Experimental verification of a conserved intronic microRNA located in the human <Emphasis Type="Italic">TrkC</Emphasis> gene with a cell type-dependent apoptotic function

Tropomyosin receptor kinase C (TrkC) is involved in cell survival, apoptosis induction and tumorigenesis. We hypothesized that, similar to p75^NTR receptor, some of the diverse functions of TrkC could be mediated by a microRNA (miRNA) embedded within the gene. Here, we experimentally verified the expression and processing of two bioinformatically predicted miRNAs named TrkC-miR1-5p and TrkC-miR1-3p. Transfecting a DNA fragment corresponding to the TrkC-premir1 sequence in HEK293t cells caused ~300-fold elevation in the level of mature TrkC-miR1 and also a significant downregulation of its predicted target genes. Furthermore, endogenous TrkC-miR1 was detected in several cell lines and brain tumors confirming its endogenous generation. Furthermore, its orthologous miRNA was detected in developing rat brain. Accordingly, TrkC-miR1 expression was increased during the course of neural differentiation of NT2 cell, whereas its suppression attenuated NT2 differentiation. Consistent with opposite functions of TrkC, TrkC-miR1 overexpression promoted survival and apoptosis in U87 and HEK293t cell lines, respectively. In conclusion, our data report the discovery of a new miRNA with overlapping function to TrkC.     

Retinoic acid regulates avian lung branching through a molecular network

Retinoic acid (RA) is of major importance during vertebrate embryonic development and its levels need to be strictly regulated otherwise congenital malformations will develop. Through the action of specific nuclear receptors, named RAR/RXR, RA regulates the expression of genes that eventually influence proliferation and tissue patterning. RA has been described as crucial for different stages of mammalian lung morphogenesis, and as part of a complex molecular network that contributes to precise organogenesis; nonetheless, nothing is known about its role in avian lung development. The current report characterizes, for the first time, the expression pattern of RA signaling members (stra6, raldh2, raldh3, cyp26a1, rarα, and rarβ) and potential RA downstream targets (sox2, sox9, meis1, meis2, tgfβ2, and id2) by in situ hybridization. In the attempt of unveiling the role of RA in chick lung branching, in vitro lung explants were performed. Supplementation studies revealed that RA stimulates lung branching in a dose-dependent manner. Moreover, the expression levels of cyp26a1, sox2, sox9, rarβ, meis2, hoxb5, tgfβ2, id2, fgf10, fgfr2, and shh were evaluated after RA treatment to disclose a putative molecular network underlying RA effect. In situ hybridization analysis showed that RA is able to alter cyp26a1, sox9, tgfβ2, and id2 spatial distribution; to increase rarβ, meis2, and hoxb5 expression levels; and has a very modest effect on sox2, fgf10, fgfr2, and shh expression levels. Overall, these findings support a role for RA in the proximal–distal patterning and branching morphogenesis of the avian lung and reveal intricate molecular interactions that ultimately orchestrate branching morphogenesis.     

Neural stem cells in Parkinson’s disease: a role for neurogenesis defects in onset and progression

Parkinson’s disease (PD) is the second most common neurodegenerative disorder, leading to a variety of motor and non-motor symptoms. Interestingly, non-motor symptoms often appear a decade or more before the first signs of motor symptoms. Some of these non-motor symptoms are remarkably similar to those observed in cases of impaired neurogenesis and several PD-related genes have been shown to play a role in embryonic or adult neurogenesis. Indeed, animal models deficient in Nurr1, Pitx3, SNCA and PINK1 display deregulated embryonic neurogenesis and LRRK2 and VPS35 have been implicated in neuronal development-related processes such as Wnt/β-catenin signaling and neurite outgrowth. Moreover, adult neurogenesis is affected in both PD patients and PD animal models and is regulated by dopamine and dopaminergic (DA) receptors, by chronic neuroinflammation, such as that observed in PD, and by differential expression of wild-type or mutant forms of PD-related genes. Indeed, an increasing number of in vivo studies demonstrate a role for SNCA and LRRK2 in adult neurogenesis and in the generation and maintenance of DA neurons. Finally, the roles of PD-related genes, SNCA, LRRK2, VPS35, Parkin, PINK1 and DJ-1 have been studied in NSCs, progenitor cells and induced pluripotent stem cells, demonstrating a role for some of these genes in stem/progenitor cell proliferation and maintenance. Together, these studies strongly suggest a link between deregulated neurogenesis and the onset and progression of PD and present strong evidence that, in addition to a neurodegenerative disorder, PD can also be regarded as a developmental disorder.     

