TRAP-seq defines markers for novel populations of hypothalamic and brainstem LepRb neurons

Abstract

ObjectiveLeptin acts via its receptor (LepRb) on multiple subpopulations of LepRb neurons in the brain, each of which controls specific aspects of energy balance. Despite the importance of LepRb-containing neurons, the transcriptome and molecular identity of many LepRb subpopulations remain undefined due to the difficulty of studying the small fraction of total cells represented by LepRb neurons in heterogeneous brain regions. Here we sought to examine the transcriptome of LepRb neurons directly and identify markers for functionally relevant LepRb subsets.MethodsWe isolated mRNA from mouse hypothalamic and brainstem LepRb cells by Translating Ribosome Affinity Purification (TRAP) and analyzed it by RNA-seq (TRAP-seq).ResultsTRAP mRNA from LepRb cells was enriched for markers of peptidergic neurons, while TRAP-depleted mRNA from non-LepRb cells was enriched for markers of glial and immune cells. Genes encoding secreted proteins that were enriched in hypothalamic and brainstem TRAP mRNA revealed subpopulations of LepRb neurons that contained neuropeptide-encoding genes (including prodynorphin, Pdyn) not previously used as functional markers for LepRb neurons. Furthermore, Pdyncre-mediated ablation of Leprflox in Pdyn-expressing neurons (LepRbPdynKO mice) blunted energy expenditure to promote obesity during high-fat feeding.ConclusionsTRAP-seq of CNS LepRb neurons defines the LepRb neuron transcriptome and reveals novel markers for previously unrecognized subpopulations of LepRb neurons.

A leptin-regulated circuit controls glucose mobilization during noxious stimuli

“…Statistical methods were not used to predetermine sample sizes, but our sample sizes are similar to those in previously published studies using similar approaches (8,17,(37)(38)(39). Sample sizes for groups that involved stereotaxic injection of specific populations of cells were 40% larger than for other groups to account for a 50%-60% bilateral hit rate.…”

Leptin Keeps Working, Even in Obesity

“…While Pomc (which encodes melanocortin peptides that are crucial for leptin action) expression is controlled by a number of convergent pathways, Socs3 is directly controlled by LepRb / STAT3 signaling and represents the best known readout of cell-autonomous transcriptional control by leptin (Allison et al, 2015;Bjørbaek et al, 1998). While LA suppresses Pomc expression similarly in DIO and lean animals, the inhibition of pSTAT3 and Socs3 expression by LA is actually greater in DIO animals than in lean controls, because (in the absence of LA) pSTAT3 and Socs3 expression are increased in DIO mice; the final (suppressed) levels are similar in the two LA-treated groups (Ottaway et al, 2015).…”

“…The present data suggest that while elevated Socs3 may limit the maximal amplitude of LepRb signaling in DIO mice, it does not reduce LepRb signaling below baseline. Indeed, since Socs3 expression is restricted to LepRb neurons in the hypothalamus and results from leptin / LepRb / STAT3 signaling ( Figure 1; Allison et al, 2015;Bjørbaek et al, 1998;Myers et al, 2012), how could Socs3 be elevated if LepRb signaling is decreased? What SOCS3 can do, though, is limit the maximum response to leptin; thus, while LepRb signaling is increased compared to lean animals in the face of elevated endogenous leptin in DIO, LepRb signaling in DIO is not as high as it might be in the absence of SOCS3, and SOCS3 likely limits the response to pharmacologic leptin (Figure 1; Bjö rnholm et al, 2007;Mori et al, 2004).…”

Guanylyl Cyclase C Hormone Axis at the Intersection of Obesity and Colorectal Cancer

“…Although the precise mechanisms mediating uroguanylininduced satiety continue to be defined (Valentino et al, 2011;Begg et al, 2014;Folgueira et al, 2016a;Kim et al, 2016), systemic uroguanylin levels increase postprandially in both mice and humans similarly to other anorexigenic hormones such as insulin, glucagon-like peptide 1, and cholecystokinin, suggesting a similar role for uroguanylin. GUCY2C expression in neurons expressing the leptin receptor (Allison et al, 2015), and activation-induced transcription of the anorexigenic second-messenger pro-opiomelanocortin, provide additional evidence supporting the role of the GUCY2C-uroguanylin gut-brain axis (Valentino et al, 2011;Kim et al, 2016). Indeed, Gucy2c 2/2 mice are hyperphagic and obese, suggesting that this regulatory pathway may play an important role in weight maintenance and the pathophysiology of obesity (Valentino et al, 2011).…”