Schizophrenia risk proteins ZNF804A and NT5C2 interact in cortical neurons.

The zinc finger protein 804A (ZNF804A) and the 5'-nucleotidase cytosolic II (NT5C2) genes are amongst the first schizophrenia susceptibility genes to have been identified in large-scale genome-wide association studies. ZNF804A has been implicated in the regulation of neuronal morphology and is required for activity-dependent changes to dendritic spines. Conversely, NT5C2 has been shown to regulate 5' adenosine monophosphate-activated protein kinase activity and has been implicated in protein synthesis in human neural progenitor cells. Schizophrenia risk genotype is associated with reduced levels of both NT5C2 and ZNF804A in the developing brain, and a yeast two-hybrid screening suggests that their encoded proteins physically interact. However, it remains unknown whether this interaction also occurs in cortical neurons and whether they could jointly regulate neuronal function. Here, we show that ZNF804A and NT5C2 colocalise and interact in HEK293T cells and that their rodent homologues, ZFP804A and NT5C2, colocalise and form a protein complex in cortical neurons. Knockdown of the Zfp804a or Nt5c2 genes resulted in a redistribution of both proteins, suggesting that both proteins influence the subcellular targeting of each other. The identified interaction between ZNF804A/ZFP804A and NT5C2 suggests a shared biological pathway pertinent to schizophrenia susceptibility within a neuronal cell type thought to be central to the neurobiology of the disorder, providing a better understanding of its genetic landscape.

Silencing of circ-NT5C2 retards the progression of IL-1ß-induced osteoarthritis in an in vitro cell model by targeting the miR-142-5p/NAMPT axis.

Osteoarthritis (OA) is a degenerative disease that occurs mostly in the elderly, and its specific pathogenesis is still unknown, but recent studies have found that circular RNA generally display aberrant expression in OA. Our study explored the expression characteristics and mechanism of action of circ-NT5C2 in OA. Circ-NT5C2, microRNA-142-5p (miR-142-5p), and nicotinamide phosphoribosyltransferase (NAMPT) mRNA levels were measured using RT-qPCR. Western blot was employed to assess the protein level of NAMPT and extracellular matrix (ECM) production-related markers. The viability, proliferation, apoptosis and inflammation were examined using 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT) assay, 5-ethynyl-2'-deoxyuridine (EdU) assay, flow cytometry, and enzyme-linked immunosorbent assay, respectively. Relationship between miR-142-5p and circ-NT5C2 or NAMPT was demonstrated by dual-luciferase reporter system and RNA immunoprecipitation assay. We reported that circ-NT5C2 and NAMPT were greatly upregulated, and miR-142-5p level was constrained in OA tissues and in a cell model. Circ-NT5C2 silencing alleviated IL-1ß-induced inhibitory effects on chondrocyte proliferation and ECM generation, meanwhile the promotional role of IL-1ß on chondrocyte apoptosis and inflammation was also weakened. The targeting relationship of miR-142-5p with either circ-NT5C2 or NAMPT was confirmed. Knockdown of miR-142-5p reversed the suppressive effects of circ-NT5C2 silencing on the OA progression in vitro, and NAMPT overexpression also attenuated the effects of miR-142-5p upregulation in an OA cell model. Collectively, circ-NT5C2 accelerated the OA process by targeting the miR-142-5p/NAMPT axis. This study provides valuable information to find a better treatment for OA.

5'-nucleotidase, cytosolic II genotype, and clinical outcome in patients with acute myeloid leukemia with intermediate-risk cytogenetics.

Acute myeloid leukemia (AML) is a complex disease, and its treatment needs to be adjusted to the risk, which is conferred by cytogenetics and molecular markers. Cytarabine is the main drug to treat AML, and it has been suggested that the genotype of cytarabine metabolizing enzymes may have a prognostic relevance in AML. Here we report the association between the 5'-nucleotidase, cytosolic II (NT5C2) rs10883841, cytidine deaminase (CDA) rs2072671 and rs532545 genotypes and the clinical outcome of 477 intermediate-risk cytogenetic AML patients receiving cytarabine-based chemotherapy. Patients younger than 50 years old with the NT5C2 rs10883841 AA genotype had lower overall survival (OS) (p: .003; HR 2.16, 95% CI 1.29-3.61) and lower disease-free survival (DFS) (p: .002; HR 2.45, 95% CI 1.41-4.27), associated to a higher relapse incidence (p: .010; HR 2.23, 95% CI 1.21-4.12). Interestingly, subgroup analysis showed that the negative effect of the NT5C2 rs10883841 AA genotype was detected in all subgroups except in patients with nucleophosmin mutation without high ratio FLT-3 internal tandem duplication. CDA polymorphisms were associated with the complete remission rate after induction chemotherapy, without influencing OS. Further studies are warranted to determine whether this pharmacogenomic approach may be helpful to individualize AML treatment.

