Identification and characterization of Necdin as a target for the Cockayne syndrome B protein in promoting neuronal differentiation and maintenance.

Cockayne syndrome (CS) is a devastating autosomal recessive genetic disorder, mainly characterized by photosensitivity, growth failure, neurological abnormalities, and premature aging. Mutations in CSB (ERCC6) are associated with almost all clinical phenotypes resembling classic CS. Using RNA-seq approach in multiple cell types, we identified Necdin (NDN) as a target of the CSB protein. Supportive of the RNA-seq results, CSB directly binds to NDN and manipulates the remodeling of active histone marks and DNA 5mC methylation on the regulatory elements of the NDN gene. Intriguingly, hyperactivation of NDN due to CSB deficiency does not interfere with nucleotide excision repair (1), but greatly affects neuronal cell differentiation. Inhibition of NDN can partially rescue the motor neuron defects in CSB mouse models. In addition to shedding light on cellular mechanisms underlying CS and pointing to future avenues for intervention, these data substantiate a reciprocal communication between CSB and NDN in the context of general transcription regulation.

Necdin, one of the important pathway proteins in the regulation of osteosarcoma progression by microRNA-200c.

MicroRNA-200c (miR-200c) generally acts as a tumor suppressor in multiple cancer types and a promising therapeutic target in tumorigenesis. However, only a few studies have explained the role of miR-200c in the development of osteosarcoma (OS). In this study, we investigated the role of miR-200c in OS progression and identified the regulatory pathway protein NDN involved in inhibiting the occurrence and development of OS. Firstly, we found that miR-200c is downregulated in OS cells and tissues. As well, in vitro and in vivo experiments showed that upregulating miR-200c inhibits the proliferation, invasion, metastasis of Saos-2 cells, promotes the apoptosis of Saos-2 cells and suppresses tumor growth in mice, indicating miR-200c plays a major role in regulating the OS progression. Furthermore, bioinformatics analysis showed that an anti-tumor protein, necdin (NDN), might be a potential target by miR-200c. To verify this hypothesis, we measured the expression level of NDN in OS cells and tissues and found NDN is downregulated, suggesting NDN is functional in OS progression. Moreover, we found that the expression levels of NDN and miR-200c in in vivo and in vitro experiments were positively correlated. However, the results of dual-luciferase reporter gene experiment showed miR-200c does not directly act on the 3' untranslated region (UTR) of NDN gene, indicating that NDN might be an important pathway protein which regulates OS progression in the presence of miR-200c. Therefore, miR-200c/NDN could be potential targets for developing effective treatment against OS.

Necdin: A purposive integrator of molecular interaction networks for mammalian neuron vitality.

Necdin was originally found in 1991 as a hypothetical protein encoded by a neural differentiation-specific gene transcript in murine embryonal carcinoma cells. Virtually all postmitotic neurons and their precursor cells express the necdin gene (Ndn) during neuronal development. Necdin mRNA is expressed only from the paternal allele through genomic imprinting, a placental mammal-specific epigenetic mechanism. Necdin and its homologous MAGE (melanoma antigen) family, which have evolved presumedly from a subcomplex component of the SMC5/6 complex, are expressed exclusively in placental mammals. Paternal Ndn-mutated mice totally lack necdin expression and exhibit various types of neuronal abnormalities throughout the nervous system. Ndn-null neurons are vulnerable to detrimental stresses such as DNA damage. Necdin also suppresses both proliferation and apoptosis of neural stem/progenitor cells. Functional analyses using Ndn-manipulated cells reveal that necdin consistently exerts antimitotic, anti-apoptotic and prosurvival effects. Necdin interacts directly with a number of regulatory proteins including E2F1, p53, neurotrophin receptors, Sirt1 and PGC-1α, which serve as major hubs of protein-protein interaction networks for mitosis, apoptosis, differentiation, neuroprotection and energy homeostasis. This review focuses on necdin as a pleiotropic protein that integrates molecular interaction networks to promote neuronal vitality in modern placental mammals.

Firing activity of locus coeruleus noradrenergic neurons decreases in necdin-deficient mice, an animal model of Prader-Willi syndrome.

