Placenta 35 (2014) 44e49

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The long non-coding RNA NEAT1 is increased in IUGR placentas, leading to potential new hypotheses of IUGR origin/development S. Gremlich a, *, F. Damnon b, D. Reymondin b, O. Braissant c, J.C. Schittny d, D. Baud b, S. Gerber b, M. Roth-Kleiner a a

Clinic of Neonatology, University Hospital and University of Lausanne, Switzerland Department of Gynecology, Obstetrics and Medical Genetics, University Hospital and University of Lausanne, Switzerland Service of Biomedicine, University Hospital and University of Lausanne, Switzerland d Institute of Anatomy, University of Bern, Switzerland b c

a r t i c l e i n f o

a b s t r a c t

Article history: Accepted 5 November 2013

Introduction: Intrauterine Growth Restriction (IUGR) is a multifactorial disease defined by an inability of the fetus to reach its growth potential. IUGR not only increases the risk of neonatal mortality/morbidity, but also the risk of metabolic syndrome during adulthood. Certain placental proteins have been shown to be implicated in IUGR development, such as proteins from the GH/IGF axis and angiogenesis/apoptosis processes. Methods: Twelve patients with term IUGR pregnancy (birth weight < 10th percentile) and 12 CTRLs were included. mRNA was extracted from the fetal part of the placenta and submitted to a subtraction method (Clontech PCR-Select cDNA Subtraction). Results: One candidate gene identified was the long non-coding RNA NEAT1 (nuclear paraspeckle assembly transcript 1). NEAT1 is the core component of a subnuclear structure called paraspeckle. This structure is responsible for the retention of hyperedited mRNAs in the nucleus. Overall, NEAT1 mRNA expression was 4.14 (
1.16)-fold increased in IUGR vs. CTRL placentas (P ¼ 0.009). NEAT1 was exclusively localized in the nuclei of the villous trophoblasts and was expressed in more nuclei and with greater intensity in IUGR placentas than in CTRLs. PSPC1, one of the three main proteins of the paraspeckle, colocalized with NEAT1 in the villous trophoblasts. The expression of NEAT1_2 mRNA, the long isoform of NEAT1, was only modestly increased in IUGR vs. CTRL placentas. Discussion/conclusion: The increase in NEAT1 and its co-localization with PSPC1 suggests an increase in paraspeckles in IUGR villous trophoblasts. This could lead to an increased retention of important mRNAs in villous trophoblasts nuclei. Given that the villous trophoblasts are crucial for the barrier function of the placenta, this could in part explain placental dysfunction in idiopathic IUGR fetuses. Ó 2013 Elsevier Ltd. All rights reserved.

Keywords: Trophoblast Non-coding RNA IUGR

1. Introduction Intrauterine Growth Restriction (IUGR) is a fetal multifactorial disease biologically defined by an inability of the fetus to reach its growth potential. IUGR increases neonatal mortality/morbidity, as well as the risk of metabolic syndrome during adulthood, a syndrome which prefigures diabetes, hypertension or cardiovascular problems [1e3]. Known risk factors for IUGR can be of maternal, fetal or placental origin [4]. However, an important proportion of IUGR cases remains idiopathic and probably arises secondary to placental insufficiency [5,6]. * Corresponding author. Clinic of Neonatology, University Hospital and University of Lausanne, Av. Pierre-Decker 2, 1011 Lausanne, Switzerland. Tel.: þ41 21 314 33 26. E-mail address: [email protected] (S. Gremlich). 0143-4004/$ e see front matter Ó 2013 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.placenta.2013.11.003

Angiogenesis is crucial for successful placental development. A reduction in angiogenesis was observed in IUGR placentas which was linked to modifications of protein expression of angiogenic factors PlGF/VEGF (Placental Growth Factor/Vascular Endothelial Growth Factor) and anti-angiogenic factor sVEGFR-1/sflt-1 (soluble receptor for VEGF) [7]. Apoptosis is involved in the maintenance of placental barrier integrity and can be detected in the syncytiotrophoblast under normal conditions. Apoptosis was shown to be increased in placentas of IUGR pregnancies [8e10]. This increase was accompanied by a reduction in anti-apoptotic gene Bcl-2 and an increase in pro-apoptotic genes p53 and Bax which still remains controversial [8,9,11,12]. Finally, protein synthesis inhibition secondary to endoplasmic reticulum stress was observed in IUGR placentas, possibly contributing to the small size of the IUGR placentas [13].