Genetic deletion of soluble 5'-nucleotidase II reduces body weight gain and insulin resistance induced by a high-fat diet.

We previously investigated whether inhibition of AMP-metabolizing enzymes could enhance AMP-activated protein kinase (AMPK) activation in skeletal muscle for the treatment of type 2 diabetes. Soluble 5'-nucleotidase II (NT5C2) hydrolyzes IMP and its inhibition could potentially lead to a rise in AMP to activate AMPK. In the present study, we investigated effects of NT5C2 deletion in mice fed a normal-chow diet (NCD) or a high-fat diet (HFD). On a NCD, NT5C2 deletion did not result in any striking metabolic phenotype. On a HFD however, NT5C2 knockout (NT5C2-/-) mice displayed reduced body/fat weight gain, improved glucose tolerance, reduced plasma insulin, triglyceride and uric acid levels compared with wild-type (WT) mice. There was a tendency towards smaller and fewer adipocytes in epididymal fat from NT5C2-/- mice compared to WT mice, consistent with a reduction in triglyceride content. Differences in fat mass under HFD could not be explained by changes in mRNA expression profiles of epididymal fat from WT versus NT5C2-/- mice. However, rates of lipolysis tended to increase in epididymal fat pads from NT5C2-/- versus WT mice, which might explain reduced fat mass. In incubated skeletal muscles, insulin-stimulated glucose uptake and associated signalling were enhanced in NT5C2-/- versus WT mice on HFD, which might contribute towards improved glycemic control. In summary, NT5C2 deletion in mice protects against HFD-induced weight gain, adiposity, insulin resistance and associated hyperglycemia.

Expanding the clinical relevance of the 5'-nucleotidase cN-II/NT5C2.

Purine metabolism is depending on a large amount of enzymes to ensure cellular homeostasis. Among these enzymes, we have been interested in the 5'-nucleotidase cN-II and its role in cancer biology and in response of cancer cells to treatments. This protein has been cited and studied in a large number of papers published during the last decade for its involvement in non-cancerous pathologies such as hereditary spastic paraplegia, schizophrenia, and blood pressure regulation. Here, we review these articles in order to give an overview of the recently discovered clinical relevance of cN-II.

Effects of genetic deletion of soluble 5'-nucleotidases NT5C1A and NT5C2 on AMPK activation and nucleotide levels in contracting mouse skeletal muscles.

AMP-activated protein kinase (AMPK) plays a key role in energy homeostasis and is activated in response to contraction-induced ATP depletion in skeletal muscle via a rise in intracellular AMP/ADP concentrations. AMP can be deaminated by AMP-deaminase (AMPD) to IMP, which is hydrolyzed to inosine by cytosolic 5'-nucleotidase II (NT5C2). AMP can also be hydrolyzed to adenosine by cytosolic 5'-nucleotidase 1A (NT5C1A). Previous gene silencing and overexpression studies indicated control of AMPK activation by NT5C enzymes. In the present study using gene knockout mouse models, we investigated the effects of NT5C1A and NT5C2 deletion on intracellular adenine nucleotide levels and AMPK activation in electrically stimulated skeletal muscles. Surprisingly, NT5C enzyme knockout did not lead to enhanced AMP or ADP concentrations in response to contraction, with no potentiation of increases in AMPK activity in extensor digitorum longus (EDL) and soleus mouse muscles. Moreover, dual blockade of AMP metabolism in EDL using an AMPD inhibitor combined with NT5C1A deletion did not enhance rises in AMP and ADP or increased AMPK activation by electrical stimulation. The results on muscles from the NT5C knockout mice contradict previous findings where AMP levels and AMPK activity were shown to be modulated by NT5C enzymes.

