Magel2 knockdown in hypothalamic POMC neurons innervating the medial amygdala reduces susceptibility to diet-induced obesity.

Hyperphagia and obesity profoundly affect the health of children with Prader-Willi syndrome (PWS). The Magel2 gene among the genes in the Prader-Willi syndrome deletion region is expressed in proopiomelanocortin (POMC) neurons in the arcuate nucleus of the hypothalamus (ARC). Knockout of the Magel2 gene disrupts POMC neuronal circuits and functions. Here, we report that loss of the Magel2 gene exclusively in ARCPOMC neurons innervating the medial amygdala (MeA) causes a reduction in body weight in both male and female mice fed with a high-fat diet. This anti-obesity effect is associated with an increased locomotor activity. There are no significant differences in glucose and insulin tolerance in mice without the Magel2 gene in ARCPOMC neurons innervating the MeA. Plasma estrogen levels are higher in female mutant mice than in controls. Blockade of the G protein-coupled estrogen receptor (GPER), but not estrogen receptor-α (ER-α), reduces locomotor activity in female mutant mice. Hence, our study provides evidence that knockdown of the Magel2 gene in ARCPOMC neurons innervating the MeA reduces susceptibility to diet-induced obesity with increased locomotor activity through activation of central GPER.

The Metabolic Efficacy of a Cannabidiolic Acid (CBDA) Derivative in Treating Diet- and Genetic-Induced Obesity.

Obesity is a global medical problem; its common form is known as diet-induced obesity (DIO); however, there are several rare genetic disorders, such as Prader-Willi syndrome (PWS), that are also associated with obesity (genetic-induced obesity, GIO). The currently available therapeutics for treating DIO and GIO are very limited, and they result in only a partial improvement. Cannabidiolic acid (CBDA), a constituent of Cannabis sativa, gradually decarboxylates to cannabidiol (CBD). Whereas the anti-obesity properties of CBD have been reasonably identified, our knowledge of the pharmacology of CBDA is more limited due to its instability. To stabilize CBDA, a new derivative, CBDA-O-methyl ester (HU-580, EPM301), was synthesized. The therapeutic potential of EPM301 in appetite reduction, weight loss, and metabolic improvements in DIO and GIO was tested in vivo. EPM301 (40 mg/kg/d, i.p.) successfully resulted in weight loss, increased ambulation, as well as improved glycemic and lipid profiles in DIO mice. Additionally, EPM301 ameliorated DIO-induced hepatic dysfunction and steatosis. Importantly, EPM301 (20 and 40 mg/kg/d, i.p.) effectively reduced body weight and hyperphagia in a high-fat diet-fed Magel2null mouse model for PWS. In addition, when given to standard-diet-fed Magel2null mice as a preventive treatment, EPM301 completely inhibited weight gain and adiposity. Lastly, EPM301 increased the oxidation of different nutrients in each strain. All together, EPM301 ameliorated obesity and its metabolic abnormalities in both DIO and GIO. These results support the idea to further promote this synthetic CBDA derivative toward clinical evaluation in humans.

Prader-Willi syndrome: Hormone therapies.

Prader-Willi syndrome (PWS) is a rare genetic neurodevelopmental disorder linked to the lack of expression of specific maternally imprinted genes located in the chromosomal region 15q11-q13. Impaired hypothalamic development and function explain most of the phenotype that is characterized by a specific trajectory from anorexia at birth to excessive weight gain at later ages, which is accompanied by hyperphagia and early severe obesity, as well as by other hormonal deficiencies, behavioral deficits, and dysautonomia. In almost all patients, their endocrine dysfunction involves growth hormone deficiency and hypogonadism, which originate from a combination of both peripheral and hypothalamic origin, central hypothyroidism in 40%, precocious adrenarche in 30% of the cases, and in rare cases, also adrenocorticotropin deficiency and precocious puberty. In addition, the oxytocin (OXT) and ghrelin systems are impaired in most patients and involved in a poor suckling response at birth, and hyperphagia with food addiction, poor social skills, and emotional dysregulation. Current hormonal replacement treatments are the same as used in classical hormonal deficiencies, and recombinant human GH treatment is registered since 2000 and has dramatically changed the phenotype of these children. OXT and OXT analogue treatments are currently investigated as well as new molecules targeting the ghrelin system. The severe condition of PWS can be seen as a model to improve the fine description and treatments of hypothalamic dysfunction.