S. Gremlich et al. / Placenta 35 (2014) 44e49

Several maternal serum proteins have been associated with IUGR. However, the association of these proteins with IUGR has never been accurate enough to be routinely used in clinical practice [4]. We recently showed that high IGFBP3 (insulin-like growth factor binding protein 3) concentrations in amniotic fluid at the beginning of the second trimester were associated with an increased risk of IUGR, whereas a certain fetal VNTR (variable number tandem repeat) polymorphism in IGF-I gene was associated with a decreased risk of IUGR [14]. We further showed that a fetal SNP (single nucleotide polymorphism) in the matrix metalloproteinase 2 (MMP2) gene was associated with an increased risk of IUGR [15]. Finally, we demonstrated that the presence and concentration of IgM antibodies to human HSP60 (heat shock protein 60) in cord blood at 29 weeks of gestation was restricted to SGAs (small for gestational age) [16]. NEAT1 (nuclear paraspeckle assembly transcript 1) is a nuclearrestricted long non-coding RNA that has two isoforms: NEAT1_1 (3.7 kb in human) and NEAT1_2 (23 kb in human). The function of this non-coding RNA was recently revealed as the core constituent of a subnuclear structure called paraspeckle, which is responsible for the retention of hyperedited mRNAs in the nucleus [17e20]. Paraspeckles are formed around the NEAT1 transcription start site during NEAT1 transcription, by the recruitment of at least three proteins of the DBHS family (drosophila behavior/human splicing): PSPC1, p54nrb/NONO and PSF/SFPQ [21e23]. In the nucleus, dsRNAs are frequently edited by dsRNA-dependent adenosine deaminases (ADARs), which catalyze the hydrolytic deamination of adenosines into inosines. In humans, more than 90% of editing occurs within inverted repeated Alu elements [17]. By sequestering hyperedited mRNAs within the nucleus, paraspeckles play an active role in the regulation of gene expression in differentiated cells [24]. Paraspeckles are however absent from embryonic stem cells and appear, together with NEAT1, during differentiation [25]. A stress signal is capable of releasing retained hyperedited mRNAs from paraspeckles, suggesting that these structures could have both beneficial and deleterious roles in gene expression regulation [26]. Knocking out NEAT1 in mice revealed that NEAT1 was not essential for mice survival under normal laboratory conditions [27]. Even microarrays from tissues of NEAT1 KO mice failed to highlight differential regulation of gene expression [28]. We show here that the long non-coding RNA NEAT1 is increased in placentas of IUGR compared to CTRL pregnancies delivered at term. 2. Methods