<Emphasis Type="Italic">Yersinia pseudotuberculosis</Emphasis> supports Th17 differentiation and limits de novo regulatory T cell induction by directly interfering with T cell receptor signaling

Adaptive immunity critically contributes to control acute infection with enteropathogenic Yersinia pseudotuberculosis; however, the role of CD4⁺ T cell subsets in establishing infection and allowing pathogen persistence remains elusive. Here, we assessed the modulatory capacity of Y. pseudotuberculosis on CD4⁺ T cell differentiation. Using in vivo assays, we report that infection with Y. pseudotuberculosis resulted in enhanced priming of IL-17-producing T cells (Th17 cells), whereas induction of Foxp3⁺ regulatory T cells (Tregs) was severely disrupted in gut-draining mesenteric lymph nodes (mLNs), in line with altered frequencies of tolerogenic and proinflammatory dendritic cell (DC) subsets within mLNs. Additionally, by using a DC-free in vitro system, we could demonstrate that Y. pseudotuberculosis can directly modulate T cell receptor (TCR) downstream signaling within naïve CD4⁺ T cells and Tregs via injection of effector molecules through the type III secretion system, thereby affecting their functional properties. Importantly, modulation of naïve CD4⁺ T cells by Y. pseudotuberculosis resulted in an enhanced Th17 differentiation and decreased induction of Foxp3⁺ Tregs in vitro. These findings shed light to the adjustment of the Th17-Treg axis in response to acute Y. pseudotuberculosis infection and highlight the direct modulation of CD4⁺ T cell subsets by altering their TCR downstream signaling.     

Sorting nexin 3 mutation impairs development and neuronal function in <Emphasis Type="Italic">Caenorhabditis elegans</Emphasis>

The sorting nexins family of proteins (SNXs) plays pleiotropic functions in protein trafficking and intracellular signaling and has been associated with several disorders, namely Alzheimer’s disease and Down’s syndrome. Despite the growing association of SNXs with neurodegeneration, not much is known about their function in the nervous system. The aim of this work was to use the nematode Caenorhabditis elegans that encodes in its genome eight SNXs orthologs, to dissect the role of distinct SNXs, particularly in the nervous system. By screening the C. elegans SNXs deletion mutants for morphological, developmental and behavioral alterations, we show here that snx-3 gene mutation leads to an array of developmental defects, such as delayed hatching, decreased brood size and life span and reduced body length. Additionally, ?snx-3 worms present increased susceptibility to osmotic, thermo and oxidative stress and distinct behavioral deficits, namely, a chemotaxis defect which is independent of the described snx-3 role in Wnt secretion. ?snx-3 animals also display abnormal GABAergic neuronal architecture and wiring and altered AIY interneuron structure. Pan-neuronal expression of C. elegans snx-3 cDNA in the ?snx-3 mutant is able to rescue its locomotion defects, as well as its chemotaxis toward isoamyl alcohol. Altogether, the present work provides the first in vivo evidence of the SNX-3 role in the nervous system.     

