{"database":"biostudies-arrayexpress","file_versions":[],"scores":null,"additional":{"submitter":["Brian Lam"],"organism":["Mus musculus"],"full_dataset_link":["https://www.ebi.ac.uk/biostudies/studies/E-MTAB-16665"],"description":["To identify the molecular signature of leucine-activated neurons in the MBH, we adopted the PhosphoTRAP assay following parenchymal injection of leucine into the MBH.  Neurons activated following MBH leucine injection (and expressing the neuronal activation marker cFos) co-express Ser240/244-phosphorylated ribosomal S6 protein , allowing selective immunoprecipitation-based capture (TRAP) and RNA sequencing of polysomes from leucine-activated neurons. Differential expression analysis between IP and input samples generated a list of enriched transcripts in leucine-activated neurons"],"repository":["biostudies-arrayexpress"],"sample_protocol":["Sequencing - Libraries were sequenced on an Illumina HiSeq 2000 Instrument at the CRUK CIGC","Nucleic Acid Extraction - RNA was extracted using the RNeasy Micro kit (Qiagen). RNA quantity was assessed using the Quant-iT RiboGreen RNA Assay kit (Thermo Fisher Scientific), and the samples’ RNA quality was assessed using Pico chips on an Agilent Bioanalyser. The samples were cleared from DNA contamination using a TURBO DNA-free kit (Thermo Fisher Scientific).","Library Construction - cDNA was prepared using a Smart Seq v4 Ultra Low Input RNA kit (Takara Clontech). Library preparation was done using Nextera XT Library Prepration kit (Illumina)","Sample Collection - To identify enriched transcripts in mediobasal hypothalamic neurons activated or inhibited by the administration leucine compared to aCSF, PhosphoTRAP assay was performed. 45 min after the MBH injection, mice were culled by overdose of anaesthetic (300 mg/kg I.P. sodium pentobarbitone; Euthatal Solution for Injection, Dopharma Research B.V.). The MBH was micro-dissected in an ice-cold Buffer B (Hanks Balanced Salt Solution (HBSS) with 2.5 mM of HEPES at pH 7.4, 4 mM of NaHCO3, 35 mM of glucose, and 100 mg/mL of cycloheximide in methanol) under a 10x dissecting microscope. The hypothalami of 5 mice were pooled as a single sample resulting in 4 replicates per experimental condition (i.e. aCSF vs. Leu). Pooled hypothalami were manually homogenized in a 1ml of buffer C (10 mM of HEPES at pH 7.4, 150 mM of KCl, 5 mM of MgCl2, 100 nM of calyculin A, 2 mM of DTT, 100 U/mL of RNasin, 100 mg/mL of cycloheximide, and Roche protease and phosphatase inhibitor cocktails) and clarified by centrifugation at 2,000 g at 4°C for 10 min. The supernatants were transferred to a new tube. 70μl of 10% NP40 and 70μl of DHPC (1,2-diheptanoyl-sn-glycero-3-phosphocholine) were added, the samples were then mixed by inversion and allowed for 2 min incubation on ice, and centrifuged at 16,100 g at 4°C for 10 min. Supernatants were transferred to a new tube. 25 μl of each sample was collected and subjected to RNA extraction as \\\"input samples\\\".  Prior to the immunoprecipitation step, 100μl Protein A Dynabeads (Invitrogen) were washed 3 times with buffer A (10mM HEPES, 150mM KCl, 5mM MgCl2 and 1% NP40 (pH 7.4) before incubation with 4μg pS6 antibody against Ser240/244 (#2215, Cell Signalling Technology) and 0.1% bovine serum albumin (BSA) in buffer A (300 μl per IP sample) at 4°C overnight with continuous mixing on an end-over-end rotator. On the next day, the antibody-bead conjugates were washed twice with wash buffer D (10 mM of HEPES, 350 mM of KCl, 5 mM of MgCl2, 2 mM of DTT, 1% NP40, 100 U/mL of RNasin, 100mg/mL of cycloheximide, and Roche protease and phosphatase