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(PFA) in PBS for histological analysis, and (3) put in RNAlater solution (Ambion, Life Technologies Ltd, Paisley, UK) and kept at 20  C for later RNA analysis. 2.3. PCR-select cDNA subtraction kit Total RNA was extracted with TRIZOL reagent according to the manufacturer’s instructions (Invitrogen, Life Technologies Ltd, Paisley, UK). Poly(Aþ) mRNA was further isolated using NucleoTrap mRNA mini columns (MachereyeNagel, GmbH&Co KG, Düren, Germany). The mRNA of five patients from each group (IUGR and CTRL) with the most homogeneous personal data was selected and pooled in order to minimize the inter-individual but not the inter-group differences in the subtraction experiment. Both pools were processed using the Clontech PCR-Select cDNA subtraction kit according to the manufacturer’s instructions (BD Biosciences Clontech, Palo Alto, CA, USA). At the end of the experiment, remaining cDNA fragments were allowed to migrate on 2% agarose gels. Each fragment was subcloned with the TOPO TA cloning kit for sequencing (Invitrogen, Life Technologies Ltd, Paisley, UK), analyzed by restriction enzyme digestion, sequenced and identified by comparison with the nucleotide database of the National Center for Biotechnology Information. 2.4. Quantitative RT-PCR (qRT-PCR) Results were verified by qRT-PCR on samples from all 24 cohort patients. Reverse transcription was carried out using First Strand cDNA synthesis kit for RT-PCR (AMV) (Roche Applied Science, Rotkreuz, Switzerland), and quantitative PCR was conducted using the Lithos qPCR Master mix-2mM (Eurogentec, Seraing, Belgium) made for the LightCycler Real-time PCR system (Roche Applied Science, Rotkreuz, Switzerland). All the results were normalized by GAPDH mRNA expression. Primers are listed in Table 1. 2.5. Histology and immunohistochemistry PFA 4%-preserved tissues were embedded in paraffin and cut in 5 mm sections. Tissue sections were deparaffinized by 15 min incubation in Histo-Clear (National Diagnostics, Charlotte, North Carolina, USA), and a series of 5 min incubation in 100%, 95% and 70% ethanol. Hematoxylin-eosin staining was performed as follows: 5 min equilibration in 1 PBS, 35 s staining in Hematoxylin (Harris), rinsing in 1 PBS and H2O, dehydration by 20 dips in 50%, 70%, 95% and 100% ethanol, 20 s staining in eosin, and finally rinsing in 100% ethanol and xylene. The slides were mounted with Eukitt quick-hardening mounting medium (Fluka, SigmaeAldrich, Switzerland). For immunohistochemistry, a heat-induced antigen retrieval method was used to unmask PSPC1 antigen. Sections were incubated for 15 min in sub-boiling 10 mM citrate buffer pH6.0 and then cooled to room temperature for 15e30 min. Immunohistochemistry was then performed as previously described [29,30]. Anti-PSPC1 antibody was purchased from Dundee Cell Products ltd, Dundee, Scotland. 2.6. In situ hybridization (ISH) Tissue sections were deparaffinized as described above and then treated with proteinase K (20 mg/ml in TE) for 30 min at room temperature. ISH was then performed as previously described [29,30]. Primers used for the digoxigenin-labeled NEAT1 probe amplification were: forward-50 -GGTAAGCCCGGGACAGTAAG-30 and reverse-50 -GACTCCATGTCTCCCGGTTC-30 . The amplified PCR fragment was subcloned in the pCR2.1-TOPO vector and two clones with the PCR fragment inserted in opposite orientations were chosen for probe marking. Tissue sections were hybridized using 0.4 mg/ml of the labeled probe.

2.1. Patients Patients were recruited at the University Hospital of Lausanne between 2003 and 2004. Inclusion criteria were all singleton pregnancies that had their follow-up and term delivery (37e41 weeks gestation) at the University Hospital of Lausanne, Switzerland. Exclusion criteria were any pathology/complication classically associated with IUGR such as hypertension, preeclampsia, intrauterine infection, fetal malformation, diabetes, chronic maternal disease, or multiple pregnancies. IUGR cases were defined by ultrasonographic follow-up during the second/third trimester showing a biometry below the 10th percentile (P10) and birth weight below P10. Controls were defined by a birth weight between P10eP90. Gestational age was based on first trimester ultrasound between 8 and 10 weeks of gestation. Twelve IUGR patients and 12 controls were included and their personal data (maternal and fetal), medical history and pregnancy follow-up were prospectively recorded and anonymized. The protocol was approved by the Clinical Research Ethical Committee of the Faculty of Biology and Medicine of the University of Lausanne. All patients were informed of the details of the study and had to give their written consent.