NT5C2 novel splicing variant expands the phenotypic spectrum of Spastic Paraplegia (SPG45): case report of a new member of thin corpus callosum SPG-Subgroup.

BACKGROUND: Hereditary Spastic Paraplegia (HSP) is a genetically heterogeneous group of neurodegenerative diseases. Thin Corpus Callosum (TCC) associated HSP is a distinguished subgroup of complex forms. Purines and pyrimidine, the basic DNA and RNA components, are regulating the cell metabolism, having roles in signal transduction, energy preservation and cellular repair. Genetic defects in nucleotide metabolism related genes have been only recently implicated in brain and neurodegenerative diseases' pathogenesis. CASE PRESENTATION: We present a consanguineous Qatari family with two brothers, 9 and 3 years, who displayed a characteristic phenotype of early onset and markedly-severe spasticity with tiptoe walking, delayed dysarthric speech, persistent truncal hypotonia, and multiple variable-sized areas of brownish skin discoloration appearing at different places on the body. A clinical diagnosis suggestive of complex hereditary spastic paraplegia (HSP) was set after the family had the second affected child. Whole genome sequencing identified a novel homozygous NT5C2 splice site mutation (NM_012229.4/NM_001134373.2: c.1159 + 1G > T) that recessively segregated in family members. Brain MRI revealed dysgenic and thin corpus callosum (TCC) with peri-trigonal white matter cystic changes in both affected boys, whereas a well-developed corpus callosum with normal white matter was shown in their apparently normal brother, who found to be a carrier for the mutant variant. This mutation led to skipping of exon 14 with removal of 58 amino acid residues at the C-terminal half. The aberrantly spliced NT5C2 showed substantial reduction in expression level in the in-vitro study, indicating marked instability of the mutant NT5C2 protein. CONCLUSION: The present report expands the phenotypic spectrum of SPG45 and confirms NT5C2-SPG45 as a member of the rare TCC SPG-subtypes. Homozygous alteration in NT5C2 seems essential to produce central white matter developmental defects. The study highlights the importance of cytosolic II 5'-nucleotidase (NT5C2) in maintaining the normal balance of purines' pool in the brain, which seems to play a pivotal role in the normal development of central white matter structures.

Novel homozygous missense mutation in NT5C2 underlying hereditary spastic paraplegia SPG45.

SPG45 is a rare form of autosomal recessive spastic paraplegia associated with mental retardation. Detailed phenotyping and mutation analysis was undertaken in three individuals with SPG45 from a consanguineous family of Arab Muslim origin. Using whole-exome sequencing, we identified a novel homozygous missense mutation in NT5C2 (c.1379T>C; p.Leu460Pro). Our data expand the molecular basis of SPG45, adding the first missense mutation to the current database of nonsense, frameshift, and splice site mutations. NT5C2 mutations seem to have a broad clinical spectrum and should be sought in patients manifesting either as uncomplicated or complicated HSP.

TRIM22 orchestrates the proliferation of GBMs and the benefits of TMZ by coordinating the modification and degradation of RIG-I.

Tripartite motif 22 (TRIM22) is an agonist of nuclear factor κB (NF-κB) that plays an important role in the proliferation and drug sensitivity of glioblastoma (GBM). However, the molecular mechanism underlying the protein network between TRIM22 and nuclear factor κB (NF-κB) in GBM remains unclear. Here, we found that knockout of TRIM22 effectively inhibited tumor proliferation and increased the sensitivity of GBM cells to temozolomide (TMZ) in vivo and in vitro. Moreover, TRIM22 forms a complex with cytosolic purine 5-nucleotidase (NT5C2) in GBM and regulates the ubiquitination of retinoic acid-inducible gene-I (RIG-I). TRIM22 promotes the K63-linked ubiquitination of RIG-I, while NT5C2 is responsible for K48-linked ubiquitination. This regulation directly affects the RIG-I/NF-κB/cell division cycle and apoptosis regulator protein 1 (CCAR1) signaling axis. Ubiquitin modification inhibitor of RIG-I restores the inhibition of tumor growth induced by TRIM22 knockout. The follow-up results showed that compared with patients with high TRIM22 expression, patients with low TRIM22 expression had a longer survival time and were more sensitive to treatment with TMZ. Our results revealed that the TRIM22-NT5C2 complex orchestrates the proliferation of GBM and benefits of TMZ through post-translational modification of RIG-I and the regulation of the RIG-I/NF-κB/CCAR1 pathway and is a promising target for single-pathway multi-target therapy.