BACKGROUND: Prader-Willi syndrome (PWS) is a neurodevelopmental disorder characterized by multiple respiratory, cognitive, endocrine, and behavioral symptoms, such as central apnea, intellectual disabilities, exaggerated stress responses, and temper tantrums. The locus coeruleus noradrenergic system (LC-NE) modulates a diverse range of behaviors, including arousal, learning, pain modulation, and stress-induced negative affective states, which are possibly correlated with the pathogenesis of PWS phenotypes. Therefore, we evaluated the LC-NE neuronal activity of necdin-deficient mice, an animal model of PWS. METHODS: Heterozygous necdin-deficient mice (B6.Cg-Ndntm1ky) were bred from wild-type (WT) females to generate WT (+m/+p) and heterozygotes (+m/-p) animals, which were examined of LC-NE neuronal activity, developmental reflexes, and plethysmography. RESULTS: On slice electrophysiology, LC-NE neurons of Ndntm1ky mice with necdin deficiency showed significantly decreased spontaneous activities and impaired excitability, which was mediated by enhanced A-type voltage-dependent potassium currents. Ndntm1ky mice also exhibited the neonatal phenotypes of PWS, such as hypotonia and blunt respiratory responses to hypercapnia. CONCLUSIONS: LC-NE neuronal firing activity decreased in necdin-deficient mice, suggesting that LC, the primary source of norepinephrine in the central nervous system, is possibly involved in PWS pathogenesis.

SENP2 Suppresses Necdin Expression to Promote Brown Adipocyte Differentiation.

Brown adipose tissue (BAT) is a thermogenic organ that maintains body temperature and energy homeostasis. Transcriptional regulation plays an important role in the program of brown adipogenesis. However, it remains unclear how the transcriptional events are controlled in this program. In this study, we analyze an SENP2 BAT conditional knockout mouse model and find that SENP2-mediated de-SUMOylation is essential for BAT development. SENP2 catalyzes de-SUMOylation of cAMP response element-binding protein (CREB) to suppress Necdin expression, which induces brown adipocyte differentiation and brown adipogenesis. Mechanistically, we find that SUMOylation enhances CREB interaction with serine/threonine protein phosphatase 2A (PP2A) to de-phosphorylate CREB, which activates Necdin transcription. SENP2 deficiency enhances the expression of Necdin to inhibit brown adipocyte differentiation. Therefore, we reveal a crucial role of SENP2-mediated de-SUMOylation of CREB in suppression of Necdin expression during brown adipose development and brown adipogenesis.

The regulators of G protein signaling RGS16 and RGS18 inhibit protease-activated receptor 2/Gi/o signaling through distinct interactions with Gα in live cells.

Protease-activated receptor 2 (PAR2) is a G protein-coupled receptor (GPCR) activated by endogenous proteases, in particular, trypsin. Although regulators of G protein signaling (RGS) are known to inhibit GPCR/Gα-mediated signaling, their specific effects on PAR2 are poorly understood at present. Here, we use a bioluminescence resonance energy transfer technique to investigate whether RGS16 and RGS18 bind PAR2 in live cells to regulate PAR2/Gαi/o -mediated signaling. Notably, we find that RGS16 binds to PAR2 in the presence of Gαi while RGS18 does not interact with PAR2, regardless of the presence of Gα. Both RGS16 and RGS18 inhibit PAR2/Gαi/o -mediated signaling. To our knowledge, the current study is the first to highlight the effects of RGS proteins on PAR2-mediated signaling.

A genetic locus for paranoia.

The psychological effects of brain-expressed imprinted genes in humans are virtually unknown. Prader-Willi syndrome (PWS) is a neurogenetic condition mediated by genomic imprinting, which involves high rates of psychosis characterized by hallucinations and paranoia, as well as autism. Altered expression of two brain-expressed imprinted genes, MAGEL2 and NDN, mediates a suite of PWS-related phenotypes, including behaviour, in mice. We phenotyped a large population of typical individuals for schizophrenia-spectrum and autism-spectrum traits, and genotyped them for the single-nucleotide polymorphism rs850807, which is putatively functional and linked with MAGEL2 and NDN Genetic variation in rs850807 was strongly and exclusively associated with the ideas of reference subscale of the schizophrenia spectrum, which is best typified as paranoia. These findings provide a single-locus genetic model for analysing the neurological and psychological bases of paranoid thinking, and implicate imprinted genes, and genomic conflicts, in human mentalistic thought.

Necdin modulates leukemia-initiating cell quiescence and chemotherapy response.