2.7. Statistical analyses Clinical characteristics were analyzed by one-way ANOVA, c2 test or Manne Whitney U test, depending on the type of variable (categorical, discrete or continuous) and variance in the samples (equal or unequal). All statistical analyses were

Table 1 Primers. Gene

Forward

Reverse

GAPDH

50 -AGATCATCAGCAATGCCTCC-30

Cytochrome b

50 -TATCCGCCATCCCATACATT-30

TncRNA¼NEAT1

50 -CCAGTTTTCCGAGAACCAAA-30

PSG1

50 -TCGACTGTCATGGATTTGGA-30

PSG3

50 -AGGGCAAATGAAGGACCTCT-30

NEAT1_2

50 -GCTGAGAAGGAAGGTGCTTG-30

50 -GTGGCAGTGATGG CATGGAC-30 50 -GGTGATTCCTAGG GGGTTGT-30 50 -ATGCTGATCTGCT GCGTATG-30 50 -CTAACCCACCGG CACAGTAT-30 50 -CCATCACCTCG CTTTACGAT-30 50 -CTGGCTAGTCCC AGTTCAGC-30

2.2. Tissue collection A placental biopsy of 1 cm2 was recovered halfway between the external rim and cord insertion. The amnion was removed, and the maternal part was separated from the fetal part. Only the fetal part was kept: (1) immediately snap-frozen in liquid nitrogen and kept at 80  C for protein analysis, (2) put in 4% paraformaldehyde

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S. Gremlich et al. / Placenta 35 (2014) 44e49

Table 2 Clinical characteristics of IUGR and CTRL pregnancies. N ¼ 24 Maternal characteristics Maternal age (years) BMI before pregnancy (kg/m2) Ethnic origin (% Caucasian) Gravidity Parity Primiparity (%) Smoking/alcohol (N) Gestational age at delivery (weeks) Fetal characteristics Newborn birth weight (g) Placenta weight (g)

CTRL N ¼ 12

IUGR N ¼ 12

29
5 (24e39) 25.0
3.2 92 1.4
0.7 (1e3) 0.3
0.6 (0e2) 83 0 39.8
1.0

32
6 (22e44) 25.6
3.0 83 1.8
1.1 (1e4) 0.6
0.8 (0e2) 58 2 38.4
1.2

3571
228 (3280e4030) 618
107 (510e890)

2507
228 (2090e2750) 440
140 (300e780)

P 0.230c 0.632c 0.537a 0.610b 0.255b 0.178a 0.140a 0.008c

2 kb) were found to be retained in specialized subnuclear compartments and to play a role in epigenetic regulation of gene expression mainly during development and differentiation processes [32,34e36]. In our experiments, the lncRNA NEAT1 was up-regulated in placentas of IUGR compared to CTRL pregnancies delivered at term. There was no linear relationship between NEAT1 mRNA expression and newborn birth weight percentile, but rather a sudden upregulation of NEAT1 mRNA below or at P10, with unfortunately no value available in our group between P10eP25 (data not shown). NEAT1 was exclusively expressed in the trophoblast cells in placental villi of pregnancies delivered at term. At the beginning of

Fig. 5. mRNA expression of the long isoform of NEAT1, NEAT1_2. The expression of this gene was evaluated by quantitative RT-PCR on samples from the 24 patients of the study. Results are presented as mean
s.e.m of 3 different experiments.

