The human Schwann cell transcriptome: species-specificity, long-term stability and changes with differentiation

Cultured Schwann cells of human origin differ from those isolated from experimental animals in both phenotype and function. However, the basis for this divergence and its significance to potential clinical applications of the primary cells are not fully understood. In this study, we used RNA-seq to comprehensively analyze the human Schwann cell transcriptome and compare it to that of ratcells. We also studied the transcriptomics profiles of human Schwann cells subjected to: (1) the pro-mitogenic effect of growth factors in cells undergoing serial passaging in vitro, and (2) the pro-differentiating action of cAMP, a signal known to promote myelin gene expression in rodent cells.Despite the human Schwann cell transcriptome differedas much as 44% from that of rat Schwann cells established under identical conditions, the human cells maintained their expected Schwann cell identity regardless of sub-culture and the continued influence of mitogenic factors. Strikingly, the transcriptomes of low passage (proliferative) and late passage (senescent) human Schwann cells were essentially undistinguishable with the exception of roughly 100 differentially expressed genes in the senescentpopulations. On the contrary, the human Schwann cell transcriptome was readily and persistently shifted in response to a single treatment with cAMP analogs as highlighted by the >1,300 genes that were upregulated and the >1,700 genes that were downregulated within 1-day post-stimulation. In sum, these results confirmed that human Schwann cellsmaintain their typical gene expression profiles in culture unless challenged with a strong pro-differentiating stimulus. The observed stability of the human Schwann celltranscriptome in the face of expansion and mitogenic stimulation adds a level of safety for theuse of these glial cells in clinical transplantation.Fil: Monje, Paula. Indiana University; Estados Unidos. University of Miami; Estados UnidosFil: Sant, David. University of Miami; Estados UnidosFil: Andersen, Natalia Denise. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Bahía Blanca. Instituto de Investigaciones Bioquímicas de Bahía Blanca. Universidad Nacional del Sur. Instituto de Investigaciones Bioquímicas de Bahía Blanca; ArgentinaFil: Camarena, Vladimir. University of Miami; Estados UnidosFil: Wang, Gaofeng. University of Miami; Estados UnidosXIV European Meeting on Glial cells in Health and DiseasePortoPortugalEuropean Meeting on Glial Cells in Health and Diseas

Magnetic separation of peripheral nerve-resident cells underscores key molecular features of human Schwann cells and fibroblasts: an immunochemical and transcriptomics approach

Nerve-derived human Schwann cell (SC) cultures are irreplaceable models for basic and translational research but their use can be limited due to the risk of fibroblast overgrowth. Fibroblasts are an ill-defined population consisting of highly proliferative cells that, contrary to human SCs, do not undergo senescence in culture. We initiated this study by performing an exhaustive immunological and functional characterization of adult nerve-derived human SCs and fibroblasts to reveal their properties and optimize a protocol of magnetic-activated cell sorting (MACS) to separate them effectively both as viable and biologically competent cells. We next used immunofluorescence microscopy imaging, flow cytometry analysis and next generation RNA sequencing (RNA-seq) to unambiguously characterize the post-MACS cell products. High resolution transcriptome profiling revealed the identity of key lineage-specific transcripts and the clearly distinct neural crest and mesenchymal origin of human SCs and fibroblasts, respectively. Our analysis underscored a progenitor- or stem cell-like molecular phenotype in SCs and fibroblasts and the heterogeneity of the fibroblast populations. In addition, pathway analysis of RNA-seq data highlighted putative bidirectional networks of fibroblast-to-SC signaling that predict a complementary, yet seemingly independent contribution of SCs and fibroblasts to nerve regeneration. In sum, combining MACS with immunochemical and transcriptomics approaches provides an ideal workflow to exhaustively assess the identity, the stage of differentiation and functional features of highly purified cells from human peripheral nerve tissues.Fil: Peng, Kaiwen. Indiana University. School of Medicine; Estados Unidos. Nanfang Hospital; ChinaFil: Sant, David. University of Utah; Estados Unidos. Miami University. School of Medicine; Estados UnidosFil: Andersen, Natalia Denise. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Bahía Blanca. Instituto de Investigaciones Bioquímicas de Bahía Blanca. Universidad Nacional del Sur. Instituto de Investigaciones Bioquímicas de Bahía Blanca; Argentina. Miami University. School of Medicine; Estados UnidosFil: Silvera, Risset. Miami University. School of Medicine; Estados UnidosFil: Camarena, Vladimir. Miami University. School of Medicine; Estados UnidosFil: Piñero, Gonzalo Miguel. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Química y Físico-Química Biológicas "Prof. Alejandro C. Paladini". Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Instituto de Química y Físico-Química Biológicas; Argentina. Miami University. School of Medicine; Estados UnidosFil: Graham, Regina. Miami University. School of Medicine; Estados UnidosFil: Khan, Aisha. Miami University. School of Medicine; Estados UnidosFil: Xu, Xiao Ming. Indiana University. School of Medicine; Estados UnidosFil: Wang, Gaofeng. Miami University. School of Medicine; Estados UnidosFil: Monje, Paula. Indiana University. School of Medicine; Estados Unidos. Miami University. School of Medicine; Estados Unido