Acute myeloid leukemia (AML) is a devastating illness which carries a very poor prognosis, with most patients living less than 18 months. Leukemia relapse may occur because current therapies eliminate proliferating leukemia cells but fail to eradicate quiescent leukemia-initiating cells (LICs) that can reinitiate the disease after a period of latency. While we demonstrated that p53 target gene Necdin maintains hematopoietic stem cell (HSC) quiescence, its roles in LIC quiescence and response to chemotherapy are unclear. In this study, we utilized two well-established murine models of human AML induced by MLL-AF9 or AML1-ETO9a to determine the role of Necdin in leukemogenesis. We found that loss of Necdin decreased the number of functional LICs and enhanced myeloid differentiation in vivo, leading to delayed development of leukemia induced by MLL-AF9. Importantly, Necdin null LICs expressing MLL-AF9 were less quiescent than wild-type LICs. Further, loss of Necdin enhanced the response of MLL-AF9+ leukemia cells to chemotherapy treatment, manifested by decreased viability and enhanced apoptosis. We observed decreased expression of Bcl2 and increased expression of p53 and its target gene Bax in Necdin null leukemia cells following chemotherapy treatment, indicating that p53-dependent apoptotic pathways may be activated in the absence of Necdin. In addition, we found that loss of Necdin decreased the engraftment of AML1-ETO9a+ hematopoietic stem and progenitor cells in transplantation assays. However, Necdin-deficiency did not affect the response of AML1-ETO9a+ hematopoietic cells to chemotherapy treatment. Thus, Necdin regulates leukemia-initiating cell quiescence and chemotherapy response in a context-dependent manner. Our findings suggest that pharmacological inhibition of Necdin may hold potential as a novel therapy for leukemia patients with MLL translocations.

Necdin shapes serotonergic development and SERT activity modulating breathing in a mouse model for Prader-Willi syndrome.

Prader-Willi syndrome (PWS) is a genetic neurodevelopmental disorder that presents with hypotonia and respiratory distress in neonates. The Necdin-deficient mouse is the only model that reproduces the respiratory phenotype of PWS (central apnea and blunted response to respiratory challenges). Here, we report that Necdin deletion disturbs the migration of serotonin (5-HT) neuronal precursors, leading to altered global serotonergic neuroarchitecture and increased spontaneous firing of 5-HT neurons. We show an increased expression and activity of 5-HT Transporter (SERT/Slc6a4) in 5-HT neurons leading to an increase of 5-HT uptake. In Necdin-KO pups, the genetic deletion of Slc6a4 or treatment with Fluoxetine, a 5-HT reuptake inhibitor, restored normal breathing. Unexpectedly, Fluoxetine administration was associated with respiratory side effects in wild-type animals. Overall, our results demonstrate that an increase of SERT activity is sufficient to cause the apneas in Necdin-KO pups, and that fluoxetine may offer therapeutic benefits to PWS patients with respiratory complications.

Necdin Overexpression Predicts Poor Prognosis in Patients with Urothelial Carcinomas of the Upper Urinary Tract and Urinary Bladder.

BACKGROUND AND AIMS: Oncogenesis is a multistep process, resulting from the accumulations of multiple mutations. Of these mutations, self-sufficiency in growth signals, i.e., disruption of cell growth regulation, is the first episode. Nonetheless, the genes associated with cell growth dysregulation have seldom been systematically evaluated in either urothelial carcinomas of upper urinary tract (UTUC) or urothelial carcinomas of urinary baldder (UBUC). By data mining a published transcriptomic dataset of UBUCs (GSE31684), we identified the NDN gene as one of the most significant of those associated with the regulation of cell growth and found this gene is associated with advanced tumor status and metastatic disease (GO:0001558). Accordingly, we analyzed NDN transcript and protein expression with their clinicopathological significance. MATERIALS AND METHODS: We used real time RT-PCR to detect NDN transcript levels in 27 UTUCs and 27 UBUCs, respectively. Immunohistochemical study was performed to determine NDN protein (a.k.a. Necdin) expression evaluated by H-score method in 340 UTUCs and 295 UBUCs. NDN expression was further correlated with clinicopathological features and disease-specific survival (DSS) and metastasis-free survival (MeFS). RESULTS: NDN transcriptional level was significantly higher in UCs of both sites with stepwise more advanced pT statuses. Through immunohistochemistry, we found NDN protein expression was significantly associated with adverse clinicopathological parameters, e.g., advanced pT status, nodal metastasis, high grade histological patterns, and frequent mitotses (all P<0.05). In univariate analysis, NDN overexpression not only predicted worse DSS and MeFS in both the UTUC and UBUC groups, it also served as an independent prognostic factor for DSS and MeFS in multivariate analysis (all P<0.05). CONCLUSIONS: NDN may play an important role in tumor progression in UC and could serve as a prognostic biomarker and a potential novel therapeutic target in UC.

