Setting the TRAP for central leptin targets

In this issue of Molecular Metabolism, Allison and her colleagues break new ground in our understanding of CNS leptin physiology [1]. By applying the sophisticated Translating Ribosome Affinity Purification (TRAP) technique in combination with RNA sequencing (TRAP-seq), they succeed in identifying novel markers for hypothalamic and brainstem subpopulations of leptin receptor (LepRb)-positive neurons with hitherto unknown function. Allison and her colleagues crossed Lepr mice with Rosa26 mice to express the ribosomal eGFP-L10a fusion protein solely in LepRb-positive cells [1]. Anti-eGFP TRAP was subsequently used on homogenates of hypothalami or brain stems from several mice to pull down mRNA attached to ribosomes of LepRb-positive cells (TRAPmRNA). RNA isolated from the TRAP-depleted homogenates (TRAPdepleted RNA) served as control. RNA sequencing and the subsequent comparative analysis of TRAP-mRNA vs. TRAP-depleted RNA revealed an enrichment of approx. 900 genes in brainstem LepRb cells and more than 1100 genes in hypothalamic LepRb cells. The presence of multiple genes known to be expressed in hypothalamic LepRb-positive neurons (e.g. Lepr, Agrp, Pomc) in the TRAP-mRNA fraction corroborated the overall TRAP-seq approach. Enrichment of marker genes for vasculature-associated cells (Flt1, Abcb1a, Tie1, Tek or Eltd1) was consistent with a role of LepRb in bloodebrain-barrier function [2]. However, the nearly complete absence of marker genes for glial or immune cells in the TRAP-mRNA fraction was surprising given the role of leptin signaling in astrocytes [3] and suggests failure of LepRbspecific TRAP to recover mRNA from many non-neuronal cell types. Overall, neuronal genes such as neuropeptides (Npw, Ucn, Prok2, Ghrh, Cartpt, Tac1) or marker genes for dopaminergic neurons (Slc6a3, Th) clearly prevailed in the hypothalamus and brainstemderived mRNA profiles. Among the highly expressed and -enriched neuropeptide-encoding transcripts were prodynorphin (Pdyn), tachykinin-1 (Tac1), corticotrophin releasing hormone (Crh), and growth hormone releasing hormone (Ghrh) that have never been associated with LepR-positive cells and may serve as novel markers for unknown neuronal LepRb subpopulations. TRAP-seq provides a unique opportunity to go beyond classical gene expression studies thate at beste reveal a blurred and heavily diluted snapshot of expression changes in a mixture of LepRb-positive and negative cells. LepRb-specific TRAP-seq overcomes those issues by solely purifying the translatome of LepRb-positive neurons. Nevertheless, a number of constraints for TRAP-seq remain. TRAP-seq doesn’t allow discriminating between mRNA from cells actively expressing