Vitamin C regulates Schwann cell myelination by promoting DNA demethylation of pro-myelinating genes

Ascorbic acid (vitamin C) is critical for Schwann cells to myelinate peripheral nerve axons during development and remyelination after injury. However, its exact mechanism remains elusive. Vitamin C is a dietary nutrient that was recently discovered to promote active DNA demethylation. Schwann cell myelination is characterized by global DNA demethylation in vivo and may therefore be regulated by vitamin C. We found that vitamin C induces a massive transcriptomic shift (n = 3,848 genes) in primary cultured Schwann cells while simultaneously producing a global increase in genomic 5-hydroxymethylcytosine (5hmC), a DNA demethylation intermediate which regulates transcription. Vitamin C up-regulates 10 pro-myelinating genes which exhibit elevated 5hmC content in both the promoter and gene body regions of these loci following treatment. Using a mouse model of human vitamin C metabolism, we found that maternal dietary vitamin C deficiency causes peripheral nerve hypomyelination throughout early development in resulting offspring. Additionally, dietary vitamin C intake regulates the expression of myelin-related proteins such as periaxin (PRX) and myelin basic protein (MBP) during development and remyelination after injury in mice. Taken together, these results suggest that vitamin C cooperatively promotes myelination through 1) increased DNA demethylation and transcription of pro-myelinating genes, and 2) its known role in stabilizing collagen helices to form the basal lamina that is necessary for myelination

Oscillatory cAMP signaling rapidly alters H3K4 methylation

receptors (GPCRs) alter H3K4 methylation via oscillatory intracellular cAMP. Activation of Gs-coupled receptors caused a rapid decrease of H3K4me3 by elevating cAMP, whereas stimulation of Gi-coupled receptors increased H3K4me3 by diminishing cAMP. H3K4me3 gradually recovered towards baseline levels after the removal of GPCR ligands, indicating that H3K4me3 oscillates in tandem with GPCR activation. cAMP increased intracellular labile Fe(II), the cofactor for histone demethylases, through a non-canonical cAMP target—Rap guanine nucleotide exchange factor-2 (RapGEF2), which subsequently enhanced endosome acidification and Fe(II) release from the endosome via vacuolar H+-ATPase assembly. Removing Fe(III) from the media blocked intracellular Fe(II) elevation after stimulation of Gs-coupled receptors. Iron chelators and inhibition of KDM5 demethylases abolished cAMP-mediated H3K4me3 demethylation. Taken together, these results suggest a novel function of cAMP signaling in modulating histone demethylation through labile Fe(II)

Partitioning the Heritability of Tourette Syndrome and Obsessive Compulsive Disorder Reveals Differences in Genetic Architecture

The direct estimation of heritability from genome-wide common variant data as implemented in the program Genome-wide Complex Trait Analysis (GCTA) has provided a means to quantify heritability attributable to all interrogated variants. We have quantified the variance in liability to disease explained

