MYCN amplification and ATRX mutations are incompatible in neuroblastoma.

Aggressive cancers often have activating mutations in growth-controlling oncogenes and inactivating mutations in tumor-suppressor genes. In neuroblastoma, amplification of the MYCN oncogene and inactivation of the ATRX tumor-suppressor gene correlate with high-risk disease and poor prognosis. Here we show that ATRX mutations and MYCN amplification are mutually exclusive across all ages and stages in neuroblastoma. Using human cell lines and mouse models, we found that elevated MYCN expression and ATRX mutations are incompatible. Elevated MYCN levels promote metabolic reprogramming, mitochondrial dysfunction, reactive-oxygen species generation, and DNA-replicative stress. The combination of replicative stress caused by defects in the ATRX-histone chaperone complex, and that induced by MYCN-mediated metabolic reprogramming, leads to synthetic lethality. Therefore, ATRX and MYCN represent an unusual example, where inactivation of a tumor-suppressor gene and activation of an oncogene are incompatible. This synthetic lethality may eventually be exploited to improve outcomes for patients with high-risk neuroblastoma.

Identification of Therapeutic Targets in Rhabdomyosarcoma through Integrated Genomic, Epigenomic, and Proteomic Analyses.

Personalized cancer therapy targeting somatic mutations in patient tumors is increasingly being incorporated into practice. Other therapeutic vulnerabilities resulting from changes in gene expression due to tumor specific epigenetic perturbations are progressively being recognized. These genomic and epigenomic changes are ultimately manifest in the tumor proteome and phosphoproteome. We integrated transcriptomic, epigenomic, and proteomic/phosphoproteomic data to elucidate the cellular origins and therapeutic vulnerabilities of rhabdomyosarcoma (RMS). We discovered that alveolar RMS occurs further along the developmental program than embryonal RMS. We also identified deregulation of the RAS/MEK/ERK/CDK4/6, G2/M, and unfolded protein response pathways through our integrated analysis. Comprehensive preclinical testing revealed that targeting the WEE1 kinase in the G2/M pathway is the most effective approach in vivo for high-risk RMS.

Quantification of Retinogenesis in 3D Cultures Reveals Epigenetic Memory and Higher Efficiency in iPSCs Derived from Rod Photoreceptors.

Cell-based therapies to treat retinal degeneration are now being tested in clinical trials. However, it is not known whether the source of stem cells is important for the production of differentiated cells suitable for transplantation. To test this, we generated induced pluripotent stem cells (iPSCs) from murine rod photoreceptors (r-iPSCs) and scored their ability to make retinae by using a standardized quantitative protocol called STEM-RET. We discovered that r-iPSCs more efficiently produced differentiated retinae than did embryonic stem cells (ESCs) or fibroblast-derived iPSCs (f-iPSCs). Retinae derived from f-iPSCs had fewer amacrine cells and other inner nuclear layer cells. Integrated epigenetic analysis showed that DNA methylation contributes to the defects in f-iPSC retinogenesis and that rod-specific CTCF insulator protein-binding sites may promote r-iPSC retinogenesis. Together, our data suggest that the source of stem cells is important for producing retinal neurons in three-dimensional (3D) organ cultures.

Muscarinic Acetylcholine Receptor Subtypes as Potential Drug Targets for the Treatment of Schizophrenia, Drug Abuse and Parkinson's Disease.

The neurotransmitter dopamine plays important roles in modulating cognitive, affective, and motor functions. Dysregulation of dopaminergic neurotransmission is thought to be involved in the pathophysiology of several psychiatric and neurological disorders, including schizophrenia, Parkinson's disease and drug abuse. Dopaminergic systems are regulated by cholinergic, especially muscarinic, input. Not surprisingly, increasing evidence implicates muscarinic acetylcholine receptor-mediated pathways as potential targets for the treatment of these disorders classically viewed as "dopamine based". There are five known muscarinic receptor subtypes (M(1) to M(5)). Due to their overlapping expression patterns and the lack of receptor subtype-specific ligands, the roles of the individual muscarinic receptors have long remained elusive. During the past decade, studies with knock-out mice lacking specific muscarinic receptor subtypes have greatly advanced our knowledge of the physiological roles of the M(1)-M(5) receptors. Recently, new ligands have been developed that can interact with allosteric sites on different muscarinic receptor subtypes, rather than the conventional (orthosteric) acetylcholine binding site. Such agents may lead to the development of novel classes of drugs useful for the treatment of psychosis, drug abuse and Parkinson's disease. The present review highlights recent studies carried out using muscarinic receptor knock-out mice and new subtype-selective allosteric ligands to assess the roles of M(1), M(4), and M(5) receptors in various central processes that are under strong dopaminergic control. The outcome of these studies opens new perspectives for the use of novel muscarinic drugs for several severe disorders of the CNS.