Transcriptomics of the Prader-Willi syndrome hypothalamus.

Prader-Willi syndrome (PWS) is a complex neurodevelopmental disorder, arising from a loss of paternity expressed genetic material on the imprinted chromosome locus 15q11-q13. Despite increasing clarity on the underlying genetic defects, the molecular basis of the condition remains poorly understood. Hypothalamic dysfunction is widely recognized as the basis of the core symptoms of PWS, which include a deficiency in growth hormone and reproductive hormones, circadian rhythm abnormalities, and a lack of satiety, leading to an extreme obesity, among others. Genome-wide gene expression analysis (transcriptomics) offers an unbiased interrogation of complex disease pathogenesis and a potential window into the dysregulated pathways involved in disease. In this chapter, we review the findings from recent work investigating the PWS hypothalamic transcriptome, discuss the significance of the findings in relation to the clinical presentation and molecular underpinnings of PWS, and highlight future research directions.

Snord116 Post-transcriptionally Increases Nhlh2 mRNA Stability: Implications for Human Prader-Willi Syndrome.

The smallest genomic region causing Prader-Willi Syndrome (PWS) deletes the non-coding RNA SNORD116 cluster; however, the function of SNORD116 remains a mystery. Previous work in the field revealed the tantalizing possibility that expression of NHLH2, a gene previously implicated in both obesity and hypogonadism, was downregulated in PWS patients and differentiated stem cells. In silico RNA: RNA modeling identified several potential interaction domains between SNORD116 and NHLH2 mRNA. One of these interaction domains was highly conserved in most vertebrate NHLH2 mRNAs examined. A construct containing the Nhlh2 mRNA, including its 3'-UTR, linked to a c-myc tag was transfected into a hypothalamic neuron cell line in the presence and absence of exogenously-expressed Snord116. Nhlh2 mRNA expression was upregulated in the presence of Snord116 dependent on the length and type of 3'UTR used on the construct. Furthermore, use of actinomycin D to stop new transcription in N29/2 cells demonstrated that the upregulation occurred through increased stability of the Nhlh2 mRNA in the 45 minutes immediately following transcription. In silico modeling also revealed that a single nucleotide variant (SNV) in the NHLH2 mRNA could reduce the predicted interaction strength of the NHLH2:SNORD116 diad. Indeed, use of an Nhlh2 mRNA construct containing this SNV significantly reduces the ability of Snord116 to increase Nhlh2 mRNA levels. For the first time, these data identify a motif and mechanism for SNORD116-mediated regulation of NHLH2, clarifying the mechanism by which deletion of the SNORD116 snoRNAs locus leads to PWS phenotypes.

Endocrine disorders in Prader-Willi syndrome: a model to understand and treat hypothalamic dysfunction.

Prader-Willi syndrome is a rare genetic neurodevelopmental disorder resulting from the loss of expression of maternally imprinted genes located in the paternal chromosomal region, 15q11-13. Impaired hypothalamic development and function is the cause of most of the phenotypes comprising the developmental trajectory of Prader-Willi syndrome: from anorexia at birth to excessive weight gain preceding hyperphagia, and early severe obesity with hormonal deficiencies, behavioural problems, and dysautonomia. Growth hormone deficiency, hypogonadism, hypothyroidism, premature adrenarche, corticotropin deficiency, precocious puberty, and glucose metabolism disorders are the main endocrine dysfunctions observed. Additionally, as a result of hypothalamic dysfunction, oxytocin and ghrelin systems are impaired in most patients. Standard pituitary and gonadal hormone replacement therapies are required. In this Review, we discuss Prader-Willi syndrome as a model of hypothalamic dysfunction, and provide a comprehensive description of the accumulated knowledge on genetics, pathophysiology, and treatment approaches of this rare disorder.

The genetic background and vitamin D supplementation can affect irisin levels in Prader-Willi syndrome.

