Hypothalamic and Cell-Specific Transcriptomes Unravel a Dynamic Neuropil Remodeling in Leptin-Induced and Typical Pubertal Transition in Female Mice

Abstract

Summary Epidemiological and genome-wide association studies (GWAS) have shown high correlation between childhood obesity and advance in puberty. Early age at menarche is associated with a series of morbidities, including breast cancer, cardiovascular diseases, type 2 diabetes, and obesity. The adipocyte hormone leptin signals the amount of fat stores to the neuroendocrine reproductive axis via direct actions in the brain. Using mouse genetics, we and others have identified the hypothalamic ventral premammillary nucleus (PMv) and the agouti-related protein (AgRP) neurons in the arcuate nucleus (Arc) as primary targets of leptin action in pubertal maturation. However, the molecular mechanisms underlying leptin's effects remain unknown. Here we assessed changes in the PMv and Arc transcriptional program during leptin-stimulated and typical pubertal development using overlapping analysis of bulk RNA sequecing, TRAP sequencing, and the published database. Our findings demonstrate that dynamic somatodendritic remodeling and extracellular space organization underlie leptin-induced and typical pubertal maturation in female mice.

Protocol to extract actively translated mRNAs from mouse hypothalamus by translating ribosome affinity purification

“…For LepRb hypothalamic neurons, we pooled 3 mice per biological replicate and used 4 biological replicates (n=12 mice/experimental group). We found that using mice homozygous for both LepRb cre/cre and Rosa eGFP-L10a/ eGFP-L10a increases efficiency of the method ( Allison et al., 2018 ; Allison et al., 2015 ; Garcia-Galiano et al., 2017 ; Han et al., 2020 ). Make sure to screen for germline recombination in your mouse model to avoid harvesting the wrong cells and that Cre is expressed in the correct or expected cells by analysis of eGFP expression.…”

“… Reverse Transcribe 3–5 μL RNA of both enriched RNAs and depleted RNAs with the Reverse Transcriptase of your choice. We used Superscript II (Invitrogen) and the reaction was primed with both oligo dT and random hexamers ( Burger et al., 2018 ; Burger et al., 2020b ; Han et al., 2020 ; Ruka et al., 2013 ). We also recommend creating a standard curve.…”

“…However, other options for determining PCR efficiency (e.g., dilution series of amplicon) are also acceptable. cDNA from the depleted and enriched RNAs were amplified for Lepr (cell specific transcript) and Tnf and Ccl3 (micro glial transcripts, potential glial contaminants), and β-actin ( Actb ; example of a housekeeping transcript) using Taqman qPCR technology ( Han et al., 2020 ). Lepr , Tnf3 and Ccl3 were normalized to Actb using normalized relative expression, comparative cycle threshold method ( Bustin, 2002 ) and enrichment calculated as normalized relative expression in the enhanced RNA sample, divided by the normalized relative expression in the depleted sample.…”

“…We only make as much lysis buffer with the RNase inhibitors as necessary, due to cost. We found that rare hypothalamic cell types (e.g., LepRb and GnRH neurons) require increased concentration of RNAse inhibitors in the lysis buffer ( Burger et al., 2018 ; Han et al., 2020 ).…”

“…We use leptin receptor-expressing neurons as an example to illustrate the protocol. For complete details on the use and execution of this protocol, please refer to Han et al. (2020) .…”