Serine protease PRSS55 is crucial for male mouse fertility via affecting sperm migration and sperm–egg binding

Testis-specific PRSS55 is a highly conserved chymotrypsin-like serine protease among mammalian species. So far, the physiological function of PRSS55 remains unknown. Here, we show that PRSS55 is a GPI-anchored membrane protein, specifically expressed in adult mouse testis and mainly observed in the luminal side of seminiferous tubules and sperm acrosome. Mice deficient for Prss55 develop male infertile with normal reproduction-related parameters observed. Interestingly, in vivo fertilization rate of Prss55^?/? males is dramatically decreased, possibly due to incapable migration of Prss55^?/? sperm from uterus into oviduct. However, in vitro fertilization rate has no difference between two genotypes although Prss55^?/? sperm presents defective recognition/binding to zona-intact or zona-free oocytes. Further study reveals that mature ADAM3 is almost undetectable in Prss55^?/? sperm, while precursor ADAM3 remains unchanged in the testis. However, it is shown that ADAM3 has no interaction with PRSS55 by immunoprecipitation with anti-PRSS55 antibody. The expression levels of several proteins known to be related to the observed phenotypes remain comparable between wt and Prss55^?/? mice. Moreover, we found that Prss55 deficiency has no effect on PRSS37 or vice versa albeit two mutant males share almost the same phenotypes. Microarray analysis reveals a total of 72 differentially expressed genes in Prss55^?/? testis, most of which are associated with cellular membrane and organelle organization, protein transport and complex assembly, and response to stimulus and signaling. In conclusion, we have demonstrated that PRSS55 plays vital roles in regulating male fertility of mice, including in vivo sperm migration and in vitro sperm–egg interaction, possibly by affecting the maturation of ADAM3 in sperm and the expression of multiple genes in testis.     

Editing porcine <Emphasis Type="Italic">IGF2</Emphasis> regulatory element improved meat production in Chinese Bama pigs

Insulin-like growth factor 2 (IGF2) is an important growth factor, which promotes growth and development in mammals during fetal and postnatal stages. Using CRISPR–Cas9 system, we generated multiple founder pigs containing 12 different mutant alleles around a regulatory element within the intron 3 of IGF2 gene. Crossing two male founders passed four mutant alleles onto F1 generation, and these mutations abolished repressor ZBED6 binding and rendered this regulatory element nonfunctional. Both founders and F1 animals showed significantly faster growth, without affecting meat quality. These results indicated that editing IGF2 intron 3–3072 site using CRISPR–Cas9 technology improved meat production in Bama pigs. This is the first demonstration that editing non-coding region can improve economic traits in livestock.     

UDP-glucose ceramide glucosyltransferase activates AKT,promoted proliferation,and doxorubicin resistance in breast cancer cells

The UDP-glucose ceramide glucosyltransferase (UGCG) is a key enzyme in the synthesis of glycosylated sphingolipids, since this enzyme generates the precursor for all complex glycosphingolipids (GSL), the GlcCer. The UGCG has been associated with several cancer-related processes such as maintaining cancer stem cell properties or multidrug resistance induction. The precise mechanisms underlying these processes are unknown. Here, we investigated the molecular mechanisms occurring after UGCG overexpression in breast cancer cells. We observed alterations of several cellular properties such as morphological changes, which enhanced proliferation and doxorubicin resistance in UGCG overexpressing MCF-7 cells. These cellular effects seem to be mediated by an altered composition of glycosphingolipid-enriched microdomains (GEMs), especially an accumulation of globotriaosylceramide (Gb3) and glucosylceramide (GlcCer), which leads to an activation of Akt and ERK1/2. The induction of the Akt and ERK1/2 signaling pathway results in an increased gene expression of multidrug resistance protein 1 (MDR1) and anti-apoptotic genes and a decrease of pro-apoptotic gene expression. Inhibition of the protein kinase C (PKC) and phosphoinositide 3 kinase (PI3K) reduced MDR1 gene expression. This study discloses how changes in UGCG expression impact several cellular signaling pathways in breast cancer cells resulting in enhanced proliferation and multidrug resistance.     

