known to promote neural development, such as NEUROG2 and NEUROD1, were enriched in regions hypermethylated in AT/RT, whereas TFs, such as SMAD2, involved in the inhibition of neural development were associated with regions hypermethylated in medulloblastoma. This suggests that DNA methylation is regulating especially the target sites for neural regulators in AT/RT tumors, thus inhibiting neural development. Expression differences did not explain the predicted decreased activity of most of these neural TFs. Low number of genes with cancer-specific expression and methylation change is at least partly explained by the different gene expression patterns in medulloblastomas and choroid plexus tumors, thus providing different references for comparison. Also differences in the measurement techniques contribute to this. Taken together, these results suggest that DNA methylation has a role as an epigenetic regulator for the oncogenesis of AT/RTs. Citation Format: Kirsi Johanna Granberg, Joonas Tuominen, Kristiina Nordfors, Meeri Pekkarinen, Ville Kytölä, Sergei Häyrynen, Ebrahim Afyounian, Olli Lohi, Pauli Helen, Juha Kesseli, Joonas Haapasalo, Hannu Haapasalo, Matti Nykter. DNA methylation analysis reveals epigenetic regulation of neural differentiation in AT/RTs [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr LB-173.

Prmt4-mediated methylation of NF-κB is critical for neural differentiation of embryonic stem cells

Neural differentiation is a complex process regulated by multiple signaling at different regulatory levels. Though great progresses have been made in understanding the mechanisms of neural differentiation, post-translational regulation of neural differentiation remains largely unknown. In this study, we found Prmt4, one of the methyltransferases catalyzing protein arginine methylation, is highly expressed in neural stem cells (NSCs) and associated with neural differentiation. Knockout of Prmt4 in mESCs blocked neural differentiation by inhibiting NF-κB activation. Mechanistically, Prmt4 interacts with NF-κB component p65 to promote its methylation, resulting in increased activation of NF-κB signaling during neural differentiation. Our study not only identified Prmt4 as novel regulator of neural differentiation, but also highlighted the importance of protein arginine methylation in cell fate transition.

Исследование роли нерецепторной тирозинкиназы c-Abl в регуляции р53-зависимой активации ERK1/2-зависимого каскада

Protein p53 is a transcription factor that induces aging, cell cycle arrest and apoptosis in response to cell stress. Published findings suggest that proapoptotic protein p53 is involved not only in apoptosis, but also in the regulation of neural differentiation along with the activation of ERK1/2 signaling cascade. However, the nature and the mechanisms of p53 effects on ERK1/2 are not completely understood. Here, we examine the intracellular mechanisms underlying p53-dependent activation of ERK1/2. The study was performed on undifferentiated PC12 cells. For short-term activation of TrkA receptors the cells were incubated with NGF. Our results demonstrate that p53 activation with Nutlin-3 results in the increase of TrkA/cRaf/ERK cascade and c-Abl activity. c-Abl inhibition under p53 activation had no effect on TrkA receptors, but was associated with enhanced activity of cRaf and ERK1/2. At the same time, combined application of Nutlin-3 with c-Abl and TrkA inhibitors evoked considerable decrease of ERK1/2 activity. Our results suggest that p53 activates ERK1/2 cascade either in complex with c-Abl or in c-Abl-independent manner by modulating the c-Raf or upstream targets.

Regulation of neural differentiation, synaptic scaling and animal behavior by MeCP2 phophorylation.

