Termination codon readthrough of NNAT mRNA regulates calcium-mediated neuronal differentiation.

Termination codon readthrough (TCR) is a process in which ribosomes continue to translate an mRNA beyond a stop codon generating a C-terminally extended protein isoform. Here, we demonstrate TCR in mammalian NNAT mRNA, which encodes NNAT, a proteolipid important for neuronal differentiation. This is a programmed event driven by cis-acting RNA sequences present immediately upstream and downstream of the canonical stop codon and is negatively regulated by NONO, an RNA-binding protein known to promote neuronal differentiation. Unlike the canonical isoform NNAT, we determined that the TCR product (NNATx) does not show detectable interaction with the sarco/endoplasmic reticulum Ca2+-ATPase isoform 2 Ca2+ pump, cannot increase cytoplasmic Ca2+ levels, and therefore does not enhance neuronal differentiation in Neuro-2a cells. Additionally, an antisense oligonucleotide that targets a region downstream of the canonical stop codon reduced TCR of NNAT and enhanced the differentiation of Neuro-2a cells to cholinergic neurons. Furthermore, NNATx-deficient Neuro-2a cells, generated using CRISPR-Cas9, showed increased cytoplasmic Ca2+ levels and enhanced neuronal differentiation. Overall, these results demonstrate regulation of neuronal differentiation by TCR of NNAT. Importantly, this process can be modulated using a synthetic antisense oligonucleotide.

Obtaining high yield recombinant Enterococcus faecium nicotinate nucleotide adenylyltransferase for X-ray crystallography and biophysical studies.

Nicotinate nucleotide adenylyltransferase (NNAT) has been a significant research focus on druggable targets, given its indispensability in the biosynthesis of NAD+, which is crucial to the survival of bacterial pathogens. However, no information is available on the structure-function of Enterococcus faecium NNAT (EfNNAT). This study established the expression and purification protocol for obtaining a high-yield recombinant EfNNAT using the E. coli expression system and a single-step IMAC purification method. Approximately 101 mg of EfNNAT was obtained per 7.8 g of wet E. coli cells, estimated to be over 98 % pure. We further characterized the biophysical structure and determined the three-dimensional structure of the EfNNAT. Biophysical studies revealed a dimeric protein with a higher α-helical composition. The highly stable protein crystalizes in multiple conditions, yielding high-quality crystals diffracting between 1.78 and 2.80 Å. Two high-resolution crystal structures of EfNNAT in its native and adenine-bound forms were determined at 1.90 Å and 1.82 Å, respectively. The X-ray structures of the EfNNAT revealed the presence of phosphate and sulfate ions occupying and interacting with conserved amino acid residues within the putative substrate binding site, hence providing insight into the probable substrate preference of EfNNAT and, consequently, why EfNNAT may not prefer ß-nicotinamide mononucleotide as a substrate. With the accessibility to high-resolution structures of EfNNAT, further structural evaluation and drug-based screening can be achieved. Hence, we anticipate that this study will provide the basis for the discovery of structure-based inhibitors against this enzyme.

Claudin 4 enhances the malignancy of glioma cells via NNAT/Wnt signaling.

Gliomas are the most common malignancies of the central nervous system and are associated with high mortality rates. However, the pathogenesis of gliomas is unclear. In this study, we show that elevated claudin-4 (CLDN4) levels in glioma tissues are associated with poor clinical outcomes. We found that upregulating the expression of CLND4 enhanced the proliferative and migratory capacities of glioma cells. Mechanistically, CLND4 upregulated Neuronatin (NNAT) by activating Wnt3A signaling, and aided in the progression of the glioma. Most importantly, our in vivo data demonstrated that CLND4 overexpression caused rapid tumor growth in mice injected with LN229 cells and reduced the survival of these mice. Our findings reveal that CLND4 modulates malignancy in glioma cells; targeting CLDN4 may open up new avenues for glioma treatment.

Regulation of beige adipocyte thermogenesis by the cold-repressed ER protein NNAT.