BACKGROUND: Prader-Willi syndrome (PWS) is associated to distinctive clinical symptoms, including obesity, cognitive and behavioral disorders, and bone impairment. Irisin is a myokine that acts on several target organs including brain adipose tissue and bone. The present study was finalized to explore circulating levels of irisin in children and adult PWS patients. METHODS: Seventy-eight subjects with PWS, 26 children (15 females, mean age 9.48 ± 3.6 years) and 52 adults (30 females, mean age 30.6 ± 10.7) were enrolled. Irisin serum levels were measured in patients and controls. Its levels were related with anthropometric and metabolic parameters, cognitive performance and bone mineral density either in pediatric or adult PWS. Multiple regression analysis was also performed. RESULTS: Irisin serum levels in PWS patients did not show different compared with controls. A more in-depth analysis showed that both pediatric and adult PWS with DEL15 displayed significantly reduced irisin levels compared to controls. Otherwise, no differences in irisin concentration were found in UPD15 patients with respect to controls. Our study revealed that in pediatric PWS the 25(OH) vitamin-D levels affected irisin serum concentration. Indeed, patients who were not supplemented with vitamin D showed lower irisin levels than controls and patients performing the supplementation. Multiple regression analysis showed that irisin levels in pediatric and adult PWS were predicted by the genetic background and 25(OH)-vitamin D levels, whereas in a group of 29 adult PWS also by intelligent quotient. CONCLUSION: We demonstrated the possible role of genetic background and vitamin-D supplementation on irisin serum levels in PWS patients.

Central neurogenetic signatures of the visuomotor integration system.

Visuomotor impairments characterize numerous neurological disorders and neurogenetic syndromes, such as autism spectrum disorder (ASD) and Dravet, Fragile X, Prader-Willi, Turner, and Williams syndromes. Despite recent advances in systems neuroscience, the biological basis underlying visuomotor functional impairments associated with these clinical conditions is poorly understood. In this study, we used neuroimaging connectomic approaches to map the visuomotor integration (VMI) system in the human brain and investigated the topology approximation of the VMI network to the Allen Human Brain Atlas, a whole-brain transcriptome-wide atlas of cortical genetic expression. We found the genetic expression of four genes-TBR1, SCN1A, MAGEL2, and CACNB4-to be prominently associated with visuomotor integrators in the human cortex. TBR1 gene transcripts, an ASD gene whose expression is related to neural development of the cortex and the hippocampus, showed a central spatial allocation within the VMI system. Our findings delineate gene expression traits underlying the VMI system in the human cortex, where specific genes, such as TBR1, are likely to play a central role in its neuronal organization, as well as on specific phenotypes of neurogenetic syndromes.

ROHHAD and Prader-Willi syndrome (PWS): clinical and genetic comparison.

BACKGROUND: Rapid-onset obesity with hypothalamic dysfunction, hypoventilation, and autonomic dysregulation (ROHHAD) is a very rare and potentially fatal pediatric disorder, the cause of which is presently unknown. ROHHAD is often compared to Prader-Willi syndrome (PWS) because both share childhood obesity as one of their most prominent and recognizable signs, and because other symptoms such as hypoventilation and autonomic dysfunction are seen in both. These phenotypic similarities suggest they might be etiologically related conditions. We performed an in-depth clinical comparison of the phenotypes of ROHHAD and PWS and used NGS and Sanger sequencing to analyze the coding regions of genes in the PWS region among seven ROHHAD probands. RESULTS: Detailed clinical comparison of ROHHAD and PWS patients revealed many important differences between the phenotypes. In particular, we highlight the fact that the areas of apparent overlap (childhood-onset obesity, hypoventilation, autonomic dysfunction) actually differ in fundamental ways, including different forms and severity of hypoventilation, different rates of obesity onset, and different manifestations of autonomic dysfunction. We did not detect any disease-causing mutations within PWS candidate genes in ROHHAD probands. CONCLUSIONS: ROHHAD and PWS are clinically distinct conditions, and do not share a genetic etiology. Our detailed clinical comparison and genetic analyses should assist physicians in timely distinction between the two disorders in obese children. Of particular importance, ROHHAD patients will have had a normal and healthy first year of life; something that is never seen in infants with PWS.

Impaired melanocortin pathway function in Prader-Willi syndrome gene-Magel2 deficient mice.