Highly expressed in the mammalian brain and widely distributed across the genome, MeCP2 is a key player in recognizing modified DNA and interpreting the epigenetic information encoded in different DNA methylation/hydroxymethylation patterns. Alterations in sequence or copy number of the X-linked human MECP2 gene cause either Rett syndrome (RTT) or MECP2 duplication syndrome. Alterations in MECP2 levels have also been identified in patients with autism. To fully understand the significant role of MECP2 in regulating the development and function of the nervous system, it is important to study all aspects of MeCP2 function. Stimulus-induced MeCP2 phosphorylation has been shown to influence the proliferation and differentiation of neural progenitor cells, synaptic scaling, excitatory synaptogenesis, and animal behavior. However, all of the previous functional evidence is from studying phospho-dead mutations. In addition, the relationship between phosphorylation events at multiple sites on the MeCP2 protein is not well understood. Here, we report the generation of a phospho-mimic knockin Mecp2 mouse line. At the synaptic and behavioral levels, the phospho-mimic Mecp2 mice show phenotypes opposite to those observed in phospho-dead mutation at the same phosphorylation site. Moreover, we report opposite phenotypes between phospho-mutants of two sites on the MeCP2 protein. Our new data further confirm the functional significance of specific MeCP2 phosphorylation event and support the opposing regulatory role between different MeCP2 phosphorylation events.

Regulation of neural differentiation and synaptogenic factors by silencing of ubiquitin ligase Dorfin

Regulation of neural differentiation and synaptogenic factors by silencing of ubiquitin ligase Dorfin

Can irisin be a linker between physical activity and brain function?

Irisin was initially discovered as a novel hormone-like myokine released from skeletal muscle during exercise to improve obesity and glucose dysfunction by stimulating the browning of white adipose tissue. Emerging evidence have indicated that irisin also affects brain function. FNDC5 mRNA and FNDC5/irisin immunoreactivity are present in various regions of the brain. Central irisin is involved in the regulation of neural differentiation and proliferation, neurobehavior, energy expenditure and cardiac function. Elevation of peripheral irisin level stimulates hippocampal genes related to neuroprotection, learning and memory. In this brief review, we summarize the current understanding on neuronal functions of irisin. In addition, we discuss the pros and cons for this molecule as a potential messenger mediating the crosstalk between skeletal muscle and central nervous system during exercise.

Human Parthenogenetic Embryonic Stem Cell-Derived Neural Stem Cells Express HLA-G and Show Unique Resistance to NK Cell-Mediated Killing.

Parent-of-origin imprints have been implicated in the regulation of neural differentiation and brain development. Previously we have shown that, despite the lack of a paternal genome, human parthenogenetic (PG) embryonic stem cells (hESCs) can form proliferating neural stem cells (NSCs) that are capable of differentiation into physiologically functional neurons while maintaining allele-specific expression of imprinted genes. Since biparental ("normal") hESC-derived NSCs (N NSCs) are targeted by immune cells, we characterized the immunogenicity of PG NSCs. Flow cytometry and immunocytochemistry revealed that both N NSCs and PG NSCs exhibited surface expression of human leukocyte antigen (HLA) class I but not HLA-DR molecules. Functional analyses using an in vitro mixed lymphocyte reaction assay resulted in less proliferation of peripheral blood mononuclear cells (PBMC) with PG compared with N NSCs. In addition, natural killer (NK) cells cytolyzed PG less than N NSCs. At a molecular level, expression analyses of immune regulatory factors revealed higher HLA-G levels in PG compared with N NSCs. In line with this finding, MIR152, which represses HLA-G expression, is less transcribed in PG compared with N cells. Blockage of HLA-G receptors ILT2 and KIR2DL4 on natural killer cell leukemia (NKL) cells increased cytolysis of PG NSCs. Together this indicates that PG NSCs have unique immunological properties due to elevated HLA-G expression.

Heparan sulfate in the regulation of neural differentiation and glioma development.