Cytosolic 5'-Nucleotidase II Is a Sensor of Energy Charge and Oxidative Stress: A Possible Function as Metabolic Regulator.

Cytosolic 5'-nucleotidase II (NT5C2) is a highly regulated enzyme involved in the maintenance of intracellular purine and the pyrimidine compound pool. It dephosphorylates mainly IMP and GMP but is also active on AMP. This enzyme is highly expressed in tumors, and its activity correlates with a high rate of proliferation. In this paper, we show that the recombinant purified NT5C2, in the presence of a physiological concentration of the inhibitor inorganic phosphate, is very sensitive to changes in the adenylate energy charge, especially from 0.4 to 0.9. The enzyme appears to be very sensitive to pro-oxidant conditions; in this regard, the possible involvement of a disulphide bridge (C175-C547) was investigated by using a C547A mutant NT5C2. Two cultured cell models were used to further assess the sensitivity of the enzyme to oxidative stress conditions. NT5C2, differently from other enzyme activities, was inactivated and not rescued by dithiothreitol in a astrocytoma cell line (ADF) incubated with hydrogen peroxide. The incubation of a human lung carcinoma cell line (A549) with 2-deoxyglucose lowered the cell energy charge and impaired the interaction of NT5C2 with the ice protease-activating factor (IPAF), a protein involved in innate immunity and inflammation.

Exome Analysis Identified Novel Homozygous Splice Site Donor Alteration in NT5C2 Gene in a Saudi Family Associated With Spastic Diplegia Cerebral Palsy, Developmental Delay, and Intellectual Disability.

Hereditary spastic paraplegias (HSPs) is a rare heterogeneous group of neurodegenerative diseases, with upper and lower limb spasticity motor neuron disintegration leading to paraplegias. NT5C2 gene (OMIM: 600417) encode a hydrolase enzyme 5'-nucleotidase, cytosolic II play an important role in maintaining the balance of purine nucleotides and free nucleobases in the spinal cord and brain. In this study we have identified a large consanguineous Saudi family segregating a novel homozygous splice site donor alteration in NT5C2 gene leading to spastic diplegia cerebral palsy, developmental delay and microcephaly. Whole exome sequencing (WES) was performed for the affected members of the family to study the novel mutation. WES data analysis, confirmed by Sanger sequencing analysis, identifies a homozygous splice site donor alteration of possible interest in NT5C2 (ENST00000343289: c.539+1G > T) at the sixth exon/intron boundaries. The mutation was further ruled out in 100 healthy control from normal population. The novel homozygous mutation observed in this study has not been reported in the literature or variant databases. The identified splicing alteration broadens the mutation spectrum of NT5C2 gene in neurodevelopmental disorders. To the best of our knowledge this is the first report from Saudi Arabia.

NT5C2 Gene Polymorphisms and the Risk of Coronary Heart Disease.

BACKGROUND: Increasing studies have reported that 5'-nucleotidase cytosolic II (NT5C2) has a strong relationship with coronary heart disease (CHD) development. This study was designed to examine the relationship between NT5C2 polymorphisms and CHD in the Chinese Han population. METHODS: We studied 501 CHD patients and 496 healthy controls from the Second Affiliated Hospital of Hainan Medical University in Hainan Province, China. Four single nucleotide polymorphisms (SNPs) in NT5C2 were selected and genotyped using Agena MassARRAY technology. Odds ratios and 95% confidence intervals were calculated using logistic regression after adjusting for age and gender. Stratification analysis was performed by age and gender in all individuals; we especially investigated the effects of NT5C2 SNPs on hypertension and diabetes among CHD patients. RESULTS: rs2148198 of NT5C2 was strongly associated with an increased risk of CHD (allele: p = 0.045; codominant: p = 0.007; additive: p = 0.016). Stratified analysis revealed that rs2148198 was associated with increased CHD risk in individuals aged ≤61 years and males. For CHD patients, rs2148198 significantly affected the risk of hypertension and diabetes (p < 0.05). Further, rs79237883 of NT5C2 was associated with decreased susceptibility to hypertension in multiple genetic models for individuals with CHD (allele: p = 0.007; codominant: p = 0.001; dominant: p = 0.001; additive: p = 0.008). CONCLUSION: This study reports the association of NT5C2 gene variants and CHD susceptibility in the Chinese Han population. Especially, NT5C2 rs2148198 was significantly associated with CHD risk in the subgroups of males, hypertension, and diabetes.