inhibitor cocktails). After the last wash, the beads were resuspended in 200 μl of homogenisation buffer C supplemented with 50 μl of 10% NP40 and 10 μl of DHPC for 1 mL of buffer C and 1 μM of ZK10 (a gift from Dr. Zackary Knight, UCSF, US). The remaining sample supernatant was added to the antibody-beads conjugates, resuspended by pipetting and mixed with a rotator at 4°C for 10 min. The beads were washed 4 times with 0.9 mL of ice-cold wash buffer D and resuspended in 350 μl of RLT buffer and allowed for 5 min incubation on ice. The supernatant was then collected as “IP sample.”"],"figure_sub":["Organization","MINSEQE Score","Assays and Data","Processed Data","MAGE-TAB Files"],"data_protocol":["Data Transformation - Data were normalised to fragments per kilobase of mapped reads (FPKM) for the analysis, raw gene-level counts provided with this submission."],"omics_type":["Unknown","Transcriptomics","Genomics","Proteomics"],"instrument_platform":["Illumina HiSeq 2000"],"pubmed_abstract":["<h4>Summary</h4>  Hypothalamic leucine sensing promotes satiety and weight loss but an understanding of how leucine regulates neuronal activity is lacking. Here we show that  Cacna1g , encoding the T-type voltage-gated calcium channel Cav3.1, is enriched in hypothalamic leucine-sensing neurons and mediates leucine sensing. Pharmacological inhibition of Cav3.1 blunts leucine-induced activation of POMC neurons as well as the anorectic response to leucine in vivo. In addition, genetic deletion of  Cacna1g in POMC neurons abolishes the appetite- and weight-suppressive effects of high-protein feeding. Mechanistically, we show that leucine binds to the voltage-sensing segment of Cav3.1, thereby reducing its threshold for voltage-dependent activation. Last, pharmacological activation of hypothalamic Cav3.1 promotes weight loss in diet-induced obese mice and potentiates the weight loss response to GLP-1 receptor agonism. These results reveal that Cav3.1 is a neuronal leucine sensor and a relevant weight loss target."],"study_type":["RNA-seq of coding RNA"],"species":["Mus musculus"],"pubmed_title":["Cav3.1 is a leucine sensor in POMC neurons mediating appetite suppression and weight loss"],"pubmed_authors":["Brian Lam","Anthony H. Tsang, Nicholas Heeley, Constanza Alcaino, Eunsang Hwang, Brian Y. Lam, Taufiq Rahman, Tamana Darwish, Danae Nuzzaci, Richard G. Kay, Amar Sarkar, Ruiyan Wang, Nihal Basha, Austin Punnoose, Peter Kirwan, Marcella Ma, Giles S. Yeo, Florian T Merkle, Fiona M. Gribble, Frank Reimann, Kevin William, Clémence Blouet","Clemence Blouet"],"additional_accession":[]},"is_claimable":false,"name":"Molecular profiling of hypothalamic leucine-sensing neurons via PhosphoTRAP","description":"To identify the molecular signature of leucine-activated neurons in the MBH, we adopted the PhosphoTRAP assay following parenchymal injection of leucine into the MBH.  Neurons activated following MBH leucine injection (and expressing the neuronal activation marker cFos) co-express Ser240/244-phosphorylated ribosomal S6 protein , allowing selective immunoprecipitation-based capture (TRAP) and RNA sequencing of polysomes from leucine-activated neurons. Differential expression analysis between IP and input samples generated a list of enriched transcripts in leucine-activated neurons","dates":{"release":"2026-04-01T00:00:00Z","modification":"2026-04-02T01:05:05.677Z","creation":"2026-02-13T15:01:56.867Z"},"accession":"E-MTAB-16665","cross_references":{"ENA":["ERP189124"],"EFO":["EFO_0002944","EFO_0004170","EFO_0005518","EFO_0003816","EFO_0003738","EFO_0004184"],"doi":["10.1101/2024.09.13.612843"]}}