Heparan sulfate proteoglycans (HSPGs) are the main components of the extracellular matrix, where they interact with a large number of physiologically important macromolecules. The sulfation pattern of heparan sulfate (HS) chains determines the interaction potential of the proteoglycans. Enzymes of the biosynthetic and degradation pathways for HS chains are thus important regulators in processes ranging from embryonic development to tissue homeostasis, but also for tumor development. Formation of the nervous system is also critically dependent on intact HSPGs, and several studies have outlined the role of HS in neural induction from embryonic stem cells. High-grade glioma is the most common malignant primary brain tumor among adults, and the outcome is poor. Neural stem cells and glioma stem cells have several common traits, such as sustained proliferation and a highly efficient migratory capacity in the brain. There are also similarities between the neurogenic niche where adult neural stem cells reside, and the tumorigenic niche. These include interactions with the extracellular matrix, and many of the matrix components are deregulated in glioma, e.g. HSPGs and enzymes implementing the biosynthesis and modification of HS. In this article, we will present how HS-regulated pathways are involved in neural differentiation, and discuss their impact on brain development. We will also review and critically discuss the important role of structural modifications of HS in glioma growth and invasion. We propose that targeting invasive mechanisms of glioma cells through modulation of HS structure and HS-mediated pathways may be an attractive alternative to other therapeutic attempts, which so far have only marginally increased survival for glioma patients.

The REST gene signature predicts drug sensitivity in neuroblastoma cell lines and is significantly associated with neuroblastoma tumor stage.

Neuroblastoma is the most common and deadly solid tumor in children, and there is currently no effective treatment available for neuroblastoma patients. The repressor element-1 silencing transcription (REST) factor has been found to play important roles in the regulation of neural differentiation and tumorigenesis. Recently, a REST signature consisting of downstream targets of REST has been reported to have clinical relevance in both breast cancer and glioblastoma. However it remains unclear how the REST signature works in neuroblastoma. Publicly available datasets were mined and bioinformatic approaches were used to investigate the utility of the REST signature in neuroblastoma with both preclinical and real patient data. The REST signature was found to be associated with drug sensitivity in neuroblastoma cell lines. Further, neuroblastoma patients with enhanced REST activity are significantly associated with higher clinical stages. Loss of heterozygosity on chromosome 11q23, which occurs in a large subset of high-risk neuroblastomas, tends to be correlated with high REST activity, with marginal significance. In conclusion, the REST signature has important implications for targeted therapy, and it is a prognostic factor in neuroblastoma patients.

MicroRNA: Novel Modulators of the Cholinergic Anti-Inflammatory Pathway

MicroRNAs (miRNAs) have emerged as key gene regulators controlling the expression of many target mRNAs. The nervous system harbors highest number of miRNAs expressed in a spatially and temporally controlled manner. Neural miRNAs have been accredited with diverse roles like regulation of neural differentiation, synaptogenesis, inflammation, memory and cognition. Their aberrant expression and/or function has been linked to various neurodegenerative, neuroinflammatory and stress related disorders. Recent evidence indicates that miRNAs are essential to the fine tuning of the immune responses. Besides controlling the maturation, proliferation and differentiation of myeloid and lymphoid lineages they participate directly by modulating the signaling pathways through the Toll-like receptors and thus the cytokine response. The miRNAs commuting between the nervous and immune systems and affecting the neuro-immune dialogue are emerging.

Implications of purinergic receptor-mediated intracellular calcium transients in neural differentiation

Purinergic receptors participate, in almost every cell type, in controlling metabolic activities and many physiological functions including signal transmission, proliferation and differentiation. While most of P2Y receptors induce transient elevations of intracellular calcium concentration by activation of intracellular calcium pools and forward these signals as waves which can also be transmitted into neighboring cells, P2X receptors produce calcium spikes which also include activation of voltage-operating calcium channels. P2Y and P2X receptors induce calcium transients that activate transcription factors responsible for the progress of differentiation through mediators including calmodulin and calcineurin. Expression of P2X2 as well as of P2X7 receptors increases in differentiating neurons and glial cells, respectively. Gene expression silencing assays indicate that these receptors are important for the progress of differentiation and neuronal or glial fate determination. Metabotropic receptors, mostly P2Y1 and P2Y2 subtypes, act on embryonic cells or cells at the neural progenitor stage by inducing proliferation as well as by regulation of neural differentiation through NFAT translocation. The scope of this review is to discuss the roles of purinergic receptor-induced calcium spike and wave activity and its codification in neurodevelopmental and neurodifferentiation processes.

