Prader-Willi syndrome (PWS) is caused by a loss of paternally expressed genes in an imprinted region of chromosome 15q. Among the canonical PWS phenotypes are hyperphagic obesity, central hypogonadism, and low growth hormone (GH). Rare microdeletions in PWS patients define a 91-kb minimum critical deletion region encompassing 3 genes, including the noncoding RNA gene SNORD116. Here, we found that protein and transcript levels of nescient helix loop helix 2 (NHLH2) and the prohormone convertase PC1 (encoded by PCSK1) were reduced in PWS patient induced pluripotent stem cell-derived (iPSC-derived) neurons. Moreover, Nhlh2 and Pcsk1 expression were reduced in hypothalami of fasted Snord116 paternal knockout (Snord116p-/m+) mice. Hypothalamic Agrp and Npy remained elevated following refeeding in association with relative hyperphagia in Snord116p-/m+ mice. Nhlh2-deficient mice display growth deficiencies as adolescents and hypogonadism, hyperphagia, and obesity as adults. Nhlh2 has also been shown to promote Pcsk1 expression. Humans and mice deficient in PC1 display hyperphagic obesity, hypogonadism, decreased GH, and hypoinsulinemic diabetes due to impaired prohormone processing. Here, we found that Snord116p-/m+ mice displayed in vivo functional defects in prohormone processing of proinsulin, pro-GH-releasing hormone, and proghrelin in association with reductions in islet, hypothalamic, and stomach PC1 content. Our findings suggest that the major neuroendocrine features of PWS are due to PC1 deficiency.

RNA transcripts encoding the 2C-subtype of serotonin (5HT 2C ) receptor undergo up to five adenosine-to-inosine editing events to encode twenty-four protein isoforms. To examine the effects of altered 5HT 2C editing in vivo, we generated mutant mice solely expressing the fullyedited (VGV) isoform of the receptor. Mutant animals present phenotypic characteristics of Prader-Willi Syndrome (PWS) including a failure to thrive, decreased somatic growth, neonatal muscular hypotonia, and reduced food consumption followed by post-weaning hyperphagia. Though previous studies have identified alterations in both 5HT 2C receptor expression and 5HT 2C -mediated behaviors in both PWS patients and mouse models of this disorder, to our knowledge the 5HT 2C gene is the first locus outside the PWS imprinted region in which mutations can phenocopy numerous aspects of this syndrome. These results not only strengthen the link between the molecular etiology of PWS and altered 5HT 2C expression, but also demonstrate the importance of normal patterns of 5HT 2C RNA editing in vivo.

RNA editing is a post-transcriptional modification in which adenosine residues are converted to inosine (adenosine-toinosine editing). Commonly used methodologies to quantify RNA editing levels involve either direct sequencing or pyrosequencing of individual cDNA clones. The limitations of these methods lead to a small number of clones characterized in comparison to the number of mRNA molecules in the original sample, thereby producing significant sampling errors and potentially erroneous conclusions. We have developed an improved method for quantifying RNA editing patterns that increases sequence analysis to an average of more than 800,000 individual cDNAs per sample, substantially increasing accuracy and sensitivity. Our method is based on the serotonin 2C receptor (5-hydroxytryptamine 2C ; 5HT 2C ) transcript, an RNA editing substrate in which up to five adenosines are modified.Using a high-throughput multiplexed transcript analysis, we were able to quantify accurately the expression of twenty 5HT 2C isoforms, each representing at least 0.25% of the total 5HT 2C transcripts. Furthermore, this approach allowed the detection of previously unobserved changes in 5HT 2C editing in RNA samples isolated from different inbred mouse strains and dissected brain regions, as well as editing differences in alternatively spliced 5HT 2C variants. This approach provides a novel and efficient strategy for large-scale analyses of RNA editing and may prove to be a valuable tool for uncovering new information regarding editing patterns in specific disease states and in response to pharmacological and physiological perturbation, further elucidating the impact of 5HT 2C RNA editing on central nervous system function.

Global neurodevelopmental delay is a prominent characteristic of individuals with Prader-Willi syndrome (PWS). The neuromolecular bases for these delays are unknown. We identified neuroanatomical changes in the brains of mice deficient for a gene in the minimal critical deletion region for PWS (Snord116p-/m+). In Snord116p-/m+ mice, reduced primary forebrain neuron cell body size is apparent in embryonic day 15.5 fetuses, and persists until postnatal day 30 in cerebellar Purkinje neurons. Snord116 is a snoRNA gene cluster of unknown function that can localize to the nucleolus. In cerebellar Purkinje neurons from postnatal day 30 Snord116p-/m+ mice the reduction in neuronal cell body size was associated with decreased neuronal nucleolar size. We also identified developmental changes in the endocrine pancreas of Snord116p-/m+ animals that persist into adulthood. Mice lacking Snord116 have smaller pancreatic islets; within the islet the percentage of δ-cells is increased, while the percentage of α-cells is reduced. The α-cell markers, Sst and Hhex, are upregulated in Snord116p-/m+ isolated islets while Ins1, Ins2, Pdx1, Nkx6-1, and Pax6 are downregulated. There is a 3-fold increase in the percentage of polyhormonal cells in the neonatal pancreata of Snord116p-/m+ mice, due primarily to an increase in cells co-positive with somatostatin. Snord116 may play a role in islet cell lineage specification. The Snord116 gene cluster is important for developmental processes in the brain as well as the endocrine pancreas.