Nerve growth factor signaling maintains herpes simplex virus latency

Many viruses infect the nervous system, where they either multiply or become silent in neurons. The decision to become latent versus lytic is not well understood. One prevalent virus in human population is Herpes simplex virus-1 (HSV-1) that establishes a life-long latent infection in peripheral neurons where productive replication is suppressed. When HSV is activated and multiplied, a range of diseases can ensue, which may lead to blisters or to more serious consequences, such inflammation in the brain or blindness. One factor that has hindered the development of new therapies against HSV latent state is the lack of understanding of cellular mechanisms that regulate the latent state. In our studies, we improved an in vitro system for viral latency in primary neurons to investigate the factors that keep HSV from multiplying. We find that nerve growth factor is essential to keep the virus latent. We show that continuous signaling through the PI3-kinase (PI3-K) pathway triggered by NGF-binding to the TrkA receptor tyrosine kinase (RTK) is instrumental to maintain latent HSV-1 in primary neuron cultures. Activity of the PI3-K p110α catalytic subunit, but not the β or δ isoforms, is specifically required to activate PDK1 and sustain latency. Disrupting this pathway, even transiently, using chemical inhibitors or RNA interference leads to reactivation. Surprisingly, EGF and GDNF, two other growth factors capable of activating PI3-K and PDK1, differ from NGF in their ability to persistently activate Akt and do not fully support HSV-1 latency. Thus the duration and intensity of RTK-signaling are critical parameters imposed by the host to regulate the HSV-1 latent-lytic switch. This work sheds light on how latency is regulated by growth factors in the nervous system, which in turn could help to develop new targeted therapies against HSV activation

Full Resistance of Herpes Simplex Virus Type 1-Infected Primary Human Cells to Alpha Interferon Requires both the Us11 and γ(1)34.5 Gene Products

The γ(1)34.5 gene product is important for the resistance of herpes simplex virus type 1 (HSV-1) to interferon. However, since the inhibition of protein synthesis observed in cells infected with a γ(1)34.5 mutant virus results from the combined loss of the γ(1)34.5 gene product and the failure to translate the late Us11 mRNA, we sought to characterize the relative interferon sensitivity of mutants unable to produce either the Us11 or the γ(1)34.5 polypeptide. We now demonstrate that primary human cells infected with a Us11 mutant virus are hypersensitive to alpha interferon, arresting translation upon entry into the late phase of the viral life cycle. Furthermore, immediate-early expression of Us11 by a γ(1)34.5 deletion mutant is sufficient to render translation resistant to alpha interferon. Finally, we establish that the Us11 gene product is required for wild-type levels of replication in alpha interferon-treated cells and, along with the γ(1)34.5 gene, is an HSV-1-encoded interferon resistance determinant

Epigenomic regulation by labile iron

Iron is an essential micronutrient metal for cellular functions but can generate highly reactive oxygen species resulting in oxidative damage. For these reasons its uptake and metabolism is highly regulated. A small but dynamic fraction of ferrous iron inside the cell, termed intracellular labile iron, is redox-reactive and ready to participate multiples reactions of intracellular enzymes. Due to its nature its determination and precise quantification has been a roadblock. However, recent progress in the development of intracellular labile iron probes are allowing the reevaluation of our current understanding and unmasking new functions. The role of intracellular labile iron in regulating the epigenome was recently discovered. This chapter examine how intracellular labile iron can modulate histone and DNA demethylation and how its pool can mediate a signaling pathway from cAMP serving as a sensor of the metabolic needs of the cells. [Display omitted] •Labile Fe(II) is redox-reactive, dynamic and required for enzymatic reactions.•Labile Fe(II), as an essential cofactor, is involved in the demethylation of DNA and histones.•Diseases that alter the intracellular labile Fe(II) pool can affect the epigenome.•cAMP signaling promotes DNA and histone demethylation by increasing intracellular labile Fe(II) pool