Identification and Characterisation of the Early Differentiating Cells in Neural Differentiation of Human Embryonic Stem Cells

One of the challenges in studying early differentiation of human embryonic stem cells (hESCs) is being able to discriminate the initial differentiated cells from the original pluripotent stem cells and their committed progenies. It remains unclear how a pluripotent stem cell becomes a lineage-specific cell type during early development, and how, or if, pluripotent genes, such as Oct4 and Sox2, play a role in this transition. Here, by studying the dynamic changes in the expression of embryonic surface antigens, we identified the sequential loss of Tra-1-81 and SSEA4 during hESC neural differentiation and isolated a transient Tra-1-81(−)/SSEA4(+) (TR−/S4+) cell population in the early stage of neural differentiation. These cells are distinct from both undifferentiated hESCs and their committed neural progenitor cells (NPCs) in their gene expression profiles and response to extracellular signalling; they co-express both the pluripotent gene Oct4 and the neural marker Pax6. Furthermore, these TR−/S4+ cells are able to produce cells of both neural and non-neural lineages, depending on their environmental cues. Our results demonstrate that expression of the pluripotent factor Oct4 is progressively downregulated and is accompanied by the gradual upregulation of neural genes, whereas the pluripotent factor Sox2 is consistently expressed at high levels, indicating that these pluripotent factors may play different roles in the regulation of neural differentiation. The identification of TR-S4+ cells provides a cell model for further elucidation of the molecular mechanisms underlying hESC neural differentiation.

Gating neural development and aging via nuclear pores

Emerging evidence suggests an involvement of nuclear pore components in the regulation of neural differentiation and aging. These findings will have far-ranging impacts on the understanding of the function of the nuclear envelope in physiological settings and in various neurological diseases.

Nuclear Factor of Activated T Cells (NFAT) Proteins Repress Canonical Wnt Signaling via Its Interaction with Dishevelled (Dvl) Protein and Participate in Regulating Neural Progenitor Cell Proliferation and Differentiation

The Ca(2+) signaling pathway appears to regulate the processes of the early development through its antagonism of canonical Wnt/β-catenin signaling pathway. However, the underlying mechanism is still poorly understood. Here, we show that nuclear factor of activated T cells (NFAT), a component of Ca(2+) signaling, interacts directly with Dishevelled (Dvl) in a Ca(2+)-dependent manner. A dominant negative form of NFAT rescued the inhibition of the Wnt/β-catenin pathway triggered by the Ca(2+) signal. NFAT functioned downstream of β-catenin without interfering with its stability, but influencing the interaction of β-catenin with Dvl by its competitively binding to Dvl. Furthermore, we demonstrate that NFAT is a regulator in the proliferation and differentiation of neural progenitor cells by modulating canonical Wnt/β-catenin signaling pathway in the neural tube of chick embryo. Our findings suggest that NFAT negatively regulates canonical Wnt/β-catenin signaling by binding to Dvl, thereby participating in vertebrate neurogenesis.

Complement expression in mice throughout neural tube closure

A role for the complement cascade in mammalian neural development is becoming apparent. There is evidence that complement factors 3a (C3a) and 5a (C5a), are regulators of neural differentiation and proliferation. Our lab has also demonstrated a very high incidence of neural tube defects in mice lacking C5a receptor (C5aR) signaling under maternal folate-deficient conditions. We are currently investigating specific complement factor and receptor knockout mice in our folate-deficiency model to elucidate their particular functional roles. These results support the hypothesis of a novel role for C3a and C5a in foetal development, with selective localisation to neural proliferative zones, and, in particular, a crucial developmental role following dietary folate deficiency.