A-to-I RNA editing is an important process for generating molecular diversity in the brain through modification of transcripts encoding several proteins important for neuronal signaling. We investigated the relationships between the extent of editing at multiple substrate transcripts (5HT2C, MGLUR4, CADPS, GLUR2, GLUR4, and GABRA3) in brain tissue obtained from adult humans and rhesus macaques. Several patterns emerged from these studies revealing conservation of editing across primate species. Additionally, variability in the human population allows us to make novel inferences about the co-regulation of editing at different editing sites and even across different brain regions.Electronic supplementary materialThe online version of this article (doi:10.1186/s13041-017-0291-1) contains supplementary material, which is available to authorized users.

Transcripts encoding ADAR1, a double-stranded, RNA-specific adenosine deaminase involved in the adenosine-to-inosine (A-to-I) editing of mammalian RNAs, can be alternatively spliced to produce an interferon-inducible protein isoform (p150) that is up-regulated in both cell culture and in vivo model systems in response to pathogen or interferon stimulation. In contrast to other tissues, p150 is expressed at extremely low levels in the brain and it is unclear what role, if any, this isoform may play in the innate immune response of the central nervous system (CNS) or whether the extent of editing for RNA substrates critical for CNS function is affected by its induction. To investigate the expression of ADAR1 isoforms in response to viral infection and subsequent alterations in A-to-I editing profiles for endogenous ADAR targets, we used a neuro-tropic strain of reovirus to infect neonatal mice and quantify A-to-I editing in discrete brain regions using a multiplexed, high-throughput sequencing strategy. While intracranial injection of reovirus resulted in a widespread increase in the expression of ADAR1 (p150) in multiple brain regions and peripheral organs, significant changes in site-specific A-to-I conversion were quite limited, suggesting that steady-state levels of p150 expression are not a primary determinant for modulating the extent of editing for numerous ADAR targets in vivo.

Type 2 diabetes (T2D) is a major risk factor for late-onset Alzheimer's disease (AD). A variety of metabolic changes related to T2D (e.g. hyperinsulinemia, hyperglycemia, and elevated branched-chain amino acids) have been proposed as mechanistic links, but the basis for this association remains unknown. Retromer-dependent trafficking is implicated in the pathogenesis of AD, and two key retromer proteins, VPS35 and VPS26, are deficient in the hippocampal formation of AD patients. We characterized VPS35 levels in five different mouse models of T2D/obesity to identify specific metabolic factors that could affect retromer in the brain. Mouse models in which hyperleucinemia was present displayed hippocampus-selective retromer deficiency. Wild-type lean mice fed a high leucine diet also developed hippocampal-selective retromer deficiency, and neuronal-like cells grown in high ambient leucine had reduced retromer complex proteins. Our results suggest that hyperleucinemia may account, in part, for the association of insulin resistance/T2D with AD.

The serotonin 2C receptor (5-HT2CR) plays a significant role in psychiatric disorders (e.g., depression) and is a target for pharmacotherapy. The 5-HT2CR is widely expressed in brain and spinal cord and is the only G-protein coupled receptor currently known to undergo mRNA editing, a post-transcriptional modification that results in translation of distinct, though closely related, protein isoforms. The 5-HT2CR RNA can be edited at five sites to alter up to three amino acids resulting in modulation of receptor:G-protein coupling and constitutive activity. To rapidly quantify changes ex vivo in individual 5-HT2CR isoform levels in response to treatment, we adapted quantitative (real-time) reverse transcription polymerase chain reaction (qRT-PCR) utilizing TaqMan® probes modified with a minor groove binder (MGB). Probes were developed for four 5-HT2CR RNA isoforms and their sensitivity and specificity were validated systematically using standard templates. Relative expression of the four isoforms was measured in cDNAs from whole brain extracted from 129S6 and C57BL/6J mice. Rank order derived from this qRT-PCR analysis matched that derived from DNA sequencing. In mutant mice solely expressing either non-edited or fully edited 5-HT2CR transcripts, only expected transcripts were detected. These data suggest this qRT-PCR method is a precise and rapid means to detect closely related mRNA sequences ex vivo without the necessity of characterizing the entire 5-HT2CR profile. Implementation of this technique will expand and expedite studies of specific brain 5-HT2CR mRNA isoforms in response to pharmacological, behavioral and genetic manipulation, particularly in ex vivo studies which require rapid collection of data on large numbers of samples.