Prader-Willi Syndrome (PWS) is a neurodevelopmental disorder causing social and learning deficits, impaired satiety and severe childhood obesity. Genetic underpinning of PWS involves deletion of a chromosomal region with several genes, including MAGEL2, which is abundantly expressed in the hypothalamus. Of appetite regulating hypothalamic cell types, both AGRP and POMC-expressing neurons contain Magel2 transcripts but the functional impact of its deletion on these cells has not been fully characterized. Here, we investigated these key neurons in Magel2-null mice in terms of the activity levels at different energy states as well as their behavioral function. Using cell type specific ex vivo electrophysiological recordings and in vivo chemogenetic activation approaches we evaluated impact of Magel2 deletion on AGRP and POMC-neuron induced changes in appetite. Our results suggest that POMC neuron activity profile as well as its communication with downstream targets is significantly compromised, while AGRP neuron function with respect to short term feeding is relatively unaffected in Magel2 deficiency.

Snord116-dependent diurnal rhythm of DNA methylation in mouse cortex.

Rhythmic oscillations of physiological processes depend on integrating the circadian clock and diurnal environment. DNA methylation is epigenetically responsive to daily rhythms, as a subset of CpG dinucleotides in brain exhibit diurnal rhythmic methylation. Here, we show a major genetic effect on rhythmic methylation in a mouse Snord116 deletion model of the imprinted disorder Prader-Willi syndrome (PWS). More than 23,000 diurnally rhythmic CpGs are identified in wild-type cortex, with nearly all lost or phase-shifted in PWS. Circadian dysregulation of a second imprinted Snord cluster at the Temple/Kagami-Ogata syndrome locus is observed at the level of methylation, transcription, and chromatin, providing mechanistic evidence of cross-talk. Genes identified by diurnal epigenetic changes in PWS mice overlapped rhythmic and PWS-specific genes in human brain and are enriched for PWS-relevant phenotypes and pathways. These results support the proposed evolutionary relationship between imprinting and sleep, and suggest possible chronotherapy in the treatment of PWS and related disorders.

A Transcriptomic Signature of the Hypothalamic Response to Fasting and BDNF Deficiency in Prader-Willi Syndrome.

Transcriptional analysis of brain tissue from people with molecularly defined causes of obesity may highlight disease mechanisms and therapeutic targets. We performed RNA sequencing of hypothalamus from individuals with Prader-Willi syndrome (PWS), a genetic obesity syndrome characterized by severe hyperphagia. We found that upregulated genes overlap with the transcriptome of mouse Agrp neurons that signal hunger, while downregulated genes overlap with the expression profile of Pomc neurons activated by feeding. Downregulated genes are expressed mainly in neuronal cells and contribute to neurogenesis, neurotransmitter release, and synaptic plasticity, while upregulated, predominantly microglial genes are involved in inflammatory responses. This transcriptional signature may be mediated by reduced brain-derived neurotrophic factor expression. Additionally, we implicate disruption of alternative splicing as a potential molecular mechanism underlying neuronal dysfunction in PWS. Transcriptomic analysis of the human hypothalamus may identify neural mechanisms involved in energy homeostasis and potential therapeutic targets for weight loss.

Hypothalamic loss of Snord116 recapitulates the hyperphagia of Prader-Willi syndrome.

Profound hyperphagia is a major disabling feature of Prader-Willi syndrome (PWS). Characterization of the mechanisms that underlie PWS-associated hyperphagia has been slowed by the paucity of animal models with increased food intake or obesity. Mice with a microdeletion encompassing the Snord116 cluster of noncoding RNAs encoded within the Prader-Willi minimal deletion critical region have previously been reported to show growth retardation and hyperphagia. Here, consistent with previous reports, we observed growth retardation in Snord116+/-P mice with a congenital paternal Snord116 deletion. However, these mice neither displayed increased food intake nor had reduced hypothalamic expression of the proprotein convertase 1 gene PCSK1 or its upstream regulator NHLH2, which have recently been suggested to be key mediators of PWS pathogenesis. Specifically, we disrupted Snord116 expression in the mediobasal hypothalamus in Snord116fl mice via bilateral stereotaxic injections of a Cre-expressing adeno-associated virus (AAV). While the Cre-injected mice had no change in measured energy expenditure, they became hyperphagic between 9 and 10 weeks after injection, with a subset of animals developing marked obesity. In conclusion, we show that selective disruption of Snord116 expression in the mediobasal hypothalamus models the hyperphagia of PWS.