Involvement of a subpopulation of neuronal M4 muscarinic acetylcholine receptors in the antipsychotic-like effects of the M1/M4 preferring muscarinic receptor agonist xanomeline.

Disturbances in central dopaminergic neurotransmission are believed to be centrally involved in the pathogenesis of schizophrenia. Central dopaminergic and cholinergic systems interact and the cholinergic muscarinic agonist xanomeline has shown antipsychotic effects in clinical studies. Preclinical studies indicate that the M(4) muscarinic cholinergic receptor subtype (mAChR) modulates the activity of the dopaminergic system and that this specific mAChR subtype is involved in mediating the antipsychotic-like effects of xanomeline. A specific neuronal subpopulation that expresses M(4) mAChRs together with D(1) dopamine receptors seems to be especially important in modulating dopamine-dependent behaviors. Using mutant mice that lack the M(4) mAChR only in D(1) dopamine receptor-expressing cells (D1-M4-KO), we investigated the role of this neuronal population in the antipsychotic-like effects of xanomeline in amphetamine-induced hyperactivity and apomorphine-induced climbing. Interestingly, the antipsychotic-like effects of xanomeline in the two models were almost completely abolished in D1-M4-KO mice, suggesting that M(4) mAChRs colocalized with D(1) dopamine receptors are centrally involved in mediating the antipsychotic-like effects of xanomeline. This is consistent with the hypothesis that activation of the M(4) mAChR represents a potential target for the future medical treatment of psychosis.

A subpopulation of neuronal M4 muscarinic acetylcholine receptors plays a critical role in modulating dopamine-dependent behaviors.

Acetylcholine (ACh) regulates many key functions of the CNS by activating cell surface receptors referred to as muscarinic ACh receptors (M(1)-M(5) mAChRs). Like other mAChR subtypes, the M(4) mAChR is widely expressed in different regions of the forebrain. Interestingly, M(4) mAChRs are coexpressed with D(1) dopamine receptors in a specific subset of striatal projection neurons. To investigate the physiological relevance of this M(4) mAChR subpopulation in modulating dopamine-dependent behaviors, we used Cre/loxP technology to generate mutant mice that lack M(4) mAChRs only in D(1) dopamine receptor-expressing cells. The newly generated mutant mice displayed several striking behavioral phenotypes, including enhanced hyperlocomotor activity and increased behavioral sensitization following treatment with psychostimulants. These behavioral changes were accompanied by a lack of muscarinic inhibition of D(1) dopamine receptor-mediated cAMP stimulation in the striatum and an increase in dopamine efflux in the nucleus accumbens. These novel findings demonstrate that a distinct subpopulation of neuronal M(4) mAChRs plays a critical role in modulating several important dopamine-dependent behaviors. Since enhanced central dopaminergic neurotransmission is a hallmark of several severe disorders of the CNS, including schizophrenia and drug addiction, our findings have substantial clinical relevance.

Neuronal M3 muscarinic acetylcholine receptors are essential for somatotroph proliferation and normal somatic growth.

The molecular pathways that promote the proliferation and maintenance of pituitary somatotrophs and other cell types of the anterior pituitary gland are not well understood at present. However, such knowledge is likely to lead to the development of novel drugs useful for the treatment of various human growth disorders. Although muscarinic cholinergic pathways have been implicated in regulating somatotroph function, the physiological relevance of this effect and the localization and nature of the receptor subtypes involved in this activity remain unclear. We report the surprising observation that mutant mice that selectively lack the M(3) muscarinic acetylcholine receptor subtype in the brain (neurons and glial cells; Br-M3-KO mice) showed a dwarf phenotype associated with a pronounced hypoplasia of the anterior pituitary gland and a marked decrease in pituitary and serum growth hormone (GH) and prolactin. Remarkably, treatment of Br-M3-KO mice with CJC-1295, a synthetic GH-releasing hormone (GHRH) analog, rescued the growth deficit displayed by Br-M3-KO mice by restoring normal pituitary size and normal serum GH and IGF-1 levels. These findings, together with results from M(3) receptor/GHRH colocalization studies and hypothalamic hormone measurements, support a model in which central (hypothalamic) M(3) receptors are required for the proper function of hypothalamic GHRH neurons. Our data reveal an unexpected and critical role for central M(3) receptors in regulating longitudinal growth by promoting the proliferation of pituitary somatotroph cells.