Studies in Zebrafish Demonstrate That CNNM2 and NT5C2 Are Most Likely the Causal Genes at the Blood Pressure-Associated Locus on Human Chromosome 10q24.32.

Background: Globally, high blood pressure (BP) is the most important risk factor for cardiovascular disease. Several genome-wide association studies (GWAS) have identified variants associated with BP traits at more than 535 chromosomal loci with genome-wide significance. The post-GWAS challenge is to annotate the most likely causal gene(s) at each locus. Chromosome 10q24.32 is a locus associated with BP that encompasses five genes: CYP17A1, BORCS7, AS3MT, CNNM2, and NT5C2 and warrants investigation to determine the specific gene or genes responsible for the phenotype. Aim: To identify the most likely causal gene(s) associated with BP at the 10q24.32 locus using zebrafish as an animal model. Results: We report significantly higher blood flow, increased arterial pulse, and elevated linear velocity in zebrafish larvae with cnnm2 and nt5c2 knocked down using gene-specific splice modification transcriptional morpholinos, compared with controls. No differences in blood-flow parameters were observed after as3mt, borcs7, or cyp17a1 knockdown. There was no effect on vessel diameter in animals with any of the four genes knocked down. At the molecular level, expression of hypertension markers (crp and ace) was significantly increased in cnnm2 and nt5c2 knockdown larvae. Further, the results obtained by morpholino knockdown were validated using zebrafish knockout (KO) lines with cnnm2 and nt5c2 deficiency, again resulting in higher blood flow, increased arterial pulse, and elevated linear velocity. Analysis of nt5c2a KO larvae demonstrated that lack of this gene resulted in reduced expression of cnnm2a, with reciprocal downregulation of nt5c2a in cnnm2a KO larvae. Staining of whole-blood smears from nt5c2 mutants revealed that KO of this gene might be associated with an acute lymphoblastic leukemia phenotype, consistent with literature reports. Additional experiments were designed based on previous literature on cnnm2a mutant zebrafish revealed impaired renal function, high levels of renin, and significantly increased expression of the ren gene, leading us to hypothesize that the observed elevated blood-flow parameters may be attributable to triggering of the renin-angiotensin-aldosterone signaling pathway. Conclusion: Our zebrafish data establish CNNM2 and NT5C2 as the most likely causal genes at the 10q24.32 BP locus and indicate that they trigger separate downstream mechanistic pathways.

Structure and Mechanisms of NT5C2 Mutations Driving Thiopurine Resistance in Relapsed Lymphoblastic Leukemia.

Activating mutations in the cytosolic 5'-nucleotidase II gene NT5C2 drive resistance to 6-mercaptopurine in acute lymphoblastic leukemia. Here we demonstrate that constitutively active NT5C2 mutations K359Q and L375F reconfigure the catalytic center for substrate access and catalysis in the absence of allosteric activator. In contrast, most relapse-associated mutations, which involve the arm segment and residues along the surface of the inter-monomeric cavity, disrupt a built-in switch-off mechanism responsible for turning off NT5C2. In addition, we show that the C-terminal acidic tail lost in the Q523X mutation functions to restrain NT5C2 activation. These results uncover dynamic mechanisms of enzyme regulation targeted by chemotherapy resistance-driving NT5C2 mutations, with important implications for the development of NT5C2 inhibitor therapies.

Can one target T-cell ALL?

Progress in our understanding of the central genes, pathways, and mechanisms in the pathobiology of T-cell acute lymphoblastic leukemia (T-ALL) has identified key drivers of the disease, opening new opportunities for therapy. Drugs targeting highly prevalent genetic alterations in NOTCH1 and CDKN2A are being explored, and multiple other targets with readily available therapeutic agents, and immunotherapies are being investigated. The molecular basis of T-ALL is reviewed here and potential targets and therapeutic targets discussed.

