George Stratigopoulos

SNORD116 is a small nucleolar RNA deficient in Prader–Willi syndrome individuals. The mechanisms by which SNORD116 conveys its effects are unknown. Here, transcriptomic analysis showed that diacylglycerol kinase kappa ( Dgkk ) expression was elevated in the hypothalamus of Snord116 -deficient mice. In silico analyses predicted a strong RNA:RNA interaction between Snord116 and Dgkk mRNA that is fully conserved in mouse and human. In vitro experiments in human cellular models demonstrated that SNORD116 mediates 2′-O-methylation of DGKK mRNA and regulates DGKK mRNA and protein expression. In Snord116 -deficient mouse neurons, increased Dgkk expression was associated with attenuated diacylglycerol-mediated activation of ERK and PKC, critical elements in neuronal signaling cascades. In mice, brain Dgkk expression peaks during late embryonic and early postnatal periods, coinciding with peak rates of neurogenesis; its expression in adults is restricted mainly to brain regions implicated in Prader–Willi syndrome pathophysiology, including the arcuate nucleus of the hypothalamus. Fasting acutely decreased hypothalamic Dgkk expression in wild-type mice. Increased Dgkk expression in Snord116 -deficient mice could reflect derangements in this pathway that affects both brain development and cell-specific functions.

Summary: Genome‐wide association studies have revealed a plethora of genetic variants that correlate with polygenic conditions. However, causal molecular mechanisms have proven challenging to fully define. Without such information, the associations are not physiologically useful or clinically actionable. By reviewing studies of the FTO locus in the genetic etiology of obesity, we wish to highlight advances in the field fueled by the evolution of technical and analytic strategies in assessing the molecular bases for genetic associations. Particular attention is drawn to extrapolating experimental findings from animal models and cell types to humans, as well as technical aspects used to identify long‐range DNA interactions and their biological relevance with regard to the associated trait. A unifying model is proposed by which independent obesogenic pathways regulated by multiple FTO variants and genes are integrated at the primary cilium, a cellular antenna where signaling molecules that control energy balance convene.

Energy balance is controlled by interconnected brain regions in the hypothalamus, brainstem, cortex, and limbic system. Gene expression signatures of these regions can help elucidate the pathophysiology underlying obesity. RNA sequencing was conducted on P56 C57BL/6NTac male mice and E14.5 C57BL/6NTac embryo punch biopsies in 16 obesity-relevant brain regions. The expression of 190 known obesity-associated genes (monogenic, rare, and low-frequency coding variants; GWAS; syndromic) was analyzed in each anatomical region. Genes associated with these genetic categories of obesity had localized expression patterns across brain regions. Known monogenic obesity causal genes were highly enriched in the arcuate nucleus of the hypothalamus and developing hypothalamus. The obesity-associated genes clustered into distinct “modules” of similar expression profile, and these were distinct from expression modules formed by similar analysis with genes known to be associated with other disease phenotypes (type 1 and type 2 diabetes, autism, breast cancer) in the same energy balance–relevant brain regions.

Bardet-Biedl syndrome (BBS) is a rare autosomal recessive disorder caused by mutations in genes encoding components of the primary cilium and is characterized by hyperphagic obesity. To investigate the molecular basis of obesity in human BBS, we developed a cellular model of BBS using induced pluripotent stem cell–derived (iPSC-derived) hypothalamic arcuate-like neurons. BBS mutations BBS1^M390R and BBS10^C91fsX95 did not affect neuronal differentiation efficiency but caused morphological defects, including impaired neurite outgrowth and longer primary cilia. Single-cell RNA sequencing of BBS1^M390R hypothalamic neurons identified several downregulated pathways, including insulin and cAMP signaling and axon guidance. Additional studies demonstrated that BBS1^M390R and BBS10^C91fsX95 mutations impaired insulin signaling in both human fibroblasts and iPSC-derived neurons. Overexpression of intact BBS10 fully restored insulin signaling by restoring insulin receptor tyrosine phosphorylation in BBS10^C91fsX95 neurons. Moreover, mutations in BBS1 and BBS10 impaired leptin-mediated p-STAT3 activation in iPSC-derived hypothalamic neurons. Correction of the BBS mutation by CRISPR rescued leptin signaling. POMC expression and neuropeptide production were decreased in BBS1^M390R and BBS10^C91fsX95 iPSC–derived hypothalamic neurons. In the aggregate, these data provide insights into the anatomic and functional mechanisms by which components of the BBSome in CNS primary cilia mediate effects on energy homeostasis.