OBJECTIVE: Cold stimuli trigger the conversion of white adipose tissue into beige adipose tissue, which is capable of non-shivering thermogenesis. However, what process drives this activation of thermogenesis in beige fat is not well understood. Here, we examine the ER protein NNAT as a regulator of thermogenesis in adipose tissue. METHODS: We investigated the regulation of adipose tissue NNAT expression in response to changes in ambient temperature. We also evaluated the functional role of NNAT in thermogenic regulation using Nnat null mice and primary adipocytes that lack or overexpress NNAT. RESULTS: Cold exposure or treatment with a ß3-adrenergic agonist reduces the expression of adipose tissue NNAT in mice. Genetic disruption of Nnat in mice enhances inguinal adipose tissue thermogenesis. Nnat null mice exhibit improved cold tolerance both in the presence and absence of UCP1. Gain-of-function studies indicate that ectopic expression of Nnat abolishes adrenergic receptor-mediated respiration in beige adipocytes. NNAT physically interacts with the ER Ca2+-ATPase (SERCA) in adipocytes and inhibits its activity, impairing Ca2+ transport and heat dissipation. We further demonstrate that NHLRC1, an E3 ubiquitin protein ligase implicated in proteasomal degradation of NNAT, is induced by cold exposure or ß3-adrenergic stimulation, thus providing regulatory control at the protein level. This serves to link cold stimuli to NNAT degradation in adipose tissue, which in turn leads to enhanced SERCA activity. CONCLUSIONS: Our study implicates NNAT in the regulation of adipocyte thermogenesis.

DNA methylation-mediated silencing of Neuronatin promotes hepatocellular carcinoma proliferation through the PI3K-Akt signaling pathway.

AIMS: To explore the methylation status, function, and underlying mechanism of the imprinted gene Neuronatin (NNAT) in hepatocellular carcinoma (HCC) progression. MAIN METHODS: Immunohistochemistry (IHC) was performed to evaluate the expression of NNAT in HCC samples. Bisulfite genomic sequencing PCR (BSP) was applied to examine the methylation status of the NNAT promoter. In addition, colony formation, 5-Ethynyl-20-deoxyuridine (EdU) assays and subcutaneous xenograft nude models were used to explore the roles of NNAT in HCC cell proliferation. Furthermore, RNA-seq and phospho-specific protein microarray assays were conducted to illustrate the underlying mechanism by which NNAT regulates HCC progression. KEY FINDINGS: NNAT was obviously downregulated in HCC tissues, and its expression level was closely associated with tumor growth and patient prognosis. The downregulation of NNAT in HCC was induced by hypermethylation of CpG islands in the promoter region, and hypermethylation was correlated with overall survival of HCC. Moreover, the enforced expression of NNAT significantly inhibited HCC cell proliferation in vitro and in vivo. Transcriptome analysis showed that the alteration of NNAT expression was mainly related to dysregulation of the PI3K-Akt signaling pathway. Finally, phospho-specific antibody microarray detection further revealed that overexpressed NNAT can increase the phosphorylation levels of LKB1, Met, and elF4E and decrease the phosphorylation levels of PTEN, which are all involved in the PI3K-Akt signaling pathway. SIGNIFICANCE: Our research provides new insights into the epigenetic regulation of imprinted genes in tumorigenesis and implies that the imprinted gene NNAT may act as a prognostic biomarker and tumor suppressor in HCC.

DNA methylation and fluoride exposure in school-age children: Epigenome-wide screening and population-based validation.