Tumor suppressor C-RASSF proteins

Human genome has ten genes that are collectedly called Ras association domain family (RASSF). RASSF is composed of two subclasses, C-RASSF and N-RASSF. Both N-RASSF and C-RASSF encode Ras association domain-containing proteins and are frequently suppressed by DNA hypermethylation in human cancers. However, C-RASSF and N-RASSF are quite different. Six C-RASSF proteins (RASSF1–6) are characterized by a C-terminal coiled-coil motif named Salvador/RASSF/Hippo domain, while four N-RASSF proteins (RASSF7–10) lack it. C-RASSF proteins interact with mammalian Ste20-like kinases—the core kinases of the tumor suppressor Hippo pathway—and cross-talk with this pathway. Some of them share the same interacting molecules such as MDM2 and exert the tumor suppressor role in similar manners. Nevertheless, each C-RASSF protein has distinct characters. In this review, we summarize our current knowledge of how C-RASSF proteins play tumor suppressor roles and discuss the similarities and differences among C-RASSF proteins.     

Defective germinal center B-cell response and reduced arthritic pathology in microRNA-29a-deficient mice

MicroRNA (miR) are short non-coding RNA sequences of 19–24 nucleotides that regulate gene expression by binding to mRNA target sequences. The miR-29 family of miR (miR-29a, b-1, b-2 and c) is a key player in T-cell differentiation and effector function, with deficiency causing thymic involution and a more inflammatory T-cell profile. However, the relative roles of different miR-29 family members in these processes have not been dissected. We studied the immunological role of the individual members of the miR-29 family using mice deficient for miR-29a/b-1 or miR-29b-2/c in homeostasis and during collagen-induced arthritis. We found a definitive hierarchy of immunological function, with the strong phenotype of miR-29a-deficiency in thymic involution and T-cell activation being reduced or absent in miR-29c-deficient mice. Strikingly, despite elevating the Th1 and Th17 responses, loss of miR-29a conferred near-complete protection from collagen-induced arthritis (CIA), with profound defects in B-cell proliferation and antibody production. Our results identify the hierarchical structure of the miR-29 family in T-cell biology, and identify miR-29a in B cells as a potential therapeutic target in arthritis.     

Germline-specific dgcr8 knockout in zebrafish using a BACK approach

Zebrafish is an important model to study developmental biology and human diseases. However, an effective approach to achieve spatial and temporal gene knockout in zebrafish has not been well established. In this study, we have developed a new approach, namely bacterial artificial chromosome-rescue-based knockout (BACK), to achieve conditional gene knockout in zebrafish using the Cre/loxP system. We have successfully deleted the DiGeorge syndrome critical region gene 8 (dgcr8) in zebrafish germ line and demonstrated that the maternal-zygotic dgcr8 (MZdgcr8) embryos exhibit MZdicer-like phenotypes with morphological defects which could be rescued by miR-430, indicating that canonical microRNAs play critical role in early development. Our findings establish that Cre/loxP-mediated tissue-specific gene knockout could be achieved using this BACK strategy and that canonical microRNAs play important roles in early embryonic development in zebrafish.     

Neuroprotective effects of the gliopeptide ODN in an in vivo model of Parkinson’s disease