Metabolic roles of the M3 muscarinic acetylcholine receptor studied with M3 receptor mutant mice: a review.

The M(3) muscarinic acetylcholine (ACh) receptor (M(3) mAChR) is expressed in many central and peripheral tissues. It is a prototypic member of the superfamily of G protein-coupled receptors and preferentially activates G proteins of the G(q) family. Recent studies involving the use of newly generated mAChR mutant mice have revealed that the M(3) mAChR plays a key role in regulating many important metabolic functions. Phenotypic analyses of mutant mice that either selectively lacked or overexpressed M(3) receptors in pancreatic beta -cells indicated that beta -cell M(3) mAChRs are essential for maintaining proper insulin release and glucose homeostasis. The experimental data also suggested that strategies aimed at enhancing signaling through beta -cell M(3) mAChRs might be beneficial for the treatment of type 2 diabetes. Recent studies with whole body M(3) mAChR knockout mice showed that the absence of M(3) receptors protected mice against various forms of experimentally or genetically induced obesity and obesity-associated metabolic deficits. Under all experimental conditions tested, M(3) receptor-deficient mice showed greatly ameliorated impairments in glucose homeostasis and insulin sensitivity, reduced food intake, and a significant elevation in basal and total energy expenditure, most likely due to increased central sympathetic outflow and increased rate of fatty acid oxidation. These findings are of potential interest for the development of novel therapeutic approaches for the treatment of obesity and associated metabolic disorders.

Learning and memory impairments in a congenic C57BL/6 strain of mice that lacks the M2 muscarinic acetylcholine receptor subtype.

The neurotransmitter acetylcholine is an important modulator of cognitive functions including attention, learning, and memory. The actions of acetylcholine are mediated by five distinct muscarinic acetylcholine receptor subtypes (M(1)-M(5)). The lack of drugs with a high degree of selectivity for these subtypes has impeded the determination of which subtypes mediate which components of cholinergic neurotransmission relevant to cognitive abilities. The present study examined the behavioral functions of the M(2) muscarinic receptor subtype by utilizing congenic C57BL/6 mice possessing a null-mutation in the M(2) muscarinic receptor gene (M(2)(-/-) mice). Comprehensive assessment of general health and the neurological function found no major differences between M(2)(-/-) and wild-type (M(2)(+/+)) mice. In the tests of learning and memory, M(2)(-/-) mice were impaired in the acquisition (trials to criterion), but not the retention (72h) of a passive avoidance task. In a novel open field, M(2)(-/-) mice were impaired in between-sessions, but not within-session habituation. In a holeboard test of spatial memory, M(2)(-/-) mice committed more errors in working memory than M(2)(+/+) mice. Reference memory did not differ between the genotypes. M(2)(-/-) mice showed no impairments in either cued or contextual fear conditioning. These findings replicate and extend earlier findings in a hybrid strain and solidify the interpretation that the M(2) receptor plays a critical role in specific components of cognitive abilities.

Beneficial metabolic effects of M3 muscarinic acetylcholine receptor deficiency.

Most animal models of obesity and hyperinsulinemia are associated with increased vagal cholinergic activity. The M3 muscarinic acetylcholine receptor subtype is widely expressed in the brain and peripheral tissues and plays a key role in mediating the physiological effects of vagal activation. Here, we tested the hypothesis that the absence of M3 receptors in mice might protect against various forms of experimentally or genetically induced obesity and obesity-associated metabolic deficits. In all cases, the lack of M3 receptors greatly ameliorated impairments in glucose homeostasis and insulin sensitivity but had less robust effects on overall adiposity. Under all experimental conditions tested, M3 receptor-deficient mice showed a significant elevation in basal and total energy expenditure, most likely due to enhanced central sympathetic outflow and increased rate of fatty-acid oxidation. These findings suggest that the M3 receptor may represent a potential pharmacologic target for the treatment of obesity and associated metabolic disorders.