The Prader-Willi syndrome proteins MAGEL2 and necdin regulate leptin receptor cell surface abundance through ubiquitination pathways.

In Prader-Willi syndrome (PWS), obesity is caused by the disruption of appetite-controlling pathways in the brain. Two PWS candidate genes encode MAGEL2 and necdin, related melanoma antigen proteins that assemble into ubiquitination complexes. Mice lacking Magel2 are obese and lack leptin sensitivity in hypothalamic pro-opiomelanocortin neurons, suggesting dysregulation of leptin receptor (LepR) activity. Hypothalamus from Magel2-null mice had less LepR and altered levels of ubiquitin pathway proteins that regulate LepR processing (Rnf41, Usp8, and Stam1). MAGEL2 increased the cell surface abundance of LepR and decreased their degradation. LepR interacts with necdin, which interacts with MAGEL2, which complexes with RNF41 and USP8. Mutations in the MAGE homology domain of MAGEL2 suppress RNF41 stabilization and prevent the MAGEL2-mediated increase of cell surface LepR. Thus, MAGEL2 and necdin together control LepR sorting and degradation through a dynamic ubiquitin-dependent pathway. Loss of MAGEL2 and necdin may uncouple LepR from ubiquitination pathways, providing a cellular mechanism for obesity in PWS.

Ambient temperature modulates the effects of the Prader-Willi syndrome candidate gene Snord116 on energy homeostasis.

Germline deletion of the Prader-Willi syndrome (PWS) candidate gene Snord116 in mice leads to some classical symptoms of human PWS, notably reductions in body weight, linear growth and bone mass. However, Snord116 deficient mice (Snord116-/-) do not develop an obese phenotype despite their increased food intake and the underlying mechanism for that is unknown. We tested the phenotypes of germline Snord116-/- as well as neuropeptide Y (NPY) neuron specific Snord116lox/lox/NPYcre/+ mice at 30°C, the thermoneutral temperature of mice, and compared these to previous reports studies conducted at normal room temperature. Snord116-/- mice at 30°C still weighed less than wild type but had increased body weight gain. Importantly, food intake and energy expenditure were no longer different at 30°C, and the reduced bone mass and nasal-anal length observed in Snord116-/- mice at room temperature were also normalized. Mechanistically, the thermoneutral condition led to the correction of the mRNA expression of NPY and pro-opiomelanocortin (POMC), which were both previously observed to be significantly up-regulated at room temperature. Importantly, almost identical phenotypes and NPY/POMC mRNA expression alterations were also observed in Snord116lox/lox/NPYcre/+ mice, which lack the Snord116 gene only in NPY neurons. These data illustrate that mild cold stress is a critical factor preventing the development of obesity in Snord116-/- mice via the NPY system. Our study highlights that the function of Snord116 in the hypothalamus may be to enhance energy expenditure, likely via the NPY system, and also indicates that Snord116 function in mice is strongly dependent on environmental conditions such as cold exposure.

Changeability of the fully methylated status of the 15q11.2 region in induced pluripotent stem cells derived from a patient with Prader-Willi syndrome.

Prader-Will syndrome (PWS) is characterized by hyperphagia, growth hormone deficiency and central hypogonadism caused by the dysfunction of the hypothalamus. Patients with PWS present with methylation abnormalities of the PWS-imprinting control region in chromosome 15q11.2, subject to parent-of-origin-specific methylation and controlling the parent-of-origin-specific expression of other paternally expressed genes flanking the region. In theory, the reversal of hypermethylation in the hypothalamic cells could be a promising strategy for the treatment of PWS patients, since cardinal symptoms of PWS patients are correlated with dysfunction of the hypothalamus. The genome-wide methylation status dramatically changes during the reprograming of somatic cells into induced pluripotent stem cells (iPSCs) and during the in vitro culture of iPSCs. Here, we tested the methylation status of the chromosome 15q11.2 region in iPSCs from a PWS patient using pyrosequencing and a more detailed method of genome-wide DNA methylation profiling to reveal whether iPSCs with a partially unmethylated status for the chromosome 15q11.2 region exhibit global methylation aberrations. As a result, we were able to show that a fully methylated status for chromosome 15q11.2 in a PWS patient could be reversed to a partially unmethylated status in at least some of the PWS-iPSC lines. Genome-wide DNA methylation profiling revealed that the partial unmethylation occurred at differentially methylated regions located in chromosome 15q11.2, but not at other differentially methylated regions associated with genome imprinting. The present data potentially opens a door to cell-based therapy for PWS patients and, possibly, patients with other disorders associated with genomic imprinting.