Parkinson’s disease (PD) is a neurodegenerative disorder characterized by a progressive loss of dopamine (DA) neurons through apoptotic, inflammatory and oxidative stress mechanisms. The octadecaneuropeptide (ODN) is a diazepam-binding inhibitor (DBI)-derived peptide, expressed by astrocytes, which protects neurons against oxidative cell damages and apoptosis in an in vitro model of PD. The present study reveals that a single intracerebroventricular injection of 10 ng ODN 1 h after the last administration of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) prevented the degeneration of DA neurons induced by the toxin in the substantia nigra pars compacta of mice, 7 days after treatment. ODN-mediated neuroprotection was associated with a reduction of the number of glial fibrillary acidic protein-positive reactive astrocytes and a strong inhibition of the expression of pro-inflammatory genes such as interleukins 1β and 6, and tumor necrosis factor-α. Moreover, ODN blocked the inhibition of the anti-apoptotic gene Bcl-2, and the stimulation of the pro-apoptotic genes Bax and caspase-3, induced by MPTP in the substantia nigra pars compacta. ODN also decreased or even in some cases abolished MPTP-induced oxidative damages, overproduction of reactive oxygen species and accumulation of lipid oxidation products in DA neurons. Furthermore, DBI knockout mice appeared to be more vulnerable than wild-type animals to MPTP neurotoxicity. Taken together, these results show that the gliopeptide ODN exerts a potent neuroprotective effect against MPTP-induced degeneration of nigrostriatal DA neurons in mice, through mechanisms involving downregulation of neuroinflammatory, oxidative and apoptotic processes. ODN may, thus, reduce neuronal damages in PD and other cerebral injuries involving oxidative neurodegeneration.     

Necroptosis and ferroptosis are alternative cell death pathways that operate in acute kidney failure

Ferroptosis is a recently recognized caspase-independent form of regulated cell death that is characterized by the accumulation of lethal lipid ROS produced through iron-dependent lipid peroxidation. Considering that regulation of fatty acid metabolism is responsible for the membrane-resident pool of oxidizable fatty acids that undergo lipid peroxidation in ferroptotic processes, we examined the contribution of the key fatty acid metabolism enzyme, acyl-CoA synthetase long-chain family member 4 (ACSL4), in regulating ferroptosis. By using CRISPR/Cas9 technology, we found that knockout of Acsl4 in ferroptosis-sensitive murine and human cells conferred protection from erastin- and RSL3-induced cell death. In the same cell types, deletion of mixed lineage kinase domain-like (Mlkl) blocked susceptibility to necroptosis, as expected. Surprisingly, these studies also revealed ferroptosis and necroptosis are alternative, in that resistance to one pathway sensitized cells to death via the other pathway. These data suggest a mechanism by which one regulated necrosis pathway compensates for another when either ferroptosis or necroptosis is compromised. We verified the synergistic contributions of ferroptosis and necroptosis to tissue damage during acute organ failure in vivo. Interestingly, in the course of pathophysiological acute ischemic kidney injury, ACSL4 was initially upregulated and its expression level correlated with the severity of tissue damage. Together, our findings reveal ACSL4 to be a reliable biomarker of the emerging cell death modality of ferroptosis, which may also serve as a novel therapeutic target in preventing pathological cell death processes.     

<Emphasis Type="Italic">O</Emphasis>-GlcNAc transferase associates with the MCM2–7 complex and its silencing destabilizes MCM–MCM interactions

O-GlcNAcylation of proteins is governed by O-GlcNAc transferase (OGT) and O-GlcNAcase (OGA). The homeostasis of O-GlcNAc cycling is regulated during cell cycle progression and is essential for proper cellular division. We previously reported the O-GlcNAcylation of the minichromosome maintenance proteins MCM2, MCM3, MCM6 and MCM7. These proteins belong to the MCM2–7 complex which is crucial for the initiation of DNA replication through its DNA helicase activity. Here we show that the six subunits of MCM2–7 are O-GlcNAcylated and that O-GlcNAcylation of MCM proteins mainly occurs in the chromatin-bound fraction of synchronized human cells. Moreover, we identify stable interaction between OGT and several MCM subunits. We also show that down-regulation of OGT decreases the chromatin binding of MCM2, MCM6 and MCM7 without affecting their steady-state level. Finally, OGT silencing or OGA inhibition destabilizes MCM2/6 and MCM4/7 interactions in the chromatin-enriched fraction. In conclusion, OGT is a new partner of the MCM2–7 complex and O-GlcNAcylation homeostasis might regulate MCM2–7 complex by regulating the chromatin loading of MCM6 and MCM7 and stabilizing MCM/MCM interactions.