Skeletal muscle regeneration in cancer cachexia.

Muscle wasting is the most important phenotypic and clinical feature of cancer cachexia, and the principal cause of impaired physical function, fatigue, and respiratory complications. Muscle loss has been attributed to a variable combination of reduced nutritional intake and an imbalance between anabolic and catabolic processes. It has been suggested that defective skeletal muscle regeneration may also contribute to muscle wasting in cancer patients. However, there is little in vitro or in vivo data available, in either animals or in humans, regarding skeletal muscle regeneration in cancer wasting. The aim of the present review is to define the role of skeletal muscle regeneration in the muscle wasting of cancer patients and to determine possible therapeutic implications.

Regulator of G protein signaling 8 inhibits protease-activated receptor 1/Gi/o signaling by forming a distinct G protein-dependent complex in live cells.

Activation of seven-transmembrane-domain-possessing G protein-coupled receptors (GPCRs) by extracellular stimuli elicits intracellular responses. One class of GPCRs-protease-activated receptors (PARs)-is activated by endogenous proteases, such as thrombin and trypsin. Members of the regulator of G protein signaling (RGS) family stimulate GTP hydrolysis of G protein alpha (Gα) subunits, thereby inhibiting GPCR/Gα-mediated signaling. We previously reported that RGS2 and RGS4 inhibit PAR1/Gα-mediated signaling by interacting with PAR1 in a Gα-dependent manner. Here, employing the bioluminescence resonance energy transfer (BRET) technique, we identified RGS8 as a novel PAR1-interacting protein. Very little BRET activity was observed between PAR1-Venus (PAR1-Ven) and RGS8-Luciferase (RGS8-Luc) in the absence of Gα. However, in the presence of Gαo, BRET activity was specifically and significantly increased. This interaction was confirmed by biochemical and immunofluorescence assays. Notably, RGS8 inhibited PAR1/Gαi/o-mediated adenylyl cyclase and ERK activation, and prevented Gαo-induced neurite outgrowth and activation of Necdin protein, a downstream target of Gαo. Our findings suggest a novel function of RGS8 and reveal cellular mechanisms by which RGS8 mediates PAR1 inhibition.

Necdin controls EGFR signaling linked to astrocyte differentiation in primary cortical progenitor cells.

Cellular signaling mediated by the EGF receptor (EGFR) plays a key role in controlling proliferation and differentiation of cortical progenitor cells (CPCs). However, regulatory mechanisms of EGFR signaling in CPCs remain largely unknown. Here we demonstrate that necdin, a MAGE (melanoma antigen) family protein, interacts with EGFR in primary CPCs and represses its downstream signaling linked to astrocyte differentiation. EGFR was autophosphorylated and interacted with necdin in EGF-stimulated CPCs. Necdin bound to autophosphorylated EGFR via its tyrosine kinase domain. EGF-induced phosphorylation of ERK was enhanced in necdin-null CPCs, where the interaction between EGFR and the adaptor protein Grb2 was strengthened, suggesting that endogenous necdin suppresses the EGFR/ERK signaling pathway in CPCs. In necdin-null CPCs, astrocyte differentiation induced by the gliogenic cytokine cardiotrophin-1 was significantly accelerated in the presence of EGF, and inhibition of EGFR/ERK signaling abolished the acceleration. Furthermore, necdin strongly suppressed astrocyte differentiation induced by overexpression of EGFR or its ligand binding-defective mutant equivalent to a glioblastoma-associated EGFR variant. These results suggest that necdin acts as an intrinsic suppressor of the EGFR/ERK signaling pathway in EGF-responsive CPCs to restrain astroglial development in a cell-autonomous manner.

Necdin is a breast cancer metastasis suppressor that regulates the transcription of c-Myc.

Metastasis is the primary cause of death in breast cancer. Earlier studies using a mammary tumorigenesis mouse model identified Necdin (Ndn)as a germline modifier of metastasis. Differential expression of Ndn induces a gene-expression signature that predicts prognosis in human breast cancer. Additionally, a non-synonymous germline single nucleotide polymorphism (T50C; V17A) in Ndn distinguishes mouse strains with differing metastatic capacities. To better understand how hereditary factors influence metastasis in breast cancer, we characterized NDN-mediated transcription. Haplotype analysis in a well-characterized breast cancer cohort revealed that NDN germline variation is associated with both NDN expression levels and patient outcome. To examine the role of NDN in mammary tumor metastasis and transcriptional regulation, mouse mammary tumor cell lines stably over-expressing either the wildtype 50T or variant 50C Ndn allele were generated. Cells over-expressing Ndn 50T, but not Ndn 50C, exhibited significant decrease in cell invasiveness and pulmonary metastases compared to control cells. Transcriptome analyses identified a 71-gene expression signature that distinguishes cells over-expressing the two Ndn allelic variants. Furthermore, ChIP assays revealed c-Myc, a target gene of NDN, to be differentially regulated by the allelic variants. These data demonstrate that NDN and the T50C allele regulate gene expression and metastasis efficiency.