pregnancy, placental villi are delineated by two layers of trophoblast cells: an external multinucleated syncytiotrophoblast and an internal mononucleated cytotrophoblast cell layer. Cytotrophoblasts are committed to differentiate and fuse with the syncytiotrophoblast to repair and renew this barrier layer when the placenta is growing. In term placentas however, the underlying cytotrophoblast cell layer almost disappears, presenting only vestigial features under the form of scattered isolated cells located immediately below the syncytiotrophoblast. NEAT1 was expressed in more nuclei, and possibly also more intensely in IUGR than in CTRL villous trophoblasts. Indeed, the punctate appearance of the staining looked more like a uniform staining in IUGR villous trophoblasts nuclei. This probably happened concomitantly with an increase in paraspeckle number. However, NEAT1 gene has two isoforms that have different cell/ tissue distribution and different roles. The short form, NEAT1_1, is highly and widely expressed in different mammalian tissues, while the long form, NEAT1_2, is restricted to particular populations of cells inside a tissue [27,37]. NEAT1_2 is essential for the formation of paraspeckles, while NEAT1_1 plays a supplementary role, increasing the number of paraspeckles when it is overexpressed [17]. Finally, only NEAT1_2 can rescue paraspeckle formation in NEAT1 knockout mice [28]. Our ISH probe was chosen on NEAT1_1 sequence in accordance with the result of the subtraction experiment. Therefore, it recognizes both isoforms. However, qRT-PCR experiments demonstrated that NEAT1_2 was also expressed in term placentas and was slightly increased in IUGR compared to CTRL placentas. PSPC1 co-localized with NEAT1 in villous trophoblasts nuclei, but was expressed in all villous trophoblasts nuclei in CTRL placentas, contrary to NEAT1. PSPC1 is one of the three DBHS proteins mainly involved in paraspeckle constitution [24]. Currently, more than 30 proteins are known to localize in paraspeckles. These

Fig. 4. Immunohistochemistry of PSPC1 in term placentas. A: anti-PSPC1 antibody; B: negative control. IHC was performed on 5 mm sections of paraffin-embedded CTRL term placentas. Magnification: 400.

S. Gremlich et al. / Placenta 35 (2014) 44e49

proteins all share a RNA binding capacity but do not possess the two RRMs (RNA Recognition Motif) followed by a conserved DBHS motif, present in PSF, P54nrb and PSPC1 [28]. It could therefore be interesting to investigate the exact protein composition of the paraspeckles found in CTRL and IUGR placentas. Up to now, NEAT1 has not been associated with many physiological and/or pathological conditions. It has been shown to be upregulated in the central nervous system of mice infected with Japanese encephalitis or rabies viruses [38], in cultured myoblast cell lines when differentiation was induced [19], in caudate nucleus in Huntington’s disease [39], in porcine skeletal muscle during post-exercise recovery [40], and very recently in HIV-1-infected Tcell lines [41]. No virus infection was found to be associated with our study population. In this paper we demonstrate that NEAT1 mRNA expression is increased in the villous trophoblasts of IUGR term placentas compared to CTRL term placentas, probably concomitantly with an increase in paraspeckle number. However, the exact significance with regards to placental gene expression remains purely speculative. Paraspeckles are able to retain hyperedited mRNAs in the nucleus. Chen et al. found roughly 333 genes potentially affected by this hyperediting activity, which are mainly implicated in essential/fundamental pathways like apoptosis and survival, DNA damage, immune response, transcription, transport or development [25]. Moreover, the increase in paraspeckle number could either be causative of IUGR development, preventing some genes to be expressed and mediate their known function, or reactive to IUGR development, trying to fight against the loss of oxygen/nutrients delivery to the fetus. In conclusion, our study opens a new field in IUGR research by suggesting the existence of an increased retention of coding mRNAs in IUGR villous trophoblasts nuclei. Since the syncytiotrophoblast is the final feto-maternal barrier, this could contribute to the pathophysiology of IUGR. Acknowledgments We would like to thank Marc Loup for his technical help concerning the in situ hybridization experiments, Dr. Maria-Chiara Osterheld for her histological analysis advice and critical reading of the manuscript and Dr. Sam Vasilevsky for his critical reading of the manuscript. References [1] Ross MG, Beall MH. Adult sequelae of intrauterine growth restriction. Semin Perinatol 2008;32(3):213e8. [2] Gluckman PD, Hanson MA, Cooper C, Thornburg KL. Effect of in utero and early-life conditions on adult health and disease. N Engl J Med 2008;359(1): 61e73. [3] Chernausek SD. Update: consequences of abnormal fetal growth. J Clin Endocrinol Metab 2012;97(3):689e95. [4] Zhong Y, Tuuli M, Odibo AO. First-trimester assessment of placenta function and the prediction of preeclampsia and intrauterine growth restriction. Prenat Diagn 2010;30(4):293e308. [5] Bamberg C, Kalache KD. Prenatal diagnosis of fetal growth restriction. Semin Fetal Neonatal Med 2004;9(5):387e94. [6] Kinzler WL, Vintzileos AM. Fetal growth restriction: a modern approach. Curr Opin Obstet Gynecol 2008;20(2):125e31. [7] Arroyo JA, Winn VD. Vasculogenesis and angiogenesis in the IUGR placenta. Semin Perinatol 2008;32(3):172e7. [8] Heazell AE, Crocker IP. Live and let die e regulation of villous trophoblast apoptosis in normal and abnormal pregnancies. Placenta 2008;29(9):772e83. [9] Heazell AE, Sharp AN, Baker PN, Crocker IP. Intra-uterine growth restriction is associated with increased apoptosis and altered expression of proteins in the p53 pathway in villous trophoblast. Apoptosis An Int J Progr Cell Death 2011;16(2):135e44. [10] Longtine MS, Chen B, Odibo AO, Zhong Y, Nelson DM. Villous trophoblast apoptosis is elevated and restricted to cytotrophoblasts in pregnancies complicated by preeclampsia, IUGR, or preeclampsia with IUGR. Placenta 2012;33(5):352e9.

