Project description:We performed a BacTRAP-based ribosome profiling of PNOC-expressing cells in the hypothalamus of mice. Therefore, we employed mice, which express a fusion protein of the ribosomal L10a protein with EGFP (L10a-EGFP) under control of the PNOC promoter (Doyle et al., 2008). Precipitation of ribosomes of hypothalamic PNOC neurons with anti-GFP antibodies and subsequent mRNA sequencing of associated mRNAs allowed for assessment of an in-depth translational profile of these cells. Affinity purification of translating ribosomes was performed as described by (Heiman et al., 2014) with minor modifications.
Project description:To identify transcripts enriched in ventomedial hypothalamus (VMH) neurons, we used translating affinity ribosome purification combined with RNA-seq (TRAP-seq) from Nr5a1-Cre mice.
Project description:To identify transcripts enriched in ventomedial hypothalamus (VMH) Lepr neurons, we used translating affinity ribosome purification combined with RNA-seq (TRAP-seq) from Lepr-Cre mice.
Project description:To identify transcripts enriched in ventomedial hypothalamus (VMH) neurons that contain both Lepr and Slc17a6, we used translating affinity ribosome purification combined with RNA-seq (TRAP-seq) from Lepr-Cre;Slc17a6-Flpo mice.
Project description:We show that leptin regulates appetite and body weight via PNOC neurons, and that loss of leptin receptor expression in PNOC-expressing neurons in the arcuate nucleus of the hypothalamus (ARC) causes hyperphagia and obesity. Lepr inactivation in PNOC neurons increases Npy expression in a subset of hypothalamic PNOC neurons that do not express Agrp and selective chemogenetic activation of PNOC/NPY neurons promotes feeding to the same extent as activating all PNOCARC neurons and overexpression of Npy in PNOCARC neurons promotes hyperphagia and obesity. Thus, we introduce PNOC/NPYARC neurons as an additional critical mediator of leptin action and a promising target for obesity therapeutics.
Project description:Localised protein translation, from subcellular localised mRNAs, has emerged as an important process of spatially-restricted cellular changes. This is especially important in cells with extremely elaborated architectures such as neurons. We present a highly sensitive TRAP protocol optimised for the specific isolation of ribosome-bound mRNAs in Drosophila from limited input samples, which enabled the isolation of axonal mRNAs from larval and adult (leg) motor neurons. RNA-seq revealed a set of axonally translated transcripts, including mRNAs encoding multiple ribosomal and mitochondrial proteins. Notably, these are among the types of transcripts found in axons of other species, indicating that axonal translation is conserved in Drosophila. Our optimised low-input TRAP method will allow the study of local translation to be explored in conjunction with Drosophila genetic manipulations, facilitating in-depth investigation of axonal translation across genetic backgrounds, developmental stages, and experimental conditions such as models of human disease. Moreover, it can be used for rare or genetically challenging genotypes and can be adapted to other tissues and model systems that may benefit from a sensitive TRAP protocol.