Excessive fluoride exposure and epigenetic change can induce numerous adverse health outcomes, but the role of epigenetics underneath the harmful health effects induced by fluoride exposure is unclear. In such gap, we evaluated the associations between fluoride exposure and genome-wide DNA methylation, and identified that novel candidate genes associated with fluoride exposure. A total of 931 school-age children (8-12 years) in Tongxu County of Henan Province (China) were recruited in 2017. Urinary fluoride (UF) concentrations were measured using the national standardized ion selective electrode method. Participants were divided into a high fluoride-exposure group (HFG) and control group (CG) according to the UF concentrations. Candidate differentially methylated regions (DMRs) were screened by Infinium-Methylation EPIC BeadChip of DNA samples collected from 16 participants (eight each from each group). Differentially methylated genes (DMGs) containing DMRs associated with skeletal and neuronal development influenced by fluoride exposure were confirmed using MethylTarget™ technology from 100 participants (fifty each from each group). DMGs were verified by quantitative methylation specific PCR from 815 participants. Serum levels of hormones were measured by auto biochemical analyzer. The mediation analysis of methylation in the effect of fluoride exposure on hormone levels was also performed. A total of 237 differentially methylated sites (DMSs) and 212 DMRs were found in different fluoride-exposure groups in the epigenome-wide phase. Methylation of the target sequences of neuronatin (NNAT), calcitonin-related polypeptide alpha (CALCA) and methylenetetrahydrofolate dehydrogenase 1 showed significant difference between the HFG and CG. Each 0.06% (95% CI: -0.11%, -0.01%) decreased in NNAT methylation status correlated with each increase of 1.0 mg/L in UF concentration in 815 school-age children using QMSP. Also, each 1.88% (95% CI: 0.04%, 3.72%) increase in CALCA methylation status correlated with each increase of 1.0 mg/L in UF concentration. The mediating effect of NNAT methylation was found in alterations of ACTH levels influenced by fluoride exposure, with a ß value of 11.7% (95% CI: 3.4%, 33.4%). In conclusion, long-term fluoride exposure affected the methylation pattern of genomic DNA. NNAT and CALCA as DMGs might be susceptible to fluoride exposure in school-age children.

Effect of Divalent Metal Ion on the Structure, Stability and Function of Klebsiella pneumoniae Nicotinate-Nucleotide Adenylyltransferase: Empirical and Computational Studies.

The continuous threat of drug-resistant Klebsiella pneumoniae justifies identifying novel targets and developing effective antibacterial agents. A potential target is nicotinate nucleotide adenylyltransferase (NNAT), an indispensable enzyme in the biosynthesis of the cell-dependent metabolite, NAD+. NNAT catalyses the adenylation of nicotinamide/nicotinate mononucleotide (NMN/NaMN), using ATP to form nicotinamide/nicotinate adenine dinucleotide (NAD+/NaAD). In addition, it employs divalent cations for co-substrate binding and catalysis and has a preference for different divalent cations. Here, the biophysical structure of NNAT from K. pneumoniae (KpNNAT) and the impact of divalent cations on its activity, conformational stability and substrate-binding are described using experimental and computational approaches. The experimental study was executed using an enzyme-coupled assay, far-UV circular dichroism, extrinsic fluorescence spectroscopy, and thermal shift assays, alongside homology modelling, molecular docking, and molecular dynamic simulation. The structure of KpNNAT revealed a predominately α-helical secondary structure content and a binding site that is partially hydrophobic. Its substrates ATP and NMN share the same binding pocket with similar affinity and exhibit an energetically favourable binding. KpNNAT showed maximum activity and minimal conformational changes with Mg2+ as a cofactor compared to Zn2+, Cu2+ and Ni2+. Overall, ATP binding affects KpNNAT dynamics, and the dynamics of ATP binding depend on the presence and type of divalent cation. The data obtained from this study would serve as a basis for further evaluation towards designing structure-based inhibitors with therapeutic potential.

Dynamic Changes in the Localization of Neuronatin-Positive Cells during Neurogenesis in the Embryonic Rat Brain.

Neuronatin (NNAT) was first identified as a gene selectively and abundantly expressed in the cytoplasm of the newborn mouse brain, and involved in neonatal neurogenesis. However, the particular roles of NNAT in the developing prenatal brain have not been identified, especially in mid to late stages. In this study, we performed immunohistochemical analyses of NNAT and SOX2 proteins, a nuclear transcription factor and neural stem/progenitor marker, in the rat brain on embryonic days 13.5, E16.5, and E20.5. NNAT signals were broadly observed across the developing brain on E13.5 and gradually more localized in later stages, eventually concentrated in the alar and basal parts of the terminal hypothalamus, the alar plate of prosomere 2 of the thalamus, and the choroid plexus in the lateral and fourth ventricles on E20.5. In particular, the mammillary body in the basal part of the terminal hypothalamus, a region with a high number of SOX2-positive cells, evidenced intense NNAT signals on E20.5. The intracellular localization of NNAT showed diverse profiles, suggesting that NNAT was involved in various cellular functions, such as cell differentiation and functional maintenance, during prenatal neurogenesis in the rat brain. Thus, the present observations suggested diverse and active roles of the NNAT protein in neurogenesis. Determining the function of this molecule may assist in the elucidation of the mechanisms involved in brain development.