Loss of Magel2 impairs the development of hypothalamic Anorexigenic circuits.

Prader-Willi syndrome (PWS) is a genetic disorder characterized by a variety of physiological and behavioral dysregulations, including hyperphagia, a condition that can lead to life-threatening obesity. Feeding behavior is a highly complex process with multiple feedback loops that involve both peripheral and central systems. The arcuate nucleus of the hypothalamus (ARH) is critical for the regulation of homeostatic processes including feeding, and this nucleus develops during neonatal life under of the influence of both environmental and genetic factors. Although much attention has focused on the metabolic and behavioral outcomes of PWS, an understanding of its effects on the development of hypothalamic circuits remains elusive. Here, we show that mice lacking Magel2, one of the genes responsible for the etiology of PWS, display an abnormal development of ARH axonal projections. Notably, the density of anorexigenic α-melanocyte-stimulating hormone axons was reduced in adult Magel2-null mice, while the density of orexigenic agouti-related peptide fibers in the mutant mice appeared identical to that in control mice. On the basis of previous findings showing a pivotal role for metabolic hormones in hypothalamic development, we also measured leptin and ghrelin levels in Magel2-null and control neonates and found that mutant mice have normal leptin and ghrelin levels. In vitro experiments show that Magel2 directly promotes axon growth. Together, these findings suggest that a loss of Magel2 leads to the disruption of hypothalamic feeding circuits, an effect that appears to be independent of the neurodevelopmental effects of leptin and ghrelin and likely involves a direct neurotrophic effect of Magel2.

SNORD116 and SNORD115 change expression of multiple genes and modify each other's activity.

The loss of two gene clusters encoding small nucleolar RNAs, SNORD115 and SNORD116 contribute to Prader-Willi syndrome (PWS), the most common syndromic form of obesity in humans. SNORD115 and SNORD116 are considered to be orphan C/D box snoRNAs (SNORDs) as they do not target rRNAs or snRNAs. SNORD115 exhibits sequence complementarity towards the serotonin receptor 2C, but SNORD116 shows no extended complementarities to known RNAs. To identify molecular targets, we performed genome-wide array analysis after overexpressing SNORD115 and SNORD116 in HEK 293T cells, either alone or together. We found that SNORD116 changes the expression of over 200 genes. SNORD116 mainly changed mRNA expression levels. Surprisingly, we found that SNORD115 changes SNORD116's influence on gene expression. In similar experiments, we compared gene expression in post-mortem hypothalamus between individuals with PWS and aged-matched controls. The synopsis of these experiments resulted in 23 genes whose expression levels were influenced by SNORD116. Together our results indicate that SNORD115 and SNORD116 influence expression levels of multiple genes and modify each other activity.

A new pathway in the control of the initiation of puberty: the MKRN3 gene.

Pubertal timing is influenced by complex interactions among genetic, nutritional, environmental, and socioeconomic factors. The role of MKRN3, an imprinted gene located in the Prader-Willi syndrome critical region (chromosome 15q11-13), in pubertal initiation was first described in 2013 after the identification of deleterious MKRN3 mutations in five families with central precocious puberty (CPP) using whole-exome sequencing analysis. Since then, additional loss-of-function mutations of MKRN3 have been associated with the inherited premature sexual development phenotype in girls and boys from different ethnic groups. In all of these families, segregation analysis clearly demonstrated autosomal dominant inheritance with complete penetrance, but with exclusive paternal transmission, consistent with the monoallelic expression of MKRN3 (a maternally imprinted gene). Interestingly, the hypothalamic Mkrn3 mRNA expression pattern in mice correlated with a putative inhibitory input on puberty initiation. Indeed, the initiation of puberty depends on a decrease in factors that inhibit the release of GnRH combined with an increase in stimulatory factors. These recent human and animal findings suggest that MKRN3 plays an inhibitory role in the reproductive axis to represent a new pathway in pubertal regulation.