Xenopus RCOR2 (REST corepressor 2) interacts with ZMYND8, which is involved in neural differentiation

Regulation of neuronal gene expression is critical to nervous system development. REST (RE1-silencing transcription factor) regulates neuronal gene expression through interacting with a group of corepressor proteins including REST corepressors (RCOR). Here we show that Xenopus RCOR2 is predominantly expressed in the developing nervous system. Through a yeast two-hybrid screen, we isolated Xenopus ZMYND8 (Zinc finger and MYND domain containing 8) as an XRCOR2 interacting factor. XRCOR2 and XZMYND8 bind each other in co-immunoprecipitation assays and both of them can function as transcriptional repressors. XZMYND8 is co-expressed with XRCOR2 in the nervous system and overexpression of XZMYND8 inhibits neural differentiation in Xenopus embryos. These data reveal a RCOR2/ZMYND8 complex which might be involved in the regulation of neural differentiation.

MicroRNAs and glioblastoma; the stem cell connection

Recent data draw close parallels between cancer, including glial brain tumors, and the biology of stem and progenitor cells. At the same time, it has become clear that one of the major roles that microRNAs play is in the regulation of stem cell biology, differentiation, and cell 'identity'. For example, microRNAs have been increasingly implicated in the regulation of neural differentiation. Interestingly, initial studies in the incurable brain tumor glioblastoma multiforme strongly suggest that microRNAs involved in neural development play a role in this disease. This encourages the idea that certain miRs allow continued tumor growth through the suppression of differentiation and the maintenance of the stem cell-like properties of tumor cells. These concepts will be explored in this article.

Inhibition of neurogenesis by SRp38, a neuroD-regulated RNA-binding protein

Although serine-arginine rich (SR) proteins have often been implicated in the positive regulation of splicing, recent studies have shown that one unusual SR protein, SRp38, serves, contrastingly, as a splicing repressor during mitosis and stress response. We have identified a novel developmental role for SRp38 in the regulation of neural differentiation. SRp38 is expressed in the neural plate during embryogenesis and is transcriptionally induced by the neurogenic bHLH protein neuroD. Overexpression of SRp38 inhibits primary neuronal differentiation at a step between neurogenin and neuroD activity. This repression of neuronal differentiation requires activation of the Notch pathway. Conversely, depletion of SRp38 activity results in a dysregulation of neurogenesis. Finally, SRp38 can interact with the peptidyltransferase center of 28S rRNA, suggesting that SRp38 activity may act, in part, via regulation of ribosome biogenesis or function. Strikingly, recent studies of several cell cycle regulators during primary neurogenesis have also revealed a crucial control step between neurogenin and neuroD. SRp38 may mediate one component of this control by maintaining splicing and translational silencing in undifferentiated neural cells.

Regulation of neural differentiation by normal and mutant (G654A, amyloidogenic) gelsolin.

Gelsolin belongs to a family of proteins that modulate the structural dynamics of cytoskeletal actin. Gelsolin activity is required for the redistribution of actin occurring during membrane ruffling, cell crawling, and platelet activation. A point mutation (G654A) in the gelsolin gene causes a dominantly inherited systemic amyloidosis called familial amyloidosis of the Finnish type (FAF). This disease is characterized by a cranial neuropathy that cannot be explained solely by amyloid deposits. To address the question of whether gelsolin has a specific role in neural cell development, we transfected cDNA for wild type and G654A point-mutated gelsolin into a neural cell line, Paju, which can be induced to differentiate by treatment with phorbol 12-myristate 13-acetate. Overexpressed wild type gelsolin inhibited neural differentiation whereas mutated gelsolin did not, indicating that appropriate gelsolin activity is essential for neural sprouting. The G654A mutant gelsolin induced stabilization of F-actin and reduced the plasticity of neural development. This provides a novel etiopathogenetic mechanism for the neuronal dysfunction in FAF.