Neuronatin is a modifier of estrogen receptor-positive breast cancer incidence and outcome.

PURPOSE: Understanding the molecular mediators of breast cancer survival is critical for accurate disease prognosis and improving therapies. Here, we identified Neuronatin (NNAT) as a novel antiproliferative modifier of estrogen receptor-alpha (ER+) breast cancer. EXPERIMENTAL DESIGN: Genomic regions harboring breast cancer modifiers were identified by congenic mapping in a rat model of carcinogen-induced mammary cancer. Tumors from susceptible and resistant congenics were analyzed by RNAseq to identify candidate genes. Candidates were prioritized by correlation with outcome, using a consensus of three breast cancer patient cohorts. NNAT was transgenically expressed in ER+ breast cancer lines (T47D and ZR75), followed by transcriptomic and phenotypic characterization. RESULTS: We identified a region on rat chromosome 3 (142-178 Mb) that modified mammary tumor incidence. RNAseq of the mammary tumors narrowed the candidate list to three differentially expressed genes: NNAT, SLC35C2, and FAM210B. NNAT mRNA and protein also correlated with survival in human breast cancer patients. Quantitative immunohistochemistry of NNAT protein revealed an inverse correlation with survival in a univariate analysis of patients with invasive ER+ breast cancer (training cohort: n = 444, HR = 0.62, p = 0.031; validation cohort: n = 430, HR = 0.48, p = 0.004). NNAT also held up as an independent predictor of survival after multivariable adjustment (HR = 0.64, p = 0.038). NNAT significantly reduced proliferation and migration of ER+ breast cancer cells, which coincided with altered expression of multiple related pathways. CONCLUSIONS: Collectively, these data implicate NNAT as a novel mediator of cell proliferation and migration, which correlates with decreased tumorigenic potential and prolonged patient survival.

Sodium fluoride disturbs DNA methylation of NNAT and declines oocyte quality by impairing glucose transport in porcine oocytes.

Sodium fluoride (NaF) is used as a medicine to prevent tooth decay; however, excessive NaF could cause a pathological damage to the health. Recent studies showed that NaF impaired mouse oocyte maturation, included of abnormal spindle configuration, actin cap formation, cortical granule-free domain formation, and the following development after fertilization. However, few studies used large animals as models to study the toxicology of NaF on oocytes maturation. We proposed a hypothesis that NaF would affect the nuclear and cytoplasmic maturation of porcine oocytes and DNA methylation pattern of imprinted genes in oocytes. Our results showed that NaF affected cumulus expansion, polar body emission, spindle morphology, cortical granule distribution, early apoptosis, and the following development after parthenogenetic activation during porcine oocyte maturation. Moreover, NaF increased the DNA methylation of NNAT and decreased its expression, which disturbed the glucose transport in oocytes. These results suggest that NaF impairs the porcine oocytes maturation epigenetically, which provides a new toxicological mechanism of NaF on the oocyte maturation. Environ. Mol. Mutagen. 59:223-233, 2018. © 2017 Wiley Periodicals, Inc.

Disruption of NNAT, NAP1L5 and MKRN3 DNA methylation and transcription in rabbit parthenogenetic fetuses.

Parthenogenetically activated oocytes cannot develop to term in mammals due to lack of paternal gene expression. Disruption of imprinted gene expression and DNA methylation status in parthenogenetic fetuses has been reported in mice and pigs, but not in rabbits. In this study, the genomic imprinting status of the paternally expressed genes Neuronatin (NNAT), Nucleosome assembly protein 1-like 5 (NAP1L5), and Makorin ring finger protein 3 (MKRN3) was compared between rabbit parthenogenetic (PA) and normally fertilized fetuses (Con) using quantitative real-time PCR (qRT-PCR) and bisulfite sequencing PCR (BSP). The results revealed a significantly reduced expression of NNAT, NAP1L5, and MKRN3 in rabbit PA fetuses compared with Con fetuses (p<0.05). In addition, the BSP results demonstrated hypermethylation in the differentially methylated regions (DMRs) of NNAT, NAP1L5, and MKRN3 in rabbit PA fetuses. Taken together, these results suggest that hypermethylation of DMRs is associated with decreased NNAT, NAP1L5, and MKRN3 expression, which may be responsible for developmental failure of rabbit PA fetuses.