Imprinted Zac1 in neural stem cells.

Neural stem cells (NSCs) and imprinted genes play an important role in brain development. On historical grounds, these two determinants have been largely studied independently of each other. Recent evidence suggests, however, that NSCs can reset select genomic imprints to prevent precocious depletion of the stem cell reservoir. Moreover, imprinted genes like the transcriptional regulator Zac1 can fine tune neuronal vs astroglial differentiation of NSCs. Zac1 binds in a sequence-specific manner to pro-neuronal and imprinted genes to confer transcriptional regulation and furthermore coregulates members of the p53-family in NSCs. At the genome scale, Zac1 is a central hub of an imprinted gene network comprising genes with an important role for NSC quiescence, proliferation and differentiation. Overall, transcriptional, epigenomic, and genomic mechanisms seem to coordinate the functional relationships of NSCs and imprinted genes from development to maturation, and possibly aging.

Novel NTRK1 Frameshift Mutation in Congenital Insensitivity to Pain With Anhidrosis.

Congenital insensitivity to pain with anhidrosis is a rare autosomal recessive disorder. It has been reported that the defect in the NTRK1 gene encoding tropomyosin-related kinase A (TrkA) can cause congenital insensitivity to pain with anhidrosis. Nerve growth factor (NGF), the product of NGFB, mediates biological effects by binding to and activating tropomyosin-related kinase A. In addition, necdin (encoded by NDN) is also essential in nerve growth factor-tropomyosin-related kinase A pathway. We performed mutation analysis in NTRK1, NGFB, and NDN genes in a Chinese Han 17-year-old female patient with congenital insensitivity to pain with anhidrosis and her healthy family members. As a result, the patient was found to have a novel insertion in exon 7 (c.727insT) of NTRK1, which causes premature termination, and a single nucleotide polymorphism (rs2192206 G>A) in NDN. Our findings imply that the genetic variations of the nerve growth factor-tropomyosin-related kinase A pathway play an important role in congenital insensitivity to pain with anhidrosis.

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.

Natural breaking of the maternal silence at the mouse and human imprinted Prader-Willi locus: A whisper with functional consequences.

Genomic imprinting is a normal process of epigenetic regulation leading some autosomal genes to be expressed from one parental allele only, the other parental allele being silenced. The reasons why this mechanism has been selected throughout evolution are not clear; however, expression dosage is critical for imprinted genes. There is a paradox between the fact that genomic imprinting is a robust mechanism controlling the expression of specific genes and the fact that this mechanism is based on epigenetic regulation that, per se, should present some flexibility. The robustness has been well studied, revealing the epigenetic modifications at the imprinted locus, but the flexibility has been poorly investigated.   Prader-Willi syndrome is the best-studied disease involving imprinted genes caused by the absence of expression of paternally inherited alleles of genes located in the human 15q11-q13 region. Until now, the silencing of the maternally inherited alleles was like a dogma. Rieusset et al. showed that in absence of the paternal Ndn allele, in Ndn +m/-p mice, the maternal Ndn allele is expressed at an extremely low level with a high degree of non-genetic heterogeneity. In about 50% of these mutant mice, this stochastic expression reduces birth lethality and severity of the breathing deficiency, correlated with a reduction in the loss of serotonergic neurons. Furthermore, using several mouse models, they reveal a competition between non-imprinted Ndn promoters, which results in monoallelic (paternal or maternal) Ndn expression, suggesting that Ndn monoallelic expression occurs in the absence of imprinting regulation. Importantly, specific expression of the maternal NDN allele is also detected in post-mortem brain samples of PWS individuals. Here, similar expression of the Magel2 maternal allele is reported in Magel2 +m/-p mice, suggesting that this loss of imprinting can be extended to other PWS genes. These data reveal an unexpected epigenetic flexibility of PWS imprinted genes that could be exploited to reactivate the functional but dormant maternal alleles in PWS.