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[11] Scifres CM, Nelson DM. Intrauterine growth restriction, human placental development and trophoblast cell death. J Physiol 2009;587(Pt 14):3453e8. [12] Borzsonyi B, Demendi C, Rigo Jr J, Szentpeteri I, Rab A, Joo JG. The regulation of apoptosis in intrauterine growth restriction: a study of Bcl-2 and Bax gene expression in human placenta. J Matern Fetal Neonatal Med 2013;26(4):347e50. [13] Burton GJ, Yung HW, Cindrova-Davies T, Charnock-Jones DS. Placental endoplasmic reticulum stress and oxidative stress in the pathophysiology of unexplained intrauterine growth restriction and early onset preeclampsia. Placenta 2009;30(Suppl. A):S43e8. [14] Murisier-Petetin G, Gremlich S, Damnon F, Reymondin D, Hohlfeld P, Gerber S. Amniotic fluid insulin-like growth factor binding protein 3 concentration as early indicator of fetal growth restriction. Eur J Obstet Gynecol Reprod Biol 2009;144(1):15e20. [15] Gremlich S, Nguyen D, Reymondin D, Hohlfeld P, Vial Y, Witkin SS, et al. Fetal MMP2/MMP9 polymorphisms and intrauterine growth restriction risk. J Reprod Immunol 2007;74(1e2):143e51. [16] Belhia F, Gremlich S, Muller-Brochut AC, Damnon F, Hohlfeld P, Witkin SS, et al. Anti-60-kDa heat shock protein antibodies in fetal serum: a biomarker for unexplained small for gestational age fetuses. Gynecol Obstet Invest 2010;70(4):299e305. [17] Clemson CM, Hutchinson JN, Sara SA, Ensminger AW, Fox AH, Chess A, et al. An architectural role for a nuclear noncoding RNA: NEAT1 RNA is essential for the structure of paraspeckles. Mol Cell 2009;33(6):717e26. [18] Sasaki YT, Ideue T, Sano M, Mituyama T, Hirose T. MENepsilon/beta noncoding RNAs are essential for structural integrity of nuclear paraspeckles. Proc Natl Acad Sci U S A 2009;106(8):2525e30. [19] Sunwoo H, Dinger ME, Wilusz JE, Amaral PP, Mattick JS, Spector DL. MEN epsilon/beta nuclear-retained non-coding RNAs are up-regulated upon muscle differentiation and are essential components of paraspeckles. Genome Res 2009;19(3):347e59. [20] Souquere S, Beauclair G, Harper F, Fox A, Pierron G. Highly ordered spatial organization of the structural long noncoding NEAT1 RNAs within paraspeckle nuclear bodies. Mol Biol Cell 2010;21(22):4020e7. [21] Fox AH, Lam YW, Leung AK, Lyon CE, Andersen J, Mann M, et al. Paraspeckles: a novel nuclear domain. Curr Biol CB 2002;12(1):13e25. [22] Fox AH, Bond CS, Lamond AI. P54nrb forms a heterodimer with PSP1 that localizes to paraspeckles in an RNA-dependent manner. Mol Biol Cell 2005;16(11):5304e15. [23] Mao YS, Sunwoo H, Zhang B, Spector DL. Direct visualization of the cotranscriptional assembly of a nuclear body