Background: SNPs in the first intron of the Fat Mass and Obesity-Associated (FTO) gene represent the strongest genome-wide associations with adiposity (BMI); the molecular basis for these associations is under intense investigation. In European populations - the focus of most GWAS conducted to date - the SNPs have indistinguishable associations due to the high level of linkage disequilibrium (LD). However, in African American (AA) individuals, reduced LD and increased haplotype diversity permit finer distinctions among obesity-associated SNPs. Such distinctions are important to mechanistic inferences and for selection of disease SNPs relevant to specific populations. Methods: To identify specific FTO SNP(s) directly related to adiposity, we performed: 1) haplotype analyses of individual-level data in 3335 AAs from the Atherosclerosis Risk in Communities Cohort (ARIC) study; as well as 2) statistical fine-mapping using summary statistics from a study of FTO in over 20 000 AAs, and over 1000 functional genomic annotations. Results: Our haplotype analyses suggest that in AAs at least two distinct signals underlie the intron 1 FTO-adiposity signal . Fine mapping showed that two SNPs have the highest posterior probability of association (PPA) with BMI: rs9927317 (PPA=0.94) and rs62033405 (PPA=0.99). These variants overlap possible enhancer sites and the 5' regions of transcribed genes in the substantia nigra, chondrocytes, and white adipocytes. Conclusions: We found two SNPs in FTO with the highest probability of direct association with BMI in AAs, as well as tissue-specific mechanisms by which these variants may contribute to the pathogenesis of obesity.

Intronic polymorphisms in the α-ketoglutarate–dependent dioxygenase gene ( FTO ) that are highly associated with increased body weight have been implicated in the transcriptional control of a nearby ciliary gene, retinitis pigmentosa GTPase regulator-interacting protein-1 like ( RPGRIP1L ). Previous studies have shown that congenital Rpgrip1l hypomorphism in murine proopiomelanocortin (Pomc) neurons causes obesity by increasing food intake. Here, we show by congenital and adult-onset Rpgrip1l deletion in Pomc-expressing neurons that the hyperphagia and obesity are likely due to neurodevelopmental effects that are characterized by a reduction in the Pomc/Neuropeptide Y (Npy) neuronal number ratio and marked increases in arcuate hypothalamic–paraventricular hypothalamic (ARH-PVH) axonal projections. Biallelic RPGRIP1L mutations result in fewer cilia-positive human induced pluripotent stem cell–derived (iPSC-derived) neurons and blunted responses to Sonic Hedgehog (SHH). Isogenic human ARH-like embryonic stem cell–derived (ESc-derived) neurons homozygous for the obesity-risk alleles at rs8050136 or rs1421085 have decreased RPGRIP1L expression and have lower numbers of POMC neurons. RPGRIP1L overexpression increases POMC cell number. These findings suggest that apparently functional intronic polymorphisms affect hypothalamic RPGRIP1L expression and impact development of POMC neurons and their derivatives, leading to hyperphagia and increased adiposity.

Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/CRISPR-associated protein-9 (Cas9) has become the tool of choice for genome editing. Despite the fact that it has evolved as a highly efficient means to edit/replace coding sequence, CRISPR/Cas9 efficiency for “clean” editing of non-coding DNA remains low. We set out to introduce a single base-pair substitution in two intronic SNPs at the FTO locus without altering nearby non-coding sequence. Substitution efficiency increased up to 10-fold by treatment of human embryonic stem cells (ESC) with non-toxic levels of DMSO (1%) before CRISPR/Cas9 delivery. Treatment with DMSO did not result in CRISPR/Cas9 off-target effects or compromise the chromosomal stability of the ESC. Twenty-four hour treatment of human ESC with DMSO before CRISPR/Cas9 delivery may prove a simple means to increase editing efficiency of non-coding DNA without incorporation of undesirable mutations.

Genetic variants within the FTO (α‐ketoglutarate‐dependent dioxygenase) gene have been strongly associated with a modest increase in adiposity as a result of increased food intake. These risk alleles are associated with decreased expression of both FTO and neighboring RPGRIP1L (retinitis pigmentosa GTPase regulator‐interacting protein 1 like). RPGRIP1L encodes a protein that is critical to the function of the primary cilium, which conveys extracellular information to the cell. Rpgrip1l ^{+/ −} mice exhibit increased adiposity, in part, as a result of hyperphagia. Here, we describe the effects of Rpgrip1l in adipocytes that may contribute to the adiposity phenotype observed in these animals and possibly in humans who segregate for FTO risk alleles. Loss of Rpgrip1l in 3T3‐L1 preadipocytes increased the number of cells that are capable of differentiating into mature adipocytes. Knockout of Rpgrip1l in mature adipocytes using Adipoq‐Cre did not increase adiposity in mice that were fed chow or a high‐fat diet. We also did not observe any effects of Rpgrip1l knockdown in mature 3T3‐L1 adipocytes. Thus, to the extent that Rpgrip1l affects cell‐autonomous adipose tissue function, it may do so as a result of the effects conveyed in preadipocytes in which the primary cilium is functionally important. We propose that decreased RPGRIP1L expression in preadipocytes in humans who segregate for FTO obesity risk alleles may increase the storage capacity of adipose tissue.—Martin Carli, J. F., LeDuc, C. A., Zhang, Y., Stratigopoulos, G., Leibel, R. L. The role of Rpgrip1l, a component of the primary cilium, in adipocyte development and function. FASEB J. 32, 3946–3956 (2018). www.fasebj.org