A critical role for beta cell M3 muscarinic acetylcholine receptors in regulating insulin release and blood glucose homeostasis in vivo.

One of the hallmarks of type 2 diabetes is that pancreatic beta cells fail to release sufficient amounts of insulin in the presence of elevated blood glucose levels. Insulin secretion is modulated by many hormones and neurotransmitters including acetylcholine, the major neurotransmitter of the peripheral parasympathetic nervous system. The physiological role of muscarinic acetylcholine receptors expressed by pancreatic beta cells remains unclear at present. Here, we demonstrate that mutant mice selectively lacking the M3 muscarinic acetylcholine receptor subtype in pancreatic beta cells display impaired glucose tolerance and greatly reduced insulin release. In contrast, transgenic mice selectively overexpressing M3 receptors in pancreatic beta cells show a profound increase in glucose tolerance and insulin release. Moreover, these mutant mice are resistant to diet-induced glucose intolerance and hyperglycemia. These findings indicate that beta cell M3 muscarinic receptors play a key role in maintaining proper insulin release and glucose homeostasis.

Ontogeny and the possible function of a novel epidermal growth factor-like repeat domain-containing protein, NELL2, in the rat brain.

In this study we investigated the mRNA expression of NELL2, a neural tissue-specific epidermal growth factor (EGF)-like repeat domain-containing protein, in the developing and adult rat CNS using in situ hybridization histochemistry and northern blot analysis. The possible candidates that interact with or be regulated by NELL2 were screened with a cDNA expression array in antisense (AS) NELL2 oligodeoxynucleotide (ODN)-injected rat hypothalami. NELL2 mRNA was detected as early as embryonic day 10, and was predominant in the CNS throughout the pre-natal stages. Its expression gradually increased during embryonic development and its strong expression was observed throughout the CNS until embryonic day 20. It was detected in the ventricular zone of the spinal cord, medulla and pons in 12-day-old-embryos, suggesting that NELL2 plays a role in the neurogenesis of these areas. After birth its expression gradually decreased, but high levels of expression could be observed in the tenia tecta, piriform cortex, hippocampus, dentate gyrus, cerebellar cortex, ambiguus nucleus, and inferior olivary nucleus of adult rat brains. The analysis of cDNA expression arrays revealed that the administration of AS NELL2 ODN markedly decreased the expression of several Ca2+-binding proteins and those involved in the transport and release of vesicles such as EF-hand Ca2+-binding protein p22 and rab7. This finding was confirmed by relative reverse transcription-polymerase chain reaction. The effect of NELL2 on synaptic vesicle content in median eminence (ME) nerve terminals was determined with synaptophysin levels as a marker protein in the AS NELL2 ODN-injected rat. It was significantly decreased by the AS ODN. These data suggest that NELL2 may play an important role in the development of the CNS as well as maintenance of neural functions, by regulating the intracellular machinery involving Ca2+ signaling, synaptic transport and/or release of vesicles.

Cloning and localization of rgpr85 encoding rat G-protein-coupled receptor.

In an attempt to isolate genes involved in the brain development using ordered differential display PCR, we cloned rgpr85 which encodes rat G-protein-coupled receptor with high degree of identity to the amine-like neurotransmitter receptors. This gene was found to be localized at rat chromosome 4q21. In situ hybridization demonstrated that rgpr85 was predominantly expressed in the developing brain and spinal cord. Hybridization signal was especially abundant within the embryonic cortical plates where postmitotic cortical neurons are localized. In the cerebral cortex, the expression of rgpr85 was gradually decreased postnatally and became undetectable by P18. However, weak but significant expression of rgpr85 was maintained in the adult hippocampal formation, olfactory bulb, and cerebellum. Interestingly, rgpr85 expression was transiently induced in the adult hippocampal formation, piriform cortex, and amygdaloid complex by kainic acid (KA) treatment. Thus, dynamic regulation of rgpr85 expression suggests an importance of rgpr85-mediated signaling in the development of cerebral cortex and in the KA-induced responses in the adult brain.