Circular RNA circ-NT5C2 acts as an oncogene in osteosarcoma proliferation and metastasis through targeting miR-448.

Circular RNAs (circRNAs) are a type of endogenous noncoding RNA which have been verified to participate in numerous pathophysiological processes. However, the underlying role of circRNAs in osteosarcoma tissue is still unidentified. Our study aims to investigate the circRNA expression profiles in osteosarcoma tissue and investigate the physiological functions of circRNAs. Human circRNAs microarray analysis showed that 785 differently expressed circRNAs were distinguished in osteosarcoma tissue and adjacent non-tumor tissue with 2 fold change. Circ-NT5C2 was validated to be up-regulated expressed in 52 pairs of osteosarcoma tissue and cell lines. Furthermore, the enforced expression of circ-NT5C2 could act as a valuable diagnostic marker for osteosarcoma detection with AUC (area under the ROC curve) value of 0.753. Functional validation experiments verified that circ-NT5C2 silencing suppressed the proliferation and invasion, and promoted apoptosis of osteosarcoma cells in vitro. In vivo, circ-NT5C2 silencing inhibited the tumor growth. Bioinformatics analysis and rescue experiments indicated that circ-NT5C2 sponged miR-448, which was confirmed by luciferase reporter assay and RT-PCR assay. Overall, our study investigates the circRNAs expression profiles and determines the function of circ-NT5C2 in osteosarcoma tumorigenesis, which might serve as a novel therapeutic target of osteosarcoma patients.

Two-stage replication of previous genome-wide association studies of AS3MT-CNNM2-NT5C2 gene cluster region in a large schizophrenia case-control sample from Han Chinese population.

Schizophrenia is a devastating psychiatric condition with high heritability. Replicating the specific genetic variants that increase susceptibility to schizophrenia in different populations is critical to better understand schizophrenia. CNNM2 and NT5C2 are genes recently identified as susceptibility genes for schizophrenia in Europeans, but the exact mechanism by which these genes confer risk for schizophrenia remains unknown. In this study, we examined the potential for genetic susceptibility to schizophrenia of a three-gene cluster region, AS3MT-CNNM2-NT5C2. We implemented a two-stage strategy to conduct association analyses of the targeted regions with schizophrenia. A total of 8218 individuals were recruited, and 45 pre-selected single nucleotide polymorphisms (SNPs) were genotyped. Both single-marker and haplotype-based analyses were conducted in addition to imputation analysis to increase the coverage of our genetic markers. Two SNPs, rs11191419 (OR=1.24, P=7.28×10(-5)) and rs11191514 (OR=1.24, P=0.0003), with significant independent effects were identified. These results were supported by the data from both the discovery and validation stages. Further haplotype and imputation analyses also validated these results, and bioinformatics analyses indicated that CALHM1, which is located approximately 630kb away from CNNM2, might be a susceptible gene for schizophrenia. Our results provide further support that AS3MT, CNNM2 and CALHM1 are involved with the etiology and pathogenesis of schizophrenia, suggesting these genes are potential targets of interest for the improvement of disease management and the development of novel pharmacological strategies.

Schizophrenia risk variants affecting microRNA function and site-specific regulation of NT5C2 by miR-206.

Despite the identification of numerous schizophrenia-associated genetic variants, few have been examined functionally to identify and characterize the causal variants. To mitigate this, we aimed at identifying functional variants affecting miRNA function. Using data from a large-scale genome-wide association study of schizophrenia, we looked for schizophrenia risk variants altering either miRNA binding sites, miRNA genes, promoters for miRNA genes, or variants that were expression quantitative trait loci (eQTLs) for miRNA genes. We hereby identified several potentially functional variants relating to miRNA function with our top finding being a schizophrenia protective allele that disrupts miR-206׳s binding to NT5C2 thus leading to increased expression of this gene. A subsequent experimental follow-up of the variant using a luciferase-based reporter assay confirmed that the allele disrupts the binding. Our study therefore suggests that miR-206 may contribute to schizophrenia risk through allele-dependent regulation of the genome-wide significant gene NT5C2.