Temporal and spatial expression pattern of Nnat during mouse eye development.

BACKGROUND: Neuronatin (Nnat) was initially identified as a highly expressed gene in neonatal mammalian brain. In this study, we analyze the spatial and temporal expression pattern of Nnat during mouse eye development as well as in the adult. METHODS: The expression of Nnat was analyzed on mRNA as well as protein level. The presence of Nnat transcripts in the adult retina was examined using reverse transcription-polymerase chain reaction (RT-PCR). Nnat protein expression was evaluated by Western blot and immunohistochemistry during eye development at embryonic day (E) 12, 15, 16 and postnatal day (P) 7, 14, 30 and 175 (adult). RESULTS: Immunohistochemical studies of the developing mouse eye revealed Nnat expression in embryonic and adult neuroretina as well as in corneal epithelial, stromal, endothelial cells and in lens epithelium. Expression of Nnat was detected from E12 onwards and was also present in adult eyes. CONCLUSIONS: The expression pattern suggests that Nnat may play an important role during eye development and in the maintenance of mature eye.

Identification of differentially methylated regions (DMRs) of neuronatin in mice.

BACKGROUND: Neuronatin (NNAT) is a paternal-inherited imprinted gene, first discovered in the rat neonatal brain, where it plays vital roles for neuronal growth, brain development, and metabolic regulation. The maternal imprint of NNAT has been identified in mice; however, the differentially methylated regions (DMRs) involved in the monoallelic expression of NNAT have not yet been investigated. RESULTS: In this study, we confirmed expression of two isoforms of the NNAT (α and ß) in the mice brain via quantitative RT-PCR. Additionally, the methylation profile of the CpG island located in the NNAT gene locus was determined in the mice liver, brain, sperm, and the MII oocyte via bisulfite sequencing PCR. CONCLUSION: In summary, we provide the first evidence for tissue- and gamete-specific methylation patterns of CpG3 that are located on exon 1, to be putative DMR of NNAT in mice.

Conservation of imprinting of Neuronatin (Nnat) in rabbits.

Although the expression and epigenetic status of imprinted genes have been extensively studied in a number of species, less is known about the genomic imprinting in rabbits. Neuronatin (Nnat) plays significant roles in the brain development and metabolic regulation and has been identified to be imprinted and paternally expressed in humans, mice and pigs; however, it has not yet been investigated in rabbits. In this study, we confirmed the expression of two isoforms of the rabbit Nnat (Nnat-a and Nnat-ß) identified in Genbank and Ensembl by quantitative real-time PCR. In addition, we also determined the methylation profile of the CpG island in the promoter region of the rabbit Nnat using bisulfite sequencing PCR and combined bisulfite restriction analysis. Here, we provide the first evidence that Nnat has two transcripts in rabbit. Additionally, the CpG island located in the promoter region shows oocyte-specific methylation and may be the differentially methylated region of Nnat in rabbits.

DNA methylation-mediated silencing of neuronatin (NNAT) in pig parthenogenetic fetuses.

It is generally believed that aberrant expression of imprinted genes participates in growth retardation of mammalian parthenogenesis. Neuronatin (NNAT), a paternally expressed gene, plays important roles in neuronal growth and metabolic regulation. Here we have compared the gene expression and promoter methylation pattern of NNAT between pig normally fertilized (Con) and parthenogenetic (PA) embryos. The results showed loss of NNAT expression (p<0.001) and hypermethylation of NNAT promoter in PA samples. Additionally, partial methylation was observed in Con fetuses, while almost full methylation and unmethylation of NNAT promoter were apparent in Metaphase II (MII) oocytes and mature sperms, respectively, which identified the CpG promoter region as a putative differentially methylated region (DMR) of NNAT. The data demonstrate that promoter hypermethylation is associated with the silencing of NNAT in pig PA fetuses, which may be related to developmental failure of pig parthenogenesis at early stages.

Memory-related gene expression profile of the male rat hippocampus induced by teeth extraction and occlusal support recovery.