by noncoding RNAs. Nat Cell Biol 2011;13(1):95e101. [24] Bond CS, Fox AH. Paraspeckles: nuclear bodies built on long noncoding RNA. J Cell Biol 2009;186(5):637e44. [25] Chen LL, DeCerbo JN, Carmichael GG. Alu element-mediated gene silencing. EMBO J 2008;27(12):1694e705. [26] Ip JY, Nakagawa S. Long non-coding RNAs in nuclear bodies. Dev Growth Differ 2012;54(1):44e54. [27] Nakagawa S, Naganuma T, Shioi G, Hirose T. Paraspeckles are subpopulationspecific nuclear bodies that are not essential in mice. J Cell Biol 2011;193(1): 31e9. [28] Nakagawa S, Hirose T. Paraspeckle nuclear bodieseuseful uselessness? Cell Mol Life Sci CMLS 2012;69(18):3027e36. [29] Braissant O, Cagnon L, Monnet-Tschudi F, Speer O, Wallimann T, Honegger P, et al. Ammonium alters creatine transport and synthesis in a 3D culture of developing brain cells, resulting in secondary cerebral creatine deficiency. Eur J Neurosci 2008;27(7):1673e85. [30] Braissant O, Beard E, Torrent C, Henry H. Dissociation of AGAT, GAMT and SLC6A8 in CNS: relevance to creatine deficiency syndromes. Neurobiol Dis 2010;37(2):423e33. [31] Mattick JS, Makunin IV. Non-coding RNA. Hum Mol Genet 2006;15(Spec No 1):R17e29. [32] Mercer TR, Dinger ME, Mattick JS. Long non-coding RNAs: insights into functions. Nat Rev Genet 2009;10(3):155e9. [33] Costa FF. Non-coding RNAs: meet thy masters. Bioessays 2010;32(7):599e 608. [34] Mohammad F, Mondal T, Kanduri C. Epigenetics of imprinted long noncoding RNAs. Epigenetics Off J DNA Methylation Society 2009;4(5):277e86. [35] Wilusz JE, Sunwoo H, Spector DL. Long noncoding RNAs: functional surprises from the RNA world. Genes Dev 2009;23(13):1494e504. [36] Saxena A, Carninci P. Long non-coding RNA modifies chromatin: epigenetic silencing by long non-coding RNAs. Bioessays 2011;33(11):830e9. [37] Hutchinson JN, Ensminger AW, Clemson CM, Lynch CR, Lawrence JB, Chess A. A screen for nuclear transcripts identifies two linked noncoding RNAs associated with SC35 splicing domains. BMC Genomics 2007;8:39. [38] Saha S, Murthy S, Rangarajan PN. Identification and characterization of a virus-inducible non-coding RNA in mouse brain. J Gen Virol 2006;87(Pt 7): 1991e5. [39] Johnson R. Long non-coding RNAs in Huntington’s disease neurodegeneration. Neurobiol Dis 2012;46(2):245e54. [40] Jensen JH, Conley LN, Hedegaard J, Nielsen M, Young JF, Oksbjerg N, et al. Gene expression profiling of porcine skeletal muscle in the early recovery phase following acute physical activity. Exp Physiol 2012;97(7):833e48. [41] Zhang Q, Chen CY, Yedavalli VS, Jeang KT. NEAT1 long noncoding RNA and paraspeckle bodies modulate HIV-1 posttranscriptional expression. MBio 2013;4(1):e00596e612.