OBJECTIVES: The present study aimed to identify the effect of memory-related genes on male rats tested for spatial memory with either molar teeth extraction or its restoration by occlusal support using experimental dentures. DESIGN: Memory-related genes were detected from hippocampi of male Wistar rats (exposed to teeth extraction with or without dentures, or no extraction (control)) (7-week old) after behavioural testing (via the radial maze task) using a DNA microarray. The time course of the expression of these genes was evaluated by quantitative real-time polymerase chain reaction (PCR) (on 49-week-old rats). RESULTS: In preliminary experiments, to determine which memory genes are affected by spatial memory training, DNA microarray analysis revealed that thyrotropin-releasing hormone (Trh) and tenascin XA (Tnxa) were up-regulated and neuronatin (Nnat) and S100a9 were down-regulated after the maze training. The expression of Tnxa, Nnat and S100a9 of 49-week-old rats (during the time course) via quantitative real-time PCR was consistent with the results of microarrays of the preliminary experiment. Expression of Trh that was evaluated by quantitative real-time PCR did not agree with the results for this gene from the microarray for all groups. Therefore, expression of Trh may have increased in only young, trained rats. The expression of S100a9 prior to the maze task was down-regulated in only the extraction group. CONCLUSION: These results demonstrated that Trh, Tnxa and Nnat genes were affected according to the degree of memory in male rats. This study also indicated that S100a9 is a memory-related gene, which is affected by the presence of occlusal support.

Sexual differences of imprinted genes' expression levels.

In mammals, genomic imprinting has evolved as a dosage-controlling mechanism for a subset of genes that play critical roles in their unusual reproduction scheme involving viviparity and placentation. As such, many imprinted genes are highly expressed in sex-specific reproductive organs. In the current study, we sought to test whether imprinted genes are differentially expressed between the two sexes. According to the results, the expression levels of the following genes differ between the two sexes of mice: Peg3, Zim1, Igf2, H19 and Zac1. The expression levels of these imprinted genes are usually greater in males than in females. This bias is most obvious in the developing brains of 14.5-dpc embryos, but also detected in the brains of postnatal-stage mice. However, this sexual bias is not obvious in 10.5-dpc embryos, a developmental stage before the sexual differentiation. Thus, the sexual bias observed in the imprinted genes is most likely attributable by gonadal hormones rather than by sex chromosome complement. Overall, the results indicate that several imprinted genes are sexually different in terms of their expression levels, and further suggest that the transcriptional regulation of these imprinted genes may be influenced by unknown mechanisms associated with sexual differentiation.

Clinical Diagnosis and Non-Verbal Ability of Primary-One School Children with LD

More school children were referred for learning difficutly (LD), especially after the introduction of LINUS sccreening programme by Ministry of Education Malaysia. To study the clinical diagnosis and non-verbal ability of primary-one school children with LD after paediatric assessment, as well as associated behavioural issues and socio-economincal background. Assessment findings by Paediatricians and Naglieri Non-Verbal Ability Test® (NNAT®) results of all primary-one school children referred in year 2010 with LD were studied retrospectively. Ninety-three children were included (62.4% male), and 72.0% of them failed the LINUS screening programme. The commonest diagnoses were Borderline Intellectual Disability (ID, 37.6%) and Mild ID (19.4%). Other diagnoses included Attention Deficit Hyperactive Disorder (ADHD, 11.8%), Specific Learning Disability (SLD, 10.8%), Autistic Spectrum Disorder (n = 5) and Severe Language Disorder (n = 3). Mean NNAT scores were 84.6 ± 11.8 (n = 85), of which 9.4% children scored less than 70 (<2nd percentile), while 63.7% scored between 71 and 90 (3rd-24th percentile). Twenty-three children(27.1%) scored 90 - 110 (25th-75th percentile) and 111-119 (76th-90th percentile). More than two-thirds of the parents never attended school, or only received education up to Form 3. Nearly 80% of mothers were housewife and 78.7% of fathers were labour or semi-skilled workers. A significant numbers of children with ADHD, Borderline ID, Mild ID and Severe Language Disorder / SLD had significant or borderline internalizing and/or externalizing behaviours.Majority of primary-one school children referred for LD do not have intellectual disability. Their clinical diagnosis and non-verbal ability were very variable. A significant number of them have poor socio-economical background and associated behavioural problems. A more realistic education system and targeted program should be offered.